Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, California.
Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California.
Cancer Immunol Res. 2023 Feb 3;11(2):150-163. doi: 10.1158/2326-6066.CIR-22-0504.
Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.
嵌合抗原受体 (CAR) 是融合蛋白，其功能结构域通常以即插即用的方式连接，从而产生多种 CAR 变体。然而，具有高度相似序列的 CAR 可能在功能上存在显著差异。因此，合理优化 CAR 蛋白的方法对于开发有效的 CAR T 细胞疗法至关重要。在这里，我们报告称，CAR 中非信号结构域中的两个氨基酸变化就能够显著增强体内抗肿瘤疗效。我们证明了膜近端丙氨酸插入和单链可变片段序列杂交是两种可以结合使用的策略，可以最大限度地提高 CAR 的功能，并描述了一种 CD20 CAR，在抗肿瘤疗效方面优于 CD19 CAR，无论是在临床前的体外和体内试验中。CAR 序列的精确变化在抗原刺激下导致了截然不同的转录组谱，最有效的 CAR 在抗原刺激下诱导高度功能性记忆 T 细胞的富集。这些发现强调了序列水平优化对 CAR T 细胞功能的重要性，并且这里描述的蛋白质工程策略可应用于针对多种抗原的其他 CAR 的开发。请参阅相关的 Spotlight 文章由 Scheller 和 Hudecek 撰写，第 142 页。
