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Sample records for antibody fab fragment

  1. Radioimmunoimaging using F(ab')2 fragment of monoclonal antibodies against human alpha-fetoprotein

    International Nuclear Information System (INIS)

    Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Koizumi, Mitsuru; Ohta, Hitoya; Torizuka, Kanji; Okada, Kenichiro; Yoshida, Osamu; Nishi, Shinzo.

    1985-01-01

    Using monoclonal antibodies against human α-fetoprotein (AFP), radioiodinated F(ab') 2 fragments were compared with whole IgG as a radiotracer for radioimmunoimaging of cancer. F(ab') 2 fragments were obtained by pepsin digestion of whole IgG (IgGl). IgG and F(ab') 2 were labeled with 125 I or 131 I by the chloramine-T method with almost full retention of antibody activity. F(ab') 2 fragments were cleared more rapidly from the circulation in normal mice with a half life of 6.3 hours than whole IgG with a half life of 5.5 days. Radioactivity of F(ab') 2 in various organs also decreased faster than IgG. In nude mice transplanted with AFP-producing human testicular tumor, F(ab') 2 fragments demonstrated superior scintigrams to whole IgG at 2 days after the injection, because of the fast disappearance of background radioactivity. Although absolute accumulation of 131 I labeled F(ab') 2 in the tumor was less than that of 131 I labeled IgG, tumor to other organ ratios were much higher with F(ab') 2 than those of IgG. The tumor to blood ratio of 131 I labeled F(ab') 2 was 1.04 at day 2, whereas tumor to blood ratio of 131 I labeled IgG was 0.55 at day 2 and 0.92 at day 4, respectively. These results indicated that for the radiolabeling of monoclonal antibodies, F(ab') 2 fragments would be superior to whole IgG in the radioimmunoimaging of cancer. (author)

  2. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-02-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.

  3. Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

    International Nuclear Information System (INIS)

    Rezkalla, S.; Kloner, R.A.; Khaw, B.A.; Haber, E.; Fallon, J.T.; Smith, F.E.; Khatib, R.

    1989-01-01

    The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I-125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection

  4. Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

    Czech Academy of Sciences Publication Activity Database

    Ohradanova-Repic, A.; Nogueira, E.; Hartl, I.; Gomes, A.C.; Preto, A.; Steinhuber, E.; Muehlgrabner, V.; Repic, M.; Kuttke, M.; Zwirzitz, A.; Prouza, M.; Suchánek, M.; Wozniak-Knopp, G.; Hořejší, Václav; Schabbauer, G.; Cavaco-Paulo, A.; Stockinger, H.

    2018-01-01

    Roč. 14, č. 1 (2018), s. 123-130 ISSN 1549-9634 Institutional support: RVO:68378050 Keywords : Active targeting * Liposome functionalization * Immunoliposome * Antibody engineering * Recombinant Fab antibody fragment Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 5.720, year: 2016

  5. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...... phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage....

  6. Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

    Science.gov (United States)

    Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

    2015-06-01

    Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

  7. Increased streptavidin uptake in tumors pretargeted with biotinylated antibody using a conjugate of streptavidin-Fab fragment

    International Nuclear Information System (INIS)

    Yao Zhengsheng; Zhang Meili; Sakahara, Harumi; Saga, Tsuneo; Kobayashi, Hisataka; Nakamoto, Yuji; Toyama, Sakuji; Konishi, Junji

    1998-01-01

    Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody

  8. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    International Nuclear Information System (INIS)

    Page, R.L.; Garg, P.K.; Gard, S.

    1994-01-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the 18 F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4'-( 18 F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T 1/2β = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of 18 F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10 -3 % injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of 18 F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs

  9. PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Page, R.L.; Garg, P.K.; Gard, S. [North Carolina State Univ., Raleigh, NC (United States)]|[Duke Univ. Medical Center, Durham, NC (United States)]|[North Carolina and Norke Radium Hospital, Oslo (Norway)] [and others

    1994-09-01

    Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor site relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.

  10. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    Science.gov (United States)

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model

    Directory of Open Access Journals (Sweden)

    Ji-Guo Su

    2015-12-01

    Full Text Available Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM. Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF of the residues, as well as the MSF in the internal distance (MSFID between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein.

  12. Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

    OpenAIRE

    Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

    2017-01-01

    Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme a...

  13. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    Directory of Open Access Journals (Sweden)

    Motedayen, M.H.

    2015-12-01

    Full Text Available Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthesis and amplification of the heavy (Fd segment and kappa light chains of IgG antibody. After digestion of the heavy chain with SpeI and XhoI and light chain with XbaI and SacI enzymes, inserted successively into the cloning vector pComb3x, and then recombinant vector transformed to TG1 bacteria to construct the Fab library. For determination insertion rate of Fab segment into cloning vector, plasmids of 12 clones of library were extracted and digested with SfiI. Length of amplified Fd and κ chains, were 650 - 750 bp. Primary library size was determined to contain 4.9×105 members out of which half of them contained the same size of Fab fragment. This result is comparable to some researchers and shows that this method could be appropriate tool for the production of human polyclonal Fab fragment antibodies for management of poisonous snake bitted victims.

  14. Radioimmunoimaging of human colon carcinoma grafted into nudemice using 131I-labeled monoclonal anticea antibody and its F(ab')2 fragments

    International Nuclear Information System (INIS)

    Liu Guangda

    1988-01-01

    131 I-labeled monoclonal anti-CEA antibody and its F(ab') 2 fragments were injected into nude mice bearing human colon carcinoma xenografts for tumor localization and radioimmunoimaging studies. Transplanted tumors were visualized in 12 hours after injection of the labeled anti-CEA or its F(ab') 2 by gamma camera. Biodistribution data indicated that F(ab') 2 fragments were cleared more rapidly from blood (T 1/2 = 13.3 h for F(ab') 2 , T 1/2 = 21.1 h for intact antibody) over 6-24 h and had higher tumor blood ratios. The intact antibody was concentrated in the tumor better than F(ab') 2 . In double-label experiments, a nonspecific localization of the control ( 125 I-labeled anti-HCG) in the tumor was also observed

  15. Localization of tumors in vivo by scintigraphic identification of Clostridium butyricum using 131I-labelled antibodies and F(ab')2-antibody fragments

    International Nuclear Information System (INIS)

    Vogt, R.; Mehnert, W.H.; Schmidt, H.E.; Altenbrunn, H.J.; Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1979-01-01

    Tumor-bearing mice injected with clostridial spores show enrichment and germination of the spores within the tumor. 131 I-labelled anti-Clostridium-antibodies and anti-Clostridium-F(ab') 2 -fragments were used for a possible localization of tumors in vivo by scintiscanning. The application of the antibody revealed increased radioactivity in the tumors of mice pretreated with spores as well as in animals without pretreatment. In using F(ab') 2 -fragments instead of total antibody neither the apparently unspecific increase of radioactivity in not pretreated mice nor the specific fixation of labelled F(ab') 2 -fragments to clostridial rods in the tumors of pretreated animals could be demonstrated. The results are discussed with respect to further investigation

  16. Serum and urine analysis of the aminoterminal procollagen peptide type III by radioimmunoassay with antibody Fab fragments.

    Science.gov (United States)

    Rohde, H; Langer, I; Krieg, T; Timpl, R

    1983-09-01

    A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.

  17. Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

    International Nuclear Information System (INIS)

    Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.

    1988-01-01

    We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging

  18. F(ab'2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

    Directory of Open Access Journals (Sweden)

    LORENA AGUILAR

    2005-01-01

    Full Text Available Trypanosoma cruzi calreticulin (TcCRT, described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host / parasite equilibrium. In an in vitro correlate of this situation, we show that the C1q / TcCRT interaction is inhibited by F(ab'2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion C1q, and this C1q is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate C1q could be inhibited by F(ab'2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process

  19. Radioimmunoimaging of human breast carcinoma xenografts in nude mouse model with 111In-labeled new monoclonal antibody EBA-1 and F(ab')2 fragments

    International Nuclear Information System (INIS)

    Yemul, Shrishailam; Leon, J.A.; Pozniakoff, Ted; Esser, P.D.; Estabrook, Alison; Met-Path Inc., Teterboro, NJ

    1993-01-01

    Radioimmunoimaging characteristics of a new monoclonal antibody EBA-1 and its F(ab') 2 fragments utilizing nu/nu mice bearing human breast carcinoma xenografts are described. 111 In-DPTA conjugates of EBA-1 localized with tumor/blood ratios of 0.99 ± 0.10 (P 2 radioconjugates at 48 h. These results suggest that EBA-1 and its F(ab') 2 might be useful reagents in radioimmunoimaging and radioimmunotherapy. (author)

  20. Radioimmunodetection (RID) with OC-125 antibody F(ab)2 fragments in the follow-up of ovarian carcinoma

    International Nuclear Information System (INIS)

    Bockisch, A.; Broeckelmann, J.; Briele, B.; Vogel, J.; Oehr, P.; Osterthun, B.; Rheinsberg, J.; Hotze, A.; Biersack, H.J.; Krebs, D.

    1990-01-01

    Radioimmunodetection (RID) using 131 I-labelled OC-125 antibody F(ab') 2 fragments proved to be sensitive and specific for detecting ovarian carcinoma metastases and recurrences. 40 investigations in patients suffering from advanced ovarian carcinoma or carcinoma of unknown origin but with elevated CA-125 serum levels were investigated. The sensitivity, specificity, and accuracy were found to be 84%, 73%, and 80%, respectively. Analysing the 37 investigations in patients with ovarian carcinomas separately, the numbers were 91%, 71%, and 84%. This imaging modality may therefore be used as an alternative to exploratory surgery. No side-effects were observed clinically. After RID the serum levels of CA-125 were unexpected high in some patients when the measurement was performed in a routine procedure using the same anti-CA-125 antibody in the in vitro test as was used for imaging. (author)

  1. 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.

  2. Kinetics and tissue distribution of the radiolabeled chimeric monoclonal antibody MOv18 IgG and F(ab')2 fragments in ovarian carcinoma patients

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; den Hollander, W.; Vermorken, J. B.; Molthoff, C. J.; Burger, C. W.; Helmerhorst, T. J.; Baak, J. P.; Roos, J. C.

    1993-01-01

    Twenty-four patients suspected of having ovarian carcinoma received i.v. injection with a combination of radiolabeled intact IgG (1 mg) and F(ab')2 fragments (1 mg) of the chimeric monoclonal antibody MOv18, each form labeled with 1.85 MBq 131I or 125I. Laparotomy was performed either 2 or 6 days

  3. Pharmacokinetics and tumor targeting of 131I-labeled F(ab')2 fragments of the chimeric monoclonal antibody G250: preclinical and clinical pilot studies.

    NARCIS (Netherlands)

    Brouwers, A.H.; Mulders, P.F.A.; Oosterwijk, E.; Buijs, W.C.A.M.; Corstens, F.H.M.; Boerman, O.C.; Oyen, W.J.G.

    2004-01-01

    INTRODUCTION: Clinical and animal studies of chimeric monoclonal antibody G250 (moAb cG250) for the targeting of clear-cell renal cell carcinoma (RCC), to date, have been with the intact IgG form. To determine whether F(ab')2 fragments are more suited for radioimmunotherapy (RIT) than intact IgG,

  4. Evaluation of tumor targeting with radiolabeled F(ab2 fragment of a humanized monoclonal antibody

    Directory of Open Access Journals (Sweden)

    "Babaei MH

    2002-08-01

    Full Text Available Humanized monoclonal antibody U36 and its F(ab'2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F(ab'2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F(ab'2 showed highly specific localization in tumor tissue. The mean tumor uptake (n=3 is expressed as the percentage of the injected dose per gram of tumor tissue (%ID/g. %ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas %ID/g of F(ab'2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios (T/B at day 1 were 0.86 for IgG and 1.32 for F(ab'2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F(ab'2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting.

  5. Immunolymphoscintigraphy and immunoscintigraphy of ovarian and fallopian tube cancer using F(ab')2 fragments of monoclonal antibody OC 125

    International Nuclear Information System (INIS)

    Lehtovirta, P.; Kairemo, K.J.; Liewendahl, K.; Seppaelae, M.

    1990-01-01

    We have used immunolymphoscintigraphy (ILS) alone or in combination with immunoscintigraphy with 131 I-labeled F(ab')2 fragments of monoclonal OC 125 antibodies to improve detection of retroperitoneal lymph node metastases of ovarian and fallopian tube cancer. ILS was carried out with bilateral dorsopedal s.c. injections on nine patients and with bilateral iliopelvic injections into the ischiorectal fossa on two other patients. Radioimaging was performed 2-4 times between 0 and 5 days. An additional dose of labeled antibody fragments was given i.v., and imaging was done 2-3 days later. Conventional immunoscintigraphy without preceding ILS was carried out on another nine patients. Dorsopedal ILS improved detection of pelvic and paraaortic lymph node metastases. Malignant lymph nodes were detectable as early as 3 h after s.c. injection of the tracer. Combined results of ILS and immunoscintigraphy in 16 surgically verified cases indicated a true positive finding in 9 patients, true negative finding in 5, false positive in one, and false negative in 1. Calculated from these figures the sensitivity, specificity, and accuracy of the method were 90, 83, and 88%, respectively. Involved lymph nodes were found more frequently in those patients whose serum CA 125 concentration was elevated demonstrating that an elevated serum CA 125 level does not preclude successful radioimmunodetection

  6. Uptake of 99mTc labelled (Fab')2 fragments of monoclonal antibody 225.28S by a benign ocular naevus

    International Nuclear Information System (INIS)

    Bomanji, J.; Granowska, M.; Britton, K.E.; Hungerford, J.L.

    1988-01-01

    Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, 99m Tc radiolabelled (Fab') 2 fragments of monoclonal antibody 225.28S raised against cutaneous melanomas have been used for imaging uveal melanomas. We report here a case where uptake of radiolabelled antibody was observed in a choroidal melanoma of the right eye and a benign choroidal naevus of the left. (orig.)

  7. Biokinetic studies and scintigraphic imaging of human pancreatic carcinoma xenografts with iodine 131-labeled F(ab')2-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Moellenstaedt, S.; Kriegel, H.; Baum, R.P.; Maul, F.D.; Hoer, G.; Wenisch, H.J.C.

    1985-01-01

    Biodistribution and tumor uptake of I-131 labeled F(ab') 2 -fragments of monoclonal antibodies to CA 125, CA 19-9 and CEA were investigated in nuce mice with human pancreatic carcinoma xenografts. To assess the immunological specificity of these antibody fragments their tumor uptake was compared with that of unspecific IgG F(ab') 2 . Additionally serum levels of CA 125 and CA 19-9 were determined in tumor bearing mice by radioimmunoassay and the expression of CA 125 and CA 19-9 in pancreatic carcinoma was demonstrated by immunhistochemicals studies. The rapid blood clearance of the antibody fragments leads to increasing tumor-to-blood ratios with time after injection for all antibody fragments. The highest ratios were found for OC 125 at all time points p.i., with mean values of 9.6 at 48 h p.i. and 11.8 at 96 h p.i. On scintigraphic images tumors with a weight of 100 mg could be localized between 24 and 48 h after injection of iodinated OC 125 F(ab') 2 . The serum levels of CA 125 and CA 19-9 were elevated only in some animals and no correlation between the antigen serum levels and the antigen expression in the tumor shown by immunhistochemical staining could be determined. (orig.) [de

  8. Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.

    1991-01-01

    Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats

  9. Preparation, biodistribution and dosimetry of copper-64-labeled anti-colorectal carcinoma monoclonal antibody fragments 1A3-F(ab')2

    International Nuclear Information System (INIS)

    Anderson, C.J.; Schwarz, S.W.; Connett, J.M.

    1995-01-01

    Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab') 2 was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of 64 Cu-BAT-2IT-1A3-F(ab') 2 was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N double-prime,N'double-prime-tetraacetic acid (BAT) was conjugated to 1A3-F(ab') 2 using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab'. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab') 2 from TETA-1A3-Fab' after conjugation prior to labeling with 64 Cu. The biodistribution of 64 Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab') 2 , the immunoreactivity of 64 Cu-labeled 1A3-F(ab') 2 was significantly improved over that of non-FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 , and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 .The improved dosimetry of 64 Cu-labeled FPLC purified BAT-2IT-1A3-F(ab') 2 should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs

  10. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  11. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    Science.gov (United States)

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-05-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  12. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples.

    Science.gov (United States)

    Stubenrauch, Kay; Wessels, Uwe; Essig, Ulrich; Kowalewsky, Frank; Vogel, Rudolf; Heinrich, Julia

    2013-01-01

    Generic immunoassay formats in animal serum have been described for pharmacokinetic (PK) analysis of human full-length antibodies, but not of human antigen binding fragment (Fab) proteins. Here we characterize two murine monoclonal antibodies (mAb) raised against human immunoglobulin G (IgG) which bind to unique epitopes in the Fab region of human IgG. mAb M-1.7.10 is directed against the constant domain of the kappa light chain and mAb M-1.19.31 binds to the constant domain 1 (CH1) of the heavy chain. Surface plasmon resonance analysis showed that mAb M-1.7.10 does not cross-react with sera from mouse, rat, rabbit, dog, marmoset, rhesus macaque, baboon and cynomolgus monkey, but binds to human and chimpanzee serum (dissociation constant K(D) of 6.8 × 10(-12) and 3.1 × 10(-11)M, respectively). mAb M-1.19.31 shows a higher K(D) for human and chimpanzee IgG (2.0 × 10(-9)M and 5.8 × 10(-10)M, respectively), but also does not bind to serum of the other species. Therefore, mAb M-1.7.10 was used as capture and mAb M-1.19.31 as detection reagent in a generic enzyme linked immunosorbent assay (ELISA) to quantify the human anti-IGF-1R Fab in mouse serum. The generic human Fab assay showed a limit of detection of 31.5 ng/mL anti-IGF-1R Fab. Intra- and inter-assay precision was less than 12% and the accuracy range for all controls was within ±20% of the target concentration. The generic human Fab ELISA was applied to determine serum levels of human anti-IGF-1R Fab after intravenous (iv) administration of 10mg/kg to mice. The resulting concentration-time profile was nearly identical to that obtained by analysis with a validated specific ELISA for anti-IGF-1R Fab. The mean relative concentration of anti-IGF-1R Fab analyzed by the generic assay was 82-118% of that of the specific assay. This equivalence was confirmed in a cynomolgus monkey study with the full length human mAb anti-TROP-2 IgG. Both specific ELISAs used mAb M-1.7.10 as detection reagent and their targets for

  13. Data on crystal organization in the structure of the Fab fragment from the NIST reference antibody, RM 8671.

    Science.gov (United States)

    Gallagher, D T; Karageorgos, I; Hudgens, J W; Galvin, C V

    2018-02-01

    The reported data describe the crystallization, crystal packing, structure determination and twinning of the unliganded Fab (antigen-binding fragment) from the NISTmAb (standard reference material 8671). The raw atomic coordinates are available as Protein Data Bank structure 5K8A and biological aspects are described in the article, (Karageorgos et al., 2017) [1]. Crystal data show that the packing is unique, and show the basis for the crystal's twinned growth. Twinning is a common and often serious problem in protein structure determination by x-ray crystallography [2]. In the present case the twinning is due to a small deviation (about 0.3 nm) from 4-fold symmetry in the primary intermolecular interface. The deviation produces pseudosymmetry, generating slightly different conformations of the protein, and alternating strong and weak forms of key packing interfaces throughout the lattice.

  14. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    OpenAIRE

    Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-01-01

    International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...

  15. Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, Thomas J.; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-04-01

    Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.

  16. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  17. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; van der Graaf, W.T.; Oyen, W.J.G.; Boerman, O.C.

    2012-01-01

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  18. Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment

    OpenAIRE

    Syvänen, Stina; Edén, Desireé; Sehlin, Dag

    2017-01-01

    Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to d...

  19. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    Science.gov (United States)

    Welfringer, Frédéric; d'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-12-20

    Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

  20. Effects of digoxin-specific antibody Fab fragment (Digibind) on blood pressure and renal water-sodium metabolism in 5/6 reduced renal mass hypertensive rats.

    Science.gov (United States)

    Kaide, J; Ura, N; Torii, T; Nakagawa, M; Takada, T; Shimamoto, K

    1999-06-01

    The importance of increased endogenous digitalis-like factor (EDLF) in volume-expanded hypertension has been generally agreed. To further clarify the role of EDLF on the development of hypertension and renal water-sodium handling in 5/6 reduced renal mass hypertensive rats (RRM), we studied the effects of acute administration of digoxin-specific antibody Fab fragment (Digibind) in the early phase and the chronic phase of hypertension in RRM. RRM and sham-operated rats were given 1% saline for 1 or 4 weeks. RRM were injected Digibind (60 mg/kg) or vehicle (0.9% saline) intravenously in the first or fourth week under thiobutabarbital anesthesia. All sham-operated rats were administered Digibind under the same condition. Digibind altered neither blood pressure, heart rate, urine volume, nor urinary sodium excretion in sham-operated rats. However, Digibind produced a gradual but significant decline in mean arterial pressure to the level slightly above that in sham-operated rats from 153 +/- 5 to 131 +/- 5 mm Hg in the first week and from 181 +/- 6 to 129 +/- 4 mm Hg in the fourth week without any significant change in heart rate. The decrease in mean arterial pressure at 160 min after Digibind administration in the fourth week (-48 +/- 5 mm Hg) was greater than that in the first week (-22 +/- 4 mm Hg). No differences were observed in urine volume, urinary sodium excretion, or plasma norepinephrine concentration between Digibind and vehicle-treated RRM in either week. These data suggest that EDLF would contribute to both the early and chronic phase in the development of hypertension in RRM.

  1. Introduction of five potentially metabolizable linking groups between 111In-cyclohexyl EDTA derivatives and F(ab')2 fragments of anti-carcinoembryonic antigen antibody--I. A new reproducible synthetic method.

    Science.gov (United States)

    Gestin, J F; Faivre-Chauvet, A; Mease, R C; Sai-Maurel, C; Thédrez, P; Slinkin, M; Meinken, G E; Srivastava, S C; Chatal, J F

    1993-08-01

    The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of 111In-labeled antibodies. A general simple synthetic method of preparing cyclohexyl EDTA (CDTA) derivatives containing a linker/spacer group is described. Linkers prepared included a diester, a six carbon aliphatic chain, two thioethers and a disulfide group. The CDTA-linker compounds were coupled to F(Ab')2 fragments of anti-carcinoembryonic antigen monoclonal antibody and labeled with 111In with good retention of immunoreactivity.

  2. 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.

  3. Ablation of human colon carcinoma in nude mice by 131I-labeled monoclonal anti-carcinoembryonic antigen antibody F(ab')2 fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Pfister, C.; Fournier, K.; Prevel, F.; Schreyer, M.; Carrel, S.; Mach, J.P.

    1989-01-01

    Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131 I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131 I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131 I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation

  4. Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice

    International Nuclear Information System (INIS)

    Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P.

    1990-01-01

    During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-( 131 I) labeled intact antibodies or 131 I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2

  5. Crystallization and diffraction properties of the Fab fragment of 3B5H10, an antibody specific for disease-causing polyglutamine stretches

    Energy Technology Data Exchange (ETDEWEB)

    Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Weisgraber, Karl [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Department of Pathology, University of California, San Francisco, CA 94143 (United States); Finkbeiner, Steven, E-mail: sfinkbeiner@gladstone.ucsf.edu [Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA 94158 (United States); Departments of Neurology, Physiology and Neuroscience and Biomedical Sciences Program, University of California, San Francisco, CA 94143 (United States)

    2005-12-01

    Optimization of crystallization conditions and cryoprotectants decreased the anisotropy of the diffraction obtained from 3B5H10 Fab crystals. Dehydration improved the resolution of cryoprotected 3B5H10 crystals from 2.6 to 1.9 Å, but changed the space group of the crystals from P2{sub 1}2{sub 1}2 to P2{sub 1}. Because it binds soluble forms of proteins with disease-associated polyglutamine expansions, the antibody 3B5H10 is a powerful tool for studying polyglutamine-related diseases. Crystals of the 3B5H10 Fab (47 kDa) were obtained by vapor diffusion at room temperature from PEG 3350. However, the initial crystals gave highly anisotropic diffraction patterns. After optimization of the crystallization conditions and cryoprotectants, a nearly isotropic diffraction pattern at 2.6 Å resolution was achieved for crystals with unit-cell parameters a = 133.26, b = 79.52, c = 41.49 Å and space group P2{sub 1}2{sub 1}2. Dehydrated crystals diffracted isotropically to 1.9 Å with unit-cell parameters a = 123.65, b = 78.25, c = 42.26 Å, β = 90.3° and space group P2{sub 1}.

  6. A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

    Science.gov (United States)

    Kramer, Karl; Fiedler, Markus; Skerra, Arne; Hock, Bertold

    2002-04-01

    Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.

  7. Crystal structure of snake venom acetylcholinesterase in complex with inhibitory antibody fragment Fab410 bound at the peripheral site: evidence for open and closed states of a back door channel.

    Science.gov (United States)

    Bourne, Yves; Renault, Ludovic; Marchot, Pascale

    2015-01-16

    The acetylcholinesterase found in the venom of Bungarus fasciatus (BfAChE) is produced as a soluble, non-amphiphilic monomer with a canonical catalytic domain but a distinct C terminus compared with the other vertebrate enzymes. Moreover, the peripheral anionic site of BfAChE, a surface site located at the active site gorge entrance, bears two substitutions altering sensitivity to cationic inhibitors. Antibody Elec410, generated against Electrophorus electricus acetylcholinesterase (EeAChE), inhibits EeAChE and BfAChE by binding to their peripheral sites. However, both complexes retain significant residual catalytic activity, suggesting incomplete gorge occlusion by bound antibody and/or high frequency back door opening. To explore a novel acetylcholinesterase species, ascertain the molecular bases of inhibition by Elec410, and document the determinants and mechanisms for back door opening, we solved a 2.7-Å resolution crystal structure of natural BfAChE in complex with antibody fragment Fab410. Crystalline BfAChE forms the canonical dimer found in all acetylcholinesterase structures. Equally represented open and closed states of a back door channel, associated with alternate positions of a tyrosine phenol ring at the active site base, coexist in each subunit. At the BfAChE molecular surface, Fab410 is seated on the long Ω-loop between two N-glycan chains and partially occludes the gorge entrance, a position that fully reflects the available mutagenesis and biochemical data. Experimentally based flexible molecular docking supports a similar Fab410 binding mode onto the EeAChE antigen. These data document the molecular and dynamic peculiarities of BfAChE with high frequency back door opening, and the mode of action of Elec410 as one of the largest peptidic inhibitors targeting the acetylcholinesterase peripheral site. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

    2016-01-01

    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Targeting human prostate cancer with (111) In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, S.; Rij, C.M. van; Franssen, G.M.; Fracasso, G.; Helfrich, W.; Eek, A.; Oyen, W.J.G.; Colombatti, M.; Boerman, O.C.

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing

  10. Targeting human prostate cancer with In-111-labeled D2B IgG, F(ab ')(2) and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.

    2014-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing

  11. 21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that consists...

  12. 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.

  13. Efficacy of IgG, Fab, and F(ab')2 fragments of horse antivenom in the ...

    African Journals Online (AJOL)

    The ability of horse antivenoms, consisting of immunoglobulin G (IgG) and its fragments F(ab')2 and Fab were comparatively studied in mice to neutralize several effects of Cerastes cerastes venom. The three antivenoms were produced from the same batch of hyperimmune horse plasma. Neutralization was only partial ...

  14. The strength of small: Improved targeting of Insulin-like Growth Factor-1 Receptor (IGF-1R) with F(ab')2-R1507 fragments in Ewing sarcomas

    NARCIS (Netherlands)

    Fleuren, Emmy D. G.; Versleijen-Jonkers, Yvonne M. H.; Heskamp, Sandra; Roeffen, Melissa H. S.; Bouwman, Wilbert H.; Molkenboer-Kuenen, Janneke D. M.; van Laarhoven, Hanneke W. M.; Oyen, Wim J. G.; Boerman, Otto C.; van der Graaf, Winette T. A.

    2013-01-01

    To investigate whether F(ab')2-fragments of the monoclonal Insulin-like Growth Factor-1 Receptor (IGF-1R) antibody R1507 (F(ab')2-R1507) can successfully target IGF-1R in Ewing sarcomas (ES). BALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human ES xenografts (EW-5 and EW-8)

  15. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage...

  16. Hybrid IgG4/IgG4 Fc antibodies form upon 'Fab-arm' exchange as demonstrated by SDS-PAGE or size-exclusion chromatography

    NARCIS (Netherlands)

    Rispens, Theo; den Bleker, Tamara H.; Aalberse, Rob C.

    2010-01-01

    Human IgG4 antibodies are dynamic molecules that in vivo exchange half-molecules to become bispecific antibodies. Here we show that IgG4 antibodies and IgG4 Fc fragments similarly exchange resulting in hybrid antibodies (a single Fab + Fc) with a molecular weight of ca. 100 kDa. These antibodies can

  17. Comparison of IgG and F(ab')2 fragments of bispecific anti-RCCxanti-DTIn-1 antibody for pretargeting purposes.

    NARCIS (Netherlands)

    Schaijk, F.G. van; Boerman, O.C.; Soede, A.C.; McBride, W.J.; Goldenberg, D.M.; Corstens, F.H.M.; Oosterwijk, E.

    2005-01-01

    PURPOSE: An effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCCxanti-DTPA(In) (bsMAb: G250xDTIn-1). Tumour uptake of a (111)In-labelled bivalent peptide after pretargeting with bsMAb G250xDTIn-1 was

  18. The preparation of F(ab')2 fragment and it's application in tumor radioimmunoimaging

    International Nuclear Information System (INIS)

    Yang Ziyi

    1991-01-01

    Monoclonal antibody against lung cancer was digested into F(ab') 2 fragment by pepsin and papain digestion. The yields of pure F(ab') 2 were 32.3 ± 5.5% and 52.3 ± 12.0% respectively. The immunoreactivity of F(ab') 2 was based on the ELISA assay and the cell binding studies was retained well. In the localization experiments, radioiodinated F(ab') 2 was injected intraperitoneally into the nude mice bearing human xenografts of lung cancer. The highest radioactivity in tumors, 1.37% of injected dose per gram, was reached on the first day after injection; its T/NT ratios were higher than those of the intact IgG in all tissues except kidney. The localization index (LI) in tumors was 4.95, while the average LI value of normal tissues was 1.25. After the injection of 131 I-F(ab') 2 intraperitoneally into lung tumor-bearing nude mice, photo scintigraphy was performed at intervals of 12 hrs. The xenografts were visualized distinctly during 36 ∼ 48 hr, and the nonspecific background was very low at 48 hr

  19. Chemical design of radiolabeled antibody fragments for low renal radioactivity levels.

    Science.gov (United States)

    Arano, Y; Fujioka, Y; Akizawa, H; Ono, M; Uehara, T; Wakisaka, K; Nakayama, M; Sakahara, H; Konishi, J; Saji, H

    1999-01-01

    The renal uptake of radiolabeled antibody fragments presents a problem in targeted imaging and therapy. We hypothesized that the renal radioactivity levels of radiolabeled antibody fragments could be reduced if radiolabeled compounds of urinary excretion were released from glomerularly filtered antibody fragments before they were incorporated into renal cells by the action of brush border enzymes, present on the lumen of renal tubules. 3'-[131I]Iodohippuryl N(epsilon)-maleoyl-L-lysine ([131I]HML) was conjugated with a thiolated Fab fragment because the glycyl-lysine sequence in HML is a substrate for a brush border enzyme and metaiodohippuric acid is released by cleavage of the linkage. Fab fragments were also radiolabeled by direct radioiodination (125I-Fab) or by conjugation with meta-[125I]-iodohippuric acid via an amide bond [N-(5-maleimidopentyl) 3'-iodohippuric acid amide ([125I]MPH-Fab)] or an ester bond [maleimidoethy 3'-iodohippurate ([125I]MIH-Fab)] by procedures similar to those used for [131I]HML-Fab. In biodistribution experiments in mice, [131I]HML-Fab demonstrated markedly low renal radioactivity levels with kidney:blood ratios of radioactivity of 1 from 10 min to 1 h due to rapid release of meta-[131I]iodohippuric acid. [125]MIH-Fab and 1251-Fab reached their peak ratios of 3.8 and 7.3 at 1 h, respectively, and [125I]MPH-Fab showed the maximum ratio of 16.8 at 6 h. In subcellular distribution studies, both [125I]MIH-Fab and 125I-Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection, whereas the majority of the radioactivity was detected only in the membrane fraction after administration of [131I]HML-Fab at both time points. In nude mice, [131I]HML-Fab showed one-quarter of the renal radioactivity of simultaneously administered 125I-Fab without impairing the target radioactivity levels 3 h after injection. These findings indicated that HML is a useful reagent for targeted

  20. The binding parameters of radiolabelled monoclonal F (ab')2 and Fab' fragments relative to immunoglobulin G in reactions with surface-bound antigens

    International Nuclear Information System (INIS)

    Fjeld, J.G.; Nustad, K.; Michaelsen, T.E.

    1992-01-01

    The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab') 2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains κ and λ, respectively. When testing the 125 I-MoAbs against monodisperse polymer particles coated with the specific antigens, the K a for the F(ab') 2 fragments were similar to that for IgG, while the K a for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab') and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG>F(ab') 2 >Fab'. The explanation of the moderate difference between the K a of the monoclonal Fab' and the divalent IgG and F(ab') 2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against humanlymphoma cells producing Ig with light chains κ or λ, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations. (orig.)

  1. Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab' fragment

    DEFF Research Database (Denmark)

    Spangfort, M D; Mirza, Osman Asghar; Gajhede, M

    1999-01-01

    of the clinical symptoms of allergy. In order to study the structural basis of allergen-antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab' fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1...

  2. Antibody Fragments as Probe in Biosensor Development

    Directory of Open Access Journals (Sweden)

    Serge Muyldermans

    2008-08-01

    Full Text Available Today’s proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

  3. Enhanced hepatic uptake and processing of cholesterol esters from low density lipoprotein by specific lactosaminated Fab fragments.

    Science.gov (United States)

    Bijsterbosch, M K; Bernini, F; Bakkeren, H F; Gotto, A M; Smith, L C; van Berkel, T J

    1991-01-01

    Reduction of the blood levels of low density lipoprotein (LDL) is important for lowering the incidence of atherosclerosis. In this study, LDL was directed to rat parenchymal liver cells by lactosaminated Fab fragments of anti-apolipoprotein B antibodies (LacFab). We followed the fate of intravenously injected complexes of LacFab and [3H]cholesteryl oleate-labeled LDL. Complexing of LacFab to LDL led to rapid disappearance of LDL from the circulation. At 30 minutes after injection, the liver contained 58.5 +/- 9.0% of the injected dose (at that time the liver contained only 5.7 +/- 2.2% of an injected dose of free LDL). Liver uptake was blocked by N-acetylgalactosamine but not by N-acetylglucosamine, which indicates that galactose-specific recognition sites are responsible for the LacFab-induced hepatic uptake. By isolating liver cells, it was found that parenchymal, endothelial, and Kupffer cells account for 87%, 3%, and 10% of the total hepatic uptake, respectively. Subcellular fractionation of the liver indicated that the complexes are rapidly internalized and transported to lysosomes. Within 1 hour after injection, virtually all the [3H]cholesteryl oleate of the internalized LDL was hydrolyzed; hydrolysis was followed by excretion of radioactivity into the bile. Compared with rats injected with native [3H]cholesteryl oleate-labeled LDL, eight times as much radioactivity was excreted into the bile during the first 4 hours after the injection of LacFab-complexed [3H]cholesteryl oleate-labeled LDL. Thus, LacFab induces enhanced hepatic uptake of LDL via galactose receptors on the parenchymal cells, followed by processing in lysosomes and excretion into the bile. In this way, LacFab induces an increased irreversible removal of LDL cholesterol from the body.

