Endogenous CD4 T cells that recognize ALK and the NPM1::ALK fusion protein can be expanded from human peripheral blood

Abstract Anaplastic lymphoma kinase (ALK) fusion proteins resulting from chromosomal rearrangements are promising targets for cancer immunotherapy. Although ALK-specific CD8+ T cells and epitopes presented on MHC class I have been identified in patients with ALK-positive malignancies, little is known about ALK-specific CD4+ T cells. We screened peripheral blood of 10 patients with ALK-positive anaplastic large-cell lymphoma in remission and six healthy donors for CD4+ T-cell responses to the whole ALK fusion protein, nucleophosmin 1 (NPM1)::ALK. ALK-specific CD4+ T cells were detected in 15 individuals after stimulation with autologous dendritic cells pulsed with long-overlapping ALK peptide pools. CD4+ T-cell epitopes were predominantly located within three specific regions (p102-188, p257-356, and p593-680) in the ALK portion of the fusion protein. We detected CD4+ T cells in one patient that recognized the NPM1::ALK fusion neoepitope and identified a corresponding T-cell receptor (TCR) by TCRαβ single-cell sequencing. The NPM1::ALK fusion–specific TCR was HLA-DR13–restricted and conferred antigen specificity when expressed in a TCR− reporter cell line (58α−β−). Together, our data provide evidence of ALK-specific CD4+ T cells in human peripheral blood, describe target epitopes in patients, and support the consideration of CD4+ T cells in the development of ALK-specific immunotherapies.