The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (V_HH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 V_HH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem V_HH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem V_HH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies.

中文翻译：

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ATTACK，一种具有三价EGFR结合和单价CD3结合的新型双特异性T细胞募集抗体，用于癌症免疫治疗

使用双特异性抗体重定向T细胞活性是目前正在开发的最有前途的癌症免疫治疗方法之一，但受到细胞因子风暴相关毒性以及当前双特异性抗体形式的药代动力学和肿瘤穿透能力的限制。这里，我们已经设计了ATTACK（甲对称串联Ť rimerbody为Ť细胞甲ctivation和Ç ancer ķ解毒，阿莫西林），其结合了三种EGFR结合的单域抗体的新的T细胞招募双特异性抗体（V _HH; 中等分子量包装中带有一个CD3结合单链可变片段（scFv;克隆OKT3）的克隆EgA1）。两种特异性指向相反的方向，以便同时与癌细胞和T细胞效应子结合，从而促进免疫突触的形成。与先前鉴定的串联双特异性抗体相比，EgA1 ATTACK被哺乳动物细胞表达为同质，非聚集的可溶性蛋白，并且与EGFR的结合增强，但与CD3的结合增强，后者每分子具有一个EgA1 V _HH和一个OKT3 scFv 。EgA1 ATTACK诱导的TCR参与下游的突触形成和早期信号传导途径的浓度低于串联V _HH-scFv双特异性抗体。此外，当将人T细胞重新定向至表达EGFR的细胞时，它表现出了极强的剂量依赖性细胞毒性，其功效比串联V _HH -scFv双特异性抗体高15倍以上。这些结果表明，ATTACK是开发下一代T细胞重定向双特异性抗体的理想形式。