Bolt S, Routledge E, Lloyd I, Chatenoud L, Pope H, Gorman S D, Clark M, Waldmann H
Department of Pathology, University of Cambridge, GB.
Eur J Immunol. 1993 Feb;23(2):403-11. doi: 10.1002/eji.1830230216.
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.
CD3抗体是经证实的免疫抑制剂，能够逆转移植排斥反应。其广泛应用受到免疫原性的限制，尤其是患者在首次注射抗体后会出现“首剂”细胞因子释放综合征。我们制备了一组人源化YTH 12.5 CD3单克隆抗体（mAb）（Routledge等人，《欧洲免疫学杂志》，1991年。21: 2717）的变体，这些变体带有不同的人重链（H）恒定区，目的是找到一种不能激活T细胞的抗体形式。在竞争性结合试验中比较具有γ1、γ2、γ3和γ4重链的变体，结果表明抗体亲和力不受IgG亚类的影响。使用FcR结合活性的敏感指标（CD3 mAb将细胞毒性T细胞重定向以杀死单核细胞系U-937的能力），我们证明了γ1 = γ4 > α2 => γ3 mAb >> γ2的功能等级。通过定点诱变（Asn297突变为Ala）产生的γ1 CD3 mAb的无糖基化版本在该试验中仍具有相当的活性（介于γ1和α2 CD3 mAb之间），尽管其水平比亲本γ1形式低约10倍。当我们在5%人血清存在的情况下测试它们在体外刺激T细胞增殖的能力时，发现所有野生型免疫球蛋白同种型都具有活性，尽管反应程度存在T细胞供体依赖性差异。然而，无糖基化γ1 mAb在所有测试的十名供体中完全无促有丝分裂作用，除非在无IgG的培养基中进行试验。尽管无刺激作用，但该mAb也能够抑制未致敏和致敏T细胞的混合淋巴细胞反应。在CD3 mAb诱导的细胞因子释放的实验小鼠模型中比较γ1和无糖基化γ1 mAb，结果表明从γ1恒定区去除碳水化合物部分可使体内肿瘤坏死因子-α反应降低至少16倍。这些数据表明无糖基化γ1 CD3 mAb是一种有前途的免疫抑制治疗候选药物，无“首剂”副作用。
