HLA class I-specific nucleolin peptides induce therapeutic T cells in triple-negative breast cancer patients

Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis and limited treatment options owing to the lack of expression of common therapeutic targets such as hormone receptors and HER2. Recent advances in immunopeptidomics have enabled the identification of tumor-associated antigen peptides that can elicit tumor-specific immune responses. We identified four novel HLA class I-restricted peptides derived from nucleolin (NCL), a protein overexpressed in TNBC that is associated with poor outcomes. We predicted and validated pNCL-01 through pNCL-04 because of its strong binding affinity to common HLA alleles (A*02:01 and B*15:01), with pNCL-04 demonstrating broad HLA-binding potential across 20 HLA alleles. Peripheral blood mononuclear cells (PBMCs) from both healthy donors and patients with TNBC were pulsed with these peptides, generating NCL-specific CD8⁺ T cells that effectively targeted and killed NCL⁺/PD-L1⁺ TNBC cell lines in vitro (MDA-MB-231 and HCC70). These T cells exhibited robust IFN-γ secretion and expressed effector memory markers (CD107a), particularly in cancer tissues of patients with high NCL expression. Notably, pNCL-04 functioned as a shared immunogenic peptide, activating T cells in the majority of TNBC patients (7/10) which was predicted to bind up to 20 different HLA class I alleles, supporting its use as a broadly applicable immunogenic peptide. Furthermore, the combination of NCL-specific T cells with atezolizumab enhanced anti-tumor activity, suggesting the potential benefit of combining NCL-targeted immunotherapy with immune checkpoint blockade. In conclusion, this study demonstrates the potential of HLA-restricted NCL peptides in combination with atezolizumab as a rational peptide-based therapeutic strategy for clinical translation in TNBC patients with high NCL.