Leucine zipper-based sorting system enables generation of multi-functional CAR T cells

Resistance to chimeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms including antigen-loss escape and tumor-induced immune suppression. Expression of multiple CARs may overcome multi-antigen-loss escape. Similarly, expression of switch receptors that convert inhibitory immune checkpoint signals into positive costimulatory signals may enhance CAR T cell activity in the tumor microenvironment. Engineering multiple features into one cell product, however, is limited by transgene packaging constraints of current vector systems. Here, we describe a leucine zipper-based cell sorting methodology that enables selective single-step immunomagnetic purification of cells co-transduced with two vectors, designed to potentially double the number of incorporated transgenes. This “Zip-sorting” system facilitated generation of T cells simultaneously expressing up to four CARs and co-expressing up to three switch receptors. These multi-CAR multi-Switch receptor arrays enabled T cells to eliminate antigenically heterogeneous syngeneic leukemia populations co-expressing multiple inhibitory ligands. Zip-sorted multi-CAR multi-Switch receptor T cells represent a combinatorial therapeutic strategy to overcome multiple mechanisms of CAR T cell resistance. Single cell RNAseq profiles of combined CD4+ and CD8+ CAR T cells sorted 24h after stimulation: dual-CAR, dual-CAR dual-Switch, dual-CAR triple-Switch

嵌合抗原受体（chimeric antigen receptor, CAR）T细胞疗法的耐药性可通过多种机制介导产生，包括抗原丢失逃逸与肿瘤诱导的免疫抑制。同时表达多种CAR或可克服多抗原丢失逃逸带来的免疫逃逸困境。类似地，可将抑制性免疫检查点信号转化为正向共刺激信号的开关受体，或可增强肿瘤微环境中CAR T细胞的抗肿瘤活性。
然而，将多种功能特征整合至单一细胞产品中，会受到当前载体系统的转基因包装容量限制。本研究开发了一种基于亮氨酸拉链的细胞分选方法，可通过单步免疫磁珠分选选择性富集共转导两种载体的细胞，理论上可使整合的转基因数量翻倍。
该“拉链分选（Zip-sorting）”系统可成功制备同时表达最多4种CAR并共表达最多3种开关受体的T细胞。这类多CAR多开关受体阵列可使T细胞有效清除同时表达多种抑制性配体的抗原异质性同基因白血病群体。
经拉链分选的多CAR多开关受体T细胞，是一种可克服CAR T细胞多重耐药机制的组合式治疗策略。
本研究对刺激24小时后分选的CD4阳性与CD8阳性CAR T细胞进行了单细胞RNA测序分析，涉及双CAR、双CAR双开关受体、双CAR三开关受体三类样本。