Abstract B032: Characterization of αPD1, αPD-L1, αCTLA4, and αLAG3 as mono and combination therapy in live tumor fragments using an ex vivo cytokine profiling platform

Abstract Background While immune checkpoint inhibitors (ICIs) provide durable clinical benefit in a subset of patients, current biomarkers to predict response lack accuracy. Ex vivo cytokine profiling has shown promise for improved accuracy but has been mostly limited to ICIs targeting PD-1 [Voabil et al, 2021]. This poses the question of whether ICIs beyond αPD-1 – such as CTLA4 blockade, which has been reported to act within the draining lymph node – would be amenable to similar approaches. The Elephas platform characterizes response to ICIs in live tumor fragments (LTFs) from core needle biopsies (CNBs) and resections using longitudinal cytokine profiling. Using a panel of 7 clinically relevant ICI regimens, we demonstrate cytokine responses across diverse tumor types, characterize similarities and differences in cytokine response profiles across different ICIs, and explore specimen variability in responses across ICI treatments. Methods CNBs were collected through 3 observational clinical trials (NCT05478538, NCT05520099, NCT0634962) and a biobank collection study, while resections were procured from NCI CHTN. LTFs were generated using an automated cutting device and encapsulated in hydrogel. LTFs were treated with mono (αPD-1, αPD-L1, αCTLA4, αLAG-3) or combination therapy (αPD-1/αCTLA4, αPD-1/αLAG-3, αPD-L1/αCTLA4) using a sequential treatment strategy in which isotype control followed by ICI were added to the same tissue in a single well. This approach enabled the comparison of control and treatment conditions while mitigating the effects of tumor heterogeneity. Production rates for 46 cytokines were measured using a multiplex assay. Remaining T-cell reactivity was also evaluated at the end of the assay to better understand the presence of functional T cells in LTFs. Results Unsupervised hierarchical clustering of cytokine profiling data from CNB LTFs across 129 patients identified defined responder and non-responder clusters. Within the response cluster, 4 subclusters with distinct immune phenotypes were identified. While tight clustering of samples treated with the same ICI was not observed, some enrichment of samples treated with combination ICIs was observed within individual subclusters. Further, cytokine responses to αCTLA4 monotherapy were observed, suggesting that CTLA4 blockade may act beyond the draining lymph node. LTFs from resections enabled the characterization of cytokine response profiles across treatment conditions within individual specimens, including the magnitude of cytokine response and variability in cytokine response profiles. Evaluation of T-cell reactivity provided further insight into the presence of functional T-cells in LTFs and the extent of T-cell activation achieved by ICI treatment. Conclusions This study demonstrates the platform’s ability to recapitulate diverse mechanisms of immune activation ex vivo, enabling the characterization of cytokine responses to clinically relevant ICI mono or combination therapy in live tumor tissue and supporting future efforts to improve ICI response prediction beyond αPD-1. Citation Format: Pichet Adstamongkonkul, Pierre Cunin, Santanu Bhattacharya, Tanmay Kulkarni, Amreen Nasreen, Nathan Marhefke, Nina Chen, T S. Ramasubramanian, Laura Hrycyniak, Julie Zweng, Erika von Euw, Christina Vivelo, Sean Caenepeel, Debabrata Mukhopadhyay, Hinco Gierman, Hilario Ramos. Characterization of αPD1, αPD-L1, αCTLA4, and αLAG3 as mono and combination therapy in live tumor fragments using an ex vivo cytokine profiling platform [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B032.