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

<h3>Background</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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&lt;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. <h3>Conclusions</h3> 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). <h3>Acknowledgements</h3> 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. <h3>References</h3> Sarnaik AA, <i>et al</i>. Lifileucel, a tumor-infiltrating lymphocyte therapy, in metastatic melanoma. <i>JCO</i> 2021;<b>39</b>:2656–2666. Creelan B, <i>et al</i>. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. <i>Nat Med</i> 2021;<b>27</b>:1410–1418. Messina JL, <i>et al</i>. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy? <i>Sci Rep</i> 2012;<b>2</b>:765. <h3>Ethics Approval</h3> The study was approved by Advarra IRB, approval number MCC20559. All patient samples were deidentified for this study.