Abstract 27: A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity

Abstract Background: Genetically engineered T cells with T cell receptor (TCR-T) cell therapy have shown great potential for cancer treatment by targeting tumor-associated antigens. Despite its success in treating various tumor types, such as synovial sarcoma, head and neck tumors, and cervical cancer, its application in liver cancer is limited. Alpha-fetoprotein (AFP), a highly expressed tumor-associated antigen in liver cancer, is an ideal target for TCR-T cell therapy. Although early human trials have confirmed the safety of AFP-targeting TCR-T products, their efficacy is still modest, highlighting the need for further research and development. Methods: We developed a robust strategy combining ex vivo stimulation and single-cell paired TCR sequencing to identify AFP-recognizing TCRs in liver cancer patients. To optimize TCR expression and minimize mispairing, we employed codon optimization and replaced the constant regions with murine counterparts. Furthermore, structural analysis guided the affinity optimization of the CDR3 region to enhance the antitumor function of the selected TCR. The anti-tumor efficacy of TCR-T cells was rigorously evaluated through in vitro cytotoxicity assays using the Real-Time Cell Analyzer (RTCA) system and in vivo antitumor assessment in immunodeficient mouse xenograft models. Antigen specificity, HLA restriction, and other potential toxicity were carefully evaluated in both in vitro and in vivo models. Results: We successfully cloned a highly effective TCR that recognizes the AFP158-166 epitope presented by HLA-A*0203. After sequence optimization, we further changed a single amino acid on the TCR vβ chain based on AI prediction. The expression and functionality of this TCR was significantly improved. When introduced into human T cells, the single amino acid mutated TCR demonstrated robust and specific anti-tumor activity in various models, including cultured tumor cells, patient-derived organoids, and mouse xenografts models. Notably, this TCR also redirected CD4 T cells to recognize tumor cells, induced the secretion of multiple cytokines, and maintained long-lasting anti-tumor effects. Our rigorous screening assays confirmed that the TCR showed no cross-reactivity with the human proteome or major HLA subtypes. Additionally, extensive preclinical safety assessments in tumor-bearing animals treated with ST08A01-AM14 TCR-T cells showed no significant safety concerns. Conclusion: In this study, we have developed an HLA-A*0203 restrict AFP158-166 specific TCR-T therapy, which exhibits exceptional anti-tumor effects while ensuring a favorable safety profile. The promising results support the further investigation against AFP-positive solid tumors in clinic. Citation Format: Chen-Song Huang, shudan Ou, Jun Li, Junjun Chu, Yanyan Han. A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 27.