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Fab Fragment (fab + fragment)
Kinds of Fab Fragment Selected AbstractsThe clinical pharmacology of therapeutic monoclonal antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Lorin K. Roskos Abstract Seventeen monoclonal antibodies are currently approved in the United States for therapeutic use in organ transplantation, percutaneous coronary intervention, prophylaxis of respiratory syncytial virus disease, rheumatoid arthritis, Crohn's disease, asthma, chronic lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, and colorectal cancer. All approved antibodies are of the IgG class. Thirteen are unconjugated intact antibodies, three are intact immunoconjugates, and one is a Fab fragment. Three of the antibodies are murine, five are chimeric, eight are humanized, and one is a fully human antibody generated by phage display technology. The antigen target and the structural and binding characteristics of the antibody determine the antibody's mechanism of action, pharmacokinetics, safety, and immunogenicity. Antibodies act through multiple mechanisms that include functional modulation of the antigen, recruitment of ADCC and CDC, and delivery of radionuclide or toxin payloads to target cells. Antibody half-life is usually governed by interaction with the FcRn receptor. In some cases, the antigen may act as a sink for antibody elimination. Safety profiles are determined by the pharmacology and tissue distribution of the target antigen, antibody isotype, the antibody payload, cytokine release, hypersensitivity reactions to xenogeneic protein, and immunogenicity. Fully human antibody technology may allow development of antibodies that have reduced risks of hypersensitivity reactions and immunogenicity, thereby enhancing safety and efficacy. The exquisite target specificity of antibodies, improvements in antibody engineering technology, and the wide availability of novel and validated therapeutic targets provide many current and future opportunities for the clinical development of therapeutic antibodies. Drug Dev. Res. 61:108,120, 2004. © 2004 Wiley-Liss, Inc. [source] Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thalianaFEBS JOURNAL, Issue 15 2001Koen Peeters To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fab fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3, regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3, regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fab fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of Fab fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants. [source] Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodiesFEBS JOURNAL, Issue 13 2001Konstantinos Poulas The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 Å resolution by X-ray crystallographic methods. The mouse anti-lysozyme Fab D1.3 was used as a search model in molecular replacement with the amore software. The complementarity determining regions (CDR)-L2, CDR-H1 and CDR-H2 belong to canonical groups. Loops CDR-L3, CDR-H2 and CDR-H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen-binding are examined. The antigen-binding site was found to be a long crescent-shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis. [source] Receptor for the globular heads of C1q (gC1q-R, p33, hyaluronan-binding protein) is preferentially expressed by adenocarcinoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Daniel B. Rubinstein Abstract Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor-associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3-D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 1010 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q-R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q-R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre-mRNA SF2. Sequencing of the gene encoding tumor-associated gC1q-R did not reveal any consistent tumor-specific mutations. However, histochemical staining with anti-gC1q-R MAb demonstrated marked differential expression of gC1q-R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q-R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q-R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q-R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell-to-cell interactions suggest that gC1q-R may have a role in tumor metastases and potentially serve in molecule-specific targeting of malignant cells. © 2004 Wiley-Liss, Inc. [source] Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT techniqueJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2007Hilke Zander Abstract Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15,mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 Fab fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x,,,P), which occurs within the so-called Nogo66 region (residues 1054,1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 Fab fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 Fab fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334,966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 Fab fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive. Copyright © 2007 John Wiley & Sons, Ltd. [source] Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2008P. OHLMANN Summary.,Objectives:,Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia-,reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Methods and results:,Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg,1) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s,1) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. Conclusion:,The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion. [source] Germline humanization of a murine A, antibody and crystal structure of the humanized recombinant Fab fragmentPROTEIN SCIENCE, Issue 2 2010Remy Robert Abstract Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The A, peptide (39,43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope (3EFRH6) in the A, peptide at the N-terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer-mediated toxicity. In this study, we describe the humanization of WO-2 using complementary determining region loop grafting onto the human germline gene and the determination