THE GENERATION OF A HUMANIZED, NON-MITOGENIC CD3 MONOCLONAL-ANTIBODY WHICH RETAINS INVITRO IMMUNOSUPPRESSIVE PROPERTIES
THE GENERATION OF A HUMANIZED, NON-MITOGENIC CD3 MONOCLONAL-ANTIBODY WHICH RETAINS INVITRO IMMUNOSUPPRESSIVE PROPERTIES
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DOI：
10.1002/eji.1830230216
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发表时间：
1993-02-01
影响因子：
5.4
通讯作者：
WALDMANN, H
中科院分区：
文献类型：
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作者：
BOLT, S;ROUTLEDGE, E;WALDMANN, H
CD3 抗体被证明是能够逆转移植排斥反应的免疫抑制剂。它们的一般应用受到其免疫原性的限制，特别是受到患者在初次施用抗体后经历的“首剂”细胞因子释放综合征的限制。我们已经生产了一组带有不同人重 (H) 链恒定区的人源化 YTH 12.5 CD3 单克隆抗体 (mAb) 变体（Routledge 等人，Eur. J.Immunol. 1991. 21: 2717），目的是寻找一种不能激活 T 细胞的抗体形式。在竞争性结合测定中比较具有 gamma1、gamma2、gamma3 和 gamma4 H 链的变体表明抗体亲合力不受 IgG 亚类的影响。使用 FcR 结合活性的敏感指标（CD3 mAb 重定向细胞毒性 T 细胞以杀死单核细胞系 U-937 的能力），我们证明了 gamma1 = gamma4 > alpha2 =/> gamma3 mb 远大于 gamma2 的功能层次。通过定点诱变（Asn297 变为 Ala）产生的 gamma1 CD3 mAb 的无糖基版本在该测定中仍然具有相当大的活性（介于 gamma1 和 alpha2 CD3 mAb 之间），尽管其水平比亲本 gamma1 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，aglusyl gamma1 mAb 在所有测试的 10 名供体中完全不具有促有丝分裂作用，除非在不含 IgG 的培养基中进行测定。尽管不具有刺激性，这种 mAb 也能够抑制初始 T 细胞和引发 T 细胞的混合淋巴细胞反应。在 CD3 mAb 诱导细胞因子释放的实验小鼠模型中比较 gamma1 和糖基 gamma1 mAb 表明，从 gamma1 恒定区去除碳水化合物部分可将体内肿瘤坏死因子-α 反应降低至少 16 倍。这些数据表明，aglusyl gamma1 CD3 mAb 是一种有前途的免疫抑制治疗候选药物，且不会产生“首剂”副作用。
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 gamma1, gamma2, gamma3 and gamma4 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 gamma1 = gamma4 > alpha2 =/> gamma3 mb much greater than gamma2. An aglycosyl version of the gamma1 CD3 mAb, produced by site-directed mutagenesis (Asn297 to Ala), still had considerable activity in this assay (intermediate to the gamma1 and alpha2 CD3 mAb), albeit at a level approximately 10-fold lower than that of the parental gamma1 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 gamma1 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 gamma1 and aglycosyl gamma1 mAb in an experimental mouse model for CD3 mAb-induced cytokine release indicated that removal of the carbohydrate moiety from the gamma1 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 gamma1 CD3 mAb is a promising candidate for immunosuppressive therapy without ''first dose'' side effects.