  4. Comparative study of intact A7 MoAc and F(ab')2 fragments for radioimmunoimaging of human colon cancer in nude mice

    International Nuclear Information System (INIS)

    Kojima, Shuji; Suzuki, Naomi; Shimura, Noriko; Kubodera, Akiko; Kubota, Kazuhiko; Yamaguchi, Toshiharu; Takahashi, Toshio; Oyamada, Hiyoshimaru

    1993-01-01

    Differences of pharmacokinetics and tumor imaging ability between intact monoclonal antibody A7 (A7 MoAb) and F(ab) 2 fragments were studied in human colon cancer (LS-174T)-bearing nude mice. The authors examined the yield and the immunoreactivity of F(ab) 2 fragments after treatment with ficin as a function of time. The yield of F(ab) 2 fragments reached about 50% after ficin treatment for 8 h, and the F(ab) 2 retained about 80% of the immunoreactivity of the corresponding MoAb. Longer digestion with ficin produced smaller fragments (less than 92 kDa) with a lower yield and most of the immunoreactivity was lost. In pharmacokinetics studies, the F(ab') 2 was preferentially taken up by the tumor, cleared more rapidly from the blood circulation and seemed to have less non-specific tissue binding than intact A7 MoAb. The tumor image obtained at an early time using 131 I-F(ab') 2 was much superior in quality to that with intact 131 I-A7 MoAb. The use of F(ab') 2 fragments may be effective for tumor diagnosis and therapy. (author)

  5. Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, V. V., E-mail: vvo@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Lapuk, V. A. [Russian Academy of Sciences, Zelinskii Institute of Organic Chemistry (Russian Federation); Shtykova, E. V.; Stepina, N. D.; Dembo, K. A.; Sokolova, A. V.; Amarantov, S. V. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Timofeev, V. P. [Russian Academy of Sciences, Engelhardt Institute of Molecular Biology (Russian Federation); Ziganshin, R. Kh. [Russian Academy of Sciences, Shemyakin Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Varlamova, E. Yu. [Russian Academy of Medical Sciences, Hematology Research Center (Russian Federation)

    2008-05-15

    The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that for Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.

  6. Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

    International Nuclear Information System (INIS)

    Volkov, V. V.; Lapuk, V. A.; Shtykova, E. V.; Stepina, N. D.; Dembo, K. A.; Sokolova, A. V.; Amarantov, S. V.; Timofeev, V. P.; Ziganshin, R. Kh.; Varlamova, E. Yu.

    2008-01-01

    The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that for Fab-RF (21±2 and 11±1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.

  7. Distribution and dosimetry of indium-111 labeled F(ab')2 fragments in humans

    International Nuclear Information System (INIS)

    Doherty, P.; Schwinger, R.; King, M.; Gionet, M.

    1986-01-01

    The purpose of this study was to obtain quantitative biodistribution data in patients injected with the indium-111 labeled F(ab') 2 fragments of mouse monoclonal antibody. From this data dosimetric calculations were made for the individual organs. The authors also evaluated the quantitative properties of SPECT in this application and compared it with the more conventional two view planar technique in both phantom and patient studies. For one antibody (19-9) the mean dose in rads/mCi for the organs of highest accumulation, namely, the liver and kidneys was 3.2 and 2.6 respectively. Preliminary data from another antibody (OC 125) showed much higher blood levels and a significantly lower liver dose of 2.3 indicating that antibody type is another significant determinant in dosimetry. The SPECT approach particularly in the presence of background activity, was more accurate in the phantom studies and resulted in larger estimated doses in the patient studies. Also, SPECT has the added advantage of providing an index of organ volume, which has to be balanced with the fact the planar is more rapid, and does not require special hardware. 24 references, 5 figures, 1 table

  8. Lactosaminated Fab fragments specific for low density lipoproteins/hepatocyte targeting and hypolipoproteinemic activity.

    Science.gov (United States)

    Bernini, F; Via, D P; Bocan, T M; Gotto, A M; Smith, L C

    1988-01-01

    We have previously reported that Fab fragments of IgGs modified by lactosamination (lac-Fab) can direct macromolecules, including low density lipoproteins (LDL), to the liver. In the present paper we demonstrate that lac-Fab that is specific for LDL is an effective and selective hypolipoprotein agent. A plasma pool of about 60 mg/dl of apoprotein B (apo B) was induced in rats by bolus injection of human LDL (hLDL), which increased the cholesterol value to about 150 mg/dl. Three hours after injection of the highest dose of lac-Fab, the total cholesterol decreased to 80 mg/dl, compared to 120 mg/dl in control animals. Studies conducted with 131I-tyramine-cellobiose-labeled LDL indicated that the liver was the only organ in which lac-Fab increased LDL uptake and degradation. The effect of lac-Fab was dose-dependent. With amounts of lac-Fab between 13 to 42 mg/kg body weight, the amount of hLDL cleared through the lac-Fab mechanism ranged from 30% to 70% of the initial pool. Analysis of the plasma lipoprotein subfractions revealed that high density lipoprotein levels were not affected. Histologic examination of liver sections after sequential injection of fluorescently labeled hLDL and lac-Fab indicated specific uptake in the hepatocytes when compared to control sections obtained from animals injected with Dil-LDL alone. The uptake of fluorescent LDL induced by lac-Fab was completely prevented by a co-injection of an excess of asialofetuin. We conclude that lac-Fab that is specific for LDL is a selective hypolipoproteinemic agent and a specific carrier to the hepatocytes.

  9. Neutralisation and binding of VHS virus by monovalent antibody fragments

    DEFF Research Database (Denmark)

    Cupit, P.M.; Lorenzen, Niels; Strachan, G.

    2001-01-01

    single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using...... the 3F1H10 variable heavy (VH) chain and variable light (V kappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured...

  10. Tumour targeting with monovalent fragments of anti-neuroblastoma antibody chCE7

    International Nuclear Information System (INIS)

    Carrel, F.; Novak-Hofer, I.; Ruch, C.; Zimmermann, K.; Amstutz, H.

    1997-01-01

    The in vitro and in vivo behaviour of the monovalent single chain (scFv) and Fab-fragments derived from anti-neuroblastoma antibody chCE7 is reported. When comparing tumour uptake and -retention of radioactivity of 67 Cu-labelled monovalent chCE7 with divalent chCE7 F(ab') 2 the advantage of the radiocopper label over the radioiodine label was more pronounced with the divalent (internalising) F(ab') 2 fragments. (author) 1 fig., 1 ref

  11. Improved radioimaging and tumor localization with monoclonal F(ab')2

    International Nuclear Information System (INIS)

    Wahl, R.L.; Parker, C.W.; Philpott, G.W.

    1983-01-01

    Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy

  12. Recombinant human antibody fragment against tetanus toxoid produced by phage display

    Science.gov (United States)

    Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

    2014-01-01

    Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

  13. Clinical usefulness of human-mouse chimeric Fab monoclonal antibody A7 for radioimmunoguided surgery

    International Nuclear Information System (INIS)

    Yamamoto, Kazuhito

    1999-01-01

    This study was designed to determine the clinical usefulness of radioimmunoguided surgery (RIGS) using the human-mouse chimeric Fab monoclonal antibody A7 (chA7Fab) for colorectal cancer patients. Whole murine monoclonal antibody A7 (whole A7) and chA7Fab were labelled with 125 I and 131 I, and their biodistributions were investigated experimentally and clinically. Radioactivities of the antibodies in the tissues were measured by a portable gamma detecting probe (GDP) purchased from Neoprobe Corp.. Of the four labelled antibodies used in a mouse model, 125 I-chA7Fab revealed the highest tumor/surrounding tissue ratio and all values were greater than 2.0. All tumor/surrounding tissue ratios of 131 I-chA7Fab were greater than 1.5, but the values were lower than those of 125 I-chA7Fab. Due to the limited clinical use of 125 I in Japan, 131 I was used as a radio-tracer for chA7Fab in the clinical trial. RIGS using 131 I-chA7Fab was performed on ten colorectal cancer patients. Tumor localization was intraoperatively determined in four of ten patients using the GDP. Liver metastasis and lymph node metastasis were identified in two patients and one patient, respectively. The GDP revealed tumor/surrounding tissue ratios of 1.5 or greater in eight of the ten resected tumors. Although radioimmunoguided surgery using chA7Fab is a promising tool to intraoperatively determine the tumor localization of colorectal cancer, 125 I and not 131 I should be used as a tracer for radioimmunoguided surgery to increase the accuracy of chA7Fab. (author)

  14. 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.

    2016-01-01

    ABSTRACT 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. PMID:27532598

  15. Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination.

    Science.gov (United States)

    Osterroth, F; Alkan, O; Mackensen, A; Lindemann, A; Fisch, P; Skerra, A; Veelken, H

    1999-10-29

    An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E. coli was developed. Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.gammakappa containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space. A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining. Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gammakappa, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested mu or kappa constant region-specific primers. Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning.

  16. In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments

    International Nuclear Information System (INIS)

    Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.; Rochester Univ., NY

    1986-01-01

    Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab') 2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radio-immunotherapy we think that Fab fragments are not useful because of their too short half-life the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab') 2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab') 2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab') 2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab') 2 fragments. (orig.) [de

  17. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  18. Human/murine chimeric 81C6 F(ab')2 fragment: preclinical evaluation of a potential construct for the targeted radiotherapy of malignant glioma

    International Nuclear Information System (INIS)

    Boskovitz, Abraham; Akabani, Gamal H.; Pegram, Charles N.; Bigner, Darrell D.; Zalutsky, Michael R.

    2004-01-01

    We have obtained encouraging responses in recent Phase I studies evaluating 131 I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab') 2 labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab') 2 labeled using N-succinimidyl 3-[ 131 I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab') 2 fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab') 2. Radiation dosimetry calculations suggest that 131 I-labeled ch81C6 F(ab') 2 may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors

  19. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to Fab-fragments enabled the development of a sensitive (LoD=3.3ng/L) immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Human kinetic distribution of I-123 F(Ab')/sub 2/ and FAb compared to the parent I-123 intact antibody

    International Nuclear Information System (INIS)

    De Nardo, S.J.; De Nardo, G.L.; O'Grady, L.F.; Peng, J.S.; Macey, D.J.; Mills, S.L.; Caridiff, R.D.; Epstein, A.L.

    1985-01-01

    Studies of radiolabeled monoclonal antibodies are being performed by many investigators using a variety of molecular forms of the antibody. This study evaluates two I-123-labeled antibodies (Lym-1 IgG2a and B6.01 IgG1) and their I-123-labeled FAb and F(Ab')/sub 2/ for relative whole body distribution and kinetics, selected organ kinetics, and site of degradation. Evidence of immunocomplex formation, circulating antigen, and HAMA, were monitored by HPLC-TSK analysis and appropriate radioassays of the plasma. Each antibody and each I-123-labeled fragment has been evaluated in 5 patients. One patient had serial comparisons of the radiolabeled parent molecule, F(Ab')/sub 2/ (100μg and 5 mg) and FAb (100μg) of Lym-1. Blood clearance, urine excretion rate, and plasma and urine HPLC fractionation of the radioactive species were determined. Whole body clearance by total body scans and organ clearance were obtained by geometric mean of anterior and posterior areas of interest. The FAb and F(Ab')/sub 2/ fragments had similar blood pool recovery 5 minutes after the 5-minute injection. These were the same as blood levels when 5mg of intact I-123-labeled antibody was given. The second phase of the blood curves for F(Ab')/sub 2/ had a T1/2 of 35-40 hours and for FAb was 15-20 hours. Urine excretion varied, but agreed with total body loss from body scan information. The patient with serial studies had a 24-hour residual dose of 82% for FAb, 62% for 10μg for F(Ab')/sub 2/ and 61% for 5mg of F(Ab')/sub 2/. Renal uptake for all patients was higher and more persistent with both fragments than intact antibody, but hepatic uptake was less

  1. Clinical utility of immunoscintigraphy with the Immu-4 99mTc-Fab' antibody

    International Nuclear Information System (INIS)

    Moffat, F.L.; Pinsky, C.M.; Hammershaimb, L.; Petrelli, N.J.; Patt, Y.Z.; Whaley, F.S.; Goldenberg, D.M.

    1996-01-01

    The purpose of these prospective, pivotal studies was to assess the performance and the potential clinical impact of a new antibody imaging agent, CEA-Scan TM , in 210 pre-surgical patients with advanced recurrent or metastatic colorectal carcinomas. CEA-Scan TM , an anti-carcinoembryonic antigen (CEA) Fab' antibody fragment labeled with 99m Tc, was injected IV and external scintigraphy performed 2-5 h and 18-24 h later. Imaging with conventional diagnostic modalities (CDM) was also performed, and findings were confirmed by surgery and histology. The sensitivity of CEA-Scan TM was superior to that of CDM in the extrahepatic abdomen (55 % vs 32 %, ρ 0,007) and pelvis (69 % vs 48 %, p = 0,005), and CEA-Scan TM findings complemented those of CDM in the liver. Among 122 patients with known disease, the positive predictive value was significantly higher when both modalities were positive (98 %) as compared to each alone (68 % - 70 %), potentially obviating the need for histological confirmation when both tests are positive. Imaging accuracy also was significantly improved y adding CEA-Scan TM to CDM. In 88 patients with occult cancer, imaging accuracy was enhanced significantly by CEA-Scan TM combined with CDM (61 % vs 33 %). Potential clinical benefit from CEA-Scan TM was demonstrated in 89 of 210 patients. Only two patients developed human anti-mouse antibodies (HAMA) to CEA-Scan TM after a single injection, and none of 19 assessable patients after two injections. (author)

  2. Structural and energetic hot-spots for the interaction between a ladder-like polycyclic ether and the anti-ciguatoxin antibody 10C9Fab.

    Science.gov (United States)

    Ui, Mihoko; Tanaka, Yoshikazu; Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Hirama, Masahiro; Tsumoto, Kouhei

    2011-03-01

    The mechanism by which anti-ciguatoxin antibody 10C9Fab recognizes a fragment of ciguatoxin CTX3C (CTX3C-ABCDE) was investigated by mutational analysis based on structural data. 10C9Fab has an extraordinarily large and deep antigen-binding pocket at the center of its variable region. We mutated several residues located at the antigen-binding pocket to Ala, and kinetic analysis of the interactions between the mutant proteins and the antigen fragment was performed. The results indicate that some residues associated with the rigid antigen-binding pocket are structural hot-spots and that L-N94 is an energetic hot-spot for association of the antibody with the antigen fragment CTX3C-ABCDE, suggesting the importance of structural complementarity and energetic hot-spot interactions for specific recognition of polycyclic ethers.

  3. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    Science.gov (United States)

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants. Copyright © 2015 Elsevier B

  4. Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.

    Science.gov (United States)

    Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

    2015-03-01

    The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Early imaging of experimental myocardial infarction by intracoronary administration of /sup 131/I-labelled anticardiac myosin (Fab')/sub 2/ fragments. [Dogs

    Energy Technology Data Exchange (ETDEWEB)

    Khaw, B.A.; Gold, H.K.; Leinbach, R.C.; Fallon, J.T.; Strauss, W.; Pohost, G.M.; Haber, E.

    1978-12-01

    The feasibility of early imaging of myocardial infarcts by intracoronary injection of /sup 131/I-labelled cardiac myosin-specific antibody (Fab')/sub 2/ was examined. The left anterior descending coronary artery was occluded for 5 hs by a balloon catheter introduced through the carotid artery in 12 dogs. The catheter was withdrawn and 1 mCi /sup 201/Tl was injected intravenously and 500 ..mu..Ci of /sup 131/I antibody were injected into the main left coronary artery. Six of these animals demonstrated evidence of myocardial infarction by ECG and subsequent triphenyl-tetrazolium chloride staining, while the others did not. In each of the infarcted animals, in vivo scintograms one-half h after injection of isotope showed uptake of /sup 131/I in the anteroapical region of the heart corresponding to the region of absent /sup 201/Tl uptake. This relationship was confirmed in the excized hearts and in heart slices. In slices, /sup 131/I uptake corresponded to regions that did not stain with triphenyltetrazolium chloride. In the six animals that did not show evidence for infarction after coronary occlusion, uptake of /sup 131/I was not demonstrated, either in vivo or in excized specimens. In four additional dogs subjected to the same procedure, /sup 125/I-labelled (Fab')/sub 2/ from nonimmune IgG was injected simultaneously into the left main coronary artery with /sup 131/I-labelled canine myosin-specific antibody (Fab')/sub 2/. The ratio of uptake between infarct center and normal tissue was 34.3 +- 1.5 (mean +- SEM) for the specific antibody fragment as contrasted to 6.6 +- 0.4 for the nonimmune IgG fragment, indicating that intracoronary injection does not favor nonspecific sequestration of protein in regions of infarction. Thus the intracoronary administration of myosin-specific antibody fragments leads to early and specific one-half h imaging of myocardial infarcts.

  6. Targeted cancer therapy through antibody fragments-decorated nanomedicines.

    Science.gov (United States)

    Alibakhshi, Abbas; Abarghooi Kahaki, Fatemeh; Ahangarzadeh, Shahrzad; Yaghoobi, Hajar; Yarian, Fatemeh; Arezumand, Roghaye; Ranjbari, Javad; Mokhtarzadeh, Ahad; de la Guardia, Miguel

    2017-12-28

    Active targeting in cancer nanomedicine, for improved delivery of agents and diagnose, has been reviewed as a successful way for facilitating active uptake of theranostic agents by the tumor cells. The application of a targeting moiety in the targeted carrier complexes can play an important role in differentiating between tumor and healthy tissues. The pharmaceutical carriers, as main part of complexes, can be polymeric nanoparticles, micelles, liposomes, nanogels and carbon nanotubes. The antibodies are among the natural ligands with highest affinity and specificity to target pharmaceutical nanoparticle conjugates. However, the limitations, such as size and long circulating half-lives, hinder reproducible manufacture in clinical studies. Therefore, novel approaches have moved towards minimizing and engineering conventional antibodies as fragments like scFv, Fab, nanobody, bispecific antibody, bifunctional antibody, diabody and minibody preserving their functional potential. Different formats of antibody fragments have been reviewed in this literature update, in terms of structure and function, as smart ligands in cancer diagnosis and therapy of tumor cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Formation of insoluble particulates in a spray-dried F(ab')(2) fragment.

    Science.gov (United States)

    Lassner, Peter; Adler, Michael; Lee, Geoffrey

    2014-04-01

    An F(ab')2 fragment of an IgG has been spray-dried on a bench-top machine to examine the presence of insoluble particulates in the redissolved powder product. Preliminary experiments showed that treatment of the F(ab')2 in aqueous solution in a water bath at 65°C for 1 h produced fragmentation and also insoluble particulates, but no alteration in soluble aggregates. On spray drying, there was, however, no fragmentation but rather formation of soluble aggregates and insoluble particulates. There is therefore a strong difference between isothermal heat-induced damage in solution and during spray drying. The insoluble particulates were detected using a particle counter as a number distribution that was converted to a weight distribution. Addition of trehalose to the liquid feed strongly reduced the formation of the insoluble particulates, indicating that these are insoluble aggregates of the protein and not particulate contamination from the spray dryer. Trehalose (100 mM) was required to suppress formation of the insoluble aggregates, whereas 10 mM was sufficient to prevent formation of soluble aggregates. The particle counter and also isoelectric focusing gave consistent detection of the insoluble particles, whereas photon correlation spectroscopy gave inconsistent results. Spray-drying of small volumes of liquid feed on the bench-top scale can be performed without introducing nonproteinaceous insoluble particulates. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  8. Radioimmunolocalization and selective delivery of radiation in a rat model system: comparison of intact and fragmented antibody

    International Nuclear Information System (INIS)

    Walker, K.Z.; Seymour-Munn, K.; Axiak, S.M.; Raison, R.L.; Basten, A.; Towson, J.E.; Bautovitch, G.J.; Morris, J.

    1988-01-01

    Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab') 2 and Fab fragments using a normal rat model system. Two days after injection of 131 I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab') 2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 ± 3.9 for Fab, 7.9 ± 1.8 for F(ab') 2 and 2.0 ± 0.3 for intact K-1-21. The improvement associated with the use of 131 I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab') 2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab') 2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates. (author)

  9. Radiolabeled monoclonal antibody 15 and its fragments for localization and imaging of xenografts of human lung cancer

    International Nuclear Information System (INIS)

    Endo, K.; Kamma, H.; Ogata, T.

    1988-01-01

    Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells

  10. Phage Fab Display Selection In Vitro and In Vivo: Novel Means to Identify New Breast Cancer Avid Compounds

    National Research Council Canada - National Science Library

    Meighan, Mark

    2001-01-01

    .... In this annual report we present preliminary results on the isolation of antibody fragments (Fabs), isolated from phage display libraries, when affinity selected against breast cancer cell lines...

  11. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.

    Science.gov (United States)

    Rea, Jennifer C; Lou, Yun; Cuzzi, Joel; Hu, Yuhua; de Jong, Isabella; Wang, Yajun Jennifer; Farnan, Dell

    2012-12-28

    Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 μL HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography.

    Science.gov (United States)

    Roque, A Cecília A; Taipa, M Angela; Lowe, Christopher R

    2005-02-04

    The development and characterization of an artificial protein L (PpL) for the affinity purification of antibodies is described. Ligand 8/7, which emerged as the lead from a de novo designed combinatorial library of ligands, inhibits the interaction of PpL with IgG and Fab by competitive ELISA and shows negligible binding to Fc. The ligand 8/7 adsorbent (Ka approximately 10(4) M(-1)) compared well with PpL in binding to immunoglobulins from different classes and sources and, in addition, bound to IgG1 with K and lambda isotypes (92% and 100% of loaded protein) and polyclonal IgG from sheep, cow, goat and chicken. These properties were also reflected in the efficient isolation of immunoglobulins from crude samples.

  13. 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.

  14. Biodistribution of anti-CEA F(ab')2 fragments conjugated with chelating polymers: influence of conjugate electron charge on tumor uptake and blood clearance

    International Nuclear Information System (INIS)

    Slinkin, M.A.; Curtet, C.; Faivre-Chauvet, A.; Sai-Maurel, C.; Gestin, J.F.; Chatal, J.F.; Torchilin, V.P.

    1993-01-01

    F(ab') 2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) were modified with three chain-terminal polysine-based chelating polymers so as to carry different electron charges. Immunoreactive conjugates labeled with 111 In up to a specific radioactivity of 120-140 μCi/μg were injected into nude mice bearing humans colorectal carcinoma, and the biodistribution patterns were compared with each other and with that of an anti-CEA F(ab') 2 -DTPA control. Immunoconjugate modified with positively-charged polymer produced the highest tumor uptake [up to 20% injected dose per gram (ID/g)], with very significant non-specific radioactivity in normal organs (particularly kidneys). When modified with a polymer carrying only a slight negative charge, the immunoconjugate also produced fairly high tumor uptake (up to 18% ID/g), with much lower non-specific radioactivity in normal organs. Highly negatively-charged conjugate produced the lowest tumor uptake (up to 8% ID/g), whereas blood and whole-body clearances were the fastest but slower than those of conventionally labeled F(ab') 2 mAb. The possible mechanisms for the effects described are discussed. (author)

  15. Biodistribution of anti-CEA F(ab')2 fragments conjugated with chelating polymers: influence of conjugate electron charge on tumor uptake and blood clearance.

    Science.gov (United States)

    Slinkin, M A; Curtet, C; Faivre-Chauvet, A; Sai-Maurel, C; Gestin, J F; Torchilin, V P; Chatal, J F

    1993-05-01

    F(ab')2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) were modified with three chain-terminal polylysine-based chelating polymers so as to carry different electron charges. Immunoreactive conjugates labeled with 111In up to a specific radioactivity of 120-140 microCi/micrograms were injected into nude mice bearing human colorectal carcinoma, and the biodistribution patterns were compared with each other and with that of an anti-CEA F(ab')2-DTPA control. Immunoconjugate modified with positively-charged polymer produced the highest tumor uptake [up to 20% injected dose per gram (ID/g)], with very significant non-specific radioactivity in normal organs (particularly kidneys). When modified with a polymer carrying only a slight negative charge, the immunoconjugate also produced fairly high tumor uptake (up to 18% ID/g), with much lower non-specific radioactivity in normal organs. Highly negatively-charged conjugate produced the lowest tumor uptake (up to 8% ID/g), whereas blood and whole-body clearances were the fastest but slower than those of conventionally labeled F(ab')2 mAb. The possible mechanisms for the effects described are discussed.

  16. Clinical experience of the radioimmunoscintigraphy with the 123-I-anti-CEA-Fab(No. 35)-fragment

    International Nuclear Information System (INIS)

    Meili, A.; Bekier, A.; Schulthess, G.K. von; Mach, J.P.

    1986-01-01

    Purified monoclonal murine 123-I-Fab-anti-CEA(Nr.35) were applied to 7 patients in a prospective study. Compared with the previous tested intact 131-I-labelled antibody (Nr. 202) the new preparation had many advantages for imaging: 2.7 times more counting efficiency of the gamma camera, higher activity utilizable and better accumulation in the tumor with sufficient image contrast in the ECT. Fifteen until sixteen hours after administration of 20 mCi 123-I the countrate in the pelvis was 130000/20 sec. In all patients the primary tumor was visualized, two patients demonstrated regional lymph node involvement and one patient liver metastasis in the left lobe. All findings were confirmed by surgery and histopathology. The average tumor/mucosa ratio was 3.5:1. The activity in the tumor 15 h after application was 0.015 per cent/g tumor/mCi 123-I used. 4/7 patients with colorectal cancer over 3.5 cm in diameter demonstrated normal plasma values of CEA < 3 microg/l. One patient with rising CEA plasma level was suspected of relapse, the focal accumulation corresponded with a mass lesion in the right sacrum in CT-scan. After the preliminary results the further applicability is recognizable: staging and screening before and after therapy, handicapped by high costs at time. A good advantage is the possibility of specific and organ independent diagnosis of tumor, which contents the corresponding surface antigen. (author)

  17. Highly Specific PET Imaging of Prostate Tumors in Mice with an Iodine-124-Labeled Antibody Fragment That Targets Phosphatidylserine

    OpenAIRE

    Stafford, Jason H.; Hao, Guiyang; Best, Anne M.; Sun, Xiankai; Thorpe, Philip E.

    2013-01-01

    Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab')2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab')2 was...

  18. 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 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.

  19. Antibody Fragments and Their Purification by Protein L Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Gustav Rodrigo

    2015-09-01

    Full Text Available Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing.

  20. LC-MS/MS quantification of free and Fab-bound colchicine in plasma, urine and organs following colchicine administration and colchicine-specific Fab fragments treatment in Göttingen minipigs.

    Science.gov (United States)

    Fabresse, Nicolas; Allard, Julien; Sardaby, Marine; Thompson, Adrian; Clutton, R Eddie; Eddleston, Michael; Alvarez, Jean-Claude

    2017-08-15

    Clinical evaluation of a colchicine specific antigen-binding fragment (Fab) in order to treat colchicine poisoning required the development of an accurate method allowing quantification of free and Fab-bound colchicine in plasma and urine, and free colchicine in tissues, to measure colchicine redistribution after Fab administration. Three methods have been developed for this purpose, and validated in plasma, urine and liver: total colchicine was determined after denaturation of Fab by dilution in water and heating; free colchicine was separated from Fab-bound colchicine by filtration with 30KDa micro-filters; tissues were homogenized in a tissue mixer. Deuterated colchicine was used as internal standard. Samples were extracted by liquid-liquid extraction and analyzed with a LC-MS/MS. LOQ were 0.5ng/mL in plasma and urine for free and total colchicine and 5pg/mg in tissues. The methods were linear in the 0.5-100ng/mL range in plasma and urine, and 5-300pg/mg in tissues with determination coefficients>0.99. Precision and accuracy of QC samples presented a CVcolchicine and treated with colchicine specific Fab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  2. Packing motifs as predictors of the propensity of antibody fragments to crystallize

    Science.gov (United States)

    Edmundson, Allen B.; DeWitt, Christina R.; Goldsteen, Benjamin Z.; Ramsland, Paul A.

    1999-01-01

    A recurring theme in the crystallization of antibody fragments in our laboratory has been a packing pattern involving formation of intermolecular, antiparallel β-pleated sheets across two-fold axes. The most common motif is the antiparallel stacking of constant (C) domains of light (L) chain dimers or Fab molecules. Here, cross-molecule six-stranded sheets are produced by hydrogen-bonding interactions of three-residue polypeptide segments (triads), in the i, i+2 and i+4 positions of the final strands (designated 3-3) of the three-chain layers from two adjacent molecules. In the variable (V) domains the triads are supplied by the first strands (4-1) of the four-chain layers and the resulting cross-molecule sheets contain eight strands. The latter type of packing is more likely to be seen in crystals of Fv fragments (V domains only) than in those of L chain dimers or Fabs. Amongst the triads from either the V or C domains, there are on average four sets of backbone carbonyl and amide groups within hydrogen bonding distance (chain dimers, Fab and Fvs are likely to crystallize in these packing patterns.

  3. Imaging of deep venous thrombosis in patients using a radiolabelled anti-D-dimer Fab' fragment (99mTc-DI-DD3B6/22-80B3): results of a phase I trial.

    Science.gov (United States)

    Macfarlane, David; Socrates, Angelides; Eisenberg, Paul; Larcos, George; Roach, Paul; Gerometta, Michael; Smart, Richard; Tsui, Wendy; Scott, Andrew M

    2009-02-01

    (99m)Tc-DI-DD3B6/22-80B3 (ThromboView, hereafter abbreviated to (99m)Tc-DI-80B3 Fab') is a radiolabelled humanised monoclonal Fab' fragment with affinity and specificity for D-dimer domains of cross-linked fibrin. Detection of thromboembolic events has been demonstrated in canine models. The study objectives were evaluation of safety and characterisation of biodistribution, immunogenicity and pharmacokinetic profile of increasing doses of (99m)Tc-DI-80B3 Fab' in subjects with acute lower-limb DVT. Twenty-six patients with acute lower limb DVT were enrolled. Of these, 21 received a single intravenous dose of 0.5 mg (n = 6), 1.0 mg (n = 9) or 2 mg (n = 6) (99m)Tc-DI-80B3 Fab'. Blood and urine samples and gamma camera images were collected to 24 h after administration for pharmacokinetic and dosimetry analysis. Vital signs, electrocardiography, hematological and biochemical data and human anti-human antibody (HAHA) levels were monitored for up to 30 days following administration. Patients were assigned to either planar or single photon emission computed tomographic (SPECT) imaging of the thorax at 4 h following injection. Thirty-five adverse events were reported in 15 of the 21 subjects. Those deemed possibly related to administration of (99m)Tc-DI-80B3 Fab' included mild hypertension, mild elevation of LD (lactate dehydrogenase) and moderate elevation of ALT (alanine transaminase). HAHA assays remained negative. Pharmacokinetics and organ dosimetry were comparable to prior normal volunteer data. Localisation of Thromboview to sites of known thrombus was evident as early as 30 min post-injection. In subjects with acute DVT, (99m)Tc-DI-80B3 Fab' was well tolerated with favourable characteristics for the detection of acute venous thrombosis.

  4. Imaging of deep venous thrombosis in patients using a radiolabelled anti-D-dimer Fab' fragment ({sup 99m}Tc-DI-DD3B6/22-80B3): results of a phase I trial

    Energy Technology Data Exchange (ETDEWEB)

    Macfarlane, David [University of Queensland, School of Medicine, Brisbane (Australia); Socrates, Angelides; Larcos, George [University of Sydney, Department of Medicine, Sydney (Australia)]|[Westmead Hospital, Department of Nuclear Medicine and Ultrasound, Westmead (Australia)]|[Westmead Hospital, Centre for Biomedical Imaging and Research, Westmead (Australia); Eisenberg, Paul [Amgen Inc, Thousand Oaks, CA (United States); Roach, Paul [University of Sydney, Department of Medicine, Sydney (Australia)]|[Royal North Shore Hospital, Nuclear Medicine, St. Leonards (Australia); Gerometta, Michael [Agen Biomedical Pty Ltd, Brisbane (Australia); Smart, Richard; Tsui, Wendy [St. George Hospital, Nuclear Medicine Department, Sydney (Australia)]|[University of New South Wales, Department of Medicine, Sydney (Australia); Scott, Andrew M. [Austin Hospital, Centre for PET, Melbourne (Australia)]|[Ludwig Institute, Melbourne (Australia)

    2009-02-15

    {sup 99m}Tc-DI-DD3B6/22-80B3 (ThromboView registered, hereafter abbreviated to {sup 99m}Tc-DI-80B3 Fab') is a radiolabelled humanised monoclonal Fab' fragment with affinity and specificity for D-dimer domains of cross-linked fibrin. Detection of thromboembolic events has been demonstrated in canine models. The study objectives were evaluation of safety and characterisation of biodistribution, immunogenicity and pharmacokinetic profile of increasing doses of {sup 99m}Tc-DI-80B3 Fab' in subjects with acute lower-limb DVT. Twenty-six patients with acute lower limb DVT were enrolled. Of these, 21 received a single intravenous dose of 0.5 mg (n = 6), 1.0 mg (n = 9) or 2 mg (n = 6) {sup 99m}Tc-DI-80B3 Fab'. Blood and urine samples and gamma camera images were collected to 24 h after administration for pharmacokinetic and dosimetry analysis. Vital signs, electrocardiography, hematological and biochemical data and human anti-human antibody (HAHA) levels were monitored for up to 30 days following administration. Patients were assigned to either planar or single photon emission computed tomographic (SPECT) imaging of the thorax at 4 h following injection. Thirty-five adverse events were reported in 15 of the 21 subjects. Those deemed possibly related to administration of {sup 99m}Tc-DI-80B3 Fab' included mild hypertension, mild elevation of LD (lactate dehydrogenase) and moderate elevation of ALT (alanine transaminase). HAHA assays remained negative. Pharmacokinetics and organ dosimetry were comparable to prior normal volunteer data. Localisation of Thromboview registered to sites of known thrombus was evident as early as 30 min post-injection. In subjects with acute DVT, {sup 99m}Tc-DI-80B3 Fab' was well tolerated with favourable characteristics for the detection of acute venous thrombosis. (orig.)

  5. Imaging of deep venous thrombosis in patients using a radiolabelled anti-D-dimer Fab' fragment (99mTc-DI-DD3B6/22-80B3): results of a phase I trial

    International Nuclear Information System (INIS)

    Macfarlane, David; Socrates, Angelides; Larcos, George; Eisenberg, Paul; Roach, Paul; Gerometta, Michael; Smart, Richard; Tsui, Wendy; Scott, Andrew M.