of the three-dimensional structure by X-ray crystallography. This humanized version retains a high affinity for the A, peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease. [source] Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineeringPROTEIN SCIENCE, Issue 8 2006Koki Makabe Abstract Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" ,-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Å resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel ,-sheet. These well-defined water molecules provide additional structural links between adjacent ,-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA. [source] Effects of molecular crowding by saccharides on ,-chymotrypsin dimerizationPROTEIN SCIENCE, Issue 5 2002Chetan N. Patel Abstract Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of ,-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the ,-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the ,-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the ,-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars. [source] Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor , in immature bovine and adult human articular cartilageARTHRITIS & RHEUMATISM, Issue 10 2009Yihong Sui Objective Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor , (TNF,), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNF,. Methods Injured or uninjured cartilage disks were incubated with or without TNF, and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6,blocking antibody Fab fragment and subjected to injury and TNF, treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. Results In bovine cartilage samples, injury combined with TNF, and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNF, exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6,blocking Fab significantly reduced proteoglycan degradation. Conclusion Our findings indicate that mechanical injury potentiates the catabolic effects of TNF, and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNF, appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6,blocking Fab. [source] Heterogeneous nucleation of three-dimensional protein nanocrystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2007Dilyana G. Georgieva Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4,Å resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hair's cuticles, explaining the strong nucleating activity of the surface. [source] Purification, crystallization, X-ray diffraction analysis and phasing of a Fab fragment of monoclonal neuroantibody ,D11 against nerve growth factorACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2004Sonia Covaceuszach The rat monoclonal neuroantibody ,D11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of ,D11 were performed. ,D11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The ,D11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7,Å, , = 82.0, , = 89.1, , = 86.0°. With two molecules in the asymmetric unit, VM is 2.3,Å3,Da,1 and the solvent content is 46%. A complete data set has been collected at 2.7,Å resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10,Å, , = 117.0°. With one molecule in the asymmetric unit, VM is 2.4,Å3,Da,1 and the solvent content is 48%. A complete data set has been collected at 1.7,Å resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern. [source] Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigenACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Shuh-Chyung Song The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293,K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4,8,mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25,Å resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75,Å resolution. Data were collected that indicated that the crystals belonged to space group P212121, with unit-cell parameters a = 57.9, b = 77.1, c = 118.1,Å and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP). [source] Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogueACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Carbamatase catalytic antibody Fab fragment Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N -(3,5-dicarboxyphenyl)- P -{N- [5,-(2,,,5,,-dioxo-1,,-pyrrolidinyl)oxy-1,,5,-dioxopentyl]-4-aminophenylmethyl}phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2,Å resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2,Å, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5,Å. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function. [source] Crystallization and preliminary X-ray analysis of a recombinant Fab fragment in complex with 17,-oestradiolACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000Urpo Lamminmäki The recombinant Fab fragment of the anti-17,-oestradiol antibody 57-2 has been a target for several protein-engineering experiments. A method for production, purification and crystallization of the Fab fragment alone (apo form) and in complex with the major female sex hormone 17,-oestradiol is reported here. Diffracting apo-form crystals were only obtained with microseeding; crystals of the Fab,steroid complex were produced by co-crystallization in the presence of oestradiol and cross-seeding with the apo-form crystals. The crystals were grown using vapour-diffusion methods with reservoir solutions containing 10,14% PEG 4000 or 8,12% PEG 8000 and Tris,HCl buffer at high pH (9.0,9.5). Both the apo and complex crystals belong to space group P212121 and diffract to 2.0,Å resolution. High-resolution X-ray data sets suitable for structure determination were collected from flash-cooled crystals using 25% glycerol as the cryoprotectant. [source] Structure of the Fab fragment from F124, a monoclonal antibody specific for hepatitis B surface antigenACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000F. A. Saul The crystal structure of the Fab fragment from the monoclonal anti-preS2 antibody F124 (IgG1,,) has been solved by molecular replacement and refined at 3.0,Å resolution. The Fab crystallizes with two independent molecules in the asymmetric unit. F124 recognizes an epitope contained within the preS2 segment between residues 120 and 132 of the surface antigen of hepatitis B virus. The antibody