Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

Abstract

Purpose: The clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4⁺ cells, including regulatory T cells (Tregs).

Methods: To determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)_257-264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model.

Results: We showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a⁺ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model.

Conclusion: The use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.

Keywords: 7-AAD, 7-amino-actinomycin D; Anti-CD4 antibody; CTL, cytotoxic T lymphocyte; Cancer; DC, dendritic cell; ELISPOT assay, enzyme-linked immunospot assay; FITC, fluorescein isothiocyanate; FOXP3, forkhead box P3; GPC3, glypican-3; HCC, hepatocellular carcinoma; IFN-γ, interferon-γ; IL-2, interleukine-2; Immunotherapy; MHC, major histocompatibility complex; Murine liver metastatic model; OVA, ovalbumin; PD-1, programmed death-1; PE, phycoerythrin; Peptide vaccine; QOL, quality of life; TGF-β, transforming growth factor-βl; TNF, tumor necrosis factor; Treg, regulatory T cell; mAb, monoclonal antibody.

Fig. 1

Number of lymphocyte subsets after administration of anti-CD4 mAb. (A) Representative flow cytometry profiles of CD4⁺ T cells in peripheral blood on days 0, 1, and 24 after administration of anti-CD4 mAb. (B) Changes in CD4⁺ T cell counts in the peripheral blood after administration of anti-CD4 mAb (GK1.5) (n=3). (C) Changes in CD8⁺ T cell counts in peripheral blood after administration of anti-CD4 mAb (GK1.5) (n=3). (D) Representative flow cytometry profiles of CD4⁺ CD25⁺ FOXP3⁺ cells in splenocytes of EG7-bearing mice on day 1 after administration of anti-CD4 mAb. (E) Statistical analysis of (D) (n=3).**P<0.01, differences are statistically significant between the two values.

Fig. 2

Comparison of peptide-specific CTL induction by peptide vaccine therapy in combination with anti-CD4 mAb. (A) Schedules for IFN-γ ELISPOT assay. I: OVA peptide vaccine alone, II: Starting from anti-CD4 mAb, and two administrations of OVA peptide vaccine, III: Starting from OVA peptide vaccine, and OVA peptide vaccination after anti-CD4 mAb administration (OVAp: OVA peptide vaccine, αCD4: anti-CD4 mAb). (B) Representative results of IFN-γ ELISPOT assay are shown. Effector cells: CD8⁺ spleen cells. Groups: I, II, III. Target cells: RMA-S cells, OVA peptide-pulsed RMA-S cells, EL4 cells, EG7 cells. Effector/target ratio=10. (C) Statistical analysis of (B). Comparisons of spot numbers between the group of OVA peptide alone (I) and groups of combination treatment (II or III) [left]. Comparisons of spot numbers between two groups of combination treatment (II and III) [right] (n=3).*P<0.05,**P<0.01, differences are statistically significant between the two value. ns, difference was not significant between the two values.

Fig. 3

Multi-function of peptide-specific CTL by peptide vaccine therapy in combination with anti-CD4 mAb. Representative data are shown. (A) Schedule for IFN-γ ELISPOT assay and CD107a assay. On days 0 and 7, mice were injected intradermally at the base of the tail with OVA peptide vaccine. On day 6, mice were injected i.p. with anti-CD4 mAb. On day 13, the CD107a assay was conducted. On day 14, the IFN-γ ELISPOT assay was conducted. (B) CD107a assay of peripheral blood cells. Effector cells: CD8⁺ peripheral blood cells. Groups: no treatment group, OVA peptide vaccine alone group, anti-CD4 mAb alone group, combination OVA peptide vaccine and anti-CD4 mAb group. Target cells: RMA-S cells, OVA peptide pulsed RMA-S cells. (C) The flow cytometric analysis of effector/memory phenotype of CD107a⁺ cells and CD107a^- cells in CD8⁺ T cells. (D) cytokine assay of peripheral blood cells. (E) IFN-γ ELISPOT assay and (F) cytokine assay of splenocytes. Effector cells: CD8⁺ spleen cells. Groups: no treatment group, OVA peptide vaccine alone group, anti-CD4 mAb alone group, combination OVA peptide vaccine and anti-CD4 mAb group. Target cells: RMA-S cells, OVA peptide pulsed RMA-S cells. Effector/target ratio=2.

Fig. 4

Suppression of tumor growth in the metastatic tumor model. The protocol for liver metastasis model was indicated in (A). All mice spleens were injected into with 1×10⁶ EG7 cells on day 0. On day −14, −7, 0, and 6, mice were injected intradermally at the base of the tail with the OVA peptide vaccine. On days −8 and 6, mice were injected i.p. with anti-CD4 mAb. On day 14, the mice were sacrificed and liver weight, spleen weight, and the major axis of splenic tumor were measured. (B) Representative liver metastasis and splenic tumor in each group. Groups: No treatment group, OVA peptide vaccine alone group, anti-CD4 mAb alone group, combination OVA peptide vaccine and anti-CD4 mAb group (n=5). (C) Statistical analysis of (B). Columns, mean number of liver weight, spleen weight, and major axis of splenic tumor.*P<0.05,**P<0.01, differences were statistically significant between the two values. ns, differences were not significant between the two values.

Fig. 5

Immunohistochemical analysis in murine liver. (A) The CD8⁺ T cells (brown color) infiltration into EG7 tumor in mice (no treatment, OVA peptide vaccine alone, and combination OVA peptide vaccine and anti-CD4 mAb) after the administration of anti-CD4 mAb. (B) CD4⁺ T cells (brown color) infiltration in liver surrounding EG7 tumor in no anti-CD4 mAb treatment group (no treatment, OVA peptide vaccine alone) and in anti-CD4 mAb treatment group (anti-CD4 mAb alone, combination OVA peptide vaccine and anti-CD4 mAb).