ALK表达可识别出一类可治疗性靶向的Merkel细胞癌亚群，其对单用ALK.CAR-T细胞疗法或联合ALK抑制剂有反应

Background: Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroendocrine skin cancer with limited treatment options. While chemo-immunotherapy remains the standard of care, nearly 50% of patients experience disease progression, with a median overall survival of 10 months. Anaplastic lymphoma kinase (ALK) has emerged as a promising therapeutic target in various malignancies, including MCC, where it is detected in 40-80% of cases. Our group recently developed novel chimeric antigen receptor T-cell against ALK (ALK.CAR-T), demonstrating potent anti-tumor activity against ALK + neuroblastoma. This ALK.CAR-T product is currently under clinical investigation in a Phase I/II clinical trial (NCT06803875). In the present study, we evaluated the relevance of ALK.CAR-T immunotherapy in ALK + MCC tumors. Methods: We tested ALK and Merkel cell polyomavirus antigen (MCPyV) expression in a cohort of 181 patients with MCC using immunohistochemistry. In addition, we characterized a panel of MCC human cell lines and two patient-derived xenografts (PDX) to identify potential immunotherapeutic targets (ALK, GD2, and HLA-I). ALK.CAR-T cells were co-cultured either in 2D with MCC cell lines or in 3D-microfluidic devices with xenograft-derived organotypic tumor spheroids (xDOTS) to assess their killing efficacy in vitro , either alone or in combination with ALK inhibitors. For in vivo testing, NSG mice were injected intravenously with human MCC cell lines and, after engraftment, treated with ALK.CAR-T cells, either alone or in combination with the ALK inhibitor lorlatinib. Tumor monitoring was performed weekly using IVIS imaging. Results: We found ALK expression in approximately 90% of MCC tumors and observed a significant association between ALK expression and poor clinical outcomes. In vitro , ALK.CAR-T treatment effectively killed ALK + MCC cell lines and xDOTS, associated with an increased production of IFNgamma and granzyme B. Moreover, the combination with an ALK inhibitor (lorlatinib or nadelalkib) enhanced ALK.CAR-T antitumor activity, even when the ALK inhibitor showed no effect alone. In vivo , ALK.CAR-T outperformed GD2.CAR-T, extending the overall survival of mice in two MCC metastatic models. Nonetheless, ALK.CAR-T efficacy was enhanced by lorlatinib, improving tumor control without altering the safety profile. Conclusions: We identified ALK as a clinically relevant target in MCC and demonstrated that ALK.CAR-T cells, especially when combined with ALK inhibitors, have a potent anti-tumor activity against ALK + MCC. Our study provides a preclinical rationale for expanding the ongoing Phase I/II clinical trial in relapsed/refractory neuroblastoma patients to include patients with MCC, thus broadening the therapeutic indication of ALK.CAR-T.