Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

International Nuclear Information System (INIS)

Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

2016-01-01

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

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...

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

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...

Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

International Nuclear Information System (INIS)

Andrew, S.M.; Pimm, M.V.; Baldwin, R.W.; Perkins, A.C.

1986-01-01

An IgG1 mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab) 2 and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA producing human tumour xenografts and injected with 131 I-labelled fragments showed earlier and superior imaging of tumours than did 131 I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody. (orig.)

Application of 99mTc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

International Nuclear Information System (INIS)

Matsumura, Hiroomi

1999-01-01

Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with 99m Tc, preventing depression of the antigen-binding activity. 99m Tc-labeled monoclonal antibody A7, 99m Tc-labeled chimeric Fab fragments of monoclonal antibody A7, 99m Tc-labeled normal mouse IgG and 99m Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Science.gov (United States)

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

Science.gov (United States)

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-01-01

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma. PMID:27009516

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.

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.)

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.

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

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

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

Science.gov (United States)

Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto 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 subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.

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.

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

Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

Science.gov (United States)

Bailey, Lucas J; Sheehy, Kimberly M; Dominik, Pawel K; Liang, Wenguang G; Rui, Huan; Clark, Michael; Jaskolowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A

2018-02-02

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. Copyright © 2017 Elsevier Ltd. All rights reserved.

Matching the Decay Half-Life with the Biological Half-Life: ImmunoPET Imaging with 44Sc-Labeled Cetuximab Fab Fragment

Science.gov (United States)

2015-01-01

Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of 44Sc–CHX-A″-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-A″-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments. PMID:25389697

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes*

Science.gov (United States)

Nelson, Alfreda D.; Hoffmann, Michele M.; Parks, Christopher A.; Dasari, Surendra; Schrum, Adam G.; Gil, Diana

2012-01-01

Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes. PMID:23109335

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

Science.gov (United States)

Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

2013-12-31

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

Science.gov (United States)

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

A kit to prepare {sup 111}In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer

Energy Technology Data Exchange (ETDEWEB)

Scollard, Deborah A.; Chan, Conrad [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Holloway, Claire M.B. [Department of Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 1H1 (Canada); Reilly, Raymond M., E-mail: raymond.reilly@utoronto.c [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3E2 (Canada); Toronto General Research Institute, University Health Network, Toronto, ON, M5G 2M9 (Canada)

2011-01-15

Introduction: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for {sup 111}In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ({sup 111}In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of {sup 111}In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. Methods: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37{sup o}C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency ({>=}85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of {sup 111}In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K{sub a}=0.6-9.6x10{sup 7} L/mol; B{sub max}=0.6-10.4x10{sup 6} sites/cell). {sup 111}In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of {sup 111}InCl{sub 3} to a single kit vial and incubating for 30 min at room temperature. {sup 111}In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for p

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

Science.gov (United States)

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

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

Science.gov (United States)

Kulmala, Antti; Huovinen, Tuomas; Lamminmäki, Urpo

2017-06-19

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia.

Science.gov (United States)

Farag, Mina; Badowski, Dominika; Koschny, Ronald; Skopp, Gisela; Brcic, Andreas; Szabo, Gabor B

2017-12-01

Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options. We report the case of a suicidal yew ingestion admitted to our hospital under prolonged cardiopulmonary resuscitation due to pulseless electrical activity. Extra-corporeal life support (ECLS) was established to maintain adequate organ perfusion. Repeated administration of digoxin-specific Fab antibody fragments, which cross-react with taxine, was associated with an immediate conversion from asystole to broad-complex bradycardia and a gradual normalization of the electrocardiogram (ECG). This was paralleled by a recovery of the cardiac function and weaning from the ECLS. The taxine metabolite 3,5-dimethoxyphenol could be detected by mass spectrometry before but not after the first Fab-fragment treatment. In contrast, the total amount of taxine (including the neutralized, Fab fragment-bound fraction) was increased after each Fab fragment administration, suggesting an accumulation of neutralized, since antibody-bound taxine in the blood by anti-digoxin Fab fragments. In conclusion, the successful clinical course of this case suggests a benefit of an early anti-digoxin Fab-fragment administration for the treatment of yew intoxication. Copyright © 2017 Elsevier Inc. All rights reserved.

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)

Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

Science.gov (United States)

Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

2015-05-01

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

[Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

Science.gov (United States)

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2016-01-01

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

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

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

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

Science.gov (United States)

2013-01-01

Background Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Results Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Conclusions Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into

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

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

Science.gov (United States)

Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

2017-05-08

The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

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 CVFab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

Radiolabeled F(ab')2-cetuximab for theranostic purposes in colorectal and skin tumor-bearing mice models.

Science.gov (United States)

Bellaye, P-S; Moreau, M; Raguin, O; Oudot, A; Bernhard, C; Vrigneaud, J-M; Dumont, L; Vandroux, D; Denat, F; Cochet, A; Brunotte, F; Collin, B

2018-05-17

This study aimed to investigate theranostic strategies in colorectal and skin cancer based on fragments of cetuximab, an anti-EGFR mAb, labeled with radionuclide with imaging and therapeutic properties, 111 In and 177 Lu, respectively. We designed F(ab') 2 -fragments of cetuximab radiolabeled with 111 In and 177 Lu. 111 In-F(ab') 2 -cetuximab tumor targeting and biodistribution were evaluated by SPECT in BalbC nude mice bearing primary colorectal tumors. The efficacy of 111 In-F(ab') 2 -cetuximab to assess therapy efficacy was performed on BalbC nude mice bearing colorectal tumors receiving 17-DMAG, an HSP90 inhibitor. Therapeutic efficacy of the radioimmunotherapy based on 177 Lu-F(ab') 2 -cetuximab was evaluated in SWISS nude mice bearing A431 tumors. Radiolabeling procedure did not change F(ab') 2 -cetuximab and cetuximab immunoreactivity nor affinity for HER1 in vitro. 111 In-DOTAGA-F(ab') 2 -cetuximab exhibited a peak tumor uptake at 24 h post-injection and showed a high tumor specificity determined by a significant decrease in tumor uptake after the addition of an excess of unlabeled-DOTAGA-F(ab') 2 -cetuximab. SPECT imaging of 111 In-DOTAGA-F(ab') 2 -cetuximab allowed an accurate evaluation of tumor growth and successfully predicted the decrease in tumor growth induced by 17-DMAG. Finally, 177 Lu-DOTAGA-F(ab') 2 -cetuximab radioimmunotherapy showed a significant reduction of tumor growth at 4 and 8 MBq doses. 111 In-DOTAGA-F(ab') 2 -cetuximab is a reliable and stable tool for specific in vivo tumor targeting and is suitable for therapy efficacy assessment. 177 Lu-DOTAGA-F(ab') 2 -cetuximab is an interesting theranostic tool allowing therapy and imaging.

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

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

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.

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

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

Science.gov (United States)

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

Science.gov (United States)

Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation

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)

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment

International Nuclear Information System (INIS)

Stangl, Stefan; Themelis, George; Friedrich, Lars; Ntziachristos, Vasilis; Sarantopoulos, Athanasios; Molls, Michael; Skerra, Arne; Multhoff, Gabriele

2011-01-01

Background and purpose: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. Materials and methods: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. Results: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 deg. C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 deg. C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. Conclusions: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

Science.gov (United States)

Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

2015-06-01

The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

Science.gov (United States)

Duan, Dongyu; Wang, Aiping; Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

Science.gov (United States)

Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

2015-01-01

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

Science.gov (United States)

Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

2015-02-02

We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

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.

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

Science.gov (United States)

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

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

International Nuclear Information System (INIS)

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D.; Graves, Stephen A.; Nickles, Robert J.; Liu, Bai; Wong, Hing C.; Cai, Weibo

2015-01-01

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 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 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 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. 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.)

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

Science.gov (United States)

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

2015-07-01

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 (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 (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 (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. (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.

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.)

Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

Science.gov (United States)

Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

2016-01-20

Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

International Nuclear Information System (INIS)

Miao, H.-Q.; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping

2006-01-01

Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies

Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

Science.gov (United States)

Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

2015-01-01

In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

Nebulized Anti-IL-13 Monoclonal Antibody Fab' Fragment Reduces Allergen-Induced Asthma

OpenAIRE

Hacha, Jonathan; Tomlinson, K; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noël, Agnès; Palframan, R; Guéders, Maud; Cataldo, Didier

2012-01-01

Rationale: Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. Objectives: We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness and remodeling in an experime...

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.

Antibody guided targeting of non-small cell lung cancer using 111In-labeled HMFG1 F(ab')2 fragments

International Nuclear Information System (INIS)

Kalofonos, H.P.; Sivolapenko, G.B.; Courtenay-Luck, N.S.

1988-01-01

Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111 In. Quality control studies showed efficient incorporation of 111 In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111 In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111 In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111 In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

Science.gov (United States)

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

Science.gov (United States)

Shatz, Whitney; Hass, Philip E; Mathieu, Mary; Kim, Hok Seon; Leach, Kim; Zhou, Michelle; Crawford, Yongping; Shen, Amy; Wang, Kathryn; Chang, Debby P; Maia, Mauricio; Crowell, Susan R; Dickmann, Leslie; Scheer, Justin M; Kelley, Robert F

2016-09-06

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Localization of 131I-labeled p97-specific Fab fragments in human melanoma as a basis for radiotherapy

International Nuclear Information System (INIS)

Larson, S.M.; Carrasquillo, J.A.; Krohn, K.A.

1983-01-01

33 patients with advanced malignant melanoma were studied after intravenous administration of 131 I-labeled Fab fragments specific for p97, an oncofetal glycoprotein of human melanoma. In all, 47 gamma camera imaging studies were performed for the purpose of localization of metastatic deposits. In addition to tumor, 131 I-Fab uptake was also seen in liver and kidney. 20 of these studies included simultaneous administration of both an 131 I-labeled Fab specific for p97, and an 125 I-labeled Fab not specific for p97. Blood clearance of p97-specific Fab was significantly more rapid than for nonspecific Fab. Eight of these patients had biopsies of subcutaneous nodules at 48 and 72 h postinjection in order to assess whether localization of radioactivity was antigen specific. Antigen-specific localization was observed with average ratios of specific/nonspecific uptake of 3.7 (48 h) and 3.4 (72 h); uptake was strongly correlated with tumor p97 concentration (r . 0.81, P less than 0.01). Also, imaging studies of the bio-distribution of 131 I-labeled anti-p97 Fab in patients selected for high p97 tumor concentration showed avid tumor uptake and more prolonged retention of labeled Fab in tumor than in normal tissues. Based on these studies, we estimated that total 131 I doses of 500 mCi could be safely given to patients before dose-limiting toxicity would be observed

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

display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured...... was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia...

Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

Science.gov (United States)

Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

2001-09-01

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

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

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

Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

Science.gov (United States)

Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

2015-05-15

The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Construction and expression of a functional monoclonal antibody SZ-51 specific for GMP-140 chimeric fab fragment in Escherichia coli

International Nuclear Information System (INIS)

Gu Jianming; Zhang Xiaomin; Xia Lijun; Wan Haiying; Liu Yue; Li Peixia; Ruan Changgeng

1996-04-01

The variable region cDNAs of a monoclonal antibody SZ-51 specific for α-granule membrane protein (GMP-140) on the surface of activated human platelets were spliced with the constant region cDNA of the heavy chain CH1 and light chain k of human Ig G by means of the gene recombination techniques. The above recombinant gene was amplified by the polymerase chain reaction (PCR). The expression vector of phage plasmid pHEN1 SZ-51 Fab/Hu was constructed. The pHEN1-51 Fab/Hu was introduced into non-suppressor E. coli HB2151. The amount of expression of SZ-51 chimeric Fab/Hu measured by quantitative ELISA was about 500 μg/L. Western blot demonstrated that the SZ-51 chimeric Fab fragment could specifically bind to GMP-140. (2 figs.)

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

International Nuclear Information System (INIS)

Baral, Pravas Kumar; Wieland, Barbara; Swayampakula, Mridula; Polymenidou, Magdalini; Aguzzi, Adriano; Kav, Nat N. V.; James, Michael N. G.

2011-01-01

The complex of MoPrP(120–232) and Fab POM1 has been crystallized (space group C2, unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°). Diffraction data to 2.30 Å resolution have been collected using synchrotron radiation. Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP c to the pathogenic isoform PrP sc . Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120–232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

Science.gov (United States)

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

Nonspecific bowel activity in imaging inflammation with Tc-99m labelled monoclonal anti-NCA-90 Fab' fragment MN3

International Nuclear Information System (INIS)

Ivancevic, V.; Wolter, A.; Munz, D.L.

2001-01-01

Since the Tc-99m labelled monoclonal anti-NCA 90 granulocyte antibody Fab' fragment MN3 (MN3 Fab') might be of interest for imaging abdominal inflammation which could be hampered by nonspecific bowel activity, we prospectively investigated the appearance of bowel activity in MN3 Fab' imaging. Methods: Eighty consecutive patients (age range 12-85 years) referred for suspected nonabdominal, mostly musculoskeletal infection, were included. Abdominal inflammation was excluded clinically and there were no signs of inflammatory bowel disease in the patients' histories. One, 5, and 24 hours after introvenous injection of up to 1.1 GBq of MN3 Fab' planar images of the abdomen were performed. Bowel activity was graded visually using a 5-point scale. Results: The one (N = 80), 5 (N = 79), and 24 (N = 52) hour images revealed 46 (10%), 162 (34%), and 173 (55%) accumulating bowel segments, respectively, in 37 (46%), 69 (87%), and 52 (100%) patients. The mean intensity score per accumulating segment was 1.1, 1.8 and 2.7 (p = 0), respectively. Relative frequencies of appearance of the small intestine were 38%, 57%, and 21%, ileocaecal region 6%, 53%, and 48%, ascending colon 5%, 67%, and 89%, transverse colon 1%, 9%, and 69%, descending colon 8%, 15%, and 67%, and rectosigmoid 0%, 4%, and 38%, respectively. Follow-up investigations in 13 patients revealed diverging uptake patterns. Conclusion: Nonspecific bowel activity is often present in the early and almost always and more intense, in the delayed images. Early imaging at one hour after administration seems feasible, but a loss in sensitivity has to be considered. Thus, nonspecific bowel activity can be anticipated to be a pitfall in imaging abdominal inflammation with MN3 Fab'. (orig.) [de

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

On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin.

Science.gov (United States)

Pont, Laura; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

2017-08-01

This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab' antibody fragments (Fab'-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (M r ~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab' fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab'-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL -1 , limits of detection (LODs) were around 0.5µgmL -1 (50-fold lower than by CE-MS, ~25µgmL -1 ) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab'-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability. Copyright © 2017. Published by Elsevier B.V.

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

Localization of mammary tumors in vivo with 131I-labeled Fab fragments of antibodies against mouse mammary epithelial (MME) antigens

International Nuclear Information System (INIS)

Wilbanks, T.; Peterson, J.A.; Miller, S.; Kaufman, L.; Ortendahl, D.; Ceriani, R.L.

1981-01-01

The Fab fragments of antibodies against cell-type-specific surface antigens of mouse mammary epithelial cells (MME-antigens) were used to localize mammary tumors successfully. The radioiodine-labeled anti-MME (Fab) was injected into mice carrying simulated mammary metastases, and after 24 hours the amount of label per gram of excised tissue was several times greater in the tumor than in liver, brain, lung, or muscle. Kidney showed considerable accumulation of label but this appeared to be nonspecific. Kinetic studies revealed a rapid elimination of labeled Fab in the urine with only 1% of the injected dose remaining in the entire blood pool after 24 hours. Wit a high-purity germanium camera, mammary tumors were clearly located ty the 131 I-labeled anti-MME (Fab), and normalization to /sup 99m/Tc-pertechnetate distribution in the animal increased the specificity. The density of 131 I-label was fourfold greater over the mammary tumor than over comparable areas of the mouse. No accumulation of 131 I-anti-MME (Fab) was observed in nonmammary tumors nor in mammary tumors when labeled nonspecific Fab was used. An analogous system using an antihuman mammary epithelial antiserum is being developed for localization of breast metastases in humans

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

Science.gov (United States)

Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2015-12-01

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

Development of radioinmunoconjugate 90Y-DOTA-nimotuzumab-Fab for therapy of EGFR over expressing tumors

International Nuclear Information System (INIS)

Rodriguez, A. M.; Alonso, L. M.; Gongora, M.; Leyva, R.; Solana, A.

2015-01-01

Many monoclonal antibodies conjugated with 1,4,7,10-tetraaza cyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA) and radiolabeled with 90 Y, have been used for radioimmunotherapy. As know IgG molecules are heavy proteins with a molecular weight of approximately 150 kDa. Accordingly, intact IgG antibodies may have significant slow kinetics biodistribution and severely limited properties of tissue penetration. Antibody fragments labeled with radio metals could be promising radiopharmaceuticals for imaging and non-invasive therapy due to its high affinity to the tumor, the lack of effector function and rapid pharmacokinetic. In this work, the nimotuzumab Fab fragment was obtained by cleavage with papain in molar excess. After separating the reaction mixture in three steps using affinity, size exclusion and ion exchange chromatography; the Fab fragment showed high values of purity, integrity and identity. The Fab fragment was derivatized with DOTA and labeled with 90 Y. The radioimmunoconjugate with high radiochemical yield was assessed by in vitro stability with an excess of 50mM DTPA. The development of 90 Y-DOTA-Nimotuzumab-Fab radioimmunoconjugate allows to count on as a potential agent for radioimmunotherapy. (Author)

Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma

International Nuclear Information System (INIS)

Carrel, Francois; Amstutz, Hanspeter; Novak-Hofer, Ilse; Schubiger, P. August

1997-01-01

Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (K d ∼10 -9 M) to target cells in vitro but formed aggregates at 37 deg. C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent 'refolding' could be achieved. In contrast, chCE7-Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (K d ∼10 -10 M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67 Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab') 2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab') 2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the radioiodinated forms, tumor uptake

Technetium-99 labelling of DD-3B6/22 antifibrin monoclonal antibody fragmented Fab' for thrombus imaging

International Nuclear Information System (INIS)

Lee, F-T.; Boniface, G.R.; Lambrecht, R.M.; Rylatt, D.B.; Bundesen, P.G.

1993-01-01

The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99m ( 99mTc ) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99m Tc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99m Tc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans. 17 refs., 3 tabs., 5 figs

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

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.

Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

International Nuclear Information System (INIS)

Obmolova, Galina; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

2014-01-01

The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization

Expression of a humanized SZ-63 McAb functional recombinant Fab fragment in E. Coli

International Nuclear Information System (INIS)

Xia Lijun; Gu Jianming; Zhang Xiaoming; Liu Yue; Wan Haiying; Li Peixia; Ruan Changgeng

1995-06-01

MRNA was selected on oligo(dT)-cellulose from total RNA isolated from SZ-63 hybridoma cells by CsCl ultracentrifugation. cDNA coding for heavy and light variable regions were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified fragments were then cloned and sequenced by the 32 P labelled sanger dideoxy-mediated chain-termination method. The nucleotides of VH and Vκ are 354 and 321 respectively, the amino acid sequence of heavy and light chain of SZ-63 were also deduced. Then, linking the variable genes of SZ-63 with human immunoglobulin γ 1 CH and κ VL genes, constructing pHEN1-63 Fab/Hu chimera for expression and transforming E. coli HB2151. The expressed chimeric SZ-63 Fab was soluble. Both ELISA and Western blot results showed the expression products could specifically bind with cross-linked fibrin and the content in expression culture was about 225 μg/L. (5 figs.)

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)

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

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT

Science.gov (United States)

2011-01-01

Background Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Methods Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. Results 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen

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

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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.

Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8

International Nuclear Information System (INIS)

Uchtenhagen, Hannes; Sourial, Samer; Friemann, Rosmarie; Ehnlund, Mariethe; Spetz, Anna-Lena; Harris, Robert A.; Madhurantakam, Chaithanya; Achour, Adnane

2009-01-01

Neutralizing Fab fragments of the HIV-2-binding murine antibody 7C8 were generated after purification from hybridoma cell-culture supernatant. Crystallization conditions were determined and diffraction data were collected to 2.7 Å resolution. 7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris–HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3 2 21, with unit-cell parameters a = b = 100.1, c = 196.8 Å, and diffracted to 2.7 Å resolution

Anti-colchicine Fab fragments prevent lethal colchicine toxicity in a porcine model: a pharmacokinetic and clinical study.

Science.gov (United States)

Eddleston, Michael; Fabresse, Nicolas; Thompson, Adrian; Al Abdulla, Ibrahim; Gregson, Rachael; King, Tim; Astier, Alain; Baud, Frederic J; Clutton, R Eddie; Alvarez, Jean-Claude

2018-08-01

Colchicine poisoning is commonly lethal. Colchicine-specific Fab fragments increase rat urinary colchicine clearance and have been associated with a good outcome in one patient. We aimed to develop a porcine model of colchicine toxicity to study the pharmacokinetics and efficacy of ovine Fab. A Göttingen minipig critical care model was established and serial blood samples taken for colchicine and Fab pharmacokinetics, clinical chemistry, and haematology. Animals were euthanised when the mean arterial pressure fell below 45 mmHg without response to vasopressor, or at study completion. Initial studies indicated that oral dosing produced variable pharmacokinetics and time-to-euthanasia. By contrast, intravenous infusion of 0.25 mg/kg colchicine over 1 h produced reproducible pharmacokinetics (AUC 0-20 343 [SD = 21] µg/L/h), acute multi-organ injury, and cardiotoxicity requiring euthanasia a mean of 22.5 (SD = 3.2) h after dosing. A full-neutralising equimolar Fab dose given 6 h after the infusion (50% first hour, 50% next 6 h [to reduce renal-loss of unbound Fab]) produced a 7.35-fold increase in plasma colchicine (AUC 0-20 2,522 [SD = 14] µg/L/h), and removed all free plasma colchicine, but did not prevent toxicity (euthanasia at 29.1 [SD = 3.4] h). Earlier administration over 1 h of the full-neutralising dose, 1 or 3 h after the colchicine, produced a 12.9-fold (AUC 0-20 4,433 [SD = 607] µg/L/h) and 6.0-fold (AUC 0-20 2,047 [SD = 51] µg/L/h) increase in plasma colchicine, respectively, absence of free plasma colchicine until 20 h, and survival to study end without marked cardiotoxicity. Colchicine-specific Fab given early, in equimolar dose, bound colchicine, eliciting its movement into the blood, and preventing severe toxicity. Clinical studies are now needed to determine how soon this antidote must be given to work in human poisoning.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Science.gov (United States)

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

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

'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

Science.gov (United States)

Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

2016-04-01

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

Science.gov (United States)

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

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

International Nuclear Information System (INIS)

Peters-Libeu, Clare; Newhouse, Yvonne; Krishnan, Preethi; Cheung, Kenneth; Brooks, Elizabeth; Weisgraber, Karl; Finkbeiner, Steven

2005-01-01

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 1 2 1 2 to P2 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 1 2 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 1

Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

Science.gov (United States)

Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

2014-01-15

Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. Copyright © 2013 Elsevier B.V. All rights reserved.

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)

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

Science.gov (United States)

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

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}.

In vitro Fab display: a cell-free system for IgG discovery

Science.gov (United States)

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Fab is the most efficient format to express functional antibodies by yeast surface display.

Science.gov (United States)

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

Science.gov (United States)

Caballero, Armando R; Foletti, Davide L; Bierdeman, Michael A; Tang, Aihua; Arana, Angela M; Hasa-Moreno, Adela; Sangalang, Emma Ruth B; O'Callaghan, Richard J

2015-08-01

To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

Science.gov (United States)

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

Synthesis and stability test of radioimmunoconjugate 177Lu-DOTA-F(ab′2-trastuzumab for theranostic agent of HER2 positive breast cancer

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Sandra Hermanto

2016-10-01

Full Text Available The use of trastuzumab as intact IgG labeling radionuclide for HER2 positive breast cancer theranostic agent is not ideal because it is slowly eliminated from the blood and normal tissues resulting in low tumor/blood (T/B and tumor/normal tissue (T/NT ratios. To overcome this limitation, we developed the trastuzumab F(ab′2 fragments and radiolabeling of the fragments by β and γ-particle of Lutetium-177. F(ab2 fragments were produced by digestion of trastuzumab IgG (Herceptin with pepsin for 18 h at 37 °C. The F(ab′2 fragment fractionated in PD-10 column, followed by the conjugation with 2-(4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA as a metal chelator and radiolabeling with 177LuCl3. Molecular weight of fragments was calculated by LCMS (Liquid Chromatography Mass Spectroscopy and the radiochemical purity was evaluated by ITLC-SG (Instan Thin Layer Chromatography. Our study showed that the purity of F(ab′2 fragment generated by PD-10 fractions was >98% and the molecular weight of F(ab′2 was 98.35 kDa. The average numbers of pSCN-Bn-DOTA chelates per antibody fragment were 5.03 ± 1.5 and the optimum conjugation reactions was performed at molar ratio 20:1 (chelator to antibody. The stability test of the radioimmunoconjugate in the human serum albumin (HSA at 37 °C showed the radiochemical purity was 91.96 ± 0.26% after 96 h storage. This indicated that the radioimmunoconjugate is relatively stable when applied to the human body's physiological condition.

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.

Fragmentation, labeling and biodistribution studies of KS1/4, a monoclonal antibody

International Nuclear Information System (INIS)

Mohd, S.B.

