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Sample records for human single-chain fv

  1. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

    Science.gov (United States)

    Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

    2015-12-01

    Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

  2. Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

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    Wu Anna M

    2009-12-01

    Full Text Available Abstract Background Antibody Directed Enzyme Prodrug Therapy (ADEPT can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent. Methods A double mutant of human purine nucleoside phosphorylase (hDM was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv, C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry. Results In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII

  3. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of

  4. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  5. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    OpenAIRE

    Matsukizono, Miho; Kamegawa, Mariko; Tanaka, Koichi; Kohra, Shinya; Arizono, Koji; Hamazoe, Yuta; Sugimura, Kazuhisa

    2013-01-01

    An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competi...

  6. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  7. Baculovirus display of single chain antibody (scFv using a novel signal peptide

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    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  8. Single Chain Fv Constructs of Anti-Ganglioside GD2 Antibodies for Radioimaging and Radioimmunotherapy

    International Nuclear Information System (INIS)

    Cheung, Nai-Kong

    2003-01-01

    because of its broad and usually homogeneous distribution in human solid tumors, and most importantly, their absence on cell membranes of normal human tissues. In separate experiments, we have shown that T-cells transduced with the herpes simplex viral thymidine kinase (HSV-tk) gene can be radiolabeled with 131 I-FIAU to a safe nuclear radiation dose. Using a dicistronic construct we are inserting chimeric immune receptor plus HSV-tk into T-cells to allow such their trafficking to be radioactively monitored. We plan to study the role of cytokines, chemoreceptors and CD4 helper T-cells in recruiting CD8+ transduced T-cells to the tumor site. These studies should provide us with an adoptive cell therapy approach to target cytotoxicity to human tumors, and a lymphocyte tracking tool to study delivery to the tumor sites, to determine if they proliferate locally and/or recirculate. Such pharmacologic information is crucial for optimizing gene-modified T-cells in future clinical trials. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging

  9. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    Directory of Open Access Journals (Sweden)

    Kazuhisa Sugimura

    2013-02-01

    Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

  10. Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

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    Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

    2002-01-01

    AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

  11. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

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    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.

  12. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Abstract. Purpose: To develop specific single chain variable fragments (scFv) against ... libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was ..... Hermelink H, Koscielniak E. A human recombinant.

  13. The use of a cocktail of single chain Fv antibody fragments to improve the in vitro and in vivo targeting of melanoma

    International Nuclear Information System (INIS)

    Pacifico, M.D.; Pearl, R.A.; Kupsch, J.M.

    2006-01-01

    Radio scintigraphy using single chain antibody fragments (scFvs) offers a potenti al means of early detection of melanoma metastases. However, previous studies have shown suboptimal levels of tumour localization and nonspecific background accumulation which may be due to antigen heterogeneity. We aimed to improve tumour localization by using a cocktail of different scFvs targeting different epitopes on melanoma cells. We have previously developed three scFvs against distinct and highly tumour-specific melanoma cell-surface antigens by chain shuffling and antibody phage selection on melanoma cells. Three scFvs, RAFT3, B3 and B4 were labeled with 1 25I odine and tested both individually and as a cocktail in a nude mouse xenograft model far human melanoma. Results demonstrated improved tumour localization in vivo when compared to the individual scFvs. Tumour uptake of the cocktail at l hour was 24.220% ID/g (injected dose/gram) compared with 2.854%, 2.263% and 1.355% far B4, RAFT3 and B3, respectively, when injected individually. In addition, the cocktail exhibited significantly superior tumour to normal tissue ratios far muscle and spleen (p<0.05). A combination or cocktail of scFv clones may have an advantage aver individual scFvs far melanoma targeting in patients because of heterogeneity in the expression of different epitopes of antigens on melanoma cells

  14. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

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    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  15. IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

    Science.gov (United States)

    Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

    2017-06-26

    IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

  16. Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

    Science.gov (United States)

    Kumada, Yoichi; Ishikawa, Yasuyuki; Fujiwara, Yusuke; Takeda, Rui; Miyamoto, Ryosuke; Niwa, Daisuke; Momose, Shun; Kang, Bongmun; Kishimoto, Michimasa

    2014-09-01

    In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results

  17. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

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    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

  18. Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.

    Science.gov (United States)

    Jung, Yuchul; Rhee, Yong; Auh, Chung-Kyoon; Shim, Hyekyung; Choi, Jung-Jin; Kwon, Suk-Tae; Yang, Joo-Sung; Kim, Donggiun; Kwon, Myung-Hee; Kim, Yong-Sung; Lee, Sukchan

    2009-10-01

    We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.

  19. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Los Alamos National Laboratory; Velappan, Nileena [Los Alamos National Laboratory; Schmidt, Jurgen G [Los Alamos National Laboratory

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  20. Expression, production and renaturation of a functional single-chain ...

    African Journals Online (AJOL)

    The single-chain variable antibody fragment (scFv) against human intercellular adhesion molecule-1 (ICAM-1) was expressed at a high level in Escherichia coli as inclusion bodies. We attempted to refold the scFv by ion-exchange chromatography (IEC), dialysis and dilution. The results show that the column ...

  1. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  2. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  3. Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

    Science.gov (United States)

    Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

  4. Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

    Science.gov (United States)

    Banisadr, Arsham; Safdari, Yaghoub; Kianmehr, Anvarsadat; Pourafshar, Mahdieh

    2018-04-03

    The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly 4 Ser) 3 ] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

  5. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    Directory of Open Access Journals (Sweden)

    Merima Bublin

    Full Text Available Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1, carp (Cyp c 1 and rainbow trout (Onc m 1 parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

  6. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

    Science.gov (United States)

    Bublin, Merima; Kostadinova, Maria; Fuchs, Julian E; Ackerbauer, Daniela; Moraes, Adolfo H; Almeida, Fabio C L; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

  7. Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

    Science.gov (United States)

    Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

    2002-06-28

    MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

  8. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

    Science.gov (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

    2017-01-01

    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  9. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

    Directory of Open Access Journals (Sweden)

    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  10. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  11. The biodistribution and pretargeting radioimmunoimaging of the fusion protein of anti-CEA single-chain antibody and core-streptavidin in human rectocolonic tumor bearing nude mice

    International Nuclear Information System (INIS)

    Yang Weidong; Li Biao; Zhu Chengmo; Jiang Xufeng; Feng Guowei; Wu Xiangpu

    2002-01-01

    Objective: To investigate the biodistribution and two-step pretargeting radioimmunoimaging of the fusion protein of anti-carcinoembryonic antigen (CEA) single-chain antibody (ScFv) and core-streptavidin in human rectocolonic tumor bearing nude mice. Methods: Before the injection of 153 Sm-biotin, the fusion protein of ScFv-core-streptavidin was pretargeted for 24 h (200 μg every nude mouse), 24 h later 153 Sm-biotin was injected. The uptake of radioactivity in tumor and normal tissues in 20 nude mice was measured at 1, 4, 8 and 24 h and the other 3 nude mice was scanned at 8 and 24 h after peritoneal injection of 153 Sm-biotin. Results: The tumor to blood ratio (tumor/blood) reached 0.49 , 1.21, 1.56 and 3.09 at 1, 4, 8 and 24 h respectively. Radioactivity concentration peaked at 8 h in tumor site and demonstrated a 'hot' area, with significant decreasing its background at 24 h. Conclusion: The fusion protein can elevate the tumor/blood ratio, shorten pretargeting and imaging process and also improve image quality

  12. Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

    Science.gov (United States)

    Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

    2017-02-01

    Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects

    NARCIS (Netherlands)

    Braat, E. A.; Levi, M. [=Marcel M.; Bos, R.; Haverkate, F.; Lassen, M. R.; de Maat, M. P.; Rijken, D. C.

    1999-01-01

    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence

  14. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  15. Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

    Science.gov (United States)

    Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

    2016-03-15

    Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  17. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  18. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  19. Isolation and characterisation of a human-like antibody fragment (scFv that inactivates VEEV in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Torsten Rülker

    Full Text Available Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv, ToR67-3B4, from a non-human primate (NHP antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

  20. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Shuli [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Nanjing Affiliated First Hospital, Nanjing Medical University, Nanjing (China); Zhao, Guangfeng; Xie, Hao; Huang, Yahong [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Hou, Yayi [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing (China)

    2012-01-27

    Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex){sub 1.3}(DOX){sub 20}. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

  1. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    International Nuclear Information System (INIS)

    Zhao, Shuli; Zhao, Guangfeng; Xie, Hao; Huang, Yahong; Hou, Yayi

    2012-01-01

    Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex) 1.3 (DOX) 20 . In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers

  2. The in vivo characteristics of genetically engineered divalent and tetravalent single-chain antibody constructs

    International Nuclear Information System (INIS)

    Wittel, Uwe A.; Jain, Maneesh; Goel, Apollina; Chauhan, Subhash C.; Colcher, David; Batra, Surinder K.

    2005-01-01

    Engineered multivalent single-chain Fv (scFv) constructs have been demonstrated to exhibit rapid blood clearance and better tumor penetration. To understand the short plasma half-life of multivalent single-chain antibody fragments, the pharmacokinetic properties of covalent dimeric scFv [sc(Fv) 2 ], noncovalent tetrameric scFv {[sc(Fv) 2 ] 2 } and IgG of MAb CC49 were examined. The scFvs displayed an ability to form higher molecular aggregates in vivo. A specific proteolytic cleavage of the linker sequence of the covalent dimeric or a deterioration of the noncovalent association of the dimeric scFv into tetravalent scFv constructs was not observed. In conclusion, sc(Fv) 2 and [sc(Fv) 2 ] 2 are stable in vivo and have significant potential for diagnostic and therapeutic applications

  3. Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

    Directory of Open Access Journals (Sweden)

    Woitok M

    2017-07-01

    Full Text Available Mira Woitok,1,2 Diana Klose,1 Stefano Di Fiore,1 Wolfgang Richter,3 Christoph Stein,1 Gerrit Gresch,1 Elena Grieger,1 Stefan Barth,1 Rainer Fischer,1,2 Katharina Kolberg,1,* Judith Niesen1,* 1Fraunhofer Institute for Molecular Biology and Applied Ecology (IME, Aachen, Germany; 2Institute of Molecular Biotechnology (Biology VII, RWTH Aachen University, Aachen, Germany; 3Tube Pharmaceuticals GmbH, Vienna, Austria *These authors contributed equally to this work Abstract: Antibody–drug conjugates (ADCs can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs with 1:1 stoichio­metry while preserving antigen binding. Two (human and mouse scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF. Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to

  4. Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

    Science.gov (United States)

    Molek, Peter; Vodnik, Miha; Strukelj, Borut; Bratkovič, Tomaž

    2014-09-26

    Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    Directory of Open Access Journals (Sweden)

    Kristensen Peter

    2009-01-01

    Full Text Available Abstract Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

  6. A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid-β oligomers.

    Science.gov (United States)

    Sebollela, Adriano; Cline, Erika N; Popova, Izolda; Luo, Kevin; Sun, Xiaoxia; Ahn, Jay; Barcelos, Milena A; Bezerra, Vanessa N; Lyra E Silva, Natalia M; Patel, Jason; Pinheiro, Nathalia R; Qin, Lei A; Kamel, Josette M; Weng, Anthea; DiNunno, Nadia; Bebenek, Adrian M; Velasco, Pauline T; Viola, Kirsten L; Lacor, Pascale N; Ferreira, Sergio T; Klein, William L

    2017-07-03

    Brain accumulation of soluble oligomers of the amyloid-β peptide (AβOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AβO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AβO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AβOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AβO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AβOs from both monomeric and fibrillar Aβ. NUsc1 readily detected AβOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AβO binding and reduced AβO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AβOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AβOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AβO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics. © 2017 International Society for Neurochemistry.

  7. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  8. Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

    Science.gov (United States)

    Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2017-04-28

    The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

  9. Species-Dependent Functionality of the Human Cytolytic Fusion Proteins Granzyme B-H22(scFv and H22(scFv-Angiogenin in Macrophages

    Directory of Open Access Journals (Sweden)

    Theo Thepen

    2013-01-01

    Full Text Available Human cytolytic fusion proteins (hCFPs are comprised of a specific cell-surface-binding moiety and an effector molecule of human origin. In contrast to common immunotoxins, including bacterial or plant toxins, they are considered not to be immunogenic. Two examples for human pro-apoptotic effector proteins are the serine protease Granzyme B and the RNase Angiogenin. Pre-clinical testing of functionality in in vitro and in vivo studies is essential for therapeutics. Establishing relevant animal models that have predictive value for therapeutic success is a great challenge in biomedical research. In this study, we investigated the species-dependent cytotoxic activity of two hCFPs prior to their application in a murine inflammation model. We found that in vitro and ex vivo either hCFP was able to kill human cells only, leaving murine cells unaffected. In contrast, no species-dependency was found for the bacterial Pseudomonas exotoxin A based immunotoxin H22(scFv-ETA’. This species-dependent functioning has to be carefully considered when performing pre-clinical studies in animal models.

  10. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    Energy Technology Data Exchange (ETDEWEB)

    Junqing, Guo; Liu, Chen; Hongwu, Ai; Jiannian, Jing; Jiyong, Zhou; Chuyu, Zhang; Shangyou, You

    2004-07-23

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-{gamma} inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V{sub H} region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

  11. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    International Nuclear Information System (INIS)

    Guo Junqing; Chen Liu; Ai Hongwu; Jing Jiannian; Zhou Jiyong; Zhang Chuyu; You Shangyou

    2004-01-01

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-γ inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V H region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo

  12. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    International Nuclear Information System (INIS)

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture

  13. Advances in single chain technology.

    Science.gov (United States)

    Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

    2015-10-07

    The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses.

  14. Targeting nanodisks via a single chain variable antibody - Apolipoprotein chimera

    International Nuclear Information System (INIS)

    Iovannisci, David M.; Beckstead, Jennifer A.; Ryan, Robert O.

    2009-01-01

    Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that α-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

  15. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  16. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  17. Construction, expression, and function of 6B11ScFv-mIL-12, a fusion protein that attacks human ovarian carcinoma.

    Science.gov (United States)

    Cheng, Hongyan; Ye, Xue; Chang, Xiaohong; Ma, Ruiqiong; Cong, Xu; Niu, Yidong; Zhang, Menglei; Liu, Kai; Cui, Heng; Sang, Jianli

    2015-04-01

    We previously produced an anti-idiotypic monoclonal antibody, 6B11, which mimics ovarian cancer antigen CA166-9 and induces cellular and humoral immunity. Here, to enhance the immunogenicity of 6B11, we constructed the 6B11ScFv-mIL-12 fusion protein (FP), by fusing single-chain fragment of 6B11 variable region (6B11ScFv) with mouse interleukin-12 (mIL-12), which was expressed in eukaryotic 293EBNA cells transfected with pSBI vectors. A binding activity assay showed 6B11ScFv-mIL-12 to have activities of both 6B11 and mIL-12-it specifically bound both ovarian monoclonal antibody COC166-9 and rabbit anti-mouse IL-12 antibody. The immune activity assay showed 6B11ScFv-mIL-12 to promote proliferation of lymphocytes stimulated by phytohemagglutinin, increase the absolute numbers and percentages of CD3(-)/CD56(+) natural killer cells and CD3(+)/CD56(+) natural killer T cells among peripheral lymphocytes, and increase interferon-γ. The FP was specifically cytotoxic to the CA166-9(+) ovarian cancer cell lines HOC1A and SKOV3 and inhibited growth of ID8 subcutaneous tumors in C57BL/6J mice. This study provides an experimental basis for clinical use of 6B11ScFv-mIL-12 in ovarian cancer therapy. To our knowledge, this is the first report of a fusion protein from an anti-idiotypic antibody and IL-12.

  18. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

    Directory of Open Access Journals (Sweden)

    Zu-Quan Hu

    2012-06-01

    Full Text Available Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10−2 μg/mL and contaminating mycelium at a quantity of 10−2 mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

  20. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    Surinder Batra

    2006-01-01

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  1. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  2. Construção de uma biblioteca de fragmentos de anticorpos monoclonais de galinhas com cadeia única (scFv por phage display com reatividade cruzada para estirpes heterólogas do vírus da bronquite infecciosa aviária Construction of an avian single chain monoclonal antibodies (scFv library by phage display that cross-reacted with heterologous avian infectious bronchitis virus strains

    Directory of Open Access Journals (Sweden)

    Camila Cesário Fernandes

    2010-06-01

    Full Text Available Anticorpos monoclonais constituem a base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI. No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por phage display contra a estirpe vacinal (H120 do VBI foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01 e IBVPR05, isoladas de surtos a campo no Brasil e a estirpe SE-17, isolada nos Estados Unidos. Após três ciclos de panning, foi identificado, pelo ELISA, um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que dois desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzidos em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus.Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of infectious bronchitis outbreaks. These antibodies are homogeneous, highly specific and fully characterizable, allowing the improvement of immunological techniques detection and antigenic characterization of avian infectious

  3. Detection of Metallothionein in Javanese Medaka (Oryzias javanicus, Using a scFv-Immobilized Protein Chip

    Directory of Open Access Journals (Sweden)

    Euiyeon Lee

    2018-04-01

    Full Text Available Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT. OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3 in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time.

  4. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

    Science.gov (United States)

    Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

    2016-08-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

  5. Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Yijia [Department; Ford, Nicole R. [Marine; Hecht, Karen A. [Marine; Roesijadi, Guritno [Marine; Department; Squier, Thomas C. [Department

    2017-10-12

    Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv or the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

  6. INCREASING OF THE EXPRESSION OF RECOMBINANT scFv-ANTIBODIES EFFICIENCY

    Directory of Open Access Journals (Sweden)

    O.V. Galkin

    2017-10-01

    Full Text Available Obtaining single-chain variable fragments (scFv of recombinant antibodies in E. coli cells is often associated with numerous problems causing low yields or inactive conformation of the product. The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins. Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

  7. Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo

    International Nuclear Information System (INIS)

    Du, Li; Zhou, Ping-Kun; Zhou, Li-Jun; Pan, Xiu-Jie; Wang, Yu-Xiao; Xu, Qin-Zhi; Yang, Zhi-Hua; Wang, Yu; Liu, Xiao-Dan; Zhu, Mao-Xiang

    2010-01-01

    Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of γH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of γH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential

  8. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  9. Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

    Science.gov (United States)

    Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

    2012-08-22

    We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

  10. Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

    Directory of Open Access Journals (Sweden)

    Witchuda Danpaiboon

    2014-05-01

    Full Text Available Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase. N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

  11. Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

    Science.gov (United States)

    Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

    2014-05-13

    Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

  12. Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

    International Nuclear Information System (INIS)

    Yazaki, Paul J.; Kassa, Thewodros; Cheung, Chia-wei; Crow, Desiree M.; Sherman, Mark A.; Bading, James R.; Anderson, Anne-Line J.; Colcher, David; Raubitschek, Andrew

    2008-01-01

    Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [ 125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [ 111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [ 64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents

  13. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    Science.gov (United States)

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  14. Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

    Science.gov (United States)

    Cui, Xiping; Vasylieva, Natalia; Wu, Panpan; Barnych, Bogdan; Yang, Jun; Shen, Ding; He, Qiyi; Gee, Shirley J; Zhao, Suqing; Hammock, Bruce D

    2017-10-17

    Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC 50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

  15. Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

    DEFF Research Database (Denmark)

    Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

    2011-01-01

    We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

  16. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee

    2017-10-01

    Full Text Available Russell’s vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs containing 3.4 × 107 and 5.5 × 107 transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  17. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis.

    Science.gov (United States)

    Lee, Chi-Hsin; Lee, Yu-Ching; Lee, Yueh-Lun; Leu, Sy-Jye; Lin, Liang-Tzung; Chen, Chi-Ching; Chiang, Jen-Ron; Mwale, Pharaoh Fellow; Tsai, Bor-Yu; Hung, Ching-Sheng; Yang, Yi-Yuan

    2017-10-27

    Russell's vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY) antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs) containing 3.4 × 10⁷ and 5.5 × 10⁷ transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  18. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

    Science.gov (United States)

    Patel, Rekha; Andrien, Bruce A

    2010-01-01

    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.

  19. Pressure effects on single chain magnets

    Energy Technology Data Exchange (ETDEWEB)

    Mito, M. E-mail: mitoh@elcs.kyutech.ac.jp; Shindo, N.; Tajiri, T.; Deguchi, H.; Takagi, S.; Miyasaka, H.; Yamashita, M.; Clerac, R.; Coulon, C

    2004-05-01

    Pressure effects on a single chain magnet [Mn{sub 2}(saltmen){sub 2}Ni(pao){sub 2}(py){sub 2}](ClO{sub 4}){sub 2} (saltmen{sup 2-}=N,N'-(1,1,2,2-tetramethylethylene)bis(salicylideneiminate), and pao{sup -}=pyridine-2-aldoximate) have been investigated through AC magnetic measurements under pressure (P). The slow relaxation of the magnetization depends on pressure. Both the blocking temperature (T{sub B}) and energy barrier ({delta}) increase by pressurization, and those enhancements saturate at around P=7 kbar.

  20. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  1. PET Imaging of 64Cu-DOTA-scFv-Anti-PSMA Lipid Nanoparticles (LNPs): Enhanced Tumor Targeting over Anti-PSMA scFv or Untargeted LNPs

    Science.gov (United States)

    Wong, Patty; Li, Lin; Chea, Junie; Delgado, Melissa K.; Crow, Desiree; Poku, Erasmus; Szpikowska, Barbara; Bowles, Nicole; Channappa, Divya; Colcher, David; Wong, Jeffrey Y.C.; Shively, John E.; Yazaki, Paul J.

    2017-01-01

    Introduction Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in Knowledge and implications for patient care Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to a LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery. PMID:28126683

  2. PET imaging of 64Cu-DOTA-scFv-anti-PSMA lipid nanoparticles (LNPs): Enhanced tumor targeting over anti-PSMA scFv or untargeted LNPs

    International Nuclear Information System (INIS)

    Wong, Patty; Li, Lin; Chea, Junie; Delgado, Melissa K.; Crow, Desiree; Poku, Erasmus; Szpikowska, Barbara; Bowles, Nicole; Channappa, Divya; Colcher, David; Wong, Jeffrey Y.C.; Shively, John E.; Yazaki, Paul J.

    2017-01-01

    Introduction: Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods: An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results: The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions: The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in knowledge and implications for patient care: Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to an LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery.

  3. Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

    International Nuclear Information System (INIS)

    Liu, Yang; Zhou, Huihao; Zhu, Juanjuan; Gao, Yongxiang; Niu, Liwen; Liu, Jing; Teng, Maikun

    2009-01-01

    An antibody–antigen complex consisting of a single-chain variable fragment of the potential therapeutic antibody chA21 and an N-terminal fragment (residues 1–192) of the human ErbB2 extracellular domain was expressed, purified and crystallized. X-ray diffraction data were collected to 2.45 Å resolution. ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody–antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1–192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 Å resolution from a single flash-cooled crystal; the crystal belonged to space group P2 1 2 1 2 1

  4. Elementary excitations in single-chain magnets

    Science.gov (United States)

    Lutz, Philipp; Aguilà, David; Mondal, Abhishake; Pinkowicz, Dawid; Marx, Raphael; Neugebauer, Petr; Fâk, Björn; Ollivier, Jacques; Clérac, Rodolphe; van Slageren, Joris

    2017-09-01

    Single-chain magnets (SCMs) are one-dimensional coordination polymers or spin chains that display slow relaxation of the magnetization. Typically their static magnetic properties are described by the Heisenberg model, while the description of their dynamic magnetic properties is based on an Ising-like model. The types of excitations predicted by these models (collective vs localized) are quite different. Therefore we probed the nature of the elementary excitations for two SCMs abbreviated Mn2Ni and Mn2Fe , as well as a mononuclear derivative of the Mn2Fe chain, by means of high-frequency electron paramagnetic resonance spectroscopy (HFEPR) and inelastic neutron scattering (INS). We find that the HFEPR spectra of the chains are clearly distinct from those of the monomer. The momentum transfer dependence of the INS intensity did not reveal significant dispersion, indicating an essentially localized nature of the excitations. At the lowest temperatures these are modified by the occurrence of short-range correlations.

  5. Domain walls in single-chain magnets

    Science.gov (United States)

    Pianet, Vivien; Urdampilleta, Matias; Colin, Thierry; Clérac, Rodolphe; Coulon, Claude

    2017-12-01

    The topology and creation energy of domain walls in different magnetic chains (called Single-Chain Magnets or SCMs) are discussed. As these domain walls, that can be seen as "defects", are known to control both static and dynamic properties of these one-dimensional systems, their study and understanding are necessary first steps before a deeper discussion of the SCM properties at finite temperature. The starting point of the paper is the simple regular ferromagnetic chain for which the characteristics of the domain walls are well known. Then two cases will be discussed (i) the "mixed chains" in which isotropic and anisotropic classical spins alternate, and (ii) the so-called "canted chains" where two different easy axis directions are present. In particular, we show that "strictly narrow" domain walls no longer exist in these more complex cases, while a cascade of phase transitions is found for canted chains as the canting angle approaches 45∘. The consequence for thermodynamic properties is briefly discussed in the last part of the paper.

  6. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

    Directory of Open Access Journals (Sweden)

    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  7. Construction of a recombinant single chain antibody recognizing nonreducing terminal mannose residues applicable to immunohistochemistry.

    Science.gov (United States)

    Yuasa, Noriyuki; Iida, Noriko; Sakaue, Hiroyuki; Zhang, Wei; Wilczynski, Sharon; Fujita-Yamaguchi, Yoko

    2007-10-01

    We recently reported characterization of 25 clones isolated from a phage library displaying human scFvs using a neoglycolipid Man3-DPPE, which was synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE). Of those, 5A3 scFv was successfully expressed and purified as a humanized scFv-Fc form (Sakai et al., Biochemistry 46:253, 2007, Zhang et al. ibid 263). To carry out immunohistochemistry (IHC) in human tissues, a HA tag sequence was introduced to the 5A3 scFv-Fc gene and the resulting construct was transfected to murine myeloma NS0 cells. The 5A3 scFv-Fc protein expressed was affinity-purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing and reducing conditions and enzyme-linked immunosorbent assay confirmed that 5A3 scFv-Fc protein is dimeric and retained the ability to recognize nonreducing terminal mannose residues. IHC staining of non-neoplastic tissues by this recombinant antibody revealed that no immunoreactivity was detectable in most of 16 tissues examined. Exceptions were found in IHC staining of kidney and pancreas, which demonstrated clear staining of proximal tubules and islet of Langerhans, respectively. These results demonstrated that nonreducing terminal mannose residues are not usually present under normal physiological conditions. This study thus provided a potentially useful tool for examination of the nonreducing terminal mannose residues, which may become exposed under certain pathophysiologycal conditions.

  8. Single-Chain Folding of Synthetic Polymers: A Critical Update.

    Science.gov (United States)

    Altintas, Ozcan; Barner-Kowollik, Christopher

    2015-11-23

    The current contribution serves as a critical update to a previous feature article from us (Macromol. Rapid Commun. 2012, 33, 958-971), and highlights the latest advances in the preparation of single chain polymeric nanoparticles and initial-yet promising-attempts towards mimicking the structure of natural biomacromolecules via single-chain folding of well-defined linear polymers via so-called single chain selective point folding and repeat unit folding. The contribution covers selected examples from the literature published up to ca. September 2015. Our aim is not to provide an exhaustive review but rather highlight a selection of new and exciting examples for single-chain folding based on advanced macromolecular precision chemistry. Initially, the discussion focuses on the synthesis and characterization of single-chain folded structures via selective point folding. The second part of the feature article addresses the folding of well-defined single-chain polymers by means of repeat unit folding. The current state of the art in the field of single-chain folding indicates that repeat unit folding-driven nanoparticle preparation is well-advanced, while initial encouraging steps towards building selective point folding systems have been taken. In addition, a summary of the-in our view-open key questions is provided that may guide future biomimetic design efforts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  10. Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

    Science.gov (United States)

    Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

    2011-08-05

    Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

  11. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    Science.gov (United States)

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. © FASEB.

  12. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. Copyright © 2015. Published by Elsevier Ltd.

  13. Arg9 facilitates the translocation and downstream signal inhibition of an anti-HER2 single chain antibody

    Directory of Open Access Journals (Sweden)

    Hu Yi

    2012-07-01

    Full Text Available Abstract Background HER2 plays a critical role in the pathogenesis of many cancers and is linked to poor prognosis or cancer metastases. Monoclonal antibodies, such as Herceptin against HER2-overexpressing cancers, have showed satisfactory clinical therapeutic effect. However, they have difficulty to surmount obstacles to enter cells or blood–brain barrier. Results In this study, a cell-penetrating peptide Arg9 was linked to the C-terminus of anti-HER2 single chain antibody (MIL5scFv. Flow cytometry, confocal microscopy and electron microscopy analysis all revealed that Arg9 peptide facilitated the penetration of MIL5scFv into HER2-negative cell line NIH3T3 and orientate in mitochondria. More interestingly, Western blot assay showed the potential enhanced bioactivity of MIL5scFv-Arg9 in HER2+ cell line SKOV3, indicating that Arg9 could help large molecules (e.g. antibody to penetrate into cells and therefore enhance its anti-neoplastic function. Conclusions Our work represented an attractive by preliminary strategy to enhance the therapeutic effect of existing antibodies by entering cells easier, or more desirable, surmounting the physical barriers, especially in hard-to-reach cancers such as brain metastases cases.

  14. ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

    Science.gov (United States)

    Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

    2014-10-01

    Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

  15. Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

    Science.gov (United States)

    Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

    2014-01-01

    Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

  16. Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

    Science.gov (United States)

    Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

    2016-11-01

    Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

  17. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

    Science.gov (United States)

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

    2015-11-01

    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  19. Development of two dimensional electrophoresis method using single chain DNA

    International Nuclear Information System (INIS)

    Ikeda, Junichi; Hidaka, So

    1998-01-01

    By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

  20. Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

    Directory of Open Access Journals (Sweden)

    Chen H

    2013-10-01

    Full Text Available Hongwei Chen,1,* Liya Wang,1,2,* Qiqi Yu,1,2 Weiping Qian,3 Diana Tiwari,1 Hong Yi,4 Andrew Y Wang,5 Jing Huang,1,2 Lily Yang,3 Hui Mao1,2 1Department of Radiology and Imaging Sciences, 2Center for Systems Imaging, 3Department of Surgery, Emory University School of Medicine, 4Robert Apkarian Electron Microscopy Core, Emory University, Atlanta, GA, 5Ocean NanoTech LLC, Springdale, AK, USA *These authors contributed equally to this work Abstract: Antifouling magnetic iron oxide nanoparticles (IONPs coated with block copolymer poly(ethylene oxide-block-poly(γ-methacryloxypropyltrimethoxysilane (PEO-b-PγMPS were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv of antibody against epidermal growth factor receptor (ScFvEGFR to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs. The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs

  1. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

    Science.gov (United States)

    Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

    2016-10-01

    We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

  2. Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo.

    Science.gov (United States)

    Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Li, Zhuang; Cheng, Yue; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2017-06-01

    Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (V H ) and light (V L ) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides V H -JUN-HIS and V L -FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of V H and V L . The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency. Copyright © 2017. Published by Elsevier B.V.

  3. Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.

    Directory of Open Access Journals (Sweden)

    Ran Taube

    Full Text Available BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

  4. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    International Nuclear Information System (INIS)

    Hu, Zu-Quan; Li, He-Ping; Zhang, Jing-Bo; Huang, Tao; Liu, Jin-Long; Xue, Sheng; Wu, Ai-Bo; Liao, Yu-Cai

    2013-01-01

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to

  5. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  6. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro

    2012-09-01

    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  7. Monte Carlo simulated dynamical magnetization of single-chain magnets

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun; Liu, Bang-Gui, E-mail: bgliu@iphy.ac.cn

    2015-03-15

    Here, a dynamical Monte-Carlo (DMC) method is used to study temperature-dependent dynamical magnetization of famous Mn{sub 2}Ni system as typical example of single-chain magnets with strong magnetic anisotropy. Simulated magnetization curves are in good agreement with experimental results under typical temperatures and sweeping rates, and simulated coercive fields as functions of temperature are also consistent with experimental curves. Further analysis indicates that the magnetization reversal is determined by both thermal-activated effects and quantum spin tunnelings. These can help explore basic properties and applications of such important magnetic systems. - Highlights: • Monte Carlo simulated magnetization curves are in good agreement with experimental results. • Simulated coercive fields as functions of temperature are consistent with experimental results. • The magnetization reversal is understood in terms of the Monte Carlo simulations.

