Innovative DNA vaccine to break immune tolerance against tumor self-antigen

Abstract

Vaccination is, in theory, a safe and effective approach for controlling disseminated or metastatic cancer due to the specificity of the mammalian immune system, yet its success in the clinic has been hampered thus far by the problem of immune tolerance to tumor self-antigen. Here we describe a DNA vaccination strategy that is able to control cancer by overcoming immune tolerance to tumor self-antigen. We engineered a DNA construct encoding a dimeric form of a secreted single-chain trimer of major histocompatibility complex class I heavy chain, β2-microglobulin, and peptide antigen linked to immunoglobulin G (SCT-Ag/IgG). The chimeric protein was able to bind to antigen-specific CD8(+) T cells with nearly 100% efficiency and strongly induce their activation and proliferation. In addition, the chimeric protein was able to coat professional antigen-presenting cells through the F(c) receptor to activate antigen-specific CD8(+) T cells. Furthermore, intradermal vaccination with DNA-encoding SCT-Ag/IgG could generate significant numbers of cytotoxic effector T cells against tumor self-antigen and leads to successful therapeutic outcomes in a preclinical model of metastatic melanoma. Our data suggest that the DNA vaccine strategy described in the current study is able to break immune tolerance against endogenous antigen from melanoma and result in potent therapeutic antitumor effects. Such strategy may be used in other antigenic systems for the control of infections and/or cancers.

FIG. 1.

Characterization of the single-chain trimer-ovalbumin/immunoglobulin G (SCT-Ova/IgG) construct. (A) Schematic diagram of the structure of the SCT-Ova/IgG protein. The SCT is composed of peptide antigen, β2 microglobulin, and major histocompatibility complex class I heavy chain. The SCT forms a dimer due to the presence of an F_c domain fused to its C-terminus. (B) Representative reducing-gel showing purified SCT-Ova/IgG protein. We transfected plasmid-encoding SCT-Ova/IgG into baby hamster kidney-21 cells and, 3 days later, collected and concentrated the culture medium. We purified SCT-Ova/IgG from this medium using protein G columns. We performed sodium dodecyl sulfate polyacrylamide gel electrophoresis using loading buffer containing 2-mercaptoethanol to confirm the reduced purified protein (S.M., standard marker). (C) Representative nonreducing gel showing purified SCT-Ova/IgG protein. Color images available online at www.liebertpub.com/hum

FIG. 2.

Characterization of Ova-specific T-cell activation and proliferation after stimulation with single chain trimer-ovalbumin/immunoglobulin G (SCT-Ova/IgG). (A) Representative flow cytometry histograms showing the binding of SCT-Ova/IgG protein to T cells. We mixed E7- and Ova-specific CD8⁺ T cells with different concentrations of purified SCT-Ova/IgG protein and stained the cells with phycoerythrin-conjugated anti-mouse IgG antibody (green line). Cells were analyzed by flow cytometry. (B) Representative flow cytometry histograms showing T-cell proliferation after stimulation with SCT-Ova/IgG protein. “Con” indicates treatment with phosphate buffered saline only. We labeled Ova-specific T cells with carboxyfluorescein succinimidyl ester (CFSE) and mixed them with different concentrations of purified SCT-Ova/IgG for 3 days in the presence of exogenous interleukin-2. We analyzed CFSE dilution by flow cytometry to assess T-cell proliferation. Right : Bar graph quantification of the data (mean+SD). (C) Representative flow cytometry dot plots showing the percentage of activated T cells after stimulation with SCT-Ova/IgG protein. We mixed E7- or Ova-specific CD8⁺ T cells with different concentrations of SCT-Ova/IgG protein. We determined the percentage of activated antigen-specific, interferon-γ-secreting CD8⁺ T cells by intracellular cytokine staining followed by flow cytometry. (D) Bar graph of the flow cytometry data (mean+SD).

FIG. 3.

Characterization of the role of F_c receptor in mediating Ova-specific T-cell activation by single chain trimer-ovalbumin/immunoglobulin G (SCT-Ova/IgG). We treated dendritic 2.4 cells with or without F_c receptor blocker for 20 min and then added SCT-Ova/IgG protein for 20 min at 37°C. (A) We incubated the dendritic 2.4 cells with Ova-specific CD8⁺ T cells (1:1 ratio) overnight and analyzed the percentage of activated T cells by intracellular cytokine staining for interferon-γ followed by flow cytometry. (B) Bar graph of the flow cytometry data (mean+SD). Color images available online at www.liebertpub.com/hum

FIG. 4.

Comparison of the Ova-specific immune response and antitumor effect generated by vaccination with DNA-encoding SCT-Ova/IgG or SCT-Ova. (A) We vaccinated C57BL/6 mice intradermally via gene gun twice at 1-week intervals with plasmid-containing SCT-Ova/IgG (pFUSE-SCT-Ova/IgG) or SCT-Ova (pIRES-SCT-Ova). We harvested splenocytes from mice 1 week after the last immunization and analyzed the number of Ova-specific, interferon (IFN)-γ-secreting CD8⁺ T cells by intracellular cytokine staining and flow cytometry analysis. (B) Bar graph of the flow cytometry data showing the number of Ova-specific, IFN-γ-secreting CD8⁺ T cells per 3×10⁵ splenocytes (mean+SD). (C) We challenged C57BL/6 mice with B16-Ova tumor cells subcutaneously in the right hind leg and vaccinated them intradermally 3 days later with pFUSE-SCT-Ova/IgG, pIRES-SCT-Ova, or empty vector control. We measured tumor size twice each week by palpation and inspection over a course of 23 days. (D) Kaplan-Meier survival curve of B16-challenged mice vaccinated with pFUSE-SCT-Ova/IgG, pIRES-SCT-Ova/IgG, or empty vector control. Data are representative of two independent experiments. Color images available online at www.liebertpub.com/hum

FIG. 5.

Characterization of the tyrosinase-related protein 2 (Trp2)-specific immune response and antitumor effect generated by DNA-encoding SCT-Trp2/IgG or SCT-Trp2. (A) We vaccinated C57BL/6 mice intradermally via gene gun twice at 1-week intervals with plasmid-containing SCT-Trp2/IgG (pFUSE-SCT-Trp2/IgG) or SCT-Trp2 (pIRES-SCT-Trp2). We harvested splenocytes from mice 1 week after the last immunization and analyzed the number of Trp2-specific, interferon (IFN)-γ-secreting CD8⁺ T cells by intracellular cytokine staining and flow cytometry analysis. (B) Bar graph of the flow cytometry data showing the number of Trp2-specific, IFN-γ-secreting CD8⁺ T cells per 3×10⁵ splenocytes (mean+SD). (C) We challenged C57BL/6 mice with B16-F10 tumor cells in the tail vein and vaccinated them intradermally 3 days later with pFUSE-SCT-Trp2/IgG, pIRES-SCT-Trp2, or no insert. We measured the number of pulmonary tumor nodules in the mice (mean+SD). (D) Kaplan-Meier survival curve of B16-challenged mice vaccinated with pFUSE-SCT-Trp2/IgG, pIRES-SCT-Trp2, or no insert. Data are representative of two independent experiments. Color images available online at www.liebertpub.com/hum