Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses in vivo

Abstract

Active cancer immunotherapy, such as cancer vaccine, is based on the fundamental knowledge that tumor‑associated antigens (TAAs) are presented on MHC molecules for recognition by specific T cells. However, most TAAs are self-antigens and are also expressed on normal tissues, including the thymus. This fact raises the issue of the tolerance of the TAA‑specific T‑cell repertoire and consequently the inability to trigger a strong and efficient antitumor immune response. In the present study, we used antigens chemically coupled to the surface of liposomes to target telomerase reverse transcriptase (TERT), a widely expressed self/tumor antigen. Taking advantage of the high homology between mouse and human TERT, we investigated immunogenicity and antitumor efficiency of the liposomal TERT peptides in HLA-A*0201 transgenic HHD mice. Using the heteroclitical peptide-modifying approach with antigen‑coupled liposomes, we identified a novel cryptic epitope with low affinity for HLA*0201 molecules derived from TERT. The heteroclitical variant derived from this novel low affinity peptide exhibited strong affinity for HLA*0201 molecules. However, it induced only weak CD8 T‑cell immune responses in HHD mice when emulsified in IFA. By contrast, when coupled to the surface of the liposomes, it induced powerful CD8 T‑cell immune responses which cross-reacted against the original cryptic epitope. The induced CD8 T cells also recognized endogenously TERT‑expressing tumor cells and inhibited their growth in HHD mice. These data suggest that heteroclitical antigen derived from low affinity epitope of tumor antigens coupled to the surface of liposome may have a role as an effective cancer vaccine candidate.

Figure 1

Immunogenicity of predicted liposomal peptides. Spleen cells from HHD mice vaccinated with a peptide pool conjugated to liposomes were restimulated in vitro with a relevant peptide for 5 h. CD8 T cells were tested for reactivity to each peptide by flow cytometric assay using the degranulation of CD107a and intracellular IFN-γ staining. Percentages of IFN-γ⁺ CD107a⁺ cells in CD8⁺ spleen cells are presented as the mean ± SD of three mice.

Figure 2

Generation of native cryptic epitope reactive CD8 T cells by vaccination with liposomal heteroclitic valiant. (A) CD8 T cells from Lip-#14 immunized mice recognized #14 as well as native epitope #13. Spleen cells from the HHD mice vaccinated with Lip-#13 (native) or Lip-#14 (heteroclitic) were restimulated in vitro with #13 or #14 peptide for 5 h. CD8 T cells were tested for specificity of each peptide by flow cytometric assay using the degranulation of CD107a and intracellular IFN-γ staining. Results are presented as the mean ± SD of three mice. (B) Flow cytometric analysis of HLA-2.1 and TERT expression on RMA and RMA-HHD mouse lymphoma cell lines. (C) CTL activities against TERT-expressing tumor cells. Spleen cells from the HHD mice vaccinated with Lip-#14 were restimulated in vitro with #14 peptide. After 6 days, the CTL-mediated cytotoxic assay was performed with TERT-expressing RMA and RMA-HHD cells as the target.

Figure 3

Comparison of native cryptic epitope specific CD8 T-cell responses in mice immunized with #14 peptide conjugated liposomes or with #14 peptide emulsified in IFA. (A) Spleen cells from naïve HHD or HHD mice vaccinated with Lip-#14 or with p-#14 were stained with #14/HLA-A2.1-dextramer. Representative FACS plot of dextramer staining and the mean ± SD of dextramer⁺ CD8⁺ cells of three mice are presented. (B) Spleen cells from HHD mice vaccinated with Lip-#14 or with P-#14 were restimulated in vitro with #13 peptide for 5 h. CD8 T cells were tested for specificity of the peptide by flow cytometric assay using the degranulation of CD107a and intracellular IFN-γ staining.

Figure 4

Induction of long-lasting memory CD8 T cells. (A) Presence of long-lasting antigen-specific CD8 T cells. HHD Mice were immunized with Lip-#14. On day 35 after immunization, spleen cells were stained with #14/HLA-A2.1-dextramer. Representative FACS plot of dextramer staining and the mean± SD of dextramer⁺ CD8⁺ cells of three mice are presented. (B and C) Immune responses of long-lasting antigen-specific CD8 T cells. The abovementioned spleen cells were restimulated in vitro with #13 peptide for 5 h. The CD8 T cells were tested for specificity of the peptide by flow cytometric assay using the degranulation of CD107a and intracellular IFN-γ staining.

Figure 5

Immunization with liposomal heteroclitic variant inhibits tumor growth in vivo. HHD mice were immunized with control liposome, Lip-#13 or -#14 and inoculated with 2×10⁴ RMA-HHD cells as indicated in Materials and methods. (A) Tumor sizes on day 24 after tumor inoculation. Tumor sizes in HHD mice vaccinated with Lip-#14 were significantly different from the control and Lip-#13-vaccinated mice (^*P<0.05, Mann-Whitney U test). (B) Survival time of tumor inoculated mice. The survival time was estimated using the Kaplan-Meier method. ^*Statistically significant survival value between the Lip-#14 group and the control liposome group (P<0.05, Log-rank test).