  8. Novel scenario of the folding transition of a single chain

    International Nuclear Information System (INIS)

    Yoshikawa, Kenichi; Yoshinaga, Natsuhiko

    2005-01-01

    Unique characteristics of a single polymer chain with the effects of stiffness and charge are discussed. It has been well established that a flexible polymer chain undergoes a continuous transition from an elongated coil to a compact globule, corresponding to the transition between disordered gas-like and disordered liquid-like states. Here, we will show that a semiflexible chain exhibits a discrete transition from coil to compact states, corresponding to a disorder-order transition to an ordered crystalline state. We will propose a novel strategy to obtain various kinds of nano-ordered structures from single chains connecting a pair of chains of different stiffness. We will also discuss the effect of charge, putting emphasis on intramolecular segregation in a single polyelectrolyte chain

  9. Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155

    Directory of Open Access Journals (Sweden)

    J. Mala

    2017-06-01

    Full Text Available Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155 were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1 was confirmed by sequencing analysis. The complementarity determining regions (CDR I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

  10. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    International Nuclear Information System (INIS)

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-01-01

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10 -9 M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  11. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  12. Characterization of a single-chain variable fragment recognizing a linear epitope of aβ: a biotechnical tool for studies on Alzheimer's disease?

    Directory of Open Access Journals (Sweden)

    Silke Dornieden

    Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs, Aβ binding single-chain variable fragments (scFvs were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8. scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD.

  13. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

    Energy Technology Data Exchange (ETDEWEB)

    Dong Jiexian; Li Zhenfeng; Lei Hongtao; Sun Yuanming [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Ducancel, Frederic [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Xu Zhenlin [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Boulain, Jean-Claude [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Yang Jinyi; Shen Yudong [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Wang Hong, E-mail: gzwhongd@63.com [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China)

    2012-07-29

    Graphical abstract: Detection model of dc-CLEIA based on anti-RAC scFv-AP fusion protein. Highlights: Black-Right-Pointing-Pointer The scFv-AP fusion protein against ractopamine (RAC) was produced. Black-Right-Pointing-Pointer A dc-CLEIA for RAC was developed based on the purified scFv-AP fusion protein. Black-Right-Pointing-Pointer The sensitivity of dc-CLEIA was 10 times as sensitive as dc-ELISA for RAC. Black-Right-Pointing-Pointer Recovery tests from pork samples were studied. Black-Right-Pointing-Pointer Good accuracy was obtained. - Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V{sub H} and V{sub L}) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V{sub H} and V{sub L} genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 {+-} 0.03 and 0.02 {+-} 0.004 ng mL{sup -1}, respectively, and the linear response range extended from 0.05 to 1.45 ng mL{sup -1}. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between

  14. [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

    Science.gov (United States)

    Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

    2017-03-01

    Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

  15. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

    Science.gov (United States)

    Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

    2018-04-01

    Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

  16. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  17. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

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    Roopali eRajput

    2015-09-01

    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

  18. Evaluation of an anti-p185{sup HER2} (scFv-C{sub H}2-C{sub H}3){sub 2} fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-D-GEEEK

    Energy Technology Data Exchange (ETDEWEB)

    Vaidyanathan, Ganesan [Duke University Medical Center, Durham, NC 27710 (United States)], E-mail: ganesan.v@duke.edu; Jestin, Emmanuelle [Duke University Medical Center, Durham, NC 27710 (United States); Olafsen, Tove; Wu, Anna M. [Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095 (United States); Zalutsky, Michael R. [Duke University Medical Center, Durham, NC 27710 (United States)

    2009-08-15

    Introduction: A 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185{sup HER2} hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods. Methods: The scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[{sup 131}I]iodobenzoate ([{sup 131}I]SGMIB) and N{sup {epsilon}}-(3-[{sup 131}I]iodobenzoyl)-Lys{sup 5}-N{sup {alpha}}- maleimido-Gly{sup 1}-GEEEK ([{sup 131}I]IB-Mal-D-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [{sup 125}I]IB-Mal-D-GEEEK and [{sup 131}I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts. Results: The scFv-Fc DM fragment was labeled with [{sup 131}I]SGMIB and [{sup 131}I]IB-Mal-D-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-D-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [{sup 125}I]IB-Mal-D-GEEEK-labeled scFv-Fc DM fragment compared with that of the [{sup 131}I]SGMIB-labeled fragment, particularly at later time points. The uptake of {sup

  19. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

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    Christiane Y Ozaki

    Full Text Available Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT and heat-stable toxin (ST, from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

  20. Enantiopure heterobimetallic single-chain magnets from the chiral Ru(III) building block.

    Science.gov (United States)

    Ru, Jing; Gao, Feng; Wu, Tao; Yao, Min-Xia; Li, Yi-Zhi; Zuo, Jing-Lin

    2014-01-21

    A pair of one-dimensional enantiomers based on the versatile chiral dicyanoruthenate(III) building block have been synthesized and they are chiral single-chain magnets with the effective spin-reversal barrier of 28.2 K.

  1. Barnase as a new therapeutic agent triggering apoptosis in human cancer cells.

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    Evelina Edelweiss

    Full Text Available BACKGROUND: RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI presented in the cytoplasm of mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50 ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50 values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50 = 1.8 nM was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

  2. Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

    Science.gov (United States)

    McElhiney, Jacqui; Drever, Mathew; Lawton, Linda A.; Porter, Andy J.

    2002-01-01

    A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples. PMID:12406716

  3. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  4. Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

    Science.gov (United States)

    de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

    2016-06-10

    The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Supramolecular Nanoparticles via Single-Chain Folding Driven by Ferrous Ions.

    Science.gov (United States)

    Wang, Fei; Pu, Hongting; Jin, Ming; Wan, Decheng

    2016-02-01

    Single-chain nanoparticles can be obtained via single-chain folding assisted by intramolecular crosslinking reversibly or irreversibly. Single-chain folding is also an efficient route to simulate biomacromolecules. In present study, poly(N-hydroxyethylacrylamide-co-4'-(propoxy urethane ethyl acrylate)-2,2':6',2''-terpyridine) (P(HEAm-co-EMA-Tpy)) is synthesized via reversible addition fragmentation chain transfer polymerization. Single-chain folding and intramolecular crosslinking of P(HEAm-co-EMA-Tpy) are achieved via metal coordination chemistry. The intramolecular interaction is characterized on ultraviolet/visible spectrophotometer (UV-vis spectroscopy), proton nuclear magnetic resonance ((1)H NMR), and differential scanning calorimetry (DSC). The supramolecular crosslinking mediated by Fe(2+) plays an important role in the intramolecular collapsing of the single-chain and the formation of the nanoparticles. The size and morphology of the nanoparticles can be controlled reversibly via metal coordination chemistry, which can be characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic force microscope (AFM). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Structure of single-chain single crystals of isotactic polystyrene and their radiation resistance

    International Nuclear Information System (INIS)

    Bu Haishan; Cao Jie; Xu Shengyong; Zhang Ze

    1997-01-01

    The structure of the single-chain single crystals of isotactic polystyrene (i-PS) was investigated by electron diffraction (ED) and high resolution electron microscopy (HREM). The nano-scale single-chain single crystals were found to be very stable to electron irradiation. According to the unit cell of i-PS crystals, the reflection rings in ED pattern and the lattice fringes in HREM images could be indexed, but the lower-index diffractions were not found. It is proposed that the single-chain single crystals are very small, thus secondary electrons may be allowed to escape and radiation damage is highly reduced, and that there are less lower-index lattice planes in the single-chain single crystals to provide sufficient diffraction intensity for recording. HREM images can be achieved at room temperature in the case of single-chain single crystals because of its stability to electron irradiation, therefore, this might be a novel experimental approach to the study of crystal structure of macromolecules

  7. The relationship between the connecting peptide of recombined single chain insulin and its biological function

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yiding; (

    2001-01-01

    [1]Straus, D. S., Growth-stimulatory of insulin in vitro and in vivo, Endocr. Rev., 1984, 5(2): 356-369.[2]Svenningsen, A. F., Kanje, M., Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H] thuymidine incorporation through their respective receptors, Glia, 1996, 18(1): 68-72.[3]Ogihara, S., Yamada, M., Saito, T. et al., Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells, Am. J. Physiol., 1996, 271(1 Pt 1): G104-121.[4]Steiner, D. F., Oyer, P. E., The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma, Proc. Nalt. Acad. Sci. USA, 1967, 57(2): 473-480.[5]King, G. L., Kahn, C. R., The growth-promoting effects of insulin, in Growth and Maturation Factors(ed. Guroff, G.), New York: John Wiley & Sons, 1984, 223-265.[6]Peavy, D. E., Brunner, M. R., Duckworth, W. C. et al., Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin, Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats, J. Biol. Chem., 1985, 260: 13989-13994.[7]Derewenda, U., Derewenda, Z., Dodson, E. J. et al., X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue, J. Mol. Biol., 1991, 220: 425-433.[8]Hua, Q. X., Shoelson, S. E., Kochoyan, M. et al., Receptor binding redefined by a structural switch in a mutant human insulin, Nature, 1991, 354: 238-241.[9]Hua, Q. X., Gozani, S. N., Chance, R. E. et al., Structure of a protein in a kinetic trap, Nat. Struc. Boil, 1995, 2: 129-138.[10]Kristensen, C., Andersen, A. S., Hach, M., A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor, Biochem. J., 1995, 305: 981-986.[11]Humbel, R. E., Insulin-like growth factors I and II, Euro. J. Biochem., 1990, 190: 445-462.[12]Cooke, R. M

  8. [Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

    Science.gov (United States)

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

    2015-10-01

    To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

  9. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain......: the medium composition in terms of ionic strengths and the medium physical parameters, such as temperature, significantly influence the formation of structures, as well as their subsequent stability. In addition, membranes composed of a single amphiphile type seem to be implausible as no potential amphiphile...... source studied to date can supply one single type of amphiphile at concentrations conducive to self-assembly. Mixtures of single-chain amphiphiles were therefore proposed to better model primitive membranes and potentially enhance their structural integrity1-3. Recently, we have established that complex...

  10. Improved scFv Anti-HIV-1 p17 Binding Affinity Guided from the Theoretical Calculation of Pairwise Decomposition Energies and Computational Alanine Scanning

    Directory of Open Access Journals (Sweden)

    Panthip Tue-ngeun

    2013-01-01

    Full Text Available Computational approaches have been used to evaluate and define important residues for protein-protein interactions, especially antigen-antibody complexes. In our previous study, pairwise decomposition of residue interaction energies of single chain Fv with HIV-1 p17 epitope variants has indicated the key specific residues in the complementary determining regions (CDRs of scFv anti-p17. In this present investigation in order to determine whether a specific side chain group of residue in CDRs plays an important role in bioactivity, computational alanine scanning has been applied. Molecular dynamics simulations were done with several complexes of original scFv anti-p17 and scFv anti-p17mutants with HIV-1 p17 epitope variants with a production run up to 10 ns. With the combination of pairwise decomposition residue interaction and alanine scanning calculations, the point mutation has been initially selected at the position MET100 to improve the residue binding affinity. The calculated docking interaction energy between a single mutation from methionine to either arginine or glycine has shown the improved binding affinity, contributed from the electrostatic interaction with the negative favorably interaction energy, compared to the wild type. Theoretical calculations agreed well with the results from the peptide ELISA results.

  11. Synthesis and functionalization of dextran-based single-chain nanoparticles in aqueous media

    OpenAIRE

    Gracia R.; Marradi M.; Cossío U.; Benito A.; Pérez-San Vicente A.; Gómez-Vallejo V.; Grande H.-J.; Llop J.; and Loinaz I.

    2017-01-01

    Water-dispersible dextran-based single-chain polymer nanoparticles (SCPNs) were prepared in aqueous media and under mild conditions. Radiolabeling of the resulting biocompatible materials allowed the study of lung deposition of aqueous aerosols after intratracheal nebulization by means of single-photon emission computed tomography (SPECT), demonstrating their potential use as imaging contrast agents.

  12. Force Spectroscopy of Hyaluronan by AFM; From H-bonded Networks Towards Single Chain Behavior

    NARCIS (Netherlands)

    Giannotti, M.I.; Rinaudo, Marguerite; Vancso, Gyula J.

    2007-01-01

    The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This comunication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures,

  13. Jet and ultrasonic nebulization of single chain urokinase plasminogen activator (scu-PA)

    DEFF Research Database (Denmark)

    Münster, Anna-Marie; Bendstrup, E; Jensen, J.I.

    2000-01-01

    locally by nebulization in a recombinant zymogen form as single chain urokinase plasminogen activator (scu-PA). We aimed to characterize the particle size distribution, drug output, and enzymatic activity of scu-PA after nebulization with a Ventstream jet nebulizer (Medic-Aid, Bognor Regis, UK) and a Syst...

  14. Surface water retardation around single-chain polymeric nanoparticles: critical for catalytic function?

    Science.gov (United States)

    Stals, Patrick J M; Cheng, Chi-Yuan; van Beek, Lotte; Wauters, Annelies C; Palmans, Anja R A; Han, Songi; Meijer, E W

    2016-03-01

    A library of water-soluble dynamic single-chain polymeric nanoparticles (SCPN) was prepared using a controlled radical polymerisation technique followed by the introduction of functional groups, including probes at targeted positions. The combined tools of electron paramagnetic resonance (EPR) and Overhauser dynamic nuclear polarization (ODNP) reveal that these SCPNs have structural and surface hydration properties resembling that of enzymes.

  15. Well-defined single-chain polymer nanoparticles via thiol-Michael addition

    NARCIS (Netherlands)

    Kröger, A. Pia P.; Boonen, Roy J.E.A.; Paulusse, Jos M.J.

    2017-01-01

    A synthetic strategy has been developed giving facile access to well-defined single-chain polymer nanoparticles (SCNPs) from styrene-, acrylate- and methacrylate-based polymers. Random copolymers (polydispersity indices 1.10–1.15) of methyl (meth)acrylate, benzyl methacrylate or styrene containing

  16. Optimization of the crystallizability of a single-chain antibody fragment

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Král, Vlastimil; Fábry, Milan; Sedláček, Juraj; Veverka, Václav; Řezáčová, Pavlína

    2014-01-01

    Roč. 70, č. 12 (2014), s. 1701-1706 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : single-chain antibody fragment * Thermofluor assay * differential scanning fluorimetry * crystallizability optimization * oligomerization * crystallization Subject RIV: CE - Biochemistry Impact factor: 0.527, year: 2014

  17. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    Directory of Open Access Journals (Sweden)

    Edyta Petters

    2015-08-01

    Full Text Available Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

  18. Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.

    Science.gov (United States)

    Arimori, Takao; Kitago, Yu; Umitsu, Masataka; Fujii, Yuki; Asaki, Ryoko; Tamura-Kawakami, Keiko; Takagi, Junichi

    2017-10-03

    Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Efficient Synthesis of Single-Chain Polymer Nanoparticles via Amide Formation

    Directory of Open Access Journals (Sweden)

    Ana Sanchez-Sanchez

    2015-01-01

    Full Text Available Single-chain technology (SCT allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles. In this work we contribute to the enlargement of the SCT toolbox by demonstrating the efficient synthesis of single-chain polymer nanoparticles (SCNPs via intrachain amide formation. In particular, we exploit cross-linking between active methylene groups and isocyanate moieties as powerful “click” chemistry driving force for SCNP construction. By employing poly(methyl methacrylate- (PMMA- based copolymers bearing β-ketoester units distributed randomly along the copolymer chains and bifunctional isocyanate cross-linkers, SCNPs were successfully synthesized at r.t. under appropriate reaction conditions. Characterization of the resulting SCNPs was carried out by means of a combination of techniques including size exclusion chromatography (SEC, infrared (IR spectroscopy, proton nuclear magnetic resonance (1H NMR spectroscopy, dynamic light scattering (DLS, and elemental analysis (EA.

  20. Cloning, bacterial expression and crystallization of Fv antibody fragments

    Science.gov (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

    1992-08-01

    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  1. Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

    Directory of Open Access Journals (Sweden)

    K. A. Chester

    2000-01-01

    Full Text Available Single chain Fv antibodies (sFvs have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA, a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2 for use in the ADEPT (antibody directed enzyme prodrug therapy system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

  2. Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

    Science.gov (United States)

    Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

    2016-10-01

    An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

  3. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Directory of Open Access Journals (Sweden)

    Ki Jung Lim

    Full Text Available Cell-penetrating peptides (CPPs have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2 was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57. The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv directed toward a mutated K-ras (G12V. BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  4. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Science.gov (United States)

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  5. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

    Science.gov (United States)

    Crivianu-Gaita, Victor; Thompson, Michael

    2016-11-15

    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N SARS-CoV protein using a phage display approach

    Directory of Open Access Journals (Sweden)

    Grasso Felicia

    2005-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

  7. Single-Chain Conformation for Interacting Poly(N-isopropylacrylamide in Aqueous Solution

    Directory of Open Access Journals (Sweden)

    Boualem Hammouda

    2015-04-01

    Full Text Available The demixing phase behavior of Poly(N-isopropylacrylamide (PNIPAM aqueous solution is investigated using small-angle neutron scattering. This polymer phase separates upon heating and demixes around 32 °C. The pre-transition temperature range is characterized by two scattering modes; a low-Q (large-scale signal and a high-Q dissolved chains signal. In order to get insight into this pre-transition region, especially the origin of the low-Q (large-scale structure, the zero average contrast method is used in order to isolate single-chain conformations even in the demixing polymers transition region. This method consists of measuring deuterated and non-deuterated polymers dissolved in mixtures of deuterated and non-deuterated water for which the polymer scattering length density matches the solvent scattering length density. A fixed 4% polymer mass fraction is used in a contrast variation series where the d-water/h-water fraction is varied in order to determine the match point. The zero average contrast (match point sample displays pure single-chain scattering with no interchain contributions. Our measurements prove that the large scale structure in this polymer solution is due to a transient polymer network formed through hydrophobic segment-segment interactions. Scattering intensity increases when the temperature gets close to the phase boundary. While the apparent radius of gyration increases substantially at the Lower Critical Solution Temperature (LCST transition due to strong interchain correlation, the single-chain true radius of gyration has been found to decrease slightly with temperature when approaching the transition.

  8. Early recognition is important when multiple magnets masquerade as a single chain after foreign body ingestion

    Directory of Open Access Journals (Sweden)

    Auriel August

    2016-10-01

    Full Text Available Ingestions of multiple magnets can lead to serious damage to the gastrointestinal tract. Moreover, these foreign bodies can take deceptive shapes such as single chains which may mislead clinicians. We report the case of a ten-year-old boy who swallowed 33 magnets, the most yet reported, which took on the appearance of a single loop in the stomach, while actually being located in the stomach, small bowel, and colon. Early recognition and prompt intervention are necessary to avoid complications of this foreign body misadventure.

  9. Finite-size effects on the static properties of a single-chain magnet

    Science.gov (United States)

    Bogani, L.; Sessoli, R.; Pini, M. G.; Rettori, A.; Novak, M. A.; Rosa, P.; Massi, M.; Fedi, M. E.; Giuntini, L.; Caneschi, A.; Gatteschi, D.

    2005-08-01

    We study the role of defects in the “single-chain magnet” CoPhOMe by inserting a controlled number of diamagnetic impurities. The samples are analyzed with unprecedented accuracy with the particle induced x-ray emission technique, and with ac and dc magnetic measurements. In an external applied field the system shows an unexpected behavior, giving rise to a double peak in the susceptibility. The static thermodynamic properties of the randomly diluted Ising chain with alternating g values are then exactly obtained via a transfer matrix approach. These results are compared to the experimental behavior of CoPhOMe, showing qualitative agreement.

  10. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

    Science.gov (United States)

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio

    2013-01-01

    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  11. Exceptionally potent anti-tumor bystander activity of an scFv : sTRAIL fusion protein with specificity for EGP2 toward target antigen-negative tumor cells

    NARCIS (Netherlands)

    Bremer, E; Samplonius, D; Kroesen, BJ; van Genne, L; de Leij, L; Helfrich, W

    2004-01-01

    Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target

  12. Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

    Energy Technology Data Exchange (ETDEWEB)

    Vindigni, Alessandro [Laboratorium fuer Festkoerperphysik, ETH Zuerich, CH-8093 Zuerich (Switzerland); Pini, Maria Gloria [Istituto dei Sistemi Complessi, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019 Sesto Fiorentino (Italy)], E-mail: vindigni@phys.ethz.ch

    2009-06-10

    The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

  13. Cell surface expression of single chain antibodies with applications to imaging of gene expression in vivo

    International Nuclear Information System (INIS)

    Northrop, Jeffrey P.; Bednarski, Mark; Li, King C.; Barbieri, Susan O.; Lu, Amy T.; Nguyen, Dee; Varadarajan, John; Osen, Maureen; Star-Lack, Josh

    2003-01-01

    Imaging of gene expression in vivo has many potential uses for biomedical research and drug discovery, ranging from the study of gene regulation and cancer to the non-invasive assessment of gene therapies. To streamline the development of imaging marker gene technologies for nuclear medicine, we propose a new approach to the design of reporter/probe pairs wherein the reporter is a cell surface-expressed single chain antibody variable fragment that has been raised against a low molecular weight imaging probe with optimized pharmacokinetic properties. Proof of concept of the approach was achieved using a single chain antibody variable fragment that binds with high affinity to fluorescein and an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine penta-acetic acid labeled with the gamma-emitter 111 In. We demonstrate specific high-affinity binding of this probe to the cell surface-expressed reporter in vitro and assess the in vivo biodistribution of the probe both in wild-type mice and in mice harboring tumor xenografts expressing the reporter. Specific uptake of the probe by, and in vivo imaging of, tumors expressing the reporter are shown. Since ScFvs with high affinities can be raised to almost any protein or small molecule, the proposed methodology may offer a new flexibility in the design of imaging tracer/reporter pairs wherein both probe pharmacokinetics and binding affinities can be readily optimized. (orig.)

  14. Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

    International Nuclear Information System (INIS)

    Vindigni, Alessandro; Pini, Maria Gloria

    2009-01-01

    The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

  15. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV

    Directory of Open Access Journals (Sweden)

    Hust Michael

    2008-09-01

    Full Text Available Abstract Background Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV and Eastern equine encephalitis virus (EEEV antigenic complex, nor did they react with Chikungunya virus (CHIKV, if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

  16. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

    Science.gov (United States)

    Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

    2013-08-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Single-ion and single-chain magnetism in triangular spin-chain oxides

    Science.gov (United States)

    Seikh, Md. Motin; Caignaert, Vincent; Perez, Olivier; Raveau, Bernard; Hardy, Vincent

    2017-05-01

    S r4 -xC axM n2Co O9 oxides (x =0 and x =2 ) are found to exhibit magnetic responses typical of single-chain magnets (SCMs) and single-ion magnets (SIMs), two features generally investigated in coordination polymers or complexes. The compound x =0 appears to be a genuine SCM, in that blocking effects associated with slow spin dynamics yield remanence and coercivity in the absence of long-range ordering (LRO). In addition, SIM signatures of nearly identical nature are detected in both compounds, coexisting with SCM in x =0 and with LRO in x =2 . It is also observed that a SCM response can be recovered in x =2 after application of magnetic field. These results suggest that purely inorganic systems could play a valuable role in the topical issue of the interplay among SIM, SCM, and LRO phenomena in low-dimensional magnetism.

  18. Screened ion-ion interaction in mercury-chain compounds: Single chain

    International Nuclear Information System (INIS)

    Mohan, M.M.; Griffin, A.

    1985-01-01

    At room temperature, the mercury chains in Hg/sub 3-delta/AsF 6 exhibit phonons characteristic of a one-dimensional lattice. We calculate the screening of the Hg ion-ion interaction in a single chain by electrons moving in a cylindrical potential of finite radius, within the random-phase approximation. The resulting Bohm-Staver-type expression for the phonon velocity is (Z 2 mN/sub I//MN/sub e/)/sup 1/2/v/sub F/, where Z is the Hg ionic charge and N/sub I/ (N/sub e/) is the number of ions (electrons) per unit length. Use of the Tomonaga-Luttinger solution for the electronic response function (keeping only the small-momentum scattering processes) just renormalizes the Fermi velocity in this expression

  19. Population pharmacokinetics of recombinant coagulation factor VIII-SingleChain in patients with severe hemophilia A.

    Science.gov (United States)

    Zhang, Y; Roberts, J; Tortorici, M; Veldman, A; St Ledger, K; Feussner, A; Sidhu, J

    2017-06-01

    Essentials rVIII-SingleChain is a unique recombinant factor VIII (FVIII) molecule. A population pharmacokinetic model was based on FVIII activity of severe hemophilia A patients. The model was used to simulate factor VIII activity-time profiles for various dosing scenarios. The model supports prolonged dosing of rVIII-SingleChain with intervals of up to twice per week. Background Single-chain recombinant coagulation factor VIII (rVIII-SingleChain) is a unique recombinant coagulation factor VIII molecule. Objectives To: (i) characterize the population pharmacokinetics (PK) of rVIII-SingleChain in patients with severe hemophilia A; (ii) identify correlates of variability in rVIII-SingleChain PK; and (iii) simulate various dosing scenarios of rVIII-SingleChain. Patients/Methods A population PK model was developed, based on FVIII activity levels of 130 patients with severe hemophilia A (n = 91 for ≥ 12-65 years; n = 39 for  85% and > 93% of patients were predicted to maintain FVIII activity level above 1 IU dL -1 , at all times with three-times-weekly dosing (given on days 0, 2, and 4.5) at the lowest (20 IU kg -1 ) and highest (50 IU kg -1 ) doses, respectively. For twice weekly dosing (days 0 and 3.5) of 50 IU kg -1 rVIII-SingleChain, 62-80% of patients across all ages were predicted to maintain a FVIII activity level above 1 IU dL -1 at day 7. Conclusions The population PK model adequately characterized rVIII-SingleChain PK, and the model can be utilized to simulate FVIII activity-time profiles for various dosing scenarios. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

  20. Pre-Discovery Detection of ASASSN-18fv by Evryscope

    Science.gov (United States)

    Corbett, H.; Law, N.; Goeke, E.; Ratzloff, J.; Howard, W.; Fors, O.; del Ser, D.; Quimby, R. M.

    2018-03-01

    We have identified pre-discovery imaging of the probable classical nova ASASSN-18fv by Evryscope-South (http://evryscope.astro.unc.edu/), an array of 6-cm telescopes continuously monitoring 8000 square degrees of sky at 2-minute cadence from CTIO, Chile.

  1. Regression of established renal cell carcinoma in nude mice using lentivirus-transduced human T cells expressing a human anti-CAIX chimeric antigen receptor

    Directory of Open Access Journals (Sweden)

    Agnes Shuk-Yee Lo

    2014-01-01

    Full Text Available Carbonic anhydrase IX (CAIX is a tumor-associated antigen and marker of hypoxia that is overexpressed on > 90% of clear-cell type renal cell carcinoma (RCC but not on neighboring normal kidney tissue. Here, we report on the construction of two chimeric antigen receptors (CARs that utilize a carbonic anhydrase (CA domain mapped, human single chain antibody (scFv G36 as a targeting moiety but differ in their capacity to provide costimulatory signaling for optimal T cell proliferation and tumor cell killing. The resulting anti-CAIX CARs were expressed on human primary T cells via lentivirus transduction. CAR-transduced T cells (CART cells expressing second-generation G36-CD28-TCRζ exhibited more potent in vitro antitumor effects on CAIX+ RCC cells than first-generation G36-CD8-TCRζ including cytotoxicity, cytokine secretion, proliferation, and clonal expansion. Adoptive G36-CD28-TCRζ CART cell therapy combined with high-dose interleukin (IL-2 injection also lead to superior regression of established RCC in nude mice with evidence of tumor cell apoptosis and tissue necrosis. These results suggest that the fully human G36-CD28-TCRζ CARs should provide substantial improvements over first-generation mouse anti-CAIX CARs in clinical use through reduced human anti-mouse antibody responses against the targeting scFv and administration of lower doses of T cells during CART cell therapy of CAIX+ RCC.

  2. Single-chain magnet features in 1D [MnR4TPP][TCNE] compounds

    International Nuclear Information System (INIS)

    Balanda, Maria; Tomkowicz, Zbigniew; Rams, Michal; Haase, Wolfgang

    2011-01-01

    Molecular chains of antiferrimagnetically coupled Mn III -ion (S = 2) and TCNE (tetracyanoethylene) radical moments (s = 1/2 ) show different behaviour depending on group R substituted to TPP (tetraphenylporphyrin) and on the substitution site. The compound with R = F in Ortho position is a Single-Chain Magnet (SCM) with blocking temperature T b = 6.6K, while that with R = F in Meta position shows both blocking (T b = 5.4 K) and magnetic ordering transition (T c = 10 K). For bulky groups R = OC n H 2n+1 , the magnetically ordered phase is observed (T c ∼ 22 K), which does not however prevent slow relaxation at T c of 2 T at 2.3 K is like that of SCM. The frequency dependent AC susceptibility in the superimposed DC field reveals common features of all systems. The energy of intrachain ferromagnetic coupling between effective spin units 3/2, relevant at low temperatures, is determined for all compounds and the interchain dipolar coupling is estimated. It is concluded that slow relaxation is inherent for all quasi one-dimensional compounds and for the magnetically ordered ones shows up in the high enough magnetic field.

  3. Reaction pathway towards formation of cobalt single chain magnets and nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Balaji, G.; Desilva, Rohini M.; Palshin, V. [Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Highway, Baton Rouge, LA 70806 (United States); Desilva, N. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Palmer, G. [Department of Biochemistry and Cell Biology, Rice University, MS 140, 6100 Main street, Houston, TX 77251 (United States); Kumar, Challa S.S.R., E-mail: ckumar1@lsu.ed [Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Highway, Baton Rouge, LA 70806 (United States)

    2010-03-15

    With the advent of molecular magnets the quest for suitable high density magnetic storage materials has fuelled further research in this area. Here in this report, we present a detailed mechanistic investigation of thermal decomposition of cyclopentadienyl cobalt [CoCp(CO){sub 2}] precursor where Cp is the cyclopentadienyl moiety. The reaction revealed the formation of cobalt nanoparticles (Co-NPs) through an isolable reaction intermediate characterized as a Single Chain Magnet (SCM), [Co(Cp){sub 2}]{sub 2}CoCl{sub 4} (1). The SQUID magnetic measurements showed the presence of very strong antiferromagnetic interactions between Co{sup 2+} ions. The zero-field cooled (ZFC) and field cooled (FC) magnetization curves branch out below 5 K and there is evidence for frequency dependent complex susceptibility along with a maximum observed around 2.5 K. The optical studies indicated that the Co{sup 2+} d-d transition is influenced by the polarity of the solvents. The cobalt nanoparticles (Co-NPs) were obtained, either directly from 1 or from its precursor. They are spherical in shape with a mean size 15 nm, have fcc crystal structure and were found to be ferromagnetic at room temperature.

  4. A family of rare-earth-based single chain magnets: playing with anisotropy.

    Science.gov (United States)

    Bernot, Kevin; Bogani, Lapo; Caneschi, Andrea; Gatteschi, Dante; Sessoli, Roberta

    2006-06-21

    The first family of rare-earth-based single chain magnets is presented. Compounds of general formula [M(hfac)3(NITPhOPh)], where M = Eu, Gd, Tb, Dy, Ho, Er, or Yb, and PhOPh is the nitronyl-nitroxide radical (2,4'-benzoxo-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), have been structurally characterized and found to be isostructural. The characterization of both static and dynamic magnetic properties of the whole family is reported. Dy, Tb, and Ho compounds display slow relaxation of the magnetization, and ac susceptibility shows a thermally activated regime with energy barriers of 69, 45, and 34 K for Dy, Tb, and Ho compounds, respectively, while only a frequency-dependent susceptibility is observed for Er below 2.0 K. In Gd and Yb derivatives, antiferromagnetic interactions dominate. The pre-exponential factors differ by about 4 orders of magnitude. Finite size effects, due to naturally occurring defects, affect the static and dynamic properties of the compounds differently.

  5. Single-chain statistics and the upper wave-vector cutoff in polymer blends

    International Nuclear Information System (INIS)

    Holyst, R.; Vilgis, T.A.

