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 CD3ζ-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 CD3ζ-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-κB 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. We pretreated EGFRvIII-targeting CAR-iMACs were incubated with U87MGEGFRvIII cells with IFN-γ/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.

嵌合抗原受体（Chimeric antigen receptor, CAR）T细胞疗法在治疗血液系统恶性肿瘤中已展现出卓越成效，但该疗法对实体瘤的治疗效果仍有待进一步优化。近年来，巨噬细胞因其能够浸润实体瘤、吞噬肿瘤细胞并具备免疫调节能力，受到了学界的广泛关注。第一代基于CD3ζ的CAR巨噬细胞研究已证实，CAR可通过肿瘤抗原依赖的方式激活巨噬细胞的吞噬功能。本研究通过基因工程技术，对靶向EGFRvIII与GPC3、携带含TLR4胞内TIR结构域CAR的诱导多能干细胞（induced pluripotent stem cell, iPSC）衍生巨噬细胞（iPSC-derived macrophages, iMACs）进行改造，结果显示该改造后的CAR-iMACs在胶质母细胞瘤与肝细胞癌两种不同实体瘤模型中均表现出显著增强的抗肿瘤活性，其中单独使用CAR-iMACs或联合抗CD47抗体即可实现肿瘤完全缓解。此外，串联CD3ζ-TIR-CAR（即基于TIR结构域的双信号CAR“第二代”设计）可赋予iMACs双重核心功能：其一，能够靶向吞噬表达对应抗原的实体瘤细胞并介导胞葬作用；其二，可通过核因子κB（NF-κB）依赖的途径，实现抗原依赖的M1型极化以及对M2型极化的抵抗。本研究还揭示了一种此前未被报道的肿瘤细胞清除机制：CAR可通过诱导针对肿瘤细胞凋亡小体的胞葬作用实现肿瘤清除。综上，本研究成功构建了新一代CAR-iMACs，其具备正交吞噬活性与极化调控能力，可在实体瘤治疗中发挥更优异的抗肿瘤功能。我们将靶向EGFRvIII的CAR-iMACs进行预处理后，与经干扰素γ/脂多糖（IFN-γ/LPS）孵育24小时的U87MG-EGFRvIII细胞共培养；随后开展10×基因组学单细胞RNA测序，结果发现，当遭遇肿瘤细胞时，基于TIR结构域的CAR可通过CAR与抗原依赖的方式，促进CAR-iMACs向促炎状态转化，并显著增强其对靶肿瘤细胞的免疫活性。