Preclinical Assessment of HLA-A*02:01-Restricted PSMA and STEAP1 Epitopes for Peptide-Based Immunotherapy in Prostate Cancer

Yueting Huang,1,* Yang Yang,2,* Yanni Xie,1,* Yue Shu,2 Siqi Yang,2 Xuezhi Long,1 Zhipeng Wen,1 Xiaolu Duan,1 Li Fu,2,* Di Gu,1 Tuxiong Huang1 1Guangdong Provincial Key Laboratory of Urological Diseases, Department of Urology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Department of Pharmacology and International Cancer Center, Shenzhen University Medical School, Shenzhen, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Tuxiong Huang, Email [email protected] Di Gu, Email [email protected]: Peptide-based immunotherapy targeting tumor-associated antigens presents a promising approach for prostate cancer intervention. In this study, we employed a preclinical approach to evaluate immunogenic epitope candidates derived from STEAP1 and PSMA, two well-characterized prostate cancer-associated antigens.Methods: High-affinity cytotoxic T lymphocyte (CTL) epitopes restricted to HLA-A*02:01 were predicted and evaluated for antigenicity, conservation, and proteasomal processing. Molecular docking with HLA-A*02:01 was performed and peptides were synthesized for experimental validation. In-vitro assays including ELISA-based MHC binding, IFN-γ ELISpot, and intracellular cytokine staining (ICS) were conducted using splenocytes isolated from HLA-A*02:01 transgenic mice.Results: Four peptides (P1, P2, P3 and P4), demonstrated strong binding to HLA-A*02:01 in-silico and were structurally compatible with the MHC class I groove. ELISA confirmed high binding for P3 and P1 at lower concentrations, while P2 and P4 showed moderate affinity. ELISpot assays showed robust IFN-γ responses in peptide-stimulated splenocytes, particularly for P3 and P1. ICS confirmed CD8⁺ T cell activation and polyfunctional cytokine expression. A comparative Nano-immunogenicity profile revealed P3 as the most potent candidate.Conclusion: This study provides preclinical evidence of antigenicity and MHC-I compatibility of four prostate cancer-derived CTL epitopes using a transgenic mouse model. These findings support the advancement of these peptides as candidates for peptide-based immunotherapy in prostate cancer.Keywords: cytotoxic T lymphocyte, epitope, HLA-A*02:01, peptide-based immunotherapy, prostate cancer