    2009-01-01

    99m Tc-DI-DD3B6/22-80B3 (ThromboView registered, hereafter abbreviated to 99m Tc-DI-80B3 Fab') is a radiolabelled humanised monoclonal Fab' fragment with affinity and specificity for D-dimer domains of cross-linked fibrin. Detection of thromboembolic events has been demonstrated in canine models. The study objectives were evaluation of safety and characterisation of biodistribution, immunogenicity and pharmacokinetic profile of increasing doses of 99m Tc-DI-80B3 Fab' in subjects with acute lower-limb DVT. Twenty-six patients with acute lower limb DVT were enrolled. Of these, 21 received a single intravenous dose of 0.5 mg (n = 6), 1.0 mg (n = 9) or 2 mg (n = 6) 99m Tc-DI-80B3 Fab'. Blood and urine samples and gamma camera images were collected to 24 h after administration for pharmacokinetic and dosimetry analysis. Vital signs, electrocardiography, hematological and biochemical data and human anti-human antibody (HAHA) levels were monitored for up to 30 days following administration. Patients were assigned to either planar or single photon emission computed tomographic (SPECT) imaging of the thorax at 4 h following injection. Thirty-five adverse events were reported in 15 of the 21 subjects. Those deemed possibly related to administration of 99m Tc-DI-80B3 Fab' included mild hypertension, mild elevation of LD (lactate dehydrogenase) and moderate elevation of ALT (alanine transaminase). HAHA assays remained negative. Pharmacokinetics and organ dosimetry were comparable to prior normal volunteer data. Localisation of Thromboview registered to sites of known thrombus was evident as early as 30 min post-injection. In subjects with acute DVT, 99m Tc-DI-80B3 Fab' was well tolerated with favourable characteristics for the detection of acute venous thrombosis. (orig.)

  6. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    Science.gov (United States)

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-09

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

  7. Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-Specific Monoclonal Antibody and Recombinant F(ab

    Directory of Open Access Journals (Sweden)

    Lawrence K. Silbart

    2013-03-01

    Full Text Available Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs such as 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

  8. Reproducibility of image interpretation in immunoscintigraphy performed with indium-111- and iodine-131-labeled OC125 F(ab')2 antibody injected into the same patients

    International Nuclear Information System (INIS)

    Vuillez, J.P.; Peltier, P.; Mayer, J.C.; Dubois, F.; Chetanneau, A.; Fumoleau, P.; Bertrand, A.; Chatal, J.F.

    1991-01-01

    An important criterion for the clinical use of a new imaging technique is the correct reproducibility of interpretation. Forty-six paired immunoscintigraphic examinations were performed on 43 patients with suspected ovarian carcinoma recurrence using F(ab')2 fragments of OC125 antibody labeled first with indium-111 and then with iodine-131. Planar scintigraphy (PS) and emission computed tomography (ECT) images were interpreted blindly and separately by three observers, and reproducibility was evaluated by a kappa concordance index. Intra- and interobserver reproducibility were generally satisfactory (kappa values of 0.6 and 0.7, respectively). Binomial analysis of kappa values for ECT showed the superiority of indium-111 for intraobserver (p = 0.035) and interobserver (p = 0.0039) study. However, for PS there was no significant difference in reproducibility with the two radionuclides

  9. How protein recognizes ladder-like polycyclic ethers. Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9.

    Science.gov (United States)

    Ui, Mihoko; Tanaka, Yoshikazu; Tsumuraya, Takeshi; Fujii, Ikuo; Inoue, Masayuki; Hirama, Masahiro; Tsumoto, Kouhei

    2008-07-11

    Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 angstroms, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 degrees C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.

  10. Study of the viability of technetium-99m labeling of whole antimyosin antibody and its fragment: development of radiopharmaceutical for cardiac survey

    International Nuclear Information System (INIS)

    Carvalho, Guilherme Luiz de Castro

    2007-01-01

    In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled

  11. Highly specific PET imaging of prostate tumors in mice with an iodine-124-labeled antibody fragment that targets phosphatidylserine.

    Directory of Open Access Journals (Sweden)

    Jason H Stafford

    Full Text Available Phosphatidylserine (PS is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab'2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab'2 was labeled with the positron-emitting isotope iodine-124 ((124I and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that (124I-PGN635 F(ab'2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that (124I-PGN635 F(ab'2 is a promising new imaging agent for predicting tumor response to therapy.

  12. Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins.

    Science.gov (United States)

    Luo, Ying-Di; Zhang, Qi-Lei; Yuan, Xiao-Ming; Shi, Wei; Yao, Shan-Jing; Lin, Dong-Qiang

    2017-01-01

    Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. 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.

  14. Effects of a superantigen-antibody recombinant fusion protein (r-C242 Fab-SEA) on toxicological responses in the anaesthetised rabbit

    International Nuclear Information System (INIS)

    Ilbaeck, Nils-Gunnar; Gunnarsson, Kjell; Persson, Robert; Lindh, Ulf; Staaalhandske, Torbjoern

    2003-01-01

    The objective was to study toxin-induced effects on physiological parameters in the rabbit and whether these parameters show dose-response and co-variation after administration of a recombinant fusion protein between staphylococcal enterotoxin (SE) and the Fab fragment of an antibody. Rabbits are very sensitive to SE toxins and the cardiovascular and immune effects are similar to those observed in septic shock in man. The test compound, r-C242 Fab-SEA, was administered intravenously to anaesthetised New Zealand white rabbits at doses in the range of 0.00005-50 μg/kg. All rabbits were checked for titres of anti-SEA antibodies before entering the experiment, since they could neutralise the effect of the test compound. Heart rate, blood pressure and body temperature were continuously monitored before and during 6 h after dosing. Immediately before the start of administration and 3 and 6 h during the experiment, blood gases (pO 2 and pCO 2 ), pH, haematology, clinical chemistry, cytokine response (TNF-α) and trace elements (Mn, Cu, Zn, Se, Ag, Cd, Hg and Pb) were measured. No mortality occurred, but at 50 μg/kg severe adverse clinical signs developed. The decrease in blood pressure was weakly dose-related. Heart rate, ECG, body temperature, pCO 2 and pH were not affected by the treatment. pO 2 tended to increase as a function of time, but not in relation to dose. WBC and PLT decreased dose dependently. TNF-α was not affected by the treatment. The major effects on clinical chemistry were a dose-dependent increase in AST and creatinine. Potassium and urea showed dose dependent increases, mainly at higher doses, though these changes were of less value for drug selection purposes. Trace element changes were observed, including an increase in Mn and a decrease of Zn at all doses. The Cu/Zn ratio decreased below normal at low doses, whereas at high doses in which adverse effects developed, it increased above normal. Post mortem examination revealed minimal to moderate

  15. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  16. An immunoglobulin E assay using radiolabelled Fab' and ammonium sulfate

    International Nuclear Information System (INIS)

    Wilcsek, R.J.; Hamburger, R.N.

    1978-01-01

    An immunochemical assay is described in which a radiolabelled antibody fragment, Fab', is bound specifically to immunoglobulin E (IgE), and precipitated with ammonium sulfate. The radioactivity in the precipitate is a measure of the amount of IgE in the sample. Results for six serum samples are compared using the double antibody and ammonium sulfate methods as well as the papωr radioimmunosorbent test (PRIST)

  17. 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...

  18. Intraspinal distribution and reaction in the grey matter with tetanus toxin of intracisternally injected anti-tetanus toxoid F(ab')2 fragments

    International Nuclear Information System (INIS)

    Erdmann, G.; Hanauske, A.; Wellhoener, H.H.

    1981-01-01

    Anti-tetanus toxoid F(ab') 2 fragments were purified using immune-affinity chromatography on tetanus toxoid-Sepharose. Fragments were labeled with 125 I. Labeled or non-labeled fragments were injected into the intrathecal space of rats. The labeled fragments were found in the spinal cord outside but not inside neurons. Tetanus toxin was injected into a muscle and 36 h later labeled fragments were injected intracisternally. After another 24 h the label was elevated in the spinal cord half segments giving neural supply to the injected muscle and in these half-segments the label was concentrated around some α-motoneurons. [ 125 I]Tetanus toxin was injected into a muscle and at different times thereafter non-labeled fragments were injected intracisternally. The development of hindlimb rigidity but not the accumulation of [ 125 I]tetanus toxin in α-motoneurons was prevented by early intracisternal injection of fragments. Injection of fragments after the appearance of hindlimb rigidity did not revert the rigidity but prevented the further development of symptoms. (Auth.)

  19. Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice.

    Science.gov (United States)

    Bugli, Francesca; Graffeo, Rosalia; Paroni Sterbini, Francesco; Torelli, Riccardo; Masucci, Luca; Sali, Michela; Grasso, Alfonso; Rufini, Stefano; Ricci, Enzo; Fadda, Giovanni; Pescatori, Mario

    2008-03-15

    Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

  20. Tuning the Hydrolytic Stability of Next Generation Maleimide Cross-Linkers Enables Access to Albumin-Antibody Fragment Conjugates and tri-scFvs.

    Science.gov (United States)

    Forte, Nafsika; Livanos, Maria; Miranda, Enrique; Morais, Maurício; Yang, Xiaoping; Rajkumar, Vineeth S; Chester, Kerry A; Chudasama, Vijay; Baker, James R

    2018-02-21

    We describe investigations to expand the scope of next generation maleimide cross-linkers for the construction of homogeneous protein-protein conjugates. Diiodomaleimides are shown to offer the ideal properties of rapid bioconjugation with reduced hydrolysis, allowing the cross-linking of even sterically hindered systems. The optimized linkers are exploited to link human serum albumin to antibody fragments (Fab or scFv) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability. Finally, a triprotein conjugate is formed, by linking scFv antibody fragments targeting carcinoembryonic antigen. This tri-scFv is shown to infer a combination of greater antigen avidity and increased in vivo half-life, representing a promising platform for antibody therapeutic development.

  1. Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

    Energy Technology Data Exchange (ETDEWEB)

    Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.; Greenberg, Harry B.; Harrison, Stephen C.; Dormitzer, Philip R.; (Stanford-MED); (CH-Boston)

    2009-06-17

    Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.

  2. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    National Research Council Canada - National Science Library

    Palys, Thomas J; Schmid, Kara E; Scherer, John M; Schoepp, Randal J

    2006-01-01

    .... Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells...

  3. Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

    Directory of Open Access Journals (Sweden)

    Katerina T. Xenaki

    2017-10-01

    Full Text Available The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors.

  4. Recombinant fragment of an antibody tailored for direct radioiodination

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj; Fábry, Milan; Sieglová, Irena; Král, Vlastimil; Uhnáková, Bronislava; Mudra, M.; Kronrád, L.; Sawicka, A.; Mikolajczak, R.; Řezáčová, Pavlína

    2012-01-01

    Roč. 55, č. 1 (2012), s. 52-56 ISSN 0362-4803 R&D Projects: GA MPO 2A-2TP1/076; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50520514 Keywords : I125 labelling * single-chain antibody variable fragment * tyrosine-rich polypeptide segment * fusion protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.240, year: 2012

  5. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  6. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    Energy Technology Data Exchange (ETDEWEB)

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue

    2009-05-13

    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  7. A pilot pharmacokinetic study of tricyclic antidepressant ovine Fab for TCA poisoning in children.

    Science.gov (United States)

    Yalindağ-Oztürk, Nilüfer; Goto, Collin S; Shepherd, Greene; Torres, Olivia Nayeli; Giroir, Brett

    2010-06-01

    A pilot study of tricyclic antidepressant (TCA)-specific antibody fragments (TCA Fab) in TCA-intoxicated adults showed a marked increase in serum total TCA concentrations following TCA Fab infusion with no worsening signs of TCA toxicity. TCA Fab pharmacokinetics (PK) was not described in this adult study. The objective of this study was to evaluate the PK of TCA Fab in children with TCA poisoning. This was an open-label, single-center, dose escalation pilot trial of three patients. Inclusion criteria were documented TCA ingestion with at least one serious complication (QRS prolongation, dysrhythmia, hypotension, seizure, or coma). Patients were assigned to either a low-dose intravenous TCA Fab regimen (15, 30, and 60 mg/kg) or a high-dose regimen (30, 60, and 120 mg/kg) as needed to reverse TCA toxicity. Following the administration of TCA Fab, samples of blood and urine were obtained for PK evaluations. The outcomes of interest were serum and urine TCA concentrations (free and total), serum and urine Fab concentrations, improvement or worsening of TCA toxicity, and adverse effects. Three study patients were 11, 11, and 14 years of age. Two patients received 15 mg/kg of TCA Fab and one patient received a total of 90 mg/kg of TCA Fab (30 + 60 mg/kg). Serum-bound TCA increased significantly following TCA Fab administration with concomitant enhanced urinary elimination. Serum-free TCA concentrations were minimal to undetectable. Fab data were available for two patients. The serum TCA Fab area under the curve was 306.12 mg/L/h for the 15 mg/kg dose and 2,198.10 mg/L/h for the 90 mg/kg dose of TCA Fab. Maximum Fab concentrations correlated with maximum bound TCA in serum. The volume of distribution (V(D)) of TCA Fab was 0.2-0.3 L/kg. The clearance was 0.036-0.05 L/kg/h and the elimination half-life was 4 h. No adverse effects were observed. The limited PK data from this study are consistent with binding of TCA to TCA Fab and redistribution of TCA from the tissue to

  8. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  9. Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding

    Directory of Open Access Journals (Sweden)

    Silverman B David

    2007-11-01

    . Conclusion With use of x-ray data from the protein data bank (PDB, these two metrics are shown to highlight, in a manner different from before, the structural changes that are induced in the overall domains as well as in the H3 loops of the complementarity-determining regions (CDR upon FAB antibody binding to a truncated and to a synthetic hemagglutinin viral antigenic target.

  10. Renal brush border enzyme-cleavable linkages for low renal radioactivity levels of radiolabeled antibody fragments.

    Science.gov (United States)

    Akizawa, Hiromichi; Imajima, Mitsuo; Hanaoka, Hirofumi; Uehara, Tomoya; Satake, Satoshi; Arano, Yasushi

    2013-02-20

    We previously demonstrated that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(ε)-maleoyl-l-lysine ([(131)I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[(131)I]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-l-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: l-tyosine O-methyl, l-asparagine, l-glutamine, and N(ε)-Boc-l-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[(125)I]iodohippuryl O-methyl-l-tyrosine (2c) liberated the highest amount of m-[(125)I]iodohippuric acid among the four substrates and the change in the linkage structure altered enzyme species responsible for the hydrolysis reaction. To further assess the applicability of the linkage, a radioiodination reagent containing a glycyl-tyrosine linkage, 3'-[(125)I]iodohippuryl O-((2-maleimidoethyl)carbamoyl)methyl-l-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [(125)I]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [(125)I]HML-Fab and directly radioiodinated Fab, while the blood clearance rates of the three were similar. The analyses of urine for 24 h postinjection of [(125)I]HMT-Fab showed that m-[(125)I]iodohippuric acid was excreted as the major radiometabolite. The findings indicated that glycyl-tyrosine linkage is also available to reduce renal radioactivity levels of radioiodinated Fab fragments, due to liberation of m-iodohippuric acid by the action of enzymes present on renal brush border membrane. These findings suggest that an appropriate selection of peptide linkages would allow the liberation of a designed radiolabeled compound from covalently conjugated

  11. Radiolabeled Antibody Fragment for Preparation of (177Lu-DOTAm-PAMAM G3.0-F(ab’2 trastuzumab as a Radiopharmaceutical for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    R.D. Haryuni

    2017-06-01

    Full Text Available Several radiolabeled monoclonal antibodies (mAbs have been used as radioimmunotherapy (RIT agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α particles, beta (β particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of (177Lu-DOTAm-PAMAM G3-F(ab'2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2. Due to its reduced molecular weight, the use of F(ab'2-trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH2 functional groups that are able to bind many DOTAs (£ 31 which in turn can bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab'2-trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab'2-trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC to produce SMCC-F(ab'2-trastuzumab. The next reaction was to conjugate SMCC-F(ab'2-trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab. Finally, the (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab was radiolabelled with 177Lu to produce (177Lu-DOTAm-PAMAM G3.0-F(ab'2-trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column.Received: 31 October 2015; Revised: 30 June 2016; Accepted: 25 September 2016

  12. Similar effectiveness of Fab and F(ab')2 antivenoms in the neutralization of hemorrhagic activity of Vipera berus snake venom in mice

    OpenAIRE

    Lomonte, Bruno; León Montero, Guillermo; Hanson, Lars Ake

    1996-01-01

    The ability of two antivenoms to Vipera spp., consisting of Fab (Therapeutic Antibodies), or of F(ab′)2 (Zagreb Institute of Immunology) antibody fragments, to neutralize the hemorrhagic activity of Vipera berus snake venom in mice, was compared. First, the neutralizing potency was determined by in vitro preincubation of venom and antivenom, followed by intradermal injection into mice and subsequent measurement of the hemorrhagic area. Both antivenoms had the same anti-hemorrhagic potency, in...

  13. Incomplete Antibodies May Reduce ABO Cross-Match Incompatibility: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Mehmet Özen

    2018-02-01

    Full Text Available Objective: Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab part of the antibody (incomplete antibody may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests. Materials and Methods: Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2 fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2 solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2 solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2 antibodies and their effects on the agglutination normally observed with complete antibodies were then measured. Results: No agglutination for the purified incomplete anti-A Fab (2 with A Rh+ erythrocyte and anti-B Fab (2 with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies. Conclusion: We determined that the Fab (2 fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an

  14. Selection of monoclonal anti-CEA antibody fragments for tumor detection by immunoscintigraphy

    International Nuclear Information System (INIS)

    Mach, J.P.; Buchegger, F.

    1986-01-01

    It is described how individual MAb directed against carcinoembryotic antigen (CEA) is selected which does not crossreact with granulocytes and gives the best tumor localization in the model of nude mice grafted with human colon carcinoma. Using this model, the superiority of F(ab')/sub 2/ and particularly Fab fragments from high affinity MAb for the localization of relatively small tumor nodules is demonstrated. These MAb fragments are also successfully used in an ongoing clinical trial for the detection of primary and metastatic colorectal carcinomas

  15. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama

  16. Immunomodulatory effects of intravenous bis-1 f(ab')(2) administration in renal-cell cancer-patients

    NARCIS (Netherlands)

    Janssen, R. A. J.; Kroesen, B. J.; Mesander, G.; Sleijfer, D. T.; The, T. Hauw; Mulder, N. H.; de Leij, L

    We report the immunomodulatory effects of an intravenous treatment with F(ab')(2) fragments of the bispecific monoclonal antibody BIS-1 during subcutaneous recombinant interleukin 2 (rIL-2) therapy of renal cell cancer (RCC) patients. BIS-1 is directed against both the CD3 antigen on T cells and the

  17. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA

  18. Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens

    International Nuclear Information System (INIS)

    Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.

    1987-01-01

    Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125 I. The 125 I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography

  19. Passive immunotherapy for influenza A H5N1 virus infection with equine hyperimmune globulin F(ab'2 in mice

    Directory of Open Access Journals (Sweden)

    Li Yanbin

    2006-03-01

    Full Text Available Abstract Background Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab'2 fragments by pepsin digestion of IgGs. Methods The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab'2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab'2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE assay and in a BALB/c mouse model by survival rate assay. Results By the protocol for 'enhanced pepsin digestion', total 16 g F(ab'2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab'2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab'2 reached 1: 2048. The in vivo assay showed that 100 μg of the F(ab'2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion The availability of highly purified H5N1-specific F(ab'2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.

  20. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  1. Stability of llama heavy chain antibody fragments under extreme conditions

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The

  2. Development of positron emission tomography imaging by 64Cu-labeled Fab for detecting ERC/mesothelin in a mesothelioma mouse model.

    Science.gov (United States)

    Yoshida, Chisato; Sogawa, Chizuru; Tsuji, Atsushi B; Sudo, Hitomi; Sugyo, Aya; Uehara, Tomoya; Hino, Okio; Yoshii, Yukie; Fujibayashi, Yasuhisa; Fukumura, Toshimitsu; Koizumi, Mitsuru; Arano, Yasushi; Saga, Tsuneo

    2010-05-01

    Malignant mesothelioma is a highly aggressive form of cancer. Curative surgery is the only effective therapy for mesothelioma, and therefore early diagnosis is important. However, early diagnosis is difficult using current diagnostic imaging techniques, and a new imaging method for early diagnosis is urgently required. We evaluated the affinity of radiolabeled monoclonal antibodies to the C-terminal fragment of ERC/mesothelin for this purpose. In-labeled or I-labeled IgG against C-terminal fragment of ERC and its Fab fragment were evaluated in vitro by cell binding, competitive inhibition, and cellular internalization assays, and in vivo by biodistribution in mice bearing ERC-expressing tumors. Next, the Fab fragment was labeled with the positron emitter Cu and evaluated by positron emission tomography (PET). Radiolabeled IgG and Fab showed specific binding to ERC-expressing mesothelioma cells with high affinity. Both radiolabeled IgG and Fab internalized into cells after binding to ERC on the cell surface. In-labeled IgG accumulated in ERC-expressing tumors and resulted in a moderate tumor-to-blood ratio at 4 days after injection. Furthermore, PET using Cu-labeled Fab visualized the tumor at 6 h after injection. Cu-labeled Fab can be useful for ERC-specific PET imaging, and can thus facilitate improved diagnosis of patients with early-stage mesothelioma.

  3. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus.

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S; Jia, Letong; Lee, Peter P; Fouts, Timothy R; Parks, Thomas P; Lagenaur, Laurel A

    Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1 BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission.

  4.  Variable fragments of heavy chain antibodies (VHHs: a new magic bullet molecule of medicine?

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek

    2012-06-01

    Full Text Available  Serum of animals belonging to the Camelidae family (camels and llamas contains fully active antibodies that are naturally devoid of light chains. Variable domains derived from heavy chain antibodies (hcAb called VHHs or nanobodies™ can bind antigens as effectively as full-length antibodies and are easy to clone and express. Because of their potential, VHHs are being intensively studied as potential therapeutic, diagnostic and imaging tools. The paper reviews the molecular background of heavy chain antibodies and describes methods of obtaining recombinant fragments of heavy chain antibodies as well as their therapeutic, diagnostic and other applications.

  5. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Construction of Recombinant Single Chain Variable Fragment (ScFv) Antibody Against Superantigen for Immunodetection Using Antibody Phage Display Technology.

    Science.gov (United States)

    Singh, Pawan Kumar; Agrawal, Ranu; Kamboj, D V; Singh, Lokendra

    2016-01-01

    Superantigens are a class of antigens that bind to the major histocompatibility complex class (MHC) II and T-cell receptor (TCR) and cause the nonspecific activation of T cells, resulting in a massive release of pro-inflammatory mediators. They are produced by the gram-positive organisms Staphylococcus aureus and Streptococcus pyogenes, and by a variety of other microbes such as viruses and mycoplasma, and cause toxic shock syndrome (TSS) and even death in some cases. The immunodetection of superantigens is difficult due to the polyclonal activation of T-cells leading to nonspecific antibody production. The production of recombinant monoclonal antibodies against superantigens can solve this problem and are far better than polyclonal antibodies in terms of detection. Here, we describe the construction of recombinant single chain variable fragments (ScFv) antibodies against superantigens with specific reference to SEB (staphylococcal enterotoxin B) using antibody phage display technology.

  9. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

    Science.gov (United States)

    Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

    2012-01-01

    Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

  10. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.

    OpenAIRE

    Hoogenboom, H R; Griffiths, A D; Johnson, K S; Chiswell, D J; Hudson, P; Winter, G

    1991-01-01

    The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that...

  11. Computational identification of antigen-binding antibody fragments.

    Science.gov (United States)

    Burkovitz, Anat; Leiderman, Olga; Sela-Culang, Inbal; Byk, Gerardo; Ofran, Yanay

    2013-03-01

    Determining which parts of the Ab are essential for Ag recognition and binding is crucial for understanding B cell-mediated immunity. Identification of fragments of Abs that maintain specificity to the Ag will also allow for the development of improved Ab-based therapy and diagnostics. In this article, we show that structural analysis of Ab-Ag complexes reveals which fragments of the Ab may bind the Ag on their own. In particular, it is possible to predict whether a given CDR is likely to bind the Ag as a peptide by analyzing the energetic contribution of each CDR to Ag binding and by assessing to what extent the interaction between that CDR and the Ag depends on other CDRs. To demonstrate this, we analyzed five Ab-Ag complexes and predicted for each of them which of the CDRs may bind the Ag on its own as a peptide. We then show that these predictions are in agreement with our experimental analysis and with previously published experimental results. These findings promote our understanding of the modular nature of Ab-Ag interactions and lay the foundation for the rational design of active CDR-derived peptides.

  12. Construction of a human recombinant polyclonal Fab fragment antibody library using peripheral blood lymphocytes of snake bitten victims

    OpenAIRE

    Motedayen, M.H.; Nikbakht, G.; Rasaee, M.J.; Zare Mirakabadi, A.

    2015-01-01

    Human snake bitten poisoning is a serious threat in many tropical and subtropical countries such as Iran. The best acceptable treatment of envenomated humans is antivenoms; however they have a series of economic and medical problems and need more improvements. In this study a combinatorial human immunoglobulin gene library against some of Iranian snakes venoms was constructed. Total RNA prepared from peripheral blood lymphocytes of two recovered snake victims. RT-PCR was used for cDNA synthes...

  13. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide......-antibody interface and the antibody intraface.the microenvironment and ecology of Acaryochloris and Prochloron, and in this thesis we attempted to further describe the distribution, growth characteristics and adaptive/regulatory mechanisms of these two cyanobacteria, both in their natural habitat and under defined...

  14. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    Directory of Open Access Journals (Sweden)

    Larissa M. Alvarenga

    2014-08-01

    Full Text Available Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.

  15. A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells

    NARCIS (Netherlands)

    Ailor, E; Pathmanathan, J; Jongbloed, JDH; Betenbaugh, MJ

    1999-01-01

    The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the

  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. 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.

  18. Pre-existing IgG antibodies cross-reacting with the Fab region of infliximab predict efficacy and safety of infliximab therapy in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Steenholdt, Casper; Palarasah, Yaseelan; Bendtzen, Klaus

    2013-01-01

    are common and may cross-react with the murine part of IFX. AIM: To investigate if Abs binding to IFX's Fab region (IFX-Fab) are present in IBD patients before exposure to IFX, and whether they predict efficacy and safety of IFX therapy. METHODS: Observational, retrospective cohort study of patients with CD...... (n = 29) and UC (n = 22). RESULTS: Pre-treatment levels of IFX-Fab reactive IgG Abs were significantly lower in CD patients in remission after 1 year of maintenance IFX (median 91 mU/L, n = 8) than in the rest of the patients (639 mU/L, n = 21; P ...

  19. 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.

  20. Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.

    Science.gov (United States)

    Dada, Oluwatosin O; Rao, Romesh; Jones, Natalie; Jaya, Nomalie; Salas-Solano, Oscar

    2017-10-25

    Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging

    Science.gov (United States)

    2016-10-01

    minibodies and cys-diabodies) can be labeled with radioisotopes for non-invasive PET imaging for use at multiple points in the prostate cancer treatment...1Crump Institute for Molecular Imaging , Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA...AWARD NUMBER: W81XWH-15-1-0725 TITLE: Multifunctional PSCA Antibody Fragments for PET and Optical Prostate Cancer Imaging PRINCIPAL

  2. Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

    International Nuclear Information System (INIS)

    Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

    1990-01-01

    Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

  3. Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S.J. (Oak Ridge National Lab., TN); Chen, J.P.; Lankford, P.K.; Foote, L.J.

    1981-01-01

    Fischer rats were immunized with fragment D (Fg-D) of human fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely wth Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 x 10/sup 9/ M/sup -1/ while antibodies from 106-59 and 106-71 (group 2) demonstrated much lower affinities of 1.0 x 10/sup 7/ and 4.7 x 10/sup 6/ M/sup -1/, respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.

  4. 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.

  5. 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.

  6. 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.

  7. Reversion of somatic mutations of the respiratory syncytial virus-specific human monoclonal antibody Fab19 reveal a direct relationship between association rate and neutralizing potency.

    Science.gov (United States)

    Bates, John T; Keefer, Christopher J; Utley, Thomas J; Correia, Bruno E; Schief, William R; Crowe, James E

    2013-04-01

    The role of affinity in determining neutralizing potency of mAbs directed against viruses is not well understood. We investigated the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in the Ab H chain V region of Fab19, a well-described neutralizing human mAb directed to respiratory syncytial virus. Comparison of the affinity-matured Ab Fab19 with recombinant Fab19 Abs that were variants containing reverted amino acids from the inferred unmutated ancestor sequence revealed the molecular basis for affinity maturation of this Ab. Enhanced binding was achieved through mutations in the third H chain CDR (HCDR3) that conferred a markedly faster on-rate and a desirable increase in antiviral neutralizing activity. In contrast, most somatic mutations in the HCDR1 and HCDR2 regions did not significantly enhance Ag binding or antiviral activity. We observed a direct relationship between the measured association rate (Kon) for F protein and antiviral activity. Modeling studies of the structure of the Ag-Ab complex suggested the HCDR3 loop interacts with the antigenic site A surface loop of the respiratory syncytial virus F protein, previously shown to contain the epitope for this Ab by experimentation. These studies define a direct relationship of affinity and neutralizing activity for a viral glycoprotein-specific human mAb.

  8. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement.

    Science.gov (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan

    2008-08-01

    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  9. Functional improvement of antibody fragments using a novel phage coat protein III fusion system

    DEFF Research Database (Denmark)

    Jensen, Kim Bak; Larsen, Martin; Pedersen, Jesper Søndergaard

    2002-01-01

    Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance...... of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria......(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity....

  10. Multifunctional PSCA antibody fragments for PET and optical prostate cancer imaging

    Science.gov (United States)

    2017-10-01

    radiolabeled antibody fragments specific for PSCA can be used for immunoPET imaging with high target-to-background at short imaging times post -injection2...1.3%ID/g) and 22Rv1 control tumor (1.2 ± 0.6%ID/g) uptake quantified by ex vivo biodistribution. The 124I-A11 cMb-Cy5.5 biodistribution was similar to...the singly labeled 124I-A11 cMb, resulting in positive-to-negative tumor ratios of 13:1 and 8:1, respectively. Fluorescent imaging post -mortem with

  11. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  12. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

  13. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    Science.gov (United States)

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. 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...

  15. Bifunctional antibodies for radioimmunotherapy.

    Science.gov (United States)

    Chatal, J F; Faivre-Chauvet, A; Bardies, M; Peltier, P; Gautherot, E; Barbet, J

    1995-04-01

    In two-step targeting technique using bifunctional antibodies, a nonradiolabeled immunoconjugate with slow uptake kinetics (several days) is initially injected, followed by a small radiolabeled hapten with fast kinetics (several hours) that binds to the bispecific immunoconjugate already taken up by the tumor target. In patients with colorectal or medullary thyroid cancer, clinical studies performed with an anti-CEA/anti-DTPA-indium bifunctional antibody and an indium-111-labeled di-DTPA-TL bivalent hapten showed that tumor uptake was not modified compared to results for F(ab')2 fragments of the same anti-CEA antibody directly labeled with indium-111, whereas the radioactivity of normal tissues was significantly reduced (3- to 6-fold). The fast tumor uptake kinetics (several hours) and high or very high tumor-to-normal tissue ratios obtained with the bifunctional antibody technique are favorable parameters for efficient radioimmunotherapy.

  16. 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.

  17. Safety, pharmacokinetic and dosimetry evaluation of the proposed thrombus imaging agent {sup 99m}Tc-DI-DD-3B6/22-80B3 Fab'

    Energy Technology Data Exchange (ETDEWEB)

    Macfarlane, David J. [Royal Brisbane and Women' s Hospital, Department of Nuclear Medicine, Brisbane (Australia); Smart, Richard C. [St George Hospital, Department of Nuclear Medicine, Sydney (Australia); Tsui, Wendy W. [St George Hospital, Department of Nuclear Medicine, Sydney (Australia); University of New South Wales, School of Medicine, Sydney (Australia); Gerometta, Michael [AGEN Biomedical Limited, Research and Development, Brisbane (Australia); Eisenberg, Paul R. [Eli Lilly Company, Lilly Research Laboratories, Indianapolis (United States); Scott, Andrew M. [Austin Health, Centre for PET, Melbourne (Australia); Ludwig Institute for Cancer Research, Melbourne (Australia)

    2006-06-15

    {sup 99m}Tc-DI-DD-3B6/22-80B3 (Thromboview, hereafter abbreviated to {sup 99m}Tc-DI-80B3 Fab') is a humanised, radiolabelled monoclonal antibody Fab' fragment with high affinity and specificity for the D-dimer domain of cross-linked fibrin. The purpose of this study was to evaluate the safety, pharmacokinetics and dosimetry of four increasing doses of {sup 99m}Tc-DI-80B3 Fab' in healthy volunteers. Thirty-two healthy volunteers (18-70 years; 16 male, 16 female) received a single intravenous injection of 0.5, 1.0, 2.0 or 4.0 mg of {sup 99m}Tc-DI-80B3 Fab'. Safety outcomes (vital signs, electrocardiography, haematology, biochemistry, adverse events and development of human anti-human antibodies) were assessed up to 30 days post injection. Blood and urine samples were collected up to 48 h post injection. Gamma camera images were acquired at 0.5, 1, 2, 4, 6 and 24 h post injection. Dosimetry was performed using standard MIRD methodology. No adverse events considered to be drug related were observed. Human anti-human antibody was not detectable in any subject during the follow-up period. {sup 99m}Tc-DI-80B3 Fab' had a rapid initial plasma clearance (t{sub 1/2}{alpha}=1 h). The pharmacokinetic profile of the Fab' fragment was generally linear across the four dose cohorts. By 24 h, 30-35% of the administered radioactivity appeared in the urine. There was marked renal accumulation with time, but no specific uptake was identified within other normal tissues. The effective dose was 9 mSv/750 MBq. (orig.)

  18. Role of Homologous Fc Fragment in the Potency and Efficacy of Anti‐Botulinum Antibody Preparations

    Directory of Open Access Journals (Sweden)

    Amram Torgeman

    2017-05-01

    Full Text Available The only approved treatment for botulism relies on passive immunity which is mostly based on antibody preparations collected from hyper‐immune horses. The IgG Fc fragment is commonly removed from these heterologous preparations to reduce the incidence of hyper‐sensitivity reactions. New‐generation therapies entering the pipeline are based on a combination of humanized monoclonal antibodies (MAbs, which exhibit improved safety and pharmacokinetics. In the current study, a systematic and quantitative approach was applied to measure the direct contribution of homologous Fc to the potency of monoclonal and polyclonal antitoxin preparations in mice. Homologous Fc increased the potency of three individual anti‐botulinum toxin MAbs by up to one order of magnitude. Moreover, Fc fragment removal almost completely abolished the synergistic potency obtained from a combined preparation of these three MAbs. The MAb mixture neutralized a 400‐mouse median lethal dose (MsLD50 of botulinum toxin, whereas the F(ab′2 combination failed to neutralize 10 MsLD50 of botulinum toxin. Notably, increased avidity did not compensate for this phenomenon, as a polyclonal, hyper‐immune, homologous preparation lost 90% of its potency as well upon Fc removal. Finally, the addition of homologous Fc arms to a heterologous pharmaceutical anti‐botulinum toxin polyclonal horse F(ab′2 preparation improved its efficacy when administered to intoxicated symptomatic mice. Our study extends the aspects by which switching from animal‐based to human‐based antitoxins will improve not only the safety but also the potency and efficacy of passive immunity against toxins.