    1994-01-01

    We derive the equation for the single-chain correlation function in polymer blends. The chains in the incompressible blend have a radius of gyration smaller than the radius of gyration for ideal chains. The chains shrink progressively as we approach the critical temperature T c . The correction responsible for shrinking is proportional to 1/ √N , where N is the polymerization index. At T=T c and for N=1000, the size of the chain has been estimated to be 10% smaller than the size of the ideal coil. The estimate relies on the appropriate cutoff. In the limit of N→∞ the chains approach the random walk limit. Additionally, we propose in this paper a self-consistent determination of the radius of gyration and the upper wave-vector cutoff. Our model is free from any divergences such as were encountered in the previous mean-field studies; we make an estimate of the chain size at the true critical temperature and not the mean-field one

  6. Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9.

    Science.gov (United States)

    Intasai, Nutjeera; Tragoolpua, Khajornsak; Pingmuang, Prakitnavin; Khunkaewla, Panida; Moonsom, Seangdeun; Kasinrerk, Watchara; Lieber, André; Tayapiwatana, Chatchai

    2008-01-01

    CD147, a multifunctional type I transmembrane glycoprotein, has been implicated in various physiological and pathological processes. It is involved in signal transduction pathways and also plays a crucial role in the invasive and metastatic activity of malignant tumor cells. Diminished expression of this molecule has been shown to be beneficial in suppression of tumor progression. In a previous study, we generated and characterized a recombinant antibody fragment, scFv, which reacted specifically to CD147. In the present study, we further investigated the biological properties, function and the effect of generated scFv on CD147 expression. The in vitro study showed that soluble scFv-M6-1B9 produced from E. coli HB2151 bound to CD147 surface molecule and inhibited OKT3-induced T cell proliferation. Furthermore, soluble lysate of scFv-M6-1B9 from 293A cells, transduced with a scFv-M6-1B9 expressing adenovirus vector, recognized both recombinant and native CD147. These results indicate that scFv-M6-1B9 binds with high efficiency and specificity. Importantly, scFv-M6-1B9 intrabody reduced the expression of CD147 on the cell surface of HeLa cells suggesting that scFv-M6-1B9 is biologically active. In conclusion, our present study demonstrated that scFv-M6-1B9 has a great potential to target both the intracellular and the extracellular CD147. The generated scFv-M6-1B9 may be an effective agent to clarify the cellular function of CD147 and may aid in efforts to develop a novel treatment in various human carcinomas.

  7. Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

    Directory of Open Access Journals (Sweden)

    Chinestra Patrick

    2008-03-01

    Full Text Available Abstract Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L, three scFvs (A8, C1 and D11 were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2, it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated

  8. Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

    Science.gov (United States)

    Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

    2015-01-01

    Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

  9. Single-chain-in-mean-field simulations of weak polyelectrolyte brushes

    Science.gov (United States)

    Léonforte, F.; Welling, U.; Müller, M.

    2016-12-01

    Structural properties of brushes which are composed of weak acidic and basic polyelectrolytes are studied in the framework of a particle-based approach that implicitly accounts for the solvent quality. Using a semi-grandcanonical partition function in the framework of the Single-Chain-in-Mean-Field (SCMF) algorithm, the weak polyelectrolyte is conceived as a supramolecular mixture of polymers in different dissociation states, which are explicitly treated in the partition function and sampled by the SCMF procedure. One obtains a local expression for the equilibrium acid-base reaction responsible for the regulation of the charged groups that is also incorporated to the SCMF sampling. Coupled to a simultaneous treatment of the electrostatics, the approach is shown to capture the main features of weak polyelectrolyte brushes as a function of the bulk pH in the solution, the salt concentration, and the grafting density. Results are compared to experimental and theoretical works from the literature using coarse-grained representations of poly(acrylic acid) (PAA) and poly(2-vinyl pyridine) (P2VP) polymer-based brushes. As the Born self-energy of ions can be straightforwardly included in the numerical approach, we also study its effect on the local charge regulation mechanism of the brush. We find that its effect becomes significant when the brush is dense and exposed to high salt concentrations. The numerical methodology is then applied (1) to the study of the kinetics of collapse/swelling of a P2VP brush and (2) to the ability of an applied voltage to induce collapse/swelling of a PAA brush in a pH range close to the pKa value of the polymer.

  10. General model of phospholipid bilayers in fluid phase within the single chain mean field theory

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yachong; Baulin, Vladimir A. [Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. dels Paisos Catalans 26, 43007 Tarragona (Spain); Pogodin, Sergey [Institute of Chemical Research of Catalonia, ICIQ, Av. Paisos Catalans 16, 43007 Tarragona (Spain)

    2014-05-07

    Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

  11. Effect of chain stiffness on the structure of single-chain polymer nanoparticles

    Science.gov (United States)

    Moreno, Angel J.; Bacova, Petra; Lo Verso, Federica; Arbe, Arantxa; Colmenero, Juan; Pomposo, José A.

    2018-01-01

    Polymeric single-chain nanoparticles (SCNPs) are soft nano-objects synthesized by purely intramolecular cross-linking of single polymer chains. By means of computer simulations, we investigate the conformational properties of SCNPs as a function of the bending stiffness of their linear polymer precursors. We investigate a broad range of characteristic ratios from the fully flexible case to those typical of bulky synthetic polymers. Increasing stiffness hinders bonding of groups separated by short contour distances and increases looping over longer distances, leading to more compact nanoparticles with a structure of highly interconnected loops. This feature is reflected in a crossover in the scaling behaviour of several structural observables. The scaling exponents change from those characteristic for Gaussian chains or rings in θ-solvents in the fully flexible limit, to values resembling fractal or ‘crumpled’ globular behaviour for very stiff SCNPs. We characterize domains in the SCNPs. These are weakly deformable regions that can be seen as disordered analogues of domains in disordered proteins. Increasing stiffness leads to bigger and less deformable domains. Surprisingly, the scaling behaviour of the domains is in all cases similar to that of Gaussian chains or rings, irrespective of the stiffness and degree of cross-linking. It is the spatial arrangement of the domains which determines the global structure of the SCNP (sparse Gaussian-like object or crumpled globule). Since intramolecular stiffness can be varied through the specific chemistry of the precursor or by introducing bulky side groups in its backbone, our results propose a new strategy to tune the global structure of SCNPs.

  12. End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

    Science.gov (United States)

    Whitmore, Mark D; Grest, Gary S; Douglas, Jack F; Kent, Michael S; Suo, Tongchuan

    2016-11-07

    An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ 1/3 , for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

  13. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    International Nuclear Information System (INIS)

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-01-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

  14. Construction and Self-Assembly of Single-Chain Polymer Nanoparticles via Coordination Association and Electrostatic Repulsion in Water.

    Science.gov (United States)

    Zhu, Zhengguang; Xu, Na; Yu, Qiuping; Guo, Lei; Cao, Hui; Lu, Xinhua; Cai, Yuanli

    2015-08-01

    Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells

    International Nuclear Information System (INIS)

    Lagrange, Magali; Boulade-Ladame, Charlotte; Mailly, Laurent; Weiss, Etienne; Orfanoudakis, Georges; Deryckere, Francois

    2007-01-01

    The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies

  16. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    Science.gov (United States)

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  17. Characterization of Fusobacterium varium Fv113-g1 isolated from a patient with ulcerative colitis based on complete genome sequence and transcriptome analysis.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Sekizuka

    Full Text Available Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn's disease and ulcerative colitis (UC. Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS and 13 Fusobacterium adhesion (FadA paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.

  18. Design and analysis of an intelligent public FV lighting system; Diseno y analisis de un alumbrado publico FV inteligente

    Energy Technology Data Exchange (ETDEWEB)

    Hanessian D, Ana V.; Gordon, Manuel [Universidad Autonoma Metropolitana, Unidad Azcapotzalco, Mexico, D.F. (Mexico)

    2009-07-01

    In the Mexico's National Energy Balance of 2008, it is considered that of the total of the electrical power consumption in our country, 18% is dedicated to lighting. Conscious of the necessity of saving energy in public lighting in this article is presented the design, construction and analysis of the power consumption of a public light fed with electricity of photovoltaic cells and the control of intensity on the light in inverse way of the natural light. A lamp constructed based in light emitting diodes (LEDs) is used. This has the quality of consuming very little energy that could be provided by the sun and be stored to use it at night. With this system, proven at scale, energy savings are obtained superior to 50% of the conventional one and, in relation to the commercial photovoltaic (FV) luminaries up to 30%. [Spanish] Del Balance Nacional de Energia, de Mexico, de 2008, se considera que del total del consumo de energia electrica en nuestro pais, el 18% esta dedicado a la iluminacion. Conscientes de la necesidad de ahorrar energia en alumbrado publico, en este articulo se presenta el diseno de construccion y analisis del consumo energetico de una luminaria publica alimentada con electricidad de celdas fotovoltaicas y el control de intensidad de la luz de manera inversa a la luz natural. Se utiliza un foco construido a base a los diodos emisores de luz (LEDs, por sus siglas en ingles). Estos tienen la cualidad de consumir muy poca energia que podra ser suministrada por el sol y almacenada para utilizarla en la noche. Con este sistema, probado a escala, se logran ahorros de energia superiores al 50% del convencional y, en relacion a las luminarias fotovoltaicas (FV) comerciales, hasa el 30%.

  19. A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

    Science.gov (United States)

    Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

    2018-02-16

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

  20. Evolution of the retroviral restriction gene Fv1: inhibition of non-MLV retroviruses.

    Directory of Open Access Journals (Sweden)

    Melvyn W Yap

    2014-03-01

    Full Text Available Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV. It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV. To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV and feline foamy virus (FFV respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.

  1. Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

    Science.gov (United States)

    Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

    2012-10-01

    Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

  2. Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

    Science.gov (United States)

    Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

    2016-01-01

    ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

  3. EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

    Science.gov (United States)

    Hausmann, Jennifer C; Wack, Allison N; Allender, Matthew C; Cranfield, Mike R; Murphy, Kevin J; Barrett, Kevin; Romero, Jennell L; Wellehan, James F X; Blum, Stella A; Zink, M Christine; Bronson, Ellen

    2015-12-01

    The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

  4. Evaluation of anti-HER2 scFv-conjugated PLGA–PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    International Nuclear Information System (INIS)

    Le, Thi Thuy Duong; Pham, Thu Hong; Ngo, Thi Hong Giang; Le, Quang Huan; Nguyen, Trong Nghia; Hoang, Thi My Nhung

    2016-01-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv–Doc–PLGA–PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv–Doc–PLGA–PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv–Doc–PLGA–PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies. (paper)

  5. Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

    Science.gov (United States)

    Panich, Tipattaraporn; Tragoolpua, Khajornsak; Pata, Supansa; Tayapiwatana, Chatchai; Intasai, Nutjeera

    2017-02-01

    Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

  6. AFSC/ABL: Origins of salmon seized from the F/V Arctic Wind

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Samples of chum (Oncorhynchus keta), sockeye (O. nerka), and chinook salmon (O. tshawytscha) seized from the F/V Arctic Wind were analyzed to determine their region...

  7. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

    Science.gov (United States)

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

  8. FvVE1 Regulates Biosynthesis of Fumonisins and Fusarins in Fusarium verticillioides

    Science.gov (United States)

    MYUNG, KYUNG; LI, SHAOJIE; BUTCHKO, ROBERT A.E.; BUSMAN, MARK; PROCTOR, ROBERT H; ABBAS, HAMED K.; CALVO, ANA M.

    2009-01-01

    The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in A. parasiticus and A. flavus. Whether veA homologs have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homolog, FvVE1, on production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen F. verticillioides. We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 play an important role in regulating mycotoxin production in F. verticillioides. PMID:19382792

  9. Xenopus-FV3 host-pathogen interactions and immune evasion.

    Science.gov (United States)

    Jacques, Robert; Edholm, Eva-Stina; Jazz, Sanchez; Odalys, Torres-Luquis; Francisco, De Jesús Andino

    2017-11-01

    We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Comparative response of platelet fV and plasma fV to activated protein C and relevance to a model of acute traumatic coagulopathy.

    Directory of Open Access Journals (Sweden)

    James E Campbell

    Full Text Available BACKGROUND: Acute traumatic coagulopathy (ATC has been linked to an increase in activated protein C (aPC from 40 pM in healthy individuals to 175 pM. aPC exerts its activity primarily through cleavage of active coagulation factor Va (fVa. Platelets reportedly possess fVa which is more resistant to aPC cleavage than plasma fVa; this work examines the hypothesis that normal platelets are sufficient to maintain coagulation in the presence of elevated aPC. METHODS: Coagulation responses of normal plasma, fV deficient plasma (fVdp, and isolated normal platelets in fVdp were conducted: prothrombin (PT tests, turbidimetry, and thromboelastography (TEG, including the dose response of aPC on the samples. RESULTS: PT and turbidimetric assays demonstrate that normal plasma is resistant to aPC at doses much higher than those found in ATC. Additionally, an average physiological number of washed normal platelets (200,000 platelets/mm3 was sufficient to eliminate the anti-coagulant effects of aPC up to 10 nM, nearly two orders of magnitude above the ATC concentration and even the steady-state pharmacological concentration of human recombinant aPC, as measured by TEG. aPC also demonstrated no significant effect on clot lysis in normal plasma samples with or without platelets. CONCLUSIONS: Although platelet fVa shows slightly superior resistance to aPC's effects compared to plasma fVa in static models, neither fVa is sufficiently cleaved in simulations of ATC or pharmacologically-delivered aPC to diminish coagulation parameters. aPC is likely a correlative indicator of ATC or may play a cooperative role with other activity altering products generated in ATC.

  11. Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Timo Heidt

    Full Text Available BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111Indium ((111In via bifunctional DTPA ( = (111In-LIBS/(111In-control. Autoradiography after incubation with (111In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2, 4010 ± 630 DLU/mm(2 and 4520 ± 293 DLU/mm(2 produced a significantly higher ligand uptake compared to (111In-control (2101 ± 76 DLU/mm(2, 1181 ± 96 DLU/mm(2 and 1866 ± 246 DLU/mm(2 indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2 vs. 17390 ± 7470 DLU/mm(2; P<0.05. These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01. CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of

  12. Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients.

    Science.gov (United States)

    Ayat, Hoda; Burrone, Oscar R; Sadghizadeh, Majid; Jahanzad, Eissa; Rastgou, Nasrin; Moghadasi, Sarrira; Arbabi, Mehdi

    2013-11-01

    Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning. Copyright © 2013 The International Alliance for Biological Standardization. All rights reserved.

  13. Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

    Science.gov (United States)

    Ueberberg, Sandra; Meier, Juris J.; Waengler, Carmen; Schechinger, Wolfgang; Dietrich, Johannes W.; Tannapfel, Andrea; Schmitz, Inge; Schirrmacher, Ralf; Köller, Manfred; Klein, Harald H.; Schneider, Stephan

    2009-01-01

    OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS We report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS Our data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo. PMID:19592622

  14. Single-chain magnet features in 1D [MnR{sub 4}TPP][TCNE] compounds

    Energy Technology Data Exchange (ETDEWEB)

    Balanda, Maria [Institute of Nuclear Physics PAN, Radzikowskiego 152, 31-342 Krakow (Poland); Tomkowicz, Zbigniew; Rams, Michal [Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krakow (Poland); Haase, Wolfgang, E-mail: Maria.Balanda@ifj.edu.pl [Institute of Physical Chemistry, Darmstadt University of Technology, 64287 Darmstadt (Germany)

    2011-07-06

    Molecular chains of antiferrimagnetically coupled Mn{sup III}-ion (S = 2) and TCNE (tetracyanoethylene) radical moments (s = 1/2 ) show different behaviour depending on group R substituted to TPP (tetraphenylporphyrin) and on the substitution site. The compound with R = F in Ortho position is a Single-Chain Magnet (SCM) with blocking temperature T{sub b} = 6.6K, while that with R = F in Meta position shows both blocking (T{sub b} = 5.4 K) and magnetic ordering transition (T{sub c} = 10 K). For bulky groups R = OC{sub n}H{sub 2n+1}, the magnetically ordered phase is observed (T{sub c} {approx} 22 K), which does not however prevent slow relaxation at T <8 K. Magnetic hysteresis with coercive field H{sub c} of 2 T at 2.3 K is like that of SCM. The frequency dependent AC susceptibility in the superimposed DC field reveals common features of all systems. The energy of intrachain ferromagnetic coupling between effective spin units 3/2, relevant at low temperatures, is determined for all compounds and the interchain dipolar coupling is estimated. It is concluded that slow relaxation is inherent for all quasi one-dimensional compounds and for the magnetically ordered ones shows up in the high enough magnetic field.

  15. A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

    International Nuclear Information System (INIS)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

    2014-01-01

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp"*_2Co] (Cp"*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K.

  16. A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

    International Nuclear Information System (INIS)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

    2014-01-01

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp*_2Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134 ±0.8 K (93 ±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T <6 K, with an impressive coercive field of 3.4 T at 2 K. (Copyright copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  17. Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

    Directory of Open Access Journals (Sweden)

    Bogen Bjarne

    2010-02-01

    Full Text Available Abstract Background Efficient expression systems exist for antibody (Ab molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs. Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc TCRs. Results The effect of 1 over-expression of the periplasmic chaperon FkpA, 2 culture conditions and 3 molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1 high yield recovery sufficient for biophysical characterization and 2 high throughput screening of such molecules following molecular engineering.

  18. Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

    Science.gov (United States)

    Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

    2016-03-03

    By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

  19. Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

    KAUST Repository

    Hou, Zhipeng; Zhang, Qiang; Xu, Guizhou; Gong, Chen; Ding, Bei; Wang, Yue; Li, Hang; Liu, Enke; Xu, Feng; Zhang, Hongwei; Yao, Yuan; Wu, Guangheng; Zhang, Xixiang; Wang, Wenhong

    2018-01-01

    Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

  20. Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

    KAUST Repository

    Hou, Zhipeng

    2018-01-04

    Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

  1. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    Science.gov (United States)

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  2. Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Zhao, Heng; Stet, René J M; Skjødt, Karsten

    2008-01-01

    Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

  3. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    Science.gov (United States)

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  4. Safety, efficacy and pharmacokinetics of rVIII-SingleChain in children with severe hemophilia A: results of a multicenter clinical trial.

    Science.gov (United States)

    Stasyshyn, O; Djambas Khayat, C; Iosava, G; Ong, J; Abdul Karim, F; Fischer, K; Veldman, A; Blackman, N; St Ledger, K; Pabinger, I

    2017-04-01

    Essentials rVIII-SingleChain is a novel recombinant factor VIII with covalently bonded heavy and light chains. Efficacy, safety and pharmacokinetics were studied in pediatric patients with severe hemophilia A. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00. rVIII-SingleChain showed excellent hemostatic efficacy and a favorable safety profile. Background rVIII-SingleChain is a novel B-domain truncated recombinant factor VIII (rFVIII) comprised of covalently bonded FVIII heavy and light chains, demonstrating a high binding affinity to von Willebrand factor. Objectives This phase III study investigated the safety, efficacy and pharmacokinetics of rVIII-SingleChain in previously treated pediatric patients hemophilia A. Patients/Methods Patients could be assigned to prophylaxis or on-demand therapy by the investigator. For patients assigned to prophylaxis, the treatment regimen and dose were based on the bleeding phenotype. For patients receiving on-demand therapy, dosing was guided by World Federation of Hemophilia recommendations. The primary endpoint was treatment success, defined as a rating of 'excellent' or 'good' on the investigator's clinical assessment of hemostatic efficacy for all treated bleeding events. Results The study enrolled 84 patients (0 to 50 EDs. In the 347 bleeds treated and evaluated by the investigator, hemostatic efficacy was rated as excellent or good in 96.3%. The median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.00, 2.20), and the median annualized bleeding rate was 3.69 (Q1, Q3: 0.00, 7.20) across all prophylaxis regimens. No participant developed an inhibitor. Conclusions rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy and a favorable safety profile in a clinical study in children hemophilia A. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and

  5. Human anti-CCR4 minibody gene transfer for the treatment of cutaneous T-cell lymphoma.

    Directory of Open Access Journals (Sweden)

    Thomas Han

    Full Text Available Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL, no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4.In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8 vector expressing a humanized single-chain variable fragment (scFv-Fc fusion (scFvFc or "minibody" of anti-CCR4 monoclonal antibody (mAb h1567 was evaluated for curative treatment. Human CCR4+ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567 minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G+ FcγRIIIa(CD16A+ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4+ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs, marked tumor infiltration of human CD16A+ CD56+ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells.Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A+ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.

  6. Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology.

    Science.gov (United States)

    Tohidkia, Mohammad R; Sepehri, Maryam; Khajeh, Shirin; Barar, Jaleh; Omidi, Yadollah

    2017-09-01

    Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the "biopanning" process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A-conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.

  7. Good Practices for Learning to Recognize Actions Using FV and VLAD.

    Science.gov (United States)

    Wu, Jianxin; Zhang, Yu; Lin, Weiyao

    2016-12-01

    High dimensional representations such as Fisher vectors (FV) and vectors of locally aggregated descriptors (VLAD) have shown state-of-the-art accuracy for action recognition in videos. The high dimensionality, on the other hand, also causes computational difficulties when scaling up to large-scale video data. This paper makes three lines of contributions to learning to recognize actions using high dimensional representations. First, we reviewed several existing techniques that improve upon FV or VLAD in image classification, and performed extensive empirical evaluations to assess their applicability for action recognition. Our analyses of these empirical results show that normality and bimodality are essential to achieve high accuracy. Second, we proposed a new pooling strategy for VLAD and three simple, efficient, and effective transformations for both FV and VLAD. Both proposed methods have shown higher accuracy than the original FV/VLAD method in extensive evaluations. Third, we proposed and evaluated new feature selection and compression methods for the FV and VLAD representations. This strategy uses only 4% of the storage of the original representation, but achieves comparable or even higher accuracy. Based on these contributions, we recommend a set of good practices for action recognition in videos for practitioners in this field.

  8. Efficient sampling of reversible cross-linking polymers: Self-assembly of single-chain polymeric nanoparticles

    Science.gov (United States)

    Oyarzún, Bernardo; Mognetti, Bortolo Matteo

    2018-03-01

    We present a new simulation technique to study systems of polymers functionalized by reactive sites that bind/unbind forming reversible linkages. Functionalized polymers feature self-assembly and responsive properties that are unmatched by the systems lacking selective interactions. The scales at which the functional properties of these materials emerge are difficult to model, especially in the reversible regime where such properties result from many binding/unbinding events. This difficulty is related to large entropic barriers associated with the formation of intra-molecular loops. In this work, we present a simulation scheme that sidesteps configurational costs by dedicated Monte Carlo moves capable of binding/unbinding reactive sites in a single step. Cross-linking reactions are implemented by trial moves that reconstruct chain sections attempting, at the same time, a dimerization reaction between pairs of reactive sites. The model is parametrized by the reaction equilibrium constant of the reactive species free in solution. This quantity can be obtained by means of experiments or atomistic/quantum simulations. We use the proposed methodology to study the self-assembly of single-chain polymeric nanoparticles, starting from flexible precursors carrying regularly or randomly distributed reactive sites. We focus on understanding differences in the morphology of chain nanoparticles when linkages are reversible as compared to the well-studied case of irreversible reactions. Intriguingly, we find that the size of regularly functionalized chains, in good solvent conditions, is non-monotonous as a function of the degree of functionalization. We clarify how this result follows from excluded volume interactions and is peculiar of reversible linkages and regular functionalizations.

  9. Spin canting in a Dy-based single-chain magnet with dominant next-nearest-neighbor antiferromagnetic interactions

    Science.gov (United States)

    Bernot, K.; Luzon, J.; Caneschi, A.; Gatteschi, D.; Sessoli, R.; Bogani, L.; Vindigni, A.; Rettori, A.; Pini, M. G.

    2009-04-01

    We investigate theoretically and experimentally the static magnetic properties of single crystals of the molecular-based single-chain magnet of formula [Dy(hfac)3NIT(C6H4OPh)]∞ comprising alternating Dy3+ and organic radicals. The magnetic molar susceptibility χM displays a strong angular variation for sample rotations around two directions perpendicular to the chain axis. A peculiar inversion between maxima and minima in the angular dependence of χM occurs on increasing temperature. Using information regarding the monomeric building block as well as an ab initio estimation of the magnetic anisotropy of the Dy3+ ion, this “anisotropy-inversion” phenomenon can be assigned to weak one-dimensional ferromagnetism along the chain axis. This indicates that antiferromagnetic next-nearest-neighbor interactions between Dy3+ ions dominate, despite the large Dy-Dy separation, over the nearest-neighbor interactions between the radicals and the Dy3+ ions. Measurements of the field dependence of the magnetization, both along and perpendicularly to the chain, and of the angular dependence of χM in a strong magnetic field confirm such an interpretation. Transfer-matrix simulations of the experimental measurements are performed using a classical one-dimensional spin model with antiferromagnetic Heisenberg exchange interaction and noncollinear uniaxial single-ion anisotropies favoring a canted antiferromagnetic spin arrangement, with a net magnetic moment along the chain axis. The fine agreement obtained with experimental data provides estimates of the Hamiltonian parameters, essential for further study of the dynamics of rare-earth-based molecular chains.

  10. Primitive-path statistics of entangled polymers: mapping multi-chain simulations onto single-chain mean-field models

    International Nuclear Information System (INIS)

    Steenbakkers, Rudi J A; Schieber, Jay D; Tzoumanekas, Christos; Li, Ying; Liu, Wing Kam; Kröger, Martin

    2014-01-01

    We present a method to map the full equilibrium distribution of the primitive-path (PP) length, obtained from multi-chain simulations of polymer melts, onto a single-chain mean-field ‘target’ model. Most previous works used the Doi–Edwards tube model as a target. However, the average number of monomers per PP segment, obtained from multi-chain PP networks, has consistently shown a discrepancy of a factor of two with respect to tube-model estimates. Part of the problem is that the tube model neglects fluctuations in the lengths of PP segments, the number of entanglements per chain and the distribution of monomers among PP segments, while all these fluctuations are observed in multi-chain simulations. Here we use a recently proposed slip-link model, which includes fluctuations in all these variables as well as in the spatial positions of the entanglements. This turns out to be essential to obtain qualitative and quantitative agreement with the equilibrium PP-length distribution obtained from multi-chain simulations. By fitting this distribution, we are able to determine two of the three parameters of the model, which govern its equilibrium properties. This mapping is executed for four different linear polymers and for different molecular weights. The two parameters are found to depend on chemistry, but not on molecular weight. The model predicts a constant plateau modulus minus a correction inversely proportional to molecular weight. The value for well-entangled chains, with the parameters determined ab initio, lies in the range of experimental data for the materials investigated. (paper)

  11. Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.

    Science.gov (United States)

    van der Steen, Sophieke C H A; van Tilborg, Angela A G; Vallen, Myrtille J E; Bulten, Johan; van Kuppevelt, Toin H; Massuger, Leon F A G

    2016-03-01

    The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology. The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status. The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status. Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas

    2017-11-01

    Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

  13. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

    Science.gov (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

    2017-01-01

    Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

  14. Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites.

    Science.gov (United States)

    Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent

    2015-05-22

    Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

  15. First study of C2491T FV mutation with ischaemic stroke risk in ...

    Indian Academy of Sciences (India)

    Faculty of Medicine and Pharmacy, Department of Genetic and Molecular Pathology Laboratory (LGPM),. Tarek Ibn Ziad, QH, Hassan II University, 9154 Casablanca, Morocco. [Diakite B., Hamzi K., Hmimech W., Nadifi S. and GMRAVC 2015 First study of C2491T FV mutation with ischaemic stroke risk in Morocco. J. Genet.

  16. BRITE nanosatellite serendipitously captures oscillatory rise and fall of ASASSN-18fv

    Science.gov (United States)

    Kuschnig, R.; Pigulski, A.; Moffat, A. F. J.; Matthews, J. M.; Zwintz, K.; Baade, D.; Handler, G.; Weiss, W. W.; Wade, G. A.; Rucinski, S. M.; Pablo, H.; Koudelka, O.; Smolec, R.; Popowicz, A.; Neiner, C.; Daszynska-Daszkiewicz, J.; Lovekin, C.; St-Louis, N.; Pamyatnykh, A. A.; Rowe, J.; Orleanski, P.; Mochnacki, S.; Schwarzenberg-Czerny, A.

    2018-04-01

    One of the five satellites in BRITE-Constellation (http://www.brite-constellation.at/) was obtaining time-series optical photometry of the star HD 92063, only 2 arcmin from the 'Possible, Very Bright Galactic Nova ASASSN-18fv' reported on 21 March 2018 (2018-03-20.32) by ATel #11454.

  17. Suspension kinematic analysis of UTeM’s FV Malaysia electric vehicle racing car

    NARCIS (Netherlands)

    Abdul Manaf, M.Z.; Latif, M.F.A.; Razak, M.S.A.; Hassan, M.Z.B.; Rosley, M.I.F.

    2016-01-01

    The purpose of this paper is to investigate the kinematic performance of students’ racing car, namely UTeM’s FV Malaysia Electric Vehicle. An elasto-kinematic analysis approach is used to predict the car’s performance during straight line drive and curvature drive. Two suspension design factors

  18. FvSet2 regulates fungal growth, pathogenicity, and secondary metabolism in Fusarium verticillioides.

    Science.gov (United States)

    Gu, Qin; Wang, Zhenzhong; Sun, Xiao; Ji, Tiantian; Huang, Hai; Yang, Yang; Zhang, Hao; Tahir, Hafiz Abdul Samad; Wu, Liming; Wu, Huijun; Gao, Xuewen

    2017-10-01

    Histone H3 lysine 36 methylation (H3K36me) is generally associated with activation of gene expression in most eukaryotic cells. However, the function of H3K36me in filamentous fungi is largely unknown. Set2 is the sole lysine histone methyltransferase (KHMTase) enzyme responsible for the methylation of H3K36 in Saccharomyces cerevisiae. In the current study, we identified a single ortholog of S. cerevisiae Set2 in Fusarium verticillioides. We report that FvSet2 is responsible for the trimethylation of H3K36 (H3K36me3). The FvSET2 deletion mutant (ΔFvSet2) showed significant defects in vegetative growth, FB 1 biosynthesis, pigmentation, and fungal virulence. Furthermore, trimethylation of H3K36 was found to be important for active transcription of genes involved in FB 1 and bikaverin biosyntheses. These data indicate that FvSet2 plays an important role in the regulation of secondary metabolism, vegetative growth and fungal virulence in F. verticillioides. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Atlantic mackerel and Horse mackerel egg survey 2016: Dutch participation on board FV Atlantic Lady: May

    NARCIS (Netherlands)

    Damme, van C.J.G.

    2016-01-01

    From 10 till 25 May 2016 IMARES carried out a mackerel and horse mackerel egg survey on board the FV Atlantic Lady. This survey was part of the international mackerel and horse mackerel egg survey coordinated by ICES. The Redersvereniging voor de Zeevisserij (RVZ) asked IMARES to carry out this

  20. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  1. The research progress and medicine application of the ScFv antibody

    International Nuclear Information System (INIS)

    Qin Lili; Zhang Chunming

    2005-01-01

    Since the scholar of England and Japan had found the diphtheria antitoxin, the research of the antibody has experienced three phases: polyclonal antibodies, monoclonal antibodies and genetically engineered antibodies. In recent years, far more attention has been paid to single-chain antibody by researchers owing to it's small molecular, strong ability of penetration, short half-lives in blood, high specificity to combine with the corresponding antibody, weak immunogenicity and possibility to be expressed in prokaryocyte. (authors)

  2. Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

    Science.gov (United States)

    Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L

    2018-05-01

    B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

  3. Interstitial loop growth in electron irradiated vacuum-remelted FV448

    International Nuclear Information System (INIS)

    Buswell, J.T.; Fisher, S.B.

    1979-05-01

    An investigation of the resistance to void swelling of the ferritic steel FV448 has been made in electron irradiation experiments in a High Voltage Electron Microscope. Overaged samples of vacuum-remelted FV448 showed zero swelling after irradiation to doses of up to 30 dpa, in the temperature range 400 to 550 0 C. The resistance to void swelling was due to the absence of mutual interaction between interstitial loops. Impurity segregation to the loops, and precipitation, prevented continuous dislocation climb, thus removing biased sinks from the system at low doses. An interstitial-impurity binding energy of 0.5 eV was measured, suggesting that the impurity responsible was carbon or nitrogen. Some effects attributable to the heating of thin foils in the HVEM environment were detected. (author)

  4. Effects of developer exhaustion on DFL Contrast FV-58 and Kodak Insight dental films.

    Science.gov (United States)

    de Carvalho, Fabiano Pachêco; da Silveira, M M F; Frazão, M A G; de Santana, S T; dos Anjos Pontual, M L

    2011-09-01

    The aim of this study was to compare the properties of the DFL Contrast FV-58 F-speed film (DFL Co., Rio de Janerio, Brazil) with the Kodak Insight E/F speed film (Eastman Kodak, Rochester, NY) in fresh and exhausted processing solutions. The parameters studied were the speed, average gradient and latitude. Five samples of each type of film were exposed under standardized conditions over 5 weeks. The films were developed in fresh and progressively exhausted processing solutions. Characteristic curves were constructed from values of optical density and radiation dose and were used to calculate the parameters. An analysis of variance was performed separately for film type and time. DFL Contrast FV-58 film has a speed and average gradient that is significantly higher than Insight film, whereas the values of latitude are lower. Exhausted processing solutions were not significant in the parameters studied. DFL Contrast FV-58 film has stable properties when exhausted manual processing solutions are used and can be recommended for use in dental practice, contributing to dose reduction.

