High-specificity CRISPR-mediated genome engineering in CB-011, an anti-BCMA allogeneic CAR-T cell, suppresses allograft rejection in preclinical models

Allogeneic chimeric antigen receptor (CAR)-T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR-T cells. Key considerations in the development of allogeneic CAR-T cell therapies include prevention of GvHD and suppression of allograft rejection. Here we describe preclinical data supporting the ongoing first-in-human clinical trial (CaMMouflage) in relapsed/refractory multiple myeloma patients evaluating CB-011, a hypoimmunogenic, allogeneic anti–B cell maturation antigen (BCMA) CAR-T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR-T cells were engineered to prevent endogenous human leukocyte antigen (HLA) class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. Additionally, T cell receptor expression was disrupted at the T cell receptor alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell co-cultures derived from patients with multiple myeloma. Additionally, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer (NK) cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. For profiling of NK and T cells following infusion in vivo, 6 different cohorts were established in NOG-Tg (Hu-IL15) mice according to the protocol described above: (i) 2 cohorts of CB-011 cells without pNK cells (CB-011 only, n = 2 mice/cohort), (ii) 2 cohorts of CB-011 cells with NK cells (CB-011+NK, n = 3 mice/cohort), (iii) 1 cohort of B2M KO CAR-T cells without NK cells (B2M KO only, n = 2 mice), and (iv) 1 cohort of B2M KO CAR-T cells with NK cells (B2M KO+NK, n = 3 mice). Additionally, NK and CB-011 cells were isolated for single-cell RNA sequencing (scRNA-Seq; NK IP and CB-011 IP, n = 1/group) prior to infusion. NK and CAR-T cells from either the CB-011 or the CAR KI B2M KO cohorts were isolated from the spleens of NOG-Tg (Hu-IL15 mice) mice 4 days post CAR-T cell infusion by FACS using a human CD45-specific antibody. Cells were resuspended in PBS + 0.4% BSA at a concentration of 1x106 cells/mL and single-cell RNA sequencing libraries were generated (Supplemental methods).

异体嵌合抗原受体（CAR）-T细胞疗法有望攻克与患者来源自体（autologous）CAR-T细胞相关的诸多挑战。异体CAR-T细胞疗法研发中的核心考量包括移植物抗宿主病（GvHD）的预防以及同种异体移植排斥反应的抑制。本文详述了支持正在开展的首个人体临床试验（CaMMouflage）的临床前数据，该试验针对复发/难治性多发性骨髓瘤患者，评估CB-011——一款低免疫原性、靶向B细胞成熟抗原（BCMA）的异体CAR-T细胞疗法候选物。CB-011细胞具备4种基因组修饰，其制备是以健康供者来源的T细胞为基础，借助Cas12a CRISPR杂合RNA-DNA（chRDNA）基因组编辑技术平台完成的。为解决同种异体移植排斥问题，研究人员对CAR-T细胞进行工程化改造，以阻断内源性人类白细胞抗原（HLA）I类复合物的表达，并过表达由β2微球蛋白（B2M）与HLA-E融合形成的单链多蛋白复合物。此外，研究人员在T细胞受体α恒定区基因座处敲除T细胞受体的表达，同时将人源化抗BCMA特异性CAR定点插入该位点。体外实验中，在源自多发性骨髓瘤患者的细胞共培养体系的混合淋巴细胞反应里，CB-011细胞展现出强劲的浆母细胞杀伤活性。此外，CB-011细胞能够抑制HLA错配T细胞对其的识别，并降低其受到的T细胞杀伤作用。由于内源性启动子驱动B2M-HLA-E的表达，CB-011细胞在体外与体内实验中均能抵御自然杀伤（NK）细胞介导的细胞毒性。该疗法结合免疫遮蔽装甲策略以减轻同种异体移植排斥反应，可实现强效的抗肿瘤功效，为多发性骨髓瘤患者提供更优化的治疗潜力。为分析体内输注后的NK细胞与T细胞的特征，研究人员按照上述实验方案，在NOG-Tg（Hu-IL15）小鼠中设立了6组实验队列：(i) 仅输注CB-011细胞的队列（不含前体NK细胞，每组2只小鼠）；(ii) 输注CB-011细胞联合NK细胞的队列（每组3只小鼠）；(iii) 仅输注B2M敲除CAR-T细胞的队列（不含NK细胞，共2只小鼠）；(iv) 输注B2M敲除CAR-T细胞联合NK细胞的队列（每组3只小鼠）。此外，在输注前，研究人员分离了NK细胞与CB-011细胞用于单细胞RNA测序（scRNA-Seq；NK细胞免疫沉淀组与CB-011细胞免疫沉淀组，每组1份样本）。CAR-T细胞输注4天后，研究人员通过荧光激活细胞分选（FACS）技术，使用人源CD45特异性抗体，从NOG-Tg（Hu-IL15）小鼠的脾脏中分离CB-011队列或CAR敲入B2M敲除队列中的NK细胞与CAR-T细胞。将细胞以1×10^6个/毫升的浓度重悬于含0.4%牛血清白蛋白（BSA）的磷酸盐缓冲液（PBS）中，并构建单细胞RNA测序文库（详见补充方法）。