  19. ECT with /sup 123/I-labeled fragments of anti-CEA monoclonal antibodies in colo-rectal cancer

    International Nuclear Information System (INIS)

    Bischof-Delaloye, A.; Delaloye, B.

    1986-01-01

    The recent progress of tumor localization with labelled antibodies can be attributed to three techniques: 1) use of I-123 as a label; 2) fragmentation of antibodies; 3) tomographic recording and evaluation of patient radiation data. Under these conditions the method yields good sensitivity and specifity indexes (15/16 for primary tumors and local recurrences, 7/10 for metastasis). A strictly prospective study, however, remains mandatory in order to assess the clinical value of this method

  20. Tumor penetration with intact MAb and fragments demonstrated in vitro on tumor spheroids and in vivo in the nude mouse model

    International Nuclear Information System (INIS)

    Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.

    1986-01-01

    Tumor spheroids grown in culture represent a good in vitro model for the study of tumor penetration phenomena of potential radiotherapeutics. Using this system, it was found that Fab-fragments penetrate tumors more quickly and deeply than complete antibodies. These results were confirmed in tumor bearing nephrectomized nude mice

  1. Generic anti-drug antibody assay with drug tolerance in serum samples from mice exposed to human antibodies.

    Science.gov (United States)

    Stubenrauch, Kay; Mackeben, Klaus; Vogel, Rudolf; Heinrich, Julia

    2012-11-15

    Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage λ immunoexpression library

    International Nuclear Information System (INIS)

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A.; Hay, B.N.; Shopes, B.

    1990-01-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and κ light-chain variable and constant region domains, were inserted into modified bacteriophase λ expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen

  3. Spectral characteristics of fluorescence and circular dichroism of aflatoxin B1 reaction with its anti-idiotypic antibody

    Science.gov (United States)

    Liu, Aiping; Yang, Hongxiu; Wang, Xiaohong; Chen, Fusheng

    2012-11-01

    Aflatoxin B1 (AFB1) is a toxic secondary metabolite and sensitive methods for its analysis have been developed. In our lab, a number of works have been carried out, including exploitation of detection methods and production of anti-idiotypic antibody (Ab2) against Fab fragment of anti-AFB1 antibody (Ab1). In this paper, Ab2 was generated upon the immunization of mice with F(ab')2 fragment, which was specific to AFB1 and obtained by pepsin digestion of Ab1. The characteristics of Ab2 was primarily investigated by indirect competitive enzyme-linked immunosorbent assay (icELISA), which indicated that Ab2, might bear an internal image of antigen AFB1 and was able to combine to F(ab')2 in competition with AFB1, and the concentration of Ab2 to cause 50% inhibition of binding (IC50) was 131.8 μg/mL. In addition, fluorescence and circular dichroism studies were designed to explore the mutual relationship among AFB1, F(ab')2 and Ab2. The fluorescence spectroscopy implied that both AFB1 and Ab2 act as a quencher upon F(ab')2, and the Ab2 could compete with AFB1 when both of Ab2 and AFB1 reacted with F(ab')2. The circular dichroism (CD) spectrum suggested that both the binding of Ab2 and AFB1 on F(ab')2 brought secondary conformation change of F(ab')2, especially in the changes of α helix and β sheet. The research performed would provide unique insight into the comprehension of interaction among AFB1, F(ab')2 and Ab2 as well as offer structural information for substitution researches of toxic antigen like AFB1.

  4. Mechanism of immunoglobulin G4 Fab-arm exchange

    NARCIS (Netherlands)

    Rispens, Theo; Ooijevaar-de Heer, Pleuni; Bende, Onno; Aalberse, Rob C.

    2011-01-01

    Immunoglobulin G (IgG) antibodies are symmetrical molecules that may be regarded as covalent dimers of 2 half-molecules, each consisting of a light chain and a heavy chain. Human IgG4 is an unusually dynamic antibody, with half-molecule exchange ("Fab-arm exchange") resulting in asymmetrical,

  5. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    International Nuclear Information System (INIS)

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-01-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80μCi of I-131-F(ab')/sub 2/ with a specific activity of 1.8μCi/μg. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice

  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. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Glaessner, H.

    1986-01-01

    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de

  8. Mapping of Monoclonal Antibody Binding Sites on CNBr Fragments of the S- Layer Protein Antigens of Rickettsia Typhi and Rickettsia Prowazekii

    Science.gov (United States)

    1992-01-01

    Security Clasification ) Mapping of monoclonal antibody binding sites on CNBr fragments o; the S-layer protein antigens of Rickettsia Typhi and...homology was found in all the viral polypeptides have long fatty acids attached to fragments which react with type I antibody (Fig. 4). A their N-termini

  9. FabIO

    DEFF Research Database (Denmark)

    Bergbäck Knudsen, Erik; Sørensen, Henning O.; Wright, Jonathan P.

    2013-01-01

    FabIO is a Python module written for easy and transparent reading of raw two-dimensional data from various X-ray detectors. The module provides a function for reading any image and returning a fabioimage object which contains both metadata (header information) and the raw data. All fabioimage...

  10. Fragmentation of Nimotuzumab for Preparation of 125I-F(ab’)2-Nimotuzumab as a Precursor for Preparing 125I-F(ab’)2-Nimotuzumab-NLS Radiopharmaceutical for Cancer Therapy

    OpenAIRE

    R.D. Haryuni; A. Bahtiar; S. Soenarjo; Y. Harahap; A. Mutalib; M. Ramli; S. Hermanto; C.N. Ardiyatno; V.Y. Susilo; D. Haffid

    2014-01-01

    Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR). Thismonoclonal antibody has a relatively high molecular weight which slowspenetration on tumor cells, making it less attractive in imaging kinetics and potentially elicits antibodies responses. Therefore, in this study nimotuzumab was fragmented to form a bivalent antibody [F(ab')2] and then labeled with 125I to form 125I-F(ab')2-nimotuzumab which can be used further as a precur...

  11. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    Science.gov (United States)

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-05-01

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

  12. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille; van Diest, Eline; Schmidt, Florian I.; Schwartz, Thomas U.; Ploegh, Hidde L. (Whitehead); (MIT)

    2016-12-13

    Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

    IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and

  13. The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment.

    Science.gov (United States)

    Hanke, Leo; Knockenhauer, Kevin E; Brewer, R Camille; van Diest, Eline; Schmidt, Florian I; Schwartz, Thomas U; Ploegh, Hidde L

    2016-12-13

    Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. Influenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and provide a well-characterized tool to

  14. A randomized, double-blind, placebo-controlled trial of a highly purified equine F(ab)2 antibody black widow spider antivenom.

    Science.gov (United States)

    Dart, Richard C; Bogdan, Gregory; Heard, Kennon; Bucher Bartelson, Becki; Garcia-Ubbelohde, Walter; Bush, Sean; Arnold, Tom; Clark, Richard C; Hendey, Gregory W; Holstege, Christopher; Spradley, Elizabeth A

    2013-04-01

    Black widow spider antivenom has never been tested in a randomized clinical trial, to our knowledge. We explore various efficacy measures for a novel F(ab)2 antivenom in patients with moderate to severe pain caused by black widow spider envenomation. A randomized, placebo-controlled, double-blind, clinical trial was conducted in 12 academic emergency departments. We included patients at least 10 years old with moderate to severe latrodectism. Subjects received either a single intravenous infusion of antivenom or placebo. Pain was assessed with the visual analog scale. The primary efficacy outcome was the difference in pre- and posttreatment visual analog scale score. Prospectively defined secondary outcomes included treatment failures and time to clinically important decrease in pain. Twenty-four subjects were enrolled between October 2005 and October 2006; 13 were randomized to antivenom and 11 to placebo. The median change in visual analog scale at 150 minutes posttreatment was -50.0 mm (Interquartile Range [IQR] -67, -41 mm) in the antivenom treatment group and -46.0 mm (IQR -51, 0 mm) in the placebo treatment group (P=.14). There were 7 treatment failures (64%; 95% confidence interval 35% to 92%) in the placebo group and 3 (23%; 95% confidence interval 0.2% to 46%) in the antivenom group (P=.06). The median time to a clinically important decrease in pain after treatment was shorter in the antivenom group compared with the placebo group (30 minutes [IQR 30, 60 minutes] versus 90 minutes [IQR 30, 90 minutes]; P=.03). No serious adverse events or deaths were reported. Although the overall reduction in pain was similar for antivenom- and placebo-treated subjects, antivenom reduced pain more rapidly than placebo. No significant adverse events occurred in either group. Copyright © 2012. Published by Mosby, Inc.

  15. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  16. Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth.

    Science.gov (United States)

    Boulding, N; Yim, S S S; Keshavarz-Moore, E; Ayazi Shamlou, P; Berry, M

    2002-08-20

    Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates. Copyright 2002 Wiley Periodicals, Inc.

  17. Immunization with recombinant enterovirus 71 viral capsid protein 1 fragment stimulated antibody responses in hamsters

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    Ch’ng Wei-Choong

    2012-08-01

    Full Text Available Abstract Enterovirus 71 (EV71 causes severe neurological diseases resulting in high mortality in young children worldwide. Development of an effective vaccine against EV71 infection is hampered by the lack of appropriate animal models for efficacy testing of candidate vaccines. Previously, we have successfully tested the immunogenicity and protectiveness of a candidate EV71 vaccine, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP11-100 protein, in a mouse model of EV71 infection. A drawback of this system is its limited window of EV71 susceptibility period, 2 weeks after birth, leading to restricted options in the evaluation of optimal dosing regimens. To address this issue, we have assessed the NPt-VP11-100 candidate vaccine in a hamster system, which offers a 4-week susceptibility period to EV71 infection. Results obtained showed that the NPt-VP11-100 candidate vaccine stimulated excellent humoral immune response in the hamsters. Despite the high level of antibody production, they failed to neutralize EV71 viruses or protect vaccinated hamsters in viral challenge studies. Nevertheless, these findings have contributed towards a better understanding of the NPt-VP11-100 recombinant protein as a candidate vaccine in an alternative animal model system.

  18. Isolation of soluble scFv antibody fragments specific for small biomarker molecule, L-Carnitine, using phage display.

    Science.gov (United States)

    Abou El-Magd, Rabab M; Vozza, Nicolas F; Tuszynski, Jack A; Wishart, David S

    2016-01-01

    Isolation of single chain antibody fragment (scFv) clones from naïve Tomlinson I+J phage display libraries that specifically bind a small biomarker molecule, L-Carnitine, was performed using iterative affinity selection procedures. L-Carnitine has been described as a conditionally essential nutrient for humans. Abnormally high concentrations of L-Carnitine in urine are related to many health disorders including diabetes mellitus type 2 and lung cancer. ELISA-based affinity characterization results indicate that selectants preferentially bind to L-Carnitine in the presence of key bioselecting component materials and closely related L-Carnitine derivatives. In addition, the affinity results were confirmed using biophysical fluorescence quenching for tyrosine residues in the V segment. Small-scale production of the soluble fragment yielded 1.3mg/L using immunopure-immobilized protein A affinity column. Circular Dichroism data revealed that the antibody fragment (Ab) represents a folded protein that mainly consists of β-sheets. These novel antibody fragments may find utility as molecular affinity interface receptors in various electrochemical biosensor platforms to provide specific L-Carnitine binding capability with potential applications in metabolomic devices for companion diagnostics and personalized medicine applications. It may also be used in any other biomedical application where detection of the L-Carnitine level is important. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Structural Basis for Enhanced HIV-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12

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    Yunji Wu

    2013-12-01

    Full Text Available The human immunoglobulin G (IgG 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs are intramolecularly domain exchanged, resulting in a rigid (Fab2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.

  20. A 10-RESIDUE FRAGMENT OF AN ANTIBODY (MINI-ANTIBODY) DIRECTED AGAINST LYSOZYME AS LIGAND IN IMMUNOAFFINITY CHROMATOGRAPHY

    NARCIS (Netherlands)

    WELLING, GW; VANGORKUM, J; DAMHOF, RA; DRIJFHOUT, JW; BLOEMHOFF, W; WELLINGWESTER, S

    1991-01-01

    The interaction between an antibody molecule and a protein antigen is an example of "natural" protein modelling. Amino acids of the antigen-binding site consisting of three hypervariable segments (L1, L2, L3) of the light (L) and three (H1, H2, H3) of the heavy (H) chain of an antibody molecule

  1. Experimental and clinical analysis of the characteristics of a chimeric monoclonal antibody, MOv18, reactive with an ovarian cancer-associated antigen

    NARCIS (Netherlands)

    Molthoff, C. F.; Buist, M. R.; Kenemans, P.; Pinedo, H. M.; Boven, E.

    1992-01-01

    Monoclonal antibody (Mab) MOv18 preferentially reacts with gynecological carcinomas. We have analyzed the characteristics of murine MOv18 (m-MOv18) and chimeric MOv18 (c-MOv18). We found no differences in affinity and binding to IGROV1 cells between c-MOv18 as IgG and F(ab')2 fragments and m-MOv18.

  2. Structure of the omalizumab Fab

    Science.gov (United States)

    Jensen, Rasmus K.; Plum, Melanie; Tjerrild, Luna; Jakob, Thilo; Spillner, Edzard; Andersen, Gregers Rom

    2015-01-01

    Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity. PMID:25849503

  3. Influence of circulating antigen on blood pool activity of a radioiodinated monoclonal antibody

    International Nuclear Information System (INIS)

    Zalutsky, M.R.; Knapp, R.C.; Bast, R.C. Jr.

    1988-01-01

    Athymic mice with and without circulating CA 125 antigen were injected with 0.1-100 μg of 131 I-labeled OC 125 F(ab') 2 antibody fragment. Both the blood clearance of 131 I activity and the change in serum CA 125 were monitored over 24 h. Influence of CA 125 on blood pool activity could be avoided only at the 100 μg dose. In patient studies, circulating CA 125 levels decreased for the first 2 h after injection of OC 125 F(ab') 2 but generally returned to preinjection levels shortly thereafter. In vitro binding studies using the sera from patients injected with 131 I-labeled OC 125 F(ab') 2 suggest that circulating CA 125 could interfere with the tumor uptake of the labeled antibody. (author)

  4. 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.

  5. Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

    Science.gov (United States)

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function. PMID:18783590

  6. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina

    2008-09-01

    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  7. Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

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    Tong Li

    Full Text Available Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc., La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect.

  8. 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.

  9. Dual display: phage selection driven by co-engagement of two targets by two different antibody fragments.

    Science.gov (United States)

    Fagète, Séverine; Botas-Perez, Ledicia; Rossito-Borlat, Irène; Adea, Kenneth; Gueneau, Franck; Ravn, Ulla; Rousseau, François; Kosco-Vilbois, Marie; Fischer, Nicolas; Hartley, Oliver

    2017-09-01

    Antibody phage display technology has supported the emergence of numerous therapeutic antibodies. The development of bispecific antibodies, a promising new frontier in antibody therapy, could be facilitated by new phage display approaches that enable pairs of antibodies to be co-selected based on co-engagement of their respective targets. We describe such an approach, making use of two complementary leucine zipper domains that heterodimerize with high affinity. Phagemids encoding a first antibody fragment (scFv) fused to phage coat protein via the first leucine zipper are rescued in bacteria expressing a second scFv fused to the second leucine zipper as a soluble periplasmic protein, so that it is acquired by phage during assembly. Using a soluble scFv specific for a human CD3-derived peptide, we show that its acquisition by phage displaying an irrelevant antibody is sufficiently robust to drive selection of rare phage (1 in 105) over three rounds of panning. We then set up a model selection experiment using a cell line expressing the chemokine receptor CCR5 fused to the CD3 peptide together with a panel of phage clones capable displaying either an anti-CCR5 scFv or an irrelevant antibody, with or without the capacity to acquire the soluble anti-CD3 scFv. In this experiment we showed that rare phage (1 in 105) capable of displaying the two different scFvs can be specifically enriched over four rounds of panning. This approach has the potential to be applied to the identification of pairs of ligands capable of co-engaging two different user-defined targets, which would facilitate the discovery of novel bispecific antibodies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

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    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  11. Isolation of populations of antipeptide antibodies directed against different epitopes of the same fragment.

    Science.gov (United States)

    Chersi, A; Greger, C; Houghten, R A

    1985-01-01

    Rabbit antibodies against small peptides may be composed by subpopulations recognizing different epitopes made likely by few amino acids. This explains the frequent crossreactivity of antipeptide antibodies with unrelated peptides. A suitable use of immunoadsorbents is suggested to obtain truly specific antibodies able to react with restricted amino acid sequences.

  12. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

    Science.gov (United States)

    Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

    2008-07-22

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

  13. Antibody-dependent cellular cytotoxicity in antimyelin antibody-induced oligodendrocyte damage in vitro.

    Science.gov (United States)

    Griot-Wenk, M; Griot, C; Pfister, H; Vandevelde, M

    1991-08-01

    Treatment of dissociated murine brain cell cultures with an antibody recognizing galactocerebroside (GalC) led to degeneration of oligodendrocytes with loss of their cell processes. F(ab')2 fragments prepared from this antibody showed no effect. The anti-GalC antibody--but not its F(ab')2 fragments b2 was able to stimulate macrophages in these cultures as seen in a chemiluminescence assay. Therefore, antibodies bound to oligodendrocytes stimulated nearby macrophages by interacting with their Fc receptors. The oligodendroglial damage coincided with the release of toxic compounds by the stimulated macrophages, since treatment of the cultures with the anti-GalC antibody and a variety of other macrophage stimulating agents led to secretion of reactive oxygen species and--in some experiments--tumor necrosis factor, both known to be toxic for oligodendrocytes. These in vitro experiments show evidence that antibody-dependent cellular cytotoxicity may be an important mechanism of tissue destruction in inflammatory demyelinating diseases.

  14. Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors.

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    Jennifer M Binning

    2018-01-01

    Full Text Available The lentiviral protein Viral Infectivity Factor (Vif counteracts the antiviral effects of host APOBEC3 (A3 proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

  15. Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

    Science.gov (United States)

    Säll, Anna; Sjöholm, Kristoffer; Waldemarson, Sofia; Happonen, Lotta; Karlsson, Christofer; Persson, Helena; Malmström, Johan

    2015-11-06

    Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

  16. Preclinical Testing of Antihuman CD28 Fab' Antibody in a Novel Nonhuman Primate Small Animal Rodent Model of Xenogenic Graft-Versus-Host Disease.

    Science.gov (United States)

    Hippen, Keli L; Watkins, Benjamin; Tkachev, Victor; Lemire, Amanda M; Lehnen, Charles; Riddle, Megan J; Singh, Karnail; Panoskaltsis-Mortari, Angela; Vanhove, Bernard; Tolar, Jakub; Kean, Leslie S; Blazar, Bruce R

    2016-12-01

    Graft-versus-host disease (GVHD) is a severe complication of hematopoietic stem cell transplantation. Current therapies to prevent alloreactive T cell activation largely cause generalized immunosuppression and may result in adverse drug, antileukemia and antipathogen responses. Recently, several immunomodulatory therapeutics have been developed that show efficacy in maintaining antileukemia responses while inhibiting GVHD in murine models. To analyze efficacy and better understand immunological tolerance, escape mechanisms, and side effects of clinical reagents, testing of species cross-reactive human agents in large animal GVHD models is critical. We have previously developed and refined a nonhuman primate (NHP) large animal GVHD model. However, this model is not readily amenable to semi-high throughput screening of candidate clinical reagents. Here, we report a novel, optimized NHP xenogeneic GVHD (xeno-GVHD) small animal model that recapitulates many aspects of NHP and human GVHD. This model was validated using a clinically available blocking, monovalent anti-CD28 antibody (FR104) whose effects in a human xeno-GVHD rodent model are known. Because human-reactive reagents may not be fully cross-reactive or effective in vivo on NHP immune cells, this NHP xeno-GVHD model provides immunological insights and direct testing on NHP-induced GVHD before committing to the intensive NHP studies that are being increasingly used for detailed evaluation of new immune therapeutic strategies before human trials.

  17. Regioselective chemical modification of monoclonal antibodies

    Science.gov (United States)

    Ranadive, Girish; Rosenzweig, Howard S.; Epperly, Michael; Bloomer, William

    1993-01-01

    A method of selectively modifying an immunoglobulin having at least one Fab region and at least one Fc region, each region having an isoelectric point wherein said isoelectric point of the Fab fragment of said immunoglobulin is different than the isoelectric point of the Fc fragment of the immunoglobulin, said method comprising modification of the immunoglobulin at a pH between the respective isoelectric points of the Fab and Fc fragments of the immunoglobulin.

  18. A universal phage display system for the seamless construction of Fab libraries.

    Science.gov (United States)

    Nelson, Renae S; Valadon, Philippe

    2017-11-01

    The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    Science.gov (United States)

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  20. Acute hypersensitivity reaction to Crotalidae polyvalent immune Fab (CroFab) as initial presentation of galactose-α-1,3-galactose (α-gal) allergy.

    Science.gov (United States)

    Rizer, Justin; Brill, Kaitlin; Charlton, Nathan; King, Joshua

    2017-08-01

    Crotalidae polyvalent immune Fab antivenom (CroFab), commonly used for the treatment of clinically significant North American crotalinae envenomation, is generally well-tolerated. A novel form of anaphylaxis due to an IgE antibody response to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) has been established following red-meat consumption as well as IV administration of cetuximab, which contain the α-gal epitope. We present a case of α-gal allergy discovered after acute hypersensitivity reaction to FabAV. A 61-year-old healthy female was bitten on her left ankle by Agkistrodon contortrix. Given the patient's rapid progression of pain and swelling, she was given FabAV. During infusion of FabAV, she developed diffuse hives over her entire body and itching, but denied respiratory or gastrointestinal symptoms and her vital signs remained stable. The FabAV was immediately discontinued and she received intravenous diphenhydramine and famotidine with gradual resolution of symptoms. On further discussion, she denied a history of α-gal or papaya allergy but rarely ate red meat and endorsed sustaining frequent tick bites. Subsequent antibody testing was significant for an α-1,3-galactose IgE concentration of 45,000 U/L (normal allergy. To our knowledge, this is the first report of FabAV hypersensitivity associated with an underlying α-gal allergy.

  1. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  2. Codon-Precise, Synthetic, Antibody Fragment Libraries Built Using Automated Hexamer Codon Additions and Validated through Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Laura Frigotto

    2015-05-01

    Full Text Available We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR in antibody fragment libraries and next generation sequencing (NGS analysis of their quality and diversity.

  3. Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

    DEFF Research Database (Denmark)

    Encinas, P.; Gomez-Casado, E.; Grandes, Fregeneda

    2011-01-01

    Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using...... refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV...

  4. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    Science.gov (United States)

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  5. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    Science.gov (United States)

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

  6. Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties

    DEFF Research Database (Denmark)

    Lundquist, Rasmus; Nielsen, Leif Kofoed; Jafarshad, Ali

    2006-01-01

    against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3...... were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been...... tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth....

  7. Evading pre-existing anti-hinge antibody binding by hinge engineering

    Science.gov (United States)

    Kim, Hok Seon; Kim, Ingrid; Zheng, Linda; Vernes, Jean-Michel; Meng, Y. Gloria; Spiess, Christoph

    2016-01-01

    ABSTRACT Antigen-binding fragments (Fab) and F(ab′)2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA. PMID:27606571

  8. Anti-idiotypic antisera in man. I. Production and immunochemical characterization of anti-idiotypic antisera to human antitetanus antibodies

    International Nuclear Information System (INIS)

    Geha, R.S.; Weinberg, R.P.

    1978-01-01

    Antisera were raised in rabbits against immunosorbent-purified F(ab') 2 fragments of IgG antitetanus toxoid (TT) antibodies obtained from three different donors. The antisera were rendered idiotype specific by absorption with insolubilized TT-nontreactive F(ab') 2 . The resulting anti-idiotypic antisera precipitated more than 85% of 125 I-radiolabeled F(ab') 2 anti-TT and were shown to be individual specific in that they reacted only with the immunizing F(ab') 2 anti-TT and not with F(ab') 2 anti-TT derived from other donors. Anti-idiotypic antiserum inhibited binding of 125 I-TT but not of 125 I-DT to IgG derived from the donor of the immunizing F(ab') 2 anti-TT, but did not inhibit 125 I-TT binding to IgG derived from other donors. Finally, binding of 125 I-F(ab') 2 anti-TT to the anti-idiotypic antiserum was completely inhibited by IgG derived from the same donor but only partially inhibited by a great excess of TT antigen, suggesting that the anti-idiotypic antiserum is recognizing nonantigen-binding idiotypic determinants on F(ab') 2 anti-TT. The data presented demonstrate that anti-idiotypic heteroantisera can be successfully raised against human antibodies to TT, indicate minimal cross-reactivity of idiotypic determinants between unrelated individuals, and suggest the presence of nonantigen binding-idiotypic determinants on the antibody molecule

  9. The effect of varying the peptide linker length in a single chain variable fragment antibody against wogonin glucuronide.

    Science.gov (United States)

    Paudel, Madan Kumar; Sakamoto, Seiichi; Van Huy, Le; Tanaka, Hiroyuki; Miyamoto, Tomofumi; Morimoto, Satoshi

    2017-06-10

    Peptide linkers of three different lengths were constructed to join the variable regions of the heavy chain (VH) and the light chain (VL) in a single-chain variable fragment antibody (scFv) specific for wogonin glucuronide (Wgn) that has the structure VH-(GGGGS) n -VL (n=3, 5, or 7). The scFv antibodies, which were expressed in Escherichia coli, were derived from an anti-Wgn monoclonal antibody (315A). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to evaluate their reactivity and sensitivity, which is also used for quantitative analysis of Wgn. Our results, showed that the reactivity and specificity of the three different scFvs were, in fact, similar. Subsequently, the scFv having a VH-(GGGGS) 3 -VL linker which was slightly better that other two scFvs against Wgn, was applied to indirect competitive ELISA (icELISA) to analyze Scutellariae Radix (S. Radix). The utility of the icELISA was demonstrated for quality control and analysis of S. Radix in this report. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. 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.

  11. 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.

  12. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  13. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  14. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Science.gov (United States)

    Tullila, Antti; Nevanen, Tarja K.

    2017-01-01

    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  15. A Role for Small Antibody Fragments to Bind and Neutralize HIV | Center for Cancer Research

    Science.gov (United States)

    The surface of the Human Immunodeficiency Virus (HIV) is studded with numerous copies of the glycoprotein Env. Each Env spike is composed of three copies of the proteins gp41, which sits in the viral membrane, and gp120, which rests on top of each gp41 molecule. Env is essential for HIV-mediated infection because the binding of gp120 to the T cell surface receptor CD4 initiates a conformational change in Env exposing the fusion peptide, which inserts into the T cell membrane and helps fuse the T cell and virus together. This makes Env an attractive target for designing therapeutic inhibitory antibodies. However, the complexities of the HIV surface proteins and the tight association of the virus and T cell during infection have hampered the identification of full-length antibodies with effective HIV neutralizing activity.

  16. Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity.

    Science.gov (United States)

    Funayama, Jaqueline C; Pucca, Manuela B; Roncolato, Eduardo C; Bertolini, Thaís B; Campos, Lucas B; Barbosa, José E

    2012-03-01

    The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

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

    Science.gov (United States)

    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. [Use of antibodies or their fragments for the treatment of tumors].

    Science.gov (United States)

    Douillard, J Y; Chatal, J F

    1998-11-01

    During the last 15 years, various antibodies specific for antigens associated with determined types of cancer have been used therapeutically, including some in unlabeled forms as immune effectors. The results of clinical studies have been unpromising for patients with colorectal cancer at the advanced metastatic stage but much more favorable in terms of increased survival for the adjuvant situation of residual microscopic disease. Antibodies have also been used as carriers for cytotoxic substances, but with rather disappointing clinical results when they were labeled with toxins or antimitotic agents. The results have been variable for labeling with radionuclides (mainly iodine-131), some-times proving quite favorable for refractory forms of non-hodgkin's lymphomas or acute leukemias. In this last indication, radioimmunotherapy has been associated with chemotherapy to enhance action before a bone-marrow graft. However, the clinical results have been disappointing in the treatment of solid tumors, showing responses only in the case of small targets. In the future, treatment with antibodies will focus on microscopic tumors, in association with other therapeutic modalities especially, chemotherapy and biotherapy.

  19. Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator.

    Science.gov (United States)

    Gakhal, Amandeep K; Jensen, Timothy J; Bozoky, Zoltan; Roldan, Ariel; Lukacs, Gergely L; Forman-Kay, Julie; Riordan, John R; Sidhu, Sachdev S

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.

  20. 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.

  1. 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

  2. Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1.

    Science.gov (United States)

    Ascione, Alessandro; Capecchi, Barbara; Campitelli, Laura; Imperiale, Valentina; Flego, Michela; Zamboni, Silvia; Gellini, Mara; Alberini, Isabella; Pittiglio, Eliana; Donatelli, Isabella; Temperton, Nigel J; Cianfriglia, Maurizio

    2009-09-01

    Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

  3. 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.)

  4. Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen

    Directory of Open Access Journals (Sweden)

    Fabio Selis

    2016-04-01

    Full Text Available PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

  5. Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen

    Science.gov (United States)

    Selis, Fabio; Focà, Giuseppina; Sandomenico, Annamaria; Marra, Carla; Di Mauro, Concetta; Saccani Jotti, Gloria; Scaramuzza, Silvia; Politano, Annalisa; Sanna, Riccardo; Ruvo, Menotti; Tonon, Giancarlo

    2016-01-01

    PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments. PMID:27043557

  6. Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

    Directory of Open Access Journals (Sweden)

    Fleur S. van de Bovenkamp

    2018-04-01

    Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

  7. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  8. Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display.

    Science.gov (United States)

    Keller, Thomas; Kalt, Romana; Raab, Ingrid; Schachner, Helga; Mayrhofer, Corina; Kerjaschki, Dontscho; Hantusch, Brigitte

    2015-01-01

    The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.

  9. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    Science.gov (United States)

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  10. Field Effect Transistor Biosensor Using Antigen Binding Fragment for Detecting Tumor Marker in Human Serum

    Science.gov (United States)

    Cheng, Shanshan; Hotani, Kaori; Hideshima, Sho; Kuroiwa, Shigeki; Nakanishi, Takuya; Hashimoto, Masahiro; Mori, Yasuro; Osaka, Tetsuya

    2014-01-01

    Detection of tumor markers is important for cancer diagnosis. Field-effect transistors (FETs) are a promising method for the label-free detection of trace amounts of biomolecules. However, detection of electrically charged proteins using antibody-immobilized FETs is limited by ionic screening by the large probe molecules adsorbed to the transistor gate surface, reducing sensor responsiveness. Here, we investigated the effect of probe molecule size on the detection of a tumor marker, α-fetoprotein (AFP) using a FET biosensor. We demonstrated that the small receptor antigen binding fragment (Fab), immobilized on a sensing surface as small as 2–3 nm, offers a higher degree of sensitivity and a wider concentration range (100 pg/mL–1 μg/mL) for the FET detection of AFP in buffer solution, compared to the whole antibody. Therefore, the use of a small Fab probe molecule instead of a whole antibody is shown to be effective for improving the sensitivity of AFP detection in FET biosensors. Furthermore, we also demonstrated that a Fab-immobilized FET subjected to a blocking treatment, to avoid non-specific interactions, could sensitively and selectively detect AFP in human serum. PMID:28788579

  11. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...... also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection...

  12. Kinetics of intralymphatically delivered monoclonal antibodies

    International Nuclear Information System (INIS)

    Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.

    1985-01-01

    Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations

  13. Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase.

    Science.gov (United States)

    Takazawa, Takeshi; Kamiya, Noriho; Ueda, Hiroshi; Nagamune, Teruyuki

    2004-05-20

    Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins. Copyright 2004 Wiley Periodicals, Inc.

  14. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Guus Koch

    2013-04-01

    Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

  15. Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

    Science.gov (United States)

    Bagheri, Salman; Yousefi, Mehdi; Safaie Qamsari, Elmira; Riazi-Rad, Farhad; Abolhassani, Mohsen; Younesi, Vahid; Dorostkar, Ruhollah; Movassaghpour, Ali Akbar; Sharifzadeh, Zahra

    2017-03-01

    The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

  16. 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.

  17. Identification and further characterization of the specific cell binding fragment from sponge aggregation factor

    OpenAIRE

    1986-01-01

    Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from t...

  18. Phage display aided improvement of a unique prostate-specific antigen (PSA) antibody unreactive with Lys(145)-Lys(146) internally cleaved forms.

    Science.gov (United States)

    Liton, Md Ferdhos Khan; Peltola, Mari T; Vehniäinen, Markus; Kuusela, Erica; Pettersson, Tiina; Lamminmäki, Urpo; Pettersson, Kim; Brockmann, Eeva-Christine

    2015-07-01

    Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome (cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys(145)-Lys(146). Due to high dissociation rate the 4D4 antibody is less than optimal for achieving a highly sensitive robust assay. In this study, we cloned the 4D4 Mab into a recombinant fragment (Fab) format and constructed three mutant libraries with the aim to increase its binding affinity. The libraries contained targeted mutations either in the CDR-H1, CDR-H2 or CDR-L3 region. PSA-I specific antibodies were enriched from the libraries by phage display technology. We identified fourteen unique clones with 1-5 mutated amino acids showing reduced dissociation of the PSA conjugate compared to the wt-4D4 Fab. Five of these mutant antibodies had 2-6 times higher binding affinity compared to the wt-4D4 Fab yet retaining the original specificity for PSA-I. The analytical sensitivity of fPSA-I assay with mutant L3-2 Fab was 0.12 μg/L compared to 4.46 μg/L with the original wt-4D4 Fab. In the method comparison study, the developed assay showed an excellent correlation to the existing fPSA-I assay. The high affinity and specificity of these mutant antibodies have potential to provide sensitive and robust detection of intact and nicked PSA from patient samples in different test formats. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Tumour localization and pharmacokinetics of iodine-125 human monoclonal IgM antibody (COU-1) and its monomeric and half-monomeric fragments analysed in nude mice grafted with human tumour

    International Nuclear Information System (INIS)

    Ditzel, H.; Erb, K.; Rasmussen, J.W.; Jensenius, J.C.

    1992-01-01

    Human monoclonal IgM antibodies reactive with cancer-associated antigens may not have the optimal imaging capability due to their large size. Fragmentation of human IgM is less than straight-forward due to the loss of immunoreactivity. From the human monoclonal IgM antibody COU-1 we have prepared monomeric and half-monomeric fragments, which retain the ability to bind to colon cancer cells in vitro. The pharmacokinetics and tumour localization were evaluated in nude mice bearing human colon adenocarcinoma and human melanoma grafts. Faster clearance from the circulation was seen for the smaller half-monomeric fragment with a half-life (rapid phase/slow phase) of 2 h/16 h compared with the intact antibody, 4 h/25 h, and the monomeric fragment, 3 h/27 h. Intact COU-1 as well as the fragments accumulated in the colon tumour graft. Higher amounts of radioactivity were found in the colon tumour as compared to normal organs for intact COU-1 at days 4 and 6, for the monomeric fragment at day 4, and for the half-monomeric fragment at day 2 after injection. This investigation demonstrates the favourable biodistribution of the half monomeric COU-1 fragment. The fast clearance of this fragment resulted in a tumour-to-muscle ratio as high as 22 on day 2 after injection. Also, only this fragment gave a positive tumour-to-blood ratio. Normal IgM and its fragments were used as controls. Radioimmunoscintigraphy demonstrated the colon tumour discriminatory properties of each of the three iodine-labelled antibody preparations. The results compare favourably with previously reported investigations of the localization of human monoclonal antibodies and suggest that fragments of human monoclonal IgM antibodies may be useful tools for the immunodetection of cancer in patients. (orig.)

