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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

Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules

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

Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas

International audienceThe rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology

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

Transducer cascades for biological literature-based discovery

International audienceG protein-coupled receptors (GPCRs) control the response of cells to many signals, and as such, are involved in most cellular processes. As membrane receptors, they are accessible at the surface of the cell. GPCRs are also the largest family of membrane receptors, with more than 800 representatives in mammal genomes. For this reason, they are ideal targets for drugs. Although about one third of approved drugs target GPCRs, only about 16% of GPCRs are targeted by drugs. One of the difficulties comes from the lack of knowledge on the intra-cellular events triggered by these molecules. In the last two decades, scientists have started mapping the signaling networks triggered by GPCRs. However, it soon appeared that the system is very complex, which led to the publication of more than 320,000 scientific papers. Clearly, a human cannot take into account such massive sources of information. These papers represent a mine of information about both ontological knowledge and experimental results related to GPCRs, which have to be exploited in order to build signaling networks. The ABLISS project aims at the automatic building of GPCRs networks usingautomated deductive reasoning, allowing to integrate all available data. Therefore, we processed the automatic extraction of network information from the literature using Natural Language Processing (NLP). We mainly focused on the experimental results about GPCRs reported in the scientific papers, as so far there is no source gathering all these experimental results. We designed a relational database in order to make them available to the scientific community later. After introducing the more general objectives of the ABLISS project, we describe the formalism in detail. We then explain the NLP program using the finite state methods (Unitex graph cascades) we implemented and discuss theextracted facts obtained. Finally, we present the design of the relational database that stores the facts extracted from the selected papers