Effect of a novel expansion process on tumor-infiltrating lymphocyte (TIL) polyfunctionality, cytotoxicity, and expansion, while preserving cells in a less differentiated and more stem-like phenotype.

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2542 Background: Adoptive cell therapy using autologous TIL has shown durable responses in patients with metastatic melanoma and some epithelial tumors (Chesney JITC 2022; Schoenfeld SITC 2021; O’Malley SITC 2021). Data suggest that maintaining a higher proportion of less differentiated and more stem-like TIL can associate with persistence and response in patients with metastatic melanoma (Krishna Science 2020; Rosenberg Clin Cancer Res 2011). Here, we describe a novel TIL expansion process that increases TIL expansion and preserves cells in a less-differentiated and more stem-like phenotype with enhanced functional output. Methods: Tumors of various histologies, including lung, breast, and renal cancer, were fragmented and expanded using either a standard process or a newly developed process that uses a different combination of cytokines during the pre-rapid expansion protocol (pre-REP) stage and another combination of cytokines and a pathway inhibitor during the REP stage to control T-cell activation and differentiation. The expansion potential, viability, and phenotypic and functional attributes of the final TIL products were evaluated by a variety of assays. Results: When compared with standard TIL expansion, the novel process increased yield and viability while preserving TIL in a less differentiated and more functional state, as evidenced by increased expression of the memory-associated markers CD27, CD28, CD62L, and IL-7R, with reduced expression of the activation markers CD38, CD39, and CD69, and lower levels of the inhibitory markers LAG3, TIM3, TIGIT, and TOX. Importantly, the novel process led to a pronounced increase in the tumor-homing marker CXCR3 as well as TIL polyfunctionality as evidenced by increased co-expression of IFNγ, TNFα, and IL-2 while showing an enrichment in genes associated with stem-like cells and a reduction in exhaustion-associated genes. Pseudotime trajectory analysis also demonstrated that the new expansion process maintained TIL in a less differentiated and more stem-like state. Together, these phenotypic and functional characteristics translated into increased cytotoxicity even after repeated stimulation. Conclusions: Our novel TIL expansion process improves multiple metrics that correlate with both TIL persistence and response, including enhanced polyfunctionality, reduced inhibitory receptor expression and a less differentiated and more stem-like phenotype, while increasing yield. These effects may translate into a more vigorous and less differentiated TIL infusion product with improved cytotoxicity and persistence.