当前，嵌合抗原受体T细胞疗法（Chimeric antigen receptor-T cell，CAR-T）在B细胞相关的恶性肿瘤（如白血病、淋巴瘤和骨髓瘤）临床治疗中表现卓著，也进而促成了CAR-T疗法在实体瘤治疗领域广泛的临床前研究。然而，截止到目前CAR-T治疗实体肿瘤效果仍然十分有限，面临多重阻碍，例如瘤内CAR-T浸润数量不足、制备中自体T细胞难以获得、CAR-T在肿瘤微环境（Tumor microenvironment，TME）内难以维持抗肿瘤活性等。当然CAR-T、CAR-NK等淋巴细胞疗法自身也在不断优化，但基于现有技术限制，改造空间较小，实质性研究进展缓慢。基于上述研究背景，嵌合抗原受体巨噬细胞或单核细胞疗法（Chimeric antigen receptor-macrophage/monocyte，CAR-M/Mo）应势而生，不同于淋巴细胞，来源于髓系细胞的生物学特性，使得CAR-M/Mo细胞疗法天生具备独特的抗癌潜力，例如自体单核细胞数量相对充足、组织浸润能力强、功能高度可塑，易于适应肿瘤微环境，维持细胞活性及抗癌活性等。

       嵌合抗原受体是人为构建的融合蛋白分子，可赋予修饰后的免疫细胞特异性识别、攻击肿瘤细胞的免疫功能，因此，巨噬细胞的吞噬能力与嵌合抗原受体十分契合。目前已有的CAR-M/Mo细胞疗法也集中于对吞噬活性的开发，其胞内信号域主要为抗原抗体复合物受体FCR通用g链或其同源蛋白CD3z，相应产品也进入了临床试验。值得注意的是，当前CAR-M/Mo细胞疗法已开发的吞噬活性及抗肿瘤活性有限，实体瘤治疗表现并不出色。但是，众所周知，巨噬细胞是机体的专职吞噬细胞，它们拥有复杂多样的吞噬受体网，而当前的CAR-M/Mo设计还不能高效调动肿瘤吞噬作用，吞噬功能尚未被深度开发。因此本研究以探索CAR-M/Mo更为强效的吞噬免疫力出发，进一步升级CAR分子设计。

       在本研究中，我们首先构建并优化了人单核细胞解螺旋DNA电转导技术平台，用以构建人源CAR-Mo细胞，体外转导效率可达50%以上。进一步，我们从由FCR-g链和补体受体、整合素、凝集素受体、清道夫受体、胞葬受体家族组成的CAR胞内信号域组合中筛选发现，其中FCR-g链+胞葬受体组成的胞内信号域所调动的CAR-Mo（CAR-BAI和CAR-MER）细胞靶向肝癌、乳腺癌细胞的吞噬效率最高，相较于胞内信号域仅包含FCR-g链的初代现行CAR-M/Mo设计，进一步增强了人单核细胞的吞噬活性。在靶点的选择上，上述CAR-Mo细胞均特异性识别新肿瘤抗原-核仁素蛋白（Nucleolin，NCL）。前期研究发现，在人健康细胞中，NCL只存在于细胞核及细胞质中，而在人肝癌、乳腺癌、子宫内膜癌中，NCL可以异位表达在肿瘤细胞表面，作为靶点，有助于提升细胞疗法的安全性和特异性。

