Abstract 3540: Proteolysis-targeting vaccines for robust combination immunotherapy of melanoma

Abstract Introduction: Protein/peptide vaccines show great promise for combination tumor immunotherapy with immune checkpoint blockade (ICB). However, conventional protein/peptide vaccines often elicit insufficient antitumor T cell responses, resulting in suboptimal therapeutic outcomes. Here, we introduced proteolysis-targeting vaccines (PROTAVs), which integrate melanoma protein/peptide antigens, E3 ligase-binding ligands, and linkers. This design enhances antigen proteolytic processing and cross-presentation, resulting in robust activation of T cell responses. By co-delivering PROTAVs with a bi-adjuvant system through lipid nanoparticles (LNPs), we aim to significantly improve the quantity and quality of specific CD8+ T cells, thereby enhancing the efficacy of ICB-based combination therapies. Method: Ovalbumin (OVA) was employed as a model antigen, with pomalidomide serving as the E3 ligase ligand, conjugated via a series of linkers. Linker optimization was conducted using an in vivo T-cell response assay. Protein degradation and antigen presentation efficiency were assessed using Western blotting, co-immunoprecipitation, and flow cytometry. Leveraging these findings, PROTAV-TgT (Trp2-gp100-Trp1), incorporating melanoma-specific antigens, was strategically developed to enable a comprehensive evaluation of its immunological mechanisms and therapeutic potential. Result: A series of PROTAV-OVA constructs incorporating linkers of varying water solubility and lengths were synthesized, eliciting robust SIINFKEL+ T cell responses in mice, with the EG4 linker achieving superior immunogenicity, inducing up to 18% SIINFKEL+ T cells. Mechanistic studies demonstrated that PROTAV-OVA facilitated enhanced antigen degradation, as evidenced by increased DQ-OVA fluorescence and OVA-ubiquitin colocalization in dendritic cells. Additionally, it significantly improved antigen presentation, as shown by elevated H2Kb-SIINFEKL staining both in vitro and in vivo. Based on these, the PROTAV-TgT was designed, incorporating melanoma-specific antigens and co-delivery with CpG using SM-102 LNPs. Immunization with PROTAV-TgT induced 4-fold increasing gp100-specific+ T cell responses, compared with TgT alone. In the B16F10 model, PROTAV-TgT combined with ICB resulted in complete tumor regression (CR) in 50% of mice treated. Remarkably, in the aggressive SM1 BrafV600E melanoma model, this combination therapy achieved 100% CR by day 24, an outcome not observed in other group at the time point. Conclusion: PROTAVs effectively elicit robust CD8+ T cell responses and alleviate tumor immunosuppression, enhancing the efficacy of ICB combination immunotherapy for melanoma. These findings highlight the potential of PROTAVs as a versatile platform for developing cancer therapeutic vaccines, offering broad applicability for robust immunotherapy across various cancer types. Citation Format: Qiyan Wang, Ting Su, Furong Chen, Shurong Zhou, Xiang Liu, You Xu, Ri Tang, Shimiao Liao, Jordan Dailey, Guolan Xiao, Guizhi Zhu. Proteolysis-targeting vaccines for robust combination immunotherapy of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3540.