Abstract 2239: FB849, a highly selective HPK1 inhibitor, reshapes the tumor immune microenvironment by modulating various immune cells

A highly unmet medical need exists for immuno-oncology agents to modulate the tumor microenvironment through broad-spectrum immune cell modulation by promoting crosstalk among T cells, B cells, and APCs, to exert a strong and durable anti-cancer immunity. Beyond T cells, other immune cells can play a crucial role in driving clinical activity, particularly in combination with current checkpoint inhibitors or patients with T cell dysfunction. We have discovered and developed FB849, a potent, highly selective, and orally bioavailable HPK1 inhibitor. FB849 has demonstrated robust anti-tumor activity across multiple preclinical models and is currently being evaluated in a Phase I/II clinical trial for advanced solid tumors (NCT05761223). To elucidate its immune-modulatory mechanisms and impact on both T cells and non-T cells, we extended our research to human primary immune cells and tumor-infiltrating lymphocytes (TILs). FB849 effectively activated T cells increasing IL-2 and IFNγ production, and stimulated B cell secreting IgG, IL-6 and IL-8. In monocyte-derived DCs, FB849 also significantly enhanced the secretion of IL-12 and IL-1β, and CD80 and CD86 expression. In vivo mouse TIL analyses revealed that FB849 increased T cell activation while re-shaped the myeloid compartment within the tumors by increasing dendritic cell infiltration with elevated cDC1, promoting tumor-associated macrophage (TAM) depletion with M1 polarization, and increasing NK cell infiltration. These create a tumor microenvironment favorable to anti-cancer immunity. Notably, FB849 retained anti-tumor activity even after CD8 T cell depletion, highlighting the pivotal role of non-T cells. To further delineate FB849’s effects on TILs, we conducted single-cell RNA sequencing and TCR sequencing with the single cell suspension samples from RCC, HCC, HNC and BRC patients. FB849 prominently expanded multiple types of T cell clones, especially the CXCL13+ CD8 TIL cluster enriched with neoantigen signature genes. Furthermore, TCR sequencing revealed a considerable overlap in between CXCL13+ CD8 and MKI67+ CD8 T cells when stimulated with autologous EpCAM+ tumor cells or NY-ESO-1 overlapping peptides, validating that CXCL13+ CD8 TILs are tumor-reactive. We hypothesize that the intra-tumoral expansion of CXCL13+ CD8+ T cells enhance interactions between T cells and various non-T cells including B cells and dendritic cells, potentially driving tertiary lymphoid structure (TLS) formation via the CXCL13 chemokine secretion, which warrants further validation in preclinical and clinical research. Altogether, our work represents a promising approach to modulate a broad spectrum of immune cells simultaneously and enhance their crosstalk, paving the way for durable clinical activity and supporting further development in the clinic. Citation Format: Seungmook Lim, A Yeong Park, Hyekyoung Kim, Seung Hyuck Jeon, Yong Joon Lee, Jamie Jae Eun Kim, Eui-Cheol Shin, Jinhwa Lee. FB849, a highly selective HPK1 inhibitor, reshapes the tumor immune microenvironment by modulating various immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2239.