Fig. 4 |. Multivalent PROTAV-TgT elicited potent antigen-specific T cell responses.

a, Sequence of the TgT multivalent antigenic peptide used in PROTAV-TgT. Minimal antigenic epitopes 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 was 2: 1. Nucleic acid phosphate over lipid nitrogen N:P ratio was 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 one-way ANOVA with Bonferroni post-test unless denoted otherwise.