  20. Possibilities in improvement of the specific biodistribution by means of radiolabelled antibodies after complexation

    International Nuclear Information System (INIS)

    Heinrich, H.

    1990-01-01

    The applicability of labeled by different nuclides poly- or monoclonal antibodies in nuclear medicine diagnostics is restricted by incidental consequences. Overhigh background activities and/or high (additional) binding reactivities in the liver are troublesome. Hence we examined if the lokalizing biodistribution of labeled antibodies regarding the target organ can be improved by complexation with metal ions. The investigations were carried out with the pancreas of four Beagle dogs. Xenogenic polyclonal antibodies were refined by passing down the gammaglobulin fraction of rabbit serum a sephadex G 200 - column three times and hence radiolabeled by Na 131 I. Then the antibodies were injected i.p. either as complete molecules or as partially digested by pepsin fragments F(ab) 2 -fragments in order to avoid nonspecific Fc interactions. In preparing the i.p. injection another part of the antibody preparations both complete and fragments had been complexed with copper ions in an alkaline buffer giving a stable copper-protein-complex (Biuret-complex). Undergoing scanning and after killing the dogs the organ deposition of the radiolabel had been observed and noticed. The complexation with copper of radiolabeled antibodies improves strikingly the specific target organ uptake. Because of inflammatory and other incidental consequences the Fc-fragments should be removed by digestion with enzymes. Higher specific organ uptake should be obtainable if the poyclonal antibody fraction is more refined specifically by immunosorption with respect to its affinity to the specific antigenic structures. (orig.) [de

  1. Neutralization effects of egg yolk immunoglobulin (IgY) and Fab ...

    African Journals Online (AJOL)

    The aim of this study is to evaluate the neutralization effects of egg yolk immunoglobulin (IgY) and Fab' fragment against lipopolysaccharide (LPS), and identify possible approaches to prevent and treat LPS related injuries. Mice with third-degree burns covering 30% of the total body surface and exposed to LPS were orally ...

  2. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

    Science.gov (United States)

    Heo, Mi-Ae; Kim, Su-Hyun; Kim, So-Yeon; Kim, Yu-Jin; Chung, Junho; Oh, Min-Kyu; Lee, Sun-Gu

    2006-05-01

    c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction

  3. Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product.

    Science.gov (United States)

    Havlik, David; Brandt, Ulrike; Bohle, Kathrin; Fleißner, André

    2017-07-25

    Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg1nr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the

  4. Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

    Science.gov (United States)

    Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K

    2016-08-02

    Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells.

    Science.gov (United States)

    Hussain, Ahmad Fawzi; Kampmeier, Florian; von Felbert, Verena; Merk, Hans-F; Tur, Mehmet Kemal; Barth, Stefan

    2011-12-21

    Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.

  6. Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

    International Nuclear Information System (INIS)

    Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P.

    1989-01-01

    Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb

  7. Structural mimicry of the dengue virus envelope glycoprotein revealed by the crystallographic study of an idiotype-anti-idiotype Fab complex.

    Science.gov (United States)

    Wong, Yee Hwa; Goh, Boon Chong; Lim, She Yah; Teo, En Wei; Lim, Angeline P C; Dedon, Pete C; Hanson, Brendon J; MacAry, Paul A; Lescar, Julien

    2017-06-21

    A detailed understanding of the fine specificity of serotype-specific human antibodies is vital for the development and evaluation of new vaccines for pathogenic Flaviviruses such as Dengue virus (DENV) and Zika virus. In this study, we thoroughly characterize the structural footprint of an anti-idiotype antibody (E1) specific for a potent, fully human DENV serotype 1-specific antibody termed HM14c10, derived from a recovered patient. The crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 provides the first detailed molecular comparison of an anti-idiotype paratope specific for a human antibody with its analogous epitope- a discontinuous quaternary structure located at the surface of the viral particle that spans adjacent envelope (E) proteins. This comparison reveals that the footprints left by E1 and E on HM14c10 largely overlap, explaining why formation of the binary complexes are mutually exclusive. Structural mimicry of the DENV E epitope by the E1 combining site is achieved via the formation of numerous interactions with heavy chain CDRs of HM14c10, while fewer interactions are observed with its light chain, compared to the E protein. We show that E1 can be utilized to detect HM14c10-like antibodies in sera from patients recovered from a DENV-1 infection suggesting that this is a public (common) idiotype. These data demonstrate the utility of employing an anti-idiotype antibody to monitor a patient's specific immune responses and suggest routes for improvement of E 'mimicry' by E1 through increasing its recognition of the FabHM14c10 light chain CDRs. IMPORTANCE A chimeric yellow fever/dengue live-attenuated tetravalent vaccine is now marketed. Dengue remains a significant public health problem, because protection conferred by this vaccine is uneven against the four circulating serotypes. Reliable tools must be developed to measure the immune response of individuals exposed to DENV, either via viral infection

  8. 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

  9. 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.

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

    Science.gov (United States)

    Flego, Michela; Di Bonito, Paola; Ascione, Alessandro; Zamboni, Silvia; Carattoli, Alessandra; Grasso, Felicia; Cassone, Antonio; Cianfriglia, Maurizio

    2005-01-01

    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. PMID:16171519

  11. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis

    1992-01-01

    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  12. The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.

    Science.gov (United States)

    Rothe, Christine; Urlinger, Stefanie; Löhning, Corinna; Prassler, Josef; Stark, Yvonne; Jäger, Ute; Hubner, Bernd; Bardroff, Michael; Pradel, Ingrid; Boss, Melanie; Bittlingmaier, Renate; Bataa, Tschimegma; Frisch, Christian; Brocks, Bodo; Honegger, Annemarie; Urban, Margit

    2008-02-29

    This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a beta-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.

  13. Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation.

    Science.gov (United States)

    Käsermann, Fabian; Boerema, David J; Rüegsegger, Monika; Hofmann, Andreas; Wymann, Sandra; Zuercher, Adrian W; Miescher, Sylvia

    2012-01-01

    It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

  14. Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG after lectin fractionation.

    Directory of Open Access Journals (Sweden)

    Fabian Käsermann

    Full Text Available It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1 or acidified lactose (E2 were analyzed for total IgG, F(ab'(2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2; again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10% of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.

  15. 99mTc-Labeling of Monoclonal Antibody to Carcinoembryonic Antigen and Biodistribution

    International Nuclear Information System (INIS)

    Moon, Dae Hyuk; Chung, June Key; Lee, Myu ng Chul; Koh, Chang Soon; Chung, Hong Keun; Park, Jae Gahb

    1992-01-01

    This study was designed to evaluate a direct method of 99m Tc labeling using β-mercaptoethanol as a reducing agent, and to investigate whether 99m Tc labeled specific monoclonal antibody against carcinoembryonic antigen (CEA-92) can be used for the scintigraphic localization of human colon cancer xenograft. Purified CEA-92 IgG was fragmented into F(ab') 2 and then labeled with 99m Tc by transchelation method using glucarate as a chelator. Labeling efficiency, immunological reactivity and in vitro stability of 99m Tc CEA-92 F(ab') 2 were measured and then injected intravenously into nude mice bearing human colon cancer (SNU-C4). Scintigrams were obtained at 24 hour after injection. Then nude mice were sacrificed and the radioactivity was measured. Labeling efficiency of injected 99m Tc CEA-92 F(ab') 2 , immunoreactive fraction and in vitro stability at 24 hour of injected 99m Tc CEA-92 F(ab') 2 was 45.2%, 32.8% and 57.4%, respectively. At 24 hour after injection, %ID/g in kidney (46.77) showed high uptake, but %ID/g in tumor (1.65) was significantly higher than spleen (0.69), muscle (0.16), intestine (0.45), stomach (0.75), heart (0.48) and blood(0.45). There was no significant difference between tumor and liver (1.81). Tumor contrast as quantitated by tumor to blood ratio of 99m Tc CEA-92 F(ab') 2 was increased significantly (p 131 I-CEA-92 F(ab') 2 . The scintigram demonstrated localization of radioactivity over transplanted tumor, but significant background radioactivity was also noted over kidney and abdomen. It is concluded that CEA-92 F(ab') 2 can be labeled with 99m Tc by a direct transchelation method using β-mercaptoethanol as a reducing agent and 99m Tc labeled CEA-92 F(ab') 2 can be used for the scintigraphic localization of human colon cancer xenograft in nude mice model.

  16. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  17. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S.J.

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  18. FAB classification of leukemia: a cytochemical study.

    Science.gov (United States)

    Sharma, Jai Shree; Mohindroo, Shobha

    2004-07-01

    The reproducibility of FAB classification for acute leukemia was assessed using the modified criteria of the FAB classification. Peripheral smears and bone marrow smears from 36 cases of acute leukemia stained with May-Graunwald-Giemsa were used for this purpose. Cytochemical stains used for this purpose were myeloperoxidase (MPO), Sudan Black B (SBB), Periodic acid Schiff's (PAS) and Alpha-naphthyl acetate esterase (ANAE). The concordance with morphology alone was 75% which improved to 92% when cytochemistry was included.

  19. An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

    Directory of Open Access Journals (Sweden)

    Urszula Jarocka

    2014-08-01

    Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

  20. 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.

  1. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor

    DEFF Research Database (Denmark)

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure...... of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro...... pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily...

  2. Immunogenicity of a Prefusion HIV-1 Envelope Trimer in Complex with a Quaternary-Structure-Specific Antibody.

    Science.gov (United States)

    Cheng, Cheng; Pancera, Marie; Bossert, Adam; Schmidt, Stephen D; Chen, Rita E; Chen, Xuejun; Druz, Aliaksandr; Narpala, Sandeep; Doria-Rose, Nicole A; McDermott, Adrian B; Kwong, Peter D; Mascola, John R

    2015-12-30

    The HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies and is being explored as a vaccine candidate to elicit protective antibodies. One of the most promising antigenic and structural mimics of HIV-1 Env is the SOSIP.664-stabilized soluble trimer from the clade A strain BG505, which is preferentially recognized by broadly neutralizing antibodies. Trimer immunization elicits high-titer neutralization of the autologous tier 2 BG505 strain; however, breadth is limited, and substantial interest has focused on understanding and improving trimer immunogenicity. We sought to improve the antigenic specificity of BG505 SOSIP.664 by reducing recognition of the variable loop 3 (V3) region, which elicits only weakly neutralizing antibodies. To stabilize the trimer in its prefusion closed conformation, we complexed trimeric BG505 SOSIP.664 with the antigen-binding fragment (Fab) of PGT145, a broadly neutralizing quaternary-structure-specific antibody. Compared to the ligand-free trimer, the PGT145 Fab-BG505 SOSIP.664 complex displayed increased melting temperature stability and reduced V3 recognition. In guinea pigs, immunization with the PGT145 Fab-BG505 SOSIP.664 complex elicited ∼100-fold lower V3-directed binding and neutralization titers than those obtained with ligand-free BG505 SOSIP.664. Both complexed and ligand-free BG505 SOSIP.664 elicited comparable neutralization of the autologous BG505 virus, and in both cases, BG505 neutralization mapped to the outer domain of gp120 for some guinea pigs. Our results indicate that it is possible to reduce immune recognition of the V3 region of the trimer while maintaining the antigenic profile needed to induce autologous neutralizing antibodies. These data suggest that appropriate modifications of trimer immunogens could further focus the immune response on key neutralization epitopes. HIV-1 Env trimers have been proposed as preferred HIV-1 vaccine immunogens. One version, BG505 SOSIP.664, a soluble

  3. Human anti-animal antibody interferences in immunological assays.

    Science.gov (United States)

    Kricka, L J

    1999-07-01

    The scope and significance of human anti-animal antibody interference in immunological assays is reviewed with an emphasis on human anti-animal immunoglobulins, particularly human anti-mouse antibodies (HAMAs). Anti-animal antibodies (IgG, IgA, IgM, IgE class, anti-isotype, and anti-idiotype specificity) arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years. Prevalence estimates for anti-animal antibodies in the general population vary widely and range from HAMA, which causes both positive and negative interferences in two-site mouse monoclonal antibody-based assays. Strategies to prevent the development of human anti-animal antibody responses include immunosuppressant therapy and the use of humanized, polyethylene glycolylated, or Fab fragments of antibody agents. Sample pretreatment or assay redesign can eliminate immunoassay interferences caused by anti-animal antibodies. Enzyme immunoassays, immunoradiometric assays, immunofluorescence, and HPLC assays have been designed to detect HAMA and other anti-animal antibodies, but intermethod comparability is complicated by differences in assay specificity and lack of standardization. Human anti-animal antibodies often go unnoticed, to the detriment of patient care. A heightened awareness on the part of laboratory staff and clinicians of the problems caused by this type of interference in routine immunoassay tests is desirable. Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference.

  4. EXPRESSION OF RECOMBINANT ANTIBODY FRAGMENT, ANTI BNP-SCFV ON THE PERIPLASM OF Escherichia coli FOR THE DETECTION OF HEART FAILURE

    Directory of Open Access Journals (Sweden)

    Shabarni Gaffar

    2017-05-01

    Full Text Available Basic natriuretic peptide (BNP is a polypeptide hormone consist of 32 amino acids that secreted by the heart ventricle to respond the excessive stretching of heart muscle cells. BNP can be used as prognostic marker for patients with heart failure. The presence of BNP in blood can be detected by BNP antibody, which is anti BNP-single chain variable fragment (Anti BNP-SCFV. The antibody is a combination of polypeptides between varying region on the heavy chain (VH and the light chain (VL of immunoglobulin. Anti BNP-SCFV will bind to BNP through the antigen-antibody interaction. Concentration of BNP in a patient’s blood can be detected through the interaction of BNP with Anti BNP-SCFV using immunosensor method. Production of recombinant Anti BNP-SCFV in Escherichia coli as host is reported in the present study. Anti BNP-SCFV was expressed in fusion form with OmpC signal peptide that direct the protein to a periplasmic space. Expression was performed under RhaBad promoter as control using L-rhamnose as inducer. SDS-PAGE characterization showed consistent band at 28 kDa, which was assumed as Anti BNP-SCFV. The optimum expression was found at four hours after induction with 4 mM inducer. Anti BNP-SCFV was secreted from the cell as characterized by the presence of the protein on periplasmic membrane and extracellular fraction.

  5. Application of xenogeneic anti-canine distemper virus antibodies in treatment of canine distemper puppies.

    Science.gov (United States)

    Liu, P C; Chen, C A; Chen, C M; Yen, C H; Lee, M H; Chuang, C K; Tu, C F; Su, B L

    2016-11-01

    The clinical feasibility of passive immunotherapy has not been demonstrated in dogs naturally infected with canine distemper. In this study, porcine anti-canine distemper virus IgG and F(ab') 2 antibody fragments were used to treat infected puppies. A total of 41 naturally infected puppies (age Äsix months) exhibiting severe respiratory signs, but lacking neurological signs, were enrolled in the study. Twenty-five puppies were treated with a combination of IgG or F(ab') 2 antibody fragments (Group 1) and supportive therapy and 16 puppies received routine supportive care only (Group 2). The survival rate of dogs in Group 1 (19/25; 76%) was significantly higher than that in Group 2 (5/16; 31·3%) (Pcanine distemper virus antibodies improved survival in puppies affected with canine distemper with minimal adverse effects. Therefore, this therapy could be considered for treatment of endangered animal species infected with canine distemper virus. © 2016 British Small Animal Veterinary Association.

  6. Distribution and pharmacokinetics of radiolabeled monoclonal antibody OC 125 after intravenous and intraperitoneal administration in gynecologic tumors

    International Nuclear Information System (INIS)

    Haisma, H.J.; Moseley, K.R.; Battaile, A.; Griffiths, T.C.; Knapp, R.C.

    1988-01-01

    Radiolabeled monoclonal antibodies may be useful for radioimmunotherapy of gynecologic tumors. Iodine 131-labeled F(ab')2 fragments of a monoclonal antibody, OC 125, with specificity for ovarian carcinoma, were used to study the distribution and pharmacokinetics of this antibody in patients with gynecologic tumors. The radiolabeled antibody was injected intravenously or intraperitoneally into 10 patients suspected of having ovarian cancer. Blood and urine samples were used for pharmacokinetic studies, and biopsy specimens were examined for the uptake of antibody. The serum half-life of the labeled antibody was 30 hours after intravenous administration, with 20% of the injected dose per liter detected at 24 hours. After intraperitoneal injection, the appearance of antibody in serum was slow, with a maximum level of 1.4% of the injected dose per liter at 24 hours. Urinary excretion of the radiolabeled antibody was similar for intravenous and intraperitoneal administration, with approximately 50% of the injected dose excreted after 48 hours. Intraperitoneal administration of the radiolabeled antibody resulted in a higher uptake of antibody in the tumor and a lower uptake of antibody in normal tissues. On the basis of this limited study, intraperitoneal administration of radiolabeled antibody is preferred over intravenous administration for radioimmunotherapy of ovarian cancer

  7. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Crystal Structures of the Pro-Inflammatory Cytokine Interleukin-23 and Its Complex with a High-Affinity Neutralizing Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Beyer, Brian M.; Ingram, Richard; Ramanathan, Lata; Reichert, Paul; Le, Hung V.; Madison, Vincent; Orth, Peter (SPRI)

    2009-06-25

    Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 {angstrom}, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.

  9. Murine CR1/2 targeted antigenized single-chain antibody fragments induce transient low affinity antibodies and negatively influence an ongoing immune response.

    Science.gov (United States)

    Prechl, József; Molnár, Eszter; Szekeres, Zsuzsanna; Isaák, Andrea; Papp, Krisztián; Balogh, Péter; Erdei, Anna

    2007-01-01

    We have generated a single-chain antibody which recognizes murine CR1/2 and carries a genetically fused influenza hemagglutinin derived peptide. Theoretically such a construct is able to crosslink the B cell antigen receptor and CR1/2 on peptide specific B cells. The construct was able to reach its CR1/2 positive target cells, yet intraperitoneal delivery of the construct elicited an IgM response only slightly exceeding that induced by the free peptide. Providing T cell help by the injection of peptide specific lymphocytes did not alter the response in essence, that is anti-peptide IgG was not detectable even after booster immunizations. When used as a booster vaccine following injection of the peptide in adjuvant, the construct even inhibited the development of IgG1 and IgG3 anti-peptide antibodies. These data indicate that although targeting of antigen to CR1/2 on B cells can enhance transient proliferation or differentiation of antigen specific B cells it cannot induce strong, longlasting humoral immune responses. Furthermore, CR1/2 targeting constructs may negatively influence an ongoing immune reaction.

  10. 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.

  11. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M

    1993-01-01

    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells......, and did not use either L(ey) epitopes on target cells for cross-linkage of virus to the cell or the Fc part of the antibody as a ligand for any cellular receptor. For enhancement to occur, binding of anti-Le(y) antibody to virus was required to take place before virus binding to its specific receptor...... also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection...

  12. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.

    Science.gov (United States)

    Kaiser, Philipp D; Maier, Julia; Traenkle, Bjoern; Emele, Felix; Rothbauer, Ulrich

    2014-11-01

    In biomedical research there is an ongoing demand for new technologies, which help to elucidate disease mechanisms and provide the basis to develop novel therapeutics. In this context a comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, posttranslational modifications and dynamic interactions of cellular components is indispensable. Besides their significant impact as therapeutic molecules, antibodies are arguably the most powerful research tools to study endogenous proteins and other cellular components. However, for cellular diagnostics their use is restricted to endpoint assays using fixed and permeabilized cells. Alternatively, live cell imaging using fluorescent protein-tagged reporters is widely used to study protein localization and dynamics in living cells. However, only artificially introduced chimeric proteins are visualized, whereas the endogenous proteins, their posttranslational modifications as well as non-protein components of the cell remain invisible and cannot be analyzed. To overcome these limitations, traceable intracellular binding molecules provide new opportunities to perform cellular diagnostics in real time. In this review we summarize recent progress in the generation of intracellular and cell penetrating antibodies and their application to target and trace cellular components in living cells. We highlight recent advances in the structural formulation of recombinant antibody formats, reliable screening protocols and sophisticated cellular targeting technologies and propose that such intrabodies will become versatile research tools for real time cell-based diagnostics including target validation and live cell imaging. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  14. Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

    Directory of Open Access Journals (Sweden)

    Els Beirnaert

    2017-07-01

    Full Text Available The activity of tumor necrosis factor (TNF, a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™ were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3 were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

  15. Immunoscintigraphy of Colorectal and Other Gastrointestinal Cancers with Radioactive Monoclonal Antibodies to CEA and CA 19 - 9

    International Nuclear Information System (INIS)

    Jang, Dae Hwan; Choi, Duck Joo; Lee, Bum Woo; Park, Won; Han, Chang Soon; Kim, Hak San; Kim, Chong Soon

    1988-01-01

    The cocktails of two 131 I labeled Monoclonal antibody (MCAB) (Anti CA 19-9 F(ab') 2 +Anti CEA F(ab') 2 fragment), which react specially, with human gastrointestinal cancers, were administered to 10 patients with colorectal (7), stomach (2) and pancreas (l) cancer for scintigraphic detection. All patients were known or postoperatively recurrent cases, and serum tumor markers, CA 19-9 and CEA, were measured with immunoradiometric assay, just before immunoscintigraphy (ISG). The tumor marker's level in serum is not correlated with positive tumor uptake in ISG. The sensitivity and specificity of ISG in detection of 21 tumor sites, based on surgery, CT, ultrasonography and pathology, were 90.5% and 100%. One case of colon cancer showed gall bladder metastasis, which was neglected on CT study. Tumor/non tumor uptake ratio of radiolabelled antibody were progressively increased from day 3 to day 7 during study. We summarized as follows: 1) The use of cocktails of CEA and CA 19-9 MCAB F(ab') 2 increased sensitivity and specificity in ISG. 2) Delayed imaging (later than 5 days) increases sensitivity and specificity due to exclusion of nonspecific iodine accumulation in stomach and lung. 3) Second tracer technique is essential for anatomical landmark by use of a double isotope scan, but subtraction technique, a possible source of artifacts, is no longer necessary when delayed imaging is performed. 4) It may be possible to use two MCAB cocktails of CA 19-9 and CEA in Radioimmunodetection of stomach and pancreas cancer. In conclusion, ISG using MCAB cocktails, F(ab') 2 fragment of anti CA 19-9 and Anti CEA, provide additional opportunity for tumor localization and detection of colorectal and other G-I cancer, such as stomach and pancreas.

  16. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody.

    Science.gov (United States)

    Lord, Dana M; Bird, Julie J; Honey, Denise M; Best, Annie; Park, Anna; Wei, Ronnie R; Qiu, Huawei

    2018-01-15

    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.

  17. Anticomplementary Activity of Horse IgG and F(ab')2 Antivenoms

    OpenAIRE

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.

    2014-01-01

    2094-01 Embargo por política editorial Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several b...

  18. Radioimmunoimaging of tumors with a pantumor antibody

    International Nuclear Information System (INIS)

    Chen, D.C.P.; Siegel, M.E.; Chen, F.; Taylor, O.R.; Epstein, A.L.

    1988-01-01

    The TNT-1 antibody was developed to bind intracellular nuclear antigens that are accessible only in degenerative or necrotic cells. Since about 50% of tumor cells are in various stages of cell degeneration or death, this antibody could serve as a pantumor antibody for tumor detection. After intravenous injection of 10 μg of TNT-1F(ab')2 fragments labeled with 20 μCi of I-131, serial images were obtained at 1 and 4 hours and daily for 6 days in mice bearing various human tumors. Accumulation of TNT-1 was imaged in a necrotic tumor as early as 4 hours after injection and because more intense at 48 hours. The tumor-muscle ratio was as high as 29:1. Intense accumulation was noted in the necrotic tumor, about nine times that of healthy tumor. In conclusion, TNT-1, a pantumor antibody, can detect necrotic tumors in animal models. It may be an ideal imaging agent for cancer detection

  19. The Human Antibody Fragment DIATHIS1 Specific for CEACAM1 Enhances Natural Killer Cell Cytotoxicity Against Melanoma Cell Lines In Vitro

    Science.gov (United States)

    Dupuis, Maria L.; Soriani, Alessandra; Ricci, Biancamaria; Dominici, Sabrina; Moricoli, Diego; Ascione, Alessandro; Santoni, Angela; Magnani, Mauro; Cianfriglia, Maurizio

    2015-01-01

    Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1+ malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1+ melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1+ melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro–expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell–mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin. PMID

  20. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

    Science.gov (United States)

    Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M. Cruz; Álvarez, Miguel A.; Hammarström, Lennart

    2015-01-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. PMID:26092449

  1. An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

    Science.gov (United States)

    Álvarez, Beatriz; Krogh-Andersen, Kasper; Tellgren-Roth, Christian; Martínez, Noelia; Günaydın, Gökçe; Lin, Yin; Martín, M Cruz; Álvarez, Miguel A; Hammarström, Lennart; Marcotte, Harold

    2015-09-01

    Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Introduction of five potentially metabolizable linking groups between 111In-cyclohexyl EDTA derivatives and F(ab')2 fragments of anti-carcinoembryonic antigen antibody--II. Comparative pharmacokinetics and biodistribution in human colorectal carcinoma-bearing nude mice.

    Science.gov (United States)

    Faivre-Chauvet, A; Gestin, J F; Mease, R C; Sai-Maurel, C; Thédrez, P; Slinkin, M; Meinken, G E; Srivastava, S C; Chatal, J F

    1993-08-01

    The five linker-containing immunoconjugates described in the preceding paper were labeled with 111In and tested for their biodistribution, pharmacokinetics and immunoscintigraphic imaging properties in tumor-xenografted nude mice. The results were compared with DTPADA and CDTAMA for reference. Results showed that, for immunoscintigraphy, the derivatives in decreasing order of effectiveness were: aliphatic (tumor/liver > 4.5 and tumor/kidney > 6.5 at 96 h), thioether (tumor/liver > 3 and tumor/kidney > 1.2 at 24 h), ethylene glycol succinate (tumor/liver > 1.7 and tumor/kidney > 0.5 at 24 h) and disulfide (tumor/liver > 0.5 and tumor/kidney > 0.6 at 96 h). Pharmacokinetic results were complementary with those of the biodistribution studies and provide a basis for the study of in vivo metabolic mechanisms of linker-immunoconjugates. Indium-111-labeled linker-immunoconjugates appear promising for tumor imaging with better contrast than what is obtained with the use of the conventional 111In-DTPA dianhydride chelate.

  3. Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

    Science.gov (United States)

    Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

    2018-04-01

    Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

  4. Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia.

    Science.gov (United States)

    Pechar, Michal; Pola, Robert; Janoušková, Olga; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Tomalová, Barbora; Kovář, Marek

    2018-01-01

    A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Construction of Rabbit Immune Antibody Libraries.

    Science.gov (United States)

    Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

    2018-01-01

    Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

  6. New Strategies Using Antibody Combinations to Increase Cancer Treatment Effectiveness

    Directory of Open Access Journals (Sweden)

    Isabel Corraliza-Gorjón

    2017-12-01

    Full Text Available Antibodies have proven their high value in antitumor therapy over the last two decades. They are currently being used as the first-choice to treat some of the most frequent metastatic cancers, like HER2+ breast cancers or colorectal cancers, currently treated with trastuzumab (Herceptin and bevacizumab (Avastin, respectively. The impressive therapeutic success of antibodies inhibiting immune checkpoints has extended the use of therapeutic antibodies to previously unanticipated tumor types. These anti-immune checkpoint antibodies allowed the cure of patients devoid of other therapeutic options, through the recovery of the patient’s own immune response against the tumor. In this review, we describe how the antibody-based therapies will evolve, including the use of antibodies in combinations, their main characteristics, advantages, and how they could contribute to significantly increase the chances of success in cancer therapy. Indeed, novel combinations will consist of mixtures of antibodies against either different epitopes of the same molecule or different targets on the same tumor cell; bispecific or multispecific antibodies able of simultaneously binding tumor cells, immune cells or extracellular molecules; immunomodulatory antibodies; antibody-based molecules, including fusion proteins between a ligand or a receptor domain and the IgG Fab or Fc fragments; autologous or heterologous cells; and different formats of vaccines. Through complementary mechanisms of action, these combinations could contribute to elude the current limitations of a single antibody which recognizes only one particular epitope. These combinations may allow the simultaneous attack of the cancer cells by using the help of the own immune cells and exerting wider therapeutic effects, based on a more specific, fast, and robust response, trying to mimic the action of the immune system.

  7. Rapportage LTO FAB II 2008 : Functionele Agro Biodiversiteit

    NARCIS (Netherlands)

    Scheele, J.; Gurp, van H.; Alebeek, van F.A.N.; Belder, den E.; Elderson, J.

    2009-01-01

    Doel van het LTO FAB II project is een gebruiksklaar FAB concept te ontwikkelen voor een aantal ziekten en plagen in een aantal gewassen die op eenvoudige wijze door telers benut kan worden en voor de toepasser kostenneutraal zijn. Uitgangspunt in het FAB project is een evenwichtige balans tussen de

  8. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    International Nuclear Information System (INIS)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-01-01

    Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  9. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); Li, Lin [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Shibo, E-mail: sjiang@nybloodcenter.org [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China)

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  10. Occurrence of anti-Leptospira spp. antibodies in Rhipidomys spp. from a forest fragment of the Brazilian Cerrado.

    Science.gov (United States)

    Gomes, D O; Ramos, G B; Alves, V B A; Ciuffa, A Z; Cuccato, L P; Dos Reis, T F M; Lima, A M C; Gonçalves, M C; Tolesano, G V; Rodrigues, V S; Szabó, M P J

    2017-03-01

    Leptospirosis is a zoonotic disease of world importance, and its transmission depends on the interaction between humans and animals. Given the necessity to investigate potential hosts of Leptospira spp., this study verified the prevalence of different serovars in the species of Rhipidomys spp., a widespread sigmodont rodent in Brazil. The studied population originates from a semi-evergreen forest located in the county of Uberlândia, in the state of Minas Gerais. The microscopic agglutination test (MAT) was performed with 14 serovars. Thirteen out of the 43 wild rodents captured showed a positive agglutination reaction, with a greater prevalence of the serovars Pyrogenes, Copenhageni, and Canicola. This study found a prevalence of 30.3% anti-Leptospira spp. antibodies; all positive animals were reactive to more than one serovar.

  11. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    Directory of Open Access Journals (Sweden)

    Soraya S Pereira

    Full Text Available In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅ of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB, surface plasmon resonance (SPR device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with

  12. Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages.

    Science.gov (United States)

    Romero-Martínez, Rafael; Curiel-Quesada, Everardo; Becerril-Luján, Baltazar; Flores-Carreón, Arturo; Pérez-Torres, Armando; Taylor, Maria Lucia

    2007-06-01

    A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.

  13. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

    2016-01-01

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  14. A High Through-put Platform for Recombinant Antibodies to Folded Proteins*

    Science.gov (United States)

    Hornsby, Michael; Paduch, Marcin; Miersch, Shane; Sääf, Annika; Matsuguchi, Tet; Lee, Brian; Wypisniak, Karolina; Doak, Allison; King, Daniel; Usatyuk, Svitlana; Perry, Kimberly; Lu, Vince; Thomas, William; Luke, Judy; Goodman, Jay; Hoey, Robert J.; Lai, Darson; Griffin, Carly; Li, Zhijian; Vizeacoumar, Franco J.; Dong, Debbie; Campbell, Elliot; Anderson, Stephen; Zhong, Nan; Gräslund, Susanne; Koide, Shohei; Moffat, Jason; Sidhu, Sachdev; Kossiakoff, Anthony; Wells, James

    2015-01-01

    Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. PMID:26290498

  15. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

    Science.gov (United States)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

  16. Investigating the interaction between the neonatal Fc receptor and monoclonal antibody variants by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein

    International Nuclear Information System (INIS)

    Su, Hua-Poo; Golden, Joseph W.; Gittis, Apostolos G.; Hooper, Jay W.; Garboczi, David N.

    2007-01-01

    Medical countermeasures to prevent or treat smallpox are needed due to the potential use of poxviruses as biological weapons. Safety concerns with the currently available smallpox vaccine indicate a need for research on alternative poxvirus vaccine strategies. Molecular vaccines involving the use of proteins and/or genes and recombinant antibodies are among the strategies under current investigation. The poxvirus L1 protein, encoded by the L1R open reading frame, is the target of neutralizing antibodies and has been successfully used as a component of both protein subunit and DNA vaccines. L1-specific monoclonal antibodies (e.g., mouse monoclonal antibody mAb-7D11, mAb-10F5) with potent neutralizing activity bind L1 in a conformation-specific manner. This suggests that proper folding of the L1 protein used in molecular vaccines will affect the production of neutralizing antibodies and protection. Here, we co-crystallized the Fab fragment of mAb-7D11 with the L1 protein. The crystal structure of the complex between Fab-7D11 and L1 reveals the basis for the conformation-specific binding as recognition of a discontinuous epitope containing two loops that are held together by a disulfide bond. The structure of this important conformational epitope of L1 will contribute to the development of molecular poxvirus vaccines and also provides a novel target for anti-poxvirus drugs. In addition, the sequence and structure of Fab-7D11 will contribute to the development of L1-targeted immunotherapeutics

  18. High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli.

    Science.gov (United States)

    Napathorn, Suchada Chanprateep; Kuroki, Motomu; Kuroki, Masahide

    2014-08-01

    The aim of this study was to establish a strategy for high-level production of single-chain variable fragment (scFv) antibodies fused with interleukin-2 (IL-2) in Escherichia coli. We constructed two fusion sequences consisting of a scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (MK-1) and human Interleukin-2(IL-2) gene, ligated the fusions into pET15b and transformed into three different E. coli strains. The effects of temperature, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and duration of IPTG induction were investigated. Employing E. coli strain Rosetta-gami B, which has an oxidizing cytoplasm that facilitates cytoplasmic disulfide bond formation, improved the level of soluble protein expression. Under optimal conditions, the highest levels of fusion protein expression and high percentages of the proteins were found in their soluble form. Specifically, 89.29% (0.28 g/l) of one fusion protein was soluble after a 10-h induction and 84.61% (0.26 g/l) of the other fusion protein was soluble after a separate 10-h induction. When analyzed by enzyme-linked immunosorbent assay, the partially-purified fusion proteins retained a specific binding activity to the cell lysate of Chinese hamster ovary (CHO) cells expressing MK-1. Taken together, the methods described herein permit the production of substantial amounts of the fusion proteins for conducting functional studies on the biological role of these fusion proteins. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    Science.gov (United States)

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  20. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ...... in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

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

    Science.gov (United States)

    Kuroki, Motomu; Kuroki, Masahide

    2017-01-01

    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. PMID:28860806

  2. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  3. Machine learning for fab automated diagnostics

    Science.gov (United States)

    Giollo, Manuel; Lam, Auguste; Gkorou, Dimitra; Liu, Xing Lan; van Haren, Richard

    2017-06-01

    Process optimization depends largely on field engineer's knowledge and expertise. However, this practice turns out to be less sustainable due to the fab complexity which is continuously increasing in order to support the extreme miniaturization of Integrated Circuits. On the one hand, process optimization and root cause analysis of tools is necessary for a smooth fab operation. On the other hand, the growth in number of wafer processing steps is adding a considerable new source of noise which may have a significant impact at the nanometer scale. This paper explores the ability of historical process data and Machine Learning to support field engineers in production analysis and monitoring. We implement an automated workflow in order to analyze a large volume of information, and build a predictive model of overlay variation. The proposed workflow addresses significant problems that are typical in fab production, like missing measurements, small number of samples, confounding effects due to heterogeneity of data, and subpopulation effects. We evaluate the proposed workflow on a real usecase and we show that it is able to predict overlay excursions observed in Integrated Circuits manufacturing. The chosen design focuses on linear and interpretable models of the wafer history, which highlight the process steps that are causing defective products. This is a fundamental feature for diagnostics, as it supports process engineers in the continuous improvement of the production line.

