Generation of two-mutation p15E mimotopes that incorporate the 3C mutation. A, Strategy of e-mimotope screening for p15E-3C. (B) Peptide and MHC-I binding prediction by NetMHC algorithms. After vaccination on days 0 and day 7 with the indicated peptides with CPQ, blood was collected on day 14 for wild-type p15E-specific CD8+ T-cell frequency (C) as measured by tetramer staining. On day 14, 1 × 106 MC38 cells were inoculated subcutaneously. Mice received 2 µg peptide of the indicated liposomal vaccine on days 0 and day 7, splenocytes were harvested on day 14 and stimulated with the p15E wild-type peptide for IFNγ production by ELISOPOT (n = 5; D). On day 19 after tumor inoculation, tumor volume (E) was recorded (n = 3). Datapoints represent individual values of 3 mice from the same group in C and E or 5 mice from the same group in D. Statistical analysis applied one-way ANOVA followed by Tukey multiple comparisons tests. *, **, *** and **** indicating P < 0.05, 0.01, 0.001, and 0.0001, respectively. Error bars show mean ± SD.
ARTICLE ABSTRACT
Mimotopes of short CD8+ T-cell epitopes generally comprise one or more mutated residues, and can increase the immunogenicity and function of peptide cancer vaccines. We recently developed a two-step approach to generate enhanced mimotopes using positional peptide microlibraries and herein applied this strategy to the broadly used H-2Kb–restricted murine leukemia p15E tumor rejection epitope. The wild-type p15E epitope (sequence: KSPWFTTL) was poorly immunogenic in mice, even when combined with a potent peptide nanoparticle vaccine system and did not delay p15E-expressing MC38 tumor growth. Following positional microlibrary functional screening of over 150 mimotope candidates, two were identified, both with mutations at residue 3 (p15E-P3C; “3C,” and p15E-P3M; “3M”) that better induced p15E-specific CD8+ T cells and led to tumor rejection. Although 3M was more immunogenic, 3C effectively delayed tumor growth in a therapeutic setting relative to the wild-type p15E. As 3C had less H-2Kb affinity relative to both p15E and 3M, 15 additional mimotope candidates (all that incorporated the 3C mutation) were assessed that maintained or improved predicted MHC-I affinity. Valine substitution at position 2 (3C2V, sequence: KVCWFTTL) led to improved p15E-specific immunogenicity, tumor rejection, and subsequent long-term antitumor immunity. 3C, 3M, and 3C2V mimotopes were more effective than p15E in controlling MC38 and B16-F10 tumors. T-cell receptor (TCR) sequencing revealed unique TCR transcripts for mimotopes, but there were no major differences in clonality. These results provide new p15E mimotopes for further vaccine use and illustrate considerations for MHC-I affinity, immunogenicity, and functional efficacy in mimotope design.
The MHC-I–restricted p15E tumor rejection epitope is expressed in multiple murine cancer lines and is used as a marker of antitumor cellular immunity, but has seen limited success as a vaccine immunogen. An in vivo screening approach based on a positional peptide microlibraries is used to identify enhanced p15E mimotopes bearing amino acid mutations that induce significantly improved functional immunogenicity relative to vaccination with the wild-type epitope.
