Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.

中文翻译：

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赋予新的基于低亲和力神经生长因子受体的间隔物的 CD44v6.CAR T 细胞的表征和功能分析

过继转移嵌合抗原受体 (CAR) T 细胞的有效性很大程度上取决于 CAR 介导的效应细胞与肿瘤细胞上的靶抗原相互作用的质量。单链可变片段 (scFv) 对抗原的亲和力和 CAR 设计在这种相互作用中起主要作用。特别是，间隔结构域可能通过影响所得 CAR 的长度和柔韧性来影响 CAR T 细胞功能。本研究通过定义源自人类低亲和力神经生长因子受体 (LNGFR) 细胞外结构域的间隔区的最佳结构，解决了改进 CD44v6 特异性 CAR T 细胞的制造过程和抗肿瘤活性的需要。我们定制了 LNGFR 垫片以调节 CAR 长度，从而有效识别远端或近端表位，并允许通过使用临床级验证制造系统选择转导的 CAR T 细胞。本研究中研究的不同 LNGFR 间隔物负责产生具有不同记忆表型的 CAR T 细胞，这主要与 CAR 表达水平和相关强直信号的程度有关。特别是，CD44v6-NWN2.CAR T 细胞富含中央记忆细胞并显示出改善 这主要与 CAR 表达水平和相关强直信号的程度有关。特别是，CD44v6-NWN2.CAR T 细胞富含中央记忆细胞并显示出改善 这主要与 CAR 表达水平和相关强直信号的程度有关。特别是，CD44v6-NWN2.CAR T 细胞富含中央记忆细胞并显示出改善在体外具有杀伤能力，以及针对血液肿瘤和实体肿瘤的体内抗肿瘤活性。临床试验注册号：clinicaltrial.gov NCT04097301；ClinicalTrials.gov，NCT00423124。