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

• Applicant: Privatdozent Dr. Uwe Thiel
• Subject Area: Pediatric and Adolescent Medicine ; Hematology, Oncology ; Immunology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 501830041

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.

• DFG Programme: Research Grants