  5. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

    Science.gov (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

    2014-02-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Potent anti-leukemia activities of humanized CD19-targeted CAR-T cells in patients with relapsed/refractory acute lymphoblastic leukemia.

    Science.gov (United States)

    Cao, Jiang; Wang, Gang; Cheng, Hai; Wei, Chen; Qi, Kunming; Sang, Wei; Zhenyu, Li; Shi, Ming; Li, Huizhong; Qiao, Jianlin; Pan, Bin; Zhao, Jing; Wu, Qingyun; Zeng, Lingyu; Niu, Mingshan; Jing, Guangjun; Zheng, Junnian; Xu, Kailin

    2018-04-10

    Chimeric antigen receptor T (CAR-T) cell therapy has shown promising results for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). The immune response induced by murine single-chain variable fragment (scFv) of the CAR may limit CAR-T cell persistence and thus increases the risk of leukemia relapse. In this study, we developed a novel humanized scFv from the murine FMC63 antibody. A total of 18 R/R ALL patients with or without prior murine CD19 CAR-T therapy were treated with humanized CD19-targeted CAR-T cells (hCART19s). After lymphodepletion chemotherapy with cyclophosphamide and fludarabine, the patients received a single dose (1 × 10 6 /kg) of autologous hCART19s infusion. Among the 14 patients without previous CAR-T therapy, 13 (92.9%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, whereas 1 of the 3 patients who failed a second murine CAR-T infusion achieved CR after hCART19s infusion. At day 180, the overall and leukemia-free survival rates were 65.8% and 71.4%, respectively. The cumulative incidence of relapse was 22.6%, and the non-relapse mortality rate was 7.1%. During treatment, 13 patients developed grade 1-2 cytokine release syndrome (CRS), 4 patients developed grade 3-5 CRS, and 1 patient experienced reversible neurotoxicity. These results indicated that hCART19s could induce remission in patients with R/R B-ALL, especially in patients who received a reinfusion of murine CAR-T. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

  7. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  8. Tandem bispecific neutralizing antibody eliminates HIV-1 infection in humanized mice.

    Science.gov (United States)

    Wu, Xilin; Guo, Jia; Niu, Mengyue; An, Minghui; Liu, Li; Wang, Hui; Jin, Xia; Zhang, Qi; Lam, Ka Shing; Wu, Tongjin; Wang, Hua; Wang, Qian; Du, Yanhua; Li, Jingjing; Cheng, Lin; Tang, Hang Ying; Shang, Hong; Zhang, Linqi; Zhou, Paul; Chen, Zhiwei

    2018-04-23

    The discovery of an HIV-1 cure remains a medical challenge because the virus rebounds quickly after the cessation of combination antiretroviral therapy (cART). Here, we investigate the potential of an engineered tandem bispecific broadly neutralizing antibody (bs-bnAb) as an innovative product for HIV-1 prophylactic and therapeutic interventions. We discovered that by preserving 2 single-chain variable fragment (scFv) binding domains of each parental bnAb, a single gene-encoded tandem bs-bnAb, BiIA-SG, displayed substantially improved breadth and potency. BiIA-SG neutralized all 124 HIV-1-pseudotyped viruses tested, including global subtypes/recombinant forms, transmitted/founder viruses, variants not susceptible to parental bnAbs and to many other bnAbs with an average IC50 value of 0.073 μg/ml (range HIV-1 stains. Moreover, whereas BiIA-SG delayed viral rebound in a short-term therapeutic setting when combined with cART, a single injection of adeno-associated virus-transferred (AAV-transferred) BiIA-SG gene resulted dose-dependently in prolonged in vivo expression of BiIA-SG, which was associated with complete viremia control and subsequent elimination of infected cells in humanized mice. These results warrant the clinical development of BiIA-SG as a promising bs-bnAb-based biomedical intervention for the prevention and treatment of HIV-1 infection.

  9. Numerical investigation on the prefabricated crack propagation of FV520B stainless steel

    Directory of Open Access Journals (Sweden)

    Juyi Pan

    Full Text Available FV520B is a common stainless steel for manufacturing centrifugal compressor impeller and shaft. The internal metal flaw destroys the continuity of the material matrix, resulting in the crack propagation fracture of the component, which seriously reduces the service life of the equipment. In this paper, Abaqus software was used to simulate the prefabricated crack propagation of FV520B specimen with unilateral gap. The results of static crack propagation simulation results show that the maximum value of stress–strain located at the tip of the crack and symmetrical distributed like a butterfly along the prefabricated crack direction, the maximum stress is 1990 MPa and the maximum strain is 9.489 × 10−3. The Mises stress and stress intensity factor KI increases with the increase of the expansion step, the critical value of crack initiation is reached at the 6th extension step. The dynamic crack propagation simulation shows that the crack propagation path is perpendicular to the load loading direction. Similarly, the maximum Mises stress located at the crack tip and is symmetrically distributed along the crack propagation direction. The critical stress range of the crack propagation is 23.3–43.4 MPa. The maximum value of stress–strain curve located at the 8th extension step, that is, the crack initiation point, the maximum stress is 55.22 MPa, and the maximum strain is 2.26 × 10−4. On the crack tip, the stress changed as 32.24–40.16 MPa, the strain is at 1.292 × 10−4–1.897 × 10−4. Keywords: FV520B, Crack propagation, Mises stress, Stress–strain, Numerical investigation

  10. Atlantic mackerel and Horse mackerel egg survey 2016: Dutch participation on board FV Atlantic Lady: May

    OpenAIRE

    Damme, van, C.J.G.

    2016-01-01

    From 10 till 25 May 2016 IMARES carried out a mackerel and horse mackerel egg survey on board the FV Atlantic Lady. This survey was part of the international mackerel and horse mackerel egg survey coordinated by ICES. The Redersvereniging voor de Zeevisserij (RVZ) asked IMARES to carry out this survey. Numbers of mackerel eggs in the samples were low, lower compared to previous surveys. Most mackerel eggs were found along the 200m depth contour of the continental slope. Most of the adult mack...

  11. Direct observation of the discrete energy spectrum of two lanthanide-based single-chain magnets by far-infrared spectroscopy

    Science.gov (United States)

    Haas, Sabrina; Heintze, Eric; Zapf, Sina; Gorshunov, Boris; Dressel, Martin; Bogani, Lapo

    2014-05-01

    The far-infrared optical transmission has been studied for two lanthanide-based single-chain magnets DyPhOPh and TbPhOPh in the frequency range between 3 and 80 cm-1. The spectra were acquired at temperatures between 2 and 80 K and magnetic fields up to 6 T. Based on their magnetic field dependence in DyPhOPh two of the observed absorption lines are identified as transitions inside the crystal field split Dy3+ ground multiplet 6H15/2, coupled to the neighboring spins. In TbPhOPh one transition was observed inside the crystal-field-split Tb3+ ground multiplet 7F6. The results allow a spectroscopic investigation of the role of single-ion anisotropy and exchange in Glauber dynamics.

  12. Structural and Functional Characterization of a Single-chain Peptide-MHC Molecule that Modulates both Naive and Activated CD8plus T Cells

    Energy Technology Data Exchange (ETDEWEB)

    D Samanta; G Mukherjee; U Ramagopal; R Chaparro; S Nathenson; T DiLorenzo; S Almo

    2011-12-31

    Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8{sup +} T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8{sup +} T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK{sup d}.IGRP) by using the class I MHC molecule H-2K{sup d} and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP{sub 206-214}), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK{sup d}.IGRP tetramers bound specifically to cognate CD8{sup +} T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-{gamma} response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8{sup +} T cells makes them a potential intervention strategy in early and late stages of disease.

  13. Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS.

    Directory of Open Access Journals (Sweden)

    Sung Sun Yim

    Full Text Available Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS. First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6. These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.

  14. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  15. Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

    Directory of Open Access Journals (Sweden)

    Mai Sabine

    2008-01-01

    Full Text Available Abstract Background Expression of intracellular antibodies (intrabodies has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer. Results The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9 reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system. Conclusion The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.

  16. Identification and production of mouse scFv to specific epitope of enterovirus-71 virion protein-2 (VP2).

    Science.gov (United States)

    Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Tongtawe, Pongsri; Glab-Ampai, Kittirat; Malik, Aijaz Ahmad; Supasorn, Oratai; Chiawwit, Phatcharaporn; Poovorawan, Yong; Chaicumpa, Wanpen

    2018-05-01

    Enterovirus-71 (EV71) and coxsackievirus-A16 (CA16) frequently cause hand-foot-mouth disease (HFMD) epidemics among infants and young children. CA16 infections are usually mild, while EV71 disease may be fatal due to neurologic complications. As such, the ability to rapidly and specifically recognize EV71 is needed to facilitate proper case management and epidemic control. Accordingly, the aim of this study was to generate antibodies to EV71-virion protein-2 (VP2) by phage display technology for further use in specific detection of EV71. A recombinant peptide sequence of EV71-VP2, carrying a predicted conserved B cell epitope fused to glutathione-S-transferase (GST) (designated GST-EV71-VP2/131-160), was produced. The fusion protein was used as bait in in-solution biopanning to separate protein-bound phages from a murine scFv (MuscFv) phage display library constructed from an immunoglobulin gene repertoire from naïve ICR mice. Three phage-transformed E. coli clones (clones 63, 82, and 83) produced MuscFvs that bound to the GST-EV71-VP2/131-160 peptide. The MuscFv of clone 83 (MuscFv83), which produced the highest ELISA signal to the target antigen, was further tested. MuscFv83 also bound to full-length EV71-VP2 and EV71 particles, but did not bind to GST, full-length EV71-VP1, or the antigenically related CA16. MuscFv83 could be a suitable reagent for rapid antigen-based immunoassay, such as immunochromatography (ICT), for the specific detection and/or diagnosis of EV71 infection as well as epidemic surveillance.

  17. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

    Science.gov (United States)

    Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

    2018-04-16

    mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

  18. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  19. Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

    Directory of Open Access Journals (Sweden)

    Chen Xu

    2010-03-01

    Full Text Available Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC, but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

  20. Mechanical Characteristic of Remanufacturing of FV520B Precipitation Hardening Stainless Steel Using MAG Surfacing Deposition

    Directory of Open Access Journals (Sweden)

    LIU Jian

    2017-10-01

    Full Text Available Surfacing deposition forming method was adopted to carry out remanufacturing experiment of FV520B precipitation hardening stainless steel. Then the mechanical property characteristic of the remanufacturing layer was tested and studied, contrasted with the corresponding property of substrate. The results show that the remanufacturing layer, formed with MAG surfacing of FV520B precipitation hardening stainless steel has mechanical characteristic with high strength and hardness, the tensile strength reaches 1195MPa, exceeds 1092MPa of substrate, yield strength is 776MPa and average hardness is 336HV, is close to the corresponding property of substrate which is 859MPa and 353HV respectively; however, the elongation and impact toughness of the remanufacturing layer is merely 8.92% and 61J/cm2 respectively, it has a large gap with the corresponding property 19.72% and 144J/cm2 respectively of substrate. Fracture and microstructure analysis on specimens shows that the microstructure of remanufacturing layer is fast cooling non-equilibrium crystallized lath martensite, and carbide precipitated strengthening phase such as NbC, MoC, M23C6,etc, which is the reason that remanufacturing layer has high strength and high hardness. But as lack of aging treatment and Cu strengthening phase, and the weak interface between contaminating brittle phase or large size spherical particles and substrate will deteriorate the deformability and induce stress concentration and cracking when the material is load-carrying, and is the main reason of the remanufacturing layer having lower static tensile elongation and impact toughness.

  1. Temporal variation in community composition, pigmentation, and Fv/Fm of desert cyanobacterial soil crusts

    Science.gov (United States)

    Bowker, M.A.; Reed, S.C.; Belnap, J.; Phillips, S.L.

    2002-01-01

    Summers on the Colorado Plateau (USA) are typified by harsh conditions such as high temperatures, brief soil hydration periods, and high UV and visible radiation. We investigated whether community composition, physiological status, and pigmentation might vary in biological soil crusts as a result of such conditions. Representative surface cores were sampled at the ENE, WSW, and top microaspects of 20 individual soil crust pedicels at a single site in Canyonlands National Park, Utah, in spring and fall of 1999. Frequency of cyanobacterial taxa, pigment concentrations, and dark adapted quantum yield (Fv/Fm) were measured for each core. The frequency of major cyanobacterial taxa was lower in the fall compared to spring. The less-pigmented cyanobacterium Microcoleus vaginatus showed significant mortality when not in the presence of Nostoc spp. and Scytonema myochrous (Dillw.) Agardh. (both synthesizers of UV radiation-linked pigments) but had little or no mortality when these species were abundant. We hypothesize that the sunscreen pigments produced by Nostoc and Scytonema in the surface of crusts protect other, less-pigmented taxa. When fall and spring samples were compared, overall cyanobacterial frequency was lower in fall, while sunscreen pigment concentrations, chlorophyll a concentration, and Fv/Fm were higher in fall. The ratio of cyanobacterial frequency/chlorophyll a concentrations was 2-3 times lower in fall than spring. Because chlorophyll a is commonly used as a surrogate measure of soil cyanobacterial biomass, these results indicate that seasonality needs to be taken into consideration. In the fall sample, most pigments associated with UV radiation protection or repair were at their highest concentrations on pedicel tops and WSW microaspects, and at their lowest concentrations on ENE microaspects. We suggest that differential pigment concentrations between microaspects are induced by varying UV radiation dosage at the soil surface on these different

  2. Combined Cytolytic Effects of a Vaccinia Virus Encoding a Single Chain Trimer of MHC-I with a Tax-Epitope and Tax-Specific CTLs on HTLV-I-Infected Cells in a Rat Model

    Directory of Open Access Journals (Sweden)

    Takashi Ohashi

    2014-01-01

    Full Text Available Adult T cell leukemia (ATL is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I. To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV, LC16m8Δ (m8Δ, and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL.

  3. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

    Directory of Open Access Journals (Sweden)

    Andrea Pecora

    2015-03-01

    Full Text Available Bovine viral diarrhea virus (BVDV is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2 was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

  4. A uranium-based UO{sub 2}{sup +}-Mn{sup 2+} single-chain magnet assembled trough cation-cation interactions

    Energy Technology Data Exchange (ETDEWEB)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella [CEA-Grenoble (France). Lab. de Reconnaissance Ionique et Chimie de Coordination; Caciuffo, Roberto; Colineau, Eric [European Commission, Karlsruhe (Germany). Inst. for Transuranium Elements; Tuna, Floriana [Manchester Univ. (United Kingdom). School of Chemistry; Magnani, Nicola [KIT Karlsruhe (Germany). Inst. of Nanotechnology; Geyer, Arnaud de [CEA-Grenoble (France). Service General des Rayons X

    2014-01-13

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]{sub n}, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO{sub 2}(salen)py][Cp{sup *}{sub 2}Co] (Cp{sup *}=pentamethylcyclopentadienyl) with Cd(NO{sub 3}){sub 2} or Mn(NO{sub 3}){sub 2} in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm{sup -1}), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K.

  5. A uranium-based UO{sub 2}{sup +}-Mn{sup 2+} single-chain magnet assembled trough cation-cation interactions

    Energy Technology Data Exchange (ETDEWEB)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella [Laboratoire de Reconnaissance Ionique et Chimie de Coordination, SCIB, UMR-E3 CEA-UJF, INAC, CEA-Grenoble (France); Caciuffo, Roberto; Colineau, Eric [European Commission, Joint Research Centre, Institute for Transuranium Elements, Karlsruhe (Germany); Tuna, Floriana [EPSRC UK EPR Facility, School of Chemistry and Photon Science Institute, The University of Manchester (United Kingdom); Magnani, Nicola [Institute of Nanotechnology, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen (Germany); Geyer, Arnaud de [Service General des Rayons X, SP2M, INAC, CEA-Grenoble (France)

    2014-01-13

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]{sub n}, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO{sub 2}(salen)py][Cp*{sub 2}Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO{sub 3}){sub 2} or Mn(NO{sub 3}){sub 2} in pyridine. The infinite UMn chain displays a high relaxation barrier of 134 ±0.8 K (93 ±0.5 cm{sup -1}), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T <6 K, with an impressive coercive field of 3.4 T at 2 K. (Copyright copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  6. Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity

    Directory of Open Access Journals (Sweden)

    Masanori Matsui

    2014-01-01

    Full Text Available We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

  7. Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement

    International Nuclear Information System (INIS)

    Grennan, Kathleen; Strachan, Gillian; Porter, Andrew J.; Killard, Anthony J.; Smyth, Malcolm R.

    2003-01-01

    This work describes the development of an electrochemical immunosensor for the analysis of atrazine using recombinant single-chain antibody (scAb) fragments. The sensors are based on carbon paste screen-printed electrodes incorporating the conducting polymer polyaniline (PANI)/poly(vinylsulphonic acid) (PVSA), which enables direct mediatorless coupling to take place between the redox centres of antigen-labelled horseradish peroxidase (HRP) and the electrode surface. Competitive immunoassays can be performed in real-time using this separation-free system. Analytical measurements based on the pseudo-linear relationship between the slope of a real-time amperometric signal and the concentration of analyte, yield a novel immunosensor set-up capable of regenerationless amperometric analysis. Multiple, sequential measurements of standards and samples can be performed on a single scAb-modified surface in a matter of minutes. No separation of bound and unbound species was necessary prior to detection. The system is capable of measuring atrazine to a detection limit of 0.1 ppb (0.1 μg l -1 ). This system offers the potential for rapid, cost-effective immunosensing for the analysis of samples of environmental, medical and pharmaceutical significance

  8. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  9. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

    Science.gov (United States)

    Islam, Kazi T; Bond, Jason P; Fakhoury, Ahmad M

    2017-08-01

    The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

  11. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  12. Allergy immunotherapy with a hypoallergenic recombinant birch pollen allergen rBet v 1-FV in a randomized controlled trial.

    Science.gov (United States)

    Klimek, Ludger; Bachert, Claus; Lukat, Karl-Friedrich; Pfaar, Oliver; Meyer, Hanns; Narkus, Annemie

    2015-01-01

    Pollen extracts and chemically modified allergoids are used successfully in allergen immunotherapy (AIT). Recombinant extracts offer potential advantages with respect to pharmaceutical quality, standardization and dosing. A hypoallergenic recombinant folding variant of the major birch pollen allergen (rBet v 1-FV) was compared with an established native birch preparation. A pre-seasonal, randomized, actively controlled phase II study was performed in birch pollen allergic rhino-conjunctivitis with or without asthma, GINA I/ II. 51 patients (24 rBet v 1-FV, 27 native extract) started therapy with subcutaneous allergen immunotherapy (SCIT). Primary end-point was a combined symptom medication score (SMS), changes in nasal provocation test, visual rating score and specific antibody responses secondary end-points. After one pre-seasonal treatment course the combined SMS was 5.86 (median; IQR: 14.02) for the rBet v 1-FV group versus 12.40 (median; IQR: 9.32) for the comparator during the three weeks pollen season (p = 0.330). After treatment in the second year, scores were 3.00 (median; IQR: 6.50) and 2.93 (4.86) respectively. Allergen tolerance in a nasal provocation test improved to a comparable extent in both groups. Significant increases in birch pollen-specific IgG1 and IgG4 were observed in both treatment groups following the first treatment phase and remained significantly raised until the end of the study. In this first in man, proof of concept phase II trial no statistical difference between rBet v 1-FV and an established natural pollen extract could be observed. rBet v 1-FV could be administered in higher doses than the native protein with no increase in adverse effects. The study was registered in clinicalTrials.gov (NCT00266526).

  13. Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals

    Directory of Open Access Journals (Sweden)

    Manuela Berto Pucca

    2011-03-01

    Full Text Available Monoclonal antibodies represent the fastest growing class of biopharmaceutical products and have a host of applications in medical research, diagnosis, therapy, and basic science. The production of recombinant monoclonal antibodies has revolutionized the generation of immunoglobulins, and their use represents a strategic breakthrough, affecting the global pharmaceutical market for therapeutic proteins. In the present work, a review of scFv, and the number of related patents, has been carried out. The results show that several countries have scFv patents, most notably the United States, China and United Kingdom. The target of these scFv antibodies was also assessed and the results demonstrate that most are directed toward cancer therapy.Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofármacos e possuem várias aplicações em pesquisa médica, diagnóstico, terapias e ciência básica. A produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas e sua utilização implica em avanço estratégico, afetando o mercado farmacêutico global de proteínas terapêuticas. No presente trabalho, uma revisão sobre scFv e a relação do seu número de patentes foi analisada. Os resultados mostram que vários países apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos também foram avaliados e as análises revelaram que a maioria é destinado a terapias contra o câncer.

  14. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

    Science.gov (United States)

    Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

    2011-10-28

    Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Three Cyanide-Bridged One-Dimensional Single Chain Co"I"I"I-Mn"I"I Complexes: Rational Design, Synthesis, Crystal Structures and Magnetic Properties

    International Nuclear Information System (INIS)

    Zhang, Daopeng; Zhao, Zengdian; Wang, Ping; Chen, Xia

    2012-01-01

    Two pyridinecarboxamide dicyanidecobalt(III) building blocks and two mononuclear seven-coordinated macrocycle manganese(II) compounds have been rationally selected to assemble cyanide-bridged heterobimetallic complexes, resulting in three cyanide-bridged Co"I"I"I-Mn"I"I complexes. Single X-ray diffraction analysis show that these complexes {[Mn(L"1)][Co(bpb)]}ClO_4·CH_3OH·0.5H_2O (1), {[Mn(L"2)][Co(bpb)]}ClO_4·0.5CH_3OH (2) and {[Mn(L"1)][Cobpmb]}ClO_4·H_2O (3) (L"1 = 3,6-diazaoctane-1,8-diamine, L"2 = 3,6-dioxaoctano-1,8- diamine: bpb"2"- = 1,2-bis(pyridine-2-carboxamido)benzenate, bpmb"2"- = 1,2-bis(pyridine-2-carboxamido)-4- methyl-benzenate) all present predictable one-dimensional single chain structures. The molecular structures of these one-dimensional complexes consists of alternating units of [Mn(L)]"2"+ (L = L"1 or L"2) and [Co(L')(CN)_2]"- (L' = bpb"2"-, or bpmb"2"-), forming a cyanide-bridged cationic polymeric chain with free ClO_4"- as the balance anion. The coordination geometry of manganese(II) ion in the three one-dimensional complexes is a slightly distorted pentagonal-bipyrimidal with two cyanide nitrogen atoms at the trans positions and N_5 or N_3O_2 coordinating mode at the equatorial plane from ligand L"1 or L"2. Investigation over magnetic properties of these complexes reveals that the very weak magnetic coupling between neighboring Mn(II) ions connected by the diamagnetic dicyanidecobalt(III) building block. A best-fit to the magnetic susceptibility of complex 1 leads to the magnetic coupling constants J = .0.084(3) cm"-"1

  16. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    Science.gov (United States)

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley

  17. Finite-size effects on the dynamic susceptibility of CoPhOMe single-chain molecular magnets in presence of a static magnetic field

    Science.gov (United States)

    Pini, M. G.; Rettori, A.; Bogani, L.; Lascialfari, A.; Mariani, M.; Caneschi, A.; Sessoli, R.

    2011-09-01

    The static and dynamic properties of the single-chain molecular magnet Co(hfac)2NITPhOMe (CoPhOMe) (hfac = hexafluoroacetylacetonate, NITPhOMe = 4'-methoxy-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) are investigated in the framework of the Ising model with Glauber dynamics, in order to take into account both the effect of an applied magnetic field and a finite size of the chains. For static fields of moderate intensity and short chain lengths, the approximation of a monoexponential decay of the magnetization fluctuations is found to be valid at low temperatures; for strong fields and long chains, a multiexponential decay should rather be assumed. The effect of an oscillating magnetic field, with intensity much smaller than that of the static one, is included in the theory in order to obtain the dynamic susceptibility χ(ω). We find that, for an open chain with N spins, χ(ω) can be written as a weighted sum of N frequency contributions, with a sum rule relating the frequency weights to the static susceptibility of the chain. Very good agreement is found between the theoretical dynamic susceptibility and the ac susceptibility measured in moderate static fields (Hdc≤2 kOe), where the approximation of a single dominating frequency for each segment length turns out to be valid. For static fields in this range, data for the relaxation time, τ versus Hdc, of the magnetization of CoPhOMe at low temperature are also qualitatively reproduced by theory, provided that finite-size effects are included.

  18. FV-100 versus valacyclovir for the prevention of post-herpetic neuralgia and the treatment of acute herpes zoster-associated pain: A randomized-controlled trial.

    Science.gov (United States)

    Tyring, Stephen K; Lee, Patricia; Hill, Gordon T; Silverfield, Joel C; Moore, Angela Yen; Matkovits, Theresa; Sullivan-Bolyai, John

    2017-07-01

    This prospective, parallel-group, randomized, double-blind, multicenter study compared the efficacy and safety of FV-100 with valacyclovir for reducing pain associated with acute herpes zoster (HZ). Patients, ≥50 years of age, diagnosed with HZ within 72 h of lesion appearance who had HZ-associated pain, were randomized 1:1:1 to a 7-day course of either FV-100 200 mg QD (n = 117), FV-100 400 mg QD (n = 116), or valacyclovir 1000 mg TID (n =117). Efficacy was evaluated on the basis of the burden of illness (BOI; Zoster Brief Pain Inventory scores); incidence and duration of clinically significant pain (CSP); pain scores; incidence and severity of post-herpetic neuralgia (PHN); and times to full lesion crusting and to lesion healing. Safety was evaluated on the basis of adverse event (AE)/SAE profiles, changes in laboratory and vital signs values, and results of electrocardiograms. The burden of illness scores for pain through 30 days were 114.5, 110.3, and 118.0 for FV-100 200 mg, FV-100 400 mg, and valacyclovir 3000 mg, respectively. The incidences of PHN at 90 days for FV-100 200 mg, FV-100 400 mg, and valacyclovir 3000 mg were 17.8%, 12.4%, and 20.2%, respectively. Adverse event and SAE profiles of the two FV-100 and the valacyclovir groups were similar and no untoward signals or trends were evident. These results demonstrate a potential for FV-100 as an antiviral for the treatment of shingles that could both reduce the pain burden of the acute episode and reduce the incidence of PHN compared with available treatments. © 2016 Wiley Periodicals, Inc.

  19. Bolus dose response characteristics of single chain urokinase plasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis.

    Science.gov (United States)

    Badylak, S F; Voytik, S; Klabunde, R E; Henkin, J; Leski, M

    1988-11-15

    Tissue plasminogen activator (t-PA) and single chain urokinase-plasminogen activator (scu-PA) are relatively "fibrin-specific" thrombolytic drugs with short plasma half lives of 6-8 minutes. Most treatment regimens with these agents utilize a bolus injection followed by continuous drug infusion, usually combined with anticoagulant therapy. The purpose of this study was to establish the dose-response characteristics for scu-PA and t-PA, when given as a single intravenous bolus injection, in a dog model of arterial thrombosis. Eight groups of 6 dogs each were given one of the following doses of scu-PA (mg/kg): 0.20, 0.50, 1.00, 2.00; or t-PA: 0.05, 0.10, 0.20; or an equivalent amount of saline (control group). All doses were given as a single bolus injection 60 minutes after formation of a totally occlusive femoral artery thrombus. Thrombolysis was measured by monitoring the continuous decrement of 125I activity from a radiolabelled thrombus. Ninety minutes after drug injection, all scu-PA treated dogs showed greater thrombolysis (30%, 45%, 56%, and 67%, respectively) than the control group (15%, p less than 0.01). The 0.10 and 0.20 mg/kg t-PA treated dogs showed greater thrombolysis (35% and 49%, respectively) than the control group (15%, p less than 0.01). Both scu-PA and t-PA caused a partial and dose-dependent decrease in alpha 2-antiplasmin activity but scu-PA caused a greater depletion (72% vs. 18%, respectively, p less than 0.05) at 60 minutes after the highest dose of drug administration. Both drugs showed a longer than expected thrombolytic effect based upon the known half lives. Neither drug caused significant changes in the prothrombin time, activated partial thromboplastin time, thrombin time, hematocrit, platelet count, or fibrin degradation product concentration. Single bolus injections of scu-PA and t-PA produce safe and effective thrombolysis in this dog model of arterial thrombosis.

  20. Magneto-structural correlations in a family of Fe(II)Re(IV)(CN)2 single-chain magnets: density functional theory and ab initio calculations.

    Science.gov (United States)

    Zhang, Yi-Quan; Luo, Cheng-Lin; Wu, Xin-Bao; Wang, Bing-Wu; Gao, Song

    2014-04-07

    Until now, the expressions of the anisotropic energy barriers Δξ and ΔA, using the uniaxial magnetic anisotropy D, the intrachain coupling strength J, and the high-spin ground state S for single-chain magnets (SCMs) in the intermediate region between the Ising and the Heisenberg limits, were unknown. To explore this relationship, we used density functional theory and ab initio methods to obtain expressions of Δξ and ΔA in terms of D, J, and S of six R4Fe(II)-Re(IV)Cl4(CN)2 (R = diethylformamide (1), dibutylformamide (2), dimethylformamide (3), dimethylbutyramide (4), dimethylpropionamide (5), and diethylacetamide (6)) SCMs in the intermediate region. The ΔA value for compounds 1-3 was very similar to the magnetic anisotropic energy of a single Fe(II), while the value of Δξ was predicted using the exchange interaction of Fe(II) with the neighboring Re(IV), which could be expressed as 2JSReSFe. Similar to compounds 1-3, the anisotropy energy barrier ΔA of compounds 4 and 5 was also equal to (Di - Ei)SFe(2), but the correlation energy Δξ was closely equal to 2JSReSFe(cos 98.4 - cos 180) due to the reversal of the spins on the opposite Fe(II). For compound 6, one unit cell of Re(IV)Fe(II) was regarded as a domain wall since it had two different Re(IV)-Fe(II) couplings. Thus, the Δξ of compound 6 was expressed as 4J″SRe1Fe1SRe2Fe2, where J″ was the coupling constant of the neighboring unit cells of Re1Fe1 and Re2Fe2, and ΔA was equal to the anisotropic energy barrier of one domain wall given by DRe1Fe1(S(2)Re1Fe1 - 1/4).

