Simultaneous, cell-intrinsic downregulation of PD-1 and TIGIT can enhance effector function of CD19-targeting CAR T cells and promote early memory phenotype

CD19-targeting chimeric antigen receptor (CAR) T cell have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, according to the recent clinical data, a significant portion of patients still do not benefit from the therapy, due various resistance mechanisms including the high expression of multiple inhibitory immune checkpoint receptors on activated CAR-T cells. Studies of checkpoint blockade immunotherapy using monoclonal antibodies have shown that simultaneously targeting inhibitory receptors that are functionally non-redundant can synergistically enhance anti-tumor responses. Here we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors can be downregulated simultaneously by a dual short-hairpin RNA (shRNA) cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations, PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT, and found that the CAR T with PD-1/TIGIT downregulation uniquely exhibited synergistic anti-tumor effect in mouse xenograft models compared to the single PD-1 downregulation and maintained cytolytic and proliferative capacity upon repeated antigen exposure. Importantly, functional and phenotypic analysis of CAR T cells as well as analysis of transcriptomic profiles suggests that downregulation of PD-1 enhances short-term effector function while downregulation of TIGIT is primarily responsible for maintaining a less-differentiated/exhausted state, providing a potential mechanism of the synergy. The PD-1/TIGIT downregulated CAR T cells generated from DLBCL patient-derived T cells following clinically applicable manufacturing process also showed a robust anti-tumor activity and significantly improved persistence in vivo compared to conventional CD19-targeting CAR T cells. Overall, our results demonstrate that the cell-intrinsic PD-1/TIGIT dual downregulation strategy may provide an effective way to overcome the immune checkpoint-mediated resistance in CAR T therapy. Overall design: RNAseq profiling of first and second restimulation CD19 specfic CAR T cells with GFP, PD-1, TIGIT, or PD-1/TIGIT downregualtion

靶向CD19的嵌合抗原受体（chimeric antigen receptor, CAR）T细胞已成为复发难治性B细胞恶性肿瘤患者的重要治疗选择。然而，根据近期临床数据，仍有相当比例的患者无法从该疗法中获益，这源于多种耐药机制，其中包括活化CAR-T细胞表面多种抑制性免疫检查点受体的高表达。已有研究采用单克隆抗体开展检查点阻断免疫疗法，结果显示同时靶向功能非冗余的抑制性受体，可协同增强抗肿瘤免疫应答。本研究报道了一种慢病毒双合一CAR-T构建策略：将双短发夹RNA（short-hairpin RNA, shRNA）表达盒整合至CAR载体中，可同时下调两种检查点受体的表达。利用该系统，我们针对PD-1/TIM-3、PD-1/LAG-3、PD-1/CTLA-4及PD-1/TIGIT四种不同的检查点组合，评估了靶向CD19的CAR-T细胞，结果发现，相较于仅下调PD-1的CAR-T细胞，同时下调PD-1/TIGIT的CAR-T细胞在小鼠异种移植模型中展现出独特的协同抗肿瘤效应，且在反复抗原刺激后仍能维持细胞溶解活性与增殖能力。重要的是，对CAR-T细胞进行功能与表型分析，以及转录组谱分析后发现，下调PD-1可增强短期效应功能，而下调TIGIT则主要负责维持细胞的低分化/耗竭状态，为协同效应的潜在作用机制提供了依据。通过临床可规模化制备工艺，从弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma, DLBCL）患者来源的T细胞中制备的PD-1/TIGIT双下调CAR-T细胞，相较于传统靶向CD19的CAR-T细胞，同样展现出强劲的抗肿瘤活性，且在体内的存续能力显著提升。综上，本研究结果表明，细胞内在性PD-1/TIGIT双下调策略或可为克服CAR-T疗法中免疫检查点介导的耐药性提供一种有效途径。总体实验设计：对分别经首次与第二次抗原刺激的、分别转染GFP、仅下调PD-1、仅下调TIGIT或同时下调PD-1/TIGIT的CD19特异性CAR-T细胞进行RNA测序（RNA-seq）谱分析。