[摘 要] 目的：开发靶向成纤维细胞活化蛋白（FAP）的CAR-T细胞，评价其通过重塑肿瘤微环境（TME）抑制乳腺癌生长的能力。方法：将识别FAP不同抗原表位的纳米抗体VHH1和VHH2串联，设计两种胞外识别区排列顺序不同的FAP-CAR1（VHH1- Linker-VHH2）和FAP-CAR2（VHH2-Linker-VHH1），构建以二价FAP抗体为胞外识别域的CAR，将其分别克隆至逆转录病毒载体并完成病毒包装。分离小鼠淋巴细胞，经抗CD3/CD28抗体激活后进行转导，制备FAP-CAR1-T和FAP-CAR2-T细胞。通过实时无标记动态细胞分析（RTCA）、流式细胞术（FCM）评估FAP-CAR-T细胞的靶向杀伤活性，ELISA检测其激活后IL-2分泌水平。 分别用FAP-CAR1-T细胞（FAP-CAR-T组）、HER2-CAR-T细胞（HER2-CAR-T组）及未转导T细胞（Mock-T组）治疗4T1-HER2荷瘤小鼠，分析FAP-CAR-T对肿瘤生长的抑制作用，并通过免疫组织化学（IHC）检测肿瘤组织中T细胞浸润情况。进一步通过转录组分析差异表达基因。结果：FAP-CAR1-T与FAP-CAR2-T细胞体外扩增稳定，CAR阳性率 > 60%。两者均能特异、高效杀伤过表达鼠源FAP的小鼠成纤维细胞NIH3T3-mFAP，且FAP-CAR1-T细胞在扩增和分泌IL-2方面优于FAP-CAR2-T细胞，因此选用FAP-CAR1-T细胞进行后续体内实验。在小鼠4T1-HER2皮下移植瘤模型中，FAP-CAR-T治疗组小鼠肿瘤生长速度显著低于Mock-T组（P < 0.05），与HER2-CAR-T组肿瘤生长速度相当（P > 0.05）；与Mock-T组相比，FAP-CAR-T治疗组小鼠肿瘤组织中CD3 + T细胞浸润增加，胶原沉积面积减少（P < 0.001或P < 0.01）。转录组分析结果显示，FAP-CAR-T组相较Mock-T组的差异表达基因富集于细胞外基质、免疫应答等GO条目及ECM-受体相互作用等KEGG通路。结论：FAP-CAR-T细胞通过特异性靶向FAP相关基质细胞，重塑肿瘤微环境，显著抑制乳腺癌生长，且在肿瘤体积控制上与HER2-CAR-T细胞相比无统计学差异。

[Abstract] Objective: To develop CAR-T cells targeting fibroblast activation protein (FAP) expressed by cancer-associated fibroblasts (CAFs) and evaluate their ability to inhibit breast cancer growth by remodeling the tumor microenvironment (TME), providing a new candidate strategy for breast cancer CAR-T cell therapy. Methods: Nanobodies VHH1 and VHH2, which recognize distinct antigenic epitopes of FAP, were linked in tandem to generate a bivalent FAP antibody and to construct two CARs with different extracellular domain arrangements: FAP-CAR1 (VHH1-Linker-VHH2) and FAP-CAR2 (VHH2-Linker-VHH1). The resulting CAR constructs, containing the bivalent FAP antibody as the extracellular antigen-recognition domain, were individually cloned into retroviral vectors and packaged into viral particles. Murine lymphocytes were isolated and activated with anti-CD3/CD28 antibodies, followed by transduction to generate FAP-CAR1-T and FAP-CAR2-T cells. Targeted cytotoxicity of FAP-CAR-T cells was evaluated using real- time label-free dynamic cell analysis (RTCA) and flow cytometry (FCM), and IL-2 secretion was measured by ELISA. In a mouse 4T1- HER2 subcutaneous tumor model, the inhibitory effects of FAP-CAR-T, HER2-CAR-T, and untransduced T cells (Mock-T) on tumor · · 611 [[PAGE_INDEX=24 FILE=202606最新正文_1-30.pdf]] 中国肿瘤生物治疗杂志, 2026, 33(6) growth were compared. Immunohistochemistry (IHC) was used to assess T cell infiltration in tumor tissues, and RNA sequencing was performed to analyze differentially expressed genes. Results: FAP-CAR1-T and FAP-CAR2-T cells expanded stably in vitro, with CAR positivity exceeding 60%. Both CAR-T cells specifically and efficiently killed NIH3T3 cells overexpressing mouse FAP (NIH3T3- mFAP), with FAP-CAR1-T showing superior expansion and IL-2 secretion compared to FAP-CAR2-T. Based on these results, FAP- CAR1-T was selected for subsequent in vivo experiments. In the 4T1-HER2 subcutaneous tumor model, FAP-CAR-T treatment significantly slowed tumor growth compared to the Mock-T group (P < 0.05), with no significant difference compared to the HER2- CAR-T group (P > 0.05). Compared to the Mock-T group, FAP-CAR-T treatment increased CD3 + T cell infiltration and reduced collagen deposition areas in tumor tissues. Transcriptomic analysis revealed that differentially expressed genes in the FAP-CAR-T group, compared to the Mock-T group (|log2FC| ≥1, P < 0.05), were primarily enriched in pathways related to extracellular matrix remodeling, immune response, and ECM-receptor interaction. Conclusion: FAP-CAR-T cells specifically target FAP-expressing stromal cells, remodel the tumor microenvironment, and significantly inhibit breast cancer growth. In terms of tumor volume control, FAP-CAR-T cells did not differ significantly from HER2-CAR-T cells, suggesting their potential as a candidate CAR-T approach for breast cancer.

[基金项目] 国家自然科学基金（82403846，82471378）