ALK-mRNA疫苗作为ALK+癌症的新型免疫疗法

Anaplastic lymphoma kinase (ALK)-driven cancers, including ALK⁺ lymphoma and non-small cell lung cancer (NSCLC), are treated with ALK tyrosine kinase inhibitors (TKIs), yet resistance and relapse remain major challenges. Vaccine-based immunotherapy has emerged as a promising approach. Our group previously developed DNA- and peptide-based vaccines that extended survival in NSCLC mouse model. However, their clinical translation is limited. The success of SARS-CoV-2 lipid nanoparticle (LNP)-mRNA vaccines has generated interest in this platform, demonstrating effective antigen delivery and strong T-cell activation. These findings support the evaluation of a new ALK-mRNA vaccination and the direct comparison with the peptide-based strategy. We designed a codon-optimized ALK cytoplasmic domain (exons 20-29) mRNA and formulated it with LNPs using Moderna technology as standard (F#1). Two additional ionizable lipids (F#2 and F#3) were tested to assess delivery efficiency. In vitro, ALK protein expression and immunogenic peptide presentation was evaluated in 293T cells (1 µg single dose) in 293T HLA-B*07:02 cells using ALK.TCR-T cell killing assay. In vivo , 1 or 10 µg ALK-mRNA or peptide vaccine was administered intramuscularly or subcutis, respectively, in three biweekly doses in BALB/c mice. ALK-specific CD8 + and CD4 + response, T cell immunophenotypes, and anti-tumor activity were evaluated. Moreover, we tested ALK peptide presentation upon ALK-mRNA vaccination in HLA-B*07:02 transgenic model. We demonstrate that the ALK-mRNA vaccine enables the delivery of the expected ALK protein (~65 kDa) without detectable phosphorylation, and antigen presentation was confirmed using an ALK.TCR-T cell killing assay in HLA-B*07:02 cells in vitro . In vivo , 1 or 10 µg ALK-mRNA vaccinations induced robust anti-ALK-specific CD8⁺ T cell responses (40-60%) against immunogenic ALK portion PGPGRVAKI in BALB/c mice, markedly surpassing peptide-based vaccines, particularly with the F#1. The 10-µg dose further elicited a CD4⁺ ALK-specific response by ELISPOT and promoted CD4⁺ and CD8⁺ T cell expansion with increased effector CD44⁺CD62⁻ expression. Notably, both ALK-mRNA doses slowed tumor growth and extended survival in a preventive low MHC-I ALK⁺ lung syngeneic tumor model. Additionally, ALK-mRNA vaccine elicited superior anti-ALK RPRPSQPSSL -CD8 + responses in HLA-B*07:02 mice compared to the peptide vaccine. No adverse effects were observed. Here, we identified F#1 as the best formulation and demonstrated that ALK-mRNA vaccine induces ALK-specific CD8 + and CD4 + strong, non-toxic immune response with an effector immunophenotype in vivo . In a preventive setting, ALK vaccination delayed tumor growth in a lung MHC-I-low ALK + model. Overall, these results outperformed the peptide vaccine and supported further investigation of the ALK-mRNA vaccine in therapeutic settings within ALK + malignancies for a future Phase I/II clinical trial.