  4. Utilisation of tracer monoclonal antibodies for the immunoscintigraphic detection of human colorectal cancers

    International Nuclear Information System (INIS)

    Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B.; Fumoleau, P.; Bourdoiseau, M.

    1983-01-01

    Two monoclonal antibodies, 17-1A and 19-9, with recognized human gastrointestinal cancers in cell cultures, were labeled with iodine 131 for immunoscintigraphic application. With the intact 131 I-17-1A antibody, 21 out of 35 (60%) primary or secondary colorectal cancer sites were visualized, whereas all 21 nonepitheliomatous colic cancer sites or noncolic cancer sites were negative. With F(ab') 2 fragments of the 19-9 antibody, 18 out of 27 (67%) colorectal cancer sites were positive. With both radioantibodies, the bestly contrasted tumor images were late, 4 to 5 days after injection. A study with paired-label technique, associating a specific iodine-131-labeled antibody with a non-specific iodine-125-labeled immunoglobulin, demonstrated, that tumor uptake was indeed specific for the 17-1A or 19-9 antibody in tumor and normal colon fragments obtained during operations on 4 patients. A preliminary prospective study showed that only immunoscintigraphy was able to confirm and localize a recurrence of rectal cancer in one patient. A larger series will be necessary to validate the clinical benefit of the technique, as compared with the results of other diagnostic techniques, before immunoscintigraphy can be proposed for routine clinical use [fr

  5. NMR Detection of Semi-Specific Antibody Interactions in Serum Environments

    Directory of Open Access Journals (Sweden)

    Saeko Yanaka

    2017-09-01

    Full Text Available Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system.

  6. Cytoreductive Chemotherapy Improves the Biodistribution of Antibodies Directed Against Tumor Necrosis in Murine Solid Tumor Models

    Science.gov (United States)

    Jang, Julie K.; Khawli, Leslie A.; Park, Ryan; Wu, Brian W.; Li, Zibo; Canter, David; Conti, Peter S.; Epstein, Alan L.

    2013-01-01

    Current strategies in cancer treatment employ combinations of different treatment modalities, which include chemotherapy, radiotherapy, immunotherapy, and surgery. Consistent with that approach, the present study demonstrates how chemotherapeutic agents can potentiate the delivery of radiolabeled, necrosis-targeting antibodies (chTNT-3, NHS76) to tumor. All chemotherapeutics in this study (5-fluorouracil, etoposide, vinblastine, paclitaxel, and doxorubicin) resulted in statistically significant increases in tumor uptake of radiolabeled antibodies and their F(ab')2 fragments compared to no pretreatment with chemotherapy. Labeled antibodies were administered at various time points following a single dose of chemotherapy in multiple tumor models, and the biodistribution of the antibodies were determined by measuring radioactivity in harvested tissues. MicroPET/CT was also done to demonstrate clinical relevancy of using chemotherapy pretreatment to increase antibody uptake. Results of biodistribution and imaging data reveal specific time frames following chemotherapy when necrosis-targeting antibodies are best delivered, either for imaging or radiotherapy. Thus, the present work offers the prospect of using cytoreductive chemotherapy to increase tumor accumulation of select therapeutic antibodies, especially when combined with other forms of immunotherapy, for the successful treatment of solid tumors. PMID:24130055

  7. Introduction of five potentially metabolizable linking groups between [sup 111]In-cyclohexyl EDTA derivatives and F(ab')[sub 2] fragments of anti-carcinoembryonic antigen antibody: Pt. 1; A new reproducible synthetic method

    Energy Technology Data Exchange (ETDEWEB)

    Gestin, J.F.; Faivre-Chauvet, A.; Sai-Maurel, C.; Thedrez, P.; Slinkin, M.; Chatal, J.F. (INSERM, 44 - Nantes (France)); Mease, R.C.; Meinken, G.E.; Srivastava, S.C. (Brookhaven National Lab., Upton, NY (United States))

    1993-08-01

    The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of [sup 111]In-labeled antibodies. A general simple synthetic method of preparing cyclohexyl EDTA (CDTA) derivatives containing a linker/spacer group is described. Linkers prepared included a diester, a six carbon aliphatic chain, two thioethers and a disulfide group. The CDTA-linker compounds were coupled to F(Ab')[sub 2] fragments of anti-carcinoembryonic antigen monoclonal antibody and labeled with [sup 111]In with good retention of immunoreactivity. (author).

  8. The binding of monoclonal antibodies to cell surface molecules. Quantitative analysis of the reactions and cross-reactions of an antibody (MB40.3) with four HLA-B molecules.

    Science.gov (United States)

    Parham, P

    1984-11-10

    The MB40.3 monoclonal antibody binds to four distinct HLA-B molecules; B7, B40, B40*, and B27. With Fab' fragments only the interaction with B7 and B40 was detected and the affinity for both was the same (1-2 X 10(8) M-1) suggesting the epitope is shared by the two molecules. Unlike many antibodies for which low affinity is due to a high-dissociation constant, that of MB40.3 results from a very low-association rate constant, coupled with a low-dissociation constant. In consequence, the affinity and avidity of Fab', F(ab')2, and IgG for B7 and B40 were found to be of a similar magnitude, soluble B7 was a more efficient competitor for antibody than cell surface B7 and in practice antibody bivalency was of little importance. The forward rate constant could be increased by removing Fc from the antibody or by removing sialic acid from the cells by treatment with neuraminidase. The neuraminidase treatment also produced an increase in the number of detectable cell surface HLA-A,B molecules. The affinity of MB40.3 for B40* and B27 was estimated to be less than 4 X 10(6) as no binding with Fab' was detected due to a high-dissociation rate. For these two HLA-B molecules bivalent attachment was critical, and it increased the strength of interaction with cell surface B40* and B27 to a point where the avidities were comparable to those obtained with B7 and B40, with B40* interacting more strongly than B27. The epitopes recognized by MB40.3 on B40* and B27 were thus shown to be structurally different from each other and from those on B7 and B40. The properties of this antibody contrast with those of other anti-HLA-A,B we have studied (Ways, J.P., and Parham, P. (1983) Biochem. J. 216, 423-432).

  9. Effects of an Aβ-antibody fragment on Aβ aggregation and astrocytic uptake are modulated by apolipoprotein E and J mimetic peptides.

    Directory of Open Access Journals (Sweden)

    Laia Montoliu-Gaya

    Full Text Available Aβ-Immunotherapy has long been studied in the treatment of Alzheimer's disease (AD, but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aβ-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE and J (apoJ concentrations to non-pathological values, and Aβ internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aβ aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs. Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aβ were studied by CD. The conformational change induced by the interaction of Aβ with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aβ complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aβ uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aβ uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides.

  10. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir; (Texas-MED); (Viral Rickettsial)

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  11. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy.

    Science.gov (United States)

    Hébert, J; Bernier, D; Mourad, W

    1990-06-01

    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  12. Expression and detection of ScFvB9 and its mutant in recombinant phage antibody system.

    Science.gov (United States)

    Yang, E J; Kwak, J W; Chang, H C

    2001-01-01

    Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL). In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E. coli. The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E. coli. The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS.

  13. Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen

    2017-08-01

    Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

  14. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

    Directory of Open Access Journals (Sweden)

    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

  15. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  16. 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

  17. Polyclonal antibodies directed against human placental Fcgamma receptor. Characterization of the antibodies and their interaction with the receptor.

    Science.gov (United States)

    Mikulska, J; Lisowski, J

    1987-01-01

    Antibodies to the putative Fc gamma receptor (Fc gamma R) of human placenta were raised by immunization of rabbits with the receptor purified form syncytiotrophoblast plasma membranes of human placenta. The rabbit antibodies were of IgG class and their F(ab')2 fragment interacted with Fc receptors in solubilized form and membrane-bound, as well. Immunological reactivity of the antibodies with Fc gamma R was demonstrated using immunodiffusion, solid-phase immunoassay, and ELISA. Studies on interaction of the antibodies with the isolated placental Fc gamma R showed that antigenic determinants of the receptor were different from the IgG-binding site. Rabbit anti-human placental Fc gamma R crossreacted, to various extent, with Fc gamma R-positive human cell lines showing antigenic relatedness of the placental receptor with Fc gamma R on other cell types. The antibodies showed only a weak crossreactivity with guinea pig peritoneal macrophage Fc gamma R. SDS-PAGE analysis of immunoprecipitates obtained by treatment of detergent lysates of 3H-labeled human placental trophoblasts membranes with the rabbit antibodies or with human IgG showed the presence of the some components which were observed in the case of the isolated, purified placental Fc gamma R: Mr of 123,000 and 52,000-56,000 under nonreducing conditions, and Mr of 64,000-67,000, 52,000-56,000, and 26,000-29,000, under reducing conditions. The polypeptide chains of the purified human placental receptor resolved in SDS-PAGE and transferred on nitrocellulose strips were able to interact both with the rabbit anti-placental receptor IgG F(ab')2 fragments and with human IgG. This gives an evidence that human placental Fc gamma R polypeptide chains Mr of approx. 64,000, 54,000, and 28,000 contain antigenic determinants of the receptor and binding sites for the Fc region of IgG, as well.

  18. Radioimmunotherapy of human colon cancer xenografts by using 131I labeled-CAb1 F(ab')2

    International Nuclear Information System (INIS)

    Li Ling; Xu Huiyun; Mi Li; Bian Huijie; Qin Jun; Xiong Hua; Feng Qiang; Wen Ning; Tian Rong; Xu Liqing; Shen Xiaomei; Tang Hao; Chen Zhinan

    2006-01-01

    Purpose: Therapeutic efficacy, suitable dose, and administration times of 131 I-CAb 1 F(ab') 2 , a new monoclonal antibody therapeutics specifically directed against a cell surface-associated glycoprotein of colon cancer, were investigated in this article. Methods and Materials: In human colon cancer xenografts, 131 I-CAb 1 F(ab') 2 at the dose of 125 μCi, 375 μCi, and 1125 μCi were administrated intraperitoneally on Days 6 and 18 after implantation of HR8348 cells with CAb 1 high reactivity. Survival time and tumor growth inhibition rate were used to evaluate the efficacy and safety of 131 I-CAb 1 F(ab') 2 in treatment of colon cancer xenografts. Results: Treatment of 125, 375, and 1125 μCi 131 I-CAb1 F(ab') 2 did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (p = 0.276, 0.865, 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 μCi and 1125 μCi 131 I-CAb1 F(ab') 2 were significantly longer than that of 5-FU-treated groups (p 0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding 131 I-labeled antibody dosage of 375 μCi and 1125 μCi. After single attack dosage, second reinforcement therapy may rise efficacy significantly. Tumor growth inhibition rates of 125 μCi, 375 μCi, and 1125 μCi 131 I-labeled antibody on Day 20 posttherapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathology examination revealed that tissue necrosis of various degrees was found in 131 I-CAb1 F(ab') 2 -treated groups. Conclusion: 131 I-CAb 1 F(ab') 2 is safe and effective for colon cancer. It may be a novel and potentially adjuvant therapeutics for colon cancer

  19. Bispecific Antibody Pretargeting for Improving Cancer Imaging and Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, Robert M.

    2005-02-04

    The main objective of this project was to evaluate pretargeting systems that use a bispecific antibody (bsMAb) to improve the detection and treatment of cancer. A bsMAb has specificity to a tumor antigen, which is used to bind the tumor, while the other specificity is to a peptide that can be radiolabeled. Pretargeting is the process by which the unlabeled bsMAb is given first, and after a sufficient time (1-2 days) is given for it to localize in the tumor and clear from the blood, a small molecular weight radiolabeled peptide is given. According to a dynamic imaging study using a 99mTc-labeled peptide, the radiolabeled peptide localizes in the tumor in less than 1 hour, with > 80% of it clearing from the blood and body within this same time. Tumor/nontumor targeting ratios that are nearly 50 times better than that with a directly radiolabeled Fab fragment have been observed (Sharkey et al., ''Signal amplification in molecular imaging by a multivalent bispecific nanobody'' submitted). The bsMAbs used in this project have been composed of 3 antibodies that will target antigens found in colorectal and pancreatic cancers (CEA, CSAp, and MUC1). For the ''peptide binding moiety'' of the bsMAb, we initially examined an antibody directed to DOTA, but subsequently focused on another antibody directed against a novel compound, HSG (histamine-succinyl-glycine).

  20. Cancer Imaging and Therapy with Bispecific Antibody Pretargeting.

    Science.gov (United States)

    Goldenberg, David M; Chatal, Jean-Francois; Barbet, Jacques; Boerman, Otto; Sharkey, Robert M

    2007-03-01

    This article reviews recent preclinical and clinical advances in the use of pretargeting methods for the radioimmunodetection and radioimmunotherapy of cancer. Whereas directly-labeled antibodies, fragments, and subfragments (minibodies and other constructs) have shown promise in both imaging and therapy applications over the past 25 years, their clinical adoption has not fulfilled the original expectations due to either poor image resolution and contrast in scanning or insufficient radiation doses delivered selectively to tumors for therapy. Pretargeting involves the separation of the localization of tumor with an anticancer antibody from the subsequent delivery of the imaging or therapeutic radionuclide. This has shown improvements in both imaging and therapy by overcoming the limitations of conventional, or 1-step, radioimmunodetection or radioimmunotherapy. We focus herein on the use of bispecific antibodies followed by radiolabeled peptide haptens as a new modality of selective delivery of radionuclides for the imaging and therapy of cancer. Our particular emphasis in pretargeting is the use of bispecific trimeric (3 Fab's) recombinant constructs made by a modular method of antibody and protein engineering of fusion molecules called Dock and Lock (DNL).

  1. Rapportage LTO FAB II 2009 : Functionele Agro Biodiversiteit (LTO FAB II)

    NARCIS (Netherlands)

    Alebeek, van F.A.N.; Korthals, G.W.; Molendijk, L.P.G.; Vlaswinkel, M.E.T.

    2010-01-01

    In 2008 startte LTO een gebruiksklaar FAB concept voor een aantal ziekten en plagen in een aantal gewassen die op eenvoudige wijze door telers benut kan worden en voor de toepasser kostenneutraal zijn. In deze rapportage zijn de resultaten en conclusies verwoord voor de deelprojecten omgeving,

  2. Evaluation of177Lu-CHX-A″-DTPA-6A10 Fab as a radioimmunotherapy agent targeting carbonic anhydrase XII.

    Science.gov (United States)

    Fiedler, L; Kellner, M; Gosewisch, A; Oos, R; Böning, G; Lindner, S; Albert, N; Bartenstein, P; Reulen, H-J; Zeidler, R; Gildehaus, F J

    2018-03-04

    Due to their infiltrative growth behavior, gliomas have, even after surgical resection, a high recurrence tendency. The approach of intracavitary radioimmunotherapy (RIT) is aimed at inhibiting tumor re-growth by directly administering drugs into the resection cavity (RC). Direct application of the radioconjugate into the RC has the advantage of bypassing the blood-brain barrier, which allows the administration of higher radiation doses than systemic application. Carbonic anhydrase XII (CA XII) is highly expressed on glioma cells while being absent from normal brain and thus an attractive target molecule for RIT. We evaluated a CA XII-specific 6A10 Fab (fragment antigen binding) labelled with 177 Lu as an agent for RIT. 6A10 Fab fragment was modified and radiolabelled with 177 Lu and characterized by MALDI-TOF, flow cytometry and radio-TLC. In vitro stability was determined under physiological conditions. Biodistribution studies, autoradiography tumor examinations and planar scintigraphy imaging were performed on SCID-mice bearing human glioma xenografts. The in vitro CA XII binding capacity of the modified Fab was confirmed. Radiochemical purity was determined to be >90% after 72 h of incubation under physiological conditions. Autoradiography experiments proved the specific binding of the Fab to CA XII on tumor cells. Biodistribution studies revealed a tumor uptake of 3.0%ID/g after 6 h and no detectable brain uptake. The tumor-to-contralateral ratio of 10/1 was confirmed by quantitative planar scintigraphy. The radiochemical stability in combination with a successful in vivo tumor uptake shows the potential suitability for future RIT applications with the 6A10 Fab. Copyright © 2018. Published by Elsevier Inc.

  3. Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

    Science.gov (United States)

    Muda, Marco; Gross, Alec W; Dawson, Jessica P; He, Chaomei; Kurosawa, Emmi; Schweickhardt, Rene; Dugas, Melanie; Soloviev, Maria; Bernhardt, Anna; Fischer, David; Wesolowski, John S; Kelton, Christie; Neuteboom, Berend; Hock, Bjoern

    2011-05-01

    The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

  4. Observations on the Biology of Callosobruchus maculatus (Fab ...

    African Journals Online (AJOL)

    Observations on the biology of Callosobruchus maculatus( Fab.) were carried out under ambient laboratory conditions. Pattern of oviposition, female adult longevity and F1 progeny emergence in Callosobruchus maculatus ( Fab. ) were observed. The results showed that over 60% of the total number of eggs was laid during ...

  5. 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.

  6. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  7. Experiments in microgravity: a comparison of crystals of a carbohydrate-binding fab grown on the ground, on space shuttle Discovery and on space station Mir.

    Science.gov (United States)

    Borisova, S N; Birnbaum, G I; Rose, D R; Evans, S V

    1996-03-01

    The Fab fragment of the hybridoma antibody (YsT9.1) specific to Brucella abortus has been crystallized on earth using both Linbro plates and ground-based models of the flight hardware, as well as in microgravity on board the space shuttle Discovery and the space station Mir. Large-scale experiments using Linbro plates gave two different crystal morphologies, pyramidal and rhomboid, depending on conditions. The pyramidal crystals proved to scatter X-rays to higher resolution, and conditions within the ground-based flight hardware for both Discovery and Mir were adjusted to produce crystals with this morphology. The experiment on Discovery produced large crystals in each of ten chambers. The experiment on Mir produced crystals in only one of the five assigned chambers, despite the fact that the simultaneous ground-based experiment produced large crystals in every corresponding chamber. Data collection was attempted for crystals from both space and ground-based experiments. Higher resolution data was obtained from crystals grown on Discovery than from either Mir or ground-based crystals, even though the crystals obtained from Discovery were smaller and forced to grow over a much shorter period of time because of the shorter length of the shuttle mission.

  8. RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

    Science.gov (United States)

    Jung, Euihan; Lee, Jungmin; Hong, Hyo Jeong; Park, Insoo; Lee, Younghoon

    2014-06-01

    Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation. © 2014 Jung et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known...... an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...

  10. Semiconductor industry wafer fab exhaust management

    CERN Document Server

    Sherer, Michael J

    2005-01-01

    Given the myriad exhaust compounds and the corresponding problems that they can pose in an exhaust management system, the proper choice of such systems is a complex task. Presenting the fundamentals, technical details, and general solutions to real-world problems, Semiconductor Industry: Wafer Fab Exhaust Management offers practical guidance on selecting an appropriate system for a given application. Using examples that provide a clear understanding of the concepts discussed, Sherer covers facility layout, support facilities operations, and semiconductor process equipment, followed by exhaust types and challenges. He reviews exhaust point-of-use devices and exhaust line requirements needed between process equipment and the centralized exhaust system. The book includes information on wet scrubbers for a centralized acid exhaust system and a centralized ammonia exhaust system and on centralized equipment to control volatile organic compounds. It concludes with a chapter devoted to emergency releases and a separ...

  11. How fabulous is Fab 5 cosmology?

    International Nuclear Information System (INIS)

    Linder, Eric V.

    2013-01-01

    Extended gravity origins for cosmic acceleration can solve some fine tuning issues and have useful characteristics, but generally have little to say regarding the cosmological constant problem. Fab 5 gravity can be ghost free and stable, have attractor solutions in the past and future, and possess self tuning that solves the original cosmological constant problem. Here we show however it does not possess all these qualities at the same time. We also demonstrate that the self tuning is so powerful that it not only cancels the cosmological constant but also all other energy density, and we derive the scalings of its approach to a renormalized de Sitter cosmology. While this strong cancellation is bad for the late universe, it greatly eases early universe inflation

  12. How fabulous is Fab 5 cosmology?

    Energy Technology Data Exchange (ETDEWEB)

    Linder, Eric V., E-mail: evlinder@lbl.gov [Berkeley Center for Cosmological Physics and Berkeley Lab, University of California, Berkeley, CA, 94720 (United States)

    2013-12-01

    Extended gravity origins for cosmic acceleration can solve some fine tuning issues and have useful characteristics, but generally have little to say regarding the cosmological constant problem. Fab 5 gravity can be ghost free and stable, have attractor solutions in the past and future, and possess self tuning that solves the original cosmological constant problem. Here we show however it does not possess all these qualities at the same time. We also demonstrate that the self tuning is so powerful that it not only cancels the cosmological constant but also all other energy density, and we derive the scalings of its approach to a renormalized de Sitter cosmology. While this strong cancellation is bad for the late universe, it greatly eases early universe inflation.

  13. Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice

    DEFF Research Database (Denmark)

    Back, T.; Haraldsson, B.; Hultborn, R

    2009-01-01

    of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals...... manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions...... in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary dose-limiting organ in systemic alpha-RIT when using (211)At-MX35-F(ab')(2) Udgivelsesdato: 2009/12...

  14. Wafer fab mask qualification techniques and limitations

    Science.gov (United States)

    Poock, Andre; Maelzer, Stephanie; Spence, Chris; Tabery, Cyrus; Lang, Michael; Schnasse, Guido; Peikert, Milko; Bhattacharyya, Kaustuve

    2006-10-01

    Mask inspection and qualification is a must for wafer fabs to ensure and guarantee high and stable yields. Single defect events can easily cause a million dollar loss through a defect duplicating onto the wafer. Several techniques and methods for mask qualification within a wafer fab are known but not all of them are neither used nor understood regarding their limitations. Increasing effort on existing tool platforms is necessary to detect the defects of interest which are at the limit of the tools specification - On the other hand next generation tools are very sensitive and therefore consume only a negligible amount of time for recipe optimization. Knowing the limits of each inspection tool helps to balance between effort and benefit. Masks with programmed defects of 90nm and 65nm design rule were used in order to compare the different available inspection techniques. During the course of this technical work, the authors concentrate mainly on two inspection techniques. The first one inspects the reticle itself using KLA-Tencor's SLF27 (TeraStar) and SL536 (TeraScan) tools. As the reticle gets inspected itself this is the so called "direct" mask defect inspection. The second inspection technique discussed is the "indirect" mask defect inspection which consists of printing the pattern on a blank wafer and use KLA-Tencor's bright-field wafer inspection tool (2xxx series) to inspect the wafer. Data of this work will include description of the techniques, inspection results, defect maps, sensitivity analysis, effort estimation as well as limitations for both techniques for the used design rule.

  15. Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Jørgensen, Mariann H; Rekvig, Ole Petter; Jacobsen, Rasmus S

    2011-01-01

    Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known....... The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect...... the development of lupus nephritis at an early stage....

  16. Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

    Science.gov (United States)

    Gadkar, Kapil; Pastuskovas, Cinthia V; Le Couter, Jennifer E; Elliott, J Michael; Zhang, Jianhuan; Lee, Chingwei V; Sanowar, Sarah; Fuh, Germaine; Kim, Hok Seon; Lombana, T Noelle; Spiess, Christoph; Nakamura, Makia; Hass, Phil; Shatz, Whitney; Meng, Y Gloria; Scheer, Justin M

    2015-08-01

    To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

  17. How a T Cell Receptor-like Antibody Recognizes Major Histocompatibility Complex-bound Peptide

    Energy Technology Data Exchange (ETDEWEB)

    Mareeva, T.; Martinez-Hackert, E; Sykulev, Y

    2008-01-01

    We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2Kb molecule. Remarkably, this antibody directly 'reads' the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.

  18. [Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25-35)].

    Science.gov (United States)

    Kolobov, V V; Davydova, T V; Fomina, V G

    2014-01-01

    Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp 1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ25-35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp 1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.

  19. Application of a single-chain fragment variable (scFv antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

    Directory of Open Access Journals (Sweden)

    Xiaobo Xu

    2017-09-01

    Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P < 0.05. The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

  20. Rasch Analysis of the Fullerton Advanced Balance (FAB) Scale

    OpenAIRE

    Klein, Penelope J.; Fiedler, Roger C.; Rose, Debra J.

    2011-01-01

    Purpose: This cross-sectional study explores the psychometric properties and dimensionality of the Fullerton Advanced Balance (FAB) Scale, a multi-item balance test for higher-functioning older adults.

  1. A Case Study of a High School Fab Lab

    Science.gov (United States)

    Lacy, Jennifer E.

    This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

  2. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    DEFF Research Database (Denmark)

    Owens, T

    1988-01-01

    on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

  3. Radiolabeled monoclonal antibodies in the diagnosis and treatment of malignant melanoma

    International Nuclear Information System (INIS)

    Divgi, C.R.; Larson, S.M.

    1989-01-01

    The use of antibodies directed against tumors has found increasing usefulness after the discovery by Kohler and Milstein of hybridoma technology, which made it possible to obtain monoclonal antibody (MoAb) that reacted specifically against a particular epitope on a particular antigen site. Relative tumor specificity and a lack of significant toxicity, together with the ability to link radionuclides (both halogens and metals) without significant deterioration of biologic behavioral characteristics such as immunoreactivity, have enabled widespread use of radiolabeled MoAbs in several malignancies, including and especially malignant melanoma. There is a significant body of data indicating that radiolabeled MoAbs directed against melanoma-associated antigens have an important role in the detection and therapy of metastatic malignant melanoma. Detection of visceral disease, while currently suboptimal, will in the future improve with optimization of SPECT imaging using 99mTc-labeled MoAb Fab fragments. This may result in an attenuated or absent antimouse response, especially after one injection, unless of course coinfused with either specific and/or nonspecific intact immunoglobulin (Ig). Radiolabeled fragments play an important role in radioimmunotherapy in metastatic melanoma. This role may be enhanced by the development of newer chelating agents that will decrease nonspecific hepatic uptake of radionuclide, enabling the use of beta-emitting radiometals such as 90Y. The recent report demonstrating diminished hepatic uptake of 99mTc-labeled anti-high molecular weight antigen (HMWA) Fab shows promise, since the same labeling technique can be used to deliver radiotherapeutic agents such as 186Re, which may be labeled to MoAb with methods similar to those used for 99mTc.82 references

  4. A High Through-put Platform for Recombinant Antibodies to Folded Proteins.

    Science.gov (United States)

    Hornsby, Michael; Paduch, Marcin; Miersch, Shane; Sääf, Annika; Matsuguchi, Tet; Lee, Brian; Wypisniak, Karolina; Doak, Allison; King, Daniel; Usatyuk, Svitlana; Perry, Kimberly; Lu, Vince; Thomas, William; Luke, Judy; Goodman, Jay; Hoey, Robert J; Lai, Darson; Griffin, Carly; Li, Zhijian; Vizeacoumar, Franco J; Dong, Debbie; Campbell, Elliot; Anderson, Stephen; Zhong, Nan; Gräslund, Susanne; Koide, Shohei; Moffat, Jason; Sidhu, Sachdev; Kossiakoff, Anthony; Wells, James

    2015-10-01

    Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Mapping the binding site of a cross-reactive Plasmodium falciparum PfEMP1 monoclonal antibody inhibitory of ICAM-1 binding

    DEFF Research Database (Denmark)

    Lennartz, Frank; Bengtsson, Anja; Olsen, Rebecca W

    2015-01-01

    of domain cassette 4 PfEMP1s. 24E9 Fab fragments bind DBLβ3_D4 with nanomolar affinity and inhibit ICAM-1 binding of domain cassette 4-expressing IE. The antigenic regions targeted by 24E9 Fab were identified by hydrogen/deuterium exchange mass spectrometry and revealed three discrete peptides...

  6. Pharmacokinetics and biodistribution of samarium-153-labelled OC125 antibody coupled to CITCDTPA in a xenograft model of ovarian cancer.

    Science.gov (United States)

    Kraeber-Bodéré, F; Mishra, A; Thédrez, P; Faivre-Chauvet, A; Bardiès, M; Imai, S; Le Boterff, J; Chatal, J F

    1996-05-01

    The use of samarium-153 in the context of radioimmunotherapy of cancers has been limited by the instability of antibody labelling, which produces high uptake concentrations in liver and bone. This study compares the pharmacokinetics and biodistribution of 153Sm-labelled OC125 monoclonal antibody, in whole or F(ab')2 fragment form and with diethylene triamine penta-acetic acid (DTPA) or 6-p-isothiocyanatobenzyl diethylene triamine penta-acetic acid (CITCDTPA) coupling, in nude mice grafted subcutaneously with an ovarian adenocarcinoma line (SHIN-3) expressing CA125 antigen. The specific activity of the immunoconjugates was 18.5-55.5 MBq/mg, and their immunoreactivity exceeded 65%. With 153Sm-DTPA-OC125F(ab')2, the stability study in serum indicated that 50% of the metal remained bound to the antibody. The pharmacokinetic study showed a retention half-life of 25.1 h and blood clearance of 0.72 ml/h. The biodistribution study indicated tumour uptake of 4.53%+/-0.49% of injected activity per gram (%ID/g) at 24 h and tumour-to-liver and tumour-to-bone ratios of 0.23+/-0.02 and 1.54+/-0.49 respectively at 24 h. With 153Sm-CITCDTPA-OC125F(ab')2, serum stability was greater (87% of the metal remaining bound to the antibody), retention half-life was 22.25 h and blood clearance was 2.23 ml/h. Tumour was better targeted (8.30%+/-3.56%ID/g at 24 h), and tumour-to-liver and tumour-to-bone ratios were 1.17+/-0.36 and 7.08+/-3.09 respectively at 24 h. However, renal retention remained elevated (29.76%+/-9. 41%ID/g at 24 h). With intact IgG, renal uptake decreased (1.41%+/-0. 49%ID/g at 24 h), but tumour uptake was lower than with fragments (1. 46%+/-0.58%ID/g at 24 h). Liver uptake was higher (tumour-to-liver ratio 0.10+/-0.05), and blood clearance was slower. The stability and distribution of 153Sm-CITCDTPA were more favourable than those of 153Sm-DTPA for application in radioimmunotherapy. Quantitative analysis performed using digitized images obtained by conventional

  7. Mapping monoclonal antibody structure by 2D 13C NMR at natural abundance.

    Science.gov (United States)

    Arbogast, Luke W; Brinson, Robert G; Marino, John P

    2015-04-07

    Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (∼150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope-labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D (13)C NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D (13)C NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.

  8. Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits

    Science.gov (United States)

    Mattera, Lucia; Bhuckory, Shashi; Wegner, K. David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J.; Hildebrandt, Niko; Reiss, Peter

    2016-05-01

    A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter 26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL-1 obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL-1) and demonstrate their direct applicability in clinical diagnostics.A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter 26 nm without biomolecules). The LODs

  9. A novel method for in Situ detection of hydrolyzable casein fragments in a cheese matrix by antibody phage display technique and CLSM

    DEFF Research Database (Denmark)

    Duan, Zhi; Brüggemann, Dagmar Adeline; Siegumfeldt, Henrik

    2009-01-01

    three small synthetic peptides of the alpha(s1)-casein sequence. These peptides traverse enzymatic cleavage sites of casein during cheese ripening. The specificity of the generated anti-peptide antibodies was determined by ELISA and Western blot. Finally, an immunofluorescent labeling protocol...

  10. The importance of tumor marker titers for the indication of immunoscintigraphy with monoclonal antibodies anti-CEA and anti-CA 19.9

    International Nuclear Information System (INIS)

    Bouvier, J.F.; Charrie, A.; Fleury-Goyon, M.C.; Chauvot, P. et; Lahneche, B.E.

    1986-01-01

    In 18 patients operated for malignant tumors 20 immunoscintigraphies were done with a monoclonal antibody cocktail (anti-CEA F(ab') 2 and anti-CA 19.9 F(ab') 2 ). Immediately before scintigraphy tumor marker titers in plasma were determined in all cases. Tumor marker levels corresponding to positive or doubtful scintigraphies are analysed. (Author)

  11. Human anti-mouse antibody response induced by anti-CD4 monoclonal antibody therapy in patients with rheumatoid arthritis.

    Science.gov (United States)

    Horneff, G; Winkler, T; Kalden, J R; Emmrich, F; Burmester, G R

    1991-04-01

    The development of human anti-mouse monoclonal antibodies (HAMAs) was investigated in 10 patients with rheumatoid arthritis (RA) who had undergone an experimental therapeutic trial with an anti-CD4 monoclonal antibody. In this patient group, the antibody 16H5 of the IgG1 isotype had been administered in a median total dosage of 140 mg per treatment cycle. Four patients took part in a second treatment regimen 6-8 weeks later. After the first treatment cycle, detectable HAMAs developed in 5 out of 10 patients. In 4 individuals undergoing a second course of therapy, increases of HAMAs were evident only in the 3 patients with previous HAMA responses. HAMAs were primarily of the IgG isotype, while the presence of rheumatoid factors usually interfered with the detectability of IgM HAMAs. However, using isolated F(ab)2 fragments of the monoclonal reagent used for therapy, HAMAs of the IgM isotype were also detectable. HAMAs of the IgG isotype did not exceed levels of 2.0 mg/liter after a single treatment cycle and 2.2 mg/liter after a repeated cycle. No IgE responses were detectable. Absorption experiments indicated that approximately 25% of the HAMA activity was directed against specific determinants of the 16H5 monoclonal antibody, presumably including anti-idiotypic reactivities. These data demonstrate that HAMAs developed only in a proportion of RA patients treated with the anti-CD4 monoclonal antibody 16H5. However, the amounts were rather low compared to other monoclonal reagents used in cancer patients and were therefore allowed for repeated applications without an apparent loss of efficacy.

  12. Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mi; Gustchina, Alla; Alexandratos, Jerry; Wlodawer, Alexander; Wünschmann, Sabina; Kepley, Christopher L.; Chapman, Martin D.; Pomes, Anna (INDOOR Bio.); (VCU); (NIH)

    2008-09-03

    The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab' fragment of a monoclonal antibody 7C11 was solved at 2.8-{angstrom} resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-{pi} interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab', Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an {alpha}-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less {beta}-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.

