Ludwig Institute for Cancer Research Lausanne, Department of Oncology, University of Lausanne and Lausanne University Hospital, Lausanne 1011, Switzerland.
Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne 1011, Switzerland.
Proc Natl Acad Sci U S A. 2024 Oct 29;121(44):e2405085121. doi: 10.1073/pnas.2405085121. Epub 2024 Oct 25.
The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients.
小分子能够远程控制嵌合抗原受体 (CAR) 的活性，从而提高基因修饰 T 细胞的安全性和疗效。分裂型 ON/OFF 开关 CAR 涉及肿瘤抗原结合从受体（R-）和信号（S-）链上的 T 细胞激活（即 CD3ζ）的解离，分别在小分子存在的情况下关联或中断。在这里，我们开发了一种包含外显子中两个链的抗凋亡 B 细胞淋巴瘤蛋白 2 蛋白的可诱导（i）ON-CAR，在 venetoclax 的存在下它们会关联。我们表明，在 venetoclax 或 BH3 模拟物 navitoclax 的存在下，诱导型 ON（iON）-CAR T 细胞以剂量依赖性方式响应靶肿瘤细胞，而药物对等效的第二代-CAR T 细胞没有影响。在 venetoclax 撤药后 48 小时内，通过干扰素-γ（IFNγ）产生评估，iON-CAR T 细胞丧失了对靶肿瘤细胞的体外响应能力，并且它们依赖 venetoclax 存在以发挥体内活性。最后，通过将降解序列融合到 iON-CAR S 链的内域，我们生成了一种一体式 ON/OFF 开关 CAR，即 iONØ-CAR，用 lenalidomide 在 4 至 6 小时内下调，使功能失活的 T 细胞（无 IFNγ产生）在 24 小时内减少。我们提出，我们的远程控制 CAR 设计可以降低临床毒性。此外，iON 和 iONØ-CAR T 细胞的定期休息可能会减轻衰竭，从而增强患者的持久性和长期肿瘤控制。
