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Analysis of anti-human CD22 human mAbs affinity and specificity on human PBMCs. a) Affinity determination of anti-human CD22 mAbs using SPR by flowing various concentration of CD22 antibody over CD22 chip-bound. b) Flow cytometry analysis of CD22 mAbs on human PBMC. Human PBMC were labeled with anti human CD19 (APC) and purified CD22 mAbs (clone γ1λ1, γ3λ3-5, γ23κ5-2 or γ27λ26) coupled with Alexa Fluor 568 fluorochrome or with a commercial mouse mAb anti-human CD22 (Ms anti-human CD22). (PPT 666 kb

Block in development at the pre-B-II to immature B cell stage in mice without lgk and iga light chain

Silencing individual C (constant region) lambda genes in a kappa(-/-) background reduces mature B cell levels, and L chain-deficient (lambda(-/-)kappa(-/-)) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal

Human antibody expression in transgenic rats: Comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions

International audienceExpression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human V[H] , D and J[H] genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human V[H]-region (comprising 22 V[H]s, all Ds and all J[H]s in natural configuration) but linked to different rat C[H]-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3′ end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes

Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies

International audienceBackground: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications andfully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied astrategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed byreconstruction of human mAbs by RT-PCR and expression cloning.Results: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbsagainst major histocompatibility complex molecules loaded with peptides (pMHC). We then implemented thistechnology using human immunoglobulin transgenic rats, which after immunization with an antigen of interestexpress high affinity-matured antibodies with human idiotypes. Using rapid immunization, followed by tetramerbasedB-cell sorting and expression cloning, we generated several fully humanized mAbs with strong affinities,which could discriminate between highly homologous proteins (eg. different pMHC complexes).Conclusions: Therefore, we describe a versatile and more effective approach as compared to hybridoma generationor phage or yeast display technologies for the generation of highly specific and discriminative fully human mAbs thatcould be useful both for basic research and immunotherapeutic purposes