Sun Shiyu, Wang Xiaojia, Chen Yongchen, Liang Ziwei, Nian Zuomin, Xu Wanni, Wang Wenzhou, Yan Li, Wu Fei, Wu Huizi, Jia Yiwei, Zhang Lu, Zhang Shuqun, Xu Yang, Ma Xingcong
The Comprehensive Breast Care Center, Xi'an Jiaotong University Second Affiliated Hospital, Xi'an, Shaanxi, China.
Department of Human Cellular Biology and Genetics, Southern University of Science and Technology, Shenzhen, Guangdong, China.
J Immunother Cancer. 2025 Sep 3;13(9):e012442. doi: 10.1136/jitc-2025-012442.
Triple-negative breast cancer (TNBC) represents a subtype of breast cancer with poorest prognosis due to limited effective targeted therapies. Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating hematological cancers, but its application in TNBC requires further development. One major obstacle is the lack of suitable tumor-specific target in TNBC. Inspired by recent success of trophoblast cell-surface antigen 2 (TROP2) antibody-drug conjugate in TNBC, we developed a second-generation CAR that specifically targets TROP2 and formally evaluated its antitumor activity and safety profile using in vitro and in vivo models.
A CAR molecule targeting TROP2 was constructed based on the clinically-validated humanized antibody Sacituzumab and expressed in primary human T cells using a retroviral vector. Tumor cytotoxicity, cytokine production and T-cell proliferation of TROP2 CAR-T cells were tested against multiple TNBC cell lines in vitro. Antitumor efficacy was evaluated using orthotopic and metastatic models of cell line-derived xenograft in NSG mice and in patient-derived xenograft (PDX) model. The safety profile of TROP2 CAR-T cells was assessed using TROP2-humanized immunocompetent mice and an "AND"-logic gated SynNotch CAR targeting B7-H3 and TROP2 was engineered to minimize off-tumor, on-target toxicity of TROP2 CAR-T cells.
Human TROP2 CAR-T cells demonstrated robust antitumor activity in vitro and in orthotopic/metastatic/PDX xenograft mouse models. TROP2 CAR-T cells caused lethal on-target, off-tumor toxicity in TROP2-humanized immunocompetent mice, causing severe tissue damage in lungs and systemic inflammation. The B7-H3/TROP2 "AND"-logic gated SynNotch CAR-T cells showed comparable antitumor efficacy without causing apparent adverse effects as in TROP2 CAR-T cells.
These data indicate that while CAR-T therapy targeting TROP2 possesses potent antitumor activity against TNBC cell lines and PDX, its potential side effects could be lethal due to TROP2 expression in vital organs such as the lung. Using an "AND"-logic gated CAR is a viable solution to overcome its in vivo toxicity. Our study lays the groundwork for future development of TROP2 CAR-T cell therapy for TNBC.
三阴性乳腺癌（TNBC）是乳腺癌的一种亚型，由于有效的靶向治疗有限，其预后最差。嵌合抗原受体T细胞（CAR-T）疗法在治疗血液系统癌症方面已显示出显著疗效，但其在TNBC中的应用需要进一步发展。一个主要障碍是TNBC中缺乏合适的肿瘤特异性靶点。受滋养层细胞表面抗原2（TROP2）抗体药物偶联物近期在TNBC中取得成功的启发，我们开发了一种特异性靶向TROP2的第二代CAR，并使用体外和体内模型正式评估了其抗肿瘤活性和安全性。
基于临床验证的人源化抗体赛托珠单抗构建靶向TROP2的CAR分子，并使用逆转录病毒载体在原代人T细胞中表达。在体外针对多种TNBC细胞系测试TROP2 CAR-T细胞的肿瘤细胞毒性、细胞因子产生和T细胞增殖。使用NSG小鼠的原位和转移细胞系来源异种移植模型以及患者来源异种移植（PDX）模型评估抗肿瘤疗效。使用TROP2人源化免疫活性小鼠评估TROP2 CAR-T细胞的安全性，并设计一种靶向B7-H3和TROP2的“与”逻辑门控SynNotch CAR，以尽量减少TROP2 CAR-T细胞的肿瘤外、靶点上毒性。
人TROP2 CAR-T细胞在体外以及原位/转移/PDX异种移植小鼠模型中表现出强大的抗肿瘤活性。TROP2 CAR-T细胞在TROP2人源化免疫活性小鼠中引起致死性的靶点上、肿瘤外毒性，导致肺部严重组织损伤和全身炎症。B7-H3/TROP2“与”逻辑门控SynNotch CAR-T细胞显示出相当的抗肿瘤疗效，且不会像TROP2 CAR-T细胞那样引起明显的不良反应。
这些数据表明，虽然靶向TROP2的CAR-T疗法对TNBC细胞系和PDX具有强大的抗肿瘤活性，但其潜在的副作用可能是致命的，因为TROP2在肺等重要器官中表达。使用“与”逻辑门控CAR是克服其体内毒性的可行解决方案。我们的研究为未来TNBC的TROP2 CAR-T细胞疗法的发展奠定了基础。
