First-in-class anaplastic lymphoma kinase (ALK)-specific TCR-T cells induce potent and selective antitumor immunity across ALK-driven human cancers

Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes for patients with ALK-rearranged non-small cell lung cancer (NSCLC) and other ALK-positive (ALK+) cancer types. However, the near-universal emergence of resistance underscores an urgent need for new, durable therapeutic strategies. Leveraging our prior identification of the ALK-derived peptide RPRPSQPSSL presented by HLA-B*07:02 (PMID: 37430060), we aimed to develop ALK-specific TCR-engineered T cells (ALK.TCR-T) that can selectively recognize and eliminate ALK-driven tumors. Methods: HLA-B*07:02 transgenic mice were vaccinated with the RPRPSQPSSL peptide. After two priming and two booster injections, CD137 + /CD8 + T cells were sorted and subjected to single-cell sequencing for TCR discovery. The most expanded TCR clonotypes were then cloned and retrovirally transduced into human T cells to generate ALK.TCR-T cells. The anti-tumor activity of ALK.TCR-T cells was evaluated against various models of ALK+ tumors both in vitro and in vivo . Results: From twelve dominant clonotypes, six ALK-specific TCRs were identified. Two (ALK.TCR-1 and ALK.TCR-2) demonstrated >95% ALK-dextramer binding. Notably, ALK.TCR-2 bound the ALK-dextramer also in CD4⁺ T cells, indicating co-receptor-independent peptide-MHC recognition. Both TCRs mediated robust, antigen-specific cytotoxicity against ALK+/HLA-B*07:02+ cell lines, with no activity against mismatched targets. Engineering either EML4;ALK variant 3 or HLA-B*07:02 into non-expressing cells restored potent killing, confirming strict peptide-MHC specificity. In vivo , a single infusion of ALK.TCR-1 or ALK.TCR-2 markedly suppressed tumor growth and extended survival in a metastatic ALK+/HLA-B*07:02+ NSCLC model. Treated animals exhibited near-complete tumor clearance by day 7 post-ALK.TCR-T infusion, while control mice experienced progressive disease. Remarkably, in a metastatic ALK+/HLA-B*07:02+ neuroblastoma model, 70% (7/10) of mice achieved complete, durable responses (>100 days tumor-free) without evidence of on-target/off-tumor or off-target toxicity. Conclusion: We report the first generation of ALK-specific TCR-T cells targeting the naturally presented ALK peptide RPRPSQPSSL in the context of HLA-B*07:02. ALK.TCR-T cells display potency, selectivity, and safety across multiple ALK-driven tumor models, providing a strong rationale for advancing ALK.TCR-T cell therapy into clinical development.