Rodrigues M L, Shalaby M R, Werther W, Presta L, Carter P
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080.
Int J Cancer Suppl. 1992;7:45-50.
We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E. coli expression of each Fab' arm followed by directed chemical coupling in vitro. BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2. Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody. This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1. In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody. Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells. One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody. BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2. This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.
我们最近通过在体外分别用大肠杆菌表达每个Fab'臂，然后进行定向化学偶联，构建了一种人源化双特异性抗体（BsF(ab')2v1）。已证明BsF(ab')2 v1（抗CD3/抗p185HER2）在体外可将人CD3+CTL的细胞毒活性重新靶向至人乳腺肿瘤细胞系SK-BR-3，该细胞系过度表达原癌基因HER2的p185HER2产物。我们的简约人源化策略是在人抗体中尽可能少地引入鼠源残基，以获得与鼠源亲本抗体相当的抗原结合亲和力和生物学特性。该策略在BsF(ab')2 v1的抗p185HER2臂上证明非常成功。相比之下，BsF(ab')2 v1通过其抗CD3臂与T细胞的结合效率远低于含有鼠源亲本抗CD3抗体可变域的嵌合BsF(ab')2。在此，我们构建了额外的BsF(ab')2片段，其含有在抗CD3臂上进行了选定氨基酸替换的变体，以试图改善抗体与T细胞的结合。其中一个变体BsF(ab')2 v9是通过将BsF(ab')2 v1抗CD3重链可变域第二高变环中的6个残基替换为鼠源亲本抗CD3抗体的对应残基而产生的。BsF(ab')2 v9与T细胞（Jurkat）的结合效率远高于BsF(ab')2 v1，几乎与嵌合BsF(ab')2一样高效。人源化BsF(ab')2在T细胞结合效率上的这种提高是其作为治疗p185HER2过表达癌症的潜在治疗剂开发中的重要一步。
