肿瘤细胞治疗研究
英文原题：Synergistic antitumor activity of CAR-NK cells combined with CAR-macrophages for pancreatic cancer treatment.
基于纳米抗体的 CAR-M 与 CAR-NK 联合疗法重编程 TME，并在巨噬细胞与 NK 细胞之间建立起由 CXCL9 驱动的前馈环路，从而产生协同的固有免疫激活和强效的肿瘤控制。
**背景：**嵌合抗原受体（CAR）过继细胞疗法在血液系统恶性肿瘤中取得显著成功，但在实体瘤中的疗效仍受限，原因包括免疫抑制性肿瘤微环境（TME）限制免疫细胞浸润和持续存在。CAR-NK细胞具有强细胞毒作用和良好安全性，但进入肿瘤的能力往往不足；相比之下，巨噬细胞具有内在的肿瘤趋向性和组织重塑能力，可能有助于克服这些障碍。 **目的：**本研究旨在开发联合先天免疫细胞疗法，将基于纳米抗体、靶向间皮素（MSLN）的CAR巨噬细胞（CAR-M）与CAR-NK细胞结合，以重塑胰腺癌TME并增强抗肿瘤免疫。 **方法：**研究采用经验证的D3纳米抗体CAR构建体，由PMA诱导分化的THP-1单核细胞制备CAR-M，并评估CAR表达、M1/M2极化、迁移、吞噬及细胞因子分泌。利用CAR-M与肿瘤共培养的条件培养液评估CAR-NK细胞迁移和细胞毒性，并在原位MSLN阳性胰腺导管腺癌（PDAC）模型中评估协同抗肿瘤活性。 **结果：**D3-CAR-M的CAR表达率高（>90%），即使与肿瘤接触后仍维持M1样表型（CD86、HLA-DR阳性，CD204阴性）。其迁移能力和对MSLN阳性PANC-1肿瘤球的浸润增强，吞噬及杀伤肿瘤的活性也提高。抗原接触后，CAR-M分泌大量CXCL9，促进CAR-NK趋化、脱颗粒和细胞毒作用。体内依次给予CAR-M和CAR-NK可显著促使肿瘤消退并产生持久应答，且无全身毒性。 **结论：**联合纳米抗体CAR-M和CAR-NK疗法可重塑TME，并在巨噬细胞与NK细胞之间建立由CXCL9驱动的正反馈环路，从而协同激活先天免疫并有力控制肿瘤。这一策略为靶向MSLN表达实体瘤的新一代CAR免疫疗法提供了有机制依据且具有转化可行性的框架。
BACKGROUND: Adoptive cell therapies employing chimeric antigen receptors (CARs) have achieved remarkable success in hematologic malignancies, yet their efficacy in solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), which restricts immune cell infiltration and persistence. While CAR-NK cells provide potent cytotoxicity with a favorable safety profile, their tumor penetration is often suboptimal. In contrast, macrophages possess intrinsic tumor-homing and tissue-remodeling abilities that could help overcome these barriers. OBJECTIVE: This study aimed to develop a combinatorial innate-cell immunotherapy integrating nanobody-based mesothelin (MSLN)-targeting CAR-macrophages (CAR-Ms) with CAR-NK cells to remodel the TME and enhance antitumor immunity in pancreatic cancer. METHODS: Using a validated D3 nanobody CAR construct, CAR-Ms were generated from PMA-differentiated THP-1 monocytes and characterized for CAR expression, M1/M2 polarization, migration, phagocytosis, and cytokine secretion. CAR-NK cell migration and cytotoxicity were evaluated using conditioned media (CM) from CAR-M/tumor co-cultures. Synergistic antitumor activity was assessed in an orthotopic MSLN pancreatic ductal adenocarcinoma (PDAC) model. RESULTS: D3-CAR-Ms exhibited robust CAR expression (> 90%) and sustained an M1-like phenotype (CD86 HLA-DR CD204 ) even after tumor engagement. Functionally, they displayed enhanced migration and infiltration into MSLN PANC-1 spheroids, along with increased phagocytic and tumoricidal activity. Upon antigen engagement, CAR-Ms secreted high levels of CXCL9, which promoted CAR-NK chemotaxis, degranulation, and cytotoxicity. Sequential administration of CAR-Ms and CAR-NK cells in vivo led to marked tumor regression and durable responses without systemic toxicity. CONCLUSION: Combinatorial nanobody-based CAR-M and CAR-NK therapy reprograms the TME and establishes a CXCL9-driven feed-forward loop between macrophages and NK cells, leading to synergistic innate immune activation and potent tumor control. This strategy provides a mechanistically grounded and translationally feasible framework for next-generation CAR-based immunotherapies targeting MSLN-expressing solid tumors.
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