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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

Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required

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

Structural motifs involved in human IgG antibody effector functions.

A humanized IgG antibody to CAMPATH-1 antigen (CDw52) is known to be lympholytic both in vitro and in vivo. So as to improve therapeutic potency through protein engineering strategies, we wish to define the structural motifs underlying some of the documented differences in function between human (h) IgG1 and IgG4 forms of the antibody. By the creation of heavy chain domain-switch and intra-domain recombinant antibodies we have established an important role for the carboxy-terminal half of the CH2 domain in determining differential behaviour in antibody-dependent cytotoxicity (ADCC) and in complement lysis. If this same region were necessary for the effector mechanisms that operate in vivo, then it might be possible to improve antibody effector functions by construction of novel antibodies that possess within the one molecule multiple copies of the crucial hinge-CH2 associated structures. Although our previous work suggested that the hIgG4 CAMPATH-1 antibody was ineffective at ADCC, we found this to be so only in some individuals. In others, IgG4, and indeed all the IgG subclasses were able to mediate ADCC. Overall, though, hIgG1 remains the best choice isotype for lytic therapy in vivo