Identification of tumour-antigen specific T cell receptors for non-viral CRISPR/Cas9 mediated orthotopic- vs. retro-viral transduction to engineer high-functional T cell responses against Ewing sarcoma

尤文氏肉瘤（EwS）是一种高度侵袭性的儿科癌症。5-转移性和复发性EwS的年生存率仍然很低。治疗是有毒的，包括局部手术，放射治疗和综合化疗。由于早期复发和/或转移性疾病患者的治疗选择已经达到平台期，因此需要创新的治疗选择，例如T细胞介导的免疫疗法。体细胞突变在EwS中是罕见的，这就是为什么它经常被认为是免疫沙漠。在我们的手中，EwS已经变得对使用肽/HLA-A2 EwS特异性同种异体限制性TCR转基因T细胞针对肿瘤相关抗原的免疫疗法敏感。目前在晚期疾病EwS患者的治愈性免疫治疗方面缺乏临床突破，这可能是由于所选TCR的功能受损以及逆转录病毒转导方案的使用导致基因组内随机插入TCR α和β链。此外，T细胞的体外培养和扩增不可避免地导致抗肿瘤反应性的耗尽和丧失。与T细胞受体α/β恒定链（TRAC/TRBC）敲除后的原位TCR置换相比，逆转录病毒TCR转导可导致T细胞功能受损和TCR与内源性TCR错配。值得注意的是，插入TCR以控制TRAC启动子可能使T细胞倾向于TCR错配，除非第二TCR β链（TRBC）也被编辑。斯科贝尔等人处理了TRAC/TRBC敲除/TRAC敲入模型，该模型显示出与生理T细胞群非常相似的TCR调节模式。本项目建议书将追求以下目标：1. 使用经典的体外和创新的体内模型（人源化小鼠）鉴定EwS以及泛癌表达的抗原用于基于过继性T细胞的免疫疗法。 使用创新技术（CRISPR/Cas9与逆转录病毒转导方案）改善基于T细胞的抗癌应答。3. 临床前准备，交叉反应性测试和非病毒转导方案的实施，用于未来使用具有良好GMP合规性的技术进行临床翻译。计划开发当地基础设施（例如，慕尼黑Christoph Priesner指导的GMP实验室综合体TUMCells），目的是开始对所有儿科癌症实体进行临床研究，作为未来的展望。

Ewing’s sarcoma (EwS) is a highly aggressive paediatric cancer. 5-year survival rates for metastatic and relapsed EwS remain dismal. Treatment is toxic and comprises local surgery, radiotherapy and poly-chemotherapy. As therapeutic options for patients with early relapse and/or metastasised disease have reached a plateau, innovative treatment options are warranted e.g. such as T cell mediated immunotherapy. Somatic mutations are rare in EwS, which is why it is often regarded upon as an immunological desert. In our hands, EwS has become susceptible to immunotherapy using peptide/HLA-A2 EwS specific allo-restricted TCR transgenic T cells against tumour-associated antigens. A clinical break-through in the curative immunotherapy of EwS patients with advanced disease is currently missing, probably both due to impaired functionality of selected TCRs and to the use of retroviral transduction protocols causing random TCR α and β chain insertion within the genome. Furthermore, in vitro culture and expansion of T cells inevitably causes exhaustion and loss of anti-tumour reactivity. Retroviral TCR transduction may cause impaired T cell function and TCR mispairing with the endogenous TCR as compared to orthotopic TCR replacement after T cell receptor α/β constant chain (TRAC/TRBC) knock-out. Of note, insertion of TCRs to the control of TRAC promoters may render T cells prone to TCR mis-pairing unless the second TCR β chain (TRBC) is also edited. Schober et al. processed TRAC/TRBC knock-out/TRAC knock-in model which showed a TCR regulation pattern very similar to that of a physiological T cell population. In this project proposal, the following objectives will be pursued:1. Identification of EwS as well as pan-cancer expressed antigens for adoptive T cell based immunotherapy using classical in vitro and innovative in vivo models (humanised mice).2. Improvement of T cell based anticancer responses using innovative techniques (CRISPR/Cas9 vs. retroviral transduction protocols). 3. Pre-clinical preparation, cross-reactivity testing and implementation of non-viral transduction protocols for future clinical translation using technologies with promising GMP conformity. Exploitation of the local infrastructure is planned (e.g. the GMP laboratory complex TUMCells directed by Christoph Priesner in Munich) with the aim to commence clinical studies on all paediatric cancer entities as a future perspective.