Therapeutic approaches are needed to promote T cell–mediated destruction of poorly immunogenic, “cold” tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8⁺ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4⁺ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell–infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8⁺ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule–mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.

需要治疗方法来促进 T 细胞介导的破坏免疫原性差的“冷”肿瘤，这些肿瘤通常与对免疫检查点阻断 (ICB) 治疗的最小反应相关。双特异性 T 细胞接合剂 (BiTE) 分子诱导多克隆 T 细胞重新定向裂解癌细胞，并在一些患者中显示出对实体瘤的有希望的临床活性。然而，人们对控制对这些疗法的临床反应的关键因素知之甚少。使用表达人源化 CD3ε 链（huCD3e 小鼠）的免疫活性小鼠模型和针对小鼠 CD19、小鼠 CLDN18.2 或人 EPCAM 抗原的 BiTE 分子，我们研究了与 BiTE 在多个同源固体中的功效相关的药代动力学和药效学参数以及免疫相关性- 肿瘤模型。⁺ T 细胞作为 BiTE 活性的重要靶点和介质，并揭示了 CD4 ⁺ T 细胞在 BiTE 功效中的拮抗作用。我们还确定了治疗组合，包括 ICB 和 4-1BB 激动剂，在 T 细胞浸润较差、免疫治疗难治性肿瘤中与 BiTE 治疗协同作用。在这些模型中，BiTE 疗效取决于肿瘤相关 CD8 ⁺T 细胞，而不是从循环中招募。我们的研究结果突出了基线 T 细胞浸润、局部 T 细胞增殖和外周 T 细胞运输对 BiTE 分子介导的疗效的相对贡献，确定了能够克服对 BiTE 治疗耐药性的组合策略，并与 BiTE 的发展具有临床相关性，其他 T 细胞参与疗法。