Knudsen Nelson H, Escobar Giulia, Korell Felix, Kienka Tamina, Nobrega Celeste, Anderson Seth, Cheng Andrew Y, Zschummel Maria, Armstrong Alexander, Bouffard Amanda, Kann Michael C, Goncalves Sadie, Pope Hans W, Pezeshki Mitra, Rojas Alexander, Suermondt Juliette S M T, Phillips Merle, Berger Trisha R, Park Sangwoo, Salas-Benito Diego, Darnell Elijah P, Birocchi Filippo, Leick Mark B, Larson Rebecca C, Doench John G, Sen Debattama, Yates Kathleen B, Manguso Robert T, Maus Marcela V
Krantz Family Center for Cancer Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
Cellular Immunotherapy Program, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
Nature. 2025 Oct;646(8086):953-962. doi: 10.1038/s41586-025-09489-8. Epub 2025 Sep 24.
Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies. However, progressive loss of CAR T cells contributes to relapse in many patients. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.
嵌合抗原受体（CAR）T细胞在血液系统恶性肿瘤中具有高效性。然而，CAR T细胞的逐渐丧失导致许多患者复发。在此，我们在靶向B细胞成熟抗原的CAR T细胞中进行了体内功能丧失型CRISPR筛选，以研究影响CAR T细胞在人多发性骨髓瘤模型中持久性和功能的基因。我们在体外以及体内早期和晚期时间点追踪CRISPR文库编辑的T细胞的扩增和持久性，以追踪基因修饰的CAR T细胞从生产到在肿瘤中存活的表现。筛选揭示了CAR T细胞扩增和持久性的背景特异性调节因子。RASA2和SOCS1的缺失增强了体外T细胞的扩增，而PTPN2、ZC3H12A和RC3H1的缺失赋予了CAR T细胞在体内早期生长优势。值得注意的是，我们确定细胞周期蛋白依赖性激酶抑制剂1B（由CDKN1B编码），一种细胞周期调节因子，是体内晚期限制CAR T细胞适应性的最重要因素。CDKN1B的缺失增加了CAR T细胞的增殖和效应功能，显著增强了肿瘤清除率和总体生存率。我们的研究结果揭示了基因扰动对CAR T细胞随时间和在不同环境中的不同影响，突出了CDKN1B作为生成用于多发性骨髓瘤的高效CAR T细胞的有前景靶点，并强调了体内筛选以鉴定增强CAR T细胞疗效的基因的潜力。
