Optimized peptide vaccines eliciting extensive CD8 T-cell responses with therapeutic antitumor effects

Abstract

A major challenge for developing effective therapeutic vaccines against cancer is overcoming immunologic tolerance to tumor-associated antigens that are expressed on both malignant cells and normal tissues. Herein, we describe a novel vaccination approach, TriVax, that uses synthetic peptides representing CD8 T-cell epitopes, Toll-like receptor agonists that function as potent immunologic adjuvants and costimulatory anti-CD40 antibodies to generate large numbers of high-avidity antigen-reactive T cells capable of recognizing and killing tumor cells. Our results show that TriVax induced huge numbers of long-lasting antigen-specific CD8 T cells that displayed significant antitumor effects in vivo. The administration of a TriVax formulation containing a CD8 T-cell epitope derived from a melanosomal antigen (Trp2(180-188)) elicited antigen-specific CD8 T cells that induced systemic autoimmunity (vitiligo). More important, TriVax immunization was effective in eliciting potent protective antitumor immunity as well as remarkable therapeutic effects against established B16 melanoma. This therapeutic effect was mediated by CD8 T cells via perforin-mediated lysis and required the participation of type-I IFN but not IFNgamma. These results suggest that similar strategies would be applicable for the design of effective vaccination for conducting clinical studies in cancer patients.

Figure 1. Effect of TLR-Ls on peptide vaccination with anit-CD40 mAb

(A), B6 mince (3 per group) were immunized i.v. with Ova _257–264 peptide and anti-CD40 mAb and one of the following TLR-L: poly-IC (TLR3-L), CpG (TLR9-L), Lps (TLR4-L), GDQ (TLR7-L), FSL1 (TLR6/2-L). Seven days later, antigen-specific CD8 T cell responses were measured in blood by using tetramer analysis. Results represent the means and SD for each group where p values compared to the no TLR group are shown inside of each bar. (B), The mice received a second identical immunization (on day 21) and the frequency of tetramer-specific CD8 T cells in peripheral blood was followed in individual mice throughout various time points. Results correspond to the man and SD for each group. Arrows, times of vaccination. P values were calculated using two-way ANOVA test (*p < 0.0001 for TriVax versus peptide+anti-CD40 mAb).

Figure 2. Immunization with Trp2_180-–88-TriVax induces strong anti-tumor Cd8 T cell responses

B6mice were vaccinated i.v. on days 0 and 14 with Trp2_180–188-TriVax/poly-IC.(A), On days 7 and 22, blood samples were evaluated by tetramer analysis. Each point represents the value for each individual mouse and horizontal line represents the average value of the group. (B), Eight days after the boost, CD8 T cells were purified from pooled splenocytes and antigen-induced IFNγ and TNFα secretion was evaluated by EliSpot. (C), Cytolytic activity of freshly isolated CD8 T cells, assessed by a ⁵¹Cr-release assay. (D), B6 mice (8 per group) were immunized on days −24 and −12 or only on day −12 with Trp2_180–188-TriVax/poly-IC. Twelve days after the last immunization, the mice received 3 × 10⁵ B16F10 melanoma cells i.v. Survival is compared against unvaccinated group (No Vax) or group that received Ova_55–63-TriVax/poly-IC (negative control). Kaplan–Mayer survival curves for all groups of mice are shown. P values were determined by log-rank tests as compared to the negative control group.

Figure 3. Therapeutic anti-tumor effects of Trp2_180–188-TriVax

(A), B6 mice (4 per group) received 3 × 10⁵ B16Fl0 cells i.v., and 3 and 11 days later the mice were vaccinated with Trp2_180–188-TriVax or Ova_55–63-TriVax. A non-vaccinated group (No Vax) was also included. On Day 24 (when the control mice appeared sick), the presence of B16 pulmonary nodules was evaluated. Results are presented as “Numbers of Lung Tumors” and “Lung Weights” for individual mice. Representative photographs of lungs of 2 mice from each group are shown. Splenocytes from mice of the 2 vaccinated groups in “A” were evaluated for Trp2-specific CD8 T cells by tetramer analysis (B), and antigen-induced IFNγ production in EliSpot assays (C). P values were calculated using unpaired Student’s test.

Figure 4. Soluble peptide is more effective than peptide emulsified in IFA in TriVal

(A), B6 mice (4 per group) received 3 × 10⁵ B16F10 cells i.v. and 3 and 11 days later were vaccinated either i.v. or s.c. with Trp2_180–188-TriVax or Ova_55–63-TriVax. Peptides were emulsified in IFA for the s.c. vaccines. A non-vaccinated group (No Vax) was included. On day 24 all the mice were the presence of B16 pulmonary tumors was evaluated as described in Figure 3A. Splenocytes from individual mice were evaluated for Trp2-specific CD8 T cells by tetramer analysis (B) or EliSpot assaya (C) P values were calculated using unpaired Student’s test.

Figure 5. Therapeutic TriVax results in increased survival

(A), B6 mice (8 per group) received 3 different doses of B16F10 cells i.v. (as indicated) and 3 days later the mice were immunized with either Trp2_180–188-TriVax, Ova_55–63-TriVax or left unvaccinated (No Vax). P values were determined by log-rank tests. (B), At the termination of the experiment presented in (A), survivor mice were pooled, randomized and received s.c. tumor challenge with either B16F10 or B16F10-kb⁻ cells (5 × 10⁵ cell/mouse). Naï ve, unvaccinated mice inoculated with the same number of tumore cells were included as control. Tumore sizes were determined in individual mice by measurements of 2 opposing diameters and are presented as tumore areas in mm². Each data point corresponds to the means and SD for each group of mice (5 per group). P value was calculated using two-ways ANOVA test.

Figure 6. Effector mechanisms involved in anti−tumor effects of TriVax

(A), B6 mice (8 per group) received with 1 × 10 ⁵ B16F10 cells i.v. Various subsets of immune effector cells (CD8 T cells, CD4 T cells or NK cells) were depleted using mAb on days −3 and −1 before receiving the TriVax injection. An addition mAb injection was administered 2 days after vaccinatioin. TriVax immunizations were administered on days 3 and 14 after tumor injections. Kaplan–Mayer survival curves for all groups of mice are shown. P values were determined by log-rank tests. (B), Therapeutic effects of Trp2_180–188-TriVax in WT B6 mice (WT), IFNγ^−/−, IFNαβR^−/−, and Prf^−/− mice were evaluated a described above. Results were evaluated for statistical significance using log-rank test. (C), In a parallel experiment freshly isolated spleen CD8 T cells from WT, IFNγ^−/−, IFNαβR^−/−, mice were tested for cytolytic activity in a 5-h ⁵¹Cr-release assay as described in Figure 2C.