1987-01-01

In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab') 2 and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab') 2 and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of 75 Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab') 2 and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma

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...

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.

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

Controlled Fab installation onto polymeric micelle nanoparticles for tuned bioactivity

Science.gov (United States)

Chen, Shaoyi; Florinas, Stelios; Teitgen, Abigail; Xu, Ze-Qi; Gao, Changshou; Wu, Herren; Kataoka, Kazunori; Cabral, Horacio; Christie, R. James

2017-12-01

Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.

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,

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.

Noninvasive detection of cardiac repair after acute myocardial infarction in rats by 111In fab fragment of monoclonal antibody specific for tenascin-C

International Nuclear Information System (INIS)

Okada, Kenichi; Uehara, Tomoya; Arano, Yasushi

2008-01-01

Left ventricular (LV) remodeling after acute myocardial infarction (MI) causes heart failure, and thus it is important to evaluate cardiac repair as the early stage of LV remodeling. Tenascin-C (TNC), an extracellular matrix glycoprotein, is transiently and abundantly expressed in the heart during the early stage of tissue remodeling after MI. However, it is not expressed in healthy adult heart. This study was undertaken to develop a new noninvasive diagnostic technique to detect cardiac repair after acute MI using 111 In Fab fragment of a monoclonal antibody specific for TNC. 111 In-anti-TNC-Fab was injected intravenously in 13 rats at 1 (D1, n=3), 3 (D3, n=5), and 5 (D5, n=5) days after producing MI and in 5 sham-operated rats (S). We performed autoradiography and dual-isotope single-photon emission computed tomography imaging (SPECT) of 111 In-anti-TNC-Fab and 99m Tc methoxyisobutyl isonitrile (MIBI). The radioactivity in the heart was significantly higher in D (D1, 0.45±0.06% injected-dose/g; D3, 0.64±0.12; D5, 0.38±0.07) than S (0.27±0.06, P 111 In-anti-TNC-Fab, which was complementary to the perfusion image. The results of the present study indicated that we can localize the infarcted region in the heart by ex vivo and in vivo imaging methods using 111 In-anti-TNC-Fab, and suggested the potential usefulness of noninvasive detection of cardiac repair. (author)

Effect of acetylation on monoclonal antibody ZCE-025 Fab': Distribution in normal and tumor-bearing mice

International Nuclear Information System (INIS)

Tarburton, J.P.; Halpern, S.E.; Hagan, P.L.; Sudora, E.; Chen, A.; Fridman, D.M.; Pfaff, A.E.

1990-01-01

Studies were performed to determine in vitro and in vivo effects of acetylation on Fab' fragments of ZCE-025, a monoclonal anti-CEA antibody. Isoelectric focusing revealed a drop in isoelectric point of 1.7 pI units following acetylation. Biodistribution studies of acetylated and nonacetylated [111In]Fab' were performed in normal BALB/c mice and in nude mice bearing the T-380 CEA-producing human colon tumor. The acetylated fragments remained in the vascular compartment longer and had significantly diminished renal uptake of 111In compared to controls. While acetylation itself effected a 50% drop in immunoreactivity, tumor uptake of the acetylated and nonacetylated 111In-labeled Fab' fragments was comparable, with the exception of one data point, through 72 h

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

In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

Science.gov (United States)

2014-01-01

Background Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours. Methods The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup. Results Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys. Conclusions We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies. PMID:24598405

A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

Science.gov (United States)

Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

2014-06-15

Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

Preparation, biodistribution, and dosimetry of 188Re-Labeled MoAb ior cea1 and its f(ab')2 fragments by avidin-biotin strategy

International Nuclear Information System (INIS)

Ferro-Flores, Guillermina; Pimentel-Gonzalez, Gilmara; Gonzalez-Zavala, Maria Antonia; Murphy, Consuelo Arteaga de; Melendez-Alafort, Laura; Tendilla, Jose I.; Croft, Barbara Y.

1999-01-01

The biotinylated monoclonal antibody (MoAb) ior cea1 and its F(ab') 2 fragments were labeled with Re-188 by combination of avidin-biotin strategy. 188 Re-MoAb, 188 Re-MoAb-biotin, 188 Re-F(ab') 2 , and 188 Re-F(ab') 2 -biotin preparations were produced for these studies with specific activities of 1.30±0.18 GBq/mg and from instant freeze-dried kit formulations using ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand. There were no significant differences (p>0.05) between the biodistribution in mice of biotinylated and unbiotinylated 188 Re-labeled immunoconjugates. When avidin was injected as a chase after injection of 188 Re-MoAb-biotin or 188 Re-F(ab') 2 -biotin, the blood radioactivity level decreased approximately 75% (cumulated activity) and the effective dose decreased almost 25% with respect to that of the radioimmunoconjugates in which the chase effect was not used. Our results suggest that 188 Re-labeled biotinylated MoAb ior cea1 and its F(ab') 2 fragments prepared by this method are stable complexes in vivo

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain

DEFF Research Database (Denmark)

Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik

2016-01-01

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one...... molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel...... of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4...

Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

Energy Technology Data Exchange (ETDEWEB)

Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

2008-05-01

Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

Neutralisation and binding of VHS virus by monovalent antibody fragments

DEFF Research Database (Denmark)

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

2001-01-01

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2...... appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial...... 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...

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.

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.

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...

Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: a prospective, blinded, multicenter, randomized clinical trial.

Science.gov (United States)

Bush, Sean P; Ruha, Anne-Michelle; Seifert, Steven A; Morgan, David L; Lewis, Brandon J; Arnold, Thomas C; Clark, Richard F; Meggs, William J; Toschlog, Eric A; Borron, Stephen W; Figge, Gary R; Sollee, Dawn R; Shirazi, Farshad M; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D; Boyer, Leslie V

2015-01-01

Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation.

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.)

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.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

Science.gov (United States)

Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

2016-11-01

Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

Science.gov (United States)

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

2016-10-01

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

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.

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.

Composition and method for detecting cancer with technetium labeled antibody fragments

International Nuclear Information System (INIS)

Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.

1984-01-01

F(ab') 2 or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab') 2 or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab') 2 or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab') 2 or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab') 2 or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased

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

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

QSAR models for prediction of chromatographic behavior of homologous Fab variants.

Science.gov (United States)

Robinson, Julie R; Karkov, Hanne S; Woo, James A; Krogh, Berit O; Cramer, Steven M

2017-06-01

While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R 2  > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

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

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.)

Radiation hardness of LuAG:Ce and LuAG:Pr scintillator crystals

CERN Document Server

Derdzyan, M V; Belsky, A; Dujardin, C; Lecoq, P; Lucchini, M; Ovanesyan, K L; Pauwels, K; Pedrini, C; Petrosyan, A G

2012-01-01

Single crystals of LuAG:Ce, LuAG:Pr and un-doped LuAG were grown by the vertical Bridgman method and studied for radiation hardness under gamma-rays with doses in the range 10-10(5) Gy (Co-60). A wide absorption band peaking at around 600 nm springs up in all three types of crystals after the irradiations. The second band peaking at around 375 nm appears in both LuAG:Pr and un-doped LuAG. Compositional variations have been done to reveal the spectral behavior of induced color centers in more detail and to understand their origin. Similarities in behavior of Yb2+ centers in as-grown garnets are found, indicating that radiation induced color centers can be associated with residual trace amounts of Yb present in the raw materials. Un-doped LuAG and LuAG:Ce demonstrate moderate radiation hardness (the induced absorption coefficients being equal to 0.05-0.08 cm(-1) for accumulated doses of 10(3)-10(4) Gy), while LuAG:Pr is less radiation hard. The ways to improve the radiation hardness are discussed.

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

Immunoscintigraphy of adenocarcinomas by means of 111In-labelled F(ab')2 fragments of anti-CEA monoclonal antibody F023C5

International Nuclear Information System (INIS)

Riva, P.; Paganelli, G.; Callegaro, L.

1988-01-01

F(ab') 2 fragments of F023C5, an anti-CEA monoclonal antibody, were conjugated to diethylenetriamine pentaacetic acid (DTPA) and converted into a ready to use reagent for instant 111 In-labelling. The resulting 111 In radiopharmaceutical was administered intravenously and tested for its ability to image (at 48-72 h after administration) 31 primary and 85 metastatic carcinoma lesions in 70 adenocarcinoma patients (26 gastrointestinal, 18 breast and 26 lung tumour patients) whose serum CEA was elevated in 43 cases and normal in the other 27. (author)

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

Science.gov (United States)

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

Science.gov (United States)

Egami, Yoko; Narushima, Yuta; Ohshima, Motohiro; Yoshida, Akira; Yoneta, Naruki; Masaki, Yasufumi; Itoh, Kunihiko

2018-01-01

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

Science.gov (United States)

Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

2018-05-01

Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

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)

Imaging Potential Evaluation of Fab Derived from the Anti-EGFRvIII Monoclonal Antibody 4G1.

Science.gov (United States)

Jing, Shen; He, Yujia; He, Yanqiong; Wang, Liang; Jia, Jianhua; Shan, Xiaomin; Liu, Shuang; Tang, Min; Peng, Zhiping; Liu, Xujie

2018-05-31

As one of the most crucial epidermal growth factor receptor (EGFR) variants, EGFRvIII can be detected in various tumors but rarely in normal tissues, making it an ideal target for prognosis, diagnosis or immune therapy. The recently developed anti-EGFRvIII monoclonal antibody (mAb), 4G1, has been validated as a promising molecular probe to detect EGFRvIII expression in tumors by single-photon emission computed tomography/computed tomography imaging. To overcome shortcomings associated with the whole antibody, including long-term retention, circulation and enhanced permeability and retention effects, the Fab fragment of 4G1 (Fab-4G1) was generated, labeled with 131 I and evaluated in vitro and in vivo to test its potential application in molecular imaging. Whole mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment, Fab-4G1. Next, SDS-PAGE, Western blot, indirect fluorescence assay, flow cytometry and enzyme-linked immunosorbent assay were performed to verify molecular weight, specificity and affinity of Fab-4G1. Finally, biodistribution planar gamma imaging was performed by injection of 131 I-labeled Fab-4G1 into xenografted EGFRvIII-overexpressed tumors in nude mice. Parallel studies were also performed with intact 4G1. The molecular weight of Fab was determined to be 35-40 kDa by SDS-PAGE. In vitro tests confirmed both intact 4G1 and Fab-4G1 specifically bound EGFRvIII but not wild-type EGFR, and Fab-4G1 showed decreased affinity. Compared to 131 I-4G1, biodistribution studies showed lower tumor uptake of 131 I-Fab-4G1 at all time points, but much faster elimination in all normal organs. As for planar gamma imaging, 131 I-Fab-4G1 and 31 I-4G1 showed similar imaging effect at 2 h after injection of tracer, while 131 I-Fab-4G1 was eliminated more quickly with time, suggesting radiolabeled Fab-4G1 could be potentially used for imaging of EGFRvIII-positive tumors at early time points. Radiolabeled

Technetium-99m-labelled monoclonal antibody fragment (99mTc-DD-3B6/22Fab') for ovarian cancer

International Nuclear Information System (INIS)

Fernandes, V.; Donaghy, T.; Prabakaran, K.; Smith, S.; Clingan, P.; Bartolo, N.; Bundesen, P.; Hillyard, C.

1998-01-01

Full text: This study is the first to assess 99m Tc-DD-3B6/22Fab'' as an agent for detecting OC recurrence. Five informed consenting volunteers with stages 3 or 4 OC (mean age 54.2 y) and with negative skin prick test to Fab'' were recruited. All had suspicion of OC recurrence, with elevated serum tumour marker CA125, and in some, OC confirmation on CT scans. Aggressive treatment was continued so that tumour regression was possible prior to 99m Tc-DD-3B6/22Fab''scan. CA125, FBC, biochemical and clot markers were measured pre-and post-study. All received IV 600-800 MBq of 99 mTc-DD-3B6/22Fab''. Temperature, pulse and BP were monitored for one hour. Data were acquired with a single head LFOV gamma camera and HIRES collimator at 1,4,6 and 24 hours. Studies were read for OC lesions which were scored as probably normal (PN) or abnormal (PAb), definitely abnormal (DAb). The gold standard was CA125 immediately post-scan and, when possible, confirmation with CT. Of 5 patients, 1 was disease negative with 99 mTc-DD-3B6/22Fab'', CA125 suggesting remission, and 4 were positive on all modalities (Sensitivity, specificity, 100% respectively). 99 mTc-DD-3B6/22Fab'' identified 15 lesions (100% DAb). CT detected 7 lesions (71% Dab) pre- 99 mTc-DD-3B6/22Fab''. Repeat CT detected 8 (100% DAb). Correlation with CA125 levels was best with 99 mTc-DD3B6/22Fab''. No side effects or adverse change in serum markers were noted. 99 mTc-DD-3B6/22Fab'' is sensitive and specific for detecting OC recurrence

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.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy.

Science.gov (United States)

Singh, Surinder M; Bandi, Swati; Jones, David N M; Mallela, Krishna M G

2017-12-01

We examined how polysorbate 20 (PS20; Tween 20) and polysorbate 80 (PS80; Tween 80) affect the higher-order structure of a monoclonal antibody (mAb) and its antigen-binding (Fab) and crystallizable (Fc) fragments, using near-UV circular dichroism and 2D nuclear magnetic resonance (NMR). Both polysorbates bind to the mAb with submillimolar affinity. Binding causes significant changes in the tertiary structure of mAb with no changes in its secondary structure. 2D 13 C- 1 H methyl NMR indicates that with increasing concentration of polysorbates, the Fab region showed a decrease in crosspeak volumes. In addition to volume changes, PS20 caused significant changes in the chemical shifts compared to no changes in the case of PS80. No such changes in crosspeak volumes or chemical shifts were observed in the case of Fc region, indicating that polysorbates predominantly affect the Fab region compared to the Fc region. This differential effect of polysorbates on the Fab and Fc regions was because of the lesser thermodynamic stability of the Fab compared to the Fc. These results further indicate that PS80 is the preferred polysorbate for this mAb formulation, because it offers higher protection against aggregation, causes lesser structural perturbation, and has weaker binding affinity with fewer binding sites compared to PS20. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

Science.gov (United States)

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-06-18

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

Characterization of Human Colorectal Cancer MDR1/P-gp Fab Antibody

Directory of Open Access Journals (Sweden)

Xuemei Zhang

2013-01-01

Full Text Available In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29. Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG. After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT comprising residues 883–898 within the transmembrane (TM domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

Science.gov (United States)

Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

2013-10-01

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Experimental study on 211At labelled monoclonal antibody 3H11 and its Fab fragment radioimmunotherapy for human gastric cancer xenografts in nude mice

International Nuclear Information System (INIS)

Jin Jiannan; Liu Ning; Zhang Shuyuan; Zhang Shiyuan; Luo Deyuan; Zhou Maolun

1996-01-01

Experimental radioimmunotherapy investigation of α-emitting radionuclide 211 At labelled anti-gastric cancer monoclonal antibody 3H11 and its Fab fragment for nude mice carrying human gastric cancer xenografts was conducted. Three i.p. injections of 14.8 or 22.2 kBq/g mouse were given, once every 5 days. The results showed that the growth of tumor xenografts was inhibited efficiently. The most evident therapy effect was observed at 15 days after treatment, and the tumor inhibition rates were 65% and 72%, respectively. No radiation injury of important organs was found

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

International Nuclear Information System (INIS)

Fand, Irwin; Sharkey, R.M.; Grundy, J.P.; Goldenberg, D.M.

1992-01-01

In this report, we have employed macroautoradiography to compare the tumor targeting of 125 I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to 125 I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab') 2 and Fab' fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3-7 days post-treatment. Treatment with F(ab') 2 and Fab' fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background 125 I-radioactivity which was well advanced by 6-24 h and complete by 24-48 h after injection. Localization of 125 I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab') 2 and Fab' fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments. (Author)

Safety, pharmacokinetic and dosimetry evaluation of the proposed thrombus imaging agent 99mTc-DI-DD-3B6/22-80B3 Fab'

International Nuclear Information System (INIS)

Macfarlane, David J.; Smart, Richard C.; Tsui, Wendy W.; Gerometta, Michael; Eisenberg, Paul R.; Scott, Andrew M.

2006-01-01

99m Tc-DI-DD-3B6/22-80B3 (Thromboview, hereafter abbreviated to 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 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 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. 99m Tc-DI-80B3 Fab' had a rapid initial plasma clearance (t 1/2 α=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.)

Beyond Fab Four

International Nuclear Information System (INIS)

Babichev, E; Charmousis, C; Langlois, D; Saito, R

2015-01-01

We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the ‘John’ and ‘Paul’ terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four. (fast track communication)

Alterations in an indium-111 Fab' under conditions of utilization

International Nuclear Information System (INIS)

Halpern, S.E.; Sudora, E.; Tarburton, J.P.; Hagar, P.

1992-01-01

This study was conducted to investigate alterations that occur in an indium/111 Fab' of a monoclonal antibody following its in vivo administration. Patients were infused with 111 In-Fab' of the monoclonal antibody ZCE-025. Serum and urine specimens were collected from these pateients. Starting materials, serum, urine and controls samples were studied by electrophoresis. Animal distribution studies were performed in normal Balb/c mice and, in some cases, nude mice bearing a carcinoembryonic antigen (CEA)/producing human colon tumour since the antibody targets CEA. The studies indicated that the molecule circulated almost totally intact for at least 4 h and to a considerable extent for 24 h, with some evidence for in vivo fragmentation by 24 h. Evidence was also obtained suggesting the formation of a high molecular weight species in some patients. Shortly after infusion, some of the 111 In in the urine appeared as the intact Fab', but within hours the majority migrated electro-phoretically as low molecular weight species. We conclude that while the majority of the 111 In-Fab' of this particular antibody remains intact and immunoreactive following its administration, the molecule is structurally changed to some degree shortly after its infusion into humans. Since each monoclonal antibody is unique, the degree and rapidity of degradation of its Fab' in vivo could vary markedly from the above and possibly adversely effect its utility as a radiopharmaceutical. (orig.)

Use of I-131 labeled, murine Fab against a high molecular weight antigen of human melanoma: Preliminary experience

International Nuclear Information System (INIS)

Larson, S.M.; Carrasquillo, J.A.; McGuffin, R.W.

1985-01-01

High molecular-weight antigen (HMWA) is tumor-associated proteoglycan of human malignant melanoma. I-131 labeled Fab fragments of these specific antibodies were used for preliminary feasibility studies for radioimmunodetection and therapy of human subjects who had inoperable metastatic melanoma. Ten patients received tracer doses of I-131 (anti-HMWA) Fab. All patients (8/8) who had melanoma lesions greater than 1 cm by correlative diagnosis methods had one or more lesions that had localization to tumor of the radiolabelled Fab. In all, 17 of 23 (74%) documented metastases were seen. Two patients who had avid uptake received potentially radiotherapeutic doses. For both of these patients, whole imaging studies showed that the localization of the high dose I-131 Fab was predominantly in tumor. On whole body images, the anti-Fab HMWA appears to be more tumor selective than Fab preparations that target the p97 antigen for melanoma, and there is less uptake in liver

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

Science.gov (United States)

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

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

Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives...

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

Science.gov (United States)

Banner, David W; Gsell, Bernard; Benz, Jörg; Bertschinger, Julian; Burger, Dominique; Brack, Simon; Cuppuleri, Simon; Debulpaep, Maja; Gast, Alain; Grabulovski, Dragan; Hennig, Michael; Hilpert, Hans; Huber, Walter; Kuglstatter, Andreas; Kusznir, Eric; Laeremans, Toon; Matile, Hugues; Miscenic, Christian; Rufer, Arne C; Schlatter, Daniel; Steyaert, Jan; Stihle, Martine; Thoma, Ralf; Weber, Martin; Ruf, Armin

2013-06-01

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

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

Re-188 labelling of DD-3B6/22 Fab' monoclonal antibody fragment for radio immuno therapy

International Nuclear Information System (INIS)

Schmidt, P.F.; Smith, S.V.; Bundesen, P.

1996-01-01

The chemical similarity of technetium and rhenium has created much interest in the nuclear medicine field to make a 'matched pair' of radiopharmaceuticals for radioimmuno- diagnosis and therapy. Clinical trials with the 99 mTc-DD-3B6/22 Fab' has shown promise in the diagnosis of ovarian cancer. The design of the analogous therapeutic agent with rhenium-188 (155 keV γ 15 % abundant, β E max 2.1 MeV, T 1/2 17 h) is under investigation. The present study describes the approach taken for direct radiolabelling of the DD-3B6/22 Fab' with carrier-free 188 Re and its biological evaluation in balb/c and nude mice. The effect of temperature, pH and antibody concentration on the amount and rate of transchelation was also evaluated. The final product had a specific activity of 35 mCi/mg with an immunoreactive fraction of 77%. Stability of the product was assessed under various conditions: temperature, presence and absence of an inert atmosphere and presence of ascorbic acid (stabilised). Pharmacokinetics of the final product was evaluated in balb/c and nude mice transplanted with both D-dimer (+Ve) and Glycine (-Ve) beads. Results show that 188 Re DD-3B6/22 Fab' clears rapidly from the blood (α = 2.4 hr, β = 3.5 hr) and is excreted through the renal system. Localisation to subcutaneous antigen beads shows specific uptake to the D-dimer (antigen) beads was achieved within 6 h (0.23% ID) and was maintained for 24 hour post injection. Specificity to antigen implants was 5:1 (P 99m Tc DD-3B6/22 Fab' in mice. The radiolabelling procedures are congenial for therapeutic levels and hence the authors believe that the 188 Re DD-3B6/22 Fab' has some potential for use in treatment of ovarian cancer

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

Science.gov (United States)

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

2016-10-01

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

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.)

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...

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.

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...

Characterization of crystals of an antibody-recognition fragment of the cancer differentiation antigen mesothelin in complex with the therapeutic antibody MORAb-009

International Nuclear Information System (INIS)

Ma, Jichun; Tang, Wai Kwan; Esser, Lothar; Pastan, Ira; Xia, Di

2012-01-01

The therapeutic antibody MORAb-009 disrupts the interaction of mesothelin and the ovarian cancer antigen CA-125. Crystals have been grown of the Fab fragment derived from MORAb-009 and of its complex with an N-terminal fragment of mesothelin. The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7–64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Å resolution and had the symmetry of space group P4 1 2 1 2, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin–Fab complex diffracted to 2.6 Å resolution and belonged to the hexagonal space group P6 4 , with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress

Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using 111In-trastuzumab (Herceptin) Fab fragments

International Nuclear Information System (INIS)

Tang Ying; Wang, Judy; Scollard, Deborah A.; Mondal, Hridya; Holloway, Claire; Kahn, Harriette J.; Reilly, Raymond M.

2005-01-01

Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with 111 In. The dissociation constant (K d ) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (K d =47 nM). 111 In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8±0.7% injected dose (ID)/g and tumor/blood ratio of 25.2±1.6 at 72 h postinjection compared with 2.7±0.7% ID/g and 7.0±0.9 for 111 In-HuM195 anti-CD33 Fab (significantly different, P 111 In-trastuzumab Fab as early as 24 h postinjection

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

Science.gov (United States)

Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

2017-10-06

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli , had unprecedentedly high binding affinities ( K d ∼10 -12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

International Nuclear Information System (INIS)

Pak, K.Y.; Nedelman, M.A.; Tam, S.H.; Wilson, E.; Daddona, P.E.

1992-01-01

The in vitro labeling and stability of 99m Tc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99m Tc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99m Tc-labeled antibody fragments after 24 h incubation at 37 o C. 99m Tc-labeled antibody fragments (IgG1, N = 2; IgG2a, n = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Using the affinity chromatography technique, two of the 99m Tc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 o C. Bonding between 99m Tc and antibody fragments was elucidated by challenging with a diamide ditholate (N 2 S 2 ) compound. The Fab' with IgG2a isotype displayed tighter binding to 99m Tc in comparison to Fab' from IgG1 and IgG3 isotype in N 2 S 2 challenge and incubation with human plasma. The in vivo biodistribution of five 99m Tc-labeled fragments were evaluated in normal mice. (Author)

Characterization of ["1"1C]Lu AE92686 as a PET radioligand for phosphodiesterase 10A in the nonhuman primate brain

International Nuclear Information System (INIS)

Yang, Kai-Chun; Stepanov, Vladimir; Amini, Nahid; Martinsson, Stefan; Takano, Akihiro; Halldin, Christer; Nielsen, Jacob; Bundgaard, Christoffer; Bang-Andersen, Benny; Grimwood, Sarah; Farde, Lars; Finnema, Sjoerd J.

2017-01-01

["1"1C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. ["1"1C]Lu AE92686 has high affinity for PDE10A (IC_5_0 = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional ["1"1C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). ["1"1C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V_T) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V_T values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V_T values increased by ∝30 % with increasing PET measurement duration from 63 to 123 min, while V_T values in target regions remained stable. Both pretreatment drugs significantly decreased ["1"1C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP_N_D) values, derived with the simplified reference tissue model (SRTM), were 13-17 in putamen and 3-5 in substantia nigra and correlated well to values from the Logan plot analysis. The method proposed for quantification of ["1"1C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that ["1"1C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia nigra. (orig.)

