Abstract A042: Uncovering the mechanism of synergistic anti-tumor effects in FAP-CD40 + PD1-IL2v combination therapy

Abstract Therapies targeting the activation and recruitment of immune cells such as T cells or DCs to the tumor microenvironment (TME) are considered as highly promising approaches to improve anti-tumor responses. Here, we evaluated the synergistic efficacy of PD1-IL2v in combination with FAP-CD40 in the syngeneic and notoriously difficult to treat KPC-4662 tumor model. PD1-IL2v drives the expansion of stem-like CD8 T cells and their differentiation into effector CD8 T cells, while FAP-CD40, a tumor targeted CD40 agonist antibody, triggers the activation of DCs. Our aim was to uncover spatial patterns of immune cell organization within the tumor tissue potentially being responsible for this synergistic activity of this combination treatment. To assess how the combination treatment influences the spatial organization of immune cells within the TME, we employed 3D immune phenotyping (3DIP). 3DIP combines multiplexed confocal imaging with a quantitative analysis pipeline, enabling the visualization and quantification of up to 12 markers in 70 μm thick tumor sections and thus the detailed characterization of complex cellular subsets and intercellular interactions within intact tumor tissues. Mice bearing subcutaneous, fibrotic and immune excluded KPC tumors were treated with FAP-CD40 and PD1-IL2v, either as monotherapies or in combination. PD1-IL2v or FAP-CD40 monotherapy resulted in delayed tumor growth compared to the vehicle. Significant tumor regression was observed only when PD1-IL2v was combined with FAP-CD40. Interestingly, 3DIP analysis of treated tumor tissues revealed strong infiltration of both CD4 and CD8 T cells in general. Only quantitative spatial analysis of tumor tissues revealed the presence of dense clusters of CD4 and CD8 T cells, co-localizing with dendritic but not B cells, throughout tumor tissues and only after combination treatment, underlining the importance of evaluating spatial organization when studying immune-modulating therapies. Taken together, these findings suggest that the observed efficacy upon combination of PD1-IL2v with FAP-CD40 is driven by the increased proliferation, recruitment and organization of CD4 and CD8 T cells in the tumor tissue. This could be mediated by dendritic cells activated by FAP-CD40, which may also play a role in reprogramming exhausted T cells into effector T cells within the intratumoral immune clusters, ultimately leading to tumor regression. Citation Format: Thuy Trinh Nguyen, Leo Kunz, Ines Grazina de Matos, Christine Trumpfheller, Pablo Umaña. Uncovering the mechanism of synergistic anti-tumor effects in FAP-CD40 + PD1-IL2v combination therapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A042.