Abstract 5250: Chimeric antigen receptor macrophages (MOTO CARs) driving repolarization of macrophages in solid tumors
Authors: Michael Boyer, Michelle Townsend, Timothy Phares, Adam Blaszczak, Rachel Garlick, Kenneth Katschke, Gaju Shrestha, Toni Mortimer, Ashlin Cowager, McCall Jensen, Abigail Cheever, Kim O'Neill
Journal: Cancer Research (2024)

Abstract Introduction: Though chimeric antigen receptor (CAR) T cells have been effective in treating hematological cancers, they have lacked in solid tumors. Due to the nature of solid tumors and how macrophages are localized there, we propose that macrophages are a better cell type for targeting solid tumors. In tumors, most of the macrophages are Tumor Associated Macrophages (TAMs) which are M2 polarized and promote tumor growth, angiogenesis, and metastases. If these macrophages could be repolarized to an anti-tumor, M1 phenotype, they could alter the tumor immune microenvironment and facilitate destruction of the tumor by immune cells. Methods: We hypothesize that CAR macrophages (MOTO-CARs) could infiltrate solid tumors, and affect the tumor immune microenvironment to repolarize (TAMs) from an M2 to M1 phenotype. Over 150 signaling domains from published sequences were cloned into a CAR constructs, comprised of an anti-mesothelin scFv, CD34 hinge, and transmembrane and activation domains from various immune cells. Functional activation assays were preformed in reporter HEK cells lines. Functional constructs were then transduced into THP-1 cells. The function of these MOTO-CARs were evaluated using a flow cytometry based phagocytosis assay (indication of an M1 phenotype) and RT-qPCR repolarization assay. Results: Of the constructs tested in HEK reporter cell lines, around one third demonstrated significant activation, that is 20% higher than the background control. The phagocytosis assay performed on the functional constructs showed that around one third of these constructs exhibited higher phagocytosis compared with a control construct with no signaling domain. However, it is noteworthy that this no signaling domain control had significantly higher rates of phagocytosis than untransduced controls. The RT-qPCR demonstrated that M2-like THP-1 cells transduced with the CAR constructs exhibited a significant shift in expression towards M1 characteristic genes when stimulated with mesothelin beads, with the TLR4 construct exhibiting the greatest shift. Conclusion: Several of the MOTO-CAR constructs, including TLR4, showed significant activation, phagocytosis, and ability to repolarize macrophages from M2 to M1 phenotype. Further analysis hopes to prove the functionality of these MOTO-CAR constructs with primary cells as well as in vivo, making MOTO-CARs a valuable treatment option for patients with solid tumors. Citation Format: Michael Boyer, Michelle Townsend, Timothy Phares, Adam Blaszczak, Rachel Garlick, Kenneth Katschke, Gaju Shrestha, Toni Mortimer, Ashlin Cowager, McCall Jensen, Abigail Cheever, Kim O'Neill. Chimeric antigen receptor macrophages (MOTO CARs) driving repolarization of macrophages in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5250.