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Archive > Volume 33 Issue 6 > 2026,33(6):611-618. DOI:10.3872/j.issn.1007-385X.2026.06.003 Prev Next
Basic Research
Preclinical evaluation of FAP-targeted CAR-T cells that inhibit breast cancer growth by remodeling the tumor microenvironment
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Abstract:
[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.
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