Abstract A028: Assessing fibroblast activation protein as an immunotherapy biomarker and CAR T cell therapeutic target in the tumor microenvironment of canine sarcomas

Abstract Canine osteosarcoma (OS) and soft tissue sarcomas (STS) have molecular, pathologic, and clinical features that closely parallel those of the human tumors. However, unlike murine sarcoma models, canine sarcomas develop spontaneously within an intact immunological context, providing a unique opportunity for developing and testing sarcoma immunotherapies within the complex native sarcoma tumor microenvironment (TME). While sarcomas are rare in people, up to 15% of malignant tumors diagnosed in dogs are sarcomas, offering an expanded patient population with accelerated clinical outcomes for incorporation into sarcoma research and drug development studies. Fibroblast activation protein-a (FAP) is a prognostic marker for cancer-associated fibroblasts (CAFs) in the carcinoma TME and is being developed as an anti-CAF therapeutic target in carcinomas. FAP expression has been reported in canine soft tissue sarcomas and some human sarcoma subtypes, but the functional role of FAP within the sarcoma TME remains unknown and FAP-targeted sarcoma therapies have been minimally explored. Our single-cell RNA sequencing of canine OS lung metastases reveals FAP+ CAFs as a prominent population in the metastatic OS TME with high expression of the pro-fibrotic and myeloid chemotactic cytokines IL-6 and CCL2 respectively. Accordingly, FAP-high sarcomas in both dogs and people show striking upregulation of matrix remodeling, immunomodulatory, and angiogenic pathways, and enrichment of myeloid cell immunotranscriptomic signatures. We therefore hypothesized that FAP-expressing stromal and/or tumor cells play a central role in shaping the sarcoma TME, making FAP both a biomarker for a myeloid-rich, T cell-excluded/dysfunctional sarcoma immune infiltrate and a cross-species therapeutic target. Both tumor and stromal compartments in OS may express FAP, thus we have developed and optimized multispectral immunofluorescence panels for simultaneously localizing and quantifying FAP protein expression within OS and stromal cells and determining its association with major immune cell abundances and spatial distribution. We have also generated canine FAP-targeting CAR T cells with robust and specific activity against FAP+ canine sarcoma cells in vitro. Since prior work in our laboratory shows that blockade of CCR2 monocyte recruitment with losartan-toceranib therapy induces tumor regression in canine metastatic OS, we are currently conducting trials of FAP CAR T cells alone or in combination with losartan-toceranib immunotherapy in dogs with STS and OS. Collectively, these studies will elucidate the impact of tumor vs. stromal FAP on immune infiltration in the sarcoma TME, assess the feasibility and efficacy of FAP CAR T cell therapy in canine STS and OS patients respectively, and inform the use of tumor-associated macrophage-depleting drugs to potentiate CAR T cell therapy in sarcomas. Citation Format: Marika L Klosowski, Alexandra McMellen, Jenna Burton, Kathryn E. Cronise, Michael Leibowitz, Daniel P Regan. Assessing fibroblast activation protein as an immunotherapy biomarker and CAR T cell therapeutic target in the tumor microenvironment of canine sarcomas [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 A028.