Abstract 3699: The "Preservation & Expansion" process: A key milestone for enhancing accessibility of autologous tumor-infiltrating lymphocyte therapy by enabling long-term cryopreservation of seed cells

Abstract Background: Tumor-infiltrating lymphocyte (TIL) therapy has shown promising efficacy in advanced solid tumors. However, its autologous nature poses a major limitation due to the ineligibility of many patients with advanced disease for repeated tumor resections, leading to a loss of potential treatment opportunities. To overcome this challenge, we developed a “Preservation & Expansion” strategy, in which TIL seed cells are generated from initial tumor tissue and cryopreserved for on-demand expansion into final products (FP) upon treatment need. Objective: This study aimed to validate the feasibility of expanding FPs from cryopreserved TIL seed cells after various storage durations. Methods: Tumor tissues were processed and initially cultured to generate seed cells. Seed cells generated from tumor tissues were either immediately expanded (G0) or cryopreserved for 3 days (G3D), 1, 2, 3, or 6 months (G1M-G6M), or 1 or 2 years (G1Y, G2Y) prior to expansion. Cell viability was assessed at the seed, intermediate, and FP stages. Phenotypic analysis (CD3+, CD8+, CD4+, CD56+) was performed on both seed cells and FPs, while functional analyses, including IFN-γ secretion and cytotoxicity via real-time cell analysis (RTCA), were conducted on the FPs. Results: Viability of cells derived from various tumor tissues post-thaw was comparable across different cryopreservation durations, with the following results (%): G0: 79.7±6.4; G3D: 80.9±5.1; G1M: 81.0±5.8; G2M: 80.2±3.9; G3M: 81.3±4.7; G6M: 81.0±5.3; G1Y: 80.4±5.6; G2Y: 80.8±4.8. Moreover, no significant differences in cell viability were observed during the REP culture phase at various time points. Phenotypic characterization by flow cytometry revealed similar profiles among seed cells cryopreserved for different durations and their corresponding final cellular products. Furthermore, cryopreservation duration had minimal impact on the effector functions of the final products, as evidenced by well-maintained levels of IFN-γ secretion and cytotoxic activity across all groups. IFN-γ secretion levels (ng/ml) were: G0: 14.5±7.0; G3D: 15.0±4.2; G1M: 13.4±5.0; G2M: 14.8±5.0; G3M: 16.5±5.0; G6M: 16.5±5.0; G1Y: 16.9±5.0; G2Y: 20.4±5.0. Cytotoxicity assessed by RTCA (%) was as follows: G0: 76.8±7.9; G3D: 78.1±4.3; G1M: 78.3±8.9; G2M: 77.4±9.0; G3M: 79.2±8.3; G6M: 79.9±3.7; G1Y: 77.2±8.7; G2Y: 83.2±3.6. Conclusion: TIL seed cells can be effectively expanded into functional FPs with consistent viability, phenotype, cytokine secretion, and cytotoxicity after long-term cryopreservation up to two years. These results validate the "Preservation & Expansion" process as a robust and feasible approach to improve the accessibility and practicality of autologous TIL therapy by decoupling tumor resection from treatment timing. Citation Format: Zhou He, Xingming Ma, Feng Yin, Shanshan Liang, Wenjia Zhuang, Hui Yan, Huajun Jin, . The "Preservation & Expansion" process: A key milestone for enhancing accessibility of autologous tumor-infiltrating lymphocyte therapy by enabling long-term cryopreservation of seed cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3699.