Proteolysis-targeting vaccines, or PROTAX, for robust tumor combination immunotherapy

Current peptide vaccines elicit limited antitumor T cell responses, leading to suboptimal tumor therapeutic efficacy. Here, we present proteolysis-targeting vaccines (PROTAX) that facilitate antigen proteolytic processing and cross-presentation to potentiate T cell responses for robust tumor immunotherapy in combination with immune checkpoint blockade (ICB). PROTAX are peptide antigens conjugated with a E3 ligase-binding ligand via linkers. In antigen-presenting cells (APCs), PROTAX bind to E3 ligases to rapidly ubiquitinate PROTAX antigens, facilitating antigen proteolytic processing by proteasome, and thereby promoting antigen cross-presentation to T cells. In mice, PROTAX promoted both the quantity and quality of CD8⁺ T cell responses against antigens, such as multivalent melanoma-associated antigen Trp2-gp100-Trp1 (TgT) and synthetic long peptide human papillomavirus (HPV)-16 E7. In melanoma-bearing mice, PROTAX-TgT + ICB remodeled the tumor immune microenvironment and enhanced tumor complete regression (CR) rates, in a manner dependent on classic type-1 dendritic cells (cDC1) and CD8 + T cells. PROTAX-TgT + ICB eradicated 100% large Braf V600E SM1 melanomas (~300 mm³), and promoted the CR rates of M3 melanoma and highly immunosuppressive Braf CA + M2 melanoma with low TgT antigen expression. In an autochthonous Braf V600E melanoma in Braf V600E/Pten-/- mice, PROTAX-TgT + ICB reduced the molecular and cellular immunosuppression in the tumor, inhibited tumor progression and metastasis, and extended mouse median survival from 21 days to 66 days post initial tumor treatment. Similarly, PROTAX-E7 + ICB resulted in 43% CR of large TC-1 tumors (~300 mm³). In human leukocyte antigen (HLA)-A02:01 human PBMCs from healthy donors and HPV-16 + Head and Neck Squamous Cell Carcinoma (HNSCC) patients, HLA-A02:01-restricted trivalent PROTAX E6-(E7)2 promoted tri-specific CD8 + T cell responses, characterized with enhanced antigen-specific T cell expansion and production of human interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Therefore, PROTAX hold the potential for robust tumor combination immunotherapy.