Characterization of [{sup 11}C]Lu AE92686 as a PET radioligand for phosphodiesterase 10A in the nonhuman primate brain

Energy Technology Data Exchange (ETDEWEB)

Yang, Kai-Chun; Stepanov, Vladimir; Amini, Nahid; Martinsson, Stefan; Takano, Akihiro; Halldin, Christer [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); Nielsen, Jacob [H. Lundbeck A/S, Synaptic Transmission, Valby (Denmark); Bundgaard, Christoffer; Bang-Andersen, Benny [H. Lundbeck A/S, Discovery Chemistry and DMPK, Valby (Denmark); Grimwood, Sarah [Pfizer Inc., Neuroscience and Pain Research Unit, Cambridge, MA (United States); Farde, Lars [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); AstraZeneca PET Science Center at Karolinska Institutet, Personalized Health Care and Biomarkers, Stockholm (Sweden); Finnema, Sjoerd J. [Karolinska Institutet, Karolinska University Hospital, Department of Clinical Neuroscience, Center for Psychiatric Research, Stockholm (Sweden); Yale University, Department of Radiology and Biomedical Imaging, New Haven, CT (United States)

2017-02-15

[{sup 11}C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. [{sup 11}C]Lu AE92686 has high affinity for PDE10A (IC{sub 50} = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional [{sup 11}C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). [{sup 11}C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V{sub T}) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V{sub T} values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V{sub T} values increased by ∝30 % with increasing PET measurement duration from 63 to 123 min, while V{sub T} values in target regions remained stable. Both pretreatment drugs significantly decreased [{sup 11}C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP{sub ND}) values, derived with the simplified reference tissue model (SRTM), were 13-17 in putamen and 3-5 in substantia nigra and correlated well to values from the Logan plot analysis. The method proposed for quantification of [{sup 11}C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that [{sup 11}C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia

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

Science.gov (United States)

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

2011-09-01

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

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

Microbial platform technology for recombinant antibody fragment production: A review.

Science.gov (United States)

Gupta, Sanjeev Kumar; Shukla, Pratyoosh

2017-02-01

Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker's ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0 g/L while scFv up to 3.5 g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.

Evaluation of the 175Lu(n,2n)174Lu, 175Lu(n,2n/sup 174m/Lu, and 175Lu(n,3n)173Lu cross sections from threshold to 20 MeV

International Nuclear Information System (INIS)

Philis, C.; Young, P.G.; Arthur, E.D.

1978-04-01

An evaluation of the 175 Lu(n,2n) 174 Lu, 175 Lu(n,2n)/sup 174m/Lu, and 175 Lu(n,3n) 173 Lu reactions from threshold to 20 MeV is presented. Available experimental data were renormalized to a consistent set of standards and were used along with results from statistical-preequilibrium model calculations to produce recommended curves for each of these reactions

Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using {sup 111}In-trastuzumab (Herceptin) Fab fragments

Energy Technology Data Exchange (ETDEWEB)

Tang Ying [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada); Wang, Judy [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Scollard, Deborah A. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Mondal, Hridya [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada); Holloway, Claire [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Kahn, Harriette J. [Sunnybrook and Women' s College Health Sciences Center, Toronto, Ontario, M4N 3M5 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, Ontario, M5G 2C4 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, M5S 2S2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, Ontario, M5S 3E2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

2005-01-01

Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with {sup 111}In. The dissociation constant (K{sub d}) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (K{sub d}=47 nM). {sup 111}In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8{+-}0.7% injected dose (ID)/g and tumor/blood ratio of 25.2{+-}1.6 at 72 h postinjection compared with 2.7{+-}0.7% ID/g and 7.0{+-}0.9 for {sup 111}In-HuM195 anti-CD33 Fab (significantly different, P<.001). Small (3-5 mm in diameter) BT-474 tumors were imaged with {sup 111}In-trastuzumab Fab as early as 24 h postinjection.

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

International Nuclear Information System (INIS)

Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

2006-01-01

The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

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

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

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

Science.gov (United States)

Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles.

Science.gov (United States)

Kennedy, Patrick J; Perreira, Ines; Ferreira, Daniel; Nestor, Marika; Oliveira, Carla; Granja, Pedro L; Sarmento, Bruno

2018-06-01

Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic-co-glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly-used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme-linked immunosorbent assay. Most importantly, Fab-decorated NP binding to the cell surface receptor was monitored by fluorescence-activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween-80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween-80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab-decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand-decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed. Copyright © 2018 Elsevier B.V. All rights reserved.

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...

Experimental half-life determination of 176Lu

International Nuclear Information System (INIS)

Kossert, Karsten; Jörg, Gerhard; Gostomski, Christoph Lierse v.

2013-01-01

The half-life of the naturally occurring long-lived rare earth isotope 176 Lu was determined by a combination of highly sophisticated experimental procedures in order to further improve the reliability and the precision of literature data. The amount of lutetium in the samples was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using a NIST reference standard. The isotopic ratio N( 176 Lu)/N(Lu) in the samples was measured by means of inductively coupled plasma high resolution mass spectrometry (ICP-HRMS). The activity divided by the mass of Lu was determined by applying liquid scintillation (LS) counting. The LS counting efficiency of the beta/gamma emitter 176 Lu was determined with the CIEMAT/NIST efficiency tracing technique with low uncertainty. The influences of colour quenching and background effects are discussed in this paper. The half-life was found to be 3.640(35)×10 10 y. The result is in good agreement with other evaluations and the relative standard uncertainty of 0.95% is among the lowest of previously published data. - Highlights: • The half-life of 176 Lu was determined by ICP-OES, ICP-HRMS and LSC. • The LSC efficiency was determined with the CIEMAT/NIST method. • The half-life was found to be 3.640(35)×10 10 y

Development of positron emission tomography probe of 64Cu-labeled anti-C-kit 12A8 Fab to measure protooncogene C-kit expression

International Nuclear Information System (INIS)

Yoshida, Chisato; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Sogawa, Chizuru; Inubushi, Masayuki; Uehara, Tomoya; Fukumura, Toshimitsu; Koizumi, Mitsuru; Arano, Yasushi; Saga, Tsuneo

2011-01-01

Introduction: C-kit is an important diagnostic and therapeutic target molecule for several malignancies, and c-kit-targeted drugs have been used clinically. Because abundant c-kit expression in tumors is a prerequisite for successful c-kit-targeted therapy, imaging of c-kit expression is expected to play a pivotal role in the therapeutic decision for each patient. We evaluated 64 Cu-labeled Fab of anti-c-kit antibody 12A8 as a positron emission tomography (PET) imaging probe. Methods: 111 In- or 125 I-Labeled 12A8 Fab was evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution in mice bearing c-kit-expressing and -non-expressing tumors. Next, Fab fragment was labeled with the positron emitter 64 Cu and evaluated by PET. Results: Radiolabeled 12A8 Fab showed specific binding to c-kit-expressing cells with high affinity and internalized into cells after binding to c-kit on cell surface. Although tumor accumulation of [ 111 In]Fab was lower than that of [ 111 In]IgG, the faster blood clearance of [ 111 In]Fab provided higher tumor-to-blood ratio at 6 h postinjection onwards. Blood clearance of 64 Cu-labeled 12A8 Fab was slower than that of [ 111 In]Fab, but PET using [ 64 Cu]Fab clearly visualized the tumor at 6 h postinjection onwards. Conclusion: The 64 Cu-labeled 12A8 Fab could be used for c-kit-specific PET imaging and might help in selecting appropriate patients for c-kit-targeted treatments.

Preparation of (177Lu-DOTAn-PAMAM-[Nimotuzumab-F(ab’2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancer Treatment

Directory of Open Access Journals (Sweden)

Titis Sekar Humani

2017-12-01

Full Text Available Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(ab’2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(ab’2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(ab’2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(ab’2-nimotuzumab was confirmed by SDS-PAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(ab’2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine] to form conjugate of (DOTAn-PAMAM-[nimotuzumab-F(ab’2]. Radiolabeling of DOTA-PAMAM-[nimotuzumab-F(ab’2] conjugate with 177Lu resulted in (177Lu-DOTAn-PAMAM-[nimotuzumab-F(ab’2] with radiochemical purity > 99% after purification with a PD-10 column. Further studies still need to be performed in order to confirm the potential of this radioimmunoconjugate as a radioimmunotherapeutic agent for EGFR overexpressed cancers.

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

Energy Technology Data Exchange (ETDEWEB)

Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

2016-07-27

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

Science.gov (United States)

Amirzargar, M A; Amirzargar, A; Basiri, A; Hajilooi, M; Roshanaei, G; Rajabi, G; Solgi, G

2015-05-01

Immunologic factors are reliable markers for allograft monitoring, because of their seminal role in rejection process. One of these factors is the immunoglobulin (Ig)A anti-Fab of the IgG antibody. This study aimed to evaluate the predictive value of pre- and posttransplant levels of this marker for kidney allograft function and survival. Sera samples of 59 living unrelated donor kidney recipients were collected before and after transplantation (days 7, 14, and 30) and investigated for IgA anti-Fab of IgG antibody levels using enzyme-linked immunosorbent assay in relation with allograft outcome. Among 59 patients, 15 cases (25%) including 10 with acute rejection and 5 with chronic rejection episodes showed graft failure during a mean of 5 years of follow-up. High posttransplant levels of IgA anti-Fab antibodies were observed more frequently in patients with stable graft function (SGF) compared with patients with graft failure (P = 2 × 10(-6)). None of patients with acute or chronic rejection episodes had high levels of IgA anti-Fab antibodies at day 30 posttransplant compared with the SGF group (P = 10(-6) and P = .01, respectively). In addition, high levels of IgA anti-Fab antibody correlated with lesser concentration of serum creatinine at 1 month posttransplantation (P = .01). Five-year graft survival was associated with high levels of pre- and posttransplant IgA anti-Fab antibodies (P = .02 and P = .003, respectively). Our findings indicate the protective effect of higher levels of IgA anti-Fab antibodies regarding to kidney allograft outcomes and long-term graft survival. Copyright © 2015 Elsevier Inc. All rights reserved.

PSMA-targeted bispecific Fab conjugates that engage T cells.

Science.gov (United States)

Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

OpenAIRE

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.; Rock, Charles O.

2014-01-01

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were...

Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

Science.gov (United States)

Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

2015-12-01

Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

Science.gov (United States)

Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-07-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

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...

radiolabeling of DOTA-substance P with 177Lu and biodistribution of 177Lu-DOTA-substance P

International Nuclear Information System (INIS)

Liang Jixin; Li Hongyu; Xiang Xueqin; Luo Zhifu; Luo Hongyi; Hu Liansheng; Chen Yang; Zhuang Ling; Deng Xinrong

2012-01-01

The aim of this project is to evaluate the biodistribution of 177 Lu-DOTA-SP in normal mice and in PANC-1 tumor bearing nude mice and to pave the way for its potentially medical application. In this study, 177 Lu-DOTA-SP was successfully prepared with labeling yield of greater than 90% at optimized conditions and more than 98% of radiochemical purity after C18 Sep-Pak purification. 177 Lu-DOTA-SP showed good stability in saline and in 5% serum while it decomposed slowly in 10% serum. Biodistribution studies in normal mice showed high uptake of 177 Lu-DOTA-SP in the kidneys, indicating the excretion mainly by renal pathway. In addition, 177 Lu-DOTA-SP was washed out from the blood quickly. Bio- distribution of 177 Lu-DOTA-SP in PANC-1 tumor bearing mice showed higher uptake in pancreatic tumor than that in normal pancreas, indicating the presence of NK-1 receptors in PANC-1 pancreatic tumor. However, from SPECT image, no radioactivity accumulation was observed in PANC-1 tumor. Further evaluation is needed to confirm its potential application for radiotherapy of pancreatic cancers. (authors)

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.

High spin K isomeric target of 177mLu

International Nuclear Information System (INIS)

Roig, O.; Belier, G.; Daugas, J.-M.; Delbourgo, P.; Maunoury, L.; Meot, V.; Morichon, E.; Sauvestre, J.-E.; Aupiais, J.; Boulin, Y.; Fioni, G.; Letourneau, A.; Marie, F.; Ridikas, D.

2004-01-01

The techniques used to produce a 177m Lu (J π =23/2 - ,T 1/2 =160.4 days) target are described in this paper. Firstly, an isotopic separation of an enriched lutetium sample was used to reach a purity of 176 Lu close to 99.993%. Afterwards, the high neutron flux of the Grenoble Institut Laue-Langevin reactor was used to produce the 177m Lu isomer by the 176 Lu(n,γ) reaction. Finally, a chemical separation was performed to extract 10 13 nuclei of 177m Lu. Thanks to this experiment, we have been able to estimate the destruction cross-section of the 177m Lu

Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

Directory of Open Access Journals (Sweden)

Orth Angela

2009-04-01

Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

Synthesis of DOTMP and biodistribution and imaging study of 177-Lu-DOTMP

International Nuclear Information System (INIS)

Deng Xinrong; Luo Zhifu; Xiang Xueqin; Li Fenglin; Fan Caiyun; Liu Zihua; Ye Zhaoyun; Li Hongyu; Chen Yang; Zhuang Ling

2012-01-01

Cyclen (1, 4, 7, 10-tetraazacyclododecane) and H 3 PO 3 was used to synthesis DOTMP (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-Tetraamino methylenephosphonate). 177 Lu was produced by irradiating enriched lutetium oxide ( 176 Lu 2 O 3 ) with thermal neutron flux of 2' × 10 13 n/cm 2 /S in swimming pool reactor (SPR) for 10 days. And then DOTMP was labelled by 177 Lu. The biodistribution of 177 Lu-DOTMP in model mice bearing S180 sarcoma and SPECT imaging in Japanese white rabbit were also carried out. The results showed that the total activity of 177 LuCl 3 solution obtained was 9.19 × 10 5 MBq after corresponding chemical process. According to the optimal condition of the labeling experiment, the labelling efficiency of 177 Lu-DOTMP was 99.4%. The results of biodistribution study indicated that 177 Lu-DOTMP eliminated rapidly from blood and was delivered to target bone. The radioactivity uptake was mainly in bone and less in other viscera. The results of SPECT imaging showed that the radioactivity was accumulated in bladder. 177 Lu-DOTMP was mainly excreted by kidney. The uptake of the activity in the skeleton was observed within 22 h postinjection and it became quite significant at 46 h post injection. It indicated that 177 Lu-DOTMP has good bone targeting and is worthy of further research. (authors)

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

Science.gov (United States)

Arora, Jayant; Hu, Yue; Esfandiary, Reza; Sathish, Hasige A; Bishop, Steven M; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Weis, David D

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

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

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

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.

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.

Spectral properties of hydrothermally-grown Nd:LuAG, Yb:LuAG, and Yb:Lu{sub 2}O{sub 3} laser materials

Energy Technology Data Exchange (ETDEWEB)

Brown, David C., E-mail: DBrown@snakecreeklasers.com [Snake Creek Lasers LLC, Friendsville, PA 18818 (United States); McMillen, Colin D.; Moore, Cheryl; Kolis, Joseph W. [Department of Chemistry and Center for Optical Materials Science and Engineering Technologies, Clemson University, Clemson, SC 29634-0973 (United States); Envid, Victoria [Snake Creek Lasers LLC, Friendsville, PA 18818 (United States)

2014-04-15

We have investigated the hydrothermal growth of, and spectrally characterized, the lutetium based laser materials Nd:LuAG, Yb:LuAG, and Yb:Lu{sub 2}O{sub 3}. Absorption cross-section data are presented for Nd:LuAG at 83, 175, and 295 K. Absorption cross-section data was also obtained for Yb:LuAG at 83, 175, and 295 K; the 295 K data was used to generate emission cross-sections using the method of reciprocity. For Yb:Lu{sub 2}O{sub 3}, we present absorption cross-sections at 295 K as well as emission cross-sections derived using reciprocity. -- Highlights: • We present spectral properties for hydrothermally-grown laser crystals. • Absorption cross-section data are presented for Nd:LuAG and Yb:LuAG at 83, 175, and 295 K. • Emission cross-sections are presented for Yb:LuAG at 295 K derived by reciprocity. • We present absorption cross-sections at 295 K as well as emission cross-sections derived using reciprocity for the laser material Yb:Lu{sub 2}O{sub 3}.

Measurement of the cross sections for the 175Lu(n,2n)174m,gLu reaction

International Nuclear Information System (INIS)

Yu Weixiang; Zhao Wenrong; Lu Hanlin; Han Xiaogang

1998-01-01

The 175 Lu(n,2n) 174m,g Lu reaction is an important indicator. There are 10 sets of measured data since 1960 (total 58 experimental values). Most of the data are finished at 14 MeV with 20%∼30% discrepancy. So the authors measured the cross section by activation method in neutron energies of 14 MeV and 10∼12 MeV. The measurement is carried out at the Cockcroft-Walton and HI-13 Tandem accelerator of CIAE

Comparative study of optical and scintillation properties of YVO4, (Lu0.5Y0.5)VO4, and LuVO4 single crystals

International Nuclear Information System (INIS)

Fujimoto, Yutaka; Yanagida, Takayuki; Yokota, Yuui; Chani, Valery; Kochurikhin, Vladimir V.; Yoshikawa, Akira

2011-01-01

Optical and scintillation properties of YVO 4 , (Lu 0.5 Y 0.5 )VO 4 , and LuVO 4 single crystals grown by the Czochralski (CZ) method with RF heating system are compared. All vanadate crystals show high transmittance (∼80%) in the 400-900 nm wavelength range. In both photo- and radio-luminescence spectra, intense peak around 400-500 nm, which was ascribed to the transition from triplet state of VO 4 3- , was clearly observed. The main decay time component was about 38 μs (YVO 4 ), 18 μs ((Lu 0.5 Y 0.5 )VO 4 ), and 17 μs (LuVO 4 ) under 340 nm excitation. The scintillation light yields of YVO 4 , (Lu 0.5 Y 0.5 )VO 4 , and LuVO 4 crystals (obtained from the 137 Cs excited pulse height spectra) were evaluated to be about 11,200, 10,700, and 10,300 ph/MeV, respectively.

The role of indium-111 antimyosin (Fab) imaging as a noninvasive surveillance method of human heart transplant rejection

International Nuclear Information System (INIS)

De Nardo, D.; Scibilia, G.; Macchiarelli, A.G.

1989-01-01

The identification of rejection after heart transplantation in patients receiving cyclosporine immunosuppressive therapy requires the endomyocardial biopsy, an invasive method associated with a finite morbidity. To evaluate the role of indium-111 antimyosin (Fab) scintigraphy as a noninvasive surveillance method of heart transplant rejection, the Fab fragment of murine monoclonal antimyosin antibodies labeled with indium-111 was administered intravenously in 30 scintigraphic studies to 10 consecutive heart transplant recipients. Endomyocardial biopsy specimens were obtained 72 hours after each scintigraphic study. Nineteen scintigraphic studies had negative findings; no false negative finding was obtained. Eleven antimyosin scintigraphic studies had positive findings, and in these studies endomyocardial biopsy revealed mild rejection in two cases, moderate acute rejection with myocyte necrosis in two cases, myocyte necrosis as a consequence of ischemic injury in six cases, and possibly cytotoxic damage in one case. Antimyosin scintigraphy may represent a reliable screening method for the surveillance of heart transplant patients. In the presence of a negative finding from antimyosin scintigraphy, it may be possible to avoid endomyocardial biopsy. Conversely, in patients who have a positive finding from antimyosin scintigraphy, the endomyocardial biopsy is mandatory to establish the definitive diagnosis by histologic examination of the myocardium

A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

Science.gov (United States)

Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

2016-12-01

Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

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

Directory of Open Access Journals (Sweden)

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.

Cloning, bacterial expression and crystallization of Fv antibody fragments

Science.gov (United States)

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

1992-08-01

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

FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

Science.gov (United States)

Pagano, John

2015-01-01

This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

Comparison of tumour and whole body absorbed doses of 177-Lu-DOTA-TATE and Lu-177-DOTA-NOC treatment in the same patient group

International Nuclear Information System (INIS)

Yeyin, N.; Kabasakal, L.; Akyel, R.; Demir, M.; Kanmaz, B.; Ocak, M.; Toklu, T.; Selcuk, N.

2015-01-01

Full text of publication follows. Peptide Receptor Radionuclide Therapy (PRRT) with Lu-177 labelled peptides in patients with neuroendocrine tumours (NETs) aroused great interest. An estimation of actual radiation doses to tumours is very important for therapy planning. There are several radiolabelled peptides, which can be used for PRRT with different biological behaviour. Aim: the aim of the study was to compare the tumour and normal organ absorbed doses in patients who have received Lu-177-DOTA-TATE and Lu-177 DOTA-NOC. Materials and methods: study was composed of 20 patients (M/F: 10/10, mean age: 51.5 ± 14.9) with histologically proven inoperable NETs. All patients received Lu-177-DOTA-NOC treatment 6 to 12 weeks after last Lu-177-DOTA-TATE treatment. Dosimetric calculations were performed using MIRD scheme and lesion doses were calculated using post therapy whole body images obtained at 4, 20, 44, and 68 hours after injection. Tumour volumes were determined from CT images. Thirteen blood samples beginning from time zero to 4 days after injection were obtained for bone marrow and whole body dosimetry. Results: There were 53 lesions in Lu-177-DOTA-TATE post-therapy whole body images and 49 lesions in Lu-177 DOTA-NOC post therapy images. Lesions were selected according to lesion delineation and superimposed lesions were excluded. Mean lesion absorbed dose is calculated to be 47.4 ± 53.4 and 42.9 ± 52.8 Gy per 370 MBq for Lu-177-DOTA-TATE and DOTA-NOC respectively (p>0.5). There were significantly higher absorbed doses for kidney and bone marrow after Lu-177-DOTA-NOC treatment as compared to Lu-177-DOTA-TATE treatment, which were 6.9 ± 2.7 vs 3.9 ± 1.7 (p<0.05) and 0.12 ± 0.0 vs 0.10 ± 0.0 (p<0.05) Gy, respectively. There was not any difference in plasma elimination times between two tracers. On the other hand the whole body absorbed dose was significantly higher after Lu-177-DOTA-NOC treatment, which was 0.24 ± 0.07 vs 0.20 ± 0.06 Gy (p<0

High spin K isomeric target of {sup 177m}Lu

Energy Technology Data Exchange (ETDEWEB)

Roig, O. E-mail: olivier.roig@cea.fr; Belier, G.; Daugas, J.-M.; Delbourgo, P.; Maunoury, L.; Meot, V.; Morichon, E.; Sauvestre, J.-E.; Aupiais, J.; Boulin, Y.; Fioni, G.; Letourneau, A.; Marie, F.; Ridikas, D

2004-03-21

The techniques used to produce a {sup 177m}Lu (J{sup {pi}}=23/2{sup -},T{sub 1/2}=160.4 days) target are described in this paper. Firstly, an isotopic separation of an enriched lutetium sample was used to reach a purity of {sup 176}Lu close to 99.993%. Afterwards, the high neutron flux of the Grenoble Institut Laue-Langevin reactor was used to produce the {sup 177m}Lu isomer by the {sup 176}Lu(n,{gamma}) reaction. Finally, a chemical separation was performed to extract 10{sup 13} nuclei of {sup 177m}Lu. Thanks to this experiment, we have been able to estimate the destruction cross-section of the {sup 177m}Lu.

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.

Science.gov (United States)

LaGraff, John R; Chu-LaGraff, Quynh

2006-05-09

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

Science.gov (United States)

Kornberger, Petra; Skerra, Arne

2014-01-01

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

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.

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...

Een inventarisatie naar de bekendheid van Functionele Agrobiodiversiteit (FAB) en de mogelijkheden om FAB met andere agroranden te combineren

NARCIS (Netherlands)

Geerts, R.H.E.M.; Meerburg, B.G.

2011-01-01

Op dit moment zijn er regio's waar het FAB concept al breed wordt toegepast en goed is ingebed (West-Brabant/Zeeland), waar het in opmars is (Flevoland en Zuid-Holland) en enkele regio's waar het FAB gedachtegoed slechts matig aanwezig is (Groningen / Drenthe).

Fragment-assisted hit investigation involving integrated HTS and fragment screening: Application to the identification of phosphodiesterase 10A (PDE10A) inhibitors.

Science.gov (United States)

Varnes, Jeffrey G; Geschwindner, Stefan; Holmquist, Christopher R; Forst, Janet; Wang, Xia; Dekker, Niek; Scott, Clay W; Tian, Gaochao; Wood, Michael W; Albert, Jeffrey S

2016-01-01

Fragment-based drug design (FBDD) relies on direct elaboration of fragment hits and typically requires high resolution structural information to guide optimization. In fragment-assisted drug discovery (FADD), fragments provide information to guide selection and design but do not serve as starting points for elaboration. We describe FADD and high-throughput screening (HTS) campaign strategies conducted in parallel against PDE10A where fragment hit co-crystallography was not available. The fragment screen led to prioritized fragment hits (IC50's ∼500μM), which were used to generate a hypothetical core scaffold. Application of this scaffold as a filter to HTS output afforded a 4μM hit, which, after preparation of a small number of analogs, was elaborated into a 16nM lead. This approach highlights the strength of FADD, as fragment methods were applied despite the absence of co-crystallographical information to efficiently identify a lead compound for further optimization. Copyright © 2015 Elsevier Ltd. All rights reserved.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

Science.gov (United States)

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

Science.gov (United States)

Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

2015-01-01

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Acute myocardial infarct imaging with indium-111-labeled monoclonal antimyosin Fab

International Nuclear Information System (INIS)

Khaw, B.A.; Yasuda, T.; Gold, H.K.; Leinbach, R.C.; Johns, J.A.; Kanke, M.; Barlai-Kovach, M.; Strauss, H.W.; Haber, E.

