Epigenetic modulation of cellular aging to improve CAR-T therapy for solid tumors

摘要 CAR-T细胞疗法在治疗血液系统恶性肿瘤方面取得了令人难以置信的临床成功。然而， 迄今为止，针对实体肿瘤的活性非常有限，尽管针对各种抗原和 肿瘤类型。重要的是，实体肿瘤占所有癌症的90%以上，影响了1600多万人 2020年的美国人。多里安治疗公司的SBIR提案的目标是调查减少 CAR-T细胞的细胞衰老以提高实体瘤靶向性。 我们已经证明，在CAR-T制造过程中，T细胞的老化时间相当于30年，这是有害的 它们扩增和杀死癌细胞的能力。此外，实体瘤的特点是有很强的 免疫抑制和促进衰老的微环境，强烈挑战T细胞效应器功能。老T字 由于恶劣的微环境，细胞特别容易受到抑制和死亡。返老还童T细胞 通过在表观遗传水平上减少细胞衰老是改善T细胞健康和临床的一种新策略 结果。这个项目的目标是识别一个以shRNA为基础的遗传盒，以表观遗传学为靶点。 调节剂Usp16增加CAR-T细胞治疗实体肿瘤的适合性。 多里安的创始人发现斯坦福大学的USP16是一种表观遗传调节因子，能够延缓衰老 通过增强干细胞在多个组织中的自我更新。Usp16是一种去泛素酶，负责 去除组蛋白H2AK119中的泛素部分，增加亲衰老染色质的可及性 程序。该公司已经开发出体外和体内的概念验证，通过以下方式靶向Usp16 CD19中共表达的shRNA CAR结构可减少T细胞老化并增强干细胞记忆 (TSCM)在制造时的频率，不影响扩散。最重要的是，经过改造的CAR-T 在白血病的临床前小鼠模型中，细胞导致了肿瘤杀伤率的提高。 本研究的目的是：(1)建立肿瘤微环境对T细胞影响的体外模型 细胞老化和功能，(2)识别针对Usp16的遗传盒并在体外增加T细胞的适合性， (3)证明表达Usp16 shRNA的CAR-T细胞具有更好的抗肿瘤作用 在骨肉瘤的肿瘤模型和患者来源的肿瘤细胞的3D系统中的活性。 在这个SBIR项目的成功完成后，我们将确定最好的靶向遗传盒 USP16和改善T细胞适合性为毒理学研究做好准备(致瘤性和细胞因子释放综合征 (CRS)研究)。

Abstract CAR-T cell therapy has had incredible clinical success in the treatment of hematological malignancies. However, very limited activity against solid tumors has been achieved so far, despite targeting a variety of antigens and tumor types. Importantly, solid tumors account for more than 90% of all cancers, affecting more than 16 million Americans in 2020. The goal of Dorian Therapeutic’s SBIR proposal is to investigate the impact of reducing cellular senescence in CAR-T cells to improve solid tumor targeting. We have proven that T cells age the equivalent of 30 years during CAR-T manufacturing, which is detrimental to their ability to expand and kill cancer cells. Moreover, solid tumors are characterized by a strong immunosuppressive and pro-senescent microenvironment, strongly challenging T cells effector functions. Old T cells are particularly prone to be suppressed and die due to the hostile microenvironment. Rejuvenating T cells by reducing cellular aging at the epigenetic level is a novel strategy to improve T cell fitness and clinical outcomes. The goal of this project is to identify a shRNA-based genetic cassette targeting the epigenetic regulator USP16 to increase CAR-T cell fitness for the treatment of solid tumors. Dorian’s founders identified USP16 at Stanford University as an epigenetic regulator able to reduce senescence by enhancing stem cell self-renewal in multiple tissues. USP16 is a deubiquitinating enzyme responsible for the removal of ubiquitin moieties from histone H2AK119, increasing chromatin accessibility to pro-senescent programs. The company has developed in vitro and in vivo proof-of-concept that targeting USP16 by means of a shRNA co-expressed within the CD19.CAR construct reduces T cell aging and increases stem cell memory (Tscm) frequency upon manufacturing, without affecting proliferation. Most importantly, the engineered CAR-T cells resulted in improved tumor killing in a preclinical mouse model of leukemia. The Aims of this proposal are to (1) Create an in vitro model to study the effect of tumor microenvironment on T cells aging and functions, (2) Identify genetic cassettes targeting USP16 and increasing T cell fitness in vitro, and (3) Demonstrate that engineered CAR-T cells expressing a shRNA for USP16 have a better anti-tumor activity in a tumor model of osteosarcoma and in a 3D system of patient-derived tumor cells. At the successful completion of this SBIR project we will have identified the best genetic cassettes targeting USP16 and improving T cell fitness ready for toxicology studies (tumorigenicity and cytokine release syndrome (CRS) studies).