Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity

Abstract

Immunization against self-tumor antigens can induce T-regulatory cells, which inhibit proliferation of type I CD4(+) T-helper (TH1) and CD8(+) cytotoxic T cells. Type I T cells are required for potent antitumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting insulin-like growth factor-binding protein (IGFBP-2) and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and interleukin (IL)-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly TH1 whereas the C-terminus stimulated TH2 and mixed TH1/TH2 responses. Epitope-specific TH2 demonstrated a higher functional avidity for antigen than epitopes, which induced IFN-γ (P = 0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T cell lines expanded from the C-terminus vaccinated animals secreted significantly more type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T cells secreted TH1 cytokines and significantly inhibited tumor growth, as compared with naïve T cells, when adoptively transferred (P = 0.005). To determine whether removal of TH2-inducing epitopes had any effect on the vaccinated antitumor response, we immunized mice with the N-terminus, C-terminus, and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (P < 0.001) as compared with the C-terminus vaccine, which had no antitumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the antitumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes.

Figure 1

The IGFBP-2 C-terminus is enriched for epitopes that induce IL-10-secreting T-cells as compared to the N-terminus. (A) ELISPOT for IFN-γ (white) and IL-10 (black) in breast cancer patient PBMC for IGFBP-2 peptides presented as interquartile box plots with Tukey whiskers (n=20). Median corrected spots per well (CSPW) are indicated by the horizontal bar. (B) Percent of PBMC stimulated with IGFBP-2 peptides that induced only an IFN-γ (white bars) response, only an IL-10 (black bars) response, or both (gray bars) in ELISPOT.

Figure 2

IGFBP-2 epitope-specific Th2 demonstrate a higher functional avidity and homology to a greater number of bacterial and self-proteins than IGFBP-2 epitope-specific Th1. Best-fit non-linear regression curves for the mean CSPW (•) generated at all peptide concentrations for each donor for (A) IL-10 (n=4) and (B) IFN-γ (n=5). Error bars: ± SEM of 4 replicates. (C) Number of homologous proteins for human (non-IGFBP family members) (gray bars) and bacteria (black bars) for the IGFBP-2 domains.

Figure 3

An N-terminus, but not C-terminus, IGFBP-2 vaccine both stimulates Type I immunity and inhibits tumor growth. (A) IFN-γ ELISPOT in splenocytes from mice immunized with the indicated vaccine. The data are presented as corrected spots per well (CSPW). The horizontal bar indicates the mean CSPW ± SEM. n=10 mice/group; *p<0.01. (B) Mean tumor volume (mm³ ± SEM) from mice injected with pUMVC3 alone (•), pUMVC3-hIGFBP2 (1-328) (■), pUMVC3-hIGFBP2 (164-328) (▲) or pUMVC3- hIGFBP2 (1-163) (○). n=5 mice/group; **p<0.001. (C) Mean tumor volume (mm³ ± SEM) from mice injected with pUMVC3 (•) or pUMVC3-hIGFBP2 (1-163) with isotype control IgG (○), CD8⁺ depletion (■) or CD4⁺ depletion (▲). #p<0.05 compared to pUMVC3-hIGFBP2 (1-163) vaccine with isotype control IgG.

Figure 4

IGFBP-2 vaccine-induced Th2 abrogates the anti-tumor effect of IGFBP-2-specific Th1. Type II cytokines IL-4 and IL-10 (A) and Type I cytokines TNFα and IFN-γ (B) secretion from T-cell lines expanded with peptides in IGFBP2 (1-163) or IGFBP2 (164-328) (mean ng/ml ± SD); **p<0.001, *p<0.01 and #p<0.05. (C) Mean tumor volume (mm³ ± SEM) from mice infused with CD3⁺ T-cells expanded from mice vaccinated with pUMVC3-hIGFBP2 (1-163) (○), pUMVC3-hIGFBP2 (164-328) (▲) or naïve T-cells (•). n=4 mice/group; *p<0.01. (D) Mean tumor volume (mm³ ± SEM) from mice injected with pUMVC3 alone (•), pUMVC3-hIGFBP2 (164-328) (▲), pUMVC3-hIGFBP2 (1-163) (○) or pUMVC3-hIGFBP2 (1-163) + pUMVC3-hIGFBP2 (164-328) (▼). n=5 mice/group; *p<0.01.