EphA2 as a glioma-associated antigen: a novel target for glioma vaccines

Abstract

EphA2 is a receptor tyrosine kinase and is frequently overexpressed in a wide array of advanced cancers. We demonstrate in the current study that the EphA2 protein is restrictedly expressed in primary glioblastoma multiforme and anaplastic astrocytoma tissues in comparison to normal brain tissues. To evaluate the possibility of targeting EphA2 in glioma vaccine strategies, we stimulated human leukocyte antigen (HLA) A2+ peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and glioma patients with autologous dendritic cells (DCs) loaded with synthetic EphA2883-891 peptide (TLADFDPRV), which has previously been reported to induce interferon-gamma in HLA-A2+ PBMCs. Stimulated PBMCs demonstrated antigen-specific cytotoxic T lymphocyte (CTL) responses as detected by specific lysis of T2 cells loaded with the EphA2883 peptide as well as HLA-A2+ glioma cells, SNB19 and U251, that express EphA2. Furthermore, in vivo immunization of HLA-A2 transgenic HHD mice with the EphA2883-891 peptide resulted in the development of an epitope-specific CTL response in splenocytes, despite the fact that EphA2883-891 is an autoantigen in these mice. Taken together, these data suggest that EphA2883-891 may be an attractive antigen epitope for molecularly targeted glioma vaccines.

Figure 1

EphA2 expression in GBM tissues. Paraffin-embedded GBM (A–C) or normal brain (D) sections were examined for EphA2 expression using anti-EphA2-specific antibody (Ab208; MedImmune, Inc.) as described in Materials and Methods section. The case in (A) was scored as strongly diffuse, and cases in (B) and (C) were scored as moderately diffuse in Table 1 (original magnification, x20).

Figure 2

Human glioma cell lines express the EphA2 protein at high levels. Expression of EphA2 protein was analyzed in three human glioma cell lines (SNB19, U251, and A172), healthy donor-derived PBLs, and two mouse lymphoma cell lines (EL-4 and EL-4-HHD) by Western blot analysis using anti-EphA2 polyclonal antibody (C-20) and anti-pan-actin mAb (ACTN05) as internal controls.

Figure 3

Stimulation of HLA-A2⁺ donor-derived PBMCs with EphA2_883–891-induced antigen-specific CTL responses. (A and B) T-cell lines generated from a healthy donor (A) or patient 1 (B) by stimulation with EphA2_883–891 peptide were tested for their ability to lyse ⁵¹Cr-labeled T2 pulsed with EphA2_883–891 (▲) or irrelevant MART-1_27–35 (AAGIGILTV) (▼) or without peptides (■). P < .05 at all E/T ratios for EphA2_883–891-pulsed T2 versus other groups. (C) T-cell lines generated from patient 2 with EphA2_883–891 peptide were incubated for 4 hours with ⁵¹Cr-labeled human glioma cell lines SNB19 (●; HLA-A2⁺, EphA2⁺), U251 (■; HLA-A2⁺, EphA2⁺), and A172 (▼; HLA-A2^-, EphA2⁺) at the indicated E/T ratios for evaluation of specific lytic ability. P < .05 at all E/T ratios for A172 vs U251 or SNB19. For the CT inhibition assay (D), ⁵¹Cr-labeled tumor target cells (5 x 10³ cells) and cold T2 cells pulsed with (○) or without (▲) EphA2_883–891 peptide were incubated with CTLs from patient 3. P < .05 at E/T ratios 50 and 100 for peptide-pulsed T2 (cold inhibition) versus other groups. These data are representative of at least three independent experiments with each of the three different donors. Bars = SD.

Figure 4

In vivo vaccination of HHD mice with EphA2_883–891 peptide induced an antigen-specific CTL activity. SPCs obtained from HHD mice that had been immunized with EphA2₈₈₃₈₉₁ peptide were tested their specific lytic activity against EL4-HHD cells pulsed with EphA2_883–891 peptide (■), nonpulsed EL-4-HHD (▲), and control EL-4S3-Rob (◆) cells by standard 4-hour ⁵¹Cr release assays. P < .05 at all E/T ratios higher than 10 for unpulsed EL-4-HHD (▲) versus other groups. These data are representative of at least two independent experiments performed. Bars = SD.