Abstract 21: Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency <4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p <0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p <0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 21.