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

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-gamma 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-gamma secretion and cytotoxic activity across all groups. IFN-gamma 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.