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Affinity Maturation (affinity + maturation)
Selected AbstractsTEM-1 ,-lactamase as a scaffold for protein recognition and assayPROTEIN SCIENCE, Issue 6 2002Daniel Legendre Abstract A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 ,-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to ,-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that ,-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to ,-lactamases binding to streptavidin, ,-lactamase clones binding to horse spleen ferritin and ,-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining ,-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for ,-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme. [source] CD4+CD25+ regulatory T,cells control the magnitude ofT-dependent humoral immune responses to exogenous antigensEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2006Fouad Eddahri Abstract CD4+CD25+ T,reg cells are critical for peripheral tolerance and prevention of autoimmunity. Here we show that CD4+CD25+ T,reg also regulate the magnitude of humoral responses against a panel of T-dependent antigens of foreign origin during both primary and secondary immune responses. Depletion of CD4+CD25+ T,cells leads to increased antigen-specific antibody production and affinity maturation but does not affect T-independent B,cell responses, suggesting that CD4+CD25+ T,reg exert a feedback mechanism on non-self antigen-specific antibody secretion by dampening the T,cell help for B,cell activation. Moreover, we show that CD4+CD25+ T,reg also suppress in vitro B,cell immunoglobulin production by inhibiting CD4+CD25, T,cell help delivery, and that blockade of TGF-, activity abolishes this suppression. [source] Life and death within germinal centres: a double-edged swordIMMUNOLOGY, Issue 2 2002Liliana Guzman-Rojas Summary Within germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unless they are positively rescued by antigen and by signals initiated by CD40,CD154 interactions. Thus, while the germinal centre microenvironment can become a virtual graveyard for most B lymphocytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli for the survival of cells that successfully accomplish affinity maturation. Such dichotomy in the physiology of germinal centre reaction that results in survival of the functional B-cell repertoire and the elimination of abnormal cells, dictates the fate towards B-cell homeostasis or disease. Consequently, the death and survival-signalling arms within germinal centres predominantly reside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 and CD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity are documented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways. The present review considers two different scenarios in the control of B-cell survival and death within germinal centres. The first is an idealistic scenario, in which a discriminatory and co-ordinate signalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of the functional B-cell repertoire and the elimination of the abnormal phenotype. The second is a gloomy scenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen as equally deleterious. [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] Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity,PROTEIN SCIENCE, Issue 9 2009Sejal S. Hall Abstract Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications. [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] BAFF: a local and systemic target in autoimmune diseasesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2009I. Moisini Summary BAFF (B lymphocyte activating factor of the tumour necrosis factor family) is a vital homeostatic cytokine for B cells that helps regulate both innate and adaptive immune responses. Increased serum levels of BAFF are found in a number of different autoimmune diseases, and BAFF is found in inflammatory sites in which there is lymphoid neogenesis. BAFF antagonism has been used in several autoimmune disease models, resulting in B cell depletion, decreased activation of T cells and dendritic cells (DC) and a reduction in the overall inflammatory burden. BAFF, through its interaction with BAFF-R, is required for survival of late transitional, marginal zone and mature naive B cells, all of which are depleted by BAFF blockade. Through their interactions with TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B cell maturation protein), BAFF and its homologue APRIL (a proliferation-inducing ligand), support the survival of at least some subsets of plasma cells; blockade of both cytokines results in a decrease in serum levels of immunoglobulin (Ig)G. In contrast, neither BAFF nor APRIL is required for the survival or reactivation of memory B cells or B1 cells. BAFF also helps DC maturation and interleukin (IL)-6 release and is required for proper formation of a follicular dendritic cell (FDC) network within germinal centres, although not for B cell affinity maturation. The clinical efficacy of BAFF blockade in animal models of autoimmunity may be caused both by the decline in the number of inflammatory cells and by the inhibition of DC maturation within target organs. Blockade of BAFF and its homologue APRIL are being explored for human use; several Phase I and II clinical trials of BAFF inhibitors for autoimmunity have been completed and Phase III trials are in progress. [source] Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display librariesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2000H. Nguyen RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities (,108 M,1) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications. [source] | |||||||||||||