  1. Experimental study on stress corrosion crack propagation rate of FV520B in carbon dioxide and hydrogen sulfide solution

    Directory of Open Access Journals (Sweden)

    Ming Qin

    Full Text Available FV520B steel is a kind of precipitation hardening Martensitic stainless steel, it has high-strength, good plasticity and good corrosion resistance. Stress corrosion cracking (SCC is one of the main corrosion failure mode for FV520B in industrial transportation of natural gas operation. For a better understanding the effect on SCC of FV520B, the improved wedge opening loading (WOL specimens and constant displacement loading methods were employed in experimental research in carbon dioxide and hydrogen sulfide solution. The test results showed that the crack propagation rate is 1.941 × 10−7–5.748 × 10−7 mm/s, the stress intensity factor KISCC is not more than 36.83 MPa m. The rate increases with the increasing of the crack opening displacement. Under the condition of different initial loading, KISCC generally shows a decreasing tendency with the increase in H2S concentration, and the crack propagation rate showed an increasing trend substantially. For the enrichment of sulfur ion in the crack tip induced the generation of pitting corrosion, promoting the surrounding metal formed the corrosion micro batteries, the pit defects gradually extended and connected with the adjacent pit to form a small crack, leading to further propagation till cracking happened. Fracture microscopic morphology displayed typical brittle fracture phenomena, accompanying with trans-granular cracking, river shape and sector, many second cracks on the fracture surface. Keywords: FV520B, Wedge opening loading specimen, Stress corrosion cracking, Hydrogen sulfide

  2. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

    Science.gov (United States)

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

    2014-01-01

    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

  3. Experimental study on stress corrosion crack propagation rate of FV520B in carbon dioxide and hydrogen sulfide solution

    Science.gov (United States)

    Qin, Ming; Li, Jianfeng; Chen, Songying; Qu, Yanpeng

    FV520B steel is a kind of precipitation hardening Martensitic stainless steel, it has high-strength, good plasticity and good corrosion resistance. Stress corrosion cracking (SCC) is one of the main corrosion failure mode for FV520B in industrial transportation of natural gas operation. For a better understanding the effect on SCC of FV520B, the improved wedge opening loading (WOL) specimens and constant displacement loading methods were employed in experimental research in carbon dioxide and hydrogen sulfide solution. The test results showed that the crack propagation rate is 1.941 × 10-7-5.748 × 10-7 mm/s, the stress intensity factor KISCC is not more than 36.83 MPa √{ m } . The rate increases with the increasing of the crack opening displacement. Under the condition of different initial loading, KISCC generally shows a decreasing tendency with the increase in H2S concentration, and the crack propagation rate showed an increasing trend substantially. For the enrichment of sulfur ion in the crack tip induced the generation of pitting corrosion, promoting the surrounding metal formed the corrosion micro batteries, the pit defects gradually extended and connected with the adjacent pit to form a small crack, leading to further propagation till cracking happened. Fracture microscopic morphology displayed typical brittle fracture phenomena, accompanying with trans-granular cracking, river shape and sector, many second cracks on the fracture surface.

  4. Coupled fvGCM-GCE Modeling System, 3D Cloud-Resolving Model and Cloud Library

    Science.gov (United States)

    Tao, Wei-Kuo

    2005-01-01

    Recent GEWEX Cloud System Study (GCSS) model comparison projects have indicated that cloud- resolving models (CRMs) agree with observations better than traditional single-column models in simulating various types of clouds and cloud systems from different geographic locations. Current and future NASA satellite programs can provide cloud, precipitation, aerosol and other data at very fine spatial and temporal scales. It requires a coupled global circulation model (GCM) and cloud-scale model (termed a super-parameterization or multi-scale modeling framework, MMF) to use these satellite data to improve the understanding of the physical processes that are responsible for the variation in global and regional climate and hydrological systems. The use of a GCM will enable global coverage, and the use of a CRM will allow for better and more sophisticated physical parameterization. NASA satellite and field campaign cloud related datasets can provide initial conditions as well as validation for both the MMF and CRMs. A seed fund is available at NASA Goddard to build a MMF based on the 2D Goddard Cumulus Ensemble (GCE) model and the Goddard finite volume general circulation model (fvGCM). A prototype MMF in being developed and production runs will be conducted at the beginning of 2005. In this talk, I will present: (1) A brief review on GCE model and its applications on precipitation processes, ( 2 ) The Goddard MMF and the major difference between two existing MMFs (CSU MMF and Goddard MMF), (3) A cloud library generated by Goddard MMF, and 3D GCE model, and (4) A brief discussion on the GCE model on developing a global cloud simulator.

  5. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    Science.gov (United States)

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  6. A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

    Directory of Open Access Journals (Sweden)

    Kumar Rajesh

    2012-11-01

    Full Text Available Abstract Background Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs against the third variable region (V3 of the clade C HIV-1 envelope. Results An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding

  7. Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

    Science.gov (United States)

    Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

    2017-10-16

    Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan)); Kawaminami, Shunro (Kao Corp., Tochigi (Japan))

    1991-07-02

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C{sub H}1 domain. A variety of Fv analogues labeled with {sup 2}H in the aromatic rings and with {sup 13}C and/or {sup 15}N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with {sup 1}H and {sup 13}C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the {sup 1}H-{sup 15}N shift correlation spectra. Use of the {beta}-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.

  9. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Odaka, Asano; Matsunaga, Chigusa; Kato, Koichi; Shimada, Ichio; Arata, Yoji; Kawaminami, Shunro

    1991-01-01

    A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire C H 1 domain. A variety of Fv analogues labeled with 2 H in the aromatic rings and with 13 C and/or 15 N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv spectra of Fv sensitively reflect the antigen binding and can be used along with 1 H and 13 C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been folowed in the absence and presence of DNS-Lys by using the 1 H- 15 N shift correlation spectra. Use of the β-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed

  10. Evidence for single-chain magnet behavior in a Mn(III)-Ni(II) chain designed with high spin magnetic units: a route to high temperature metastable magnets.

    Science.gov (United States)

    Clérac, Rodolphe; Miyasaka, Hitoshi; Yamashita, Masahiro; Coulon, Claude

    2002-10-30

    . This result indicates the presence of a metastable state without magnetic long-range order. This material is the first experimental design of a heterometallic chain with ST = 3 magnetic units showing a "single-chain magnet" behavior predicted in 1963 by R. J. Glauber for an Ising one-dimensional system. This work opens new perspectives for one-dimensional systems to obtain high temperature metastable magnets by combining high spin magnetic units, strong interunit interactions, and uniaxial anisotropy.

  11. ATON - Informe sobre el estado del arte de la FV integrada en edificación, y productos y tecnologías FV actuales con enfoque en la tecnología de capa fina

    OpenAIRE

    Masseck, Torsten; Fonseca Casas, Antoni; Godoy Muñoz, Alfonso

    2012-01-01

    Informe sobre el estado del arte de la FV integrada en edificación en el marco del projecto ATON (CENIT-E), convenio UPC-ACCIONA Instalaciones El proyecto ATON en su Actividad 10: Integración arquitectónica persigue la búsqueda de conceptos innovadores para la integración de sistemas fotovoltaicos de lámina delgada o Thin-film en entornos urbanos, con énfasis en edificios. En colaboración con Acciona, el equipo de trabajo de la UPC ha realizado estudios de integraciones singulares de in...

  12. Wheat cultivars selected for high Fv /Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter.

    Science.gov (United States)

    Sharma, Dew Kumari; Andersen, Sven Bode; Ottosen, Carl-Otto; Rosenqvist, Eva

    2015-02-01

    The chlorophyll fluorescence parameter Fv /Fm reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry and has been widely used for early stress detection in plants. Previously, we have used a three-tiered approach of phenotyping by Fv /Fm to identify naturally existing genetic variation for tolerance to severe heat stress (3 days at 40°C in controlled conditions) in wheat (Triticum aestivum L.). Here we investigated the performance of the previously selected cultivars (high and low group based on Fv /Fm value) in terms of growth and photosynthetic traits under moderate heat stress (1 week at 36/30°C day/night temperature in greenhouse) closer to natural heat waves in North-Western Europe. Dry matter accumulation after 7 days of heat stress was positively correlated to Fv /Fm . The high Fv /Fm group maintained significantly higher total chlorophyll and net photosynthetic rate (PN ) than the low group, accompanied by higher stomatal conductance (gs ), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv /Fm and intracellular CO2 (Ci ) was non-significant under the given heat stress. This study validated that our three-tiered approach of phenotyping by Fv /Fm performed under increasing severity of heat was successful in identifying wheat cultivars differing in photosynthesis under moderate and agronomically more relevant heat stress. The identified cultivars may serve as a valuable resource for further studies to understand the physiological mechanisms underlying the genetic variability in heat sensitivity of photosynthesis. © 2014 Scandinavian Plant Physiology Society.

  13. Expression and characterization of recombinant human serum ...

    African Journals Online (AJOL)

    C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host ...

  14. Expression and characterization of recombinant human serum ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-14

    Nov 14, 2011 ... C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog. [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and.

  15. Prevalence of 1691G>A FV mutation in Poland compared with that in other Central, Eastern and South-Eastern European countries.

    Science.gov (United States)

    Adler, Grażyna; Clark, Jeremy S C; Loniewska, Beata; Czerska, Ewa; Salkic, Nermin N; Ciechanowicz, Andrzej

    2012-05-01

    The 1691G>A FV variant has been described as a common genetic risk factor in venous thromboembolism. The purpose of this study was to provide a further frequency value for 1691G>A FV in Poland and to collate summary data from Central (Poland, Czech, Slovakia), Eastern (Russia, Belarus, Ukraine) and South-Eastern (Slovenia, Croatia, Bosnia and Herzegovina, Serbia, Montenegro, Macedonia, Bulgaria) European countries. For this purpose in 2007 the 1691G>A FV variant was analyzed by polymerase chain reaction-restriction fragment length polymorphism from DNA collected in 2005-2006. We studied 650 subjects: 400 newborns and 250 older individuals (mean age 46.1 y) from Poland and compared results with reports from other countries, as well as with the frequency trend of 845G>A HFE across South-Eastern European countries using centroid cities. From our 1691G>A FV study we identified 626 GG homozygotes, 23 GA heterozygotes, and 1 AA homozygote (n = 650), giving an A allele frequency of 1.9%, and a summed frequency value for Poland of 2.0% (n = 1588); the frequency in Central European countries was 3.9% (n = 4559), mostly due to the high value in the Czech Republic: 5.1% (n = 2819); the South-Eastern European countries had 2.5% (n = 2410). Among the Eastern European countries the 1691G>A FV allele frequency was 1.9% (n=791), between the South-Eastern and Eastern European countries there was no significant difference (p=0.17). We confirm that the 1691G>A FV allele frequency in Poland, as well as other countries compared, is significantly lower than that in Czech.

  16. Analysis on the stress corrosion crack inception based on pit shape and size of the FV520B tensile specimen

    Science.gov (United States)

    Xiang, Longhao; Pan, Juyi; Chen, Songying

    2018-06-01

    The influence of pit shape and size on local stress concentration in the tensile specimen and the stress corrosion cracks inception was studied by employing the element remove technique. The maximum stress located in the bottom of pit on FV520B tensile specimen. The location of maximum strain was near the mouth of the pit or the shoulder and plastic strain existed in this region. Stress concentration factor and plastic deformation on four different geometrical shape pits of hemisphere, semi-ellipsoid, bullet and butterfly were numerically investigated, respectively. The simulation results showed that butterfly pit got the biggest stress concentration factor. The plastic strain rate during pit growth was in the sensitivity range of stress corrosion cracks inception, indicating that stress corrosion cracks were more likely to nucleate near the pit tip or the shoulder.

  17. Self-assembling complexes of quantum dots and scFv antibodies for cancer cell targeting and imaging.

    Directory of Open Access Journals (Sweden)

    Tatiana A Zdobnova

    Full Text Available Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex.

  18. Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional 1H NMR analyses fo the antigen-antibody interactions

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Igarashi, Takako; Shimada, Ichio; Arata, Yoji

    1991-01-01

    The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire C H 1 domain is deleted. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides 1 H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2'-H and Tyr C3',5'-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansly group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, the authors have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed

  19. Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional sup 1 H NMR analyses fo the antigen-antibody interactions

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hideo; Igarashi, Takako; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan))

    1991-03-19

    The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire C{sub H}1 domain is deleted. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides {sup 1}H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2{prime}-H and Tyr C3{prime},5{prime}-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansly group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, the authors have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed.

  20. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Science.gov (United States)

    Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

    2013-01-01

    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

  1. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

    Science.gov (United States)

    2016-08-01

    ECBC-TR-1395 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR... ANTIBODY CHARACTERIZATION: CHARACTERIZATION OF AN MS2 SCFV ANTIBODY PRODUCED BY ILLUMINA Patricia E. Buckley Alena M. Calm Heather Welsh Roy...4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv

  2. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  3. Wheat cultivars selected for high Fv/Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter

    DEFF Research Database (Denmark)

    Sharma, Dew Kumari; Andersen, Sven Bode; Ottosen, Carl Otto

    2015-01-01

    ) than the low group, accompanied by higher stomatal conductance (gs), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv/Fm and intracellular CO2 (Ci) was non...

  4. FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

    Directory of Open Access Journals (Sweden)

    Chengkang eZhang

    2015-10-01

    Full Text Available Fusarium verticillioides (formerly F. moniliforme is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

  5. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2013-02-01

    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  6. Cultivation of Pichia pastoris carrying the scFv anti LDL (- antibody fragment. Effect of preculture carbon source

    Directory of Open Access Journals (Sweden)

    Cesar Andres Diaz Arias

    Full Text Available Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (- under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

  7. Preclinical pharmacokinetics, interspecies scaling, and pharmacokinetics of a Phase I clinical trial of TTAC-0001, a fully human monoclonal antibody against vascular endothelial growth factor 2

    Directory of Open Access Journals (Sweden)

    Lee WS

    2018-03-01

    Full Text Available Weon Sup Lee,1 Sang Ryeol Shim,1 Seon Young Lee,1 Jin San Yoo,1 Sung Kweon Cho2 1PharmAbcine, Inc., Daejeon, Republic of Korea; 2Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea Background: VEGF is a highly selective mitogen that serves as the central regulator of tumor angiogenesis by mediating endothelial proliferation, permeability, and survival. Tanibirumab (TTAC-0001 is a fully human IgG1 monoclonal antibody derived from a fully human naïve single-chain variable fragment (ScFv phage library that was developed to inhibit the effects of VEGF in the treatment of solid tumors, especially those of the brain. Methods: In the present study, we conducted intravenous pharmacokinetic studies of TTAC-0001 in mice, rats, and cynomolgus monkeys. At the doses studied (3 mg/kg, 10 mg/kg, 30 mg/kg, TTAC-0001 exhibited dose proportionality in mice and monkeys. At a dose of ~10 mg/kg, the clearance of TTAC-0001 from serum was 0.017 mL/h in mice, 0.35 mL/h in rats, and 2.19 mL/h in cynomolgus monkeys, and the terminal half-life ranged from 20–30 h among the three species. Pharmacokinetic data in mice, rats, and cynomolgus monkeys were used to predict the pharmacokinetics of TTAC-0001 in humans using allometric scaling. The predicted serum clearance of TTAC-0001 in humans was 102.45 mL/h and the terminal half-life was 27.52 h. Results: The maximum life span-corrected clearance value was 72.92 mL/h. The observed clearance in humans was more similar to the predicted scaled clearance. Conclusion: We investigated the pharmacokinetics of TTAC-0001 in mice, rats, and cynomolgus monkeys after intravenous administration. At the doses studied, TTAC-0001 exhibited dose proportionality in mice and monkeys. The scaled pharmacokinetics of TTAC-0001 reported here was useful for designing first-in-human studies. Allometric scaling in the therapeutic antibody is feasible. Keywords: VEGF2, tanibirumab, pharmacokinetics

  8. Development of a Targeted anti-HER2 scFv Chimeric Peptide for Gene Delivery into HER2-Positive Breast Cancer Cells.

    Science.gov (United States)

    Cheraghi, Roya; Nazari, Mahboobeh; Alipour, Mohsen; Majidi, Asia; Hosseinkhani, Saman

    2016-12-30

    Chimeric polymers are known as suitable carriers for gene delivery. Certain properties are critical for a polymer to be used as a gene delivery vector. A new polymer was designed for the targeted delivery of genes into breast cancer cell lines, based on MPG peptide. It is composed of different functional domains, including HIV gp41, nuclear localization sequence of SV40 T-antigen, two C-terminus repeats of histone H1, and the scFv of anti-HER2 antibody. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with an average size of 250nm. Moreover, fusion of the scFv portion to the carrier brought about the specific recognition of HER2. Overall, the transfection efficiency of the vector demonstrated that it could deliver the desired gene into BT-474 HER2-positive breast cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

    Directory of Open Access Journals (Sweden)

    Liang Xu

    2017-09-01

    Full Text Available Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26, a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action

  10. Identification and characterisation of the proteins bound by specific phage-displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts.

    Science.gov (United States)

    de la Cruz, Silvia; Madrid, Raquel; García-García, Aina; Alcocer, Marcos; Martín, Rosario; González, Isabel; García, Teresa

    2018-03-01

    Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene

    International Nuclear Information System (INIS)

    Hoatlin, M.E.; Kozak, S.L.; Kabat, D.; Lilly, F.; Chakraborti, A.; Kozak, C.A.

    1990-01-01

    The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) to stimulate erythroblastosis. To directly compare the effects of gp55 with erythropoietin (Epo), the authors produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125 I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2 r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemia. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein

  12. Profile data from CTD casts aboard the F/V Ocean Explorer in the Arctic Ocean and Beaufort Sea from 2008-08-06 to 2008-08-22 (NODC Accession 0001920)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This profile data aboard the F/V Ocean Explorer in the Arctic Ocean and Beaufort Sea from August 6, 2008 to August 22, 2008 was supported by the Minerals Management...

  13. BASALIT trial: double-blind placebo-controlled allergen immunotherapy with rBet v 1-FV in birch-related soya allergy.

    Science.gov (United States)

    Treudler, R; Franke, A; Schmiedeknecht, A; Ballmer-Weber, B; Worm, M; Werfel, T; Jappe, U; Biedermann, T; Schmitt, J; Brehler, R; Kleinheinz, A; Kleine-Tebbe, J; Brüning, H; Ruëff, F; Ring, J; Saloga, J; Schäkel, K; Holzhauser, T; Vieths, S; Simon, J C

    2017-08-01

    Conflicting results exist on the effect of allergen immunotherapy (AIT) on pollen-related food allergy. We aimed to investigate the efficacy of one-year AIT with the folding variant (FV) of recombinant (r) Bet v 1 on birch-related soya allergy. Of 138 subjects with Bet v 1 sensitization, 82 were positive at double-blind placebo-controlled food challenge (DBPCFC) with soya. A total of 56 of 82 were randomized in the ratio of 2:1 (active: placebo). Per-protocol population (PPP) had received ≥150 μg of allergen or placebo preparation. lowest observed adverse effect levels (LOAEL), postinterventional occurrence of objective signs (objS) at any dose level, sIgE/IgG4 against Bet v 1 and Gly m 4. Between-group changes were investigated (ancova, Mann-Whitney U-test, Fisher exact test). Baseline characteristics including LOAELs were comparable in both groups with objS and subjS occurring in 82% and 95% of active (n = 38) vs 78% and 83% of placebo group (n = 18). After AIT, objS occurred in 24% and 47%, respectively. LOAEL group differences showed a beneficial tendency (P = 0.081) for LOAEL objective in PPP (30 active, 15 placebo). sIgG4 raised only in active group (Bet v 1: P = 0.054, Gly m 4: P = 0.037), and no relevant changes occurred for sIgE. Only 56% of the intended sample size was recruited. For the first time, we present data on the effect of rBet v 1-FV on birch-related soya allergy. rBet v 1-FV AIT induced significant immunogenic effects. Clinical assessment showed a tendency in favour of the active group but did not reach statistical significance. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. ScFv anti-heparan sulfate antibodies unexpectedly activate endothelial and cancer cells through p38 MAPK: implications for antibody-based targeting of heparan sulfate proteoglycans in cancer.

    Directory of Open Access Journals (Sweden)

    Helena C Christianson

    Full Text Available Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS as a more rational approach to target HSPG function in endothelial cells (ECs. αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer.

  15. An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Schulz, D.

    2012-01-01

    -reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses. CONCLUSIONS: The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the Ig...... generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1. METHODS: Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107 mononuclear cells. An obtained scFv was converted into human immunoglobulin...

  16. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Michelle A Favila

    2015-12-01

    Full Text Available Leishmania major infection induces robust interleukin-12 (IL12 production in human dendritic cells (hDC, ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG and other phosphoglycan-containing molecules (PGs, making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS responsible for IL12 induction.Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-, or generally deficient for all PGs, (FV1 lpg2-. Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB and Interferon Regulatory Factor (IRF mediated transcription.These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

  17. EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3β1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2.

    Science.gov (United States)

    Sangboonruang, S; Thammasit, P; Intasai, N; Kasinrerk, W; Tayapiwatana, C; Tragoolpua, K

    2014-06-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3β1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3β1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

  18. tavg3_3d_chm_Fv: MERRA Chem 3D IAU States Cloud Precip, Time average 3-hourly 1.25 x 1.25 degree V5.2.0 (MAT3FVCHM) at GES DISC

    Data.gov (United States)

    National Aeronautics and Space Administration — The MAT3FVCHM or tavg3_3d_chm_Fv data product is the MERRA Data Assimilation System Chemistry 3-Dimensional chemistry on layers file that is time averaged, 3D model...

  19. FV peptide induces apoptosis in HEp 2 and HeLa cells: an insight into the mechanism of induction

    Directory of Open Access Journals (Sweden)

    Karthigayan S

    2006-01-01

    Full Text Available Abstract The present study is an attempt to evaluate the antiproliferative potential of peptide (7.6 kDa from lionfish (Pterios volitans venom on cultured HEp2 and HeLa cells. Different dose of purified peptide (1, 2 and 4 μg/ml at different time points (12, 24 and 36 hrs were tested for antiproliferative index of the peptide. Among them, 2 μg/ml at 24 hrs was found to effectively inhibit cancer cell growth in vitro and did not cause any adverse effect on normal human lymphocytes. Apoptosis was examined by propidium iodide staining, confirmed by the expression of caspase-8 and caspase-3, down regulation of Bcl-2 expression and DNA fragmentation in treated cells, when compared to untreated HEp2 and HeLa cells. Thus fish venom peptide was found to selectively induce apoptosis in cancer cell.

  20. FV peptide induces apoptosis in HEp 2 and HeLa cells: an insight into the mechanism of induction

    Science.gov (United States)

    Sri Balasubashini, M; Karthigayan, S; Somasundaram, ST; Balasubramanian, T; Rukkumani, R; Menon, Venugopal P

    2006-01-01

    The present study is an attempt to evaluate the antiproliferative potential of peptide (7.6 kDa) from lionfish (Pterios volitans) venom on cultured HEp2 and HeLa cells. Different dose of purified peptide (1, 2 and 4 μg/ml) at different time points (12, 24 and 36 hrs) were tested for antiproliferative index of the peptide. Among them, 2 μg/ml at 24 hrs was found to effectively inhibit cancer cell growth in vitro and did not cause any adverse effect on normal human lymphocytes. Apoptosis was examined by propidium iodide staining, confirmed by the expression of caspase-8 and caspase-3, down regulation of Bcl-2 expression and DNA fragmentation in treated cells, when compared to untreated HEp2 and HeLa cells. Thus fish venom peptide was found to selectively induce apoptosis in cancer cell. PMID:17137521

  1. Leaf gas exchange, fv/fm ratio, ion content and growth conditions of the two moringa species under magnetic water treatment

    International Nuclear Information System (INIS)

    Hasan, M.M.; Alharby, H.F.; Hajar, A.; Hakeem, K.R.

    2017-01-01

    The current greenhouse experiment investigates the role of magnetic water on the two Moringa species (Moringa oleifera and Moringa peregrina). Both species were exposed to the magnetic field (30 mT). The magnetic water increased the plant height, leaf number, leaflet number, and internode distances in both the species, respectively. Relative water content (RWC) and leaf area in both the species showed changes under magnetic water treatment. The results showed in magnetic water treatment, the leaf gas exchange parameters such as assimilation (A), stomatal conductance (gs), transpiration rate (E), and vapor pressure deficit (VPD) were increased. Similarly, Photosynthetic pigments (Chl a, Chl b, Chl (a+b), Carotenoids), photosynthetic water use efficiency (WUE) were also increased significantly. Magnetized water had also significant effects on the maximal efficiency of PSII photochemistry (Fv/Fm). Our study suggested that magnetic water treatment could be used as an environment-friendly technology for improving the growth and physiology of Moringa species. In addition, this technology could be further incorporated into the traditional methods of agriculture for the improvement of crop plants, particularly in the arid and sub-arid areas of the world. (author)

  2. Targeted delivery of CD44s-siRNA by ScFv overcomes de novo resistance to cetuximab in triple negative breast cancer.

    Science.gov (United States)

    Fu, Wenyan; Sun, Hefen; Zhao, Yang; Chen, Mengting; Yang, Lipeng; Yang, Xueli; Jin, Wei

    2018-05-16

    The overexpression of EGFR often occurs in TNBC, and the anti-EGFR receptor antibody cetuximab is used widely to treat metastatic cancer in the clinic. However, EGFR-targeted therapies have been developed for TNBC without clinical success. In this study, we show that impaired EGFR degradation is crucial for resistance to cetuximab, which depends on the cell surface molecule CD44. To further investigate the role of CD44 in EGFR signaling and its treatment potential, we developed a targeting fusion protein composed of an anti-EGFR scFv generated from cetuximab and truncated protamine, called Ce-tP. CD44 siRNA can be specifically delivered into EGFR-positive TNBC cells by Ce-tP. Efficient knockdown of CD44 and suppression of both EGFR and downstream signaling by the Ce-tP/siRNA complex were observed in EGFR-positive TNBC cells. More importantly, our results also showed that targeted delivery of siRNA specific for CD44 can efficiently overcome resistance to EGFR targeting in TNBC cells both in vitro and in vivo. Overall, our results establish a new principle to achieve EGFR inhibition in TNBC and limit drug resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Buus, S

    1999-01-01

    of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule...... electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained....

  4. Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin

    International Nuclear Information System (INIS)

    Demartis, S.; Tarli, L.; Neri, D.; Borsi, L.; Zardi, L.

    2001-01-01

    Angiogenesis is a characteristic feature of many aggressive tumours and other disorders. Antibodies capable of binding to new blood vessels, but not to mature vessels, could be used as selective targeting agents for immunoscintigraphic and radioimmunotherapeutic applications. Here we show that scFv(L19), a recombinant human antibody fragment with sub-nanomolar affinity for the ED-B domain of fibronectin, a marker of angiogenesis, can be stably labelled with iodine-125 and astatine-211 with full retention of immunoreactivity, using a trimethyl-stannyl benzoate bifunctional derivative. Biodistribution studies in mice bearing two different types of tumour grafted subcutaneously, followed by ex vivo micro-autoradiographic analysis, revealed that scFv(L19) rapidly localises around tumour blood vessels, but not around normal vessels. Four hours after intravenous injection of the stably radioiodinated scFv(L19), tumour to blood ratios were 6:1 in mice bearing the F9 murine teratocarcinoma and 9:1 in mice bearing an FE8 rat sarcoma. As expected, all other organs (including kidney) contained significantly less radioactivity than the tumour. Since the ED-B domain of fibronectin has an identical sequence in mouse and man, scFv(L19) is a pan-species antibody and the results presented here suggest clinical utility of radiolabelled scFv(L19) for the scintigraphic detection of angiogenesis in vivo. Furthermore, it should now be possible to investigate scFv(L19) for the selective delivery of 211 At to the tumour neovasculature, causing the selective death of tumour endothelial cells and tumour collapse. (orig.)

  5. Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

    Science.gov (United States)

    Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

    2017-11-01

    The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Numerical study of steady/unsteady flow and heat transfer in porous media using a characteristics-based matrix-free implicit FV method on unstructured grids

    International Nuclear Information System (INIS)

    Chiem, Kok Siong; Zhao Yong

    2004-01-01

    In this study, a high-resolution characteristic-based finite-volume (FV) method on unstructured grids [Int. J. Numer. Method Eng. 50 (2001) 11; Int. J. Heat Fluid Flow 21 (2000) 432] is extended by a matrix-free implicit dual-time stepping scheme for the numerical simulation of steady and unsteady flow and heat transfer with porous media. The method has been used to study the characteristics of a complex problem: flow and heat transfer in a channel with multiple discrete porous blocks, which was originally proposed by Huang and Vafai [J. Thermophys. Heat Transfer 8 (3) (1994) 563]. In addition, flow and heat transfer in a channel partially or fully filled with porous layers and containing solid protruding blocks with constant heat flux on its lower surface are also investigated in details. Hydrodynamic and heat transfer results are reported for both steady and transient flow cases. In particular, the effects of Darcy and Reynolds numbers on heat transfer augmentation and pressure loss are studied. An in-depth discussion of the formation and variation of recirculation is presented and the existence of optimum porous insert is demonstrated. At high Reynolds numbers the flow in the porous channel exhibits a cyclic characteristics although unlike the non-porous channel flow, the cyclic vortex development is only restricted to a small area behind the last solid block, while temperature changes more slowly and does not exhibit cyclic variations over a long period of time. It is shown that for all the cases studied altering some parametric values can have significant and interesting effects on both flow pattern as well as heat transfer characteristics

  7. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Directory of Open Access Journals (Sweden)

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  8. A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

    2014-10-31

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Performance of the FV3-powered Next Generation Global Prediction System for Harvey and Irma, and a vision for a "beyond weather timescale" prediction system for long-range hurricane track and intensity predictions

    Science.gov (United States)

    Lin, S. J.; Bender, M.; Harris, L.; Hazelton, A.

    2017-12-01

    The performance of a GFDL developed FV3-based Next Generation Global Prediction System (NGGPS) for Harvey and Irma will be reported. We will report on aspects of track and intensity errors (vs operational models), heavy precipitation (Harvey), rapid intensification, and simulated structure (in comparison with ground based radar), and point to a need of a future long-range (from day-5 up to 30 days) physically based ensemble hurricane prediction system for providing useful information to the forecasters, beyond the usual weather timescale.

  10. Mechanics of biopolymer materials: Single chains to bulk properties

    NARCIS (Netherlands)

    Amuasi, H.E.; Storm, C.

    2010-01-01

    We outline the first stages in the multiscale modeling of biopolymer materials, starting with the statistical mechanics of single stiff chains. In the first coarse graining step, we demonstrate how to integrate out the single polymer degrees of freedom in supramolecular assemblies of such

  11. Calculation of single chain cellulose elasticity using fully atomistic modeling

    Science.gov (United States)

    Xiawa Wu; Robert J. Moon; Ashlie Martini

    2011-01-01

    Cellulose nanocrystals, a potential base material for green nanocomposites, are ordered bundles of cellulose chains. The properties of these chains have been studied for many years using atomic-scale modeling. However, model predictions are difficult to interpret because of the significant dependence of predicted properties on model details. The goal of this study is...

  12. Resonance Raman Spectroscopic Evaluation of Skin Carotenoids as a Biomarker of Carotenoid Status for Human Studies

    Science.gov (United States)

    Mayne, Susan T.; Cartmel, Brenda; Scarmo, Stephanie; Jahns, Lisa; Ermakov, Igor V.; Gellermann, Werner

    2013-01-01

    Resonance Raman Spectroscopy (RRS) is a non-invasive method that has been developed to assess carotenoid status in human tissues including human skin in vivo. Skin carotenoid status has been suggested as a promising biomarker for human studies. This manuscript describes research done relevant to the development of this biomarker, including its reproducibility, validity, feasibility for use in field settings, and factors that affect the biomarker such as diet, smoking, and adiposity. Recent studies have evaluated the response of the biomarker to controlled carotenoid interventions, both supplement-based and dietary [e.g., provision of a high-carotenoid fruit and vegetable (F/V)-enriched diet], demonstrating consistent response to intervention. The totality of evidence supports the use of skin carotenoid status as an objective biomarker of F/V intake, although in the cross-sectional setting, diet explains only some of the variation in this biomarker. However, this limitation is also a strength in that skin carotenoids may effectively serve as an integrated biomarker of health, with higher status reflecting greater F/V intake, lack of smoking, and lack of adiposity. Thus, this biomarker holds promise as both a health biomarker and an objective indicator of F/V intake, supporting its further development and utilization for medical and public health purposes. PMID:23823930

  13. Frequent and recent human acquisition of simian foamy viruses through apes' bites in central Africa.

    Directory of Open Access Journals (Sweden)

    Edouard Betsem

    2011-10-01

    Full Text Available Human infection by simian foamy viruses (SFV can be acquired by persons occupationally exposed to non-human primates (NHP or in natural settings. This study aimed at getting better knowledge on SFV transmission dynamics, risk factors for such a zoonotic infection and, searching for intra-familial dissemination and the level of peripheral blood (proviral loads in infected individuals. We studied 1,321 people from the general adult population (mean age 49 yrs, 640 women and 681 men and 198 individuals, mostly men, all of whom had encountered a NHP with a resulting bite or scratch. All of these, either Pygmies (436 or Bantus (1085 live in villages in South Cameroon. A specific SFV Western blot was used and two nested PCRs (polymerase, and LTR were done on all the positive/borderline samples by serology. In the general population, 2/1,321 (0.2% persons were found to be infected. In the second group, 37/198 (18.6% persons were SFV positive. They were mostly infected by apes (37/39 FV (mainly gorilla. Infection by monkey FV was less frequent (2/39. The viral origin of the amplified sequences matched with the history reported by the hunters, most of which (83% are aged 20 to 40 years and acquired the infection during the last twenty years. The (proviral load in 33 individuals infected by a gorilla FV was quite low (<1 to 145 copies per 10(5 cells in the peripheral blood leucocytes. Of the 30 wives and 12 children from families of FV infected persons, only one woman was seropositive in WB without subsequent viral DNA amplification. We demonstrate a high level of recent transmission of SFVs to humans in natural settings specifically following severe gorilla bites during hunting activities. The virus was found to persist over several years, with low SFV loads in infected persons. Secondary transmission remains an open question.