1987-01-01

Indium-111 monoclonal antimyosin Fab scintigraphy was used to detect myocardial necrosis in 52 of 54 patients (96.3%) with acute myocardial infarction. Infarcts were visualized when coronary arteries were persistently occluded (n = 10), became patent after thrombolysis (n = 33), or became patent after spontaneous reperfusion (n = 7). Posteroinferolateral visualizations were obtained in two patients with clinical and enzymatic evidence of infarction but normal electrocardiograms. Of the two patients in whom no infarcts were visualized, one had an anterior myocardial infarct. This patient underwent successful thrombolytic therapy, with attendant minimization of creatine kinase release. The other patient had a small, nonreperfused inferior myocardial infarct. Five patients with a history of remote infarction and acute necrosis showed antimyosin uptake only in regions concordant with the acute episodes of infarction, and radiolabeled antimyosin Fab localized in neither old infarcts nor normal, noninfarcted myocardium. Antimyosin Fab scintigraphy, thus, appears to be a highly specific means of delineating necrotic myocardium, at least in this limited and selected group of patients

177 Lu-Dota-octreotate radionuclide therapy of advanced gastrointestinal neuroendocrine tumors: results from a phase II study

Energy Technology Data Exchange (ETDEWEB)

Paganelli, Giovanni; Sansovini, Maddalena; Ambrosetti, Alice; Severi, Stefano; Ianniello, Annarita; Matteucci, Federica [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Nuclear Medicine and Radiometabolic Units, Meldola, FC (Italy); Monti, Manuela; Scarpi, Emanuela [IRST IRCCS, Unit of Biostatistics and Clinical Trials, Meldola (Italy); Donati, Caterina [IRST IRCCS, Oncology Pharmacy Laboratory, Meldola (Italy); Amadori, Dino [IRST IRCCS, Department of Medical Oncology, Meldola (Italy)

2014-10-15

We evaluated the activity and safety profile of {sup 177}Lu-Dotatate peptide receptor radionuclide therapy (Lu-PRRT) in patients with advanced, well-differentiated (G1-G2) gastrointestinal neuroendocrine tumors (GI-NETs). Forty-three patients with radiological tumor progression at baseline and a positive Octreoscan registered completed the treatment with Lu-PRRT, resulting in the cumulative activity of 18.5 or 27.8 GBq in five cycles. Total activity was scheduled on the basis of kidney function or bone marrow reserve. Twenty-five (58 %) patients were treated with a ''standard'' Lu-PRRT full dosage (FD) of 25.7 GBq (range 22.2-27.8), while the remaining 18 patients (42 %) who, at enrolment, showed a higher probability of developing kidney or bone marrow toxicity received a reduced dosage (RD) of 18.4 GBq (range 14.4-20.4). According to SWOG criteria, the overall response was complete response (CR) in (7 %) cases and stable disease (SD) in 33 (77 %), with a disease control rate (DCR) of 84 %. Median response duration was 25 months (range 7-50). Median progression-free survival (PFS) was 36 months (95 % CI 24-nr), and median overall survival (OS) has not yet been reached. Remarkably, none of the patients, including those at a higher risk of toxicity, showed side-effects after either dosage of Lu-PRRT. Lu-PRRT was shown to be an effective therapeutic option in our patients with advanced progressive GI-NETs, showing an 84 % DCR (95 % CI 73-95) that lasted for 25 months and a PFS of 36 months. Both activities of 27.8 GBq and 18.5 GBq proved safe and effective in all patients, including those with a higher probability of developing kidney or bone marrow toxicity. (orig.)

Alternative method to determine Specific Activity of (177)Lu by HPLC.

Science.gov (United States)

Breeman, Wouter A P; de Zanger, Rory M S; Chan, Ho Sze; de Blois, Erik

2015-01-01

Peptide Receptor Radionuclide Therapy (PRRT) with (177)Lu-DOTA-peptides requires (177)Lu with high specific activity (SA) and values >740 GBq (177)Lu per mg Lu to maximise the atom% of (177)Lu over total Lu. Vendors provide SA values which are based on activity and mass of the target, whereas due to "burn-up" of target, these SA values are not accurate. For a radiochemist the SA of (177)Lu is of interest prior to radiolabeling. An alternative method to determine SA was developed by HPLC, which includes a metal titration of a known amount of DOTA-peptide with a known amount of activity ((177)Lu), and a unknown amount of metal ((177+nat)Lu). Based on an HPLC separation of radiometal-DOTA-peptide and DOTA-peptide, and the concordant ratio of these components the metal content ((177+nat)Lu) can be calculated, and eventually the SA of (177)Lu can be accurately determined. These experimentally determined SA values exceeded the estimated values provided by vendors by 27 ± 16%, (range 6-73 %). The deviation of SA values for samples from the same Lu batch was <2% (n ≥ 10). the SA of (177)Lu is apparently often higher as stated by vendors in comparison to the experimentally determined actual values. For this reason, the SA of (177)Lu-DOTA-TATE and other Lu-DOTA-peptides could be increased accordingly.

Phase equilibria and crystalline structure of compounds in the Lu-Al and Lu-Cu-Al systems

International Nuclear Information System (INIS)

Kuz'ma, Yu.B.; Stel'makhovich, B.M.; Galamushka, L.I.

1992-01-01

Phase equilibria and crystal structure of compounds in Lu-Al and Lu-Cu-Al systems were studied. Existence of Lu 2 Al compound having the structure of the PbCl 2 type is ascertained. Diagram of phase equilibria of Lu-Cu-Al system at 870 K is plotted. Compounds Lu 2 (Cu,Al) 17 (the Th 2 Zn 17 type structure), Lu(Cu,Al) 5 (CaCu 5 type structure), Lu 6 (Cu,Al) 23 (Th 6 Mn 23 type structure) and ∼ LuCuAl 2 have been prepared for the first time. Investigation of component interaction in Lu-Cu-Al system shows that the system is similar to previously studied systems Dy-Cu-Al and Er-Cu-Al. The main difference consists in the absence of LuCuAl 3 compound with rhombic structure of the CeNi 2+x Sb 2-x type in the system investigated

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.

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

Imaging of low-grade bone infection with a technetium-99m labelled monoclonal anti-NCA-90 Fab' fragment in patients with previous joint surgery

Energy Technology Data Exchange (ETDEWEB)

Ivaneeviae, V.; Sandrock, D.; Munz, D.L. [Clinic for Nuclear Medicine, University Hospital Charite, Humboldt University of Berlin (Germany); Perka, C.; Hasart, O. [Orthopaedic Clinic, University Hospital Charite, Humboldt University of Berlin (Germany)

2002-04-01

We analysed 38 scans in 30 consecutive patients (age range, 30-85 years; median age, 62 years) referred for suspected low-grade bone infection. There were 17 patients (21 scans) with total hip arthroplasty (THA), six with total knee arthroplasty (TKA), three who had undergone hip or knee surgery for trauma and five (seven scans) with resected hips and no endoprostheses (Girdlestone situations); There were no patients with rheumatoid arthritis as the underlying disease. Results were verified by means of bacteriological cultures, histopathological findings and/or follow-up and compared with the respective Zimmerli scores, which were used for clinical assessment of inflammatory activity. In one patient, the final diagnosis could not be established. One, 5 and 24 h after intravenous injection of up to 1.1 GBq of MN3 Fab', whole-body and planar scans were performed using a dual-head gamma camera. Scans were analysed visually and semiquantitatively adopting an arbitrary score ranging from 0 to 3. There were 13 true positive, 14 true negative and 10 false positive outcomes, yielding an overall sensitivity of 100%, an overall specificity of 58%, an accuracy of 73% and positive and negative predictive values of 57% and 100%, respectively. In patients with THA or TKA, accuracy was 81% and 80%, respectively, while it dropped to 43% in patients with Girdlestone situations owing to a high proportion of false positive findings (4/7) in this subgroup. Scintigraphic score was 1 in all of the false positive and in 11/13 true positive findings. The two remaining true positive findings displayed scintigraphic scores of 2 and 3, respectively. Scintigraphic and Zimmerli scores were loosely correlated (Spearman {rho}=0.38, P<0.05). Infection was excluded in 22/24 investigations with Zimmerli scores of <6. In this group, there were 13 scintigraphically true negative, nine false positive outcomes, and just two true positive outcomes. In 11/12 investigations with Zimmerli scores of 6

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

OpenAIRE

Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

Analysis list: NR3C1 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NR3C1 Blood,Bone,Breast,Liver,Others,Prostate,Uterus + hg19 http://dbarchive.biosci...encedbc.jp/kyushu-u/hg19/target/NR3C1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/NR3C1.5.tsv http:...//dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/NR3C1.10.tsv http://dbarchive.biosciencedbc.jp/kyu...shu-u/hg19/colo/NR3C1.Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/NR3C1.Bone.tsv,http:...//dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/NR3C1.Breast.tsv,http://dbarchive.bi

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Science.gov (United States)

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

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

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

Pollution prevention opportunity assessment for MicroFab and SiFab facilities at Sandia National Laboratories.

Energy Technology Data Exchange (ETDEWEB)

Gerard, Morgan Evan

2011-12-01

This Pollution Prevention Opportunity Assessment (PPOA) was conducted for the MicroFab and SiFab facilities at Sandia National Laboratories/New Mexico in Fiscal Year 2011. The primary purpose of this PPOA is to provide recommendations to assist organizations in reducing the generation of waste and improving the efficiency of their processes and procedures. This report contains a summary of the information collected, the analyses performed, and recommended options for implementation. The Sandia National Laboratories Environmental Management System (EMS) and Pollution Prevention (P2) staff will continue to work with the organizations to implement the recommendations.

The antigen-binding fragment of human gamma immunoglobulin prevents amyloid β-peptide folding into β-sheet to form oligomers

Science.gov (United States)

Valls-Comamala, Victòria; Guivernau, Biuse; Bonet, Jaume; Puig, Marta; Perálvarez-Marín, Alex; Palomer, Ernest; Fernàndez-Busquets, Xavier; Altafaj, Xavier; Tajes, Marta; Puig-Pijoan, Albert; Vicente, Rubén; Oliva, Baldomero; Muñoz, Francisco J.

2017-01-01

The amyloid beta-peptide (Aβ) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aβ aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aβ aggregation and its neuroprotective role. PMID:28467807

Analysis list: Nr3c1 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Nr3c1 Adipocyte,Blood,Breast,Embryo,Embryonic fibroblast,Liver,Neural + mm9 http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/target/Nr3c1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/ta...rget/Nr3c1.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Nr3c1.10.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Nr3c1.Adipocyte.tsv,http://dbarchive.biosciencedbc.jp.../kyushu-u/mm9/colo/Nr3c1.Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Nr3c1.Breast.tsv,http://dbarchive.bioscience

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 ...

Immunocytochemistry by electron spectroscopic imaging using well defined boronated monovalent antibody fragments.

Science.gov (United States)

Kessels, M M; Qualmann, B; Sierralta, W D

1996-01-01

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI)--an electron microscopic technique with high-resolution potential. Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not affected by the attachment of the markers and the resulting immunoprobes exhibited an elongated shape with the antigen combining site and the label located at opposite ends. The labeling densities observed with these reagents were found to be significantly higher than those obtained by using conventional colloidal gold methods. Combined with digital image processing and analysis systems, boron-based ESI proved to be a powerful approach in ultrastructural immunocytochemistry employing pre- and post-embedding methods.

Evidence for a magnetic moment at the Lu site of LuFe2

International Nuclear Information System (INIS)

Kasamatsu, Y.; Armitage, J.G.M.; Lord, J.S.; Riedi, P.C.; Fort, D.

1995-01-01

High pressure 175 Lu NMR measurements of LuFe 2 at 4.2 K suggest that Lu, like Y and Zr, carries a negative magnetic moment, in agreement with self-consistent energy band calculations. The observed spectrum is more complicated than would be expected for a Laves phase compound. ((orig.))

Tumour detection and localization using 99Tcm-labelled OV-TL 3 Fab' in patients suspected of ovarian cancer

International Nuclear Information System (INIS)

Tibben, J.G.; Massuger, L.F.A.G.; Claessens, R.A.M.J.; Schijf, C.P.T.; Strijk, S.P.; Kenemans, P.; Corstens, F.H.M.; Pak, K.Y.

1992-01-01

Fab' fragments of the monoclonal antibody OV-TL 3, that recognizes an ovarian carcinoma-associated antigen (OA3), were labelled with 99 Tc m using D-glucarate as a ligand. Twenty patients suspected of having primary or recurrent ovarian cancer received intravenously 1 mg of the Fab' labelled with 740 MBq 99 Tc m . Both planar and single photon emission computed tomographic (SPECT) scintigraphy were performed up to 30 h after intravenous infusion. Imaging results were compared with X-ray computed tomography (CT), ultrasonography (US) and CA 125 serum level. Blood clearance was fast (t 1/2 of 9.5 h). Thirty-seven per cent of the injected dose (% ID) was excreted in the urine within the first 24 h, whereas 7% ID was excreted in the 24 h faeces. In one patient with an OA3 negative ovarian carcinoma, radioimmunoscintigraphy (RIS) did not visualize the tumour. In two other patients a benign ovarian cyst was found, also showing no elevated uptake. In 13 out of 17 patients ovarian cancer lesions were detected with RIS, whereas CT and US detected lesions in, respectively, 15 and 12 patients. Of 36 surgically defined tumour deposits larger than 1 cm in diameter, 53% were detected and localized with RIS, whereas CT and US detected 61 and 40%, respectively. Radioimmunoscintigraphy with 99 Tc m -OV-TL 3 Fab' is less distressing for the patients but the overall imaging performance is not improved when compared with 111 In-OV-TL 3 F(ab') 2 . (Author)

Meditope-Fab interaction: threading the hole.

Science.gov (United States)

Bzymek, Krzysztof P; Ma, Yuelong; Avery, Kendra N; Horne, David A; Williams, John C

2017-12-01

Meditope, a cyclic 12-residue peptide, binds to a unique binding side between the light and heavy chains of the cetuximab Fab. In an effort to improve the affinity of the interaction, it was sought to extend the side chain of Arg8 in the meditope, a residue that is accessible from the other side of the meditope binding site, in order to increase the number of interactions. These modifications included an n-butyl and n-octyl extension as well as hydroxyl, amine and carboxyl substitutions. The atomic structures of the complexes and the binding kinetics for each modified meditope indicated that each extension threaded through the Fab `hole' and that the carboxyethylarginine substitution makes a favorable interaction with the Fab, increasing the half-life of the complex by threefold compared with the unmodified meditope. Taken together, these studies provide a basis for the design of additional modifications to enhance the overall affinity of this unique interaction.

Comparative study of optical and scintillation properties of YVO{sub 4}, (Lu{sub 0.5}Y{sub 0.5})VO{sub 4}, and LuVO{sub 4} single crystals

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, Yutaka, E-mail: fuji-you@tagen.tohoku.ac.j [IMRAM, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Yanagida, Takayuki; Yokota, Yuui; Chani, Valery [IMRAM, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Kochurikhin, Vladimir V. [General Physics Institute, 38 Vavilov Street, 119991, Federation, Moscow (Russian Federation); Yoshikawa, Akira [IMRAM, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); NICHe, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan)

2011-04-11

Optical and scintillation properties of YVO{sub 4}, (Lu{sub 0.5}Y{sub 0.5})VO{sub 4}, and LuVO{sub 4} single crystals grown by the Czochralski (CZ) method with RF heating system are compared. All vanadate crystals show high transmittance ({approx}80%) in the 400-900 nm wavelength range. In both photo- and radio-luminescence spectra, intense peak around 400-500 nm, which was ascribed to the transition from triplet state of VO{sub 4}{sup 3-}, was clearly observed. The main decay time component was about 38 {mu}s (YVO{sub 4}), 18 {mu}s ((Lu{sub 0.5}Y{sub 0.5})VO{sub 4}), and 17 {mu}s (LuVO{sub 4}) under 340 nm excitation. The scintillation light yields of YVO{sub 4}, (Lu{sub 0.5}Y{sub 0.5})VO{sub 4}, and LuVO{sub 4} crystals (obtained from the {sup 137}Cs excited pulse height spectra) were evaluated to be about 11,200, 10,700, and 10,300 ph/MeV, respectively.

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

Directory of Open Access Journals (Sweden)

Nan Zhong

Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

Analysis list: NR1H3 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available NR1H3 Adipocyte,Blood + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target.../NR1H3.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/NR1H3.5.tsv http://dbarchive.bioscienced...bc.jp/kyushu-u/hg19/target/NR1H3.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/NR1H3.Adipocyte....tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/NR1H3.Blood.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Adipocyte.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Blood.gml ...

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

Science.gov (United States)

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

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.

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.

Data-driven discovery of energy materials: efficient BaM2Si3O10 : Eu2+ (M = Sc, Lu) phosphors for application in solid state white lighting.

Science.gov (United States)

Brgoch, Jakoah; Hasz, Kathryn; Denault, Kristin A; Borg, Christopher K H; Mikhailovsky, Alexander A; Seshadri, Ram

2014-01-01

In developing phosphors for application in solid state lighting, it is advantageous to target structures from databases with highly condensed polyhedral networks that produce rigid host compounds. Rigidity limits channels for non-radiative decay that will decrease the luminescence quantum yield. BaM(2)Si(3)O(10) (M = Sc, Lu) follows this design criterion and is studied here as an efficient Eu(2+)-based phosphor. M = Sc(3+) and Lu(3+) compounds with Eu(2+) substitution were prepared and characterized using synchrotron X-ray powder diffraction and photoluminescence spectroscopy. Substitution with Eu(2+) according to Ba(1-x)Eu(x)Sc(2)Si(3)O(10) and Ba(1-x)Eu(x)Lu(2)Si(3)O(10) results in UV-to-blue and UV-to-blue-green phosphors, respectively. Interestingly, substitution with Eu(2+) in the Lu(3+) containing material produces two emission peaks at low temperature and with 365 nm excitation, as allowed by the two substitution sites. The photoluminescence of the Sc(3+) compound is robust at high temperature, decreasing by only 25% of its room temperature intensity at 503 K, while the Lu-analogue suffers a large drop (75%) from its room temperature intensity. The decrease in emission intensity is explained as stemming from charge transfer quenching due to the short distances separating the luminescent centers on the Lu(3+) substitution site. The correlation between structure and optical response in these two compounds indicates that even though the structures are three-dimensionally connected, high symmetry is required to prevent structural distortions that could impact photoluminescence.

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.

Family of Advanced Beyond Line-of-Sight Terminals (FAB-T)

Science.gov (United States)

2015-12-01

Selected Acquisition Report (SAR) RCS: DD-A&T(Q&A)823-199 Family of Advanced Beyond Line-of-Sight Terminals ( FAB -T) As of FY 2017 President’s...Budget Defense Acquisition Management Information Retrieval (DAMIR) March 21, 2016 15:24:15 UNCLASSIFIED FAB -T December 2015 SAR March 21, 2016 15:24...Operational Requirements Document OSD - Office of the Secretary of Defense O&S - Operating and Support PAUC - Program Acquisition Unit Cost FAB -T December 2015

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.

Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody.

Science.gov (United States)

Cignetto, Simona; Modica, Chiara; Chiriaco, Cristina; Fontani, Lara; Milla, Paola; Michieli, Paolo; Comoglio, Paolo M; Vigna, Elisa

2016-06-01

The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Long-term follow-up and role of FDG PET in advanced pancreatic neuroendocrine patients treated with {sup 177}Lu-D OTATATE

Energy Technology Data Exchange (ETDEWEB)

Sansovini, Maddalena; Severi, Stefano; Ianniello, Annarita; Nicolini, Silvia; Fantini, Lorenzo; Paganelli, Giovanni [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Nuclear Medicine Unit, Meldola (Italy); Mezzenga, Emilio [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Medical Physics Unit, Meldola (Italy); Ferroni, Fabio [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Radiology Unit, Meldola (Italy); Scarpi, Emanuela; Monti, Manuela [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Unit of Biostatistics and Clinical Trials, Meldola (Italy); Bongiovanni, Alberto [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Osteoncology and Rare Tumors Center, Meldola (Italy); Cingarlini, Sara [University of Verona, Department of Oncology, Verona Comprehensive Cancer Network, G.B. Rossi Hospital, Verona (Italy); Grana, Chiara Maria; Bodei, Lisa [European Institute of Oncology Milan (IEO), Division of Nuclear Medicine, Milan (Italy)

2017-03-15

Lu-DOTATATE (Lu-PRRT) is a valid therapeutic option in differentiated pancreatic neuroendocrine tumors (P-NETs). FDG PET seems to be an important prognostic factor in P-NETs. We evaluated the efficacy of Lu-PRRT and the role of FDG PET in 60 patients with advanced P-NETs. From March 2008 to June 2011, 60 consecutive patients with P-NETs were enrolled in the study. Follow-up lasted until March 2016. Eligible patients were treated with two different total cumulative activities (18.5 or 27.8 GBq in 5 cycles every 6-8 weeks), according to kidney and bone marrow parameters. Twenty-eight patients received a mean full activity (FA) of 25.9 GBq and 32 a mean reduced activity (RA) of 18.5 GBq. The disease control rate (DCR), defined as the sum of CR+PR+SD was 85.7 % in the FA group and 78.1 % in the RA group. Median progression-free survival (mPFS) was 53.4 months in the FA group and 21.7 months in the RA group (P = 0.353). Median overall survival (mOS) was not reached (nr) in FA patients and was 63.8 months in the RA group (P = 0.007). Fifty-five patients underwent an FDG PET scan before Lu-PRRT, 32 (58 %) showing an increased FDG uptake in tumor sites. mPFS was 21.1 months in FDG PET-positive patients and 68.7 months in the FDG PET-negative group (P < 0.0002), regardless of the total activity administered. Both FA and RA are active in patients undergoing Lu-PRRT. However, an FA of 27.8 GBq of Lu-PRRT prolongs PFS and OS compared to an RA of 18.5 GBq. Our results indicate that FDG PET is an independent prognostic factor in this patient setting. (orig.)

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.

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.

CW laser properties of Nd:GdYAG, Nd:LuYAG, and Nd:GdLuAG mixed crystals

Science.gov (United States)

Di, J. Q.; Xu, X. D.; Li, D. Z.; Zhou, D. H.; Wu, F.; Zhao, Z. W.; Xu, J.; Tang, D. Y.

2011-10-01

Three mixed crystals, Nd:GdYAG, Nd:LuYAG, and Nd:GdLuAG, were grown by Czochralski method. We report the continuous-wave (CW) Nd:GdYAG, Nd:LuYAG, and Nd:GdLuAG laser operation under laser diode pumping. The maximum output powers are 4.11, 5.31, and 7.47 W, with slope efficiency of 73.0, 55.3, and 57.1%, respectively. With replacing Lu3+ or Y3+ ions with large Gd3+ ions, the pump efficiency increases.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

Science.gov (United States)

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Karch, Francois

2015-01-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. PMID:26303531

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

Science.gov (United States)

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

2015-11-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Study of the production of 177Lu through 176Yb (n, γ) 177Yb → 177Lu nuclear reaction

International Nuclear Information System (INIS)

Silva, Giovana Pasqualini da; Osso Junior, Joao Alberto

2007-01-01

The beta minus emitter 177 Lu is a promising therapeutic radioisotope for the curative treatment of cancer using labelled proteins. It has a half - life of T 1/2 = 6.71 day and maximum and average β - energies of 421 and 133 keV, resulting in a short range of radiation in tissue. The decay is accompanied by the emission of low energy γ-radiation with 208.3 keV (11%) and 113 keV (6.4%) suitable for simultaneous imaging, 177 Lu can be produced by two different routes, namely, by irradiation of natural Lu 2 O 3 target ( 176 Lu, 2.6%) or enriched (in 176 Lu) Lu 2 O 3 target, as also by irradiation of Yb target (Yb 2 O 3 ) followed by radiochemical separation of 177 Lu from Yb isotopes. The objective of this work is to study the production of 177 Lu through the indirect 176 Yb(n,γ) 177 Yb → 177 Lu nuclear reaction. The results of the production yield of 177 Lu will be shown and compared with the direct reaction. The method of choice for the chemical separation between Lu and Yb was the ion exchange, using an cation exchange resin in Cl - form and α-HIBA as eluent. Preliminary results showed a good separation of 177 Lu from Yb 2 O 3 indirect targets. (author)

Progress in the development of LuAlO$_{3}$ based scintillators

CERN Document Server

Belsky, A; Lecoq, P; Dujardin, C; Garnier, N; Canibano, H; Pédrini, C; Petrosian, A

2000-01-01

LuAlO/sub 3/:Ce/sup 3+/ (LuAP) and Lu/sub x/Y/sub 1/-xAlO/sub 3/:Ce /sup 3+/ (LuYAP) crystals are used as scintillation materials for positron emission tomography. The actual study of these scintillators develops in three directions: (i) growth of large size LuAP crystals with stable properties, (ii) the relationship between the composition of LuYAP crystals and scintillation properties, and (iii) scintillation mechanisms in lutetium compounds. After improving of growth conditions a large size samples (length >40 mm) have been prepared. Crystals show a good correlation between growth parameters, light yield and transmission spectra. We studied a series of samples with calibrated size (2*2*10 mm3) and compare the light yield with standard BGO and LSO samples. Mixed crystals with composition of 0.6

Memories of Lu Jingqing

Institute of Scientific and Technical Information of China (English)

1994-01-01

Lu Jingqing was a woman of letters who wrote poems when she was young and also worked as an editor and teacher. Her main works are Women poets of the Tang Dynasty (1931), Whisper (a collection of poems, 1932), Plain Writing (a collection of verses, 1930) and The Wandering (1933). In March 1993, 86-year-old Lu Jingqing, Professor of the Shanghai College of Finance and Economics, died in Shanghai. Zhu Bokang, an old friend of Lu and her husband wrote the following article in her memory.

Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients

DEFF Research Database (Denmark)

Cederkrantz, Elin; Andersson, Håkan; Bernhardt, Peter

2015-01-01

dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation...... 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective...

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.

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

Science.gov (United States)

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-01-01

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Time-dependent transcriptional response of GOT1 human small intestine neuroendocrine tumor after 177Lu[Lu]-octreotate therapy.

Science.gov (United States)

Spetz, Johan; Rudqvist, Nils; Langen, Britta; Parris, Toshima Z; Dalmo, Johanna; Schüler, Emil; Wängberg, Bo; Nilsson, Ola; Helou, Khalil; Forssell-Aronsson, Eva

2018-05-01

Patients with neuroendocrine tumors expressing somatostatin receptors are often treated with 177 Lu[Lu]-octreotate. Despite being highly effective in animal models, 177 Lu[Lu]-octreotate-based therapies in the clinical setting can be optimized further. The aims of the study were to identify and elucidate possible optimization venues for 177 Lu[Lu]-octreotate tumor therapy by characterizing transcriptional responses in the GOT1 small intestine neuroendocrine tumor model in nude mice. GOT1-bearing female BALB/c nude mice were intravenously injected with 15 MBq 177 Lu[Lu]-octreotate (non-curative amount) or mock-treated with saline solution. Animals were killed 1, 3, 7 or 41 d after injection. Total RNA was extracted from the tumor samples and profiled using Illumina microarray expression analysis. Differentially expressed genes were identified (treated vs. control) and pathway analysis was performed. Distribution of differentially expressed transcripts indicated a time-dependent treatment response in GOT1 tumors after 177 Lu[Lu]-octreotate administration. Regulation of CDKN1A, BCAT1 and PAM at 1 d after injection was compatible with growth arrest as the initial response to treatment. Upregulation of APOE and BAX at 3 d, and ADORA2A, BNIP3, BNIP3L and HSPB1 at 41 d after injection suggests first activation and then inhibition of the intrinsic apoptotic pathway during tumor regression and regrowth, respectively. Transcriptional analysis showed radiation-induced apoptosis as an early response after 177 Lu[Lu]-octreotate administration, followed by pro-survival transcriptional changes in the tumor during the regrowth phase. Time-dependent changes in cell cycle and apoptosis-related processes suggest different time points after radionuclide therapy when tumor cells may be more susceptible to additional treatment, highlighting the importance of timing when administering multiple therapeutic agents. Copyright © 2018 The Authors. Published by Elsevier Inc. All

Luminescent properties of LuPO4-Pr and LuPO4-Eu nanoparticles

International Nuclear Information System (INIS)

Vistovskyy, V.; Malyi, T.; Vas’kiv, A.; Chylii, M.; Mitina, N.; Zaichenko, A.; Gektin, A.; Voloshinovskii, A.

2016-01-01

Spectral-luminescence parameters of LuPO 4 -Eu and LuPO 4 -Pr nanoparticles of different sizes are studied upon excitation by the synchrotron radiation with photon energies 4–40 eV. Influence of the nanoparticle size on Eu 3+ and Pr 3+ impurity luminescence is analyzed for intracenter and recombination excitation. It is shown that the luminescence intensity of impurities in the case of recombination excitation significantly stronger decreases with decreasing of nanoparticle size compared to intracenter excitation. This feature is explained by the influence of thermalization length to nanoparticle size ratio on the recombination luminescence. Electron recombination luminescence inherent for LuPO 4 -Eu nanoparticles shows a weaker dependence on the nanoparticle size than the hole one in LuPO 4 -Pr nanoparticles. The difference between energy states of praseodymium impurity ions in nanoparticles of different sizes is revealed.

The reliability and validity of the Turkish version of Fullerton Advanced Balance (FAB-T) scale.

Science.gov (United States)

Iyigun, Gozde; Kirmizigil, Berkiye; Angin, Ender; Oksuz, Sevim; Can, Filiz; Eker, Levent; Rose, Debra J

2018-06-04

The aim of this study was to evaluate the reliability and validity of the Turkish version of the FAB(FAB-T) scale in the older Turkish adults. The reliability and validity of the scale was tested on 200 community-dwelling older adults. FAB-T scale was scored by different physiotherapists on different days to evaluate inter-rater and intrarater reliability. The Berg Balance Scale (BBS) was used for the evaluation of convergent validity, and the content validity of the FAB-T scale was investigated. The FAB-T scale showed very high inter- and intra-rater reliability. For inter-rater agreement, on the individual test items and total score ICC values were 0.92 (95 %CI; 0.90-0.94) and 0.96 (95% CI; 0.95-0.97) respectively. The intra-rater agreement, on the individual test items and total score ICC values were 0.93 (95 %CI; 0.91- 0.95) and 0.96 (95% CI; 0.95- 0.97) respectively. There was a good agreement between the FAB-T and BBS scales. A high correlation was found between the BBS and FAB-T scales [rho = 0.70 (%95 CI; 0.62-0.76)] indicating good convergent validity. Considering the content validity of the FAB-T scale, no floor (floor score: 0%) or ceiling (ceiling score: 6.5%) effect was detected. The FAB-T scale was successfully translated from the original English version (FAB) and demonstrated strong psychometric features. It was found that the FAB-T scale has very high inter-rater and intra-rater reliability. Considering the convergent validity, the scale has high correlation with the BBS. The FAB-T has no floor and ceiling effect. Copyright © 2018 Elsevier B.V. All rights reserved.

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)

Use of isothiocyanatobenzyl-DTPA derivatized monoclonal antimyosin fab for enhanced in vivo target localization

International Nuclear Information System (INIS)

Khaw, B.A.; Gansow, O.; Brechbiel, M.W.; O'Donnell, S.M.; Nossiff, N.

1990-01-01

Monoclonal antimyosin Fab (AM-Fab) was radiolabeled with 111In via a new bifunctional chelating agent, isothiocyanatobenzyl-DTPA (SCN-DTPA), and used to visualize acute reperfused experimental myocardial infarction. Antibody localization was compared to 201 Tl (0.6 mCi) distribution in nine animals. Each animal was injected intravenously with 0.5 mCi of 111 In-SCN-DTPA AM-Fab preparations (Prep 1 [n = 5] and 2 [n = 4]). The biodistribution was compared to that of 111 In-labeled conventional bicyclic anhydride DTPA-AM-Fab (n = 5). 111 In-SCN-DTPA AM-Fab Prep 1 (lowest specific activity) showed highest specific target localization (31.6 +/- 3.5, MEAN infarct[0-20% Tl-201] to normal ration +/- SE) and lowest hepatic sequestration (0.0108 +/- 0.002% ID/g). Prep 2 showed similar infarct localization (18.4 +/- 1.2) to control 111 In-DTPA AM-Fab (16.9 +/- 1.1), but had higher hepatic activity (0.0326 +/- 0.014 and 0.0267 +/- 0.006 respectively). This difference in in vivo localization occurred despite the lack of changes in in vitro immunoreactivities of the AM-Fab preparations. The enhanced target localization with minimal hepatic activity may permit a more sensitive diagnostic application of 111 In-labeled AM-Fab in future clinical studies

Structural and dynamical aspects of Streptococcus gordonii FabH through molecular docking and MD simulations.

Science.gov (United States)

Shamim, Amen; Abbasi, Sumra Wajid; Azam, Syed Sikander

2015-07-01

β-Ketoacyl-ACP-synthase III (FabH or KAS III) has become an attractive target for the development of new antibacterial agents which can overcome the multidrug resistance. Unraveling the fatty acid biosynthesis (FAB) metabolic pathway and understanding structural coordinates of FabH will provide valuable insights to target Streptococcus gordonii for curing oral infection. In this study, we designed inhibitors against therapeutic target FabH, in order to block the FAB pathway. As compared to other targets, FabH has more interactions with other proteins, located on the leading strand with higher codon adaptation index value and associated with lipid metabolism category of COG. Current study aims to gain in silico insights into the structural and dynamical aspect of S. gordonii FabH via molecular docking and molecular dynamics (MD) simulations. The FabH protein is catalytically active in dimerization while it can lock in monomeric state. Current study highlights two residues Pro88 and Leu315 that are close to each other by dimerization. The active site of FabH is composed of the catalytic triad formed by residues Cys112, His249, and Asn279 in which Cys112 is involved in acetyl transfer, while His249 and Asn279 play an active role in decarboxylation. Docking analysis revealed that among the studied compounds, methyl-CoA disulfide has highest GOLD score (82.75), binding affinity (-11 kcal/mol) and exhibited consistently better interactions. During MD simulations, the FabH structure remained stable with the average RMSD value of 1.7 Å and 1.6 Å for undocked protein and docked complex, respectively. Further, crucial hydrogen bonding of the conserved catalytic triad for exhibiting high affinity between the FabH protein and ligand is observed by RDF analysis. The MD simulation results clearly demonstrated that binding of the inhibitor with S. gordonii FabH enhanced the structure and stabilized the dimeric FabH protein. Therefore, the inhibitor has the potential to become

Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

Directory of Open Access Journals (Sweden)

Justin M Scheer

Full Text Available The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab'(2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

Acute adverse events associated with the administration of Crotalidae polyvalent immune Fab antivenom within the North American Snakebite Registry.

Science.gov (United States)

Kleinschmidt, Kurt; Ruha, Anne-Michelle; Campleman, Sharan; Brent, Jeffrey; Wax, Paul

2018-04-24

Crotalidae Polyvalent Immune Fab (Fab Antivenom) is the primary Viperid antivenom used in the United States since 2000. Adverse event data associated with its use are limited. The purpose of this study is to describe the prevalence of acute adverse events associated with the use of Fab antivenom. The American College of Medical Toxicology's Toxicology Investigators Consortium maintains a prospective case registry of poisoned and envenomated patients managed by medical toxicologists at the bedside. This registry includes the North American Snakebite sub-registry. We performed a review of 438 cases entered into the Snakebite sub-registry. A total of 373 (85.2%) received at least one vial of Fab Antivenom. Forty percent were children. Adverse events occurred in 10 patients (2.7%) of whom six were adults. Rash was the most common adverse event. More severe adverse events (hypotension, bronchospasm, and/or angioedema) occurred in four (1.1%) patients. Prophylaxis was administered prior to Fab antivenom in 4.0%. Eight patients received various treatments for their adverse events. Neither the initial number of Fab antivenom vials, atopic history, nor prior envenomation correlated with the prevalence of adverse events. This prevalence of adverse events was lower than in previous studies and in a meta-analysis of 11 studies. The types of adverse events and treatments used are consistent with those in previous reports. There were no prior reports of prophylaxis use with which to compare. The prevalence of Fab antivenom adverse events in the North American Snakebite Registry was 2.7%.

The research on biodistribution of bearing sarcoma mice and rabbit SPECT imaging of 177Lu-DOTMP

International Nuclear Information System (INIS)

Deng Xinrong; Xiang Xueqin; Li Fenglin; Fan Caiyun; Liu Zihua; Luo Zhifu; Chen Yang

2012-01-01

Cyclen (1, 4, 7, 10-tetraazacyclododecane) and H 3 PO 3 were used to synthesis DOTMP (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-Tetraaminomethylenephosphonate), and then DOTMP was labelled with 177 Lu. The research of biodistribution of 177 Lu-DOTMP in model mice bearing S180 sarcoma and SPECT imaging in Japanese white rabbit were also carried out. The results of biodistribution of bearing S180 mice indicated that 177 Lu-DOTMP cleared rapidly from blood and was selectively delivered to target bone. The radioactivity uptake was mainly in bone and less in other organs and tissues. The results of SPECT imaging of Japanese white rabbit showed that the radioactivity was accumulated in bladder. 177 Lu-DOTMP was mainly excreted by kidney. The uptake of the activity in the skeleton was observed significantly within 22 h post-injection and it became quite significant at 46 h post-injection. It indicated that 177 Lu-DOTMP has good bone targeting and is worthy of further study. (authors)

Mixed Messages from Garnet Lu-Hf and Sm-Nd Geochronology

Science.gov (United States)

Vervoort, J. D.; Wang, D.; Johnson, T. A.

2017-12-01

Garnet geochronology provides important information on the timing and conditions of metamorphism. As a major indicator mineral formed during metamorphism, its direct dating can not only help establish the timing of metamorphism, provide the "t" for P-T-t paths, but also, if the dated garnet can be placed in a textural context, can provide information on the timing of deformational features. With advances in chemistry and mass spectrometry, garnet Lu-Hf and Sm-Nd geochronology has become an important geochronological tool and we can now reliably (if not routinely) date a wide variety of garnet compositions formed under diverse conditions. In the course of dating a variety of lithologies using both Lu-Hf and Sm-Nd isotope systems, however, some intriguing results have emerged. Although there are many examples where the Lu-Hf and Sm-Nd systems give the same date within uncertainty, there are also many cases where these systems yield significantly different dates, and the differences between these dates can be considerable—many 10's of Ma of and even 100's of Ma. For example, in garnet-bearing Mesoproterozoic gneisses from across the Blue Ridge Province in Virginia, both Lu-Hf and Sm-Nd analyses (determined on the same solutions) define narrow time spans, but with the Sm-Nd dates systematically younger (for orthogneisses Lu-Hf dates are 1032 to 1019 Ma whereas Sm-Nd dates are 965 to 949 Ma—a difference of 67 to 80 Ma). There are many other examples of systematically younger Sm-Nd garnet dates in both the literature and with our ongoing research. Potential explanations for these differences include: 1) strong partitioning of Lu into garnet during growth yielding ages weighted toward the beginning of growth; 2) faster Lu diffusion from high Lu regions after garnet formation, potentially leading to isochron rotation and anomalously old Lu-Hf dates; and 3) differences in closure temperatures of the two isotope systems. We will review several examples of divergent Lu

Roussel. Sinfonien Nr. 3 g-Moll op. 42 und Nr. 4 A-Dur op. 5, Neeme Järvi / Christoph Schlüren

Index Scriptorium Estoniae

Schlüren, Christoph

1995-01-01

Uuest heliplaadist "Roussel. Sinfonien Nr. 3 g-Moll op. 42 und Nr. 4 A-Dur op. 53, Bacchus et Ariane op. 43 (Zweite Orchestersuite). Sinfonietta für Streichorchester d-Moll op. 52. Detroit Symphony Orchester, N. Järvi". Chandos/Koch CD 7007(WD: 70'10") DDD

Enhancement of tumor contrast on radioimmunoscans by using mixtures of monoclonal antibody F(ab')2 fragements

International Nuclear Information System (INIS)

Munz, D.L.; Alavi, A.; Koprowski, H.; Herlyn, D.; Pennsylvania Univ., Philadelphia

1986-01-01

F(ab') 2 fragments of MAbs GA 73-3 (IgG 2 a) and CO 29.11 (IgG 1), which detect distinct antigenic determinants on adenocarcinoma cells of the gastrointestinal tract, were labeled with 131 I using the iodogen method. 41 nude mice bearing SW-948 CRC tumores were injected either with a mixture of 100 μCi (11 μg) each (n=9) of the two 131 I-F(ab') 2 fragments or with either fragment alone at various doses (each group consisting of 8 mice): GA 73-3, 100 μCi (11 μg) and 200 μCi (25 μg); CO 29.11, 100 μCi (11 μg) and 200 μCi (26 μg). Whole-body images of the mice were obtained daily for up to six days after injection. Ratios of cpm/pixel in the tumor to those in the rest of the body (rob), representing tumor contrast, were significantly (p 1/2 biol. ) f the mixture (44.8±14.5 h) in the CRC tumors was significantly (p 1/2 biol. determined in the groups given either fragment alone. T 1/2 biol. in the rob was similar in all groups of mice examined. (orig.) [de

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

OpenAIRE

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2013-01-01

In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynt...

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.

Frontal assessment battery (FAB) performance following traumatic brain injury hospitalized in an acute care setting.

Science.gov (United States)

Rojas, Natalia; Laguë-Beauvais, Maude; Belisle, Arielle; Lamoureux, Julie; AlSideiri, Ghusn; Marcoux, Judith; Maleki, Mohammed; Alturki, Abdulrahman Y; Anchouche, Sonia; Alquraini, Hanan; Feyz, Mitra; Guise, Elaine de

2018-01-19

The Frontal Assessment Battery (FAB) has been shown to be useful in several clinical settings. The aim of the present study was to examine the performance of patients with traumatic brain injury (TBI) on the FAB and to predict their acute outcome. The FAB was administered to 89 patients with mild (27 = uncomplicated and 39 = complicated) and moderate (n = 23) TBI during hospitalization in an acute care setting. The length of stay in days (LOS), Glasgow Outcome Scale-Revised score (GOSE) and Disability Rating Scale (DRS) score were collected. Results showed no significant differences between the three groups on the FAB score, but age and education were significantly associated with the FAB score. Parietal lesions were associated with lower total FAB score, and with the Similarities, Motor series and Conflicting instructions subscales, while frontal lesions were associated with lower performance on the Motor series and Conflicting instructions subscales. Total FAB score was significantly correlated with all outcome measures, and together the FAB total score and the Glasgow Coma Scale (GCS) score explained 30.8% of the variance in the DRS score. The FAB may be useful clinically to acutely assess frontal and parietal lobe functions at bedside in patients with TBI and, in combination with the GCS score to measure TBI severity, can enable clinicians to predict early outcome.

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).

Science.gov (United States)

Hobson, Neil; Deyholos, Michael K

2013-04-01

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Complex Binding of the FabR Repressor of Bacterial Unsaturated Fatty Acid Biosynthesis to its Cognate Promoters

OpenAIRE

Feng, Youjun; Cronan, John E.

2011-01-01

Two transcriptional regulators, the FadR activator and the FabR repressor control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was bloc...

Coagulation parameters in copperhead compared to other Crotalinae envenomation: secondary analysis of the F(ab')2 versus Fab antivenom trial.

Science.gov (United States)

Gerardo, Charles J; Vissoci, Joao R Nickenig; Brown, Michael W J; Bush, Sean P

2017-02-01

Coagulation derangements in copperhead envenomation are considered less severe than other crotaline envenomations, resulting in recommendations to limit both coagulation testing and antivenom treatment. A prospective, blinded, multicenter, randomized clinical trial comparing the effectiveness of F(ab') 2 versus Fab antivenom in crotaline envenomation patients was completed in 2011. We determined the difference between coagulation parameters in copperhead compared to other crotaline envenomations. We performed a post hoc analysis comparing the coagulation parameters (platelets and fibrinogen) prospectively obtained in the aforementioned trial. All the patients received antivenom in one of three treatment arms [F(ab') 2 with maintenance, F(ab') 2 with placebo maintenance, or Fab with maintenance]. Coagulation parameters were measured at pretreatment baseline, during acute hospitalization, day 5, day 8, and day 15 post-envenomation. Mean platelet count and fibrinogen levels for the copperhead and other crotaline groups were compared. The platelet and fibrinogen point estimates with distribution are presented graphically over time. 122 patients were enrolled in the study. There were 22 patients with copperhead envenomation, 93 with other crotaline envenomations, and 7 that could not be definitively determined. The mean age was 42 (SD 20) years. There was a minor pretreatment difference in mean baseline platelet count between the copperhead group (246 × 109/L 95% CI 215, 277) compared to other crotaline envenomation patients (184 × 109/L 95% CI 167, 202). There was a modest pretreatment difference in mean fibrinogen level between copperhead patients (345 mg/dL 95% CI 277, 415) and other crotaline patients (261mg/dL 95% CI 241, 281). Pretreatment coagulation parameter means were normal and converged post treatment. On average, copperhead envenomations have less severe initial coagulation derangements. However, in mild envenomations, differences in laboratory

20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

Science.gov (United States)

2010-04-01

... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of such...

Progress in the development of LuAlO3 based scintillators

CERN Document Server

Belsky, A; Lecoq, P; Dujardin, C; Garnier, N; Canibano, H; Pédrini, C; Petrosian, A

2000-01-01

LuAlO3:Ce3+ (LuAP) and LuxY1-xAlO3:Ce3+ (LuYAP) crystals are the promote scintillation materials for Positron Emission Tomography. Actual study of these scintillators develops in the tree directions: (i) growth of large size LuAP crystals with stable properties, (ii) relationship between composition of LuYAP crystals and scintillation properties, and (iii) scintillation mechanisms in lutetium compounds. After improving of growth conditions a large size samples (length greater than 40 mm) have been prepared. Crystals show a good correlation between growth parameters, light yield and transmission spectra. We performed a series of samples with calibrated size (2x2x10 mm3) and compare the light yield with a standard BGO and LSO samples. Mixed crystals with composition of 0.6 less than x less than 0.8 show a significant increase of light yield. We suggest that the short order clusterisation in mixed crystals may by playing an important role in governing the scintillation efficiency. In order to clarify the scintil...

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.

Fragmentation and Multifragmentation of 10.6 A GeV Gold Nuclei

CERN Document Server

Adamovich, M I

1999-01-01

We present the results of a study performed on the interactions of 10.6A GeV gold nuclei in nuclear emulsions. In a minimum bias sample of 1311 interac- tions, 5260 helium nuclei and 2622 heavy fragments were observed as Au projec- tile fragments. The experimental data are analyzed with particular emphasis of target separation interactions in emulsions and study of criticalexponents. Multiplicity distributions of the fast-moving projectile fragments are inves- tigated. Charged fragment moments, conditional moments as well as two and three -body asymmetries of the fast moving projectile particles are determined in terms of the total charge remaining bound in the multiply charged projectile fragments. Some differences in the average yields of helium nuclei and heavier fragments are observed, which may be attributed to a target effect. However, two and three-body asymmetries and conditional moments indicate that the breakup mechanism of the projectile seems to be independent of target mass. We looked for evidenc...

Optimalization activity of ZnO NR/TiO2 NR-P3HT as an active layer based on hybrid bulk heterojunction on dye sensitized solar cell (DSSC)

International Nuclear Information System (INIS)

Saputri, Liya Nikmatul Maula Zulfa; Ramelan, Ari Handono; Hanif, Qonita Awliya; Hasanah, Yesi Ihdina Fityatal; Prajanira, Lau Bekti; Wahyuningsih, Sayekti

2016-01-01

Dye sensitized solar cell (DSSC) with metal inorganic and conjugated organic polymer mixture, ZnO NR/TiO 2 NR-P3HT as an active layer based on hybrid bulk heterojunction has been studied. The hybrid material was used to optimize DSSC performs for better efficiency than only TiO 2 as an electrode. Synthesis of TiO 2 nanorods (NR) was conducted by ball milling 1000 rpm for 4 hours and strong base reaction by hydrothermal process at 120 °C overnight. And the ZnO NR was synthesized from Zn(NO 3 ) 2 .4H 2 O precusor by hydrotermal process at 90 °C for 5 hours and calcined on various temperature s of 400, 600, and 800 °C. ZnO NR was coated into an Tndium Tin Oxide (TTO) glass to collecting electron s effectively, where TiO 2 NR were incorporated with poly(3 -hexylthiophene) (P3HT) on various concentration s of 5, 10, 15 mg/mL to obtain a larger surface area. Material characterization were performed by X -Ray Diffraction (XRD) and Uv-Vis spectrophotometer. For an application of DSSC were measured by T-V Keithley Multimeter and the efficiency of DSSC at various P3HT’s concentrations of 5, 10, 15 mg/mL were 7.44 × 10 −3 , 0.0114, 0.0104, respectively. The maximum efficiency of DSSC was showed when TiO 2 NR-P3HT’s concentration was 10 mg/mL.

Optimalization activity of ZnO NR/TiO2 NR-P3HT as an active layer based on hybrid bulk heterojunction on dye sensitized solar cell (DSSC)

Energy Technology Data Exchange (ETDEWEB)

Saputri, Liya Nikmatul Maula Zulfa; Ramelan, Ari Handono; Hanif, Qonita Awliya; Hasanah, Yesi Ihdina Fityatal; Prajanira, Lau Bekti; Wahyuningsih, Sayekti, E-mail: sayektiw@mipa.uns.ac.id [Chemistry Department, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Ir.Sutami 36A Kentingan Surakarta 57/26, Central Java (Indonesia)

2016-04-19

Dye sensitized solar cell (DSSC) with metal inorganic and conjugated organic polymer mixture, ZnO NR/TiO{sub 2} NR-P3HT as an active layer based on hybrid bulk heterojunction has been studied. The hybrid material was used to optimize DSSC performs for better efficiency than only TiO{sub 2} as an electrode. Synthesis of TiO{sub 2} nanorods (NR) was conducted by ball milling 1000 rpm for 4 hours and strong base reaction by hydrothermal process at 120 °C overnight. And the ZnO NR was synthesized from Zn(NO{sub 3}){sub 2}.4H{sub 2}O precusor by hydrotermal process at 90 °C for 5 hours and calcined on various temperature s of 400, 600, and 800 °C. ZnO NR was coated into an Tndium Tin Oxide (TTO) glass to collecting electron s effectively, where TiO{sup 2} NR were incorporated with poly(3 -hexylthiophene) (P3HT) on various concentration s of 5, 10, 15 mg/mL to obtain a larger surface area. Material characterization were performed by X -Ray Diffraction (XRD) and Uv-Vis spectrophotometer. For an application of DSSC were measured by T-V Keithley Multimeter and the efficiency of DSSC at various P3HT’s concentrations of 5, 10, 15 mg/mL were 7.44 × 10{sup −3}, 0.0114, 0.0104, respectively. The maximum efficiency of DSSC was showed when TiO{sup 2} NR-P3HT’s concentration was 10 mg/mL.

