PD1/CD28或IL4/IL7R嵌合体修饰的热点突变特异性TCR-T细胞治疗结直肠癌的效果评价及机制研究

靶向新抗原的TCR-T细胞治疗肿瘤的研究备受关注，但存在高度个体化和肿瘤微环境抑制的挑战。热点突变是通用性治疗的理想靶点，但目前热点突变特异性TCR有限。结合前期研究基础和文献，我们拟筛选目前尚未报道的PIK3CA E545K、BRAF V600E等热点突变特异性TCR，建立PD1/CD28或IL-4/IL7R嵌合体修饰的热点突变特异性TCR-T疗法，以期通用性治疗肿瘤同时扭转微环境抑制。为实现以上假说，本项目拟通过筛选热点突变特异性T细胞并解析其TCR，制备联合表达PD1/CD28或IL4/IL7R的TCR-T细胞，采用体内外实验分别探索其对微卫星不稳定型（MSI）或微卫星稳定型（MSS）结直肠癌的治疗效果，并阐明嵌合体扭转抑制性微环境的调控机制，为新型结直肠癌治疗策略提供新思路。本项目将为嵌合体修饰的热点突变特异性TCR-T疗法应用于结直肠癌提供理论与实验依据，具有重要的临床转化意义。

Increasing evidences have indicated the promising therapeutic application of neoantigen-specific TCR-T therapy, however, the major challenges lie in the highly individualized treating strategy and the immune-suppressed tumor microenvironment (TME). Hotspot mutation is thought as ideal target for general therapy, but hotspot mutation-specific TCRs are limited.Thus, based on previous studies and our research experience, we intend to establish TCR-T therapies targeting unexplored hotspot mutations in colorectal cancer (CRC) such as PIK3CA (E545K) and BRAF (V600E) to extend as general application. Besides, to recover the suppression of TME, PD-1/CD28 or IL4/IL7R chimeric receptors are also introduced into TCR-T cells. To prove the hypothesis, the hotspot mutation-specific T cells are induced and expanded, whose paired full-length TCR genes could be evaluated. And then the specific TCR gene and chimeric receptor (PD-1/CD28 or IL4/IL7R) would be introduced into peripheral T cells to construct chimeric receptor-modified TCR-T cells. Finally, we would validate the efficacy of PD1/CD28-modified TCR-T cells against microsatellite instability (MSI)-CRC or IL4/IL7R-modified TCR-T cells against microsatellite stability (MSS)-CRC, respectively, by in vivo and in vitro functional assay. Meanwhile, the mechanism of reversing suppressing TME through chimeric receptors is primarily clarified. In summary, we provide an efficient TCR-T therapy targeting hotspot mutation, modified with chimeric receptors, which could provide solid theoretical and experimental basises on general application in CRC. This novel strategy would be of great significance in the future treatments for colorectal cancer.