  13. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  14. Structural Basis for Treating Tumor Necrosis Factor α (TNFα)-associated Diseases with the Therapeutic Antibody Infliximab*

    Science.gov (United States)

    Liang, Shuaiyi; Dai, Jianxin; Hou, Sheng; Su, Lishu; Zhang, Dapeng; Guo, Huaizu; Hu, Shi; Wang, Hao; Rao, Zihe; Guo, Yajun; Lou, Zhiyong

    2013-01-01

    Monoclonal antibody (mAb) drugs have been widely used for treating tumor necrosis factor α (TNFα)-related diseases for over 10 years. Although their action has been hypothesized to depend in part on their ability to bind precursor cell surface TNFα, the precise mechanism and the epitope bound on TNFα remain unclear. In the present work, we report the crystal structure of the infliximab Fab fragment in complex with TNFα at a resolution of 2.6 Å. The key features of the TNFα E-F loop region in this complex distinguish the interaction between infliximab and TNFα from other TNF-receptor structures, revealing the mechanism of TNFα inhibition by overlapping with the TNFα-receptor interface and indicating the crucial role of the E-F loop in the action of this therapeutic antibody. This structure also indicates the formation of an aggregated network for the activation of complement-dependent cytolysis and antibody-dependent cell-mediated cytotoxicity, which result in development of granulomatous infections through TNFα blockage. These results provide the first experimental model for the interaction of TNFα with therapeutic antibodies and offer useful information for antibody optimization by understanding the precise molecular mechanism of TNFα inhibition. PMID:23504311

  15. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. ELISAs and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35–220.5 nM) dynamic range. Herceptin denatures and forms significant amount of aggregates when heated. UV-Vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 1013 M−2. The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  16. High-resolution mask inspection in advanced fab

    Science.gov (United States)

    Maelzer, Stephanie; Poock, Andre; Reese, Bryan; Bhattacharyya, Kaustuve; Mirzaagha, Farzin; Cox, Stephen; Lang, Michael

    2006-10-01

    High resolution mask inspection in advanced wafer fabs is a necessity. Initial and progressive mask defect problem still remains an industry wide mask reliability issue. Defect incidences and its criticality vary significantly among the type of masks, technology node and layer, fab environment and mask usage. A usage and layer based qualification strategy for masks in production need to be adopted in wafer fabs. With the help of a high-resolution direct reticle inspection, early detection of critical and also non-critical defects at high capture rates is possible. A high-resolution inspection that is capable of providing necessary sensitivity to critical emerging defects (near edge) is very important in advanced nodes. At the same time, a way to disposition (make a go / no-go decision) on these defective masks is also very important. As the impact of these defects will depend on not only their size, but also on their transmission and MEEF, various defect types and characteristics have to be considered. In this technical report the adoption of such a high-resolution mask inspection system in wafer fab production is presented and discussed. Data on this work will include inspection results from advanced masks, layer and product based inspection pixel assignment, defect disposition and overall wafer fab strategies in day-to-day production towards mask inspection.

  17. The presence of both antivirus and antiself antibodies in sera from patients with adenovirus and influenza B.

    Science.gov (United States)

    Portocala, R; Spyrou, N; Lambropoulou, V; Pateraki, E

    1988-01-01

    Using the ELISA method we examined serum samples from 62 male patients aged 19-23 infected with adenovirus (serotype 7), 22 children aged 7-14 infected with influenza B (B/Norway 1/84) and 113 normal subjects aged 5-30. The infections were diagnosed serologically by complement fixation, by inhibition of hemagglutination, by ELISA and by viral culture. Moreover using enzyme-linked short-time culture assay, the production of specific antivirus antibodies and autoantibodies in vitro by spleen cells (1 x 10(6) cells/well) from normal mice and from mice immunized with adenovirus and influenza B was studied. At the same time their sera antibody titers were determined. All the serum samples were tested against the following antigens: adenovirus, influenza B, ds-DNA, actin, myosin, myoglobin, thyroglobulin, H. transferrin, H. interferon a and BSA. FV. For the further characterization of positive sera, an evaluation of specificity by competitive ELISA-test and by preparations of F(ab')2 fragments from patients' sera was also carried out. It was found that the percentage of positivity for the specific virus and other antigens was higher in the patients' samples than in the samples from the normal subjects. The specific antivirus antibody was of IgG class and their titers ranged from 1/4, 800 up to 1/19,200. Autoantibodies belonged to IgM, IgA, IgG classes and their titers ranged from 1/400 to 1/1,600. In comparison, titers of normal subjects' sera ranged from 1/150 to 1/600 and 1/150 to 1/300, respectively and both were IgG classes. Both specific virus antibodies and autoantibodies appeared at the same time. The competitive ELISA-test showed a marked inhibition (95-98%) of antivirus antibodies with the specific antigen, whereas autoantibodies were less inhibited (40-50%) by homologous antigens. The antigen-antibody reaction occurred at the Fab portion of the immunoglobulin molecule, since these fragments inhibited antibody reactivity. The same results were observed with

  18. Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake

    International Nuclear Information System (INIS)

    Rotman, Maarten; Welling, Mick M.; Boogaard, Marlinde L. van den; Moursel, Laure Grand; Graaf, Linda M. van der; Buchem, Mark A. van; Maarel, Silvère M. van der; Weerd, Louise van der

    2015-01-01

    Introduction: Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab′)2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. Methods: To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ( 111 In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 μg VHH-pa2H-Fc-DTPA- 111 In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. Results: We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50 mg/L growth medium. The fusion protein showed homodimerization – necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA- 111 In and the VHH-Fc-DTPA- 111 In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA- 111 In

  19. Elucidating Substrate Promiscuity within the FabI Enzyme Family.

    Science.gov (United States)

    Freund, Gabriel S; O'Brien, Terrence E; Vinson, Logan; Carlin, Dylan Alexander; Yao, Andrew; Mak, Wai Shun; Tagkopoulos, Ilias; Facciotti, Marc T; Tantillo, Dean J; Siegel, Justin B

    2017-09-15

    The rapidly growing appreciation of enzymes' catalytic and substrate promiscuity may lead to their expanded use in the fields of chemical synthesis and industrial biotechnology. Here, we explore the substrate promiscuity of enoyl-acyl carrier protein reductases (commonly known as FabI) and how that promiscuity is a function of inherent reactivity and the geometric demands of the enzyme's active site. We demonstrate that these enzymes catalyze the reduction of a wide range of substrates, particularly α,β-unsaturated aldehydes. In addition, we demonstrate that a combination of quantum mechanical hydride affinity calculations and molecular docking can be used to rapidly categorize compounds that FabI can use as substrates. The results here provide new insight into the determinants of catalysis for FabI and set the stage for the development of a new assay for drug discovery, organic synthesis, and novel biocatalysts.

  20. Preparation of monoclonal antibodies against cardiac myosin and some radiolabelling studies

    International Nuclear Information System (INIS)

    Bapat, K.; Venkatesh, M.; Pillai, M.R.A.; Sarma, H.D.; Sainis, K.B.

    1998-01-01

    Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125 I and 99m Tc. Human cardiac myosin was isolated from normal human heart and was used for raising the monoclonal antibodies by the hybridoma technique. Antibody producing clones were identified by ELISA and cloning was done by the limiting dilution technique. Of the 13 clones obtained, 4 were deemed suitable for further studies. The antibodies were grown in ascites, purified, isotyped and their cross reactions with other forms of myosin were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted to different extents with bovine skeletal myosin. The most avid antibody Mab-4G4 was chosen for further labelling studies. Mab-4G4 was labelled with 125 I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. Purified radioiodinated antibody with radiochemical purity >95% could be obtained by gel filtration. Immunoreactivity was retained as tested by binding to myosin immobilised on a solid support. Mab-4G4 was also labelled with 99m Tc using stannous tartrate as the reducing agent. Radiolabelling yield was ∼60%, the purity was >95% and the immunoreactivity was retained. Both the labelled preparations were tested for bio-distribution in normal and infarcted rats. The activity accumulation in the infarcted region was ∼ 1.5 and 3.5 times as that in normal heart muscle for 125 I and 99m Tc labelled Mab-4G4 respectively. The major problem with the iodinated antibody was the in vivo deiodination resulting in very high percentage of activity in the thyroid. Although the fraction of the total activity associated with the infarcted heart is not very impressive, the fact that the activities with the infarcted and normal hearths are significantly different is heartening. With further optimisation of labelling and use of F(ab)'2 fragments, better delineation of the infarct sites is aspired. (author)

  1. Technetium-99m labeled monoclonal antibodies in the detection of metastatic melanoma

    International Nuclear Information System (INIS)

    Serafini, A.N.; Kotler, J.; Feun, L.; Dewanjee, M.; Robinson, D.; Salk, D.; Sfakianakis, G.; Abrams, P.; Savaraj, N.; Goodwin, D.

    1989-01-01

    Twenty-six stage II/III malignant melanoma patients with 321 measurable metastatic lesions were imaged using Fab fragments of an IgG murine monoclonal antibody labeled specifically with 10-30 mCi Tc-99m with a bi-functional chelating method (NeoRx, Seattle, WA). There were no side effects or adverse reactions. Immunoscintigraphy demonstrated 66.6% of lesions larger than 1 cm and 92.5% of lesions larger than 3 cm. Most frequently detected metastases were in lymph nodes, subcutaneous areas, and bone. Of lesions less than 1 cm, 23.6% were detected if superficial cutaneous lesions were excluded. The smallest detectable lesion was 4 mm. Twenty-one additional clinically unsuspected sites were visualized in 12 of the 26 patients studied. Of these, 56% were confirmed as metastasis by other tests. There were apparent nonspecific localizations owing to other causes, including fracture, varicosities, skin abscess and pneumonitis. Increased experience in image analysis facilitates correct interpretation of these localizations. This study demonstrates that imaging with Tc-99m labeled antibody fragments detects melanoma lesions in organs routinely surveyed and in other areas not routinely assessed by other imaging techniques. The procedure is readily performed and safe. The principal advantage of the test is its ability to survey the entire body and all organs with a single test. Its principal limitation, in common with other diagnostic imaging procedures, is its poor sensitivity for detecting lesions less than 1 cm

  2. 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.

  3. Working mechanism of immunoglobulin A1 (IgA1) protease: cleavage of IgA1 antibody to Neisseria meningitidis PorA requires de novo synthesis of IgA1 Protease

    DEFF Research Database (Denmark)

    Vidarsson, G; Overbeeke, N; Stemerding, AM

    2005-01-01

    Neisseria meningitidis secretes a protease that specifically cleaves the hinge region of immunoglobulin A1 (IgA1), releasing the effector (Fc) domain of IgA1 from the antigen binding (Fab) determinants. Theoretically, the remaining Fab fragments can block pathogen receptors or toxins and still...

  4. The future of monoclonal antibody technology

    OpenAIRE

    Zider, Alexander; Drakeman, Donald L

    2010-01-01

    With the rapid growth of monoclonal antibody-based products, new technologies have emerged for creating modified forms of antibodies, including fragments, conjugates and multi-specific antibodies. We created a database of 450 therapeutic antibodies in development to determine which technologies and indications will constitute the “next generation” of antibody products. We conclude that the antibodies of the future will closely resemble the antibodies that have already been approved for commer...

  5. Opening the Arms: The FAB Projects and Digital Resilience

    Science.gov (United States)

    Longden, Alison; Monaghan, Tom; Mycroft, Lou; Kelly, Claire

    2018-01-01

    A chance observation in a staff training session led to a series of action research projects, which dug into why some educators are digitally resistant whilst others, however experienced, are not. The Education and Training Foundation-funded "FAB Projects" have so far spanned three years and three small-scale research investigations and…

  6. Scatterometry on pelliclized masks: an option for wafer fabs

    Science.gov (United States)

    Gallagher, Emily; Benson, Craig; Higuchi, Masaru; Okumoto, Yasuhiro; Kwon, Michael; Yedur, Sanjay; Li, Shifang; Lee, Sangbong; Tabet, Milad

    2007-03-01

    Optical scatterometry-based metrology is now widely used in wafer fabs for lithography, etch, and CMP applications. This acceptance of a new metrology method occurred despite the abundance of wellestablished CD-SEM and AFM methods. It was driven by the desire to make measurements faster and with a lower cost of ownership. Over the last year, scatterometry has also been introduced in advanced mask shops for mask measurements. Binary and phase shift masks have been successfully measured at all desired points during photomask production before the pellicle is mounted. There is a significant benefit to measuring masks with the pellicle in place. From the wafer fab's perspective, through-pellicle metrology would verify mask effects on the same features that are characterized on wafer. On-site mask verification would enable quality control and trouble-shooting without returning the mask to a mask house. Another potential application is monitoring changes to mask films once the mask has been delivered to the fab (haze, oxide growth, etc.). Similar opportunities apply to the mask metrologist receiving line returns from a wafer fab. The ability to make line-return measurements without risking defect introduction is clearly attractive. This paper will evaluate the feasibility of collecting scatterometry data on pelliclized masks. We explore the effects of several different pellicle types on scatterometry measurements made with broadband light in the range of 320-780 nm. The complexity introduced by the pellicles' optical behavior will be studied.

  7. Nuclear fragmentation

    International Nuclear Information System (INIS)

    Chung, K.C.

    1989-01-01

    An introduction to nuclear fragmentation, with emphasis in percolation ideas, is presented. The main theoretical models are discussed and as an application, the uniform expansion approximation is presented and the statistical multifragmentation model is used to calculate the fragment energy spectra. (L.C.)

  8. Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis

    Directory of Open Access Journals (Sweden)

    Jing Jin

    2015-12-01

    Full Text Available We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV infection. Potently neutralizing antibodies (NAbs blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

  9. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    Science.gov (United States)

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  10. A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody.

    Directory of Open Access Journals (Sweden)

    Benny Chain

    Full Text Available The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively, and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.

  11. Identification of endogenous opioid receptor components in rat brain using a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bero, L.A.; Roy, S.; Lee, N.M.

    1988-11-01

    A monoclonal antibody generated against the tertiary structure of a partially purified opioid binding protein was used to probe the structure of the dynorphin and beta-endorphin receptors. The Fab fragment 3B4F11 inhibited completely the binding of 125I-beta-endorphin and (3H)dynorphin to rat brain P2 membranes with IC50 values of 26 ng/ml and 40 ng/ml, respectively. To explore further the interaction of 3B4F11 with the beta-endorphin receptor, the effect of the Fab fragment on 125I-beta-endorphin cross-linking to rat brain membranes was examined. 125I-beta-endorphin was covalently bound to three major species of approximate molecular weights 108,000, 73,000, and 49,000. The delta-selective ligand D-Pen2, D-pen5enkephalin was least effective at inhibiting the cross-linking of beta-endorphin, whereas the micro-selective ligand Tyr-D-Ala-Gly-NMe-Phe-Gly-ol and kappa-selective ligand U50488 inhibited beta-endorphin cross-linking to the 108,000 and 73,000 Da species. Both 3B4F11 and beta-endorphin prevented the covalent binding of 125I-beta-endorphin to all three labeled species. These findings suggest that micro and kappa receptor types might have some structural similarities, whereas the delta receptor type might differ in molecular size. In addition, the micro, kappa, and delta ligands might have different primary sequences, whereas their tertiary structures might share regions of molecular homology with all three receptor constituents labeled by 125I-beta-endorphin. 3B4F11 will be a valuable tool for the purification and isolation of the several components of the beta-endorphin receptor complex.

  12. Pre-clinical studies with CDR-grafted humanised anti-colorectal monoclonal antibody A33

    Energy Technology Data Exchange (ETDEWEB)

    Scott, A.M.; Lee, F.T.; Hutchinson, D.; Cebon, J. [Ludwig Inst. for Cancer Research, Austin Hospital, Heidelberg, VIC (Australia); Sturrock, S.; Pollock, D.; Armes, J.; Sinclair, R. [Austin Hospital, Heilderberg, VIC (Australia). Dept. of Anatomical Pathology; Welt, S.; Old, L.J. [Ludwig Inst. for Cancer Research, New York, NY (United States)

    1998-03-01

    Full text: Monoclonal antibodies (mAbs) can be raised to selected antigens preferentially expressed on cancer cells compared to normal tissues, and therefore have attractive properties for targeting of cancers for therapy. Numerous strategies can be employed for cell kill, including conjugation of isotopes or toxins, and utilisation of intrinsic immune effector function of mAbs. To date, immunogenicity of murine antibodies (HAMA) has been a major limitation in effectiveness of mAb therapy. The anticolorectal mAb A33 in its murine form has been studied in Phase l/ll trials. A CDR-grafted humanised versions of A33 (huA33) has been developed, and we are evaluating this for clinical trials. The intact mAb huA33 has been radiolabelled and shown to have retained immunoreactivity of 80% specific binding to SW-1222 colon cells (Ka of 1.4 x 10{sup -9} M). An F(ab``)2 fragment has been produced, and demonstrates similar affinity to the intact version with immunoreactivity of 74% specific binding, a Ka of 2 x 10{sup -9} M, and similar molar displacement by intact huA33 in in-vitro displacement assays. Immunohistochemistry with biotinylated huA33 has demonstrated similar normal tissue and colon cancer specificity to the murine A33. Biodistribution studies with {sup 125}I labelled huA33 and {sup 125}I-F(ab``)2 huA33 have been performed in SW-1222 xenografted balb-c nude mice, and have demonstrated excellent targeting to tumours with maximal tumour: blood and tumour: liver ratios ranging from 37:1 and 62:1, respectively. Clinical trials with intact and F(ab``)2 huA33 are planned based on the results of these pre-clinical studies.

  13. Pre-clinical studies with CDR-grafted humanised anti-colorectal monoclonal antibody A33

    International Nuclear Information System (INIS)

    Scott, A.M.; Lee, F.T.; Hutchinson, D.; Cebon, J.; Sturrock, S.; Pollock, D.; Armes, J.; Sinclair, R.

    1998-01-01

    Full text: Monoclonal antibodies (mAbs) can be raised to selected antigens preferentially expressed on cancer cells compared to normal tissues, and therefore have attractive properties for targeting of cancers for therapy. Numerous strategies can be employed for cell kill, including conjugation of isotopes or toxins, and utilisation of intrinsic immune effector function of mAbs. To date, immunogenicity of murine antibodies (HAMA) has been a major limitation in effectiveness of mAb therapy. The anticolorectal mAb A33 in its murine form has been studied in Phase l/ll trials. A CDR-grafted humanised versions of A33 (huA33) has been developed, and we are evaluating this for clinical trials. The intact mAb huA33 has been radiolabelled and shown to have retained immunoreactivity of 80% specific binding to SW-1222 colon cells (Ka of 1.4 x 10 -9 M). An F(ab'')2 fragment has been produced, and demonstrates similar affinity to the intact version with immunoreactivity of 74% specific binding, a Ka of 2 x 10 -9 M, and similar molar displacement by intact huA33 in in-vitro displacement assays. Immunohistochemistry with biotinylated huA33 has demonstrated similar normal tissue and colon cancer specificity to the murine A33. Biodistribution studies with 125 I labelled huA33 and 125 I-F(ab'')2 huA33 have been performed in SW-1222 xenografted balb-c nude mice, and have demonstrated excellent targeting to tumours with maximal tumour: blood and tumour: liver ratios ranging from 37:1 and 62:1, respectively. Clinical trials with intact and F(ab'')2 huA33 are planned based on the results of these pre-clinical studies

  14. Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    2010-11-01

    Full Text Available The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  15. In Vivo HER2-Targeted Magnetic Resonance Tumor Imaging Using Iron Oxide Nanoparticles Conjugated with Anti-HER2 Fragment Antibody.

    Science.gov (United States)

    Ding, Ning; Sano, Kohei; Kanazaki, Kengo; Ohashi, Manami; Deguchi, Jun; Kanada, Yuko; Ono, Masahiro; Saji, Hideo

    2016-12-01

    The feasibility of iron oxide nanoparticles (IONPs) conjugated with anti-epidermal growth factor receptor 2 (HER2) single-chain antibody (scFv-IONPs) as novel HER2-targeted magnetic resonance (MR) contrast agents was investigated. The scFv-IONPs were prepared and identified. For in vitro MRI, NCI-N87 (HER2 high expression) and SUIT2 (low expression) cells were incubated with scFv-IONPs. For in vivo MRI, NCI-N87 and SUIT2 tumor-bearing mice were intravenously injected with scFv-IONPs and imaged before and 24 h post-injection. The scFv-IONPs demonstrated high transverse relaxivity (296.3 s -1  mM -1 ) and affinity toward HER2 (K D  = 11.7 nM). In the in vitro MRI, NCI-N87 cells treated with scFv-IONPs exhibited significant MR signal reduction (44.6 %) than SUIT2 cells (6.8 %). In the in vivo MRI, decrease of MR signals in NCI-N87 tumors (19.3 %) was more notable than that in SUIT2 tumors (6.2 %). The scFv-IONPs enabled HER2-specific tumor MR imaging, suggesting the potential of scFv-IONPs as a robust HER2-targeted MR contrast agent.

  16. Recombinant monovalent llama-derived antibody fragments (VHH to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Directory of Open Access Journals (Sweden)

    Celina G Vega

    Full Text Available Group A Rotavirus (RVA is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH to protect against human rotavirus in gnotobiotic (Gn piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256 for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  17. Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.

    Science.gov (United States)

    Vega, Celina G; Bok, Marina; Vlasova, Anastasia N; Chattha, Kuldeep S; Gómez-Sebastián, Silvia; Nuñez, Carmen; Alvarado, Carmen; Lasa, Rodrigo; Escribano, José M; Garaicoechea, Lorena L; Fernandez, Fernando; Bok, Karin; Wigdorovitz, Andrés; Saif, Linda J; Parreño, Viviana

    2013-01-01

    Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

  18. Administered activity and metastatic cure probability during radioimmunotherapy of ovarian cancer in nude mice with 211At-MX35 F(ab')2.

    Science.gov (United States)

    Elgqvist, Jörgen; Andersson, Håkan; Bernhardt, Peter; Bäck, Tom; Claesson, Ingela; Hultborn, Ragnar; Jensen, Holger; Johansson, Bengt R; Lindegren, Sture; Olsson, Marita; Palm, Stig; Warnhammar, Elisabet; Jacobsson, Lars

    2006-11-15

    To elucidate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice. This study: (i) estimated the minimum required activity (MRA), giving a reasonable high therapeutic efficacy; and (ii) calculated the specific energy to tumor cell nuclei and the metastatic cure probability (MCP) using various assumptions regarding monoclonal-antibody (mAb) distribution in measured tumors. The study was performed using the alpha-particle emitter Astatine-211 (211At) labeled to the mAb MX35 F(ab')2. Animals were inoculated intraperitoneally with approximately 1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later animals were treated with 25, 50, 100, or 200 kBq 211At-MX35 F(ab')2 (n = 74). Another group of animals was treated with a nonspecific mAb: 100 kBq 211At-Rituximab F(ab')2 (n = 18). Eight weeks after treatment the animals were sacrificed and presence of macro- and microscopic tumors and ascites was determined. An MCP model was developed and compared with the experimentally determined tumor-free fraction (TFF). When treatment was given 4 weeks after cell inoculation, the TFFs were 25%, 22%, 50%, and 61% after treatment with 25, 50, 100, or 200 kBq (211)At-MX35 F(ab')2, respectively, the specific energy to irradiated cell nuclei varying between approximately 2 and approximately 400 Gy. As a significant increase in the therapeutic efficacy was observed between the activity levels of 50 and 100 kBq (TFF increase from 22% to 50%), the conclusion was that the MRA is approximately 100 kBq (211)At-MX35 F(ab')2. MCP was most consistent with the TFF when assuming a diffusion depth of 30 mum of the mAbs in the tumors.

  19. Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera.

    Directory of Open Access Journals (Sweden)

    Ruklanthi de Alwis

    2014-10-01

    Full Text Available Dengue viruses (DENV are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to

  20. Radiolabeled antibody imaging

    International Nuclear Information System (INIS)

    Wahl, R.L.

    1987-01-01

    Radiolabeled antibodies, in particular monoclonal antibodies, offer the potential for the specific nuclear imaging of malignant and benign diseases in man. If this imaging potential is realized, they may also have a large role in cancer treatment. This paper reviews: (1) what monoclonal antibodies are and how they differ from polyclonal antibodies, (2) how they are produced and radiolabeled, (3) the results of preclinical and clinical trials in cancer imaging, including the utility of SPECT and antibody fragments, (4) the role of antibodies in the diagnosis of benign diseases, (5) alternate routes of antibody delivery, (6) the role of these agents in therapy, and (7) whether this technology ''revolutionizes'' the practice of nuclear radiology, or has a more limited complementary role in the imaging department

  1. Biodistribution and dosimetric study in medullary thyroid cancer xenograft using bispecific antibody and iodine-125-labeled bivalent hapten.

    Science.gov (United States)

    Hosono, M; Hosono, M N; Kraeber-Bodéré, F; Devys, A; Thédrez, P; Fiche, M; Gautherot, E; Barbet, J; Chatal, J F

    1998-09-01

    The purpose of this study was to evaluate biodistributions and absorbed doses of anti-carcinoembryonic antigen (CEA)/anti-diethylenetriamine pentaacetic acid (DTPA)-indium (anti-DTPA-In) bispecific monoclonal antibody (BsMAb) F6-734 and 125I-labeled DTPA-indium dimer hapten (125I-di-DTPA-In hapten) in athymic mice xenografted with human medullary thyroid cancer. Bispecific monoclonal antibodies F6-679 (anti-CEA/antihistamine) and G7A5-734 (antimelanoma/anti-di-DTPA-In) were used as irrelevant BsMAbs. Athymic mice inoculated with TT medullary thyroid cancer cells expressing CEA were administered BsMAbs F6-734, F6-679 or G7A5-734 and then, 48 hr later, 125I-di-DTPA-In hapten. Iodine-125-labeled F6 F(ab')2 fragment was injected into other groups of mice. Biodistributions were examined at 30 min and 5, 24, 48 and 96 hr after injection of 125I-di-DTPA-In hapten or 125I-labeled F6 F(ab')2. In mice injected with BsMAb F6-734 and 125I-di-DTPA-In hapten, tumor uptake was 9.1%+/-2.1%, 8.7%+/-3.5%, 8.0%+/-2.3%, 5.1%+/-0.9% and 3.5%+/-1.5% of the injected dose/g at 30 min and 5, 24, 48 and 96 hr, and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios were 37.0+/-12.5, 32.3+/-10.9 and 10.4+/-2.7 at 24 hr. Iodine-125-F6 F(ab')2 fragment showed a tumor uptake of 7.39% injected dose/g and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios of 1.8+/-0.6, 7.3+/-2.9 and 3.6+/-1.6 at 24 hr. In mice injected with F6-679 or G7A5-734, tumor uptake and tumor-to-normal tissue ratios were much lower than in the mice injected with F6-734. These results were confirmed by autoradiographic studies that demonstrated clear tumor-to-normal tissue contrast. This two-step targeting method seems very potent for the diagnosis and therapy of human medullary thyroid cancer and other CEA-producing tumors because it combines high tumor uptake and low normal tissue background.

  2. Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

    Directory of Open Access Journals (Sweden)

    Janina Jamasbi, RPh

    2016-04-01

    Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

  3. Characterization of molecular mechanisms controlling fabAB transcription in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Herbert P Schweizer

    Full Text Available BACKGROUND: The FabAB pathway is one of the unsaturated fatty acid (UFA synthesis pathways for Pseudomonas aeruginosa. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt fabA upstream sequence, which is conserved in P. aeruginosa, P. putida, and P. syringae, led to a significant decrease in fabA transcription, suggesting positive regulation by an unknown positive regulatory mechanism. METHODS/PRINCIPAL FINDINGS: Here, genetic and biochemical approaches were employed to identify a potential fabAB activator. Deletion of candidate genes such as PA1611 or PA1627 was performed to determine if any of these gene products act as a fabAB activator. However, none of these genes were involved in the regulation of fabAB transcription. Use of mariner-based random mutagenesis to screen for fabA activator(s showed that several genes encoding unknown functions, rpoN and DesA may be involved in fabA regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of fabAB operon. CONCLUSION/SIGNIFICANCE: The data suggest that fabA expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism.

  4. Novel antibody conjugates for enhanced tumor uptake. Final report, September 1, 1992--August 31, 1997

    International Nuclear Information System (INIS)

    Thakur, M.

    1997-01-01

    Progress in three areas of research is summarized. These are as follows: Labeling Monoclonal antibodies (MAbs) with Tc-99m and Re-186; human melanoma tumors and specific MAbs; evaluation of biological response modifiers (BRM). The techniques of labeling MAbs (IgM, IgG, F(ab') 2 or F(ab')) with Tc-99m was developed in the author's laboratory in 1989 and that with Re-186 in 1992. The techniques are in daily use in the laboratory since then and are adapted to a convenient kit formulation. The metal ions are bound at MAb sulfhydryls generated by a controlled reduction of a pair of disulfide groups. At least two types of MAbs labeled with Tc-99m by this method have been administered into patients and excellent diagnostic results have been obtained. Over the past two and a half years the author has been successfully growing human melanoma tumors in athymic Balb/c nude mice. The cell LINE, WM-9, was obtained from Dr. D Herlyn's laboratory at Wistar Institute in Philadelphia. Sufficient quantities of antihuman melanoma specific antibodies ME 31.3 (Wistar, IgG-1) and MEM-136 (Hybritech, IgG-2A) and their F(ab') 2 fragments are also available in the laboratory. The use of BRM is a rapidly evolving field. Over the past four years, the author has evaluated a number of BRMs in a quest for agents that may augment MAb tumor uptake. These included interferon-α; a pokeweed mitogen and Ukrain, an alkaloid separated from a plant Chelideonium Majis. In these preliminary studies, normal Balb/c mice were used and the BRMs were given i.p. one hour prior to the i.v. administration of tumor necrosis factor or an MAb (TNT-F(ab') 2 ) labeled with Tc-99m which served as an imaging agent. Animals were sacrificed at 1.5 hr or 4 hrs post-injection. Highlights of the work are given here in a table

  5. Wind Fab: Manufacturer of water pumping windmills in India

    International Nuclear Information System (INIS)

    Gurumoorthy, S.

    1991-01-01

    Wind Fab is one of the four manufacturers appointed recently by the Indian government to produce a classic gear type wind pump for deep well pumping (H = 50-100 meter). In various regions, the ground water table has been lowering considerably and a deep well wind pump is required. Wind Fab is still producing the 12PU500 windmill. The main problem is that the wind pumps are applied in a wide range of climatological, hydrological and agricultural conditions. Different types of windmills suitable for the different application ranges should be produced. It is explained that the drawings of the 12PU500 windmills were easily available and consequently the windmills were produced all over the country. There was no quality control and the 12PU500 was applied under low lift to high lift conditions. 5 figs., 5 tabs

  6. Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

    Science.gov (United States)

    Alanen, Heli I; Walker, Kelly L; Lourdes Velez Suberbie, M; Matos, Cristina F R O; Bönisch, Sarah; Freedman, Robert B; Keshavarz-Moore, Eli; Ruddock, Lloyd W; Robinson, Colin

    2015-03-01

    Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.

    Science.gov (United States)

    Gramzow, M; Bachmann, M; Uhlenbruck, G; Dorn, A; Müller, W E

    1986-04-01

    Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.

  8. Fab Four self-interaction in quantum regime

    Science.gov (United States)

    Arbuzov, A. B.; Latosh, B. N.

    2017-10-01

    Quantum behavior of the John Lagrangian from the Fab Four class of covariant Galileons is studied. We consider one-loop corrections to the John interaction due to cubic scalar field interaction. Counter terms are calculated, one appears because of massless scalar field theory infrared issues, another one lies in the George class, and the rest of them can be reduced to the initial Lagrangian up to surface terms. The role of quantum corrections in the context of cosmological applications is discussed.

  9. Fab Four self-interaction in quantum regime

    Energy Technology Data Exchange (ETDEWEB)

    Arbuzov, A.B.; Latosh, B.N. [JINR, Bogoliubov Laboratory for Theoretical Physics, Dubna (Russian Federation); Dubna State University, Dubna, Moscow Region (Russian Federation)

    2017-10-15

    Quantum behavior of the John Lagrangian from the Fab Four class of covariant Galileons is studied. We consider one-loop corrections to the John interaction due to cubic scalar field interaction. Counter terms are calculated, one appears because of massless scalar field theory infrared issues, another one lies in the George class, and the rest of them can be reduced to the initial Lagrangian up to surface terms. The role of quantum corrections in the context of cosmological applications is discussed. (orig.)

  10. DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

    Science.gov (United States)

    Takahashi, S; Maecker, H T; Levy, R

    1989-10-01

    An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

  11. Serum concentration of immunoglobulin G-type antibodies against the whole Epstein-Barr nuclear antigen 1 and its aa35-58 or aa398-404 fragments in the sera of patients with systemic lupus erythematosus and multiple sclerosis.

    Science.gov (United States)

    Csuka, D; Simon, D; Hóbor, R; Uray, K; Prohászka, Z; Bánlaki, Z; Jani, P K; Szilágyi, Á; Hudecz, F; Rajczy, K; Beke, G; Boros Major, A; Tordai, A; Illés, Z; Berki, T; Czirják, L; Füst, G

    2013-03-01

    Several studies suggest that infection by Epstein-Barr virus (EBV) might be one of the environmental factors which facilitates the development of autoimmune disorders in genetically susceptible individuals. Recent data indicate that high anti-Epstein-Barr nuclear antigen 1 (EBNA)-1 immunoglobulin (Ig)G titre is a strong risk factor for multiple sclerosis (MS) in patients both with and without the main genetic predisposing trait, human leucocyte antigen (HLA)-DRB1*15:01. Because no similar studies have been published in systemic lupus erythematosus (SLE) patients, we determined the HLA-DRB1*15:01 carrier state and the serum titres against the whole EBNA-1 and its small fragments aa35-58 and aa398-404 in 301 SLE patients, 135 MS patients and in 345 healthy controls. The carrier state of the HLA-DRB1*15:01 allele was deduced from genotyping of a tagSNP (rs3135388) by applying a Taqman-based assay. The serum concentrations of antibodies to EBNA-1 and its aa35-58 or aa398-404 fragments were determined using a commercial assay (ETI-EBNA-G) and home-made enzyme-linked immunosorbent assays, respectively. The serum concentration of anti-EBNA-1 antibodies was significantly (P HLA-DRB1*15:01 carriers and non-carriers. Furthermore, titres of antibodies against the aa35-58 EBNA-1 fragment were elevated both in MS and SLE patients. By contrast, the levels of aa398-404 EBNA-1 antibodies were elevated significantly only in the SLE patients. These findings indicate that high anti-EBNA-1 IgG titres are HLA-DRB1*15:01-independent risk factors not only for MS, but also for SLE, while high antibody titres against the aa398-404 fragment are characteristic for SLE. © 2012 British Society for Immunology.

  12. Rasch Analysis of the Fullerton Advanced Balance (FAB) Scale

    Science.gov (United States)

    Fiedler, Roger C.; Rose, Debra J.

