(P 239) INVESTIGATING HOW TUMOUR ASSOCIATED MACROPHAGES INFLUENCE THE EFFICACY OF CAR T THERAPY IN A MURINE MODEL OF SOFT TISSUE SARCOMA

Objective: 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. 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 enhance the efficacy of CAR T therapy. Herein, we investigated blocking TAMs from the STS TME in combination CAR T therapy.

Methods: We have developed a syngeneic murine model of STS that expresses GD2, which is a known CAR T target used in neuroblastoma and is also expressed in many 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 (Figure 1A). 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 (Figure 1B). 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 or non-targeting HER2 CAR T cells as a control. We will explore various doses of CAR T cell infusions (1x10^4 – 1x10^7 cells). We monitored tumor growth using caliper measurements and assessed immune cells in murine STS using flow cytometry.

Results: Ccr2-/- mice bearing STS showed significantly smaller tumor size in comparison to C57Bl/6 mice (Figures 2A & 2B). In assessing the TME, we found a significant reduction in the proportion of monocytes and TAMs (Figure 3A & 3B), and an increased proportion CD3e+ and CD8+ T cells in the TME (Figures 3C & 3D). Thus, reducing TAMs may provide an avenue to increase CAR T cell infiltration in the TME. We have demonstrated that our CAR T cells display robust cytotoxicity to murine STS cells expressing GD2 in vitro (Figure 4A). Experiments assessing CAR T therapy in Ccr2-/- mice are ongoing and scheduled to be completed by September 2024.

Conclusion: CCR2 deficiency provided both a significant reduction to TAMs and tumor size in the T1M6 model of STS. Given the increased proportion of T cells, we believe this strategy may also improve the efficacy of CAR T therapy, which is under experimental assessment.