Abstract 3254: MOTO-CARs™: A novel macrophage-based chimeric antigen receptor technology

Abstract

Abstract We report the development of a novel macrophage-mediated immunotherapy targeting mesothelin-expressing solid tumors. Although the advent of immunotherapy heralded a new era in cancer treatment, it has been accompanied by severe limitations, including antigen escape, cytokine release syndrome (CRS), and the inability to penetrate solid tumors. We sought to ameliorate these issues by developing a macrophage toll-like chimeric antigen receptor (MOTO-CAR™) designed to target and activate upon cancer antigen recognition. Here we show evidence supporting this proof-of-concept MOTO-CAR™ technology in a murine macrophage cell line (RAW 264.7) targeting a mesothelin-expressing, triple-negative, mammary gland squamous carcinoma cell line (HCC-1806). The MOTO-CAR™ construct was transfected into macrophage cells with an average efficiency above 50%. The presence of the chimeric construct was confirmed via mesothelin scFv amplification of isolated mRNA, and the engineered macrophages stably expressed the chimeric receptor for over 7 days, with the highest level of expression approximately 24 hours post transfection. We found that upon exposure to HCC-1806 cells, MOTO-CARs™ are not only effective at eliminating the cancer cells, but also secrete significant levels of TNF-α when compared to mock controls (p= 0.009), indicating activation and polarization to an M1 phenotype upon target recognition. In vitro cytotoxicity assays confirmed that macrophages containing the MOTO-CAR™ construct increasingly targeted and killed HCC-1806 cells when compared to the mock control (p < 0.0001) in three different effector to target ratios. Furthermore, in NSG mice containing HCC-1806-derived xenografts, tumor burdens were significantly lower in mice treated with MOTO-CARs™ when compared to the mock control (p = 0.006). In these models, the MOTO-CARs were administered through the tail vein, which indicates the MOTO-CARs™ can traffic effectively to the tumor site in vivo. Both TLR4 and TLR2 based toll-like receptor signaling domains were evaluated with TLR4 vectors showing higher killing and better efficacy against target cancer cells. Following initial testing in RAW 264.7 macrophages, similar results were observed when MOTO-CARs™ derived from primary human monocytes were co-incubated with the same target cells. These data illustrate the potential for MOTO-CARs™ to be a powerful alternative to current immunotherapies targeting solid tumors. Citation Format: Michelle Hannah Townsend, Zachary D. Ewell, Kelsey A. Bennion, Guoying Wang, Jonathan Skidmore, David Lum, Michael Boyer, Kim L. O'Neill. MOTO-CARs™: A novel macrophage-based chimeric antigen receptor technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3254.