PROJECT SUMMARY About 5% of non-small cell lung cancers (NSCLCs) are driven by chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. ALK-positive NSCLC is highly sensitive to ALK tyrosine kinase inhibitors (TKIs), and there are currently five FDA-approved ALK TKIs (crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib) for clinical use. While patients initially benefit from these ALK-targeting therapies, essentially all cancers will develop drug resistance. Upon relapse, standard cytotoxic chemotherapy has modest activity and immune checkpoint inhibitors (ICIs) do not provide any substantial benefit despite evidence that the expression of ALK by tumor cells induces a spontaneous immune response in patients. In preclinical mouse models, we demonstrated that the spontaneous ALK-specific immune response generated by ALK-driven lung cancers is insufficient to build an efficacious anti-tumor response. However, a vaccine that potentiates ALK-specific immune responses in mouse models can achieve a significant therapeutic effect including complete cure. Building on these studies, we propose to develop the first therapeutic ALK vaccine for clinical use in patients with advanced ALK+ NSCLC. To achieve this goal, through immunoproteomic analysis, we have identified the precise ALK peptides that are processed by tumor cells and presented by two major MHC class I molecules in ALK-driven cell lines. We confirmed that these peptides are immunogenic in transgenic mice, patients, and healthy donors. These validated ALK peptides will be included in the formulation of an ALK vaccine for clinical use together with a novel highly potent vaccine adjuvant. The clinical trial will also be supported with additional funding from industry and foundation grants. Leveraging these data and support, we will launch a first-in-human Phase I clinical trial to test the efficacy of the ALK vaccine in NSCLCs that have progressed after treatment with ALK TKIs. In this trial, the safety, tolerability, and efficacy of the ALK vaccine will be tested in two cohorts of patients expressing either HLA-A*0201 or HLA-B*0702 MHC-I molecules. The ALK vaccine will be added to ALK TKIs (cohort 1) or administered together with an ICI (cohort 2) at the time of acquired TKI resistance. Circulating and intratumoral immune responses to the ALK vaccine will be determined in treated patients. Potential mechanisms of escape will be also studied in patients and mouse models. Finally, a discovery effort will allow the identification of ALK peptides presented on additional MHC class I molecules to support a future design of a vaccine with broader application to patients with different MHC-I molecules. Through this project we expect to develop the first cancer vaccine to treat ALK-driven lung cancers that could be eventually use to treat any ALK-driven tumor.