DataSheet_1_Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.pdf

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a “cytokine window” defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.

T细胞衔接型双特异性抗体（T cell engaging bispecific antibodies, TCEs）已在血液系统恶性肿瘤领域获得临床概念验证。但细胞因子释放综合征（cytokine release syndrome, CRS）、神经毒性以及靶标相关肿瘤外毒性，尤其是在实体瘤治疗场景中，仍是制约其应用的主要障碍。现有研究已报道第二代TCE可通过降低对CD3及/或肿瘤相关抗原（tumor-associated antigen, TAA）的表观结合亲和力，实现细胞毒性与细胞因子释放的解偶联；不过此类工程化改造通常仅能降低细胞因子释放的最大诱导水平，且往往以削弱最大细胞毒性为代价。本研究以ROR1为模型TAA，并依托高度模块化的骆驼科纳米抗体，报道了下一代解偶联型TCE的工程化策略：该抗体引入了“细胞因子窗口”这一概念，即存在一个可达到最大杀伤效果的剂量区间，在此区间内细胞因子释放水平可从安全的极低水平，调节至接近第一代TCE的诱导水平。这一特性可支持包括旁观者杀伤效应在内的促炎性抗肿瘤活性，临床医师可借此安全调整患者给药剂量，使其达到据推测优于标准第二代策略所能实现的最大疗效水平。研究团队结合优化TCE介导的突触距离与TAA结合臂的表观亲和力调控两种手段，构建了一种相对长效且持久的突触结构，该结构可支撑宽泛的细胞因子窗口、强效杀伤效果，并降低免疫耗竭倾向。尤为关键的是，这款下一代TCE在体内可显著抑制肿瘤生长；与诱导致死性CRS的第一代非解偶联对照TCE不同，本研究未观察到任何不良反应迹象。