Dual stimulation of CD40 and 41BB pathways during ex-vivo TIL expansion enhances CD8+ T cell expansion

Tumor-infiltrating lymphocyte (TIL) therapy has demonstrated clinical efficacy in malignant melanoma; however, inefficient ex vivo expansion remains a major limitation. We previously showed that stimulation of tumor-infiltrating B cells via CD40-CD40L axis improves TIL expansion, and that direct activation of the 41BB-41BBL pathway on T cells enhances CD8⁺ T cell outgrowth. We hypothesized that adding simultaneous targeting of both pathways would augment the growth and activity of CD8+ cytotoxic T cells. We conducted a study with the objective of determining the feasibility of dual stimulation with human tumors as justification for a Phase I trial.

CD40L variants were generated by yeast display selection and evaluated for B cell binding and activation. The effects of CD40L variants on TIL expansion were evaluated using tumors derived from standard of care resections using fragment method. Based on these findings, a bi-specific molecule was designed and generated fusing a CD40L variant and 41BB to the N- and C-termini of a trimeric leucine zipper. The effects of the bi-specific molecule (termed CD40L^EPC6-41BBL) on TIL expansion were evaluated in TIL cultures derived from lung tumor and melanoma fragments. TIL phenotypes were assessed by flow cytometry, including high-dimensional FlowSOM analysis, and tumor reactivity by autologous tumor co-culture assays.

Each of our engineered CD40L variants bound B cells and induced CD80/CD86 expression at levels comparable to wild-type CD40L. Supplementation of TIL cultures with CD40L variants increased the success rate of TIL expansion compared to control. We then developed a bi-specific CD40L^EPC6-41BBL molecule capable of binding to both B and T cells. Addition of CD40L^EPC6-41BBL significantly increased total TIL yield and improved expansion success rates in both lung tumor and melanoma cultures. In particular, CD40L^EPC6-41BBL promoted preferential expansion of CD8⁺ T cells. High-dimensional analysis revealed enrichment of CD8⁺ T cell clusters expressing CD39, CD69, TIM3, and CD56 in cultures supplemented with CD40L^EPC6-41BBL. Furthermore, treated cultures displayed increased frequencies of CD27⁺ CD4⁺ T cells. Functional assessment suggested a trend toward enhanced tumor reactivity in melanoma-derived TIL products expanded with CD40L^EPC6-41BBL.

Simultaneous stimulation of CD40 and 41BB pathways using a novel bi-specific molecule resulted in qualitative and quantitative enhancement of TIL products. These findings support dual targeting of tumor-infiltrating B cells and T cells as a promising strategy to optimize TIL manufacturing for adoptive cell therapy in Phase I trials.