Enhancement in specific CD8+ T cell recognition of EphA2+ tumors in vitro and in vivo after treatment with ligand agonists.

The EphA2 receptor tyrosine kinase is an attractive therapeutic target that is commonly overexpressed on solid tumors, with the degree of overexpression associated with disease progression, metastatic potential, and poor prognosis. Agonistic mAbs or ligand (ephrinA1)-Fc fusion protein are capable of inducing EphA2 internalization and degradation, thereby (at least transiently) eliminating the influence of this oncoprotein. We and others have also shown that EphA2 contains multiple peptide epitopes that can be recognized by effector CD4(+) and CD8(+) T cells isolated from tumor-bearing patients. Herein, we show that "agonist" reagents that trigger the proteasome-dependent degradation of tumor cell EphA2 result in the improved presentation of peptides derived from (both the extracellular and intracellular domains of) EphA2 in MHC class I complexes expressed on the tumor cell membrane for at least 48 h, as manifested by increased recognition by EphA2-specific CD8(+) T cells in vitro. We also observed that while delivery of ephrinA1-Fc fusion protein or agonist mAb into EphA2(+) tumor lesions promotes EphA2 degradation in situ, this single administration of agent does not dramatically alter tumor progression in a humanized SCID model. However, when combined with the adoptive transfer of normally nontherapeutic (human) anti-EphA2 CD8(+) CTL, this dual-agent regimen results in complete tumor eradication. These results suggest that strategies targeting the conditional proteasome-mediated destruction of tumor cell EphA2 may enable EphA2-specific CD8(+) T cells (of modest functional avidity) to realize improved therapeutic potential.

Expression of EphA2 is prognostic of disease-free interval and overall survival in surgically treated patients with renal cell carcinoma.

Whereas normally expressed at sites of cell-to-cell contact in adult epithelial tissues, recent studies have shown that the receptor tyrosine kinase EphA2 is overexpressed in numerous epithelial-type carcinomas, with the greatest level of EphA2 expression observed in metastatic lesions. In the current study, we have assessed EphA2 expression in archived renal cell carcinoma (RCC) tissues as it relates to patient disease course. Using specific anti-EphA2 monoclonal antibody 208 and immunohistochemistry, we evaluated EphA2 protein expression levels in RCC specimens surgically resected from 34 patients (including 30 conventional clear-cell RCC, 3 papillary, and 1 chromophobic RCC cases) resulting in clinical cures. Regardless of histopathologic subtype, RCC lesions expressing higher levels of EphA2 tended to be of a higher grade (P < 0.05) and larger (P = 0.093), more-highly-vascularized tumors (P = 0.005). Perhaps most notable, the degree of EphA2 overexpression (versus normal matched autologous kidney tissue) seemed predictive of short-term (<1 year) versus longer-term (> or =1 year) disease-free interval (P < 0.001) and of overall survival (P < 0.001) among the RCC patients evaluated. These data suggest that EphA2 expression level may serve as a useful prognostic tool in the clinical management of patients who have been successfully treated with surgery, but who are at greater risk for accelerated disease recurrence and who have a poorer prognosis.

Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma.

We have evaluated CD8+ and CD4+ T-cell responses against a new tumor-associated antigen, the receptor tyrosine kinase EphA2, which is broadly expressed in diverse cancer histologies and is frequently overexpressed in advanced stage/metastatic disease. We report herein that EphA2 is overexpressed in renal cell carcinoma (RCC) cell lines and clinical specimens of RCC, and find that the highest levels of EphA2 are consistently found in the most advanced stages of the disease. We identified and synthesized five putative HLA class I-binding and three class II-binding peptides derived from EphA2 that might serve as targets for immune reactivity. Each peptide induced specific, tumor-reactive CD8+ or CD4+T-cell responses as measured using IFN-gamma enzyme-linked immunospot assays. The EphA2 peptides elicited relatively weak responses from CD8+ T cells derived from normal healthy volunteers or from RCC patients with active disease. In marked contrast, immune reactivity to EphA2-derived epitopes was greatly enhanced in CD8+ T cells that had been isolated from patients who were rendered disease-free, after surgery. Furthermore, enzyme-linked immunospot analyses demonstrated prominent EphA2-restricted T-helper 1-type CD4+ T cell activity in patients with early stage disease, whereas T-helper 2-type and T regulatory-type responses predominated in patients with more advanced forms of RCC. These data suggest that the immune system of cancer patients actively monitors EphA2-derived epitopes, and that the magnitude and character of T-cell responses to EphA2 epitopes may convey much-needed predictive information about disease stage and outcome.