shows a high affinity for the glycan N-linked to Asn123, but it also cross-reacts with the non-glycosylated peptide fragment 120,132. Although crystallization was performed in the presence of an eightfold excess of the cross-reactive peptide, no evidence for the ligand was found in the antigen-binding site, which is close to a neighbouring molecule in the crystal lattice. The antigen-binding site has a groove-like topology which is modulated with pocket-like cavities. It is characterized by a large number of tyrosine and aspartate residues. The importance of germ-line mutations at the binding site is discussed. [source] Crystallization of BMP receptor type IA bound to the antibody Fab fragment AbD1556ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Stefan Harth An antibody Fab fragment, AbD1556, was selected against the extracellular domain of BMP receptor type IA, which blocks the binding of BMP-2 to BMPR-IA and thereby neutralizes BMP-2 activity. To study the mechanism by which BMPR-IA is recognized and bound by the Fab fragment, the complex of AbD1556 bound to BMPR-IA was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group P21, with unit-cell parameters a = 89.32, b = 129.25, c = 100.24,Å, , = 92.27°. [source] Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Lei Jin NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0,Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2,Å resolution and a clear molecular-replacement solution was obtained for solution of the structure. [source] Crystallization and preliminary X-ray diffraction analysis of the complex of a human anti-ephrin type-A receptor 2 antibody fragment and its cognate antigenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Vaheh Oganesyan The recombinant N-terminal domain of human ephrin type-A receptor 2 (rEphA2) has been crystallized in complex with the recombinantly produced Fab fragment of a fully human antibody (1C1; IgG1/,). These are the first reported crystals of an ephrin receptor bound to an antibody. The orthorhombic crystals belonged to space group C2221 (the 00l reflections obey the l = 2n rule), with unit-cell parameters a = 78.93, b = 120.79, c = 286.20,Å. The diffraction of the crystals extended to 2.0,Å resolution. However, only data to 2.55,Å resolution were considered to be useful owing to spot overlap caused by the long unit-cell parameter. The asymmetric unit is most likely to contain two 1C1 Fab,rEphA2 complexes. This corresponds to a crystal volume per protein weight (VM) of 2.4,Å3,Da,1 and a solvent content of 49.5%. The three-dimensional structure of this complex will shed light on the molecular basis of 1C1 specificity. This will also contribute to a better understanding of the mechanism of action of this antibody, the current evaluation of which as an antibody,drug conjugate in cancer therapy makes it a particularly interesting case study. [source] Noncanonical conformation of CDR L1 in the anti-IL-23 antibody CNTO4088ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Alexey Teplyakov CNTO4088 is a monoclonal antibody to human IL-23. The X-ray structure of the Fab fragment revealed an unusual noncanonical conformation of CDR L1. Most antibodies with the , light chain exhibit a canonical structure for CDR L1 in which residue 29 anchors the CDR loop to the framework. Analysis of the residues believed to define the conformation of CDR L1 did not explain why it should not adopt a canonical conformation in this antibody. This makes CNTO4088 a benchmark case for developing prediction methods and structure-modeling tools. [source] Crystallization of the receptor-binding domain of parathyroid hormone-related protein in complex with a neutralizing monoclonal antibody Fab fragmentACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009William J. McKinstry Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5,Å, and diffracted to 2.0,Å resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone. [source] Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon ,-2AACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Vaheh Oganesyan Recombinant human interferon ,-2A (rhIFN-,-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/,) which targets several human interferon , subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I212121, with unit-cell parameters a = 134.82, b = 153.26, c = 163.49,Å. The diffraction of the crystals extended to 3.0,Å resolution. The asymmetric unit contained two Fab,rhIFN-,-2A complexes. This corresponded to a crystal volume per protein weight (VM) of 3.02,Å3,Da,1 and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity. [source] Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the A, peptides associated with Alzheimer's diseaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2008Kwok S. Wun The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid , peptide (A,) associated with Alzheimer's disease. This region of A, 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 A, peptides A,1,16 and A,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 ZnSO4; they belonged to the orthorhombic space group P212121 and diffracted to 1.6,Å resolution. The complexes of WO2 Fab with either A,1,16 or A,1,28 were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P212121, and diffracted to 1.6,Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble A,1,42 in PEG 550 MME. This second form belonged to space group P21 and diffracted to 1.9,Å resolution. [source] CEACAM1 (CD66a) mediates delay of spontaneous and Fas ligand-induced apoptosis in granulocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2005Bernhard Abstract Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro-apoptotic and pro-inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen-related cell adhesion molecule,1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin,V staining revealed that binding of CEACAM1-specific antibodies, Fab fragments and soluble CEACAM1-Fc constructs to cell surface-expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)-induced apoptosis. Tyrosine phosphorylation of CEACAM1-L, its association with SHP-1, the activation of Erk1/2 and caspase-3 appeared to be crucial for