title: The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties.
abstract: CD3 antibodies are proven immunosuppressants capable of reversing transplant rejection episodes. Their general application has been limited both by their immunogenicity and, in particular, by the "first-dose" cytokine-release syndrome experienced by patients after the initial administration of antibody. We have produced a set of variants of the humanized YTH 12.5 CD3 monoclonal antibody (mAb) (Routledge et al., Eur. J. Immunol. 1991. 21: 2717) bearing different human heavy (H) chain constant regions, with the intention of finding a form of the antibody that is not able to activate T cells. Comparison of the variants having gamma 1, gamma 2, gamma 3 and gamma 4 H chains in a competitive binding assay showed that antibody avidity was not affected by IgG subclass. Using a sensitive indicator of FcR binding activity (the capacity of the CD3 mAb to redirect cytotoxic T cells to kill the monocytic cell line U-937) we demonstrated a functional hierarchy of gamma 1 = gamma 4 > alpha 2 =/> gamma 3 mb >> gamma 2. An aglycosyl version of the gamma 1 CD3 mAb, produced by site-directed mutagenesis (Asn297 to Ala), still had considerable activity in this assay (intermediate to the gamma 1 and alpha 2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental gamma 1 form. When we tested their ability to stimulate T cell proliferation in vitro in the presence of 5% human serum, all of the wild-type immunoglobulin isotypes were found to be active, although there were T cell donor-dependent variations in the extent of the responses. The aglycosyl gamma 1 mAb was, however, completely non-mitogenic in all of ten donors tested, unless the assay was performed in IgG-free medium. Despite being non-stimulatory, this mAb was also able to inhibit the mixed lymphocyte reaction responses of both naive and primed T cells. Comparison of the gamma 1 and aglycosyl gamma 1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the gamma 1 constant region reduced the in vivo tumor necrosis factor-alpha response by a factor of at least 16-fold. These data suggest that the aglycosyl gamma 1 CD3 mAb is a promising candidate for immunosuppressive therapy without "first dose" side effects.
authors.affiliation: Department of Pathology, University of Cambridge, GB.
authors.forename: S
authors.initials: S
authors.lastname: Bolt
authors.forename: E
authors.initials: E
authors.lastname: Routledge
authors.forename: I
authors.initials: I
authors.lastname: Lloyd
authors.forename: L
authors.initials: L
authors.lastname: Chatenoud
authors.forename: H
authors.initials: H
authors.lastname: Pope
authors.forename: S D
authors.initials: SD
authors.lastname: Gorman
authors.forename: M
authors.initials: M
authors.lastname: Clark
authors.forename: H
authors.initials: H
authors.lastname: Waldmann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005803:Genes, Immunoglobulin / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007165:Immunosuppression Therapy; D008213:Lymphocyte Activation / Q000276:immunology; D008934:Mitogens; D016297:Mutagenesis, Site-Directed; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D016207:Cytokines; D007143:Immunoglobulin Heavy Chains; D008934:Mitogens