Optimization of Peptide Vaccines to Induce Robust Antitumor CD4 T-cell Responses

Abstract

Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor-associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell-based immunotherapies. Cancer Immunol Res; 5(1); 72-83. ©2016 AACR.

Figure 1. Combination of poly-IC and CD40 agonist with peptide vaccine induces robust CD4 T-cell responses

(A) Mice were immunized with Ova_265-280 peptide on days 0 and 14. TriVax (poly-IC and CD40 mAb), TiterMax^®, or CFA-IFA were used as adjuvants. TriVax was administered i.v. and TiterMax and CFA-IFA were injected s.c. EliSpot assay was done on day 21 using purified CD4 T cells from spleens (APCs: Ova_265-280 peptide-pulsed LB27.4 cells). Less than 100 spots were observed using unpulsed APCs (E. Celis, unpublished observations). (B) Mice were vaccinated i.v. with Ova_265-280 peptide with or w/o CD40 mAb, poly-IC, or with the combination of CD40 mAb and poly-IC (TriVax) on days 0 and 14. Mice were sacrificed on day 21, and the responses were measured as described in Fig. 1A. (C) Different doses of Ova_265-280 peptide (10 μg, 50 μg, 100 μg, and 200 μg) were injected i.v. with TriVax protocol. EliSpot assay was done on day 21. (D) Mice were injected i.v. with peptide 2W1S (50 μg or 200 μg) combined with LPS or TriVax on days 0 and 14. Mice were sacrificed on day 6 after the boost and splenocytes (SP) and bone marrow cells (BM) were isolated for 2W1S/I-Ab tetramer staining. Splenocytes were incubated with peptide (1 μg/ml) (pep) in an intracellular IFNγ staining assay. Data shown are representative of three experiments with similar results. (E) Purified HTLs from splenocytes were cocultured with 2W1S peptide-pulsed LB27.4 cells for the EliSpot assay. Data represents the average of three experiments including 3–5 mice/experiment. Results are presented as mean ± SD. (*P < 0.05)

Figure 2. OX40 agonist synergizes with TriVax to further expand CD4 T cells

(A) Mice received TriVax (Ova_265-280 peptide, poly-IC: 50 μg, and CD40 mAb) on days 0 and 14 and mice also received α4-1BB, αGITR, or αOX40 mAbs on days 0, 2, 12, and 14. Mice were sacrificed on day 21 and splenocytes were incubated with 1 μg/ml Ova_265-280 peptide (+pep) or w/o peptide (−) in an intracellular cytokine-staining assay. The percentages of IFNγ⁺ CD4 T cells are shown in the upper right quadrants. The dot plots represent the results from one of the three separate experiments with similar results. (B) The number of IFNγ⁺ spots by CD4 T cells cocultured with Ova_265-280 peptide-pulsed LB27.4 cells. Less than 100 spots were observed in the absence of peptide (E. Celis, unpublished observations). (C) Mice were vaccinated with TriVax (2W1S peptide, poly-IC, and CD40 mAb) combined with or w/o OX40 mAb (“prime & boost”: Day 0 and day 12, “boost”: day 12). The percentage of 2W1S tetramer+ cells in CD4 T cells was examined in blood or spleen. (D) The total number of 2W1S tetramer+ CD4 T cells in spleen. (*P < 0.05, n.s.: not significant). These experiments were repeated at least 2 times with similar results.

Figure 3. TLR7 agonist is a suitable adjuvant for CD4-targeted peptide vaccine, which requires type 1 IFN

(A) Mice were immunized on days 0, 14, and 65 (arrows) with TriVax (VV H3L_273-286 peptide, CD40 mAb, and indicated TLR agonists; LPS, CpG, poly-IC, GDQ or no TLR agonist) combined with OX40 mAb. Antigen-specific CD4 T-cell responses were evaluated in blood using intracellular cytokine staining. (B) Mice received TriVax (2W1S peptide, CD40 mAb, and poly-IC or GDQ), with or w/o OX40 mAb on days 0 and 12. The percentages of 2W1S tetramer + CD4 T cells in blood on days 7 and 19 were examined by flow cytometry. (C) IFNαβ-KO and WT-B6 mice were vaccinated with TriVax (VV H3L_273-286 peptide, Gardiquimod, CD40 mAb) and OX40 mAb on days 0 and 14, and responses were measured in blood on day 21. (D) IFNγ KO (GKO) mice and WT-B6 mice were vaccinated on day 0 and 14, and responses were measured in blood on day 21. (*P < 0.05, n.s.: not significant). These results represent example of three independent experiments.

