Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

Abstract

BACKGROUND: A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. METHODS: C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+ (mEphA2671-679, mEphA2682-689) and CD4+ (mEphA230-44) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6) establishment/progression was then assessed. RESULTS: Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA) peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. CONCLUSIONS: These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells.

Figure 1

Expression of EphA2 in murine tumor cell lines and tumor/normal tissues. (A) Aliquots of protein lysates (10 μg/lane) were analyzed by Western Blotting for expression of mEphA2 protein using a specific monoclonal antibody (C-20 Ab; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Lysate samples were obtained from mouse tumor lines including the B16, B16V1 and B16BL6 melanomas and the KR129, KR130, KR233 and KR158D glioma cell lines derived from spontaneously developed glioma tissues in NPcis mice. Control β-actin labeling demonstrated equal amount of protein loading in each lane. (B) Protein lysates isolated from normal mouse brain, spleen, liver, lung, heart, skeletal muscle, and the G261 glioma and MCA 205 sarcoma cell lines were separated by SDS-PAGE, blotted onto nitrocellulose and analyzed for expression of mEphA2 using C-20 anti-EphA2 monoclonal antibody.

Figure 2

Immunizations with mEphA2-derived peptides elicit anti-tumor CTLs. C57BL/6 mice received two cycles of s.c. immunization with 5 × 10⁵ DCs loaded with mEphA2-derived peptides or control OVA-derived peptides. One week after the second immunization, the animals were sacrificed, and SPCs were re-stimulated in vitro with the indicated peptides in the presence of 20 IU/ml hIL-2 for 7 days. Standard 4-hr (upper panel) and 20-hr (lower panel) ⁵¹Cr-release assays were used to assess cytotoxicity of the responder SPCs against the EphA2+ MCA205, GL261 and KR158D tumor cell lines. Each value represents the average of triplicate determinations for each group. YAC cells were evaluated as non-specific target cells, while EphA2-negative B16 melanoma cells were examined as negative control target cells in all groups, with the lysis of each of these targets constantly less than 10% (data not shown).

Figure 3

Immunizations with DCs loaded with Eph-A2 derived peptides inhibit the growth of both EphA2-positive and EphA2-negative tumors. (A) C57BL/6 mice received 1 × 10⁵ MCA205 cells s.c. on day 0. These animals were then immunized with 1 × 10⁶ DCs loaded with the indicated peptides on days 3, 10. Tumor growth was monitored for 28 days following the tumor inoculation (N = 5/group). P < 0.05 for both mEphA2_{671–679/30–44} and mEphA2_{682–689/30–44} treatments in comparison to OVA control for the tumor based on a two-tailed Student-t test (*). (B) C57BL/6 mice received s.c. pre-immunizations with 1 × 10⁶ DCs loaded with either mEphA2_671–679, mEphA2_682–689, mEphA2_30–44 or control OVA_257–264 on days -14 and -7 (N = 5 /group), followed by s.c. challenge with 5 × 10⁴ B16 melanoma cells on day 0. On day 14, the size of tumors and the number of animals that had measurable tumors were assessed. P < 0.01 for EphA2_671–679, EphA2_682–689 and EphA2_30–44 treatments in comparison to OVA_257–264 treated group (*).

Figure 4

Inhibition of B16-BL6 lung metastasis and VEGF-induced angiogenesis by s.c. immunizations with DCs loaded with EphA2-derived peptides. (A and B), C57BL/6 mice received i.v. injections of 2 × 10⁵ B16-BL6 cells followed by s.c. immunizations with 1 × 10⁶ DCs loaded with the indicated peptides on days 3, 10 and 17 (N = 4/group). The animals were sacrificed on day 28, with representative macroscopic pictures of the harvested lungs from each group (A) and the numbers of pulmonary surface metastases counted (B) P < 0.001 for both mEphA2_{671–679/30–44} (*) and mEphA2_{682–689/30–44} (**) treatments in comparison to OVA control for the number of metastasis based on two-tailed Student-t test. (C), mEphA2-targeted immunizations inhibited the vascular formation in the Matrigel plug assay. Syngeneic mice pre-immunized with control OVA- (left) or EphA2- (right) peptides were injected s.c. with Matrigel containing VEGF. After 10 days, the plugs were removed and photographed. The picture shows representative VEGF-containing plugs excised from total 5 mice/group.