T cell–bispecific antibodies (BsAbs) couple cytotoxic T lymphocytes to tumor cells, inducing their destruction. Although there are more than 60 classes of BsAbs in development, the relative importance of parameters such as interdomain spacing or spatial configuration is largely unknown. Here, we dissected a symmetric dual bivalent BsAb platform (IgG-[L]-scFv: antitumor IgG with anti-CD3 scFv fused to the light chains) to explore the importance of valency and spatial configuration for BsAb-induced T cell cytotoxicity. Our results revealed that placing tumor and T cell binding domains on the same side of a BsAb (cis-configuration) elicited substantially stronger antitumor activity, in vitro and in vivo, compared to positioning them on opposite sides (trans-configuration). Moreover, using two cis-modules in the same BsAb further improved cytotoxicity (up to 2000-fold). In addition, separating antigen-binding components with a single Ig domain (C_L) markedly enhanced cytokine release and in vivo tumor responses compared to smaller (G₄S₁) or larger (C_H1-C_H2-C_H3) spacers. These findings provide guidelines for improving BsAb function and highlight the importance of spatial configuration and dual bivalency as development parameters.

T细胞双特异性抗体（BsAbs）将细胞毒性T淋巴细胞与肿瘤细胞结合，诱导其破坏。尽管正在开发的BsAb类别超过60种，但是诸如域间间距或空间配置等参数的相对重要性仍然未知。在这里，我们解剖了对称的双二价BsAb平台（IgG- [L] -scFv：将抗CD3 scFv融合到轻链上的抗肿瘤IgG）来探索价和空间构型对于BsAb诱导的T细胞细胞毒性的重要性。我们的研究结果表明，与将肿瘤和T细胞结合域置于BsAb的同一侧（顺式构型）相比，将其在体外和体内的抗肿瘤活性大大增强（在体外和体内）。此外，在同一BsAb中使用两个顺式模块可进一步改善细胞毒性（最高达2000倍）。此外，用单个Ig结构域分离抗原结合成分（C_大号）显着增强的细胞因子释放和体内肿瘤反应相比更小的（G ₄小号₁）或更大（C _H1 -C _H2 -C _H3）隔离物。这些发现为改善BsAb功能提供了指导，并强调了空间构型和双重双价作为发育参数的重要性。