{
  "abstract": "Background Tyrosine kinase inhibitors (TKIs) represent the standard treatment for anaplastic lymphoma kinase (ALK)-driven malignancies, including ALK + lymphoma and non-small cell lung cancer (NSCLC); however, resistance and relapse remain major challenges.1 Immunotherapy vaccine-based strategies have been emerging as a powerful option for cancer treatment. Our group developed DNA-and peptide-based vaccines against the ALK oncoantigen. These ALK vaccine formulations cured or significantly extended survival in mouse models.2–4 However, DNA and peptide vaccines have shown limited clinical efficacy. The success of SARS-CoV-2 lipid nanoparticle (LNP)-mRNA vaccines has generate interest in this platform, with early studies showing tumor-specific antigen delivery and T cell activation in solid tumors.5–7 Here, we aimed to assess the immunogenicity of mRNA and peptide vaccines to the same ALK target epitope.Methods We designed a codon-optimized ALK cytoplasmic domain (exons 20–29) mRNA and formulated it with LNPs using Moderna mRNA-1273 technology as standard (F#1). Two additional ionizable lipids—F#2 and F#3—were tested to assess delivery efficiency ( figure 1A). ALK protein expression was evaluated in 293T cells (1 µg single dose). In vitro, ALK.TCR-T cell killing assay was performed to investigate ALK immunogenic peptide presentation in WaGa HLA-B*07:02 cells after IFN-γ-induced HLA stimulation.8 In vivo, 1 or 10 µg ALK-mRNA or peptide vaccines were administrated respectively intramuscular or subcutis in three biweekly doses in BALB/c mice and dextramer/ALKPGPRVAKI peptide CD8+ response and T cell immunophenotypes were analyzed by FACS (figure 2A).Results We demonstrated that ALK-mRNA vaccines are efficiently expressed and processed into immunogenic peptides. In vitro delivery produced the expected ALK protein (~65 kDa), with no detectable phosphorylation (figure 1B). Antigen presentation was validated by specific ALK.TCR-T cell killing assay that recognizes HLA-B*07:02 (figure 1C-F). In vivo, administration of either 1 or 10 µg doses of ALK-mRNA vaccines resulted in up to 60% of ALKPGPRVAKI-specific CD8+ T cell responses in the peripheral blood, outperforming peptide vaccines (~12%). Notably, 10 µg mRNA vaccination resulted in peripheral CD4+CD8+ T cell expansion with increased CD44+CD62-, indicating effector commitment. There were no adverse effects observed following vaccination (figure 2D-F).Conclusions We demonstrated that ALK-mRNA vaccines strongly express ALK. Furthermore, in vitro ALK immunogenic peptides presentation was confirmed. In vivo, we discovered the best ALK-mRNA formulation that is capable of eliciting a stronger, non-toxic CD8+ ALK-specific response with an effector immunophenotype, largely outperforming the peptide vaccine. Overall, these results support further investigation of the ALK-mRNA vaccine in clinical trials against ALK+ tumors.Acknowledgements We thank the philanthropic donations that support this project. G.S. is funded by a fellowship from the American Italian Cancer Foundation.References Voena C , Ambrogio C, Iannelli F, Chiarle R. ALK in cancer: from function to therapeutic targeting. Nat Rev Cancer. 2025,doi:10.1038/s41568-025-00797-9.Chiarle R, Martinengo C, Mastini C, Ambrogio C, D’Escamard V, Forni G, Inghirami G. The anaplastic lymphoma kinase is an effective oncoantigen for lymphoma vaccination. Nat Med. 2008;14:676–680. doi:10.1038/nm1769.Voena C, Menotti M, Mastini C, Di Giacomo F, Longo DL, Castella B, Merlo MEB, Ambrogio C, Wang Q, Minero VG, et al. Efficacy of a cancer vaccine against ALK-rearranged lung tumors. Cancer Immunol Res. 2015;3:1333–1343.doi:10.1158/2326-6066.CIR-15-0089Mota I, Patrucco E, Mastini C, Mahadevan NR, Thai TC, Bergaggio E, Cheong TC, Leonardi G, Karaca-Atabay E, Campisi M, et al. ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer. Nat Cancer. 2023;4:1016–1035. doi:10.1038/s43018-023-00591-2Fang E, Liu X, Li M, Zhang Z, Song L, Zhu B, Wu X, Liu J, Zhao D, Li Y. Advances in COVID-19 mRNA vaccine development. Signal Transduct Target Ther. 2022;7:94. doi:10.1038/s41392-022-00950-yWeber JS, Carlino MS, Khattak A, Meniawy T, Ansstas G, Taylor MH, Kim KB, McKean M, Long GV, Sullivan RJ, et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. In Lancet; © 2024 Elsevier Ltd: England, 2024;403:632–644.Yaremenko AV, Khan MM, Zhen X, Tang Y, Tao W. Clinical advances of mRNA vaccines for cancer immunotherapy. Med. 2025;6:100562. doi:10.1016/j.medj.2024.11.015.Mecca C , Piane S, Azambuja A , et al. Development of ALK TCR-T therapy against ALK-positive human cancers. 2024;12. doi:10.1136/jitc-2024-SITC2024.0438.Ethics Approval All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston children’s Hospital animal facility- Protocol ID 00002434- and conducted in accordance with ethical guidelines, adhering to the principles of the 3Rs (Replacement, Reduction, and Refinement) to ensure humane and responsible use of mice.Abstract 1297 Figure 1ALK mRNA vaccine in vitro validation. A) ALK-mRNA vaccine scheme design. B) ALK(D5F3) and pALK(1278) western blot after vaccine delivery in 293T cells.C)ALK (D5F3) western blot in WaGa cells. D) HLA-B*07 FACS expression in WaGa cell line. E-F) ALK.TCR killing assay on WaGa cell line. E:T =1:1Abstract 1297 Figure 2ALK mRNA in vivo vaccine administration induces a T cell response in BALB/c mouse model. A) ALK vaccination schedule. B-C) Quantification and FACS of CD8+Dextramer+ cells after vaccine administration at 1 and 10 µg doses.Mean ± SD. D)Mice weight (g).E-F) PB CD4+ and CD8+ quantification and immunophenotype characterization-CD44 and CD62L",
  "authors": [
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA"
      ],
      "name": "Gabriele Saccu"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA",
        "University of Torino, Turin, Italy"
      ],
      "name": "Alessandro Gasparetto"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA",
        "Harvard Cancer Center, Boston, MA, USA"
      ],
      "name": "Carmen Mecca"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA"
      ],
      "name": "Elisa Bergaggio"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA"
      ],
      "name": "Taek Chin Cheong"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA",
        "European Institute of Oncology, Milano, Italy"
      ],
      "name": "Simone Piane"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA"
      ],
      "name": "Nirmala Tilija Pun"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA"
      ],
      "name": "Phuc Bao Chi Nguyen"
    },
    {
      "affiliations": [
        "Boston Children’s Hospital, Boston, MA, USA",
        "University of Torino, Turin, Italy",
        "European Institute of Oncology, Milano, Italy"
      ],
      "name": "Roberto Chiarle"
    }
  ],
  "title": "1297 ALK-mRNA vaccine as a new immunotherapy for ALK+ cancers",
  "uid": "82d675a4-452d-5d51-844d-4771b604d48c"
}