438 Development of ALK TCR-T therapy against ALK-positive human cancers

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Background Although the development of ALK tyrosine kinase inhibitors (TKIs) has significantly improved clinical outcomes in patients with Anaplastic Lymphoma Kinase (ALK)-rearranged tumors, including non-small cell lung cancers (NSCLCs) and anaplastic large cell lymphoma (ALCL), acquired resistance inevitably develops within 2–3 years, limiting the survival of these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes targeting two human ALK immunogenic peptides presented by the human HLA-B*07:02 that we previously identified by mass spectrometry in ALK-positive cell lines and a biopsy of a patient with NSCLCs1 and to develop TCR-T cell therapy for ALK-rearranged cancers. Methods Transgenic mice expressing the human HLA-B*07:02 were vaccinated either with the human ALK peptide RPRPSQPSSL or IVRCIGVSL. Mice received two subcutis priming injections on days 0 and 14, followed by two boosters on days 28 and 56. On day 62 activated CD137(4-1BB)+/CD8+ T cells were sorted and subjected to single-cell sequencing. The expanded TCR clonotypes identified (VDJ count ≥ 4) were then cloned in a retroviral vector that was used to produce ALK TCR-T cells. Results We identified 353 and 742 unique TCR clonotypes from transgenic HLA-B*07:02 mice vaccinated with the human ALK peptides RPRPSQPSSL and IVRCIGVSL respectively. 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 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p < 0.05). Gene set enrichment analysis (GSEA) confirmed the upregulation of pathways of adaptive immune response, including T cell activation and proliferation, IFN-γ signaling, and cytokine release (adjusted p < 0.05). The in vitro functional validation of the expanded TCR clonotypes isolated from transgenic mice vaccinated with the RPRPSQPSSL peptide, revealed efficient binding to RPRPSQPSSL-dextramer (figure 1A). Clonotype 2.2, which showed 94% dextramer binding, did not kill tumor cells that were ALK-negative and HLA-B*07:02-positive, or tumor cells that were ALK-positive and HLA-B*07:02 negative. In contrast, we observed 95% killing activity against ALK-positive and HLA-B*07:02-positive lung and lymphoma human tumor cells (figure 1B). These data prove that clonotype 2.2 recognizes the ALK RPRPSQPSSL peptide specifically presented by HLA-B*07:02 on tumor cells, exerting robust anti-tumor activity. Conclusions Here, we demonstrated that human HLA-B*07:02 restricted TCR-T identified via HLA-B*07:02 transgenic mice vaccination exerted potent anti-tumor activity in vitro. These findings pave the way for the development of ALK TCR-T cell therapy for patients with ALK-rearranged tumors. Reference Mota I, Patrucco E, Mastini C, Mahadevan NR, Thai TC, Bergaggio E, Cheong TC, Leonardi G, Karaca-Atabay E, Campisi M, Poggio T, Menotti M, Ambrogio C, Longo DL, Klaeger S, Keshishian H, Sztupinszki ZM, Szallasi Z, Keskin DB, Duke-Cohan JS, Reinhold B, Carr SA, Wu CJ, Moynihan KD, Irvine DJ, Barbie DA, Reinherz EL, Voena C, Awad MM, Blasco EB, Chiarle R. ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer. Nat Cancer 2023;4:1016–1035.

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