Abstract 6593: Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy
Authors: Vizcardo Raul , Huang Yin , Fioravanti Jessica , Maeda Takuya , Tamaoki Naritaka , Yamazaki Yasuhiro , Kutlu Burak , Bahl Kriti , Wang Biao , Zhong Zheng , Potluri Shobha , Restifo Nicholas P. , Lee Gary 
Published: March 2024

Abstract
TIL therapy is a promising approach for the treatment of metastatic solid tumors, but its efficacy is limited by T-cell exhaustion and terminal differentiation. In addition, recent studies have underscored the adverse effects of aging on T cells. Reprogramming T cells into induced pluripotent stem cells (iPSC) and differentiating them back to the T-cell lineage has proven to be complex and time-consuming. In particular for TIL applications, de novo T cell production via iPSCs requires each TCR in the final product to be derived from an independently established iPSC clone. To overcome these barriers, we developed a novel technology called Rejuvenation, which bypasses the need for reprogramming of T cells into iPSCs. Rejuvenated TIL products retain their TCR diversity while offering several significant advantages, including reduced T-cell epigenetic age, improved expansion capacity, stem cell-like phenotype, and enhanced secretion of cytokines upon activation by target antigens. In this study, we used TIL derived from patients with melanoma and NSCLC. Rejuvenated TIL were generated by the transient expression of transcription factors in a process called ‘Partial Reprogramming’ and returned back to a T-cell lineage phenotype by a process called ‘Redirection’. Partially reprogrammed TIL showed an altered morphology resembling adherent stromal cells and down-regulated conventional T-cell markers including CD3, CD4, and CD8. Redirected T cells reacquired a conventional CD4/CD8 phenotype including morphology, surface markers and function, and demonstrated decreased epigenetic age (&gt;7 years younger, N=3). We also observed a higher proliferative capacity, improved metabolic state, and increased expression of biomarkers associated with T-cell stemness, including CCR7 and CD62L, when compared with untreated TIL. Rejuvenated TIL demonstrated improved cytokine secretion, including IFNγ, TNFα and IL-2 in functional assays. Immune repertoire analysis revealed a significant polyclonal population of putative tumor-reactive TCRs. In vivo functionality was also evaluated using a xenograft model of engineered A375 melanoma cell line that agonizes T-cells in hIL2-NOG mice. Adoptive transfer of rejuvenated TIL resulted in significantly improved suppression of tumor growth and prolonged survival compared with TIL expanded by conventional methods. These results were also confirmed in other surrogate models, including rejuvenated PBMC, TCR and CAR T cells, demonstrating the broad applicability of Rejuvenation technology. Our results indicate TIL Rejuvenation enhances the functionality and antitumor characteristics of T cells, while preserving a broad TCR repertoire. This technology holds substantial promise and may pave the way for the development of the first rejuvenated autologous polyclonal TIL therapy for treatment of advanced solid tumors.
Citation Format: Raul Vizcardo, Yin Huang, Jessica Fioravanti, Takuya Maeda, Naritaka Tamaoki, Yasuhiro Yamazaki, Burak Kutlu, Kriti Bahl, Biao Wang, Zheng Zhong, Shobha Potluri, Nicholas P. Restifo, Gary Lee. Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy [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(6_Suppl):Abstract nr 6593.