461 Leveraging tumor infiltrating B-cells to enhance TIL immunotherapy using CD40 and 41BB agonism

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Background Tumor infiltrating lymphocyte (TIL) therapy has been approved for melanoma1 and has shown promise in lung cancer.2 To broaden the use of TIL, a detailed understanding of the factors conditioning TIL performance is paramount. We herein investigate the role of tumor infiltrating B cells (TIL-B).3 Specifically, we hypothesized that activation of TIL-B ex vivo would result in enhancement of the TIL products. Methods We studied the abundance and phenotype of TIL-B, by flow, in association with the success of TIL expansion. We also characterized the changes induced by a CD40 agonist (as TIL-B activator) using flow and single cell RNA sequencing (scRNAseq, 10x Genomics; n=7). ImmunoSEQ CDR3 analysis was used to trace the expansion of individual neoantigen-reactive lung TIL clones. Novel bifunctional CD40- and 41BB-agonistic molecules were generated for simultaneous TIL and TIL-B stimulation. Finally, TIL tumor reactivity was assessed through IFNg release following coculture with autologous tumor digests. Results A higher percentage of TIL-B, specially CD27+IgD- cells, was associated with successful melanoma TIL expansion (p≤0.05). Further, higher proportion of non-lymphoid cells (p<0.05) and of CD27- IgD- B cells (p≤0.001) was associated with poor TIL expansion. Stimulation of TIL-B using soluble CD40L increased the success rate of TIL expansion from frozen melanoma digests (from 33% to 67%, p≤0.05); improved the expansion of lung TIL( from 71% to 83%, p≤0.01); and increased the proportion of CD39- CD69- TIL in melanoma (median difference= 11%, p≤0.05). CD40L induced gene expression changes in TIL-B after 48h in culture (126 differentially expressed genes, DEG), with minimal to no changes observed in other cell types (including no DEG in monocytes, 11 DEG in dendritic cells) suggesting a central role for B cells in the mechanism of action of CD40L. Importantly, the abundance of neoantigen-responsive clonotypes was also increased in the CD40L-treated cultures (p≤0.01). Autologous tumor reactivity was preserved in CD40L-enhanced TIL. Finally, we found that the use of bifunctional CD40L-41BBL fusion protein increased CD8+ proportion in lung (1.5-fold, p≤0.01) and melanoma (2.1-fold, p≤0.05) TIL. Conclusions The presence of TIL-B can be leveraged to improve ex vivo TIL expansion, and novel bifunctional molecules were generated to simultaneously stimulate TIL-B and TIL, resulting in increased CD8+ T cell content. These approaches are currently under study in clinical trials NCT05681780 (lung) and MCC23082 (melanoma). Acknowledgements This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence, the Suzi Q Foundation, and the Mark Foundation for the financial support. References Sarnaik AA, et al. Lifileucel, a tumor-infiltrating lymphocyte therapy, in metastatic melanoma. JCO 2021;39:2656–2666. Creelan B, et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat Med 2021;27:1410–1418. Messina JL, et al. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy? Sci Rep 2012;2:765. Ethics Approval The study was approved by Advarra IRB, approval number MCC20559. All patient samples were deidentified for this study.

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