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.

"Fab 13": The Learning Factory.

Science.gov (United States)

Crooks, Steven M.; Eucker, Tom R.

2001-01-01

Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

Cross-talk between the NR3B and NR4A families of orphan nuclear receptors

International Nuclear Information System (INIS)

Lammi, Johanna; Rajalin, Ann-Marie; Huppunen, Johanna; Aarnisalo, Piia

2007-01-01

Estrogen-related receptors (NR3B family) and Nurr1, NGFI-B, and Nor1 (NR4A family) are orphan nuclear receptors lacking identified natural ligands. The mechanisms regulating their transcriptional activities have remained elusive. We have previously observed that the members of NR3B and NR4A families are coexpressed in certain cell types such as osteoblasts and that the ability of Nurr1 to transactivate the osteopontin promoter is repressed by ERRs. We have now studied the cross-talk between NR3B and NR4A receptors. We show that NR3B and NR4A receptors mutually repress each others' transcriptional activity. The repression involves intact DNA-binding domains and dimerization interfaces but does not result from competition for DNA binding or from heterodimerization. The activation functions of NR3B and NR4A receptors are dispensable for the cross-talk. In conclusion, we report that cross-talk between NR3B and NR4A receptors is a mechanism modulating the transcriptional activities of these orphan nuclear receptors

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.

Process monitoring in high volume semiconductor production environment with in-fab TXRF

International Nuclear Information System (INIS)

Ghatak-Roy, A.R.; Hossain, T.Z.

2000-01-01

After its introduction in the 80's, TXRF has become an important tool for surface contamination analysis. This is particularly true for the semiconductor industries, where monitoring trace level contamination in ultra clean environment is absolutely necessary for successful device production with reasonable yield. In FAB 25 of the Advanced Micro Devices in Austin, we have installed two TXRF tools, which are model TXRF3750 manufactured by Rigaku. They contain rotating tungsten anodes with three beam capability for wide selection of elements. One of the beams (WM) is used for monitoring of low Z elements such as Na, Mg and Al. The standard output is 9 kW with 300 mA at 30 kV. The tool runs 24 hours a day, 7 days a week, except for maintenance and breakdowns. We have been using TXRF for in-fab monitoring of various tools and processes for trace contamination and some quantification of materials. This in-fab operation is important because it gives real time monitoring without the necessity of bringing the wafers out of the fab. Secondly, being in ultra clean fab environment, the risk of background contamination is minimized. Since TXRF measurement is fast and does not need any sample preparation, this works very well as production support tool. Several wafer fab technicians have been trained to use the tool for round the clock operation. We have successfully monitored tools and processes in our fab. One example is the monitoring of numerous sinks used in the cleaning of production wafers after various processes. Monitor wafers are run after sink cleaning and solvent changes and they are then analyzed for any contamination. Another example is the monitoring of tools that use Ferrofluidic seals so as to prevent any contamination from Fe and Cr. Other tools using TXRF include diffusion furnaces, etchers and plasma cleaning tools. We have also been monitoring processes such as ion implantation, metal deposition and rapid thermal annealing. In this presentation, we will

Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

Science.gov (United States)

Nanson, Jeffrey D; Forwood, Jade K

2015-01-01

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

Directory of Open Access Journals (Sweden)

Jeffrey D Nanson

Full Text Available Ketoacyl-acyl carrier protein reductases (FabG are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP linked thioesters within the bacterial type II fatty acid synthesis (FASII pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG, the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

Science.gov (United States)

Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

2018-05-01

Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

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

Science.gov (United States)

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

2017-10-03

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

Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Lydie Michaillat

Full Text Available The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

The key point of fragmentation of quasiparticle-phonon configurations in the order-disorder transformations of atomic nuclei

International Nuclear Information System (INIS)

Solov'ev, V.G.

1993-01-01

To find out at what excitation energies the order-disorder transformations occur in intermediate and heavy nuclei, it is suggested to study fragmentation of multiquasiparticle and quasiparticle-phonon configurations. One-nucleon transfer reactions on odd-odd targets, for instance on 176 Lu and 180 Ta, should be taken as a particular case of fragmentation of three-quasiparticle configurations on the long living isomer 178 m 2 Hf-fragmentation of five-quasiparticle configurations. From the analysis of γ-decay of high-spin isomers one can information on fragmentation of quasi-phonon configurations

Blood clearance and occupational exposure for {sup 177}Lu-DOTATATE compared to {sup 177}Lu-PSMA radionuclide therapy

Energy Technology Data Exchange (ETDEWEB)

Abuqbeitah, Mohammad; Demir, Mustafa; Uslu-Besli, Lebriz; Yeyin, Nami; Soenmezoglu, Kerim [Istanbul University, Department of Nuclear Medicine, Cerrahpasa Faculty of Medicine, Istanbul (Turkey)

2018-03-15

The main target of this work is to examine blood clearance and external exposure for {sup 177}Lu-DOTATATE compared with new emerging {sup 177}Lu-PSMA therapy. Blood clearance and radiation exposure of 31 patients treated with 5.5 ± 1.1 GBq {sup 177}Lu-DOTATATE were compared to those of 23 patients treated with 7.4 GBq {sup 177}Lu-PSMA. Dose rates were measured at several distances and time points up to 120 h after treatment. Blood samples were collected conjunctively after infusion. Caregiver's cumulative dose was measured by means of an OSL (optically stimulated luminescence) dosimeter for 4-5 days and medical staff's dose was also estimated using electronic personal dosimeters. Finger dose was determined via ring TLD (Thermoluminescence Dosimeter) for radiopharmacists and nurses. Dose rates due to {sup 177}Lu-DOTATATE at a distance of 1 m, 4 h and 6 h after infusion, were 3.0 ± 2.8 and 2 ± 1.9 μSv/(h GBq), respectively, while those due to {sup 177}Lu-PSMA were 3.1 ± 0.8 and 2.2 ± 0.9 μSv/(h GBq). Total effective dose of 17 caregivers was 100-200 μSv for {sup 177}Lu-DOTATATE therapy. Mean effective doses to nurses and radiopharmacists were 5 and 4 μSv per patient, respectively, while those for physicists and physicians were 2 μSv per patient. For {sup 177}Lu-DOTATATE, effective half-life in blood and early elimination phase were 0.31 ± 0.13 and 4.5 ± 1 h, while they were found as 0.4 ± 0.1 and 5 ± 1 h, respectively, for {sup 177}Lu-PSMA. The first micturition time following {sup 177}Lu-DOTATATE infusion was noted after 36 ± 14 min, while the second and third voiding times were after 74 ± 9 and 128 ± 41 min, respectively. It is concluded that blood clearance and radiation exposure for {sup 177}Lu-DOTATATE are very similar to those for {sup 177}Lu-PSMA, and both treatment modalities are reasonably reliable for outpatient treatment, since the mean dose rate [2.1 μSv/(h GBq)] decreased below the dose rate that allows release of the patient

Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

Science.gov (United States)

Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

2016-01-01

Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

Potentiation of glycine-gated NR1/NR3A NMDA receptors relieves Ca2+-dependent outward rectification

Directory of Open Access Journals (Sweden)

Christian Madry

2010-03-01

Full Text Available Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named excitatory glycine receptors. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I-V dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I-V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I-V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors.

Analysis of the spectrum of four-times-ionized lutetium (Lu V)

International Nuclear Information System (INIS)

Kaufman, V.; Sugar, J.

1978-01-01

Spectra of Lu obtained with a sliding spark discharge at peak currents of 50--500 A were recorded with a 10.7 m normal incidence spectrograph in the range of 500--2100 A. Intercomparison of spectra revealed a distinct separation of Lu III, IV, and V, the first two of which have already been anlayzed. The present work contains an interpretation of Lu V in which 419 lines are classified as transitions among 136 energy levels of the 4f 13 , 4f 12 5d, 4f 12 6s, and 4f 12 6p configurations. Calculated energy levels and eigenvectors, obtained with fitted values for the radial integrals, are given

An electro-amalgamation approach to isolate no-carrier-added 177Lu from neutron irradiated Yb for biomedical applications

International Nuclear Information System (INIS)

Chakravarty, Rubel; Das, Tapas; Dash, Ashutosh; Venkatesh, Meera

2010-01-01

Introduction: A novel two-step separation process for the production of no-carrier-added (NCA) 177 Lu from neutron irradiated Yb target through an electrochemical pathway employing mercury-pool cathode has been developed. Methods: A two-cycle electrolysis procedure was adopted for separation of 177 Lu from 177 Lu/Yb mixture in lithium citrate medium. The influence of different experimental parameters on the separation process was investigated and optimized for the quantitative deposition of Yb in presence of 177 Lu. The first electrolysis was performed for 50 min in the 177 Lu/Yb feed solution at pH 6 applying a potential of 8 V using platinum electrode as anode and mercury as the cathode. The second electrolysis was performed under the same conditions using fresh electrodes. The radionuclidic and chemical purity of 177 Lu was determined by using gamma ray spectrometry and atomic absorption spectrometry. The suitability of 177 Lu for biomedical applications was ascertained by labeling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid D-Phe 1 -Tyr 3 -octreotate(DOTA-TATE) with 177 Lu. Results: This process could provide NCA 177 Lu with >99.99% radionuclidic purity and an overall separation yield of ∼99% was achieved within 3-4 h. The Hg content in the product was determined to be 98% was obtained with DOTA-TATE under the optimized reaction conditions. Conclusions: An efficient strategy for the separation of NCA 177 Lu, suitable for biomedical applications, has been developed.

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

Severe adverse drug reaction following Crotalidae Polyvalent Immune Fab (Ovine) administration for copperhead snakebite.

Science.gov (United States)

Lepak, Maryjoy R; Bochenek, Samantha H; Bush, Sean P

2015-01-01

To present the case of a severe anaphylactic/anaphylactoid reaction to Crotalidae Polyvalent Immune Fab (Ovine) in a patient bitten by a copperhead snake. A 68-year-old man presented with progressive envenomation after receiving a copperhead snakebite on each hand. Crotalinae Fab antivenom was administered. While the initial and only dose was partially infusing, the patient developed an adverse drug reaction (ADR) of urticaria and hypotension, which resolved with cessation of the infusion, recurred with resumption of the infusion, and ultimately was completed with supportive care. An additional episode of hypotension, urticaria, and angioedema occurred shortly after antivenom therapy completion. Epinephrine was administered, resolving the reaction with complete patient recovery. The event received a Naranjo score of 10, indicating a definite ADR. Treating copperhead snakebites with antivenom is a matter of debate. Concern over adverse events and cost induce some physicians to manage copperhead bites without antivenom because they are generally milder in severity. As demonstrated in this case, severe ADR can occur with Crotalinae Fab antivenom, and its efficacy for copperhead envenoming needs to be better established via placebo-controlled, randomized trials. © The Author(s) 2014.

Reference metrology in a research fab: the NIST clean calibrations thrust

Science.gov (United States)

Dixson, Ronald; Fu, Joe; Orji, Ndubuisi; Renegar, Thomas; Zheng, Alan; Vorburger, Theodore; Hilton, Al; Cangemi, Marc; Chen, Lei; Hernandez, Mike; Hajdaj, Russell; Bishop, Michael; Cordes, Aaron

2009-03-01

In 2004, the National Institute of Standards and Technology (NIST) commissioned the Advanced Measurement Laboratory (AML) - a state-of-the-art, five-wing laboratory complex for leading edge NIST research. The NIST NanoFab - a 1765 m2 (19,000 ft2) clean room with 743 m2 (8000 ft2) of class 100 space - is the anchor of this facility and an integral component of the new Center for Nanoscale Science and Technology (CNST) at NIST. Although the CNST/NanoFab is a nanotechnology research facility with a different strategic focus than a current high volume semiconductor fab, metrology tools still play an important role in the nanofabrication research conducted here. Some of the metrology tools available to users of the NanoFab include stylus profiling, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Since 2001, NIST has collaborated with SEMATECH to implement a reference measurement system (RMS) using critical dimension atomic force microscopy (CD-AFM). NIST brought metrology expertise to the table and SEMATECH provided access to leading edge metrology tools in their clean room facility in Austin. Now, in the newly launched "clean calibrations" thrust at NIST, we are implementing the reference metrology paradigm on several tools in the CNST/NanoFab. Initially, we have focused on calibration, monitoring, and uncertainty analysis for a three-tool set consisting of a stylus profiler, an SEM, and an AFM. Our larger goal is the development of new and supplemental calibrations and standards that will benefit from the Class 100 environment available in the NanoFab and offering our customers calibration options that do not require exposing their samples to less clean environments. Toward this end, we have completed a preliminary evaluation of the performance of these instruments. The results of these evaluations suggest that the achievable uncertainties are generally consistent with our measurement goals.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

Science.gov (United States)

Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Reducing renal uptake of 9Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues

International Nuclear Information System (INIS)

Miao Yubin; Fisher, Darrell R.; Quinn, Thomas P.

2006-01-01

Objective: The purpose of this study was to improve the tumor-to-kidney uptake ratios of 9 Y- and 177 Lu-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Re-Cys 3,4,1 , D-Phe 7 , Arg 11 ]α-melanocyte stimulating hormone 3-13 {DOTA-Re(Arg 11 )CCMSH} through coupling a negatively charged glutamic acid (Glu) to the peptide sequence. Methods: A new peptide of DOTA-Re(Glu 2 , Arg 11 )CCMSH was designed, synthesized and labeled with 9 Y and 177 Lu. Pharmacokinetics of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH was determined in B16/F1 murine melanoma-bearing C57 mice. Results: 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH exhibited significantly (P 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The renal uptake values of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH were 28.16% and 28.81% of those of 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH, respectively, at 4 h postinjection. 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH displayed higher tumor-to-kidney uptake ratios than 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The tumor-to-kidney uptake ratio of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH was 2.28 and 1.69 times of 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH, respectively, at 4 h postinjection. The 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH activity accumulation was low in normal organs except for kidney. Conclusions: Coupling a negatively charged amino acid (Glu) to the CCMSH peptide sequence dramatically reduced the renal uptake values and increased the tumor-to-kidney uptake ratios of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH, facilitating their potential applications as radiopharmaceuticals for targeted radionuclide therapy of melanoma

Technetium-99m labeling of antibodies to cardiac myosin Fab and to human fibrinogen

International Nuclear Information System (INIS)

Khaw, B.A.; Strauss, H.W.; Carvalho, A.; Locke, E.; Gold, H.K.; Haber, E.

1982-01-01

A method of labeling biologically active labile macromolecules, such as human fibrinogen (HF) and anticardiac-myosin Fab (AM-Fab), with Tc-99m at neutral pH was developed. This method uses dithionite reduction of pertechnetate and subsequent labeling to test the method with acid-labile macromolecules. Complexes of diethylene triamine pentaacetic acid with macromolecules such as human fibrinogen (D-HF) and anticardiac-myosin Fab (D-AM-Fab) were labeled and utilized in in vitro and in vivo studies. In biodistribution studies, the Tc-99m D-HF had a two-component blood clearance (half-times 1 hr and 15 hr) and was 80-88% coagulable. The Tc-99m AM-Fab retained its immunoreactivity as tested by affinity chromatography; also during in vivo localization in experimental myocardial infarction. This labeling technique provides an easy and efficient approach to the Tc-99m labeling of other biologically active and acid-labile macromolecules

20 CFR 30.316 - How does the FAB issue a final decision on a claim?

Science.gov (United States)

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final decision...

Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators

Science.gov (United States)

Scheer, Justin M.; Sandoval, Wendy; Elliott, J. Michael; Shao, Lily; Luis, Elizabeth; Lewin-Koh, Sock-Cheng; Schaefer, Gabriele; Vandlen, Richard

2012-01-01

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules. PMID:23284778

The experimental study on biodistribution and radioimmunoimaging of 131I labeled anti-lymphoma Fab antibody in nude mice bearing human B cell lymphoma

International Nuclear Information System (INIS)

Yang Xiaochun; Zhang Meihua; Shen Junkang; Shen Yongmei; Shi Yizhen; Liu Zengli

2008-01-01

Objective: Radioimmunoimaging is still an interesting study in the domain of nuclear medicine. The aim of this study was to evaluate the biodistribution and radioimmunoimaging of 131 I-Fab anti- body in nude mice beating human B cell lymphoma. Methods: The immunoreactivity of Fab antibody to Raji cells was analyzed by immunohistochemistry and flow cytometry. Fab antibody and CD20 monoclonal antibody (as control) were labeled with 131 I using Iodogen method. 131 I-Fab antibody or 131 I-CD20 was injected into nude mice bearing B cell lymphoma via tail veins. The biodistribution and radioimmunoimaging results were obtained at 2, 4, 8 and 24 h postinjection, respectively. Results: The results of immunohistochemistry and flow cytometry indicated that both Fab antibody and 131 I-Fab antibody could bind strongly with membrane antigens on Raji cells, and the binding rate reached above 87%. Clear tumor image was obtained at 8 h after injection with 131 I-Fab and elimination was observed at 24 h postinjection. The clear tumor image for 131 I-CD20 antibody was obtained at 24 h post injection. The biodistribution in vivo showed that the percentage activities of injection dose per gram of tumor (% ID/g) of 131 I-Fab group at 2, 4, 8 h postinjection were higher than that of 131 I-CD20 antibody [(1.37±0.28), (1.84±0.13), (2.21±0.15)% ID/g vs (0.33±0.06), (0.62±0.08), (1.46±0.24)% ID/g, respectively; F=52.22, 278.42 and 29.00, all P 131 I-Fad and 131 I-CD20 groups at 2, 4, 8 and 24 h were [(0.22±0.03)-(5.44± 0.31)] vs[(0.04±0.01)-(3.10±0.29)], [(0.43±0.11) - (21.01±3.97)] vs [(0.11±0.05) - (7.99±1.81)], [(1.09±0.07) -(20.28±2.77)] vs [(0.48±0.06) - (23.55±1.69)], [(1.12± 0.02) - (10.29±1.78)] vs [(2.32 ± 0.34) - (33.23±6.83)], respectively. Conclusion: 131 I-Fab anti- body has advantages of small molecular weight, excellent targeting characteristics, early imaging and fast elimination, which indicates the potential application value in diagnosing B cell

Late night activity regarding stroke codes: LuNAR strokes.

Science.gov (United States)

Tafreshi, Gilda; Raman, Rema; Ernstrom, Karin; Rapp, Karen; Meyer, Brett C

2012-08-01

There is diurnal variation for cardiac arrest and sudden cardiac death. Stroke may show a similar pattern. We assessed whether strokes presenting during a particular time of day or night are more likely of vascular etiology. To compare emergency department stroke codes arriving between 22:00 and 8:00 hours (LuNAR strokes) vs. others (n-LuNAR strokes). The purpose was to determine if late night strokes are more likely to be true strokes or warrant acute tissue plasminogen activator evaluations. We reviewed prospectively collected cases in the University of California, San Diego Stroke Team database gathered over a four-year period. Stroke codes at six emergency departments were classified based on arrival time. Those arriving between 22:00 and 8:00 hours were classified as LuNAR stroke codes, the remainder were classified as 'n-LuNAR'. Patients were further classified as intracerebral hemorrhage, acute ischemic stroke not receiving tissue plasminogen activator, acute ischemic stroke receiving tissue plasminogen activator, transient ischemic attack, and nonstroke. Categorical outcomes were compared using Fisher's Exact test. Continuous outcomes were compared using Wilcoxon's Rank-sum test. A total of 1607 patients were included in our study, of which, 299 (19%) were LuNAR code strokes. The overall median NIHSS was five, higher in the LuNAR group (n-LuNAR 5, LuNAR 7; P=0·022). There was no overall differences in patient diagnoses between LuNAR and n-LuNAR strokes (P=0·169) or diagnosis of acute ischemic stroke receiving tissue plasminogen activator (n-LuNAR 191 (14·6%), LuNAR 42 (14·0%); P=0·86). Mean arrival to computed tomography scan time was longer during LuNAR hours (n-LuNAR 54·9±76·3 min, LuNAR 62·5±87·7 min; P=0·027). There was no significant difference in 90-day mortality (n-LuNAR 15·0%, LuNAR 13·2%; P=0·45). Our stroke center experience showed no difference in diagnosis of acute ischemic stroke between day and night stroke codes. This

Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

Directory of Open Access Journals (Sweden)

Takayoshi Matsuda

Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

An electro-amalgamation approach to isolate no-carrier-added 177Lu from neutron irradiated Yb for biomedical applications.

Science.gov (United States)

Chakravarty, Rubel; Das, Tapas; Dash, Ashutosh; Venkatesh, Meera

2010-10-01

A novel two-step separation process for the production of no-carrier-added (NCA) (177)Lu from neutron irradiated Yb target through an electrochemical pathway employing mercury-pool cathode has been developed. A two-cycle electrolysis procedure was adopted for separation of (177)Lu from (177)Lu/Yb mixture in lithium citrate medium. The influence of different experimental parameters on the separation process was investigated and optimized for the quantitative deposition of Yb in presence of (177)Lu. The first electrolysis was performed for 50 min in the (177)Lu/Yb feed solution at pH 6 applying a potential of 8 V using platinum electrode as anode and mercury as the cathode. The second electrolysis was performed under the same conditions using fresh electrodes. The radionuclidic and chemical purity of (177)Lu was determined by using gamma ray spectrometry and atomic absorption spectrometry. The suitability of (177)Lu for biomedical applications was ascertained by labeling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid D-Phe(1)-Tyr(3)-octreotate(DOTA-TATE) with (177)Lu. This process could provide NCA (177)Lu with >99.99% radionuclidic purity and an overall separation yield of ∼99% was achieved within 3-4 h. The Hg content in the product was determined to be 98% was obtained with DOTA-TATE under the optimized reaction conditions. An efficient strategy for the separation of NCA (177)Lu, suitable for biomedical applications, has been developed. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

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

Science.gov (United States)

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

2016-01-01

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

75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

Science.gov (United States)

2010-04-23

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,642] Intel Corporation, Fab 20... of Intel Corporation, Fab 20 Division, including on-site leased workers of Volt Technical Resources... Precision, Inc. were employed on-site at the Hillsboro, Oregon location of Intel Corporation, Fab 20...

Development of 177Lu-DOTA-anti-CD20 for radioimmunotherapy

International Nuclear Information System (INIS)

Hassan Yousefnia; Amir Reza Jalilian; Ali Bahrami-Samani; Simindokht Shirvani-Arani; Mohammad Ghannadi-Maragheh; Azim Arbabi; Edalat Radfar

2011-01-01

Rituximab was successively labeled with 177 Lu-lutetium chloride. 177 Lu chloride was obtained by thermal neutron flux (4 x 1013 n cm -2 s -1 ) of natural Lu 2 O 3 sample with a specific activity of 2.6-3 GBq/mg. The macrocyclic bifunctional chelating agent, N-succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 deg C using DOTA, N-hydroxy succinimide (NHS) in CH 2 Cl 2 . DOTA-rituximab was obtained by the addition of 1 mL of a rituximab pharmaceutical solution (5 mg/mL, in phosphate buffer, pH 7.8) to a glass tube pre-coated with DOTA-NHS (0.01-0.1 mg) at 25 deg C with continuous mild stirring for 15 h. Radiolabeling was performed at 37 deg C in 24 h. Radio-thin layer chromatography showed an overall radiochemical purity of >98% at optimized conditions (specific activity = 444 MBq/mg, labeling efficacy; 82%). The final isotonic 177 Lu-DOTA-rituximab complex was checked by gel electrophoresis for structure integrity control. Radio-TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary biodistribution studies in normal rats were carried out to determine complex distribution of the radioimmunoconjugate up to 168 h. The biodistribution data were in accordance with other antiCD20 radioimmunoconjugates already reported. (author)

Improving methyl ketone production in Escherichia coli by heterologous expression of NADH-dependent FabG

DEFF Research Database (Denmark)

Goh, Ee Been; Chen, Yan; Petzold, Christopher J.

2018-01-01

balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance...... by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed...... for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain...

Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

Energy Technology Data Exchange (ETDEWEB)

An, Xiuli; Gauthier, Emilie; Zhang, Xihui; Guo, Xinhua; Anstee, David; Mohandas, Narla; Anne Chasis, Joel

2008-03-18

The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of {alpha}-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

Study of the production of {sup 177}Lu through {sup 176}Yb (n, {gamma}) {sup 177}Yb {yields} {sup 177}Lu nuclear reaction

Energy Technology Data Exchange (ETDEWEB)

Silva, Giovana Pasqualini da; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: gpsilva@ipen.br; jaosso@ipen.br

2007-07-01

The beta minus emitter {sup 177}Lu is a promising therapeutic radioisotope for the curative treatment of cancer using labelled proteins. It has a half - life of T{sub 1/2} = 6.71 day and maximum and average {beta}{sup -} energies of 421 and 133 keV, resulting in a short range of radiation in tissue. The decay is accompanied by the emission of low energy {gamma}-radiation with 208.3 keV (11%) and 113 keV (6.4%) suitable for simultaneous imaging, {sup 177}Lu can be produced by two different routes, namely, by irradiation of natural Lu{sub 2}O{sub 3} target ({sup 176}Lu, 2.6%) or enriched (in {sup 176}Lu) Lu{sub 2}O{sub 3} target, as also by irradiation of Yb target (Yb{sub 2}O{sub 3}) followed by radiochemical separation of {sup 177}Lu from Yb isotopes. The objective of this work is to study the production of {sup 177}Lu through the indirect {sup 176}Yb(n,{gamma}){sup 177}Yb {yields} {sup 177}Lu nuclear reaction. The results of the production yield of {sup 177}Lu will be shown and compared with the direct reaction. The method of choice for the chemical separation between Lu and Yb was the ion exchange, using an cation exchange resin in Cl{sup -} form and {alpha}-HIBA as eluent. Preliminary results showed a good separation of {sup 177}Lu from Yb{sub 2}O{sub 3} indirect targets. (author)

3D printing in social education: Eki-Fab and student PBL

Science.gov (United States)

Makino, Masato; Saito, Azusa; Kodama, Mai; Takamatsu, Kyuuichiro; Tamate, Hideaki; Sakai, Kazuyuki; Wada, Masato; Khosla, Ajit; Kawakami, Masaru; Furukawa, Hidemitsu

2017-04-01

Additive manufacturing or 3D printer is one of the most innovative material processing methods. We are considering that human resources for 3D printing would be needed in the future. To educate the abilities of the digital fabrication, we have the public digital fabrication space "Eki-Fab" for junior and high school students and Project Based Learning (PBL) class for undergraduate students. Eki-Fab is held on every Saturday at the Yonezawa train station. In the "Eki-Fab", anybody can study the utilizing of 3D printer and modeling technics under the instruction of staff in Yamagata University. In the PBL class, we have the class every Thursday. The students get the techniques of the digital fabrication through the PBL.

Crystallization and preliminary X-ray crystallographic analysis of enoyl-ACP reductase III (FabL) from Bacillus subtilis

International Nuclear Information System (INIS)

Kim, Kook-Han; Park, Joon Kyu; Ha, Byung Hak; Moon, Jin Ho; Kim, Eunice EunKyeong

2007-01-01

Enoyl-ACP reductase III (FabL) from B. subtilis has been overexpressed, purified and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120°, and data were collected to 2.5 Å resolution using synchrotron radiation. Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120° and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 Å resolution

NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

Science.gov (United States)

Prince, Lynne R; Prosseda, Svenja D; Higgins, Kathryn; Carlring, Jennifer; Prestwich, Elizabeth C; Ogryzko, Nikolay V; Rahman, Atiqur; Basran, Alexander; Falciani, Francesco; Taylor, Philip; Renshaw, Stephen A; Whyte, Moira K B; Sabroe, Ian

2017-08-24

The lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis. © 2017 by The American Society of Hematology.

Comparison of Spectral and Scintillation Properties of LuAP:Ce and LuAP:Ce,Sc Single Crystals

Science.gov (United States)

Petrosyan, Ashot G.; Derdzyan, Marina; Ovanesyan, Karine; Shirinyan, Grigori; Lecoq, Paul; Auffray, Etiennette; Kronberger, Matthias; Frisch, Benjamin; Pedrini, Christian; Dujardin, Christophe

2009-10-01

Scintillation properties of LuAP:Ce and LuAP:Ce,Sc crystal series were studied under excitation by gamma-rays from a 137Cs source. Both series demonstrated comparable optical quality in terms of underlying absorption at 260 nm, slope of the optical edge and transmission in the range of emission. The light yield of LuAP:Ce crystals measured in 0.2 cm times 0.2 cm times 0.8 cm pixels increases linearly with the Ce concentration reaching at 0.58 at. % 6448 plusmn 322 ph/MeV and 9911 plusmn 496 ph/MeV in the long and in the short directions respectively (the light yield ratio is 65%) and shows no sign of light saturation. The energy resolution is found to depend, among other factors, on the uniformity of Ce concentration within the pixels and is improved to 7.1 plusmn 0.4% (I = 0.2 cm), 9.5 plusmn 0.5% (I = 0.8 cm). Intentional co-doping with Sc + ions was tested and resulted in increase of the Ce distribution coefficient to about 0.3. This enabled to increase the concentration of Ce in LuAP:Ce,Sc crystals up to 0.7 at. %, while conserving high optical quality. In contrast to LuAP:Ce, the light yield in LuAP:Ce,Sc crystals does not increase with Ce concentration, the photo peak being gradually suppressed. The involved mechanisms are discussed basing on measurements of the unit cell volumes, Ce concentration uniformity, x-ray rocking spectra, absorption spectra of pure and variously doped LuAP crystals, and emission spectra under different excitations.

Partnerstwo publiczno – prywatne w świetle Krajowego Standardu Rachunkowości nr 10

Directory of Open Access Journals (Sweden)

Dorota Adamek-Hyska

2017-09-01

Full Text Available Partnerstwo publiczno – prywatne w świetle Krajowego Standardu Rachunkowości nr 10 In Poland, the concept of public-private partnership generates many dilemmas in valuation, accounting records and the reporting of essential assets and liabilities. This issue is addressed by National Accounting Standard No. 10, which outlines the principles of PPP accounting in public and private accounting systems. The purpose of this paper is to analyze and evaluate the solutions adopted in National Accounting Standard No. 10 against the background of synthetic PPP characteristics. The research methods used are based on a critical analysis of national legislation, a comparative analysis of national and international accounting standards, and a literature analysis of public-private partnerships, public finances and financial accounting. The paper uses the conclusions from the observations of financial aspects of selected public-private partnership contracts as well as concession contracts for construction works and services in Poland

The Female Athlete Body (FAB) study: Rationale, design, and baseline characteristics.

Science.gov (United States)

Stewart, Tiffany M; Pollard, Tarryn; Hildebrandt, Tom; Beyl, Robbie; Wesley, Nicole; Kilpela, Lisa Smith; Becker, Carolyn Black

2017-09-01

Eating Disorders (EDs) are serious psychiatric illnesses marked by psychiatric comorbidity, medical complications, and functional impairment. Research indicates that female athletes are often at greater risk for developing ED pathology versus non-athlete females. The Female Athlete Body (FAB) study is a three-site, randomized controlled trial (RCT) designed to assess the efficacy of a behavioral ED prevention program for female collegiate athletes when implemented by community providers. This paper describes the design, intervention, and participant baseline characteristics. Future papers will discuss outcomes. Female collegiate athletes (N=481) aged 17-21 were randomized by site, team, and sport type to either FAB or a waitlist control group. FAB consisted of three sessions (1.3h each) of a behavioral ED prevention program. Assessments were conducted at baseline (pre-intervention), post-intervention (3weeks), and six-, 12-, and 18-month follow-ups. This study achieved 96% (N=481) of target recruitment (N=500). Few group differences emerged at baseline. Total sample analyses revealed moderately low baseline instances of ED symptoms and clinical cases. Health risks associated with EDs necessitate interventions for female athletes. The FAB study is the largest existing RCT for female athletes aimed at both reduction of ED risk factors and ED prevention. The methods presented and population recruited for this study represent an ideal intervention for assessing the effects of FAB on both the aforementioned outcomes. We anticipate that findings of this study (reported in future papers) will make a significant contribution to the ED risk factor reduction and prevention literature. Copyright © 2017 Elsevier Inc. All rights reserved.

An electro-amalgamation approach to isolate no-carrier-added {sup 177}Lu from neutron irradiated Yb for biomedical applications

Energy Technology Data Exchange (ETDEWEB)

Chakravarty, Rubel; Das, Tapas [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Dash, Ashutosh, E-mail: adash@barc.gov.i [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Venkatesh, Meera [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

2010-10-15

Introduction: A novel two-step separation process for the production of no-carrier-added (NCA) {sup 177}Lu from neutron irradiated Yb target through an electrochemical pathway employing mercury-pool cathode has been developed. Methods: A two-cycle electrolysis procedure was adopted for separation of {sup 177}Lu from {sup 177}Lu/Yb mixture in lithium citrate medium. The influence of different experimental parameters on the separation process was investigated and optimized for the quantitative deposition of Yb in presence of {sup 177}Lu. The first electrolysis was performed for 50 min in the {sup 177}Lu/Yb feed solution at pH 6 applying a potential of 8 V using platinum electrode as anode and mercury as the cathode. The second electrolysis was performed under the same conditions using fresh electrodes. The radionuclidic and chemical purity of {sup 177}Lu was determined by using gamma ray spectrometry and atomic absorption spectrometry. The suitability of {sup 177}Lu for biomedical applications was ascertained by labeling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid D-Phe{sup 1}-Tyr{sup 3}-octreotate(DOTA-TATE) with {sup 177}Lu. Results: This process could provide NCA {sup 177}Lu with >99.99% radionuclidic purity and an overall separation yield of {approx}99% was achieved within 3-4 h. The Hg content in the product was determined to be <1 ppm. Radiolabeling yield of >98% was obtained with DOTA-TATE under the optimized reaction conditions. Conclusions: An efficient strategy for the separation of NCA {sup 177}Lu, suitable for biomedical applications, has been developed.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

Science.gov (United States)

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

/sup 99m/Tc labeling of antibodies to cardiac myosin Fab and to human fibrinogen

International Nuclear Information System (INIS)

Khaw, B.A.; Strauss, H.W.; Carvalho, A.; Locke, E.; Gold, H.K.; Haber, E.

1982-01-01

We have developed a method of labeling biologically active labile macromolecules, such as human fibrinogen (HF) and anticardiac-myosin Fab (AM-Fab), with /sup 99m/Tc at neutral pH. This method uses dithionite reduction of pertechnetate and subsequent labeling, to test the method with acid-labile macromolecules. Complexes of diethylene triamine pentaacetic acid with macromolecules such as human fibrinogen (D-HF) and anticardiac-myosin Fab (D-AM-Fab) were labeled and utilized in in vitro and in vivo studies. In biodistribution studies, the /sup 99m/Tc D-HF had a two-component blood clearance (half-times 1 hr and 15 hr) and was 80--88% coagulable. The /sup 99m/Tc AM-Fab retained its immunoreactivity as tested by affinity chromatography; also during in vivo localization in experimental myocardial infarction. This labeling technique provides an easy and efficient approach to the /sup 99m/Tc labeling of other biologically active and acid-labile macromolecules

Preparation and bioevaluation of {sup 177}Lu-labelled anti-CD44 for radioimmunotherapy of colon cancer

Energy Technology Data Exchange (ETDEWEB)

Lee, So Young; Hong, Young Don; Jung, Sung Hee; Choi, Sun Ju [Radioisotope Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2015-12-15

CD44 is a particular adhesion molecule and facilitates both cell-cell and cell-matrix interactions. In particular, splice variants of CD44 are particularly overexpressed in a large number of malignancies and carcinomas. In this study, the {sup 177}Lu-labelled CD44 targeting antibody was prepared and bioevaluated in vitro and in vivo. Anti-CD44 was immunoconjugated with the equivalent molar ratio of cysteine-based dtPA-ncS and radioimmunoconjugated with {sup 177}Lu at room temperature within 15 minutes. the stability was tested in human serum. An in vitro study was carried out in Ht-29 human colon cancer cell lines. For the biodistribution study {sup 177}Lu-labelled anti-CD44 was injected in xenograft mice. Anti-CD44 was immunoconjugated with cysteinebased dtPA-ncS and purified by a centricon filter system having a molecular cut-off of 50 kda. radioimmunoconjugation with {sup 177}Lu was reacted for 15 min at room temperature. the radiolabeling yield was >99%, and it was stable in human serum without any fragmentation or degradation. The radioimmunoconjugate showed a high binding affinity on HT-29 colon cancer cell surfaces. In a biodistribution study, the tumor-to-blood ratio of the radioimmunoconjugate was 43 : 1 at 1 day post injection (p.i) in human colon cancer bearing mice. the anti-CD44 monoclonal antibody for the targeting of colon cancer was effectively radioimmunoconjugated with {sup 177}Lu. the in vitro high immunoactivity of this radioimmunoconjugate was determined by a cell binding assay. In addition, the antibody's tumor targeting ability was demonstrated with very high uptake in tumors. this radioimmunoconjugate is applicable to therapy in human colon cancer with highly expressed CD44.

The mechanical properties of radiation-vulcanized NR/BR blending system

Energy Technology Data Exchange (ETDEWEB)

Yan Aoshuang E-mail: yanas@public3.bta.net.cn; Guo Zhengtao; Li Li; Zhai Ying; Zhou Peng

2002-03-01

The effect of radiation dose on the mechanical properties of NR/BR blending system is reported in this paper. A comparison was made between sulphur vulcanization and radiation vulcanization for an optimal nature rubber (NR)/ butyl rubber (BR) blending ratio (60/40) at dose range from 10 to 150 kGy. The result shows that the mechanical properties, especially, tensile strength, elongation at break, and tear strength have been improved significantly by radiation-vulcanization. This finding was also proved by thermal aging experiment on a selected NR/BR blend at 70 deg. C for up to 168 h.

Progress and improvement of the manufacturing process of snake antivenom

Directory of Open Access Journals (Sweden)

Zolfagharian H.

2013-05-01

Full Text Available Antivenoms have been used successfully for more than a century and up to now constitute the only effectivetreatment for snakebites .The production of antivenin started long time ago when the calmette was preparedthe antivenom in 1894.The method currently used to prepare antivenom by most of the manufacturers areoriginated from the method of Pope which was develop in 1938. Several new approaches in the production ofantivenom have been proposed to produce IgG, F(ab2, F(ab antivenin to improve their quality .Theseimprovement include complete or partial modification in the antivenom production regarding animal,immunization protocols , new adjuvants in hyperimmunization of animals , purification processes ( caprylicacid ,chromatography , diafiltration and ulterafiltration ,enzymatic digestion of IgG (pepsin, papain andfractionation of venom .When the IgG is digested enzymatically, different fragments are obtained depending on the enzyme used, that is, if papain is used, three fragments are obtained, the crystallizing fragment (Fc and two antigen-binding fragments F(ab and, if pepsin is used, one F(ab'2 fragment is obtained, while thecrystallizing fragment is digested. Fab and F(ab2 fragments conserve their capacity to specifically bind to the antigen that gave rise to them.

⁸⁹Zr-Labeled Versus ¹²⁴I-Labeled αHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo.

Science.gov (United States)

Mendler, Claudia T; Gehring, Torben; Wester, Hans-Jürgen; Schwaiger, Markus; Skerra, Arne

2015-07-01

Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide (89)Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide (124)I. To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS200) residues was either conjugated with (124)I via an iodination reagent or coupled with deferoxamine (Df) and complexed with (89)Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1(nu) mice bearing HER2-positive human tumor xenografts. (89)Zr⋅Df-Fab-PAS200 and (124)I-Fab-PAS200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt (124)I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for (89)Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Both (89)Zr- and (124)I

Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

International Nuclear Information System (INIS)

Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C.

2016-01-01

An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex

Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

Energy Technology Data Exchange (ETDEWEB)

Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.; Horne, David A.; Williams, John C., E-mail: jcwilliams@coh.org [Beckman Research Institute of City of Hope, 1710 Flower Street, Duarte, CA 91010 (United States)

2016-05-23

An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented. The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.

Modernization of control system of the beam critical parameters at a lu-10 industrial electron accelerator

International Nuclear Information System (INIS)

Pomatsalyuk, R.I.; Uvarov, V.L.; Shevchenko, V.A.; Shlyakhov, I.N.

2017-01-01

Continuous control and monitoring of critical parameters of radiation processing of products is one of the requirements of the international standard ISO 11137. The current system to monitoring the parameters of radiation treatment of products at the LU-10 accelerator is being in operation for more than 15 years. The life-time of the mayor part of measuring modules is over, and those modules are no longer produced. Modernization of monitoring system with the use of the multi-functional USB modules, single-board mini-computers and EPICS control system (Experimental Physics and Industrial Control System) is considered. The architecture and software for a new monitoring system have been developed. Debugging and operation of the system in a test mode is performed

20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

Science.gov (United States)

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the FAB...

Rational optimization of drug-target residence time: Insights from inhibitor binding to the S. aureus FabI enzyme-product complex

Science.gov (United States)

Chang, Andrew; Schiebel, Johannes; Yu, Weixuan; Bommineni, Gopal R.; Pan, Pan; Baxter, Michael V.; Khanna, Avinash; Sotriffer, Christoph A.; Kisker, Caroline; Tonge, Peter J.

2013-01-01

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme. PMID:23697754

Rare earth-rich cadmium compounds RE{sub 10}TCd{sub 3} (RE = Y, Tb, Dy, Ho, Er, Tm, Lu; T = Rh, Pd, Ir, Pt) with an ordered Co{sub 2}Al{sub 5}-type structure

Energy Technology Data Exchange (ETDEWEB)

Block, Theresa; Klenner, Steffen; Heletta, Lukas; Poettgen, Rainer [Muenster Univ. (Germany). Inst. fuer Anorganische und Analytische Chemie

2018-04-01

Eighteen new rare earth-rich intermetallic phases RE{sub 10}TCd{sub 3} (RE = Y, Tb, Dy, Ho, Er, Tm, Lu; T = Rh, Pd, Ir, Pt) were obtained by induction melting of the elements in sealed niobium ampoules followed by annealing in muffle furnaces. All samples were characterized by X-ray powder diffraction. The structures of four representatives were refined from single-crystal X-ray diffractometer data: ordered Co{sub 2}Al{sub 5} type, P6{sub 3}/mmc, a = 951.2(1), c = 962.9(2) pm, wR = 0.0460, 595 F{sup 2} values, 20 parameters for Er{sub 10}RhCd{sub 3}; a = 945.17(4), c = 943.33(4), wR = 0.0395, 582 F{sup 2} values, 21 parameters for Lu{sub 9.89}PdCd{sub 3.11}; a = 964.16(6), c = 974.93(6) pm, wR = 0.0463, 614 F{sup 2} values, 21 parameters for Y{sub 10}Ir{sub 1.09}Cd{sub 2.91}; a = 955.33(3), c = 974.56(3) pm, wR = 0.0508, 607 F{sup 2} values, 22 refined parameters for Dy{sub 9.92}IrCd{sub 3.08}. Refinements of the occupancy parameters revealed small homogeneity ranges resulting from RE/Cd, respectively T/Cd mixing. The basic building units of the RE{sub 10}TCd{sub 3} phases are transition metal-centered RE{sub 6} trigonal prisms (TP) that are condensed with double-pairs of empty RE{sub 6} octahedra via common triangular faces. A second type of rods is formed by slightly distorted RE3 rate at Cd{sub 6}RE{sub 6} icosahedra which are condensed via Cd{sub 3} triangular faces. The shortest interatomic distances occur for RE-T, compatible with strong covalent bonding interactions. Temperature dependent magnetic susceptibility measurements were performed for RE{sub 10}RhCd{sub 3} (RE = Dy-Tm, Lu), RE{sub 10}IrCd{sub 3} (RE = Er, Tm, Lu) and RE{sub 10}PtCd{sub 3} (RE = Y, Lu). While Y{sub 10}PtCd{sub 3} and Lu{sub 10}TCd{sub 3} (T = Rh, Ir, Pt) show Pauli paramagnetic behavior, the compounds containing paramagnetic rare earth elements show Curie-Weiss behavior (the experimental magnetic moments indicate stable trivalent RE{sup 3+}) and magnetic ordering at low temperatures

Fragment-Based Discovery of Pyrimido[1,2-b]indazole PDE10A Inhibitors.

Science.gov (United States)

Chino, Ayaka; Seo, Ryushi; Amano, Yasushi; Namatame, Ichiji; Hamaguchi, Wataru; Honbou, Kazuya; Mihara, Takuma; Yamazaki, Mayako; Tomishima, Masaki; Masuda, Naoyuki

2018-01-01

In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.

Hybrid synchronization of hyperchaotic Lu system

Indian Academy of Sciences (India)

In this paper, we study the hybrid synchronization between two identical hyperchaotic Lu systems. Hybrid synchronization of hyperchaotic Lu system is achieved through synchronization of two pairs of states and anti-synchronization of the other two pairs of states. Active controls are designed to achieve hybrid ...

Somatostatin receptor expression in the human spleen - Answer to an enigma by ex-vivo and in-vitro autoradiography after 177Lu-DOTA-octreotate administration

International Nuclear Information System (INIS)

Melis, M.; Swart, J. de; Groen, H.C.; Konijnenberg, M.W.; Van der Graaf, L.M.; Kaemmerer, D.; Kulkami, H.R.; Baum, R.P.; Lupp, A.; Saenger, J.; Jong, M. de

2015-01-01

Full text of publication follows. Aim: radiolabelled somatostatin analogues are being used for diagnostic and therapeutic (PRRT) purposes in patients with somatostatin receptor (SSTR) expressing tumours. During PRRT a significant spleen uptake may lead to radiation doses of > 20 Gy. Yet, the threshold dose for spleen radiation induced toxicity is currently unknown. Based on previous 68 Ga-DOTATOC PET/CT studies, demonstrating higher uptake in spleen than in splenosis, white pulp (WP) localization of radioactivity was suggested. This hypothesis was investigated in the current pilot study using the longer lived 177 Lu-DOTA-octreotate. Methods: a patient diagnosed with neuroendocrine neoplasm of the pancreatic tail (SUV max on 68 Ga-DOTATOC PET/CT 100.4) with liver metastasis (SUV 47.3, normal liver SUV 12.5) and uptake in the spleen (SUV 41.0) received 1 GBq 177 Lu-DOTA-octreotate. 2 h after administration whole-body planar scintigraphy and SPECT/CT of the upper abdomen was performed, followed by laparoscopic resection of the pancreatic tumour and splenectomy the next day. After spleen transport from Bad Berka to Rotterdam ex-vivo micro-SPECT of the removed spleen was acquired for 73 min using 2.5 mm diameter pinholes. Spleen fragments (∼10 * 10 * 5 mm) were either snap-frozen in liquid nitrogen or fixed in 10% formalin and paraffin embedded. Ex-vivo autoradiography of 10 μm cryo-sections was performed and serial sections were used for 111 In-DOTA-octreotate in-vitro autoradiography after decay of 177 Lu. FFPE sections were used for HE- and immunostaining for SSTR2A and cell subsets CD4 (Th-cell), CD8 (Ts-cell), CD20 (B-cell) and CD68 (macrophage). Results: 177 Lu-DOTA-octreotate scintigraphy and SPECT/CT demonstrated high uptake in the pancreatic tumor, hepatic metastasis and homogeneously in the normal spleen. High resolution micro-SPECT imaging of the isolated spleen also revealed a relatively homogeneous uptake (calculated rest activity 60 MBq 177 Lu). The

İki uçlu bozukluk 1 ve iki uçlu bozukluk 2 depresyon tedavisi arasındaki farklar

OpenAIRE

Sayar, Kemal

2013-01-01

İki uçlu bozukluk 1 ve 2 depresyonları, klinik açıdan farklılıklar gösterebilmektedir. İki uçlu bozukluk 2 ve iki uçlu bozukluk 1 depresyonların tedavisi arasındaki farklar ise tartışmalı bir alandır. Henüz İki uçlu bozukluk 2 depresyon tedavisi konusunda yeterince kanıt birikmemişse de ilk veriler her iki durumun tedavisinde farklar olması gerektiğini düşündürmektedir. Özellikle antidepresan kullanımı konusunda dikkatli olmak gerekmektedir.

The Bad Berka dose protocol: comparative results of dosimetry in peptide receptor radionuclide therapy using (177)Lu-DOTATATE, (177)Lu-DOTANOC, and (177)Lu-DOTATOC.

Science.gov (United States)

Schuchardt, Christiane; Kulkarni, Harshad R; Prasad, Vikas; Zachert, Carolin; Müller, Dirk; Baum, Richard P

2013-01-01

The objective of this study is to analyze the in vivo behavior of the (177)Lu-labeled peptides DOTATATE, DOTANOC, and DOTATOC used for peptide receptor radionuclide therapy (PRRNT) of neuroendocrine tumors (NETs), by measuring organ and tumor kinetics and by performing dosimetric calculations. Two hundred fifty-three patients (group 1) with metastasized NET who underwent PRRNT were examined. Out of these, 185 patients received (177)Lu-DOTATATE, 9 were treated with (177)Lu-DOTANOC, and 59 with (177)Lu-DOTATOC. Additionally, 25 patients receiving, in consecutive PRRNT cycles, DOTATATE followed by DOTATOC (group 2) and 3 patients receiving DOTATATE and DOTANOC (group 3) were analyzed. Dosimetric calculations (according to MIRD scheme) were performed using OLINDA software. In group 1, DOTATOC exhibited the lowest and DOTANOC the highest uptake and therefore mean absorbed dose in normal organs (whole body, kidney, and spleen). In group 2, there was a significant difference between DOTATATE and DOTATOC concerning kinetics and normal organ doses. (177)Lu-DOTATOC had the lowest uptake/dose delivered to normal organs and highest tumor-to-kidney ratio. There were no significant differences between the three peptides concerning tumor kinetics and mean absorbed tumor dose. The study demonstrates a correlation between high affinity of DOTANOC in vitro and high uptake in normal organs/whole body in vivo, resulting in a higher whole-body dose. DOTATOC exhibited the lowest uptake and dose delivered to normal tissues and the best tumor-to-kidney ratio. Due to large interpatient variability, individual dosimetry should be performed for each therapy cycle.