  14. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer

    2016-01-01

    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  15. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  16. Combining EL4-B5-based B-cell stimulation and phage display technology for the successful isolation of human anti-Scl-70 autoantibody fragments.

    Science.gov (United States)

    Weber, Malte; Weiss, Etienne; Engel, Alfred M

    2003-07-01

    Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.

  17. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

    Science.gov (United States)

    Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

    2018-04-01

    Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

  18. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  19. Photopatterning of self assembled monolayers on oxide surfaces for the selective attachment of biomolecules.

    Science.gov (United States)

    Hazarika, Pompi; Behrendt, Jonathan M; Petersson, Linn; Wingren, Christer; Turner, Michael L

    2014-03-15

    The immobilization of functional biomolecules to surfaces is a critical process for the development of biosensors for disease diagnostics. In this work we report the patterned attachment of single chain fragment variable (scFv) antibodies to the surface of metal oxides by the photodeprotection of self-assembled monolayers, using near-UV light. The photodeprotection step alters the functionality at the surface; revealing amino groups that are utilized to bind biomolecules in the exposed regions of the substrate only. The patterned antibodies are used for the detection of specific disease biomarker proteins in buffer and in complex samples such as human serum. © 2013 Elsevier B.V. All rights reserved.

  20. Quantitative estimation of compliance of human systemic veins by occlusion plethysmography with radionuclide

    International Nuclear Information System (INIS)

    Takatsu, Hisato; Gotoh, Kohshi; Suzuki, Takahiko; Ohsumi, Yukio; Yagi, Yasuo; Tsukamoto, Tatsuo; Terashima, Yasushi; Nagashima, Kenshi; Hirakawa, Senri

    1989-01-01

    Volume-pressure relationship and compliance of human systemic veins were estimated quantitatively and noninvasively using radionuclide. The effect of nitroglycerin (NTG) on these parameters was examined. Plethysmography with radionuclide (RN) was performed using the occlusion method on the forearm in 56 patients with various cardiac diseases after RN angiocardiography with 99m Tc-RBC. The RN counts-venous pressure curve was constructed from (1) the changes in radioactivity from region of interest on the forearm that were considered to reflect the changes in the blood volume of the forearm, and (2) the changes in the pressure of the forearm vein (fv) due to venous occlusion. The specific compliance of the forearm veins (Csp.fv; (1/V)·(ΔV/ΔP)) was obtained graphically from this curve at each patient's venous pressure (Pv). Csp.fv was 0.044±0.012 mmHg -1 in class I (mean±SD; n=13), 0.033±0.007 mmHg -1 in class II (n=30), and 0.019±0.007 mmHg -1 in class III (n=13), of the previous NYHA classification of work tolerance. There were significant differences in Csp.fv among the three classes. The systemic venous blood volume (Vsv) was determined by subtracting the central blood volume, measured by RN-angiocardiography, from total blood volume, measured by the indicator dilution method utilizing 131 I-human serum albumin. Systemic venous compliance (Csv) was calculated from Csv=Csp.fv·Vsv. The Csv was 127.2±24.8 ml·mmHg -1 (mean±SD) in class I, 101.1±24.1 ml·mmHg -1 in class II and 62.2±28.1 ml·mmHg -1 in class III. There were significant differences in Csv among the three classes. The class I Csv value was calculated to be 127.2±24.8 ml·mmHg -1 and the Csv/body weight was calculated to be 2.3±0.7 ml·mmHg -1 ·kg -1 of body weight. The administration of NTG increased Csv significantly in all cases. (J.P.N.)

  1. Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

    Science.gov (United States)

    Kawabe, Yoshinori; Komatsu, Shinya; Komatsu, Shodai; Murakami, Mai; Ito, Akira; Sakuma, Tetsushi; Nakamura, Takahiro; Yamamoto, Takashi; Kamihira, Masamichi

    2018-05-01

    Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Structural and functional studies on human coagulation factor V

    OpenAIRE

    Neut Kolfschoten, Marijn van der

    2005-01-01

    The aim of this research was to obtain a better insight into the structure and functioning of clotting factor V (FV), a protein that plays an important role in the regulation of clotting. Congenital defects in FV can greatly disturb the coagulation system, and can lead to symptoms ranging from para-haemophilia to thrombosis. One example of a congenital defect in FV I is the R506Q mutation (an aminoacid change at position 506 in the aminoacid chain of FV). This deviating FV molecule (also know...

  3. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  4. Polyphenolic glycoconjugates from medical plants of Rosaceae/Asteraceae family protect human lymphocytes against γ-radiation-induced damage.

    Science.gov (United States)

    Szejk, Magdalena; Poplawski, Tomasz; Sarnik, Joanna; Pawlaczyk-Graja, Izabela; Czechowski, Franciszek; Olejnik, Alicja Klaudia; Gancarz, Roman; Zbikowska, Halina Malgorzata

    2017-01-01

    Radioprotective effects of the water-soluble polyphenolic glycoconjugates, isolated from flowers of Sanguisorba officinalis L.(SO) and Erigeron canadensis L.(EC), and from leaves of Fragaria vesca L. (FV) and Rubus plicatus Whe. Et N. E. (RP), against γ-radiation-induced toxicity in human peripheral blood lymphocytes were investigated. Cell treatment with glycoconjugates (1, 5 and 25μg/mL) prior exposure to 10/15Gy radiation resulted in concentration-dependent reduction of DNA damage including oxidative DNA lesions (comet assay), substantial inhibition of lipid peroxidation (TBARS) and restoration of superoxide dismutase and S-glutathione transferase activities. Glycoconjugates isolated from SO and EC ensured better protection versus these from RP and FV, with the SO product potential comparable to that of the reference quercetin. Strong antioxidant/radioprotective activity of the SO and EC glycoconjugates could be attributed to high abundance of syringol-type and ferulic acid units in their matrices, respectively. Moreover, polyphenolic glycoconjugates (25μg/mL), including RP and FV products, significantly decreased DNA damage when applied post-radiation suggesting their modulating effects on DNA repair pathways. Preliminary data on the glycoconjugate phenolic structural units, based on GLC/MS of the products of pyrolysis and in situ methylation, in relation to application of plant products as potential radioprotectors is promising and deserves further investigation. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

    Science.gov (United States)

    Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

    2017-10-06

    Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli , had unprecedentedly high binding affinities ( K d ∼10 -12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

  6. Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

    Science.gov (United States)

    Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

    2017-01-01

    Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

  7. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

    Directory of Open Access Journals (Sweden)

    Xiaocong Yu

    2016-01-01

    Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

  8. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Science.gov (United States)

    Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

    2010-11-18

    The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  9. Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

    Science.gov (United States)

    Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

    2013-02-01

    We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

  10. Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

    Science.gov (United States)

    Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

    2003-05-01

    Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

  11. Effects of genetic engineering on the pharmacokinetics of antibodies

    International Nuclear Information System (INIS)

    Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K.

    1999-01-01

    Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment

  12. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

    Science.gov (United States)

    Garcia-Rodriguez, Consuelo; Razai, Ali; Geren, Isin N; Lou, Jianlong; Conrad, Fraser; Wen, Wei-Hua; Farr-Jones, Shauna; Smith, Theresa J; Brown, Jennifer L; Skerry, Janet C; Smith, Leonard A; Marks, James D

    2018-03-01

    Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (K D ). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had K D values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

  13. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes

    Directory of Open Access Journals (Sweden)

    Consuelo Garcia-Rodriguez

    2018-03-01

    Full Text Available Human botulism is most commonly caused by botulinum neurotoxin (BoNT serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS. A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD. By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633 is in pre-clinical development with an investigational new drug (IND application filing expected in 2018.

  14. Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

    Science.gov (United States)

    Khajeh, Shirin; Tohidkia, Mohammad Reza; Aghanejad, Ayuob; Mehdipour, Tayebeh; Fathi, Farzaneh; Omidi, Yadollah

    2018-06-09

    Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 V L antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed K D values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and V L G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and V L antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

  15. Insight into the potential for DNA idiotypic fusion vaccines designed for patients by analysing xenogeneic anti-idiotypic antibody responses

    Science.gov (United States)

    Forconi, Francesco; King, Catherine A; Sahota, Surinder S; Kennaway, Christopher K; Russell, Nigel H; Stevenson, Freda K

    2002-01-01

    DNA vaccines induce immune responses against encoded proteins, and have clear potential for cancer vaccines. For B-cell tumours, idiotypic (Id) immunoglobulin encoded by the variable region genes provides a target antigen. When assembled as single chain Fv (scFv), and fused to an immunoenhancing sequence from tetanus toxin (TT), DNA fusion vaccines induce anti-Id antibodies. In lymphoma models, these antibodies have a critical role in mediating protection. For application to patients with lymphoma, two questions arise: first, whether pre-existing antibody against TT affects induction of anti-scFv antibodies; second, whether individual human scFv fusion sequences are able to fold consistently to generate antibodies able to recognize private conformational Id determinants expressed by tumour cells. Using xenogeneic vaccination with scFv sequences from four patients, we have shown that pre-existing anti-TT immunity slows, but does not prevent, anti-Id antibody responses. To determine folding, we have monitored the ability of nine DNAscFv–FrC patients' vaccines to induce xenogeneic anti-Id antibodies. Antibodies were induced in all cases, and were strikingly specific for each patient's immunoglobulin with little cross-reactivity between patients, even when similar VH or VL genes were involved. Blocking experiments with human serum confirmed reactivity against private determinants in 26–97% of total antibody. Both immunoglobulin G1 (IgG1) and IgG2a subclasses were present at 1·3 : 1–15 : 1 consistent with a T helper 2-dominated response. Xenogeneic vaccination provides a simple route for testing individual patients' DNAscFv–FrC fusion vaccines, and offers a strategy for production of anti-Id antibodies. The findings underpin the approach of DNA idiotypic fusion vaccination for patients with B-cell tumours. PMID:12225361

  16. Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

    Directory of Open Access Journals (Sweden)

    Leonie Schnell

    2016-07-01

    Full Text Available Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenylsemicarbazone (EGA has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT. Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.

  17. Production and properties of monoclonal antibodies to human blood coagulation factor VII and factor VIIa

    International Nuclear Information System (INIS)

    Mann, P.; Nesbitt, J.A.; Ge, M.; Kisiel, W.

    1986-01-01

    Human factor VII is a trace vitamin K-dependent protein that circulates in blood as a single-chain precursor to a serine protease. Upon activation, two-chain factor VIIa activates factor x in the presence of tissue factor and calcium. Purified preparations of single-chain (SC) human factor VII and two-chain (TC) factor VIIa were utilized to immunize Balb/c mice. Spleen cells from these immunized mice were fused to a non-secreting NS-1 derivative of X63-Ag8 myeloma cells and grown in selective medium. Analysis of culture supernatants by EIA revealed several hybridomas that were secreting IgG specific for Sc-factor VII and TC-factor VIIa. In addition, several hybridomas secreted IgG that reacted equally well with factor VII and factor VIIa. One of the latter McAb (A-29) reacted with the heavy chain of factor VIIa and the intact factor VII molecule equally as judged by Western blotting. A-29 was produced in ascites fluid, purified and coupled to activated CH-Sepharose. Application of one liter of normal human plasma to 10 ml of this immunoadsorbent column, elution of factor VII and subsequent Western blot using 125 I-rabbit anti-human factor VII indicated a single species of factor VII(M/sub r/ = 50 KDa) in normal plasma. These specific factor VII/VIIa McAbs may prove useful in the analysis of these factors, and in the separation of SC-factor VII from TC-factor VIIa

  18. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  19. Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

    Directory of Open Access Journals (Sweden)

    Phelps Amanda L

    2009-11-01

    Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

  20. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Xiong Hairong [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Medicine, Wuhan (China); Lee, Robert J. [Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH (United States); Haura, Eric B. [Thoracic Oncology and Experimental Therapeutics Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Edwards, John G. [Apeliotus Technologies, Inc., Atlanta, GA (United States); Dynan, William S. [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Li Shuyi, E-mail: sli@georgiahealth.edu [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Apeliotus Technologies, Inc., Atlanta, GA (United States)

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  1. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

    Science.gov (United States)

    Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

    2017-10-01

    The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Epitope mapping of the domains of human angiotensin converting enzyme.

    Science.gov (United States)

    Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

    2006-06-01

    Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

  4. The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure

    DEFF Research Database (Denmark)

    Gaster, M; Schrøder, H D; Handberg, A

    2001-01-01

    results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin......There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic...... parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased...

  5. Application of dimensional regularization to single chain polymer static properties: Conformational space renormalization of polymers. III

    International Nuclear Information System (INIS)

    Oono, Y.; Ohta, T.; Freed, K.F.

    1981-01-01

    A dimensional regularization approach to the renormalization group treatment of polymer excluded volume is formulated in chain conformation space where monomers are specified by their spatial positions and their positions along the chain and the polymers may be taken to be monodisperse. The method utilizes basic scale invariance considerations. First, it is recognized that long wavelength macroscopic descriptions must be well defined in the limit that the minimum atomic or molecular scale L is set to zero. Secondly, the microscopic theory is independent of the conveniently chosen macroscopic scale of length k. The freedom of choice of k is exploited along with the assumed renormalizability of the theory to provide the renormalization group equations which directly imply the universal scaling laws for macroscopic properties. The renormalizability of the model implies the existence of the general relations between the basic macroparameters, such as chain length, excluded volume, etc., and their microscopic counterparts in the microscopic model for the system. These macro--micro relations are defined through the condition that macroscopic quantities be well defined for polymer chains for any spatial dimensionality. The method is illustrated by calculating the end vector distribution function for all values of end vectors R. The evaluation of this distribution function currently requires the use of expansions in e = 4-d. In this case our distribution reduces to known limits for R→0 or infinity. Subsequent papers will present calculations of the polymer coherent scattering function, the monomer spatial distribution function, and concentration dependent properties

  6. Magnetic hysteresis and domain wall dynamics in single chain magnets with antiferromagnetic interchain coupling

    Energy Technology Data Exchange (ETDEWEB)

    Bukharov, A A; Ovchinnikov, A S; Baranov, N V [Department of Physics, Ural State University, Ekaterinburg, 620083 (Russian Federation); Inoue, K [Institute for Advanced Materials Research, Hiroshima University, Hiroshima (Japan)

    2010-11-03

    Using Monte Carlo simulations we investigate magnetic hysteresis in two- and three-dimensional systems of weakly antiferromagnetically coupled spin chains based on a scenario of domain wall (kink) motion within the chains. By adapting the model of walkers to simulate the domain wall dynamics and using the Ising-like dipole-dipole model, we study the effects of interchain coupling, temperature and anisotropy axis direction on hysteresis curves.

  7. Mapping the q-voter model: From a single chain to complex networks

    Science.gov (United States)

    Jȩdrzejewski, Arkadiusz; Sznajd-Weron, Katarzyna; Szwabiński, Janusz

    2016-03-01

    We propose and compare six different ways of mapping the modified q-voter model to complex networks. Considering square lattices, Barabási-Albert, Watts-Strogatz and real Twitter networks, we ask the question if always a particular choice of the group of influence of a fixed size q leads to different behavior at the macroscopic level. Using Monte Carlo simulations we show that the answer depends on the relative average path length of the network and for real-life topologies the differences between the considered mappings may be negligible.

  8. Improved affinity of a chicken single-chain antibody to avian ...

    African Journals Online (AJOL)

    linyuan

    2011-12-12

    Dec 12, 2011 ... Jianhua Wang. 1. , Ying Zhang ... et al., 1991; Dougan et al., 1998; Chang et al., 2009). The site-directed ... Analysis of the three-dimensional structure of the antigen-antibody ..... Altering the fine specificity of an anti-Legionella ...

  9. Single-chain vascular endothelial growth factor variant with antagonist activity

    DEFF Research Database (Denmark)

    Boesen, Thomas P; Soni, Bobby; Schwartz, Thue W

    2002-01-01

    receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce...... dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing...

  10. Effect of chain stiffness on the structure of single-chain polymer nanoparticles

    DEFF Research Database (Denmark)

    Moreno, Angel J; Bacova, Petra; Lo Verso, Federica

    2018-01-01

    of the domains is in all cases similar to that of Gaussian chains or rings, irrespective of the stiffness and degree of cross-linking. It is the spatial arrangement of the domains which determines the global structure of the SCNP (sparse Gaussian-like object or crumpled globule). Since intramolecular stiffness...... or 'crumpled' globular behaviour for very stiff SCNPs. We characterize domains in the SCNPs. These are weakly deformable regions that can be seen as disordered analogues of domains in disordered proteins. Increasing stiffness leads to bigger and less deformable domains. Surprisingly, the scaling behaviour...... can be varied through the specific chemistry of the precursor or by introducing bulky side groups in its backbone, our results propose a new strategy to tune the global structure of SCNPs. ....

  11. Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

    Science.gov (United States)

    2011-01-01

    QuickChange R© Site-Directed Mutagenesis Kit, were stored in 50 μl aliquots at –80◦C for up to 6 months (see Note 3). 4. NZY broth: 0.01% (w/v) NZ amine ( casein ...glutamine, 17 mM sodium bicarbonate, 1 mM sodium pyruvate, 1% antibiotic/antimycotic (v/v), 10% fetal bovine serum. The media can be stored at 4◦C for...FluoReporter FITC protein labeling kit (Molecular Probes) was stored at 4◦C shielded from light for up to 6 months. 2. 1 M sodium bicarbonate buffer pH9

  12. Finite-Size Effects in Single Chain Magnets: An Experimental and Theoretical Study

    Science.gov (United States)

    Bogani, L.; Caneschi, A.; Fedi, M.; Gatteschi, D.; Massi, M.; Novak, M. A.; Pini, M. G.; Rettori, A.; Sessoli, R.; Vindigni, A.

    2004-05-01

    The problem of finite-size effects in s=1/2 Ising systems showing slow dynamics of the magnetization is investigated introducing diamagnetic impurities in a Co2+-radical chain. The static magnetic properties have been measured and analyzed considering the peculiarities induced by the ferrimagnetic character of the compound. The dynamic susceptibility shows that an Arrhenius law is observed with the same energy barrier for the pure and the doped compounds while the prefactor decreases, as theoretically predicted. Multiple spin reversal has also been investigated.

  13. [Fe(III)(dmbpy)(CN)4]-: a new building block for designing single-chain magnets.

    Science.gov (United States)

    Toma, Luminita Marilena; Pasán, Jorge; Ruiz-Pérez, Catalina; Lloret, Francesc; Julve, Miguel

    2012-11-28

    We herein present the synthesis and magneto-structural study of a new family of heterobimetallic chains of general formula {[Fe(III)(dmbpy)(CN)(4)](2)M(II)(H(2)O)(2)}(n)·pnH(2)O [dmbpy = 4,4'-dimethyl-2,2'-bipyridine; M = Mn (2), Cu (3), Ni (4) and Co (5) with p = 4 (2), 3 (3), 9 (4) and 3.5 (5)] which were prepared by using the mononuclear PPh(4)[Fe(III)(dmbpy)(CN)(4)]·3H(2)O (1) building block (PPh(4)(+) = tetraphenylphosphonium) as a ligand toward fully solvated M(II) ions. The structure of 1 consists of discrete [Fe(III)(dmbpy)(CN)(4)](-) anions, tetraphenylphosphonium cations and noncoordinated water molecules. Complexes 2-5 are isostructural compounds whose structure consists of neutral 4,2-wave like heterobimetallic chains of formula {[Fe(III)(dmbpy)(CN)(4)](2)M(II)(H(2)O)(2)}(n) where the [Fe(III)(dmbpy)(CN)(4)](-) entity adopts a bis-monodentate coordination mode toward trans-[M(II)(H(2)O)(2)] units through two of its four cyanide groups in cis positions. 1 exhibits the magnetic behaviour of magnetically isolated six-coordinate low-spin Fe(III) complexes with an important orbital contribution. 2 behaves as ferrimagnetic Fe(III)(2)Mn(II) chains, whereas 3-5 exhibit intrachain ferromagnetic couplings between the low-spin Fe(III) and either Cu(II) (3), Ni (4) or Co(II) (5) as well as frequency-dependence of the out-of-phase ac susceptibility signals below 3.0 (3), 5.5 (4) and 5.0 K (5). The relaxation time and the energy to reverse the magnetization of 3-5 are related to the anisotropy of the M(II) center and to the intra- and interchain magnetic interactions. Unprecedentedly in the world of cyanide-bearing complexes, 5 exhibits a double slow relaxation of the magnetization.

  14. Block copolymer systems: from single chain to self-assembled nanostructures.

    Science.gov (United States)

    Giacomelli, Cristiano; Schmidt, Vanessa; Aissou, Karim; Borsali, Redouane

    2010-10-19

    Recent advances in the field of macromolecular engineering applied to the fabrication of nanostructured materials using block copolymer chains as elementary building blocks are described in this feature article. By highlighting some of our work in the area and accounting for the contribution of other groups, we discuss the relationship between the physical-chemical properties of copolymer chains and the characteristics of nano-objects originating from their self-assembly in solution and in bulk, with emphasis on convenient strategies that allow for the control of composition, functionality, and topology at different levels of sophistication. In the case of micellar nanoparticles in solution, in particular, we present approaches leading to morphology selection via macromolecular architectural design, the functionalization of external solvent-philic shells with biomolecules (polysaccharides and proteins), and the maximization of micelle loading capacity by the suitable choice of solvent-phobic polymer segments. The fabrication of nanomaterials mediated by thin block copolymer films is also discussed. In this case, we emphasize the development of novel polymer chain manipulation strategies that ultimately allow for the preparation of precisely positioned nanodomains with a reduced number of defects via block-selective chemical reactivity. The challenges facing the soft matter community, the urgent demand to convert huge public and private investments into consumer products, and future possible directions in the field are also considered herein.

  15. Impact of charge carrier injection on single-chain photophysics of conjugated polymers

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, Felix J.; Vogelsang, Jan, E-mail: jan.vogelsang@physik.uni-regensburg.de; Lupton, John M. [Institut für Experimentelle und Angewandte Physik, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg (Germany)

    2016-06-27

    Charges in conjugated polymer materials have a strong impact on the photophysics and their interaction with the primary excited state species has to be taken into account in understanding device properties. Here, we employ single-molecule spectroscopy to unravel the influence of charges on several photoluminescence (PL) observables. The charges are injected either stochastically by a photochemical process or deterministically in a hole-injection sandwich device configuration. We find that upon charge injection, besides a blue-shift of the PL emission and a shortening of the PL lifetime due to quenching and blocking of the lowest-energy chromophores, the non-classical photon arrival time distribution of the multichromophoric chain is modified towards a more classical distribution. Surprisingly, the fidelity of photon antibunching deteriorates upon charging, whereas one would actually expect the opposite: the number of chromophores to be reduced. A qualitative model is presented to explain the observed PL changes. The results are of interest to developing a microscopic understanding of the intrinsic charge-exciton quenching interaction in devices.

  16. Comparison of Boltzmann and Gibbs entropies for the analysis of single-chain phase transitions

    Science.gov (United States)

    Shakirov, T.; Zablotskiy, S.; Böker, A.; Ivanov, V.; Paul, W.

    2017-03-01

    In the last 10 years, flat histogram Monte Carlo simulations have contributed strongly to our understanding of the phase behavior of simple generic models of polymers. These simulations result in an estimate for the density of states of a model system. To connect this result with thermodynamics, one has to relate the density of states to the microcanonical entropy. In a series of publications, Dunkel, Hilbert and Hänggi argued that it would lead to a more consistent thermodynamic description of small systems, when one uses the Gibbs definition of entropy instead of the Boltzmann one. The latter is the logarithm of the density of states at a certain energy, the former is the logarithm of the integral of the density of states over all energies smaller than or equal to this energy. We will compare the predictions using these two definitions for two polymer models, a coarse-grained model of a flexible-semiflexible multiblock copolymer and a coarse-grained model of the protein poly-alanine. Additionally, it is important to note that while Monte Carlo techniques are normally concerned with the configurational energy only, the microcanonical ensemble is defined for the complete energy. We will show how taking the kinetic energy into account alters the predictions from the analysis. Finally, the microcanonical ensemble is supposed to represent a closed mechanical N-particle system. But due to Galilei invariance such a system has two additional conservation laws, in general: momentum and angular momentum. We will also show, how taking these conservation laws into account alters the results.

  17. Single-Chain Antibody Fragment VEGF Inhibitor RTH258 for Neovascular Age-Related Macular Degeneration

    DEFF Research Database (Denmark)

    Holz, Frank G; Dugel, Pravin U.; Weissgerber, Georges

    2016-01-01

    Purpose To assess the safety and efficacy of different doses of RTH258 applied as single intravitreal administration compared with ranibizumab 0.5 mg in patients with neovascular age-related macular degeneration (AMD). Design Six-month, phase 1/2, prospective, multicenter, double-masked, randomized...

  18. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  19. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

    2011-09-01

    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  20. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Directory of Open Access Journals (Sweden)

    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  1. Primary structure of the human follistatin precursor and its genomic organization

    International Nuclear Information System (INIS)

    Shimasaki, Shunichi; Koga, Makoto; Esch, F.

    1988-01-01

    Follistatin is a single-chain gonadal protein that specifically inhibits follicle-stimulating hormone release. By use of the recently characterized porcine follistatin cDNA as a probe to screen a human testis cDNA library and a genomic library, the structure of the complete human follistatin precursor as well as its genomic organization have been determined. Three of eight cDNA clones that were sequenced predicted a precursor with 344 amino acids, whereas the remaining five cDNA clones encoded a 317 amino acid precursor, resulting from alternative splicing of the precursor mRNA. Mature follistatins contain four contiguous domains that are encoded by precisely separated exons; three of the domains are highly similar to each other, as well as to human epidermal growth factor and human pancreatic secretory trypsin inhibitor. The genomic organization of the human follistatin is similar to that of the human epidermal growth factor gene and thus supports the notion of exon shuffling during evolution

  2. Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

    Science.gov (United States)

    Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2018-01-01

    Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

  3. Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter

    1993-01-01

    Fetal antigen 1 was purified from second trimester human amniotic fluid by immunospecific affinity chromatography followed by reversed-phase chromatography. Fetal antigen 1 is a single chain glycoprotein with a M(r) of 32-38 kDa. The amino acid composition revealed a high content of cysteines......, prolines and amino acids (aa) with acidic side-chains indicating that fetal antigen 1 is a compactly folded, strongly hydrophilic molecule. The N-terminal amino acid sequence (37 aa) revealed no homology to other known protein sequences, implying that fetal antigen 1 is a 'novel' human protein. When the aa...... sequence was back-translated into the appropriate degenerate sequence of nucleic acids, fetal antigen 1 could be partially aligned to a 'human adrenal-specific mRNA, pG2'. The indirect immunoperoxidase technique demonstrated fetal antigen 1 in fetal hepatocytes, glandular cells of fetal pancreas...

  4. A potential amyloid-imaging probe for Alzheimer's disease

    International Nuclear Information System (INIS)

    Cai Jiong; Wang Shizhen; Yuan Jiangang; Qiang Boqin

    2004-01-01

    Purpose: To screen out the human single-chain fragment variable (scFv) against amyloid β peptide 40 from a human synthetic antibody library, sub-clone its gene into E. coli expression system, and express and purify it for amyloid peptide imaging research. The overload of amyloid β peptide and the appearance of senile plaques in the human brain tissue is one of the hallmark of the Alzheimer's disease, and in vivo imaging of amyloidβ peptide is valuable for the earlier diagnosis of Alzheimer's disease. Methods: Amyloid β peptide 40 was bound on the solid surface of Nunc plates as antigen and a human antibody library constructed with human antibody heavy and light chain variable gene and nucleotides sequence coded (Gly4Ser)3 linker and displayed on the protein surface of filamentous phage was used to screen the binding clones. After five rounds of bio-panning, the host E. coli TG1 was infected with eluted filamentous phage from the last turn of selection. 55 well-separated colonies were picked randomly from the plates and several specific positive clones were identified by ELISA testing, and their binding sites were determined by competitive ELISA with amyloid 13 peptide 40, 1-16, 25-35. The single-chain Fv antibody gene was sequenced and their amino acids sequence was deduced. The scFv antibody gene was sub-cloned into a protokayotic expression vector pET-22b(+) and transformed into bacteria strain BL21 to express the His6-tagged single-chain antibody and the whole cell culture was subjected to SDS-PAGE analysis. The antibody was expressed in inclusion bodies and purified with serial buffers and verified with western blotting and their activity was tested by ELISA against amyloid β peptide 40. Results: ELISA testing showed that 33 clones could bind amyloid β peptide 40 and 10 of these clones could be inhibited by amyloid β peptide 40 itself to below 50% of its original binding activities. Five clones could also be inhibited by amyloid β peptide 1-16. DNA

  5. Evaluation of glycodendron and synthetically-modified dextran clearing agents for multi-step targeting of radioisotopes for molecular imaging and radioimmunotherapy

    Science.gov (United States)

    Cheal, Sarah M.; Yoo, Barney; Boughdad, Sarah; Punzalan, Blesida; Yang, Guangbin; Dilhas, Anna; Torchon, Geralda; Pu, Jun; Axworthy, Don B.; Zanzonico, Pat; Ouerfelli, Ouathek; Larson, Steven M.

    2014-01-01

    A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific anti-tumor antibody (scFv4) streptavidin (SA) fusion protein (scFv4-SA) in vivo, to improve tumor to normal tissue concentration ratios for targeted radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with average molecular weight (MW) of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogs (e.g. CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of 124I-labelled-scFv4-SA clearance and dual-label biodistribution studies following multi-step targeting (MST) directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a bispecific antibody specific for an anti-tumor associated glycoprotein (TAG-72) single chain genetically-fused with SA (scFv4-SA); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of 124I-labelled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e. biotin) can impact the binding to antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor- to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin. PMID:24219178

  6. More Human than Human.

    Science.gov (United States)

    Lawrence, David

    2017-07-01

    Within the literature surrounding nonhuman animals on the one hand and cognitively disabled humans on the other, there is much discussion of where beings that do not satisfy the criteria for personhood fit in our moral deliberations. In the future, we may face a different but related problem: that we might create (or cause the creation of) beings that not only satisfy but exceed these criteria. The question becomes whether these are minimal criteria, or hierarchical, such that those who fulfill them to greater degree should be afforded greater consideration. This article questions the validity and necessity of drawing divisions among beings that satisfy the minimum requirements for personhood; considering how future beings-intelligent androids, synthezoids, even alternate-substrate sentiences-might fit alongside the "baseline" human. I ask whether these alternate beings ought to be considered different to us, and why this may or may not matter in terms of a notion of "human community." The film Blade Runner, concerned in large part with humanity and its key synthezoid antagonist Roy Batty, forms a framing touchstone for my discussion. Batty is stronger, faster, more resilient, and more intelligent than Homo sapiens. His exploits, far beyond the capability of normal humans, are contrasted with his frailty and transient lifespan, his aesthetic appreciation of the sights he has seen, and his burgeoning empathy. Not for nothing does his creator within the mythos term him "more human than human."

  7. Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

    Science.gov (United States)

    Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

    2014-06-01

    We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both

  8. Attachment of a Genetically Engineered Antibody to a Carbon Nanotube Transistor for Detection of Prostate Cancer Biomarkers

    Science.gov (United States)

    Lerner, Mitchell; Dailey, Jennifer; Goldsmith, Brett; Robinson, Matthew; Johnson, A. T. Charlie

    2011-03-01

    We have developed a novel detection method for osteopontin (OPN) by attaching an engineered single chain variable fragment (scFv) protein with high binding affinity for OPN to a carbon nanotube transistor. Osteopontin is a potential new biomarker for prostate cancer; its presence in humans is already associated with several forms of cancer, arthritis, osteoporosis and stress. Prostate cancer is the most commonly diagnosed cancer and second leading cause of cancer deaths among American men and as such represents a major public health issue. Detection of early-stage cancer often results in successful treatment, with long term disease-free survival in 60-90% of patients. Electronic transport measurements are used to detect the presence of OPN in solution at clinically relevant concentrations.

