Karapetyan, L. | bioRxiv Science

bioRxiv2026

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

BackgroundTumor-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. MethodsCD40L 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 CD40LEPC6-41BBL) 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. ResultsEach 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 CD40LEPC6-41BBL molecule capable of binding to both B and T cells. Addition of CD40LEPC6-41BBL significantly increased total TIL yield and improved expansion success rates in both lung tumor and melanoma cultures. In particular, CD40LEPC6-41BBL 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 CD40LEPC6-41BBL. 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 CD40LEPC6-41BBL. ConclusionsSimultaneous 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.

immunology↗

bioRxiv2023

Defining the mechanisms of action and resistance to the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain models

BackgroundAlthough the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap. MethodsWe used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination. ResultsThe anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFN{gamma} secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFN{gamma} release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFN{gamma} release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner. ConclusionsTogether these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

immunology↗