    2011-01-01

    ABSTRACT Purpose: This cross-sectional study explores the psychometric properties and dimensionality of the Fullerton Advanced Balance (FAB) Scale, a multi-item balance test for higher-functioning older adults. Methods: Participants (n=480) were community-dwelling adults able to ambulate independently. Data gathering consisted of survey and balance performance assessment. Psychometric properties were assessed using Rasch analysis. Results: Mean age of participants was 76.4 (SD=7.1) years. Mean FAB Scale scores were 24.7/40 (SD=7.5). Analyses for scale dimensionality showed that 9 of the 10 items fit a unidimensional measure of balance. Item 10 (Reactive Postural Control) did not fit the model. The reliability of the scale to separate persons was 0.81 out of 1.00; the reliability of the scale to separate items in terms of their difficulty was 0.99 out of 1.00. Cronbach's alpha for a 10-item model was 0.805. Items of differing difficulties formed a useful ordinal hierarchy for scaling patterns of expected balance ability scoring for a normative population. Conclusion: The FAB Scale appears to be a reliable and valid tool to assess balance function in higher-functioning older adults. The test was found to discriminate among participants of varying balance abilities. Further exploration of concurrent validity of Rasch-generated expected item scoring patterns should be undertaken to determine the test's diagnostic and prescriptive utility. PMID:22210989

  13. Rasch Analysis of the Fullerton Advanced Balance (FAB) Scale.

    Science.gov (United States)

    Klein, Penelope J; Fiedler, Roger C; Rose, Debra J

    2011-01-01

    This cross-sectional study explores the psychometric properties and dimensionality of the Fullerton Advanced Balance (FAB) Scale, a multi-item balance test for higher-functioning older adults. Participants (n=480) were community-dwelling adults able to ambulate independently. Data gathering consisted of survey and balance performance assessment. Psychometric properties were assessed using Rasch analysis. Mean age of participants was 76.4 (SD=7.1) years. Mean FAB Scale scores were 24.7/40 (SD=7.5). Analyses for scale dimensionality showed that 9 of the 10 items fit a unidimensional measure of balance. Item 10 (Reactive Postural Control) did not fit the model. The reliability of the scale to separate persons was 0.81 out of 1.00; the reliability of the scale to separate items in terms of their difficulty was 0.99 out of 1.00. Cronbach's alpha for a 10-item model was 0.805. Items of differing difficulties formed a useful ordinal hierarchy for scaling patterns of expected balance ability scoring for a normative population. The FAB Scale appears to be a reliable and valid tool to assess balance function in higher-functioning older adults. The test was found to discriminate among participants of varying balance abilities. Further exploration of concurrent validity of Rasch-generated expected item scoring patterns should be undertaken to determine the test's diagnostic and prescriptive utility.

  14. Chameleon fragmentation

    International Nuclear Information System (INIS)

    Brax, Philippe; Upadhye, Amol

    2014-01-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ 4 and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments

  15. Chameleon fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brax, Philippe [Institut de Physique Théorique, CEA, IPhT, CNRS, URA 2306, F-91191Gif/Yvette Cedex (France); Upadhye, Amol, E-mail: philippe.brax@cea.fr, E-mail: aupadhye@anl.gov [Institute for the Early Universe, Ewha University, International Education, Building #601, 11-1, Daehyun-Dong Seodaemun-Gu, Seoul 120-750 (Korea, Republic of)

    2014-02-01

    A scalar field dark energy candidate could couple to ordinary matter and photons, enabling its detection in laboratory experiments. Here we study the quantum properties of the chameleon field, one such dark energy candidate, in an ''afterglow'' experiment designed to produce, trap, and detect chameleon particles. In particular, we investigate the possible fragmentation of a beam of chameleon particles into multiple particle states due to the highly non-linear interaction terms in the chameleon Lagrangian. Fragmentation could weaken the constraints of an afterglow experiment by reducing the energy of the regenerated photons, but this energy reduction also provides a unique signature which could be detected by a properly-designed experiment. We show that constraints from the CHASE experiment are essentially unaffected by fragmentation for φ{sup 4} and 1/φ potentials, but are weakened for steeper potentials, and we discuss possible future afterglow experiments.

  16. Cloning and molecular characterization of a human recombinant IgG Fab binding to the Tat protein of human immunodeficiency virus type 1 (HIV-1) derived from the repertoire of a seronegative patient.

    Science.gov (United States)

    Mancini, Nicasio; Perotti, Mario; Carletti, Silvia; Canducci, Filippo; Sampaolo, Michela; Clementi, Massimo; Burioni, Roberto

    2006-03-01

    A study aiming at cloning and characterizing natural antibodies to human immunodeficiency virus type 1 (HIV-1) targets is described. In particular, we report the molecular cloning of a Fab molecule binding the HIV-1/Tat protein from a seronegative patient. The Fab was characterized for its binding specificity and investigated in regards to its molecular structure. Furthermore, to evaluate the role played by the heavy and light chains in the binding to the antigen, hybrid Fabs were constructed combining the heavy and the light chain of the natural anti-Tat clone with a control high-affinity Fab derived from the repertoire of the same patient. The results indicate that the natural immunoglobulin under study: (i) is a polyreactive antibody of IgG1 isotype, and not an IgM as usually described for anti-HIV natural clones, (ii) shows a pattern of mutations compatible with an antigen-driven mechanisms, (iii) its heavy chain derives from a V-gene subfamily (V3-23) highly represented in fetal life, and (iv) its heavy chain variable region exhibits several characteristics, including an extremely long, hydrophilic CDR3, that are unusual and theoretically important in determining the polyreactive capacity of the molecule.

  17. Aerial imaging for FABs: productivity and yield aspects

    Science.gov (United States)

    Englard, Ilan; Cohen, Yaron; Elblinger, Yair; Attal, Shay; Berns, Neil; Shoval, Lior; Ben-Yishai, Michael; Mangan, Shmoolik

    2009-03-01

    The economy of wafer fabs is changing faster for 3x geometry requirements and below. Mask set and exposure tool costs are almost certain to increase the overall cost per die requiring manufacturers to develop productivity and yield improvements to defray the lithography cell economic burden. Lithography cell cost effectiveness can be significantly improved by increasing mask availability while reducing the amount of mask sets needed during a product life cycle. Further efficiency can be gained from reducing send-ahead wafers and qualification cycle time, and elimination of inefficient metrology. Yield is the overriding die cost modulator and is significantly more sensitive to lithography as a result of masking steps required to fabricate the integrated circuit. Thus, for productivity to increase with minimal yield risk, the sample space of reticle induced source of variations should be large, with shortest measurement acquisition time possible. This paper presents the latest introduction of mask aerial imaging technology for the fab, Aera2TM for Lithography with IntenCTM, as an enabler for efficient lithography manufacturing. IntenCD is a high throughput, high density mask-based critical dimension (CD) mapping technology, with the potential for increasing productivity and yield in a wafer production environment. Connecting IntenCD to a feed forward advance process control (APC) reduces significantly the amount of traditional CD metrology required for robust wafer CD uniformity (CDU) correction and increases wafer CD uniformity. This in turn improves the lithography process window and yield and contributes to cost reduction and cycle time reduction of new reticles qualification. Advanced mask technology has introduced a new challenge. Exposure to 193nm wavelength stimulates haze growth on the mask and imposes a regular cleaning schedule. Cleaning eventually causes mask degradation. Haze growth impacts mask CD uniformity and induce global transmission fingerprint

  18. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    Science.gov (United States)

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  19. Fragmentation of Nimotuzumab for Preparation of 125I-F(ab’2-Nimotuzumab as a Precursor for Preparing 125I-F(ab’2-Nimotuzumab-NLS Radiopharmaceutical for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    R.D. Haryuni

    2014-04-01

    Full Text Available Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR. This monoclonal antibody has a relatively high molecular weight which makes slow penetration on tumor cell, as concequence, it is less attractive in imaging kinetics, and potentially elicits antibodies respons. Therefore in this study nimotuzumab was fragmented to form bivalent antibody [F(ab’2] and then labeled with 125I to form 125I-F(ab’2-nimotuzumab which can be used further as a precursor for preparing 125I-F(ab’2-nimotuzumab-NLS (NLS = nuclear localizing sequences radiopharmaceutical for radioimmunotherapy. The aims of this study were to obtain characteristics of 125I-F(ab’2-nimotuzumab by comparing with the 125I labeled-intact nimotuzumab (125I-nimotuzumab. This study was initiated by purifying nimotuzumab by mean of dialysis. The purified nimotuzumab was then fragmented by using pepsin. The F(ab'2-nimotuzumab formed was then purified from its by-products which formed in fragmentation process by using a PD-10 column (consisted Sephadex G25. The intact nimotuzumab and its F(ab’2 fragment were then labeled with the 125I to form 125I-nimotuzumab and 125I-F(ab’2-nimotuzumab. The radiochemical purity are 98.27 % and 93.24 % ,respectively. Stability test results show that, both of 125I-nimotuzumab and 125I-F(ab’2-nimotuzumab more stable at 4 °C than at room temperature storage and 37 °C

  20. Fragmentation of Nimotuzumab for Preparation of 125I-F(ab’)2-Nimotuzumab as a Precursor for Preparing 125I-F(ab’)2-Nimotuzumab-NLS Radiopharmaceutical for Cancer Therapy

    International Nuclear Information System (INIS)

    Haryuni, R.D.; Bahtiar, A.; Soenarjo, S.; Harahap, Y.; Mutalib, A.; Ramli, M.; Hermanto, S.; Ardiyatno, C.N.; Susilo, V.Y.; Haffid, D.

    2014-01-01

    Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR). This monoclonal antibody has a relatively high molecular weight which slows penetration on tumor cells, making it less attractive in imaging kinetics and potentially elicits antibodies responses. Therefore, in this study nimotuzumab was fragmented to form a bivalent antibody [F(ab’) 2 ] and then labeled with 125 I to form 125 I-F(ab’) 2 -nimotuzumab which can be used further as a precursor for preparing 125 I-F(ab’) 2 -nimotuzumab-NLS (NLS = nuclear localization sequence) radiopharmaceutical for radioimmunotherapy. The aims of this study was to obtain characteristics of 125 I-F(ab’) 2 -nimotuzumab by comparing with the 125 I labeled-intact nimotuzumab ( 125 I-nimotuzumab). This study was initiated by purifying nimotuzumab by mean of dialysis. The purified nimotuzumab was then fragmented by using pepsin. The F(ab') 2 -nimotuzumab formed was then purified from its by-products which formed in fragmentation process by using a PD-10 column (consisted Sephadex G25). The intact nimotuzumab and its F(ab’)2 fragment were then labeled with the 125 I to form 125 I-nimotuzumab and 125 I-F(ab’) 2 -nimotuzumab. The radiochemical purity are 98.27 % and 93.24 %, respectively. Stability test results show that, both 125 I-nimotuzumab and 125 I-F(ab’) 2 -nimotuzumab are more stable at 4 °C than at room temperature storage and 37 °C. (author)

  1. Intermediate Fragment

    DEFF Research Database (Denmark)

    Kruse Aagaard, Anders

    2015-01-01

    ‘Engaging Through Architecture’ in 2015 by Aarhus School of Architecture as a part of the Ventura Lambrate Milan Design Week, where it was exhibited under the name Concrete. The fundamental pool of techniques and knowledge that set the agenda for the fragment was established before the intentions...

  2. Framing Fragmentation

    DEFF Research Database (Denmark)

    Bundgaard, Charlotte

    2009-01-01

    to create architectural meaning and give character to an architecture of fragmentation. Layers are both seen as conceptual as well as material frames which define certain strong properties or meanings in the architectural work. Defining layers is a way of separating and organizing; it both defines...

  3. Rock fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Brown, W.S.; Green, S.J.; Hakala, W.W.; Hustrulid, W.A.; Maurer, W.C. (eds.)

    1976-01-01

    Experts in rock mechanics, mining, excavation, drilling, tunneling and use of underground space met to discuss the relative merits of a wide variety of rock fragmentation schemes. Information is presented on novel rock fracturing techniques; tunneling using electron beams, thermocorer, electric spark drills, water jets, and diamond drills; and rock fracturing research needs for mining and underground construction. (LCL)

  4. Separation of cell-dependent antibody (CDA) and inhibitory antibody by protein-A affinity chromatography and the effect of fractions on antibody-dependent cellular cytotoxicity (ADCC).

    Science.gov (United States)

    Sato, N; Yabuki, Y; Toh, K; Ishii, Y; Kikuchi, K

    1979-01-01

    The nature of cell-dependent antibody (CDA) and the mechanism of inhibition of antibody-dependent cellular cytotoxicity (ADCC) were studied in the ADCC assay system in which culture cells of methylcholanthrene-induced rat fibrosarcoma (KMT-50) were used as target cells, xenogeneic antiserum (rabbit anti-KMT-50) as the CDA, and human peripheral blood leucocytes (PBL) as effector cells, respectively. By using protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-KMT-50 serum, CDA was shown to bind protein A. Complement dependent-cytotoxicity (CDC), however, was demonstrated in both the adsorbed fraction (eluate) and the non-adsorbed fraction (effluent) to protein A from the same affinity column chromatography. These data confirmed that CDA was IgG with an intact Fc portion. Inhibition of ADCC occurred by pretreatment of effector cells with rabbit anti-effector (human PBL) serum even with extremely small amounts of antiserum. Such inhibition was demonstrated with the eluate but not with the effluent from protein-A Sepharose CL-4B affinity column chromatography of rabbit anti-effector serum. F(ab')2 fragments of the same eluate (IgG) did not inhibit the ADCC activity. These data showed that the inhibition of ADCC was induced by the blocking of Fc receptors of effector cells with the Fc portions of IgG in anti-effector serum. The data obtained indicate the usefulness of protein A in separation and analysis of CDA and in investigation of the inhibitory mechanisms of ADCC. Images Figure 2 PMID:437836

  5. Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

    Science.gov (United States)

    Boyles, Jeffrey S; Atwell, Shane; Druzina, Zhanna; Heuer, Josef G; Witcher, Derrick R

    2016-11-01

    Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala 41 , Glu 44 , and His 45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859. © 2016 The Protein Society.

  6. Feasibility study of radioimmunoguided surgery of colorectal carcinomas using indium-111 CEA-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Curtet, C.; Aillet, G.; Kremer, M.; Thedrez, P.; Chatal, J.F.; Vuillez, J.P.; Daniel, G.; Chetanneau, A.; Visset, J.

    1990-01-01

    The study was undertaken to define the potential use of 111 In carcinoembryonic antigen-specific antibody labelled [CEA F(ab')2] for the radioimmunodetection of colorectal carcinoma using an intraoperative hand-held gamma probe. The use of a linear radioactive source allowed optimization of physical characteristics. The best results regarding sensitivity and resolution were obtained using a 5-mm thick tungsten alloy collimator. A simulation study with a liver phantom (22 MBq or 0.6 mCi) was performed to determine the effect of side scatter as opposed to direct background and showed that it is possible to detect small radioactive targets (3.7 KBq or 0.1 μCi) 4 cm from the phantom. A clinical study performed with ten patients showed that tumours with good uptake of CEA-specific antibody could be detected with sufficient contrast in two patients when the probe was used. Results of a biodistribution study performed after tumour fragment or normal tissue countings in a well counter showed high tumour uptake (above 8x10 -3 injected dose/g) and tumour-to-normal tissue ratios (between 2.5 and 20) in five patients. Results with the probe showed markedly lower ratios. There was no correlation between absolute tumour uptake and the count rates of tumour measured intraoperatively. This can be attributed to the degradation of depth resolution resulting from the high energy photopeak of gamma-emitting indium 111. (orig.)

  7. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    or from a position as business leader to a position in the state apparatus or in the Party and vice versa. To conceptualize the coexistence of the contradicting forces for further enterprise autonomy and continued central control that characterizes the evolving relationship between business groups...... and the Party-state, I suggest the notion of integrated fragmentation....

  8. Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto using anti-fab affinity chromatography reveals the low abundance of IgA.

    Directory of Open Access Journals (Sweden)

    James W Wynne

    Full Text Available There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

  9. Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (Pteropus alecto) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

    Science.gov (United States)

    Shiell, Brian J.; Beddome, Gary; Cowled, Christopher; Peck, Grantley R.; Huang, Jing; Grimley, Samantha L.; Baker, Michelle L.; Michalski, Wojtek P.

    2013-01-01

    There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. PMID:23308125

  10. A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220.

    Science.gov (United States)

    Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo

    2011-03-01

    A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Two-step targeting and dosimetry for small cell lung cancer xenograft with anti-NCAM/antihistamine bispecific antibody and radioiodinated bivalent hapten.

    Science.gov (United States)

    Hosono, M; Hosono, M N; Kraeber-Bodéré, F; Devys, A; Thédrez, P; Faivre-Chauvet, A; Gautherot, E; Barbet, J; Chatal, J F

    1999-07-01

    The "affinity enhancement system," a two-step targeting technique using bispecific antibody and radiolabeled bivalent hapten, has been reported to be useful for carcinoembryonic antigen-expressing tumors. The purpose of this study was to evaluate the efficacy of this method for targeting human small cell lung cancer using an antineural cell adhesion molecule antibody. Antineural cell adhesion molecule/antihistamine bispecific antibody NK1NBL1-679 was prepared by coupling an equimolecular quantity of a Fab' fragment of NK1NBL1 to a Fab fragment of antihistamine 679. Athymic mice inoculated with NCI-H69 small cell lung cancer cells expressing neural cell adhesion molecule were administered bispecific antibody and then 48 h later 125I-labeled bivalent histamine hapten. 125I-labeled intact NK1NBL1 was injected into other groups of mice. Biodistributions were examined as a function of time. In mice of the two-step targeting, tumor uptake was 2.5 +/- 0.2, 3.2 +/- 0.4, 6.4 +/- 2.0, 7.2 +/- 2.7, 6.1 +/- 2.1 and 2.2 +/- 0.4 %ID/g at 5, 30 min, 5, 24, 48 and 96 h, and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios were 1.4 +/- 1.1, 10.8 +/- 13.2 and 4.6 +/- 4.7, respectively, at 5 h, whereas 125I-labeled NK1NBL1 showed a tumor uptake of 5.7 +/- 0.4 %ID/g and tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios of 0.3 +/- 0.1, 1.1 +/- 0.2 and 0.9 +/- 0.1, respectively, at 5 h. These results were confirmed by autoradiographic studies, which demonstrated clear tumor-to-normal tissue contrast. Dosimetry showed that the affinity enhancement system could enhance the therapeutic potential of the antineural cell adhesion molecule antibody NK1NBL1. This two-step targeting method seems promising for the diagnosis and therapy of small cell lung cancer.

  12. Antibodies Damage the Resilience of Fimbriae, Causing Them To Be Stiff and Tangled.

    Science.gov (United States)

    Singh, Bhupender; Mortezaei, Narges; Savarino, Stephen J; Uhlin, Bernt Eric; Bullitt, Esther; Andersson, Magnus

    2017-01-01

    As adhesion fimbriae are a major virulence factor for many pathogenic Gram-negative bacteria, they are also potential targets for antibodies. Fimbriae are commonly required for initiating the colonization that leads to disease, and their success as adhesion organelles lies in their ability to both initiate and sustain bacterial attachment to epithelial cells. The ability of fimbriae to unwind and rewind their helical filaments presumably reduces their detachment from tissue surfaces with the shear forces that accompany significant fluid flow. Therefore, the disruption of functional fimbriae by inhibiting this resilience should have high potential for use as a vaccine to prevent disease. In this study, we show that two characteristic biomechanical features of fimbrial resilience, namely, the extension force and the extension length, are significantly altered by the binding of antibodies to fimbriae. The fimbriae that were studied are normally expressed on enterotoxigenic Escherichia coli, which are a major cause of diarrheal disease. This alteration in biomechanical properties was observed with bivalent polyclonal antifimbrial antibodies that recognize major pilin subunits but not with the Fab fragments of these antibodies. Thus, we propose that the mechanism by which bound antibodies disrupt the uncoiling of natural fimbria under force is by clamping together layers of the helical filament, thereby increasing their stiffness and reducing their resilience during fluid flow. In addition, we propose that antibodies tangle fimbriae via bivalent binding, i.e., by binding to two individual fimbriae and linking them together. Use of antibodies to disrupt physical properties of fimbriae may be generally applicable to the large number of Gram-negative bacteria that rely on these surface-adhesion molecules as an essential virulence factor. Our study shows that the resiliency of colonization factor antigen I (CFA/I) and coli surface antigen 2 (CS2) fimbriae, which are current

  13. Extending green technology innovations to enable greener fabs

    Science.gov (United States)

    Takahisa, Kenji; Yoo, Young Sun; Fukuda, Hitomi; Minegishi, Yuji; Enami, Tatsuo

    2015-03-01

    Semiconductor manufacturing industry has growing concerns over future environmental impacts as fabs expand and new generations of equipment become more powerful. Especially rare gases supply and price are one of prime concerns for operation of high volume manufacturing (HVM) fabs. Over the past year it has come to our attention that Helium and Neon gas supplies could be unstable and become a threat to HVM fabs. To address these concerns, Gigaphoton has implemented various green technologies under its EcoPhoton program. One of the initiatives is GigaTwin deep ultraviolet (DUV) lithography laser design which enables highly efficient and stable operation. Under this design laser systems run with 50% less electric energy and gas consumption compared to conventional laser designs. In 2014 we have developed two technologies to further reduce electric energy and gas efficiency. The electric energy reduction technology is called eGRYCOS (enhanced Gigaphoton Recycled Chamber Operation System), and it reduces electric energy by 15% without compromising any of laser performances. eGRYCOS system has a sophisticated gas flow design so that we can reduce cross-flow-fan rotation speed. The gas reduction technology is called eTGM (enhanced Total gas Manager) and it improves gas management system optimizing the gas injection and exhaust amount based on laser performances, resulting in 50% gas savings. The next steps in our roadmap technologies are indicated and we call for potential partners to work with us based on OPEN INNOVATION concept to successfully develop faster and better solutions in all possible areas where green innovation may exist.

  14. High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC.

    Directory of Open Access Journals (Sweden)

    Alexey V Gribenko

    2016-09-01

    Full Text Available The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 - group 1, mAB 305-78-7 - group 2, and mAB 305-101-8 - group 3 were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS. All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7 with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.

  15. Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice

    DEFF Research Database (Denmark)

    Back, T.; Haraldsson, B.; Hultborn, R

    2009-01-01

    Besides bone marrow, the kidneys are often dose-limiting organs in internal radiotherapy. The effects of high-linear energy transfer (LET) radiation on the kidneys after alpha-radioimmunotherapy (alpha-RIT) with the alpha-particle emitter, (211)At, were studied in nude mice by serial measurements...... of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals...

  16. Fragmentation based

    Directory of Open Access Journals (Sweden)

    Shashank Srivastava

    2014-01-01

    Gaining the understanding of mobile agent architecture and the security concerns, in this paper, we proposed a security protocol which addresses security with mitigated computational cost. The protocol is a combination of self decryption, co-operation and obfuscation technique. To circumvent the risk of malicious code execution in attacking environment, we have proposed fragmentation based encryption technique. Our encryption technique suits the general mobile agent size and provides hard and thorny obfuscation increasing attacker’s challenge on the same plane providing better performance with respect to computational cost as compared to existing AES encryption.

  17. Architectural fragments

    DEFF Research Database (Denmark)

    Bang, Jacob Sebastian

    2018-01-01

    the photographs as a starting point for a series of paintings. This way of creating representations of something that already exists is for me to see a way forward in the "decoding" of my own models into other depictions. The models are analyzed through a series of representations in different types of drawings....... I try to invent the ways of drawing the models - that decode and unfold them into architectural fragments- into future buildings or constructions in the landscape. [1] Luigi Moretti: Italian architect, 1907 - 1973 [2] Man Ray: American artist, 1890 - 1976. in 2015, I saw the wonderful exhibition...

  18. Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

    Science.gov (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

    2017-04-01

    Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  19. Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma.

    Science.gov (United States)

    Haylock, Anna-Karin; Spiegelberg, Diana; Mortensen, Anja C; Selvaraju, Ram K; Nilvebrant, Johan; Eriksson, Olof; Tolmachev, Vladimir; Nestor, Marika V

    2016-02-01

    We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodistribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using

  20. Defect reduction in different periods of a fab's life

    Science.gov (United States)

    Kallis, Norbert

    1997-08-01

    The reduction of yield loss caused by particles is a key topic of productivity enhancement. It deals with many components of a fab's structure: (1) Cultural mindset of managers, engineers, and operators -- to have champions for that mission, (2) selection of defect metrology tools -- with high throughput and reliable data, (3) single equipment characterization -- to set challenging specs according to the best performing tool, (4) inline reaction procedures -- to transfer knowledge and responsibility to the operators, (5) statistical evaluation of large datasets -- including correlation to electrical yield, (6) and to trace back the way to the single tools or the low performing processes. This paper discusses some topics to be considered during process and equipment planning, ramp up, and mature production. It has been proven to be successful during a very aggressive ramp up in wafer starts and an even more convincing yield increase of a fab, that now started the production of DRAM's in quarter micron technology. It will conclude in an understanding, that besides a reliable statistical methodology the human performance is the key: can we really integrate people of different groups and levels of responsibility and motivate them to take joint actions for yield goals?

  1. Engineered antibodies for monitoring of polynuclear aromatic hydrocarbons

    International Nuclear Information System (INIS)

    Karu, A.E.; Li, Q.X.; Roberts, V.A.

    1998-01-01

    'The long-term goal of this project is to develop antibodies and antibody-based methods for detection and recovery of polynuclear aromatic hydrocarbons (PAHs) and PAH adducts that are potential biomarkers in environmental and biological samples. The inherent cross-reactivity will be exploited by pattern recognition methods. Dr. Karu''s laboratory uses new haptens representing key PAHs to derive recombinant Fab (rFab) and single-chain Fv (scFv) antibodies from hybridoma lines and combinatorial phage display libraries. Computational models of the haptens and combining sites made by Dr. Roberts''s group are used to guide antibody engineering by mutagenesis. Dr. Li''s laboratory develops enzyme immunoassays (EIAs), sensors, and immunoaffinity methods that make use of the novel haptens and antibodies for practical analytical applications in support of DOE''s mission. This report summarizes work completed in one and one-half years of a 3-year project, with close collaboration between the three research groups. Dr. Alexander Karu''s laboratory: the authors proceeded with the two strategies described in the original proposal. Site-directed mutagenesis was used to correct differences in the rFab N-terminal amino acids that were introduced by the degenerate PCR primers used for gene amplification. The binding constants of the rFabs with the corrected sequences will be compared with those of the parent MAbs, and should be very similar. The 4D5 and 10C10 heavy and light chain sequences are being moved to the pCOMB3H phagemid vector to facilitate selection of new engineered mutants.'

  2. CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro

    NARCIS (Netherlands)

    Molema, G; Tervaert, JWC; Kroesen, BJ; Helfrich, W; Meijer, DKF; de Leij, LFMH

    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells; intravenous administration of BIS-1 F(ab')(2) to carcinoma patients in a phase I/II clinical trial, caused

  3. Structural basis of activation-dependent binding of ligand-mimetic antibody AL-57 to integrin LFA-1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongmin; Liu, Jin-huan; Yang, Wei; Springer, Timothy; Shimaoka, Motomu; Wang, Jia-huai; (CH-Boston); (DFCI)

    2010-09-21

    The activity of integrin LFA-1 ({alpha}{sub L}{beta}{sub 2}) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of {alpha}{sub L} chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.

  4. Hepatic targeting and hypocholesterolemic effect of lactosaminated Fab against low density lipoproteins

    International Nuclear Information System (INIS)

    Bernini, F.; Bocan, T.M.A.; Via, D.P.; Gotto, A.M. Jr.; Smith, L.C.

    1986-01-01

    Lactosaminated Fab (lac-Fab) specific for human LDL induces plasma clearance and uptake of circulating [ 125 -I]-iodo-LDL in rat, a process mediated by galactose receptors of the liver. This study demonstrates that lac-Fab is a specific carrier of LDL to the liver parenchymal cells and exhibits hypocholesterolemic activity in vivo. Rats were injected with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-LDL (diI-LDL) or 6 mg of LDL plus tracer amounts of [ 125 I]-iodo-LDL. After 10-20 min, the animals received 3-10 mg of lac-Fab. Histologic examination of the liver sections showed the uptake of diI-LDL in the parenchymal cells, as compared to diI-acetyl-LDL which was localized in sinusoidal cells. More than 85% of human LDL disappeared within 2.5 hr after lac-Fab injection, reducing plasma cholesterol from 133.0 +/- 12.6 mg/dl to 66.4 +/- 8.0 mg/dl, the basal value in the rat. In control rats, only about 20% of radioactivity and cholesterol disappeared at 2.5 hr. HDL levels were unaffected. The authors conclude that lac-Fab is a specific carrier of LDL to hepatocytes and can lower plasma LDL-cholesterol in vivo. Lac-Fab specific for other antigens may act as specific carriers of molecule or macromolecules to hepatocytes

  5. The community FabLab platform: applications and implications in biomedical engineering.

    Science.gov (United States)

    Stephenson, Makeda K; Dow, Douglas E

    2014-01-01

    Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

  6. Fragmented Authoritarianism or Integrated Fragmentation

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik

    proved their influence by obstructing the creation of new ministries and regulatory commissions that would limit their powers. The heads of these business groups often outrank their counterparts in state administrative organs and bureaus that are supposed to regulate their activities. The increased role...... of these business leaders prompts the question of whether we are seeing the development of distinct interest groups that could challenge Party and state authority and create a fragmented polity. However, through the nomenklatura system the Party has an important instrument of control to wield over business groups....... Through this system the Party controls the appointment and promotion of the heads of the most important state-owned enterprises. The nomenklatura system also enables the Party to rotate leaders in big business from a position as CEO in one company to a similar position in another state-owned company...

  7. Meleagrin, a new FabI inhibitor from Penicillium chryosogenum with at least one additional mode of action.

    Directory of Open Access Journals (Sweden)

    Chang Ji Zheng

    Full Text Available Bacterial enoyl-acyl carrier protein reductase (FabI is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.

  8. Automated reticle inspection data analysis for wafer fabs

    Science.gov (United States)

    Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

    2009-04-01

    To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity Defect(R) data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

  9. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    International Nuclear Information System (INIS)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

    1982-01-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

  10. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  11. Anti-sulfotyrosine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  12. Antitumor activity of anti-C-ERC/mesothelin monoclonal antibody in vivo.

    Science.gov (United States)

    Inami, Koichi; Abe, Masaaki; Takeda, Kazuyoshi; Hagiwara, Yoshiaki; Maeda, Masahiro; Segawa, Tatsuya; Suyama, Masafumi; Watanabe, Sumio; Hino, Okio

    2010-04-01

    Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.

  13. 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

  14. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

    Directory of Open Access Journals (Sweden)

    Davide Corti

    2010-01-01

    Full Text Available The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

  15. Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification.

    Science.gov (United States)

    Zhang, Qingchun; Goetze, Andrew M; Cui, Huanchun; Wylie, Jenna; Tillotson, Ben; Hewig, Art; Hall, Michael P; Flynn, Gregory C

    2016-05-01

    Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co-purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D-LC-HDMS(E) approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (∼10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co-purification with individual Fc and (Fab')2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708-717, 2016. © 2016 American Institute of Chemical Engineers.

  16. Design of Fab-based chimeric antibodies against Bothrops asper toxins

    DEFF Research Database (Denmark)

    M. Haack, Aleksander; B. Hallgren, Malte; U. W. Friis, Rasmus

    Snakebite is one of the world’s most neglected tropical diseases, with an estimated 5 million bites per year, resulting in about 125.000 deaths. The only current treatment for snakebite envenoming is antiserum derived from the blood of immunized mammals(typically horses). These antisera are expen...... are expensive to produce and carry a high risk of causing hyper-allergic reactions in human recipients due to their heterologous origin. Here we report the discovery of chimeric scFvs against Bothrops asper toxins....

  17. SpiderFab: Process for On-Orbit Construction of Kilometer-Scale Apertures

    Data.gov (United States)

    National Aeronautics and Space Administration — SpiderFab is a novel additive manufacturing technique which combines the techniques of fused deposition modeling (FDM) with methods derived from automated composite...

  18. SpiderFab: Architecture for On-Orbit Construction of Kilometer-Scale Apertures

    Data.gov (United States)

    National Aeronautics and Space Administration — The SpiderFab effort has investigated the value proposition and technical feasibility of radically changing the way we build and deploy spacecraft in order to escape...

  19. Functional characterization of triclosan-resistant enoyl-acyl-carrier protein reductase (FabV in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Yong-Heng Huang

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR, FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholera fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acylhomoserine lactones (AHLs production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore to the fabV mutant strain the ability to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity.

  20. Polyvalent horse F(Ab`) 2 snake antivenom: Development of ...

    African Journals Online (AJOL)

    A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica crude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired ...

  1. Crystal Structure of HIV-1 Primary Receptor CD4 i Complex with a Potent Antiviral Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, M.M.; Hong, X.; Seaman, M.S.; Rits-Vollock, S.p Kao, C.Y.; Ho, D.D.; Chen, B.

    2010-06-18

    Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 {angstrom} resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.

  2. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A. (Scripps); (IAVI)

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  3. Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

    Science.gov (United States)

    Karau, Melissa J; Tilahun, Mulualem E; Krogman, Ashton; Osborne, Barbara A; Goldsby, Richard A; David, Chella S; Mandrekar, Jayawant N; Patel, Robin; Rajagopalan, Govindarajan

    2017-10-03

    Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

  4. Single mutation induced H3N2 hemagglutinin antibody neutralization: a free energy perturbation study.

    Science.gov (United States)

    Zhou, Ruhong; Das, Payel; Royyuru, Ajay K

    2008-12-11

    The single mutation effect on the binding affinity of H3N2 viral protein hemagglutinin (HA) with the monoclonical antibody fragment (Fab) is studied in this paper using the free energy perturbation (FEP) simulations. An all-atom protein model with explicit solvents is used to perform an aggregate of several microsecond FEP molecular dynamics simulations. A recent experiment shows that a single mutation in H3N2 HA, T131I, increases the antibody-antigen dissociation constant Kd by a factor of approximately 4000 (equivalent to a binding affinity decrease of approximately 5 kcal/mol), thus introducing an escape of the antibody (Ab) neutralization. Our FEP result confirms this experimental finding by estimating the HA-Ab binding affinity decrease of 5.2 +/- 0.9 kcal/mol but with a somewhat different molecular mechanism from the experimental findings. Detailed analysis reveals that this large binding affinity decrease in the T131I mutant is mainly due to the displacement of two bridge water molecules otherwise present in the wild-type HA/Ab interface. The decomposition of the binding free energy supports this observation, as the major contribution to the binding affinity is from the electrostatic interactions. In addition, we find that the loss of the binding affinity is also related to the large conformational distortion of one loop (loop 155-161) in the unbound state of the mutant. We then simulate all other possible mutations for this specific mutation site T131, and predict a few more mutations with even larger decreases in the binding affinity (i.e., better candidates for antibody neutralization), such as T131W, T131Y, and T131F. As for further validation, we have also modeled another mutation, S157L, with experimental binding affinity available (Kd increasing approximately 500 times), and found a binding affinity decrease of 4.1 +/- 1.0 kcal/mol, which is again in excellent agreement with experiment. These large scale simulations might provide new insights into the

  5. Molecular perspective of antibody aggregates and their adsorption on Protein A resin.

    Science.gov (United States)

    Yu, Deqiang; Song, Yuanli; Huang, Richard Y-C; Swanson, Ryan K; Tan, Zhijun; Schutsky, Elizabeth; Lewandowski, Angela; Chen, Guodong; Li, Zheng Jian

    2016-07-29

    Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-