  9. Monoclonal TCR-redirected tumor cell killing.

    Science.gov (United States)

    Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

    2012-06-01

    T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

  10. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity {sup 86}Y- or {sup 177}Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

    Energy Technology Data Exchange (ETDEWEB)

    Cheal, Sarah M.; Lee, Sang-gyu; Punzalan, Blesida; Larson, Steven M. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Xu, Hong; Guo, Hong-fen [Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States); Chalasani, Sandhya; Carrasquillo, Jorge A. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Fung, Edward K. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Jungbluth, Achim [Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, NY (United States); Zanzonico, Pat B.; O' Donoghue, Joseph [Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Smith-Jones, Peter M. [Stony Brook University, Department of Psychiatry and Behavioral Science, Stony Brook, NY (United States); Stony Brook University, Department of Radiology, Stony Brook, NY (United States); Wittrup, K.D. [Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA (United States); Cheung, Nai-Kong V. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States)

    2016-05-15

    GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens {sup 177}Lu-or {sup 86}Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq {sup 177}Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm{sup 3}) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm{sup 3} tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten {sup 86}Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts. (orig.)

  11. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity 86Y- or 177Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

    International Nuclear Information System (INIS)

    Cheal, Sarah M.; Lee, Sang-gyu; Punzalan, Blesida; Larson, Steven M.; Xu, Hong; Guo, Hong-fen; Chalasani, Sandhya; Carrasquillo, Jorge A.; Fung, Edward K.; Jungbluth, Achim; Zanzonico, Pat B.; O'Donoghue, Joseph; Smith-Jones, Peter M.; Wittrup, K.D.; Cheung, Nai-Kong V.

    2016-01-01

    GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens 177 Lu-or 86 Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq 177 Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm 3 ) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm 3 tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten 86 Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts. (orig.)

  12. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity Y-86- or Lu-177-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

    Science.gov (United States)

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Lee, Sang-gyu; Punzalan, Blesida; Chalasani, Sandhya; Fung, Edward K.; Jungbluth, Achim; Zanzonico, Pat B.; Carrasquillo, Jorge A.; O’Donoghue, Joseph; Smith-Jones, Peter M.; Wittrup, K. Dane; Cheung, Nai-Kong V.; Larson, Steven M.

    2015-01-01

    Purpose GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pre-targeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn (radiolanthanide metal) complex. Methods PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent (CA), and the C825-haptens 177Lu-or 86Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Results Using optimized PRIT, therapeutic indices (TIs) for tumor radiation absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI: 73), 6.3 (TI: 10), 6.6 (TI: 10), and 5.3 (TI: 12), respectively. Two cycles of PRIT treatment (66.6 or 111 MBq 177Lu-DOTA-Bn) were safe and effective, with 9/9 complete responses of established s.c. tumors (100–700 mm3) and 2/9 alive without recurrence >140 d. Tumor log kill in this model was estimated to be 2.1–3.0 based time to 500-mm3 tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA-hapten 86Y-DOTA-Bn. Conclusions We have developed anti-GPA33 PRIT, as a triple-step theranostic strategy for pre-clinical detection, dosimetry and safe targeted radiotherapy of established human colorectal mouse xenografts. PMID:26596724

  13. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    International Nuclear Information System (INIS)

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

    2012-01-01

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id + tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id + single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id + fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id + tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id + scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

  14. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    Energy Technology Data Exchange (ETDEWEB)

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne, E-mail: bjarne.bogen@medisin.uio.no [Centre for Immune Regulation, Institute of Immunology, University of Oslo and Oslo University Hospital, Oslo (Norway)

    2012-10-30

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id{sup +} tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id{sup +} single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id{sup +} fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id{sup +} tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id{sup +} scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

  15. Synthesis, purification, and characterization of an Arg152 → Glu site-directed mutant of recombinant human blood clotting factor VII

    International Nuclear Information System (INIS)

    Wildgoose, P.; Kisiel, W.; Berkner, K.L.

    1990-01-01

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg 152 -Ile 153 . Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg 152 → Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M r ∼40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX

  16. Synthesis, purification, and characterization of an Arg sub 152 yields Glu site-directed mutant of recombinant human blood clotting factor VII

    Energy Technology Data Exchange (ETDEWEB)

    Wildgoose, P.; Kisiel, W. (Univ. of New Mexico, Albuquerque (USA)); Berkner, K.L. (ZymoGenetics, Inc., Seattle, WA (USA))

    1990-04-03

    Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg{sub 152}-Ile{sub 153}. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, the authors have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg{sub 152} {yields} Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. The clotting activity of mutant factor VII was completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease. Immunoblots of mutant factor VII with human factor IXa revealed no cleavage, whereas incubation of mutant factor VII with human factor Xa resulted in cleavage of mutant factor VII and the formation of a lower molecular weight degradation product migrating at M{sup r}{approx}40 000. The results are consistent with the proposal that zymogen factor VII possesses no intrinsic proteolytic activity toward factor X or factor IX.

  17. Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells

    International Nuclear Information System (INIS)

    Accardi, Luisa; Tommasino, Massimo; Banks, Lawrence; Chirullo, Barbara; Giorgi, Colomba; Donà, Maria Gabriella; Mileo, Anna M; Paggi, Marco G; Federico, Antonio; Torreri, Paola; Petrucci, Tamara C; Accardi, Rosita; Pim, David

    2011-01-01

    'High risk' Human Papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The 'intracellular antibody' technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to 'knock out' the function of specific proteins. In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the 'high-risk' HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™technology and Surface Plasmon Resonance. The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic

  18. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Clipboard: Monitoring the energy status of a living organism in real time ... Homology modelling and bivalent single-chain Fv construction of anti-HepG2 .... for the validation of inhibitors of the epidermal growth factor receptor/Ras pathway.

  19. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  20. Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Krogh, Thomas N; Højrup, Peter

    1994-01-01

    The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second-trimester human amniotic fluid. FA1 is a single-chained, heterogeneous glycoprotein of 225-262 amino acid residues. FA1 has six well conserved epidermal...... extends with minor corrections to the human adrenal-specific mRNA, pG2 as well. Immunohistochemical analysis demonstrated the presence of FA1 in 10 out of 14 lung tumors containing neuroendocrine elements, and in the placental villi where FA1 was exclusively seen in stromal cells in close contact...... to the vascular structure. In the pancreas, FA1 co-localized with insulin in the insulin secretory granules of the beta cells within the islets of Langerhans. Our findings suggest that FA1 is synthesized as a membrane anchored protein and released into the circulation after enzymic cleavage, and that circulating...

  1. Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

    Science.gov (United States)

    McElhiney, J; Lawton, L A; Porter, A J

    2000-12-01

    Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

  2. CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth.

    Science.gov (United States)

    Golubovskaya, Vita; Berahovich, Robert; Zhou, Hua; Xu, Shirley; Harto, Hizkia; Li, Le; Chao, Cheng-Chi; Mao, Mike Ming; Wu, Lijun

    2017-10-21

    CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

  3. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  4. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2.

    Science.gov (United States)

    Tripathi, Amit; Lammers, Karen M; Goldblum, Simeon; Shea-Donohue, Terez; Netzel-Arnett, Sarah; Buzza, Marguerite S; Antalis, Toni M; Vogel, Stefanie N; Zhao, Aiping; Yang, Shiqi; Arrietta, Marie-Claire; Meddings, Jon B; Fasano, Alessio

    2009-09-29

    Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR(2)) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR(2) or the use of PAR(2)(-/-) mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its alpha(2)- and beta-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.

  5. Human engineering

    International Nuclear Information System (INIS)

    Yang, Seong Hwan; Park, Bum; Gang, Yeong Sik; Gal, Won Mo; Baek, Seung Ryeol; Choe, Jeong Hwa; Kim, Dae Sung

    2006-07-01

    This book mentions human engineering, which deals with introduction of human engineering, Man-Machine system like system design, and analysis and evaluation of Man-Machine system, data processing and data input, display, system control of man, human mistake and reliability, human measurement and design of working place, human working, hand tool and manual material handling, condition of working circumstance, working management, working analysis, motion analysis working measurement, and working improvement and design in human engineering.

  6. Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Cui, Huaqing; Wu, Feng; Sun, Yanling; Fan, Guocai; Wang, Qingming

    2010-01-01

    Hepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown. Recombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry. We found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased. This work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed

  7. Economical analysis of FV power plants according installed performance

    Directory of Open Access Journals (Sweden)

    Peter Tauš

    2009-09-01

    Full Text Available According prognosis of future development of power capacities in Slovakia till 2030 there is assumed increasing of the electricalenergy volume, produced from renewable energy sources from present 260 MW approximately to 2100 MW (Petrovič, 2008, thatpresents almost 800 % increasing! In Slovakia position of photovoltaic in this sector was due to the high investment cost and on the lastplace due to the low system efficiency. Only possible way for implementation of the photovoltaic to the energetic system of the stateis 0donation. Slovakia will go this way also.

  8. Human rights

    NARCIS (Netherlands)

    Gaay Fortman, B. de

    2006-01-01

    Human rights reflect a determined effort to protect the dignity of each and every human being against abuse of power. This endeavour is as old as human history. What is relatively new is the international venture for the protection of human dignity through internationally accepted legal standards

  9. Human reliability

    International Nuclear Information System (INIS)

    Embrey, D.E.

    1987-01-01

    Concepts and techniques of human reliability have been developed and are used mostly in probabilistic risk assessment. For this, the major application of human reliability assessment has been to identify the human errors which have a significant effect on the overall safety of the system and to quantify the probability of their occurrence. Some of the major issues within human reliability studies are reviewed and it is shown how these are applied to the assessment of human failures in systems. This is done under the following headings; models of human performance used in human reliability assessment, the nature of human error, classification of errors in man-machine systems, practical aspects, human reliability modelling in complex situations, quantification and examination of human reliability, judgement based approaches, holistic techniques and decision analytic approaches. (UK)

  10. The preparation of albumin as a biological drug from human plasma by fiber filtration

    Directory of Open Access Journals (Sweden)

    Mousavi Hosseini K

    2011-08-01

    Full Text Available "nBackground: In recent years, consumption of whole-blood for the treatment of patients has decreased but use of biological plasma-derived medicines such as albumin, immunoglobulin and coagulation factors have increased instead. Paying attention to albumin molecular structure is important for its isolation from human plasma. Albumin is a single-chain protein consisting of about 585 amino acids and a molecular weight of 66500 Daltons. Albumin is a stable molecule and it is spherical in shape. There are different methods for human albumin preparation. Considering the large consumption of this biological drug in clinical settings, methods with fewer steps in production line are of big advantage in saving time and manufacturing more products."n "nMethods: In this project, we prepared human albumin using hollow fiber cartridges in order to omit the rework on fraction V+VI. Human albumin is usually produced by the application of cold ethanol method, where albumin is obtained from fraction V by doing a rework on fraction V+VI to separate fraction V."n "nResults: In the current work, human albumin was prepared from fraction V+VI by the help of hollow fiber cartridges. With a concentration of 20%, the obtained albumin had 96.5% of monomer and 3.5% of polymer and polymer aggregate."n "nConclusion: Comparing the obtained human albumin with a number of commercial human albumin samples by the use of SDS-page, the results were satisfactory regarding the 3.5 percent polymer and aggregate rate for the prepared albumin.

  11. Human Rights, Human Needs, Human Development, Human Security

    OpenAIRE

    Gasper, Des

    2009-01-01

    Human rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and politics; each has emerged within the United Nations world; each relies implicitly on a conceptualisation of human need; each has specific strengths. Yet mutual communication, understanding and co-operation are deficient, espec...

  12. Re-engineering and evaluation of anti-DNA autoantibody 3E10 for therapeutic applications.

    Science.gov (United States)

    Rattray, Zahra; Dubljevic, Valentina; Rattray, Nicholas J W; Greenwood, Deanne L; Johnson, Caroline H; Campbell, James A; Hansen, James E

    2018-02-12

    A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair. A murine divalent single chain variable fragment of 3E10 with mutations for improved DNA binding affinity, 3E10 (D31N) di-scFv, has previously been produced in P. pastoris and yielded promising pre-clinical findings, but is unsuitable for clinical testing. The present study reports the design, expression and testing of a panel of humanized 3E10 (D31N) di-scFvs, some of which contain CDR substitution. These variants were expressed in a modified CHO system and evaluated for their physicochemical attributes and ability to penetrate nuclei to selectively cause DNA damage accumulation in and kill cancer cells with DNA repair defects. Secondary structure was conserved and most variants retained the key characteristics of the murine 3E10 (D31N) di-scFv produced in P. pastoris. Moreover, several variants with CDR substitutions outperformed the murine prototype. In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Xikun Zhou

    2013-05-01

    Full Text Available Epidermal growth factor receptor (EGFR is overexpressed in a variety of human malignancies, including pancreatic cancer, breast cancer, colon cancer, and non-small cell lung cancer. Overexpression of EGFR is a predictive marker of therapeutic response and several lines of evidence suggest that EGFR is an excellent target for tumor therapy. However, the effective antitumor capacity of EGFR-specific T cells against EGFR-overexpressing tumor cells has not been fully elucidated. In our previous study, we identified an anti-EGFR single-chain variable fragment (scFv with specific and high affinity after screening by ribosome display. In this study, the anticancer potential of anti-EGFR scFv was investigated on the basis of cell-targeted therapy. A chimeric antigen receptor (CAR targeting EGFR was constructed and expressed on the cell membrane of T lymphocytes. These CAR-modified T cells demonstrated antitumor efficacy both in vitro and in vivo. In addition, the safety evaluation showed that CAR-modified lymphocytes have no or very minimal acute systemic toxicity. Taken together, our study provided the experimental basis for clinical application of genetically engineered lymphocytes; moreover, we also evaluate a new and interesting cell therapy protocol.

  14. Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

    Science.gov (United States)

    Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

    2010-01-01

    The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

  15. The novel immunotoxin HM1.24-ETA′ induces apoptosis in multiple myeloma cells

    International Nuclear Information System (INIS)

    Staudinger, M; Glorius, P; Burger, R; Kellner, C; Klausz, K; Günther, A; Repp, R; Klapper, W; Gramatzki, M; Peipp, M

    2014-01-01

    Despite new treatment modalities, the clinical outcome in a substantial number of patients with multiple myeloma (MM) has yet to be improved. Antibody-based targeted therapies for myeloma patients could make use of the HM1.24 antigen (CD317), a surface molecule overexpressed on malignant plasma cells and efficiently internalized. Here, a novel immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). HM1.24-ETA′ inhibited growth of interleukin 6 (IL-6)-dependent and -independent myeloma cell lines. Half-maximal growth inhibition was observed at concentrations as low as 0.3 nM. Target cell killing occurred via induction of apoptosis and was unaffected in co-culture experiments with bone marrow stromal cells. HM1.24-ETA′ efficiently triggered apoptosis of freshly isolated/cryopreserved cells of patients with plasma cell leukemia and MM and was active in a preclinical severe combined immunodeficiency (SCID) mouse xenograft model. Importantly, HM1.24-ETA′ was not cytotoxic against CD317-positive cells from healthy tissue (monocytes, human umbilical vein endothelial cells). These results indicate that CD317 may represent a promising target structure for specific and efficient immunotoxin therapy for patients with plasma cell tumors

  16. Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Zineb Lakhrif

    2018-02-01

    Full Text Available Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites.

  17. Human niche, human behaviour, human nature.

    Science.gov (United States)

    Fuentes, Agustin

    2017-10-06

    The concept of a 'human nature' or 'human natures' retains a central role in theorizing about the human experience. In Homo sapiens it is clear that we have a suite of capacities generated via our evolutionary past, and present, and a flexible capacity to create and sustain particular kinds of cultures and to be shaped by them. Regardless of whether we label these capacities 'human natures' or not, humans occupy a distinctive niche and an evolutionary approach to examining it is critical. At present we are faced with a few different narratives as to exactly what such an evolutionary approach entails. There is a need for a robust and dynamic theoretical toolkit in order to develop a richer, and more nuanced, understanding of the cognitively sophisticated genus Homo and the diverse sorts of niches humans constructed and occupied across the Pleistocene, Holocene, and into the Anthropocene. Here I review current evolutionary approaches to 'human nature', arguing that we benefit from re-framing our investigations via the concept of the human niche and in the context of the extended evolutionary synthesis (EES). While not a replacement of standard evolutionary approaches, this is an expansion and enhancement of our toolkit. I offer brief examples from human evolution in support of these assertions.

  18. Human Smuggling

    NARCIS (Netherlands)

    Siegel - Rozenblit, Dina; Zaitch, Damian

    2014-01-01

    Human smuggling is based on a consensus between smuggler, smuggled, and his/her family (which usually guarantees or effectuates payment). However, unauthorized immigrants are violating immigration laws and human smugglers are profiting from enabling illegal immigration. Both human smuggling and its

  19. Human intrusion

    International Nuclear Information System (INIS)

    Hora, S.; Neill, R.; Williams, R.; Bauser, M.; Channell, J.

    1993-01-01

    This paper focused on the possible approaches to evaluating the impacts of human intrusion on nuclear waste disposal. Several major issues were reviewed. First, it was noted that human intrusion could be addressed either quantitatively through performance assessments or qualitatively through design requirements. Second, it was decided that it was impossible to construct a complete set of possible future human intrusion scenarios. Third, the question of when the effect of possible human intrusion should be considered, before or after site selection was reviewed. Finally, the time frame over which human intrusion should be considered was discussed

  20. Human Technology and Human Affects

    DEFF Research Database (Denmark)

    Fausing, Bent

    2009-01-01

    Human Technology and Human Affects  This year Samsung introduced a mobile phone with "Soul". It was made with a human touch and included itself a magical touch. Which function does technology and affects get in everyday aesthetics like this, its images and interactions included this presentation...... will ask and try to answer. The mobile phone and its devices are depicted as being able to make a unique human presence, interaction, and affect. The medium, the technology is a necessary helper to get towards this very special and lost humanity. Without the technology, no special humanity - soul....... The paper will investigate how technology, humanity, affects, and synaesthesia are presented and combined with examples from everyday aesthetics, e.g. early computer tv-commercial, net-commercial for mobile phones. Technology and affects point, is the conclusion, towards a forgotten pre-human and not he...

  1. Human Parvoviruses

    Science.gov (United States)

    Söderlund-Venermo, Maria; Young, Neal S.

    2016-01-01

    SUMMARY Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large Parvoviridae family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable in vitro, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology. PMID:27806994

  2. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  3. Optimizing the Delivery of Short-Lived Alpha Particle-Emitting Isotopes to Solid Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Gregory P.

    2004-11-24

    The underlying hypothesis of this project was that optimal alpha emitter-based radioimmunotherapy (RAIT) could be achieved by pairing the physical half-life of the radioisotope to the biological half-life of the targeting vehicle. The project had two specific aims. The first aim was to create and optimize the therapeutic efficacy of 211At-SAPS-C6.5 diabody conjugates. The second aim was to develop bispecific-targeting strategies that increase the specificity and efficacy of alpha-emitter-based RAIT. In the performance of the first aim, we created 211At-SAPS-C6.5 diabody conjugates that specifically targeted the HER2 tumor associated antigen. In evaluating these immunoconjugates we determined that they were capable of efficient tumor targeting and therapeutic efficacy of established human tumor xenografts growing in immunodeficient mice. We also determined that therapeutic doses were associated with late renal toxicity, likely due to the role of the kidneys in the systemic elimination o f these agents. We are currently performing more studies focused on better understanding the observed toxicity. In the second aim, we successfully generated bispecific single-chain Fv (bs-scFv) molecules that co-targeted HER2 and HER3 or HER2 and HER4. The in vitro kinetics and in vivo tumor-targeting properties of these molecules were evaluated. These studies revealed that the bs-scFv molecules selectively localized in vitro on tumor cells that expressed both antigens and were capable of effective tumor localization in in vivo studies.

  4. Optimizing the Delivery of Short-Lived Alpha Particle-Emitting Isotopes to Solid Tumors

    International Nuclear Information System (INIS)

    Adams, Gregory P.

    2004-01-01

    The underlying hypothesis of this project was that optimal alpha emitter-based radioimmunotherapy (RAIT) could be achieved by pairing the physical half-life of the radioisotope to the biological half-life of the targeting vehicle. The project had two specific aims. The first aim was to create and optimize the therapeutic efficacy of 211At-SAPS-C6.5 diabody conjugates. The second aim was to develop bispecific-targeting strategies that increase the specificity and efficacy of alpha-emitter-based RAIT. In the performance of the first aim, we created 211At-SAPS-C6.5 diabody conjugates that specifically targeted the HER2 tumor associated antigen. In evaluating these immunoconjugates we determined that they were capable of efficient tumor targeting and therapeutic efficacy of established human tumor xenografts growing in immunodeficient mice. We also determined that therapeutic doses were associated with late renal toxicity, likely due to the role of the kidneys in the systemic elimination o f these agents. We are currently performing more studies focused on better understanding the observed toxicity. In the second aim, we successfully generated bispecific single-chain Fv (bs-scFv) molecules that co-targeted HER2 and HER3 or HER2 and HER4. The in vitro kinetics and in vivo tumor-targeting properties of these molecules were evaluated. These studies revealed that the bs-scFv molecules selectively localized in vitro on tumor cells that expressed both antigens and were capable of effective tumor localization in in vivo studies

  5. Human Rights/Human Needs.

    Science.gov (United States)

    Canning, Cynthia

    1978-01-01

    The faculty of Holy Names High School developed an interdisciplinary human rights program with school-wide activities focusing on three selected themes: the United Nations Universal Declaration of Human Rights, in conjunction with Human Rights Week; Food; and Women. This article outlines major program activities. (SJL)

  6. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  7. Vesicular stomatitis virus modified with single chain IL-23 exhibits oncolytic activity against tumor cells in vitro and in vivo

    OpenAIRE

    Reiss, Carol Shoshkes

    2010-01-01

    James M Miller1, Sarah McNulty Bidula1,5, Troels Mygind Jensen1,6, Carol Shoshkes Reiss1,2,3,41Department of Biology, New York University, New York, NY, USA; 2Center for Neural Science, NYU; 3NYU Cancer Institute; 4Departments of Microbiology, NYU School of Medicine and Mt Sinai School of Medicine, New York, NY, USA; 5Present address Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA,USA 6Present address: Univercity of Copenhagen, Copenhagen, DenmarkAbstract: Viruse...

  8. Novel single-chain antibody GD3A10 defines a chondroitin sulfate biomarker for ovarian cancer

    NARCIS (Netherlands)

    Vallen, M.J.E.; Tilborg, A.G. van; Tesselaar, M.H.; Dam, G.B. ten; Bulten, J.; Kuppevelt, T.H. van; Massuger, L.F.A.G.

    2014-01-01

    AIMS: Ovarian cancer has the highest case-to-fatality-index of all gynecological cancers. In this study, tumor-related alterations in the extracellular matrix, especially regarding chondroitin sulfate glycosaminoglycans, are proposed as a novel biomarker in ovarian cancer. MATERIALS & METHODS: Phage

  9. Prodrug Therapy for Breast Cancer Targeted by Single-Chain Antibodies F19 and 3S193

    National Research Council Canada - National Science Library

    Deckert, Peter

    2003-01-01

    ... of E.coli CD with the yeast isoenzyme. In addition, a second line of fusion constructs with green fluorescent protein instead of cytosine deaminase was designed for histological and intracellular distribution studies...

  10. Follistatin is a novel biomarker for lung adenocarcinoma in humans.

    Directory of Open Access Journals (Sweden)

    Fangfang Chen

    Full Text Available Follistatin (FST, a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear.The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80, which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40 using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis.These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma.

  11. Human Rights, Human Needs, Human Development, Human Security - Relationships between four international human discourses.

    NARCIS (Netherlands)

    D.R. Gasper (Des)

    2007-01-01

    markdownabstractAbstract: Human rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and

  12. A simple and robust approach to immobilization of antibody fragments.

    Science.gov (United States)

    Ikonomova, Svetlana P; He, Ziming; Karlsson, Amy J

    2016-08-01

    Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Human evolution

    DEFF Research Database (Denmark)

    Llamas, Bastien; Willerslev, Eske; Orlando, Ludovic Antoine Alexandre

    2017-01-01

    The field of human ancient DNA (aDNA) has moved from mitochondrial sequencing that suffered from contamination and provided limited biological insights, to become a fully genomic discipline that is changing our conception of human history. Recent successes include the sequencing of extinct homini...

  14. Think Human

    DEFF Research Database (Denmark)

    Nielsen, Charlotte Marie Bisgaard

    2013-01-01

    years' campaigns suggests that the theory of communication underlying the campaign has its basis in mechanical action rather than in human communication. The practice of 'Communication design' is investigated in relation to this metaphorical 'machine thinking' model of communication and contrasted...... with the human-centered theory of communication advocated by integrationism....

  15. Human kapital

    DEFF Research Database (Denmark)

    Grosen, Anders; Nielsen, Peder Harbjerg

    2007-01-01

    finansiel og human kapital. Den traditionelle rådgivnings snævre synsvinkel kan føre til forkerte investeringsråd. Der skal derfor opfordres til, at de finansielle virksomheder i tilrettelæggelsen af deres rådgivning af private kunder systematisk inddrager den humane kapitals størrelse og karakteristika i...

  16. Human trichuriasis

    DEFF Research Database (Denmark)

    Betson, Martha; Søe, Martin Jensen; Nejsum, Peter

    2015-01-01

    Human trichuriasis is a neglected tropical disease which affects hundreds of millions of people worldwide and is particularly prevalent among children living in areas where sanitation is poor. This review examines the current knowledge on the taxonomy, genetics and phylogeography of human Trichuris...

  17. Digital Humanities

    DEFF Research Database (Denmark)

    Brügger, Niels

    2016-01-01

    , and preserving material to study, as an object of study in its own right, as an analytical tool, or for collaborating, and for disseminating results. The term "digital humanities" was coined around 2001, and gained currency within academia in the following years. However, computers had been used within......Digital humanities is an umbrella term for theories, methodologies, and practices related to humanities scholarship that use the digital computer as an integrated and essential part of its research and teaching activities. The computer can be used for establishing, finding, collecting...

  18. Human Computation

    CERN Multimedia

    CERN. Geneva

    2008-01-01

    What if people could play computer games and accomplish work without even realizing it? What if billions of people collaborated to solve important problems for humanity or generate training data for computers? My work aims at a general paradigm for doing exactly that: utilizing human processing power to solve computational problems in a distributed manner. In particular, I focus on harnessing human time and energy for addressing problems that computers cannot yet solve. Although computers have advanced dramatically in many respects over the last 50 years, they still do not possess the basic conceptual intelligence or perceptual capabilities...

  19. Effect of Human Milk Appetite Hormones, Macronutrients, and Infant Characteristics on Gastric Emptying and Breastfeeding Patterns of Term Fully Breastfed Infants

    Directory of Open Access Journals (Sweden)

    Zoya Gridneva

    2016-12-01

    Full Text Available Human milk (HM components influence infant feeding patterns and nutrient intake, yet it is unclear how they influence gastric emptying (GE, a key component of appetite regulation. This study analyzed GE of a single breastfeed, HM appetite hormones/macronutrients and demographics/anthropometrics/body composition of term fully breastfed infants (n = 41, 2 and/or 5 mo. Stomach volumes (SV were calculated from pre-/post-feed ultrasound scans, then repeatedly until the next feed. Feed volume (FV was measured by the test-weigh method. HM samples were analyzed for adiponectin, leptin, fat, lactose, total carbohydrate, lysozyme, and total/whey/casein protein. Linear regression/mixed effect models were used to determine associations between GE/feed variables and HM components/infant anthropometrics/adiposity. Higher FVs were associated with faster (−0.07 [−0.10, −0.03], p < 0.001 GE rate, higher post-feed SVs (0.82 [0.53, 1.12], p < 0.001, and longer GE times (0.24 [0.03, 0.46], p = 0.033. Higher whey protein concentration was associated with higher post-feed SVs (4.99 [0.84, 9.13], p = 0.023. Longer GE time was associated with higher adiponectin concentration (2.29 [0.92, 3.66], p = 0.002 and dose (0.02 [0.01, 0.03], p = 0.005, and lower casein:whey ratio (−65.89 [−107.13, −2.66], p = 0.003. FV and HM composition influence GE and breastfeeding patterns in term breastfed infants.

  20. Effect of Human Milk Appetite Hormones, Macronutrients, and Infant Characteristics on Gastric Emptying and Breastfeeding Patterns of Term Fully Breastfed Infants.

    Science.gov (United States)

    Gridneva, Zoya; Kugananthan, Sambavi; Hepworth, Anna R; Tie, Wan J; Lai, Ching T; Ward, Leigh C; Hartmann, Peter E; Geddes, Donna T

    2016-12-28

    Human milk (HM) components influence infant feeding patterns and nutrient intake, yet it is unclear how they influence gastric emptying (GE), a key component of appetite regulation. This study analyzed GE of a single breastfeed, HM appetite hormones/macronutrients and demographics/anthropometrics/body composition of term fully breastfed infants ( n = 41, 2 and/or 5 mo). Stomach volumes (SV) were calculated from pre-/post-feed ultrasound scans, then repeatedly until the next feed. Feed volume (FV) was measured by the test-weigh method. HM samples were analyzed for adiponectin, leptin, fat, lactose, total carbohydrate, lysozyme, and total/whey/casein protein. Linear regression/mixed effect models were used to determine associations between GE/feed variables and HM components/infant anthropometrics/adiposity. Higher FVs were associated with faster (-0.07 [-0.10, -0.03], p < 0.001) GE rate, higher post-feed SVs (0.82 [0.53, 1.12], p < 0.001), and longer GE times (0.24 [0.03, 0.46], p = 0.033). Higher whey protein concentration was associated with higher post-feed SVs (4.99 [0.84, 9.13], p = 0.023). Longer GE time was associated with higher adiponectin concentration (2.29 [0.92, 3.66], p = 0.002) and dose (0.02 [0.01, 0.03], p = 0.005), and lower casein:whey ratio (-65.89 [-107.13, -2.66], p = 0.003). FV and HM composition influence GE and breastfeeding patterns in term breastfed infants.

  1. Human expunction

    Science.gov (United States)

    Klee, Robert

    2017-10-01

    Thomas Nagel in `The Absurd' (Nagel 1971) mentions the future expunction of the human species as a `metaphor' for our ability to see our lives from the outside, which he claims is one source of our sense of life's absurdity. I argue that the future expunction (not to be confused with extinction) of everything human - indeed of everything biological in a terran sense - is not a mere metaphor but a physical certainty under the laws of nature. The causal processes by which human expunction will take place are presented in some empirical detail, so that philosophers cannot dismiss it as merely speculative. I also argue that appeals to anthropic principles or to forms of mystical cosmology are of no plausible avail in the face of human expunction under the laws of physics.

  2. Human Cloning

    National Research Council Canada - National Science Library

    Johnson, Judith A; Williams, Erin D

    2006-01-01

    .... Scientists in other labs, including Harvard University and the University of California at San Francisco, intend to produce cloned human embryos in order to derive stem cells for medical research...

  3. Human brucellosis

    NARCIS (Netherlands)

    Franco, María Pía; Mulder, Maximilian; Gilman, Robert H.; Smits, Henk L.

    2007-01-01

    Human brucellosis still presents scientists and clinicians with several challenges, such as the understanding of pathogenic mechanisms of Brucella spp, the identification of markers for disease severity, progression, and treatment response, and the development of improved treatment regimens.

  4. Human settlements

    CSIR Research Space (South Africa)

    Van Niekerk, Cornelia W

    2017-09-01

    Full Text Available risk of deaths and injuries by drowning in floods and migration- related health effects. • Increased migration, which can result in human suffering, human rights violations, conflicts and political instability. • Loss of property and livelihoods.... The vulnerability of settlements in southern Africa is impacted by various and complex socio-economic processes related to the cultural, political and institutional contexts and demographic pressure, as well as specific high-risk zones susceptible to flash floods...

