An In Vivo Screen to Identify Short Peptide Mimotopes with Enhanced Antitumor Immunogenicity

Abstract

Tumor-associated self-antigens are potential cancer vaccine targets but suffer from limited immunogenicity. There are examples of mutated, short self-peptides inducing epitope-specific CD8+ T cells more efficiently than the wild-type epitope, but current approaches cannot yet reliably identify such epitopes, which are referred to as enhanced mimotopes ("e-mimotopes"). Here, we present a generalized strategy to develop e-mimotopes, using the tyrosinase-related protein 2 (Trp2) peptide Trp2180-188, which is a murine MHC class I (MHC-I) epitope, as a test case. Using a vaccine adjuvant that induces peptide particle formation and strong cellular responses with nanogram antigen doses, a two-step method systematically identified e-mimotope candidates with murine immunization. First, position-scanning peptide microlibraries were generated in which each position of the wild-type epitope sequence was randomized. Randomization of only one specific residue of the Trp2 epitope increased antitumor immunogenicity. Second, all 20 amino acids were individually substituted and tested at that position, enabling the identification of two e-mimotopes with single amino acid mutations. Despite similar MHC-I affinity compared with the wild-type epitope, e-mimotope immunization elicited improved Trp2-specific cytotoxic T-cell phenotypes and improved T-cell receptor affinity for both the e-mimotopes and the native epitope, resulting in better outcomes in multiple prophylactic and therapeutic tumor models. The screening method was also applied to other targets with other murine MHC-I restriction elements, including epitopes within glycoprotein 70 and Wilms' Tumor Gene 1, to identify additional e-mimotopes with enhanced potency.

Figure 1.. A screening method identifies Trp2 e-mimotopes with improved function.

A) A two-step approach for developing of e-mimotopes using positional micro-libraries with in vivo screening and tumor challenge guiding the sequence selection. C57BL/6 mice were immunized with CPQ admixed with peptide libraries or single peptides on days 0 and 7, then were challenged with B16-F10 cells on day 14 (n=3 mice per group for B and C). B) Day 21 tumor volume of mice vaccinated with CPQ and indicated peptide libraries. C) Day 21 tumor volume of mice vaccinated with CPQ and indicated position 8 substituted peptides. Tumor growth (D) and day 23 tumor volume (E) of untreated mice or mice vaccinated with CPQ or 2HPQ and e-mimotopes (Trp2–8C and Trp2–8Y) or the wild-type sequence (Trp2–8W). F) Percentage of mice with tumor sizes smaller than 1 cm. Error bars show mean +/−std. dev. for n=5 mice per group (for D–F). * p < 0.05, and ** p < 0.01 and **** p < 0.0001, analyzed by one-way ANOVA with Dunnett’s multiple comparisons post-test (D) by comparing all groups to control group or one-way ANOVA with Bonferroni multiple comparisons post-test (E) or long rank test (F).

Figure 2.. Immunization with e-mimotope (Trp2–8C and 8Y) particles enhances T-cell profiles and improves immune recognition of the wild-type epitope (Trp2–8W).

A) Peptide binding to CPQ and 2HPQ liposomes after 1 hr incubation at room temperature. B) Liposome size with or without peptide binding. Error bars show mean +/−std. dev. for n=3 triplicate experiments (A, B). C) Cryo-EM images of CPQ admixed with Trp2–8W, Trp2–8Y and Trp2–8C. (D, E, F, G) C57BL/6 mice were vaccinated with CPQ/Trp2–8W or CPQ/Trp2–8Y or CPQ/Trp2–8C on days 0 and 7, and splenocytes were collected on day 14. n=5 mice per group. Percentage of T_EM cells (D) and T_CM cells (E) in the CD8⁺ T-cell population. Percentage of IFNγ (F) and TNFα (G) producing cells in the CD8⁺ T-cell population after 10 μg/mL Trp2–8W stimulation in vitro. Splenocytes from CPQ/Trp2–8C (H) or CPQ/Trp2–8Y (I) or CPQ/Trp2–8W (J) vaccinated mice were stimulated with different concentrations of Trp2–8W, Trp2–8Y or Trp2–8C peptides and assessed for intracellular IFNγ production. K) Splenocytes were prepared from untreated mice or mice vaccinated with CPQ/Trp2–8C and CPQ/Trp2–8W and stimulated with Trp2–8W for 72 hr, then the supernatant of cell culture medium was collected for IFNγ ELISA assay. Error bars show mean +/−std. dev. for n=2 splenocyte samples pooled from 5 mice (H, I, J, K). * p < 0.05, ** p < 0.01, **** p < 0.0001, and **** p < 0.0001, analyzed by (D, E, F, G) one-way ANOVA with Bonferroni multiple comparisons post-test.

Figure 3.. Identified e-mimotopes (Trp2–8C and Trp2–8Y) and wild-type (Trp2–8W) epitopes effectively bind MHC-I.

