Abstract 2193: Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype
Authors: Xiaohong Wang, Konrad Gabrusiewicz, David M. Spencer, Aaron E. Foster, J. Henri Bayle
Journal: Cancer Research (2020)

Abstract Background: Effective chimeric antigen receptor (CAR) T cell therapy against solid tumors must overcome a hostile, tumor microenvironment that includes tumor-associated macrophages (TAM). Pancreatic adenocarcinomas (PDAC) are commonly infiltrated with TAMs polarized to a tumor-promoting M2 phenotype rather than a T cell-stimulatory and tumor-inhibitory M1 phenotype. The GoCAR platform combines an inducible MyD88/CD40 (iMC) costimulation protein with a 1st generation CAR. Our previously published results demonstrated that iMC costimulation, activated by the small molecule dimerizer, rimiducid (Rim), enhanced CAR-T proliferation and anti-tumor efficacy. Here, we examined the extrinsic effects of iMC signaling on CAR-T cell immune-activating ligands, cytokine production and their ability to polarize M2 macrophage to an anti-tumor phenotype. Methods: Macrophages were prepared from peripheral blood monocytes of four or more random blood donors from the Gulf Coast Regional Blood Center (Houston, TX) and differentiated in vitro to an M2 phenotype with TGF-β and IL-10, or an M1 phenotype (as a positive control). GoCAR-T cells targeting prostate stem cell antigen (PSCA) were prepared by retroviral transduction from the autologous donors. To test the effects of iMC activation on macrophage polarization, contact-dependent or independent (i.e., separation of cell populations with transwell inserts) coculture assays were performed with and without activation of iMC with 1 nM Rim and/or surface-bound PSCA. Anti-tumor cytotoxicity was measured by coculture with PSCA+ Panc1-GFP cells. Results: CAR activation by PSCA antigen recognition or iMC activation with Rim decreased CD163, an M2 macrophage marker, and increased CD80, expressed on M1 macrophage. Full activation of the GoCAR-T cells with both Rim and the target antigen fully repolarized M2 macrophage to an M1 marked phenotype (CD163lowCD80high). This repolarization could be directed partially in the absence of cell-cell contact by diffusion of soluble factors through Transwell membranes. M2 macrophages repolarized by conditioning media from activated GoCAR-T cells also exhibited the functionality of M1 macrophages and acquired cytotoxicity against tumor cells. Furthermore, cultures of conditioned macrophages and limiting dilutions of GoCAR-T cells demonstrated a cooperative enhancement of the cytotoxicity of PSCA GoCAR-T cells toward Panc-1 targets. This cooperation was more effective for GoCAR-T cells than CD28- or 4-1BB-enhanced 2nd generation PSCA CAR-T cells. Conclusions: These results predict that GoCAR-T activation with Rim will convert TAMs within a solid tumor microenvironment from T cell inhibitors to tumor-caustic agents. Citation Format: Xiaohong Wang, Konrad Gabrusiewicz, David M. Spencer, Aaron E. Foster, J. Henri Bayle. Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype [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 2193.