       其次，体外验证发现FCR-g链+胞葬受体所调动的高效吞噬活性来源于这两种吞噬受体合作的协同信号，缺一不可。不论是靶向新抗原NCL或是靶向经典抗原HER2，CAR-BAI和CAR-MER（二者胞内信号域为 FCR-g链+胞葬受体）细胞均发挥了针对肝癌SK-Hep1、乳腺癌MDA-MB-231或卵巢癌SKOV3细胞的高效吞噬及清除功能。相应地，靶向人肝癌、卵巢癌异位肿瘤模型，回输入体的CAR-BAI和CAR-MER亦展现了高效的抗肿瘤能力，肿瘤抑制率保持在40%左右。进一步，基于在多类吞噬受体信号域组合中必须具有FCR-g链+胞葬受体的组合才可协同高效调动人单核细胞吞噬活性，因此为寻求具备更强吞噬免疫力的CAR-Mo设计，我们正交设计了多种由FCR-g链、胞葬受体（BAI1、MERTK）信号域组成的双嵌合抗原受体单核细胞（Dual-CAR-Mo）。经体外实验探究发现，其中胞内信号域由FCR-g链-BAI1+ FCR-g链-MERTK组成的Dual-CAR-Mo拥有最高的肿瘤吞噬和清除活性，优于上述初级设计CAR-BAI和CAR-MER。同样地，当靶向人单靶点或双靶点肝癌、卵巢癌异位肿瘤模型时，该Dual-CAR-Mo细胞通过瘤内或静脉注射入体后都可发挥远强于CAR-BAI和CAR-MER的抗肿瘤活性，肿瘤抑制率提升至60%-90%。此外，我们观测了上述Dual-CAR-Mo的安全性，荷瘤小鼠在治疗期间未见毒副反应，脏器重量、形态、结构正常。

       最后，我们通过转录组分析，从机制层面进一步阐述Dual-CAR-Mo免疫表型。与对照细胞CAR-MCS的差异基因（富集）分析指出，活化Dual-CAR-Mo细胞胞内吞噬信号上调激活，不吃我信号全面下调，在分子层面阐明Dual-CAR-Mo强效吞噬活性来源。此外，活化Dual-CAR-Mo细胞中与巨噬细胞抗癌免疫相关的代谢通路上调，伴随免疫抑制因子的下调及细胞毒效应分子的上调，共同表明其抗肿瘤免疫表型活化，结合强效的吞噬活性及肿瘤浸润性，有望在临床试验或应用中表现出较好的抗实体肿瘤的效果。

       总结本研究结果，我们认为Dual-CAR-Mo（FCR-g链-BAI1+ FCR-g链-MERTK）可高效调动人单核巨噬细胞多效抗肿瘤免疫力，相较现有设计，更具备疗效优势，利于靶向治疗实体瘤，具有极大的应用潜力。

Currently, chimeric antigen receptor T cell therapy (CAR-T) has demonstrated remarkable clinical efficacy in treating B-cell-associated malignancies, including leukemia, lymphoma, and myeloma. Additionally, it has spurred extensive preclinical research into the application of CAR-T therapy for solid tumors. However, the effectiveness of CAR-T therapy for solid tumors remains limited due to several challenges, such as inadequate CAR-T infiltration within tumors, difficulties in obtaining autologous T cells for preparation, and the challenge of sustaining CAR-T anti-tumor activity in the tumor microenvironment (TME). While CAR-T, CAR-NK, and other lymphocyte therapies are continually being optimized, existing technological limitations restrict significant transformation, resulting in slow progress in substantive research. In light of this background, the development of chimeric antigen receptor macrophage or monocytic cell therapy (CAR-M/Mo) emerges as a natural response. Unlike lymphocytes, CAR-M/Mo therapy leverages the inherent biological characteristics of myeloid cells, providing unique anti-cancer potential. For instance, autologous monocytes are relatively abundant, possess strong tissue infiltration capabilities, exhibit high functional plasticity, and can readily adapt to the tumor microenvironment, thereby maintaining both cellular activity and anti-cancer efficacy.

Chimeric antigen receptors (CARs) are artificially constructed fusion protein molecules that endow modified immune cells with specific functions to recognize and attack tumor cells. Consequently, the phagocytic ability of macrophages is more compatible with chimeric antigen receptors. Currently, existing CAR-M/Mo cell therapies primarily focus on enhancing phagocytic activity, utilizing the common g chain of the antigen-antibody complex receptor FCR or its homologous protein CD3z as their intracellular signaling domain. Corresponding products have also entered clinical trials. However, it is important to note that the phagocytic and anti-tumor activities developed by current CAR-M/Mo cell therapies are limited, and their efficacy against solid tumors remains suboptimal. Macrophages, as professional phagocytic cells, possess a complex and diverse array of phagocytic receptors. Unfortunately, the current design of CAR-M/Mo has not effectively harnessed the tumor phagocytic function, and the potential for enhancing this phagocytic capability has not been thoroughly explored. Therefore, to investigate the potential for a more robust phagocytic immunity within CAR-M/Mo, this study aims to further refine the molecular design of CAR.