Pharmacokinetics of the FO23C5 anti-CEA antibody fragment labelled with 99Tcm and 111In: a comparison in patients

International Nuclear Information System (INIS)

Hnatowich, D.J.; Mardirossian, G.; Rusckowski, M.; Roy, S.; Busche, H.; Griffin, T.W.; Brill, A.B.

1993-01-01

The FO23C5 anti-carcinoembryonic antigen (CEA) F(ab') 2 antibody was radiolabelled with sup(111)In via diethylenetriaminepentaacetic acid (DTPA) and directly with 99 Tc m by stannous ion and mercaptoethanol antibody reduction to compare the pharmacokinetics of these three agents. Four patients received 15 mCi 99 Tc m -Fab' 1 week before receiving 1 mCi 111 In-F(ab') 2 . Five additional patients received only the 99 Tc m -Fab'. The biodistribution of 99 Tc m was as expected for a labelled Fab' fragment: relative to 111 In, 99 Tc m cleared rapidly from circulation and into kidneys and urine. Liver levels of 111 In and 99 Tc m were surprisingly similar at 1 day although initial 111 In levels were lower and increased while 99 Tc m levels were higher and decreased. Spleen levels were also similar. In 4/9 patients receiving 99 Tc m , hepatobiliary clearance was observed at levels which could confuse interpretation whereas this mode of clearance was observed in only 1/4 patients receiving 111 In. Image quality was superior with 111 In versus 99 Tc m at 1 day postadministration as judged by counting rates and background activity whereas the opposite was true at 2-3 h postadministration. (author)

Mälu defragmenteerimine / Ilja Sundelevitsh

Index Scriptorium Estoniae

Sundelevitsh, Ilja

2007-01-01

Viktor Misiano kureeritud projekt "Mälu tagasitulek" Kumu Kunstimuuseumis kuni 29. VII. Dmitri Gutovi projektist, mis koosneb videotest "Sula" ja "Kajakad" ning kolmest maalist, Valentin Arhipovi projektist "Vassili Grigorjevitsh Arhipovi looming aastail 1962-2006", Juri Leidermani heliinstallatsioonist "Isa jutustus", Jevgeni Fiksi projektist, Gljuklja & Tzaplja (Olga Jegorova, Natalja Pershina) installatsioonist "Punased purjed", Igor Shtsherbini tööst "Defragmenteerimine. Stirlitzi mälu" ja Sergei Bratkovi tööst

Targeting angiogenesis for radioimmunotherapy with a {sup 177}Lu-labeled antibody

Energy Technology Data Exchange (ETDEWEB)

Ehlerding, Emily B.; Hernandez, Reinier [University of Wisconsin - Madison, Department of Medical Physics, Madison, WI (United States); Lacognata, Saige; Jiang, Dawei [University of Wisconsin - Madison, Department of Radiology, Madison, WI (United States); Ferreira, Carolina A. [University of Wisconsin - Madison, Department of Biomedical Engineering, Madison, WI (United States); Goel, Shreya [University of Wisconsin - Madison, Department of Materials Science and Engineering, Madison, WI (United States); Jeffery, Justin J. [University of Wisconsin - Madison, Small Animal Imaging Facility, Madison, WI (United States); Theuer, Charles P. [TRACON Pharmaceuticals, Inc., San Diego, CA (United States); Cai, Weibo [University of Wisconsin - Madison, Department of Medical Physics, Madison, WI (United States); University of Wisconsin - Madison, Department of Radiology, Madison, WI (United States); University of Wisconsin - Madison, Department of Biomedical Engineering, Madison, WI (United States); University of Wisconsin - Madison, Department of Materials Science and Engineering, Madison, WI (United States)

2018-01-15

Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with {sup 177}Lu (t{sub 1/2} 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: {sup 177}Lu only, TRC105 only, {sup 177}Lu-DTPA-IgG (a nonspecific antibody), {sup 177}Lu-DTPA-TRC105 low-dose, and {sup 177}Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of {sup 177}Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose {sup 177}Lu-DTPA-TRC105. Biodistribution studies indicated steady uptake of {sup 177}Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p < 0.001, all groups). In most study groups (all except the nonspecific IgG group), the body weights of the mice did not decrease by more than 10%, indicating the safety of the injected agents. Serum alanine transaminase levels remained nearly constant indicating no damage to the liver (a primary clearance organ of the agent), and this was confirmed by ex vivo histological analyses. {sup 177}Lu-DTPA-TRC105, when administered at a sufficient dose, is able to curtail tumor growth and provide a

Radionuclide therapy with tissue factor targeting Lu-177-FVIIai inhibits growth in an experimental mouse model of human pancreatic cancer

DEFF Research Database (Denmark)

Nielsen, Carsten; Jensen, Mette; Fonslet, Jesper

2017-01-01

in several organs at 1, 4, 24, 72 and 168 hours after injection. The in vivo biodistribution of 177Lu-FVIIai was evaluated by SPECT/CT imaging. Furthermore, competition and dose escalation experiments (1-30 MBq) were performed. In a parallel set of NMRI mice, toxic effects of 177Lu-FVIIai were evaluated...... by hematology, histology and 99mTc-DMSA scintigraphy. Results: FVIIai was successfully radiolabeled with 177Lu with a specific activity of 10-25 GBq/µmol after EDTA scavenging and PD-10 purification. Treatment with FVIIai did not change tumor growth compared to the vehicle groups. The mice that received 15 MBq...... uptake of 177Lu-FVIIai measured ex vivo was 1.16±0.04, 1.97±0.18, 1.95±0.07, 1.01±0.06, 0.31±0.02 percent injected dose per gram (%ID/g) at 1, 4, 24, 72 and 168 hours post-injection, respectively. Injection with unlabeled FVIIai 10 minutes before 177Lu-FVIIai injection significantly reduced tumor uptake...

Reclassification of leukemia among A-bomb survivors by French-American-British (FAB) classification, 1

International Nuclear Information System (INIS)

Matsuo, Tatsuki; Tomonaga, Masao; Bennett, J.M.

1987-09-01

The concordance rate for the French-American-British (FAB) reclassification diagnoses of atomic bomb-related cases of leukemia in Nagasaki was determined by a group of RERF hematologists and one of the members of the FAB cooperative gruop. The peripheral blood and/or bone marrow smears from 193 persons with leukemia or related disorder were reviewed. There was 85% agreement in the identification of leukemia types and subtypes. There was almost complete agreement for the diagnosis of non-FAB disorders (chronic myeloid leukemia and others) resulting in overall concordance of 88.2%. The conclusion from this remarkably high rate of concordance is that it is feasible to accurately apply the FAB classification system to the cases of A-bomb-related leukemia. These preliminary observations suggest that the previously established leukemia types for about a quarter of the cases of acute leukemia and related disorders should be changed. (author)

20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

Science.gov (United States)

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316, and...

Luís de Camões então e agoradoi:10.5007/2176-8552.2010n10p15

Directory of Open Access Journals (Sweden)

Helder Macedo

2010-01-01

Full Text Available Luís de Camões viveu num mundo em transição entre verdades recebidas e certezas ainda por conhecer. Esta leitura analítica da sua obra visa a caracterizar a arrojada modernidade da sua demanda de uma significação universal para a sua experiência pessoal.

First evidence for triaxial superdeformation in sup 1 sup 6 sup 1 Lu and sup 1 sup 6 sup 2 Lu

CERN Document Server

Bringel, P; Domscheit, J; Neusser, A; Schoenwasser, G; Singh, A K; Amro, H; Axiotis, M; Kröll, T; Napoli, D R; Bazzacco, D; Lunardi, S; Ur, C; Bhattacharya, S; Bhowmik, R; Hagemann, G B; Jensen, D R; Pancholi, S C; Petrache, C M

2003-01-01

High-spin states in sup 1 sup 6 sup 1 Lu and sup 1 sup 6 sup 2 Lu have been investigated using the GASP gamma-ray spectrometer array. Excited states in these nuclei have been populated through the sup 1 sup 0 sup 0 Mo( sup 6 sup 5 Cu, xn) reaction at a beam energy of 260 MeV. Four presumably triaxial superdeformed bands, three in sup 1 sup 6 sup 2 Lu and one in sup 1 sup 6 sup 1 Lu, have been observed. This is the first evidence for triaxial superdeformation in the two isotopes. (orig.)

Differential functions of NR2A and NR2B in short-term and long-term memory in rats.

Science.gov (United States)

Jung, Ye-Ha; Suh, Yoo-Hun

2010-08-23

N-methyl-D-aspartate receptors (NMDARs) are glutamate receptors implicated in synaptic plasticity and memory function. The specific functions of NMDA receptor subunits NR2A and NR2B have not yet been fully determined in the different types of memory. Nine Wistar rats (8-weeks-old) were subjected to the Morris water maze task to evaluate the memory behaviorally. Quantitative analysis of NR1, NR2A, and NR2B levels in the right and left forebrain of rats was performed and subunit associations with different types of memory were investigated using the Morris water maze task. Right forebrain NR2A expression was significantly increased and correlated with faster escape time onto a hidden platform, indicating involvement of short-term memory, because of the training time interval. Right forebrain NR2B expression was positively associated with long-term memory lasting 24-h (h). In the left forebrain, NR2B expression was positively related to 72-h long-term memory. In conclusion, the functions of NR2A and NR2B receptors were differentially specialized in short-term and long-term memory, depending on the right or left forebrain.

Neutron capture reactions on Lu isotopes at DANCE

CERN Document Server

Roig, O

2010-01-01

The DANCE (Detector for Advanced Neutron Capture Experiments) array located at the Los Alamos national laboratory has been used to obtain the neutron capture cross sections for 175Lu and 176Lu with neutron energies from thermal up to 100 keV. Both isotopes are of current interest for the nucleosynthesis s-process in astrophysics and for applications as in reactor physics or in nuclear medicine. Three targets were used to perform these measurements. One was natLu foil and the other two were isotope-enriched targets of 175Lu and 176Lu. The cross sections are obtained for now through a precise neutron flux determination and a normalization at the thermal neutron cross section value. A comparison with the recent experimental data and the evaluated data of ENDF/B-VII.0 will be presented. In addition, resonances parameters and spin assignments for some resonances will be featured.

Fast biodegradation of toxic bisphenol a by Pseudomonas aeruginosa NR.22 (Ps.NR.22) isolated from Malaysian local lake

Science.gov (United States)

Him, Nik Raikhan Nik; Zainuddin, Mohammad Fiqri; Basha, Anuar Zain Anuar

2017-12-01

The paper focused on microbial degradation of Bisphenol A (BPA) as a safe and fast method to reduce BPA contamination in water. BPA is found in waste water, sea water and home water pipeline and it is nondegradable pollutant. Biodegradation is suggested to be practical solution for large volume of BPA. Biodegradation plays an important role and the effect of low concentration significantly decreased the degradation rate. Pseudomonas aeruginosa NR.22 (Ps.NR.22) which has been isolated from a lake at Seksyen 2, Shah Alam, was used. In Malaysia, Ps.NR.22 isolation agar is used for the BPA degradation process. It was stained with Gram negative-rod shaped bacteria that confirmed to carry a 16S rRNA gene. BPA as a sole carbon has been tested at various concentrations. The research showed that BPA was degraded at 10, 20, 30, 40 and 50 ppm and the bacteria growth rate was excellent in 20 ppm BPA. Degradation of BPA was carried out for 9 hours to 18 hours and the maximum degradation was recorded at 9 hours. The value of the highest peak of growth at 540 nm was 2.0617 using 20 ppm BPA. This novel Pseudomonas aeruginosa NR.22 has great potential to be used in waste water treatment.

Co-Application of Corticosterone and Growth Hormone Upregulates NR2B Protein and Increases the NR2B:NR2A Ratio and Synaptic Transmission in the Hippocampus

Directory of Open Access Journals (Sweden)

Ghada S. Mahmoud

2014-10-01

Full Text Available Objectives: This in vitro study aimed to investigate the possible mechanism underlying the protective effect of growth hormone (GH on hippocampal function during periods of heightened glucocorticoid exposure. Methods: This study was conducted between January and June 2005 at the Joan C. Edwards School of Medicine, Marshall University, in Huntington, West Virginia, USA. The effects of the co-application of GH and corticosterone (CORT were tested at different concentrations on the field excitatory postsynaptic potentials (fEPSPs of the hippocampal slices of rats in two different age groups. Changes in the protein expression of N-methyl-D-aspartate receptor (NMDAR subunits NR1, NR2B and NR2A were measured in hippocampal brain slices treated with either artificial cerebrospinal fluid (ACSF, low doses of CORT alone or both CORT and GH for three hours. Results: The co-application of CORT and GH was found to have an additive effect on hippocampal synaptic transmission compared to either drug alone. Furthermore, the combined use of low concentrations of GH and CORT was found to have significantly higher effects on the enhancement of fEPSPs in older rats compared to young ones. Both GH and CORT enhanced the protein expression of the NR2A subunit. Simultaneous exposure to low concentrations of GH and CORT significantly enhanced NR2B expression and increased the NR2B:NR2A ratio. In contrast, perfusion with CORT alone caused significant suppression in the NR1 and NR2B protein expression and a decrease in the NR2B:NR2A ratio. Conclusion: These results suggest that NMDARs provide a potential target for mediating the GH potential protective effect against stress and age-related memory and cognitive impairment.

Thermodynamic model of binding of flexible bivalent haptens to antibody

Energy Technology Data Exchange (ETDEWEB)

Dembo, M; Goldstein, B

1978-01-01

Studies by Wilder et al. of the binding of Fab' fragments to small haptens have shown that the cross-linking constant (the equilibrium constant for binding an additional Fab' fragment to a hapten-Fab' complex) is strongly dependent on the length of the hapten. We present a simple model for predicting the relationship between the intermolecular cross-linking constant and the monovalent hapten-antibody binding constant. In particular we used the model to obtain the dependence of the cross-linking constant on the length of th hapten, the depth to which the hapten fills th Fab' binding site, and the size of the Fab' fragment. To test the model, we devised expressions which allowed us to analyze the data of Wilder et al. From their data we determined the values of two parameters which we took to be unknown in the theory, the size of the Fab' fragment and the depth to which the hapten fills the Fab' binding site. The values arrived at in this way agreed well with published measurements of these parameters.

A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

DEFF Research Database (Denmark)

Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

2002-01-01

The fab1 mutant of Arabidopsis is partially deficient in activity of ß-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a c......DNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

Spherical cows in the sky with fab four

Energy Technology Data Exchange (ETDEWEB)

Kaloper, Nemanja; Sandora, McCullen, E-mail: kaloper@physics.ucdavis.edu, E-mail: mesandora@ucdavis.edu [Department of Physics, University of California, Davis, CA 95616 (United States)

2014-05-01

We explore spherically symmetric static solutions in a subclass of unitary scalar-tensor theories of gravity, called the 'Fab Four' models. The weak field large distance solutions may be phenomenologically viable, but only if the Gauss-Bonnet term is negligible. Only in this limit will the Vainshtein mechanism work consistently. Further, classical constraints and unitarity bounds constrain the models quite tightly. Nevertheless, in the limits where the range of individual terms at large scales is respectively Kinetic Braiding, Horndeski, and Gauss-Bonnet, the horizon scale effects may occur while the theory satisfies Solar system constraints and, marginally, unitarity bounds. On the other hand, to bring the cutoff down to below a millimeter constrains all the couplings scales such that 'Fab Fours' can't be heard outside of the Solar system.

Internal radiotherapy and dosimetric study for 111In/177Lu-pegylated liposomes conjugates in tumor-bearing mice

International Nuclear Information System (INIS)

Wang, H.-E.; Yu, H.-M.; Lu, Y.-C.; Heish, N.-N.; Tseng, Yun-Long; Huang, K.-L.; Chuang, K.-T.; Chen, Chin-Hsiung; Hwang, J.-J.; Lin, W.-J.; Wang, Shyh-Jen; Ting, G.; Whang-Peng, Jacqueline; Deng, W.-P.

2006-01-01

In vivo characterization and dosimetric analysis has been performed to evaluate the potential of pegylated liposomes as carriers of radionuclides in tumor internal radiotherapy. Methods: The DTPA/PEG-liposomes were synthesized with a medium size of 110 nm, conjugated with 111 In/ 177 Lu-(oxine) 3 to afford 111 In/ 177 Lu-liposome. The stability of 111 In/ 177 Lu-liposome in serum was investigated. The biodistribution, scintigraphic imaging and pharmacokinetics of 111 In/ 177 Lu-liposomes after intravenous(i.v.) injection into C-26 tumor-bearing BALB/cByJ mice were studied. Radiation dose was estimated by MIRD-III program. Results: The incorporation efficiency of 111 In/ 177 Lu into liposomes was 95%. After incubation at 37 o C for 72 h in serum, more than 83% of radioactivity was still retained in the intact 111 In/ 177 Lu-liposomes. The biodistribution of 111 In-liposomes showed that the radioactivity in the blood decreased from 23.14±8.16%ID/g at 1 h to 0.02±0.00%ID/g at 72 h post-injection (p.i.), while reaching its maximum accumulation in tumors at 48 h p.i., with half-life in blood of 10.2 h. The results were supported by that of 177 Lu-liposomes. Scintigraphic imaging with 111 In-liposomes showed unambiguous tumor images at 48 h p.i. Dose estimation showed that the absorbed dose in tumor from 177 Lu-liposomes was 5.74x10 -5 Gy/MBq. Conclusions: This study provides an in vivo characterization and dosimetric evaluation for the use of liposome systems as carriers in targeted radionuclide therapy. The results suggest that adequate tumor targeting as well as dose delivered to tumors could be achieved by the use of radionuclide targeted liposomes

Crotaline Fab antivenom appears to be effective in cases of severe North American pit viper envenomation: An integrative review

Directory of Open Access Journals (Sweden)

Mlynarchek Sara L

2009-06-01

Full Text Available Abstract Background In 2000, the United States Food and Drug Administration approved Crotalidae Polyvalent Immune Fab (Ovine (hereafter, FabAV, "for the management of patients with minimal to moderate North American Crotalid envenomation." Because whole-IgG pit viper antivenom is no longer available in the United States, FabAV is currently the only specific treatment option available to United States clinicians treating snakebite victims of any severity. No clinical trial data are available concerning the effectiveness of FabAV for treatment of severe snakebite, but several published articles describe its use in this setting. Methods We performed a comprehensive review of the English-language medical literature to identify all publications (1996 to July, 2008 containing data about the administration of FabAV. Two trained reviewers separately extracted case-level data concerning the administration of FabAV to patients with severe envenomation by North American crotaline snakes to a standardized form. Descriptive statistics were used. In addition, we hand-searched the US National Poison Data System reports for the years 2000–2006 to identify and describe any reports of death that occurred after FabAV administration. Results The literature review found 147 unique publications regarding FabAV. Twenty-four evaluable cases of severe human envenomation treated with FabAV were identified in 19 publications. Seven cases were described in five cohort studies, and 17 cases were described in 14 single patient case reports or non-cohort case series. Sixty-five specific severe venom effects were reported in these 24 patients, of which 50 effects (77% improved or resolved after FabAV therapy. Initial control of all severe venom effects was achieved in 12 patients (50%. The rate at which initial control was achieved was significantly higher among patients reported in the cohort series than in the case series and non-cohort reports (100% vs. 29%, P = 0

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

Science.gov (United States)

Lu, Danfeng; Li, Zhuoran; Li, Lingling; Yang, Liping; Chen, Guijun; Yang, Deying; Zhang, Yue; Singh, Vikrant; Smith, Sheryl; Xiao, Yu; Wang, Erlin; Ye, Yunshuang; Zhang, Wei; Zhou, Lei; Rong, Yikang; Zhou, Jumin

2018-01-01

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights

Tumoral fibrosis effect on the radiation absorbed dose of 177Lu-Tyr3-octreotate-gold nanoparticles and 177Lu-Tyr3-octreotate radiopharmaceuticals

International Nuclear Information System (INIS)

Zambrano R, O. D.

2015-01-01

In this work was comparatively evaluated the effect of tumoral fibrosis in the radiation absorbed dose of the radiopharmaceutical 177 Lu-Tyr 3 -octreotate with and without gold nanoparticles. For this, was used an experimental array of tumoral fibrosis and computer models based on Monte Carlo calculations to simulate tumoral micro environments without fibrosis and with fibrosis. The computer simulation code Penelope (Penetration Energy Loss of Positron and Electrons) and MCNP (Monte Carlo N-particle Transport Code System) which are based on the Monte Carlo methodology were used to create the computer models for the simulation of the transport of particles (emitted by 177 Lu) in the micro environments (without fibrosis and with fibrosis) with the purpose of calculating the radiation absorbed dose in the interstitial space and in the nucleus of cancer cells. The first computational model consisted of multiple concentric spheres (as onion shells) with the radioactive source homogeneously distributed in the shell between 5 and 10 μm in diameter which represents the internalization of the radioactive source into the cell cytoplasm as it occurs in target specific radiotherapy. The concentric spheres were useful to calculate the radiation absorbed dose in depth in the models without fibrosis and with fibrosis. Furthermore, there were constructed other computer models using two different codes that simulate the transport of radiation (Penelope and MCNP). These models consist of seven spheres that represent cancer cells (HeLa cells) of 10 μm in diameter and each one of them contain another smaller sphere in the center that represents the cell nucleus. A comparison was done of the radiation absorbed dose in the nucleus of the cells, calculated with both codes, Penelope and MCNP. The radioactive source ( 177 Lu) used for the simulations was given to the codes by means of a convoluted spectrum of the most important beta particles (high percentage emission). To this spectrum

The coordination number of Lu(III) in a mixed system of methanol and water

International Nuclear Information System (INIS)

Arisaka, M.; Takuwa, N.; Suganuma, H.

2000-01-01

The stability constants (β 1(F) ) of the monofluoro complex of Lu(III) and those (β 1(Cl) ) of the monochloride solvent-shared ion-pair of Lu(III) have been determined in mixed solvents of methanol and water at 0.10 and 1.00 mol x dm -3 ionic strengths, respectively. The variation in lnβ 1(F) with an increase in the mole fraction of methanol (X s ) in the mixed solvent system showed an acute-angled convex inflection point at X s ∼ 0.12, an acute-angled concave inflection point at X s ∼ 0.22, and another acute-angled convex inflection point at X s ∼ 0.27. It was concluded that the first and the second convex inflection points denoted the CN of Lu 3+ from CN = 8 to a mixture of CN = 8 and 7 and from CN = 8 and 7 to a mixture containing CN = 6, respectively. The concave point is the starting point of a change in the CN of Lu(III) in LuF 2+ from CN = 8 to a mixture of CN = 8 and 7. The values at two inflection points of the CN around Lu 3+ are consistent with the inflection points of the variation in the values of lnβ 1(Cl ) versus the dielectric constant of the mixed solvent. (author)

{sup 177}Lu-DOTMP: a viable agent for palliative radiotherapy of painful bone metastasis

Energy Technology Data Exchange (ETDEWEB)

Das, T.; Chakraborty, S.; Banerjee, S. [Radiopharmaceuticals Div., Bhabha Atomic Research Centre, Mumbai (India); Sarma, H.D. [Radiation Biology and Health Sciences Div., Bhabha Atomic Research Centre, Mumbai (India)

2008-07-01

The suitable nuclear decay characteristics [T{sub 1/2} = 6.73 d, E{sub {beta}}{sub (max)} = 497 keV, E{sub {gamma}} = 113 keV (6.4%), 208 keV (11%)] as well as the feasibility of large-scale production with adequate specific activity and radionuclidic purity using a moderate flux reactor are important attributes towards {sup 177}Lu to be considered as a promising radionuclide for palliative care in painful bone metastasis. The present study describes the preparation of {sup 177}Lu complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene phosphonic acid (DOTMP) and its preliminary biological evaluation in animal models with an aim to proposing it as a viable radiopharmaceutical for bone pain palliation. The choice DOTMP as the polyaminophosphonic acid carrier ligand is based on the enhanced thermodynamic stability and kinetic inertness of the metal-ligand complexes with macrocyclic chelators. {sup 177}Lu was produced with a specific activity of {proportional_to} 12 GBq/mg ({proportional_to} 324 mCi/mg) and radionuclidic purity of 99.98% by irradiation of natural Lu{sub 2}O{sub 3} target at a thermal neutron flux of {proportional_to} 6 x 10{sup 13} n/cm{sup 2} s for 21 d. {sup 177}Lu-DOTMP complex was prepared in high yield and excellent radiochemical purity (> 99%) using DOTMP synthesized and characterized in-house. The complex exhibited excellent in-vitro stability at room temperature. Biodistribution studies in Wistar rats showed rapid skeletal accumulation of the injected activity [(1.60{+-}0.19)% per gram in femur at 3 h post-injection] with fast clearance from blood and minimal uptake in any of the major organs. Scintigraphic studies carried out in normal Wistar rats and New Zealand white rabbits also demonstrated significant accumulation of the agent in skeleton and almost no retention in any other vital organs. (orig.)

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.

Peptide receptor radionuclide therapy (PRRT) using Lu-177 DOTA-NOC and Lu-177 DOTA-TATE: Comparative results

International Nuclear Information System (INIS)