Authors: Qiyan Wang, Ting Su, Furong Cheng, Shurong Zhou, Xiang Liu, Mi Wang, Ri Tang, You Xu, Shimiao Liao, Jordan Dailey, Guolan Xiao, Hanning Wen, Weijia Zheng, Bo Wen, Katarzyna M Tyc, Venkatesha Basrur, Jinze Liu, Duxin Sun, Shaomeng Wang, and Guizhi Zhu
Abstract
Protein/peptide vaccines can improve the tumor therapeutic efficacy of immune checkpoint blockade (ICB). However, current protein/peptide vaccines elicit limited T cell responses pivotal for tumor immunotherapy. Here, we present proteolysis-targeting vaccines (PROTAVs) that facilitate antigen proteolytic processing and crosspresentation to potentiate T cell responses for robust ICB combination immunotherapy of tumors. PROTAVs are modular conjugates of protein/peptide antigens, E3 ligase-binding ligands, and linkers. In antigen-presenting cells (APCs), PROTAVs bind to E3 ligases to rapidly ubiquitinate PROTAV antigens, thereby facilitating antigen proteolytic processing by proteasome and promoting antigen cross-presentation to T cells. In mice, when codelivered with bi-adjuvants activating cyclic-GMP-AMP synthase (cGAS) and Toll-like receptor 9 (TLR9) using lipid nanoparticles (LNPs), PROTAVs promoted the quantity and quality of CD8+ T cells against antigens of proteins, synthetic long peptides, and multivalent fusion peptides. Combining PROTAVs and ICB remodels tumor immune microenvironment, promotes the complete regression rates of murine melanoma and human papillomavirus (HPV)-associated tumors, and eradicates 50% ICB-resistant large tumors (300 mm3). Overall, PROTAVs represent a simple and broadly applicable platform for robust tumor combination immunotherapy.
Fig 1. Schematic illustration of PROTAVs that facilitate antigen proteolytic processing and antigen crosspresentation to potentiate antitumor T cell responses for robust ICB combination immunotherapy of tumor. A PROTAV is a covalent conjugate of a protein/peptide antigen and an E3 ligase-binding ligand, with a chemical linker in between. PROTAVs are broadly applicable for common types of tumor protein/peptide antigens, including proteins, synthetic long peptides, and multivalent fusion peptides. Upon delivery into APCs by LNPs, PROTAVs are bound to endogenous E3 ligases, which promoted the ubiquitination of the antigens in PROTAVs, facilitated antigen proteolytic processing by proteasome, promoted antigen cross-presentation to T cells, thereby potentiating antigen-specific T cell responses. Peptide antigen-based PROTAVs were co-loaded with two oligonucleotide immunostimulant adjuvants, Svg3 and CpG, in LNPs for optimal PROTAV/adjuvant codelivery into APCs. The bi-adjuvants of Svg3 and CpG activate cGAS and TLR9, respectively, to elicit proinflammatory responses, including type-I interferon (IFN) responses. As a result, PROTAVs potentiated antitumor CD8+ cytotoxic T cell responses. The combination of PROTAVs with ICB remodeled the tumor immune microenvironment, facilitated tumor regression, and promoted the CR rates of tumors, including ICB-resistant large tumors. LNP: lipid nanoparticle; cGAS: cyclic GMP-AMP synthase; Svg3: an oligonucleotide agonist for cGAS; TLR9: Toll-like receptor 9; CpG: an oligonucleotide agonist for TLR9; E2: E2 ligase; E3: E3 ligase; Ub: ubiquitin; APC: antigen-presenting cell; MHC: major histocompatibility complex; ICB: immune checkpoint blockade. Created in BioRender.
Fig 2. Multivalent PROTAV-TgT elicited potent antigen-specific T cell responses. (A) Sequence of the TgT multivalent antigenic peptide used in PROTAV-TgT. Minimal peptides were underlined. Multiple terminal K and C were added to promote pomalidomide conjugation via maleimide-thiol conjugation and ubiquitination on lysine, respectively. (B) Structure of TgT predicted by AlphFold3. (C) DLS data and a cryo-EM image showing the hydrodynamic sizes and morphology of SM-102 LNPs co-loaded with PROTAV-TgT, CpG, and Svg3. CpG: Svg3 molar ratio: 2: 1. Nucleic acid over lipids N:P ratio: 6:1. (D) Timeline of PROTAV-TgT immunization study in mice. C57BL/6 mice (6-8 weeks) were immunized with PROTAV-TgT and TgT vaccines, respectively (s.c. at tail base, day 0 and day 14). Dose: 20 ug antigen, 2 nmole CpG, and 1 nmole Svg3. e-j, PROTAV-TgT elicited potent multivalent T cell responses while upregulating immune checkpoint levels on T cells in mice. (E-F) Representative flow cytometry graphs (E) and quantified flow cytometry data for tetramer staining results showing that relative to TgT, PROTAV-TgT promoted gp100-specific CD8+ T cell responses (day 21). (G-H) Representative flow cytometry graphs (G) and quantified intracellular cytokine staining results (H) of CD8+ T cells upon ex vivo restimulation of PBMC CD8+ T cells with gp100 and Trp2 peptides, respectively, suggesting that relative to TgT, PROTAV-TgT promoted multifunctional T cell responses producing antitumor cytokines. (I-J) MFI of PD-1 (I) and Tim-3 (J) on total live PBMC CD8+ T cells in the as-immunized mice, suggesting that PROTAV-TgT upregulated these immune checkpoint levels on T cells. Data represent mean ± s.e.m. (n = 5); statistical analysis was conducted using oneway ANOVA with Bonferroni post-test unless denoted otherwise.
Selected Figures
Keywords: lipid nanoparticles; PROTAV; melanoma; immunotheraphy; immune checkpoint blockade; Flex-S
bioRxiv 2024