In this study, we first constructed and optimized an uncoiled DNA electrical transduction technology platform to create human CAR-Monocytes, achieving a transduction efficiency exceeding 50% in vitro. Furthermore, we screened a combination of CAR intracellular signal domains, which included the FCR-g chain with complement receptor, integrin, lectin receptor, scavenger receptor, and efferocytic receptor family. Among these, CAR-Mo cells (CAR-BAI and CAR-MER) mobilized by the intracellular signal domain composed of the FCR-g chain and cytochrome receptor exhibited the highest phagocytic efficiency against liver and breast cancer cells. In comparison to the original CAR-M/Mo design, which contained only the FCR-g chain in the intracellular signal domain, the phagocytic activity of human monocytes was further enhanced. Notably, these CAR-Mo cells specifically recognize the novel tumor antigen nucleolin protein (NCL). Previous studies have demonstrated that in healthy human cells, NCL is localized exclusively in the nucleus and cytoplasm; however, in cases of liver cancer, breast cancer, and endometrial cancer, NCL can be ectopically expressed on the surface of tumor cells, serving as a target that enhances the safety and specificity of cell therapy.

In vitro verification revealed that the highly efficient phagocytic activity mobilized by the FCR-g chain and efferocytic receptors stemmed from the cooperative signaling of these two phagocytosis receptors, which was indispensable. Whether targeting the neoantigen NCL or the classical antigen HER2, CAR-BAI and CAR-MER (both of which possess intracellular signaling domains comprising the FCR-g chain and efferocytic receptor) demonstrated effective phagocytosis and clearance against SK-Hep1 cells of liver cancer, MDA-MB-231 cells of breast cancer, and SKOV3 cells of ovarian cancer. Accordingly, in ectopic tumor models of human liver cancer and ovarian cancer, CAR-BAI and CAR-MER also exhibited potent anti-tumor capabilities, with a tumor inhibition rate maintained at approximately 40%. Furthermore, among various combinations of phagocytic receptor signaling domains, only the pairing of the FCR-g chain with the efferocytic receptor could synergistically and efficiently mobilize the phagocytic activity of human monocytes. To enhance the phagocytic immunity of CAR-Mo, we orthogonally designed several Dual-chimeric antigen receptor-monocytes (Dual-CAR-Mo) incorporating FCR-g chain and BAI1, as well as MERTK signaling domains. In vitro experiments indicated that Dual-CAR-Mo featuring an intracellular signaling domain composed of FCR-g chain-BAI1 and FCR-g chain-MERTK exhibited the highest phagocytosis and clearance activity, surpassing the previously designed CAR-BAI and CAR-MER. Similarly, when targeting single- or dual-antigen ectopic tumor models of human liver cancer and ovarian cancer, Dual-CAR-Mo cells demonstrated significantly stronger anti-tumor activity compared to CAR-BAI and CAR-MER following intratumoral or intravenous injection, with the tumor inhibition rate escalating to 60%-90%. Moreover, the treatment was safe, as no toxic side effects were observed in the tumor-bearing mice throughout the study, and both organ weight and morphology remained normal.

And then, we further elucidated the mechanism of the Dual-CAR-Mo immunophenotype through transcriptome analysis. Differential gene enrichment analysis comparing CAR-MCS with control cells revealed an upregulation of intracellular phagocytosis signaling in activated Dual-CAR-Mo cells, “don’t eat me” signals were downregulated as well，which clarified the molecular basis for the robust phagocytic activity observed in Dual-CAR-Mo cells. Furthermore, metabolic pathways associated with macrophage anti-cancer immunity were upregulated in activated Dual-CAR-Mo cells. This was accompanied by a downregulation of immunosuppressive factors and an upregulation of cytotoxic effector molecules, collectively indicating an activation of anti-tumor immunophenotype. The combination of anti-tumor immunophenotype with potent phagocytic activity and tumor infiltration suggested promising outcomes in clinical trials and applications.

In summary, our findings indicate that Dual-CAR-Mo (FCR-g chain-BAI1 + FCR-g chain-MERTK) can effectively harness the pleiotropic immunity of human monocytes, demonstrating significant anti-cancer potential in the targeted treatment of solid tumors, which may offer therapeutic advantages over existing strategies.