  5. Human Cloning

    Science.gov (United States)

    2006-07-20

    Human Fertilization and Embryology Authority (HFEA). A team of scientists headed by Alison Murdoch at the University of Newcastle received permission...not yet reported success in isolating stem cells from a cloned human embryo. A research team headed by Ian Wilmut at the University of Edinburgh...research group, headed by Douglas Melton and Kevin Eggan, submitted their proposal to a Harvard committee composed of ethicists, scientists and public

  6. Discovery of human scFvs that cross-neutralize the toxic effects of B. jararacussu and C. d. terrificus venoms.

    Science.gov (United States)

    Silva, Luciano C; Pucca, Manuela B; Pessenda, Gabriela; Campos, Lucas B; Martinez, Edson Z; Cerni, Felipe A; Barbosa, José E

    2018-01-01

    Accidents involving venomous snakes are a public health problem worldwide, causing a large number of deaths per year. In Brazil, the majority of accidents are caused by the Bothrops and Crotalus genera, which are responsible for approximately 80% of severe envenoming cases. The cross-neutralization of snake venoms by antibodies is an important issue for development of more effective treatments. Our group has previously reported the construction of human monoclonal antibody fragments towards Bothrops jararacussu and Crotalus durissus terrificus' venoms. This study aimed to select human single-chain variable fragments (scFvs) that recognize both bothropic and crotalic crude venoms following venoms neutralizing capacity in vitro and in vivo. The cross-reactivity of Cro-Bothrumabs were demonstrated by ELISA and in vitro and in vivo experiments showed that a combination of scFvs neutralizes in vitro toxic activities (e.g. indirect hemolysis and plasma-clotting) of crotalic and bothropic venoms as well as prolonged survival time of envenomed animals. Our results may contribute to the development of the first human polyvalent antivenom against Bothrops jararacussu and Crotalus durissus terrificus venoms, overcoming some undesirable effects caused by conventional serotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Functionalized Magnetic Resonance Contrast Agent Selectively Binds to Glycoprotein IIb/IIIa on Activated Human Platelets under Flow Conditions and Is Detectable at Clinically Relevant Field Strengths

    Directory of Open Access Journals (Sweden)

    Constantin von zur Mühlen

    2008-03-01

    Full Text Available Recent progress in molecular magnetic resonance imaging (MRI provides the opportunity to image cells and cellular receptors using microparticles of iron oxide (MPIOs. However, imaging targets on vessel walls remains challenging owing to the quantity of contrast agents delivered to areas of interest under shear stress conditions. We evaluated ex vivo binding characteristics of a functional MRI contrast agent to ligand-induced binding sites (LIBSs on activated glycoprotein IIb/IIIa receptors of human platelets, which were lining rupture-prone atherosclerotic plaques and could therefore facilitate detection of platelet-mediated pathology in atherothrombotic disease. MPIOs were conjugated to anti-LIBS single-chain antibodies (LIBS-MPIO or control antibodies (control MPIO. Ex vivo binding to human platelet-rich clots in a dose-dependent manner was confirmed on a 3 T clinical MRI scanner and by histology (p < .05 for LIBS-MPIO vs control MPIO. By using a flow chamber setup, significant binding of LIBS-MPIO to a platelet matrix was observed under venous and arterial flow conditions, but not for control MPIO (p < .001. A newly generated MRI contrast agent detects activated human platelets at clinically relevant magnetic field strengths and binds to platelets under venous and arterial flow conditions, conveying high payloads of contrast to specific molecular targets. This may provide the opportunity to identify vulnerable, rupture-prone atherosclerotic plaques via noninvasive MRI.

  8. An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Green David E

    2010-02-01

    Full Text Available Abstract Background Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC, shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs such as Shiga-like Toxin 1 (SLT-1 represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP derived from the cytotoxic A subunit of SLT-1 (SLT-1A, harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM allowing the toxin variant to selectively target and kill human melanoma cells. Results SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values IYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. Conclusions These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.

  9. Human cognition

    International Nuclear Information System (INIS)

    Norman, D.A.

    1982-01-01

    The study of human cognition encompasses the study of all mental phenomena, from the receipt and interpretation of sensory information to the final control of the motor system in the performance of action. The cognitive scientist examines all intermediary processes, including thought, decision making, and memory and including the effects of motivation, states of arousal and stress, the study of language, and the effects of social factors. The field therefore ranges over an enormous territory, covering all that is known or that should be known about human behavior. It is not possible to summarize the current state of knowledge about cognition with any great confidence that we know the correct answer about any aspect of the work. Nontheless, models provide good characterizations of certain aspects of the data and situations. Even if these models should prove to be incorrect, they do provide good approximate descriptions of people's behavior in some situations, and these approximations will still apply even when the underlying theories have changed. A quick description is provided of models within a number of areas of human cognition and skill and some general theoretical frameworks with which to view human cognition. The frameworks are qualitative descriptions that provide a way to view the development of more detailed, quantitative models and, most important, a way of thinking about human performance and skill

  10. Beyond Humanisms

    OpenAIRE

    Capurro, Rafael

    2012-01-01

    In the first part of this paper a short history of Western humanisms (Socrates, Pico della Mirandola, Descartes, Kant) is presented. As far as these humanisms rest on a fixation of the ‘humanum’ they are metaphysical, although they might radically differ from each other. The second part deals with the present debate on trans- and posthumanism in the context of some breath-taking developments in science and technology.Angeletics, a theory of messengers and messages, intends to give an answer t...

  11. Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

    Science.gov (United States)

    Matlawska-Wasowska, K; Ward, E; Stevens, S; Wang, Y; Herbst, R; Winter, S S; Wilson, B S

    2013-06-01

    This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

  12. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

    Directory of Open Access Journals (Sweden)

    Yanxing Han

    2008-07-01

    Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

  13. Human Parechoviruses

    DEFF Research Database (Denmark)

    Fischer, Thea Kølsen; Harvala, Heli; Midgley, Sofie

    2017-01-01

    Infections with human parechoviruses (HPeV) are highly prevalent, particularly in neonates, where they may cause substantial morbidity and mortality. The clinical presentation of HPeV infection is often indistinguishable from that of enterovirus (EV) infection and may vary from mild disease...

  14. Practicing Humanities

    DEFF Research Database (Denmark)

    Gimmler, Antje

    2016-01-01

    and self-reflective democracy. Contemporary humanities have adopted a new orientation towards practices, and it is not clear how this fits with the ideals of ‘Bildung’ and ‘pure science’. A possible theoretical framework for this orientation towards practices could be found in John Dewey’s pragmatic...

  15. Human waste

    NARCIS (Netherlands)

    Amin, Md Nurul; Kroeze, Carolien; Strokal, Maryna

    2017-01-01

    Many people practice open defecation in south Asia. As a result, lot of human waste containing nutrients such as nitrogen (N) and phosphorus (P) enter rivers. Rivers transport these nutrients to coastal waters, resulting in marine pollution. This source of nutrient pollution is, however, ignored in

  16. Human Trafficking

    Science.gov (United States)

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  17. Think Human

    DEFF Research Database (Denmark)

    Nielsen, Charlotte Marie Bisgaard

    2013-01-01

    years' campaigns suggests that the theory of communication underlying the campaign has its basis in mechanical action rather than in human communication. The practice of 'Communication design' is investigated in relation to this metaphorical 'machine thinking' model of communication and contrasted...

  18. Nothing Human

    Science.gov (United States)

    Wharram, C. C.

    2014-01-01

    In this essay C. C. Wharram argues that Terence's concept of translation as a form of "contamination" anticipates recent developments in philosophy, ecology, and translation studies. Placing these divergent fields of inquiry into dialogue enables us read Terence's well-known statement "I am a human being--I deem nothing…

  19. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  20. Human Rights and Human Function

    Directory of Open Access Journals (Sweden)

    Mohsen Javadi

    2006-03-01

    Full Text Available This paper firstly explores some theories of Human Rights justification and then assents to the theory that Human Rights is based on justified moral values. In order to justify moral values, Aristotle’s approach called “Function Argument” is reviewed. Propounding this argument, the writer attempts to show that all analysis of human identity will directly contribute to the man’s view of his rights. Not only Human rights is really determined by human function or human distinguishing characteristic i.e. human identity, but in the world of knowledge the proper method to know human rights is to know human being himself. n cloning violates man’s rights due to two reasons: damage of human identity and violation of the right to be unique. Attempting to clarify the nature of human cloning, this article examines the aspects to be claimed to violate human rights and evaluates the strength of the reasons for this claim. این مقاله پس از بررسی اجمالی برخی از نظریه‌های توجیه حقوق بشر، نظریة ابتنای آن بر ارزش‌های اخلاقی موجّه را می‌پذیرد. دربارة چگونگی توجیه ارزش اخلاقی، رویکرد ارسطو که به «برهان ارگن» موسوم است، مورد بحث و بررسی قرار می‌گیرد. مؤلف با طرح این برهان می‌کوشد نشان دهد ارائه هرگونه تحلیل از هویت انسان در نگرش آدمی به حقوق خود تأثیر مستقیم خواهد گذاشت. حقوق آدمی نه فقط از ناحیة کارویژه یا فصل ممیز وی (هویت انسان تعیّن واقعی می‌گیرد، بلکه در عالم معرفت هم راه درست شناخت حقوق بشر، شناخت خود انسان است.

  1. Human Rights, Human Needs, Human Development, Human Security : Relationships between four international 'human' discourses

    NARCIS (Netherlands)

    D.R. Gasper (Des)

    2007-01-01

    textabstractHuman rights, human development and human security form increasingly important, partly interconnected, partly competitive and misunderstood ethical and policy discourses. Each tries to humanize a pre-existing and unavoidable major discourse of everyday life, policy and politics; each

  2. Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents

    International Nuclear Information System (INIS)

    Minta, J.O.

    1988-01-01

    The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [ 35 S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression

  3. Human endogenous retroviruses and cancer prevention: evidence and prospects

    Directory of Open Access Journals (Sweden)

    Cegolon Luca

    2013-01-01

    as other tumors like sarcoma, lymphoma, bladder and breast cancer. An amino acid sequence similar to HERV-K-MEL, recognized to cause a significant protective effect against melanoma, is shared by the antigenic determinants expressed by some vaccines such as BCG, vaccinia virus and the yellow fever virus. HERV-K are also reactivated in the majority of human breast cancers. Monoclonal and single-chain antibodies against the HERV-K Env protein recently proved capable of blocking the proliferation of human breast cancer cells in vitro, inhibiting tumor growth in mice bearing xenograft tumors. Summary A recent epidemiological study provided provisional evidence of how melanoma risk could possibly be reduced if the yellow fever virus vaccine (YFV were received at least 10 years before, possibly preventing tumor initiation rather than culling melanoma cells already compromised. Further research is recommended to confirm the temporal pattern of this protection and eliminate/attenuate the potential role of relevant confounders as socio-economic status and other vaccinations. It appears also appropriate to examine the potential protective effect of YFV against other malignancies expressing high levels of HERV-K antigens, namely breast cancer, sarcoma, lymphoma and bladder cancer. Tumor immune-therapy, as described for the monoclonal antibodies against breast cancer, is indeed considered more complex and less advantageous than immune-prevention. Cellular immunity possibly triggered by vaccines as for YFV might also be involved in anti-cancer response, in addition to humoral immunity.

  4. Human endogenous retroviruses and cancer prevention: evidence and prospects.

    Science.gov (United States)

    Cegolon, Luca; Salata, Cristiano; Weiderpass, Elisabete; Vineis, Paolo; Palù, Giorgio; Mastrangelo, Giuseppe

    2013-01-03

    breast cancer. An amino acid sequence similar to HERV-K-MEL, recognized to cause a significant protective effect against melanoma, is shared by the antigenic determinants expressed by some vaccines such as BCG, vaccinia virus and the yellow fever virus.HERV-K are also reactivated in the majority of human breast cancers. Monoclonal and single-chain antibodies against the HERV-K Env protein recently proved capable of blocking the proliferation of human breast cancer cells in vitro, inhibiting tumor growth in mice bearing xenograft tumors. A recent epidemiological study provided provisional evidence of how melanoma risk could possibly be reduced if the yellow fever virus vaccine (YFV) were received at least 10 years before, possibly preventing tumor initiation rather than culling melanoma cells already compromised. Further research is recommended to confirm the temporal pattern of this protection and eliminate/attenuate the potential role of relevant confounders as socio-economic status and other vaccinations.It appears also appropriate to examine the potential protective effect of YFV against other malignancies expressing high levels of HERV-K antigens, namely breast cancer, sarcoma, lymphoma and bladder cancer.Tumor immune-therapy, as described for the monoclonal antibodies against breast cancer, is indeed considered more complex and less advantageous than immune-prevention. Cellular immunity possibly triggered by vaccines as for YFV might also be involved in anti-cancer response, in addition to humoral immunity.

  5. Human endogenous retroviruses and cancer prevention: evidence and prospects

    International Nuclear Information System (INIS)

    Cegolon, Luca; Salata, Cristiano; Weiderpass, Elisabete; Vineis, Paolo; Palù, Giorgio; Mastrangelo, Giuseppe

    2013-01-01

    and breast cancer. An amino acid sequence similar to HERV-K-MEL, recognized to cause a significant protective effect against melanoma, is shared by the antigenic determinants expressed by some vaccines such as BCG, vaccinia virus and the yellow fever virus. HERV-K are also reactivated in the majority of human breast cancers. Monoclonal and single-chain antibodies against the HERV-K Env protein recently proved capable of blocking the proliferation of human breast cancer cells in vitro, inhibiting tumor growth in mice bearing xenograft tumors. A recent epidemiological study provided provisional evidence of how melanoma risk could possibly be reduced if the yellow fever virus vaccine (YFV) were received at least 10 years before, possibly preventing tumor initiation rather than culling melanoma cells already compromised. Further research is recommended to confirm the temporal pattern of this protection and eliminate/attenuate the potential role of relevant confounders as socio-economic status and other vaccinations. It appears also appropriate to examine the potential protective effect of YFV against other malignancies expressing high levels of HERV-K antigens, namely breast cancer, sarcoma, lymphoma and bladder cancer. Tumor immune-therapy, as described for the monoclonal antibodies against breast cancer, is indeed considered more complex and less advantageous than immune-prevention. Cellular immunity possibly triggered by vaccines as for YFV might also be involved in anti-cancer response, in addition to humoral immunity

  6. Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Erik Kvam

    2009-05-01

    Full Text Available Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to

  7. Human Face as human single identity

    OpenAIRE

    Warnars, Spits

    2014-01-01

    Human face as a physical human recognition can be used as a unique identity for computer to recognize human by transforming human face with face algorithm as simple text number which can be primary key for human. Human face as single identity for human will be done by making a huge and large world centre human face database, where the human face around the world will be recorded from time to time and from generation to generation. Architecture database will be divided become human face image ...

  8. Human Rights in the Humanities

    Science.gov (United States)

    Harpham, Geoffrey

    2012-01-01

    Human rights are rapidly entering the academic curriculum, with programs appearing all over the country--including at Duke, Harvard, Northeastern, and Stanford Universities; the Massachusetts Institute of Technology; the Universities of Chicago, of Connecticut, of California at Berkeley, and of Minnesota; and Trinity College. Most of these…

  9. Human reliability

    International Nuclear Information System (INIS)

    Bubb, H.

    1992-01-01

    This book resulted from the activity of Task Force 4.2 - 'Human Reliability'. This group was established on February 27th, 1986, at the plenary meeting of the Technical Reliability Committee of VDI, within the framework of the joint committee of VDI on industrial systems technology - GIS. It is composed of representatives of industry, representatives of research institutes, of technical control boards and universities, whose job it is to study how man fits into the technical side of the world of work and to optimize this interaction. In a total of 17 sessions, information from the part of ergonomy dealing with human reliability in using technical systems at work was exchanged, and different methods for its evaluation were examined and analyzed. The outcome of this work was systematized and compiled in this book. (orig.) [de

  10. Human paleoneurology

    CERN Document Server

    2015-01-01

    The book presents an integrative review of paleoneurology, the study of endocranial morphology in fossil species. The main focus is on showing how computed methods can be used to support advances in evolutionary neuroanatomy, paleoanthropology and archaeology and how they have contributed to creating a completely new perspective in cognitive neuroscience. Moreover, thanks to its multidisciplinary approach, the book addresses students and researchers approaching human paleoneurology from different angles and for different purposes, such as biologists, physicians, anthropologists, archaeologists

  11. Human universe

    CERN Document Server

    Cox, Brian

    2014-01-01

    Human life is a staggeringly strange thing. On the surface of a ball of rock falling around a nuclear fireball in the blackness of a vacuum the laws of nature conspired to create a naked ape that can look up at the stars and wonder where it came from. What is a human being? Objectively, nothing of consequence. Particles of dust in an infinite arena, present for an instant in eternity. Clumps of atoms in a universe with more galaxies than people. And yet a human being is necessary for the question itself to exist, and the presence of a question in the universe - any question - is the most wonderful thing. Questions require minds, and minds bring meaning. What is meaning? I don't know, except that the universe and every pointless speck inside it means something to me. I am astonished by the existence of a single atom, and find my civilisation to be an outrageous imprint on reality. I don't understand it. Nobody does, but it makes me smile. This book asks questions about our origins, our destiny, and our place i...

  12. Recombinant Immunotoxin Therapy of Solid Tumors: Challenges and Strategies.

    Science.gov (United States)

    Shan, Liang; Liu, Yuanyi; Wang, Paul

    2013-01-01

    Immunotoxins are a group of protein-based therapeutics, basically comprising two functional moieties: one is the antibody or antibody Fv fragment that allows the immunotoxin to bind specifically to target cells; another is the plant or bacterial toxin that kills the cells upon internalization. Immunotoxins have several unique features which are superior to conventional chemotherapeutics, including high specificity, extraordinary potency, and no known drug resistance. Development of immunotoxins evolves with time and technology, but significant progress has been achieved in the past 20 years after introduction of recombinant DNA technique and generation of the first single-chain variable fragment of monoclonal antibodies. Since then, more than 1,000 recombinant immunotoxins have been generated against cancer. However, most success in immunotoxin therapy has been achieved against hematological malignancies, several issues persist to be significant barriers for effective therapy of human solid tumors. Further development of immunotoxins will largely focus on the improvement of penetration capability to solid tumor mass and elimination of immunogenicity occurred when given repeatedly to patients. Promising strategies may include construction of recombinant antibody fragments with higher binding affinity and stability, elimination of immunodominant T- and B-cell epitopes of toxins, modification of immunotoxins with macromolecules like poly(ethylene glycol) and liposomes, and generation of immunotoxins with humanized antibody fragments and human endogenous cytotoxic enzymes. In this paper, we briefly reviewed the evolution of immunotoxin development and then discussed the challenges of immunotoxin therapy for human solid tumors and the potential strategies we may seek to overcome the challenges.

  13. Introduction: Digital Humanities, Public Humanities

    Directory of Open Access Journals (Sweden)

    Alex Christie

    2014-07-01

    Full Text Available NANO: New American Notes Online: An Interdisciplinary Academic Journal for Big Ideas in a Small World. This special issue shows how both public and digital humanities research can be rendered more persuasive through engagement with cultures beyond the academy. More specifically, the aim of this special issue is to demonstrate how investments in technologies and computation are not necessarily antithetical to investments in critical theory and social justice.

  14. Human Capital, (Human) Capabilities and Higher Education

    Science.gov (United States)

    Le Grange, L.

    2011-01-01

    In this article I initiate a debate into the (de)merits of human capital theory and human capability theory and discuss implications of the debate for higher education. Human capital theory holds that economic growth depends on investment in education and that economic growth is the basis for improving the quality of human life. Human capable…

  15. Humanizing Architecture

    DEFF Research Database (Denmark)

    Toft, Tanya Søndergaard

    2015-01-01

    The article proposes the urban digital gallery as an opportunity to explore the relationship between ‘human’ and ‘technology,’ through the programming of media architecture. It takes a curatorial perspective when proposing an ontological shift from considering media facades as visual spectacles...... agency and a sense of being by way of dematerializing architecture. This is achieved by way of programming the symbolic to provide new emotional realizations and situations of enlightenment in the public audience. This reflects a greater potential to humanize the digital in media architecture....

  16. Antiviral Activity of HIV gp120 Targeting Bispecific T Cell Engager (BiTE®) Antibody Constructs.

    Science.gov (United States)

    Brozy, Johannes; Schlaepfer, Erika; Mueller, Christina K S; Rochat, Mary-Aude; Rampini, Silvana K; Myburgh, Renier; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A; Muenz, Markus; Speck, Roberto F

    2018-05-02

    Today's gold standard in HIV therapy is the combined antiretroviral therapy (cART). It requires strict adherence by patients and life-long medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency, but cannot cure patients. The bispecific T cell engaging (BiTE®) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. We here generated BiTE® antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1+2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ϵ scFv. These engineered human BiTE® antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in PBMCs as well as in macrophages co-cultured with autologous CD8+ T-cells, the most potent being the human CD4(1+2) BiTE® antibody construct and the CD4(1+2)L17b BiTE® antibody construct. The CD4(1+2) h BiTE® antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE® antibody constructs as well as the CD4(1+2)L17b BiTE® antibody construct did not. Thus, BiTE® antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential. Importance HIV is a chronic infection well controlled with the current cART. However, we lack cure of HIV, and the HIV pandemic goes on. Here we showed in vitro and ex vivo t hat a bispecific T-cell engaging (BiTE®) antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE® antibody constructs display efficient killing of gp120 expressing cells and inhibited replication in ex vivo

  17. Humanized mouse models: Application to human diseases.

    Science.gov (United States)

    Ito, Ryoji; Takahashi, Takeshi; Ito, Mamoru

    2018-05-01

    Humanized mice are superior to rodents for preclinical evaluation of the efficacy and safety of drug candidates using human cells or tissues. During the past decade, humanized mouse technology has been greatly advanced by the establishment of novel platforms of genetically modified immunodeficient mice. Several human diseases can be recapitulated using humanized mice due to the improved engraftment and differentiation capacity of human cells or tissues. In this review, we discuss current advanced humanized mouse models that recapitulate human diseases including cancer, allergy, and graft-versus-host disease. © 2017 Wiley Periodicals, Inc.

  18. Microbial platform technology for recombinant antibody fragment production: A review.

    Science.gov (United States)

    Gupta, Sanjeev Kumar; Shukla, Pratyoosh

    2017-02-01

    Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

  19. Human steroidogenesis

    DEFF Research Database (Denmark)

    Andersen, Claus Y; Ezcurra, Diego

    2014-01-01

    In the menstrual cycle, the mid-cycle surge of gonadotropins (both luteinising hormone [LH] and follicle-stimulating hormone [FSH]) signals the initiation of the periovulatory interval, during which the follicle augments progesterone production and begins to luteinise, ultimately leading to the r......In the menstrual cycle, the mid-cycle surge of gonadotropins (both luteinising hormone [LH] and follicle-stimulating hormone [FSH]) signals the initiation of the periovulatory interval, during which the follicle augments progesterone production and begins to luteinise, ultimately leading...... reviews current knowledge of the regulation of progesterone in the human ovary during the follicular phase and highlights areas where knowledge remains limited. In this review, we provide in-depth information outlining the regulation and function of gonadotropins in the complicated area of steroidogenesis...

  20. NATO Human View Architecture and Human Networks

    Science.gov (United States)

    Handley, Holly A. H.; Houston, Nancy P.

    2010-01-01

    The NATO Human View is a system architectural viewpoint that focuses on the human as part of a system. Its purpose is to capture the human requirements and to inform on how the human impacts the system design. The viewpoint contains seven static models that include different aspects of the human element, such as roles, tasks, constraints, training and metrics. It also includes a Human Dynamics component to perform simulations of the human system under design. One of the static models, termed Human Networks, focuses on the human-to-human communication patterns that occur as a result of ad hoc or deliberate team formation, especially teams distributed across space and time. Parameters of human teams that effect system performance can be captured in this model. Human centered aspects of networks, such as differences in operational tempo (sense of urgency), priorities (common goal), and team history (knowledge of the other team members), can be incorporated. The information captured in the Human Network static model can then be included in the Human Dynamics component so that the impact of distributed teams is represented in the simulation. As the NATO militaries transform to a more networked force, the Human View architecture is an important tool that can be used to make recommendations on the proper mix of technological innovations and human interactions.

  1. The Digital Humanities as a Humanities Project

    Science.gov (United States)

    Svensson, Patrik

    2012-01-01

    This article argues that the digital humanities can be seen as a humanities project in a time of significant change in the academy. The background is a number of scholarly, educational and technical challenges, the multiple epistemic traditions linked to the digital humanities, the potential reach of the field across and outside the humanities,…

  2. Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

    Directory of Open Access Journals (Sweden)

    Anna Bochicchio

    2017-02-01

    Full Text Available Targeted human cytolytic fusion proteins (hCFPs are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv specifically binding to e.g., tumor associated antigens (TAAs. After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to upregulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively, reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD simulations and enhanced sampling methods (ESM. MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.

  3. Managing the Human in Human Brands

    Directory of Open Access Journals (Sweden)

    Fournier Susan

    2018-05-01

    Full Text Available The physical and social realities, mental biases and limitations of being human differentiate human brands from others. It is their very humanness that introduces risk while generating the ability for enhanced returns. Four particular human characteristics can create imbalance or inconsistency between the person and the brand: mortality, hubris, unpredictability and social embeddedness. None of these qualities manifest in traditional non-human brands, and all of them present risks requiring active managerial attention. Rather than treating humans as brands and making humans into brands for sale in the commercial marketplace, our framework forces a focus on keeping a balance between the person and the personified object.

  4. Human cloning and human dignity

    Directory of Open Access Journals (Sweden)

    Hasan Eslami

    2006-12-01

    Full Text Available Catholic Church and most of Muslims believe that human cloning is in contrast with human rights. They argue that applying Somatic Nuclear Transfer Technique or so-called cloning to humans is against human dignity. Their main reason is that the cloned person would be a copy or shadow of another person and lack his or her identity and uniqueness. They also argue that in the process of cloning human beings would be treated as laboratory mice. This article tries to evaluate this kind of argumentation and shows that the "human dignity" expression in the relevant writings is vague and has been used inappropriately. مسیحیان و برخی از مسلمانان استدلال می‌کنند که کاربست تکنیک شبیه‌سازی ناقض کرامت انسانی است. این دلیل خود به صورت‌های مختلفی بیان می‌شود، مانند آنکه انسان موضوع آزمایش‌های علمی قرار می‌گیرد و با او مانند حیوانات رفتار می‌شود. گاه نیز تغییر نحوة تولید مثل، مایة نقض کرامت انسانی قلمداد می‌گردد و گاه به مسئلة از بین رفتن هویت فردی اشاره می‌شود. نگارنده در دو قسمت، دیدگاه مسیحیان و مسلمانان را در این باره نقل و تحلیل کرده است و کوشیده است نشان دهد که استناد به مفهوم کرامت انسانی در این جا مبهم و ناگویاست و مخالفان کوشش دقیقی در جهت تبیین دلیل خود به عمل نیاورده‌اند.

  5. Digital Humanities

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørn

    2015-01-01

    overgangen fra trykkekultur til digital kultur. For det første problemstillingen omkring digitalisering af litterær kulturarv med fokus på kodning og tagging af teksten samt organisering i hypertekststrukturer. For det andet reorganiseringen af det digitale dokument i dataelementer og database. For det......Artiklen præsenterer først nogle generelle problemstillinger omkring Digital Humanities (DH) med det formål at undersøge dem nærmere i relation til konkrete eksempler på forskellige digitaliseringsmåder og ændringer i dokumentproduktion. I en nærmere afgrænsning vælger artiklen den tendens i DH......, der betragter DH som forbundet med "making" og "building" af digitale objekter og former. Dette kan også karakteriseres som DH som praktisk-produktiv vending. Artiklen har valgt tre typer af digitalisering. De er valgt ud fra, at de skal repræsentere forskellige måder at håndtere digitaliseringen på...

  6. The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

    Science.gov (United States)

    Karmohapatra, Soumendra K; Chakraborty, Kushal; Kahn, Nighat N; Sinha, Asru K

    2007-11-01

    Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture. (c) 2007 Wiley-Liss, Inc.

  7. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  8. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT).

    Science.gov (United States)

    Francis, R J; Mather, S J; Chester, K; Sharma, S K; Bhatia, J; Pedley, R B; Waibel, R; Green, A J; Begent, R H J

    2004-08-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

  9. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    International Nuclear Information System (INIS)

    Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J.; Mather, S.J.; Waibel, R.

    2004-01-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99m Tc-carbonyl [ 99m Tc(H 2 O) 3 (CO) 3 ] + (abbreviated to TcCO) mediated labelling of 99m Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  10. Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs, named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig domains 1-4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn domains 1-3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H(2O(2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1-4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1-3 trigger L1 functions of cultured neuroblastoma cells.

  11. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  12. Modern Human Engineering

    International Nuclear Information System (INIS)

    Jeong, Byeong Yong; Lee Dong Kyeong

    2005-08-01

    These are the titles of each chapter. They are as in the following; design of human-centerdness, human machine system, information processing process, sense of human, user interface, elements of human body, vital dynamics, measurement of reaction of human body, estimation and management of working environment, mental characteristic of human, human error, group, organization and leadership, safety supervision, process analysis, time studying, work sampling, work factor and methods time measurement, introduction of muscular skeletal disease and program of preventive management.

  13. Modern Human Engineering

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Byeong Yong; Lee Dong Kyeong

    2005-08-15

    These are the titles of each chapter. They are as in the following; design of human-centerdness, human machine system, information processing process, sense of human, user interface, elements of human body, vital dynamics, measurement of reaction of human body, estimation and management of working environment, mental characteristic of human, human error, group, organization and leadership, safety supervision, process analysis, time studying, work sampling, work factor and methods time measurement, introduction of muscular skeletal disease and program of preventive management.

  14. HUMANISM OF ANTROPOCENTRISM AND ANTROPOCENTRISM WITHOUT HUMANISM

    Directory of Open Access Journals (Sweden)

    N. S. Shilovskaya

    2014-01-01

    Full Text Available Article is devoted to the distinction of humanism and anthropocentrism which is based on the parity of the person and being. Genetic communication of humanism and anthropocentrism and their historical break comes to light.

  15. Superintelligence, Humans, and War

    Science.gov (United States)

    2015-04-13

    Recent studies of the human mind debunk the myth that humans only use 10-20 percent of the human mind. A healthy human mind uses up to 90 percent...way. They will eat what is in front of them to satiate their appetite not knowing if there is anymore food for the future. Humans can predict

  16. Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

    Science.gov (United States)

    Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

    2014-12-01

    Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

  17. Comparative Response of Platelet fV and Plasma fV to Activated Protein C and Relevance to a Model of Acute Traumatic Coagulopathy

    Science.gov (United States)

    2014-06-12

    through a series of centrifugation steps: 200 g for 10 min with minimal braking separated platelet rich plasma (PRP) which was collected and stored at...localization of fVa and phospholipid as a result of platelet deposition accelerates thrombin generation and fibrin formation/crosslinking at sites of...is ineffective at inhibiting both platelet adhesion and fibrin formation in blood flow models except at extreme Figure 7. aPC concentrations below 10

  18. The golden triangle of human dignity: human security, human development and human rights

    NARCIS (Netherlands)

    Gaay Fortman, B. de

    2004-01-01

    The success or failure of processes of democratization cannot be detached from processes of development related to the aspirations of people at the grassroots. Human rights, in a more theoretical terminology, require human development in order to enhance human security.

  19. Human factors in training

    International Nuclear Information System (INIS)

    Dutton, J.W.; Brown, W.R.

    1981-01-01

    The Human Factors concept is a focused effort directed at those activities which require human involvement. Training is, by its nature, an activity totally dependent on the Human Factor. This paper identifies several concerns significant to training situations and discusses how Human Factor awareness can increase the quality of learning. Psychology in the training arena is applied Human Factors. Training is a method of communication represented by sender, medium, and receiver. Two-thirds of this communications model involves the human element directly

  20. Human-machine interactions

    Science.gov (United States)

    Forsythe, J Chris [Sandia Park, NM; Xavier, Patrick G [Albuquerque, NM; Abbott, Robert G [Albuquerque, NM; Brannon, Nathan G [Albuquerque, NM; Bernard, Michael L [Tijeras, NM; Speed, Ann E [Albuquerque, NM

    2009-04-28

    Digital technology utilizing a cognitive model based on human naturalistic decision-making processes, including pattern recognition and episodic memory, can reduce the dependency of human-machine interactions on the abilities of a human user and can enable a machine to more closely emulate human-like responses. Such a cognitive model can enable digital technology to use cognitive capacities fundamental to human-like communication and cooperation to interact with humans.

  1. Complementary DNA and derived amino acid sequence of the α subunit of human complement protein C8: evidence for the existence of a separate α subunit messenger RNA

    International Nuclear Information System (INIS)

    Rao, A.G.; Howard, O.M.Z.; Ng, S.C.; Whitehead, A.S.; Colten, H.R.; Sodetz, J.M.

    1987-01-01

    The entire amino acid sequence of the α subunit (M/sub r/ 64,000) of the eight component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire α coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence con