通过双特异性抗体将 T 细胞重定向至肿瘤细胞是治疗癌症的有效方法，而 T 细胞依赖性双特异性抗体 (TDBA) 是一类新兴的有效免疫治疗剂。通过同时靶向肿瘤细胞和T细胞上的抗原，T细胞被激活以杀死肿瘤细胞。在此，我们报告了一个平台，用于生成一类新型 2:1 结构的 T 细胞依赖性双特异性抗体，该抗体对肿瘤细胞上的 HER2 受体具有二价性，对 T 细胞上的 CD3 受体具有单价性。为此，我们在基因编码的酪氨酸残基上使用生物源逆电子需求狄尔斯-阿尔德 (IEDDA) 点击反应，在治疗批准的抗体曲妥珠单抗上安装一个 TCO 手柄。随后用四嗪功能化的 CD3 结合 Fab 进行 TCO-四嗪点击，产生 2:1 HER2 × CD3 TDBA，在皮摩尔浓度下表现出肿瘤杀伤能力。 T 细胞上 CD3 受体的单价可以降低细胞因子释放综合征的几率，这是此类药物的常见副作用。我们的半合成方法可以通过几个化学酶促和合成步骤生成高效的 TDBA 结构。

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.