A) NetMHC predicted MHC binding percentile of Trp2 positional peptide library members to H-2K^b. B) NetMHC predicted H-2K^b binding percentile of peptides from Trp2-Pos8. C) RMA-S cells were incubated with or without 15 μg/mL (D) or 1.5 μg/mL (E) Trp2–8W, Trp2–8Y or Trp2–8C at 26°C for 2 hr and at 37°C for 2 hr and then stained for the surface expression of H-2K^b. The image shows flow cytometry histogram of H-2K^b staining. Error bars show mean +/−std. dev. for n=4 independent experiments (D, E). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, analyzed by (D, E) one-way ANOVA with Bonferroni multiple comparisons post-test.

Figure 4.. Trp2–8C and Trp2–8Y induce different TCR repertoire.

Mice were vaccinated with CPQ/Trp2–8C or CPQ/Trp2–8Y on days 0 and 7. On Day 14, splenocytes were restimulated with 10 μg/mL antigen and IFNγ–producing CD8⁺ T cells were sorted, DNA was extracted from these cells and TCRs were sequenced. A) The 30 most abundant amino acid clonotype frequencies from each sample are multi-colored and the remaining cumulative clone frequency is shown in purple. The total number of unique CDR3 clonotypes observed in each sample is noted. B) CDR3 sequence length comparison based on the amino acid sequence. C) TRBJ segment usage. D)TRBV segment usage. E) Similarity matrix of TCR repertoires from IFNγ⁺CD8⁺ T cells from CPQ/Trp2–8C and CPQ/Trp2–8Y vaccinated mice. F) Venn diagrams of unique CDR3 nucleotide sequences found in splenocytes of each animal from the respective treatment group. n=3 independent mice. * p < 0.05, ** p < 0.01, and *** p < 0.001, analyzed by unpaired student t-test (B, C, D).

Figure 5.. Trp2 e-mimotopes as therapeutic vaccine antigens.

C57BL/6 mice were inoculated with B16-F10 tumor cells on day 0 and injected with CPQ liposomal vaccines with the indicated Trp2 epitopes (0.5 μg peptide per mouse) on days 4 and 11 (n=10 mice per group). Tumor growth in individual mice (A) and day 28-tumor volumes (B) are shown. C57BL/6 mice were injected with 1×10⁵ B16-F10 cells intravenously on day 0 and then vaccinated on days 2 and 9 (n=5 mice per group). C) Image of lungs, D) lung nodules and lung weights (E) were assessed on day 23. * p < 0.05, ** p < 0.01, analyzed by one-way ANOVA with Bonferroni multiple comparisons post-test (B, D. E).

Figure 6.. Enhanced efficacy of Trp2 vaccines with immune checkpoint blockade using anti-PD-1 and anti-CTLA-4 antibodies.

C57BL/6 mice were inoculated with B16-F10 cells subcutaneously on day 0, then vaccine was given on days 4 and 11 and spleen and tumor were collected on day 28 day for analysis. Percentage of CD45⁺ T (A) and CD8⁺ T (B) cells of the live cells in the tumor. C) Percentage of Trp2_180–188 tetramer⁺ cells in the CD8⁺ T-cell population. D) Percentage of PD-1⁺ cells in the tetramer⁺CD8⁺ T cell population. C57BL/6 mice were inoculated with B16-F10 cells subcutaneously on day 0, then vaccine was given on days 4 and 11 intramuscularly and anti–PD-1 and anti–CTLA-4 were injected intraperitoneally on days 6, 8, 13 and 15. Tumor growth (E, G) and percentage of mice with tumor sizes smaller than 1 cm (F, H). Error bars show mean +/−std. dev. for n=5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, analyzed by one-way ANOVA with Bonferroni multiple comparisons post-test (A, B, C, D) or two-tailed unpaired Student’s t-test (E, G) or long-rank comparison (F, H).

Figure 7.. Identification of the AH1–5C e-mimotope.

A) Strategy used to identify the AH1–5C e-mimotope. BALB/c mice were inoculated subcutaneously with CT26 tumor cells on day 0 and then vaccinated with CPQ vaccine either on days 5 and 12 or days 8 and 15. B) Tumor sizes of mice on day 60. C) Tumor growth of untreated mice or mice vaccinated with CPQ/5C or CPQ/AH1 on days 8 and 15. D) Percentage of mice with tumor sizes smaller than 1 cm. BALB/c mice were inoculated with CT26 subcutaneously with CT26 tumor cells on day 0 and then vaccinated with CPQ vaccine on days 8 and 15. Splenocytes were collected on day 28 for analysis. Percentage of AH1 tetramer⁺ cells (E) T_EM cells (F) in the CD8⁺ T-cell population. Percentage of IFNγ (G) and granzyme-B (H) producing cells in the CD8⁺ T-cell population after 10 μg/mL stimulation with the wild-type AH1 peptide. Error bars show mean +/−std. dev. for n=5 mice per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 analyzed by long-rank (D) or one-way ANOVA with Bonferroni multiple comparisons post-test (E, F, G, H).