Figure 4. CD4 T-cell responses to a non-immunogenic melanosomal antigen in wild-type or Trp1 KO mice

WT-B6 or Trp1-KO mice received TriVax (Trp1_113-127 peptide, GDQ, and αCD40 mAb) and αOX40 mAb on days 0 and 12. (A) Splenocytes were harvested on day 19 and were stimulated with Trp1_113-127 peptide (1 μg/ml) in intracellular cytokine staining assay. (B) EliSpot assay and (C) flow cytometry-based cytotoxicity assays were performed using purified CD4 T cells from splenocytes. (D) WT-B6 mice received TriVax and αOX40 mAb on days 0 and 12, mice were sacrificed on day 31 and splenocytes were used in intracellular cytokine staining assays; (*P < 0.05). These experiments were repeated 3 times with similar results.

Figure 5. Endogenous CD4 T cells induced by TriVax/OX40 have direct antitumor activity

(A and B) WT-B6 mice (10 per group) were inoculated subcutaneously on day 0 with 3 x 10⁵ B16F10 melanoma cells. Mice were untreated (No Vac), received a control vaccine (mock Vac: GDQ and CD40 mAb) or TriVax with Trp1_113-127 peptide, GDQ and CD40 mAb and OX40 mAb on days 3 and 15 (Vac). Tumor size (A) and the percent survival (time to euthanasia) of mice (B) are shown. Results are presented as mean ± SD. Individual tumor growth curves for each group are also presented. (C and D) WT-B6 mice (10 per group) were inoculated subcutaneously on day 0 with 3 x 10⁵ B16F10 melanoma cells. Mice were untreated, received TriVax with Trp1_113-127 peptide, GDQ, and CD40 mAb and OX40 mAb on days 3 and 15 (Vac). One group of vaccinated mice received αCD8 mAb on days 3, 5, 15, and 17 (Vac+CD8). The tumor size (C) and the percent survival of mice (D) are shown. Results are presented as mean ± SD. Arrows denote times of vaccination. These experiments were repeated 2 independent times and similar results were obtained

Figure 6. TriVax/OX40 expands adoptively transferred CD4 T cells without need of lymphodepletion

(A) B6 CD45.1 mice received 1 x 10⁵ TRP1-TCR (CD45.2) splenocytes (~3 x 10⁴ CD4 T cells) one day before the vaccination. The components of the vaccine (Trp1_113-127 peptide with adjuvants) are indicated in the figure. The percentages of TRP1-TCR cells T cells (CD45.2⁺/CD4⁺) were measured in blood on day 7 after vaccination. Images of the representative results are shown. (B) Peptide-induced intracellular cytokine profiles of Trp1-TCR cells after vaccination. (C) Splenocytes were harvested from mice, which received 1 x 10⁵ Trp1-TCR cells ACT (1 day before vaccination) and TriVax (Trp1_113-127 peptide, GDQ, and CD40 mAb) and OX40 mAb on day 7 after vaccination. CD4 T cells were purified from the splenocytes and used in EliSpot assay with various target cells. (D) B6 mice received 1 x 10⁵ TRP1-TCR (CD45.2) splenocytes (~3 x 10⁴ CD4 T cells) one day before the TriVax with or without OX40 mAb agonist. Seven days later, the expression of Bcl-xL or Ki-67 in TRP1-TCR CD4 T cells from naïve TRP1-TCR or vaccinated mice were examined. (E) Splenocytes were harvested from mice, which received 1 x 10⁵ Trp1-TCR cells ACT (1 day before vaccination) and TriVax (Trp1_113-127 peptide, GDQ, and CD40 mAb) and OX40 mAb on day 7 after vaccination. CD4 T cells were purified from the splenocytes and used in EliSpot assay with various target cells; (*P < 0.05).

Figure 7. TriVax/OX40 potentiates the therapeutic antitumor effect of adoptively transferred CD4 T cells

(A and B) WT-B6 mice (10 per group) were inoculated subcutaneously on day 0 with 3 x 10⁵ B16F10 melanoma cells. Mice received 1 x 10⁵ TRP1-TCR cells on day 10 and TriVax (Trp1_113-127 peptide, GDQ, and CD40 mAb) and OX40 mAb on day 11 (red arrows). Some mice received 2 injections of αPD-L1 following vaccination (black arrows). The size of tumor (A) and the percent survival of mice (B) are shown. Individual tumor growth curves for each group are also presented. (C) The percentages of TRP1-TCR cells TCR Vβ14⁺/CD4⁺) in blood after vaccine were measured in WT-B6 and Trp1-KO mice. The dotted line indicates the normal percentage of Vβ14+ CD4 T cells in WT-B6 mice. The blood was withdrawn on days 7, 12, and 14 after vaccination. Results are presented as mean ± SD; (*P < 0.05).