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Development of a new monoclonal antibody specific to mouse V?6 chain.

ABSTRACT: There are seven V? gene segments in the TCR ? chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the V?6 chain (Heilig & Tonegawa nomenclature). By immunizing V?4/6 KO mice with complementarity-determining region peptides in V?6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of V?6/V?1 gene-transfected T-cell line lacking TCR as well as of V?1^- V?4^- V?5^- V?7^- ?? T cells and the CD3^high TCR?^int ?? T cells in various organs. The location of V?6⁺ ?? T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of V?6 ?? T cells in mice.

SUBMITTER: Hatano S

PROVIDER: S-EPMC6504751 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Development of a new monoclonal antibody specific to mouse Vγ6 chain.

Hatano Shinya S Tun Xin X Noguchi Naoto N Yue Dan D Yamada Hisakata H Sun Xun X Matsumoto Mitsuru M Yoshikai Yasunobu Y

Life science alliance 20190507 3

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and ...[more]

PMID: 31064767

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Project description:BackgroundPlasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates.Material and methodsUsing antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1-3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays.ResultsThe selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2.ConclusionWe have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

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Development of a new monoclonal antibody specific to mouse V?6 chain.

Publications

Development of a new monoclonal antibody specific to mouse Vγ6 chain.

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