The Second Generation of CAR-iMACs with Enhanced Polarization, CAR-mediated Efferocytosis and Superior Potency against Solid Tumors

Chimeric antigen receptor (CAR)-T cell therapies have shown great success in treating hematologic malignancies. Nonetheless, their therapeutic effect on solid tumors remains to be improved. Recently, macrophages have attracted great attention, given their ability to infiltrate solid tumors, phagocytize tumor cells as well as their immunomodulatory capacities. The first generation of CD3?-based CAR-macrophages demonstrated that the CAR could stimulate macrophage phagocytosis in a tumor antigen-dependent way. Here, we genetically engineered induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) with TLR4 intracellular TIR domain-containing CARs against EGFRvIII and GPC3, which yielded markedly enhanced antitumor effect in two different solid tumor models including glioblastoma, and hepatocellular carcinoma in which complete remission was achieved with CAR-iMACs alone or in combination with CD47 antibody. Moreover, the tandem CD3?-TIR-CAR, or the “second-generation” design of TIR-based dual signaling CAR, endowed iMACs with both target engulfment/efferocytosis capacity against antigen-expressing solid tumor cells, and potency of antigen-dependent M1 state polarization and M2 state resistance in an NF-?B dependent manner. We also illustrated a surprising mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we established the next generation CAR-iMACs equipped with orthogonal phagocytosis and polarization capacity for better antitumor functions in treating solid tumors. Overall design: We pretreated EGFRvIII-targeting CAR-iMACs were incubated with U87MGEGFRvIII cells with IFN-?/LPS for 24 hours. Subsequently, we performed 10×genomics single-cell RNA-sequencing and found that TIR-based CARs contributed to the proinflammatory state of CAR-iMACs in a CAR- and antigen-dependent manner when encountering tumor cells and improved immune activity against target tumor cells.