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Variable Fragment (variable + fragment)
Kinds of Variable Fragment Selected AbstractsStructural basis for preferential binding of non- ortho -substituted polychlorinated biphenyls by the monoclonal antibody S2B1JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2005Jean-Luc Pellequer Abstract Polychlorinated biphenyls (PCBs) are a family of 209 isomers (congeners) with a wide range of toxic effects. In structural terms, they are of two types: those with and those without chlorines at the ortho positions (2, 2,, 6 and 6,). Only 20 congeners have no ortho chlorines. Three of these are bound by the aryl hydrocarbon receptor and are one to four orders of magnitude more toxic than all others. A monoclonal antibody, S2B1, and its recombinant Fab have high selectivity and nanomolar binding affinities for two of the most toxic non- ortho -chlorinated PCBs, 3,4,3,,4,-tetrachlorobiphenyl and 3,4,3,,4,,5,-pentachlorobiphenyl. To investigate the basis for these properties, we built a three-dimensional structure model of the S2B1 variable fragment (Fv) based on the high-resolution crystallographic structures of antibodies 48G7 and N1G9. Two plausible conformations for the complementarity-determining region (CDR) H3 loop led to two putative PCB-binding pockets with very different shapes (models A and B). Docking studies using molecular mechanics and potentials of mean force (PMF) indicated that model B was most consistent with the selectivity observed for S2B1 in competition ELISAs. The binding site in model B had a deep, narrow pocket between VL and VH, with a slight constriction at the top that opened into a wider pocket between CDRs H1 and H3 on the antibody surface. This binding site resembles those of esterolytic antibodies that bind haptens with phenyl rings. One phenyl ring of the PCB fits into the deep pocket, and the other ring is bound in the shallower one. The bound PCB is surrounded by the side chains of TyrL91, TyrL96 and TrpH98, and it has a ,-cation interaction with ArgL46. The tight fit of the binding pocket around the ortho positions of the bound PCBs indicates that steric hindrance of ortho chlorines in the binding site, rather than induced conformational change of the PCBs, is responsible for the selectivity of S2B1. Copyright © 2005 John Wiley & Sons, Ltd. [source] Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowthJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2002Ling Dong Abstract The neural cell adhesion molecule CHL1 (close homolog of L1) plays important roles in neurite outgrowth and neuronal survival in vitro. Reproducible and functionally active CHL1 antibodies are critical for a better understanding of the functional properties of CHL1 in vitro and in vivo. We have isolated human single-chain variable fragment (scFv) antibodies against mouse CHL1 from a human synthetic phage display library. To improve the binding activity of such antibodies, a clone (C12) was selected for affinity maturation by combined random mutagenesis of the VH gene and site-directed cassette mutagenesis to introduce random mutations in the complementarity determining region 3 (CDR3) of the VL gene. From the mutant phage display library, we selected a clone (6C2) that gave the strongest signal as determined by ELISA. The dissociation constant of 6C2 (Kd 2.28 × 10,8 M) was increased approximately 85-fold compared with the wild-type clone C12 (Kd 1.93 × 10,6 M). 6C2 detected CHL1 by Western blot analysis in mouse brain homogenates and detected CHL1 in CHL1-transfected cells by immunofluorescence. Furthermore, the wild-type and affinity-matured antibodies promoted neurite outgrowth of hippocampal and cerebellar neurons in vitro. Our results suggest that the affinity-matured CHL1 scFv antibody will serve a range of applications in vitro and in vivo. © 2002 Wiley-Liss, Inc. [source] Human single-chain variable fragment that specifically targets arthritic cartilageARTHRITIS & RHEUMATISM, Issue 4 2010Chris Hughes Objective To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. Methods We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti,ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti,ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti,ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II,Fc fusion protein (mTNFRII-Fc) was also investigated. Results The anti,ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti,ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. Conclusion Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis. [source] Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yang Liu ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody,antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1,192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45,Å resolution from a single flash-cooled crystal; the crystal belonged to space group P212121. [source] Promoting ,-secretase cleavage of beta-amyloid with engineered proteolytic antibody fragmentsBIOTECHNOLOGY PROGRESS, Issue 4 2009Srinath Kasturirangan Abstract Deposition of beta-amyloid (A,) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of A, levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A, at its ,-secretase site. We have previously identified a light chain fragment, mk18, with ,-secretase-like catalytic activity, producing the 1,16 and 17,40 amino acid fragments of A,40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking ,-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A,. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the ,-secretase site of A,. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3- and 6-fold increase in catalytic activity (kcat/KM) toward the synthetic A, substrate compared to the original scFv primarily due to an expected decrease in KM rather than an increase in kcat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A,. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A, levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009 [source] Diverting a protein from its cellular location by intracellular antibodiesFEBS JOURNAL, Issue 4 2000The case of p21Ras We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic. [source] Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experimentsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2001Florence Bettsworth Abstract Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC),bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed. Copyright © 2001 John Wiley & Sons, Ltd. [source] Human single-chain variable fragment that specifically targets arthritic cartilageARTHRITIS & RHEUMATISM, Issue 4 2010Chris Hughes Objective To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. Methods We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti,ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti,ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti,ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II,Fc fusion protein (mTNFRII-Fc) was also investigated. Results The anti,ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti,ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. Conclusion Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis. [source] Humanized immunotoxins: A new generation of immunotoxins for targeted cancer therapyCANCER SCIENCE, Issue 8 2009Mrudula Mathew Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non-specificity leading to peripheral toxicity, and non-resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA-approved fusion protein. In spite of the promise shown by bacterial- and plant toxin-based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non-specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored. (Cancer Sci 2009) [source] Antitumor activity of chimeric immunoreceptor gene-modified Tc1 and Th1 cells against autologous carcinoembryonic antigen-expressing colon cancer cellsCANCER SCIENCE, Issue 9 2006Takeshi Sasaki To generate tumor-specific and interferon (IFN)-,-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3,. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2,/, mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-, in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer. (Cancer Sci 2006; 97: 920,927) [source] | ||||||||||||||||