TARGETING TUMOUR-ASSOCIATED MACROPHAGES TO ENHANCE CART THERAPY IN A MOUSE MODEL OF SOFT-TISSUE SARCOMA

Soft tissue sarcomas (STS) are rare and aggressive tumors that are notoriously difficult to treat as they rarely respond to conventional systemic therapies and require extensive operations. Chimeric antigen receptor (CAR) T therapy is a novel immunotherapy where a patient's T cells are engineered to recognize and kill malignant cells based on a target antigen. CAR T cell therapy has revolutionized treatment of hematological malignancies. However, STS generally do not respond to this therapy despite expressing known CAR T target antigens. This is thought to be due to the immune suppressive tumor microenvironment (TME) characteristic of many STS, which diminishes CAR T cells infiltrating the TME, preventing CAR T mediated killing of malignant cells. Additionally, studies suggest tumor associated macrophages (TAMs) are highly abundant in STS and may be driving immune suppression in the TME. Thus, targeting TAMs may alleviate the immune suppression in the TME and enhance the efficacy of CAR T therapy in STS. The purpose of this study investigating diminishing TAMs in combination CAR T therapy in a murine model of STS. We used a previously published murine model of STS developed by our laboratory. The immune cells within this tumor model are predominantly monocyte-TAM lineage cells. This model also expresses GD2, which is a known CAR T target used in neuroblastoma and is also expressed in many STS. Thus, this provides a clinically relevant murine model of STS. We engrafted STS into wild type C57Bl/6 mice and Ccr2−/− mice, which have a deficiency in the classical monocyte recruitment pathway, which is essential for macrophages to infiltrate inflamed tissues. For CAR T therapy, we have generated a second generation CD28-CD3z mouse GD2 CAR using the 14G2a scFV currently being utilized in GD2 clinical trials. This construct contains a scFV proximal MYC tag to measure CAR cell surface expression, and a Thy1.1 cell surface receptor to measure T cell transduction efficiency. We have demonstrated that our CAR T cells display robust cytotoxicity to murine STS cells expressing GD2 in vitro. Mice bearing STS will undergo nonmyeloablative lymphodepletion with a single 200mg/kg dose of cyclophosphamide as standard part of CAR T cell therapy, followed by an intravenous delivery of GD2 CAR T cells. We monitored tumor growth using caliper measurements and assessed immune cells in murine STS using flow cytometry. Ccr2−/− mice bearing STS showed significantly smaller tumor size in comparison to C57Bl/6 mice. In assessing the TME, we found a significant reduction in the proportion of monocytes and TAMs, and an increased proportion CD3e+ and CD8+ T cells in the TME. Thus, reducing TAMs may provide an avenue to increase CAR T cell infiltration in the TME. Next, we administered CAR T cells to Ccr2−/− mice and C57Bl/6 mice. This experiment is currently ongoing. CCR2 deficiency provided both a significant reduction to TAMs and tumor size in this murine model of STS. Given the increased proportion of CD8 T cells, we believe this strategy may also improve the efficacy of CAR T therapy, which is under experimental assessment.