the CEACAM1-mediated anti-apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes. [source] Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thalianaFEBS JOURNAL, Issue 15 2001Koen Peeters To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fab fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3, regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3, regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fab fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of Fab fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants. [source] Regulation of epithelial cell cytokine responses by the ,3,1 integrinIMMUNOLOGY, Issue 2 2003Farah D. Lubin Summary Epithelial cells (EC) from various tissues can produce important cytokines and chemokines when stimulated by proinflammatory cytokines. These EC also receive signals from cell surface integrins, like the ,3,1 integrin, which is important in cell migration and wound healing of epithelial monolayers. However, little is known of the effect of integrin signals on cytokine responses by EC. Colonic Caco-2 cells treated with an anti-,3 integrin antibody prior to stimulation with the proinflammatory cytokine interleukin (IL)-1 yielded suppressed levels of mRNA and secreted IL-6, IL-8 and monocyte chemoattractant protein-1 as compared to cells treated with normal mouse immunoglobulin G. Lung A549 cells also showed a similar suppression of cytokine secretion. Likewise, treatment of the Caco-2 cells with the same antibody suppressed tumour necrosis factor-,-stimulated IL-6 secretion. Fab fragments of the anti-,3 integrin antibody did not induce the suppressive effect but did block the suppressive effect of the whole antibody suggesting that the effect of the antibody required cross-linking of the integrins. Finally, culture of the Caco-2 cells on laminin type 5 (the major ligand for this integrin) yielded depressed levels of IL-1-induced IL-6 secretion as compared to cells on laminin type 1. These data are the first indication that the ,3,1 integrin may cause a suppression of cytokine responses by EC which may be important in regulating the capacity of EC to respond during inflammation or wound healing. [source] Specific Fab fragments recovered by phage display technique recognizing human spermatozoaINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 5 2009Dorota Fiszer Summary Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding ,/, and , chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine. [source] Low-resolution structure of immunoglobulins IgG1, IgM and rheumatoid factor IgM-RF from solution X-ray scattering dataJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3-1 2003Vladimir V. Volkov Low-resolution structures of immunoglobulins IgG, IgM and rheumatoid factor IgM-RF in solution were analyzed using synchrotron radiation small-angle X-ray scattering and the macromolecular shapes were restored ab initio from the scattering data. The shape of IgG agrees well with the distorted Y-type crystallographic model but has a swollen appearance reflecting flexibility of the molecule in solution. The structures of pentameric IgM and IgM-RF were reconstructed assuming a five-fold symmetry. The IgM displays a flat star-like shape with observable F(ab)2 regions. The overall shape of the IgM-RF is similar to that of the IgM but with distinctly asymmetric F(ab)2 regions. This result agrees with the earlier observed functional dissimilarity of the Fab fragments in the rheumatoid factor and points to their structural dissimilarity. [source] Application of pharmacokinetic,pharmacodynamic modeling to predict the kinetic and dynamic effects of anti-methotrexate antibodies in miceJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2003Evelyn D. Lobo Abstract We have shown that intravenous (iv) administration of anti-methotrexate (MTX) antibodies (AMAb) reduces the systemic exposure of intraperitoneal (ip) MTX therapy, and we have proposed that AMAb effects on MTX systemic exposure would allow a reduction in MTX-induced systemic toxicity (i.e., producing a desirable antagonistic effect). However, many literature reports have shown that anti-toxin antibodies occasionally demonstrate unexpected agonist-like activity, increasing the extent of toxicity induced by their ligand. In this report, we have utilized a pharmacokinetic,pharmacodynamic (PKPD) model to predict the potential of AMAb to increase or decrease the magnitude of MTX-induced body weight loss in mice. Simulations predicted that both anti-MTX immunoglobulin G (AMI) and anti-MTX Fab fragments (AMF) would lead to increases or decreases in MTX toxicity, with effects dependent on the dosing protocol used. Based on the computer simulations, two protocols were selected for in vivo evaluation of predicted agonistic or antagonistic effects. Murine monoclonal AMI and AMF were produced, purified, and characterized. Agonistic effects were tested after 24-h infusion of ip MTX (10 mg/kg) and iv administration of an equimolar dose of AMI. Antagonistic effects were tested after 72-h infusion of ip MTX (5 mg/kg) and iv infusion of an equimolar dose of AMF. Consistent with model predictions of agonist-like activity, the 24-h AMI protocol led to significantly increased animal mortality (all animals died, p,<,0.005) and mean nadir weight loss (p,<,0.005). Also consistent with the predictions of the PKPD model, the 72-h AMF protocol significantly decreased animal mortality and mean nadir body weight loss (p,<,0.01). Thus, these studies demonstrate that agonistic and antagonistic effects of anti-toxin antibodies may be predicted through the use of an integrated PKPD model. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1665,1676, 2003 [source] Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineeringPROTEIN SCIENCE, Issue 8 2006Koki Makabe Abstract Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" ,-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Å resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel ,-sheet. These well-defined water molecules provide additional structural links between adjacent ,-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA. [source] | ||||||||||||||||