Figure 1.

Rationally designed mutant peptide-based vaccines. (A–E) Contributions of mutated and non-mutated epitopes to Anti-Tumor efficacy were determined when DTT was used as a carrier protein. (A) DTT is the diphtheria toxin T-domain, corresponding to amino acids 202–378 of DT. FD^mut represents KRAS G domain (residues 2–164) carrying the G12D mutation. DTT-FD^mut and DTT-FD^wt were constructed by fusing KRAS G domain (residues 2–164) to the C-terminal of DTT through a GS linker. DTT-FD^mut and DTT-FD^wt represent presence and absence of KRAS G12D mutation, respectively, in the fusion construct. (B) Flow chart of immunization and tumor inoculation. (C–E) DTT-FD^mut, DTT-FD^wt, and PBS were separately formulated in Alum and CpG. Female BALB/c mice (n = 5–8) received the formulated vaccines three times at 2-week intervals. Mice were injected with 2 × 10⁵ cells/mouse 10 days after the last immunization. (C) Tumor growth curves of DTT-FD^mut and DTT-FD^wt were plotted by averaging tumor size over time in each group. Data are presented as means ± SD. ^****p < 0.0001, Student's t-test (D) Kaplan–Meier survival curve. ^**p < 0.01, ns, not significant, log-rank (Mantel–Cox) test for significance. (E) Tumor growth curves for DTT-FD^wt and PBS. Data are plotted as means ± SD. ns, not significant. (F,G) Design and purification of mutant peptide-based vaccines. (F) Schematic representation of DTT-SP₄ and DTSP. SP represents residues 5–21 of KRAS containing the G12D mutation. DTSP^wt represents wild-type DTSP lacking the KRAS G12D mutation. The texture box stands for the linker sequence (GG). The white box denotes the position of DTT from 88 to 95 replaced with SP. (G) Expression levels of DTT-SP₄ (lane 1) and DTSP (lane 2).