[Show abstract][Hide abstract] ABSTRACT: Overexpression and altered function of EphA2 receptor tyrosine kinase are critical in the progression of breast cancer and provide a target for breast cancer therapy. We have previously demonstrated that EphA2 overexpression decreases estrogen dependence and Tamoxifen sensitivity both in vitro and in vivo. EA5, a novel monoclonal antibody that mimicks the binding of ephrin A to EphA2, reverses the effect of EphA2 overexpression and restores Tamoxifen sensitivity in EphA2-transfected MCF-7 cells in vitro. To explore the role of EphA2 overexpression on ER-dependent mechanisms, we used two different ER+/EphA2-transfected cell line models (MCF-7(neo)/MCF-7(EphA2) and T47D(neo)/T47D(EphA2)). EA5 inhibits primary tumor growth and restores Tamoxifen sensitivity in the MCF-7(EphA2) xenografts. Using the T47D(EphA2) in vitro model, we verified that EphA2 decreases ER activation in response to E2 stimulation consistent with our earlier results in MCF-7(EphA2) model. We found no direct interaction between ER and EphA2 and no difference in expression of canonical ER-dependent proteins or ER co-regulators. However, E2 stimulation phosphorylates FAK(Tyr925) only in ER+/EphA2+ cell lines. Treatment of T47D(EphA2) cells with EA5 and Tamoxifen leads to dephosphorylation of FAK(Tyr925) in response to E2. Our data demonstrate that dual targeting of EphA2 and ER is a promising approach for delaying resistance to Tamoxifen. The data support our hypothesis that EphA2 impacts ER function via a FAK dependent pathway.
Full-text · Article · Jun 2011 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: EphA2 overexpression predicts poor prognosis in endometrial cancer. To explore mechanisms for this association and assess its potential as therapeutic target, the relationship of EphA2 expression to markers of angiogenesis was examined using patient samples and an orthotopic mouse model of uterine cancer.
Expression of EphA2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was evaluated using immunohistochemistry in 85 endometrioid endometrial adenocarcinomas (EEC) by two independent investigators. Results were correlated with clinicopathological characteristics. The effect of EphA2- agonist monoclonal antibody EA5, alone or in combination with docetaxel was studied in vitro and in vivo. Samples were analyzed for markers of angiogenesis, proliferation and apoptosis.
Of 85 EEC samples, EphA2 was overexpressed in 47% of tumors and was significantly associated with high VEGF expression (p=0.001) and high MVD counts (p=0.02). High EphA2 expression, high VEGF expression and high MVD counts were significantly associated with shorter disease-specific survival. EA5 led to decrease in EphA2 expression and phosphorylation in vitro. In the murine model, while EA5 (33-88%) and docetaxel (23-55%) individually led to tumor inhibition over controls, combination therapy had the greatest efficacy (78-92%, p.
Full-text · Article · Jan 2011 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Dec 2009 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.
Full-text · Article · Jul 2009 · Neoplasia (New York, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV-PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV-PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.
Full-text · Article · Feb 2008 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: The EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in malignant cells and thus provides opportunities for selective targeting of tumor cells. We describe here the development of a novel, bispecific single-chain antibody (bscAb) referred to as bscEphA2xCD3. This molecule simultaneously targets EphA2 on tumor cells and the T-cell receptor/CD3 complex on T cells and possesses structural and functional characteristics of the recently developed BiTE technology. An EphA2-specific single-chain antibody was selected for recognition of an epitope that is preferentially exposed on malignant cells based on the concept of epitope exclusion; this was fused to a CD3-specific single-chain antibody to generate bscEphA2xCD3. The resultant bscAb redirected unstimulated human T cells to lyse EphA2-expressing tumor cells both in vitro and in vivo. In separate experiments, efficient tumor cell lysis was achieved in vitro at drug concentrations <or=1 microg/mL, at a low T-cell effector-to-tumor target cell ratio (1:1), and with tumor cells that possess few available binding sites (2,400 per cell) for bscEphA2xCD3. Time-lapsed microscopy revealed potent cytotoxic activity of bscEphA2xCD3-activated T cells against monolayers of malignant cells but not against monolayers of nontransformed EphA2-positive cells except at the edges of the monolayer where the target epitope was exposed. BscEphA2xCD3 was also efficacious in human xenograft mouse models modified to show human T-cell killing of tumors. Together, our results reveal opportunities for redirecting the potent activity of cytotoxic T cells towards tumor cells that express selectively accessible epitopes and establish EphA2-specific bscAb molecules as novel and potent therapeutics with selectivity for tumor cells.
[Show abstract][Hide abstract] ABSTRACT: EphA2 is an oncoprotein and tyrosine kinase receptor that is overexpressed in ovarian and many other cancers. We investigated the effects of reduced EphA2 levels on tumor growth and the tumor microenvironment in an orthotopic ovarian cancer model.
The effect of the EphA2-agonistic monoclonal antibody EA5, alone or in combination with paclitaxel, on the growth of ovarian cancer cells (SKOV3ip1, HeyA8, and HeyA8MDR [taxane-platinum resistant]) was determined in vitro and in vivo by immunoblotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and immunohistochemical analysis. Expression of EphA2 and markers of angiogenesis (CD31, vascular endothelial growth factor [VEGF], and basic fibroblast growth factor), proliferation (proliferating cell nuclear antigen), and endothelial cell apoptosis (CD31-terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick-end labeling colocalization) and phosphorylation of Src were analyzed by immunoblotting, immunohistochemistry, immunofluorescence, and in situ hybridization in tumors from treated mice. Statistical tests were two-sided.
EA5 antibody treatment led to a more than 90% reduction in EphA2 expression in HeyA8 tumors in vivo. In mice bearing orthotopic SKOV3ip1 or HeyA8 tumors, 4 weeks of EA5 treatment resulted in tumors that weighed 31% and 45% less, respectively, than those in control (IgG-treated) mice (95% confidence interval [CI] = -0.09% to 71% and 20% to 70%, P = .27 and .01, respectively). Combination therapy with EA5 and paclitaxel reduced tumor weight by 77% and 80% (95% CI = 63% to 91% and 68% to 91%), respectively, compared with paclitaxel alone and by 92% and 88% (95% CI = 87% to 97% and 80% to 94%), respectively, compared with IgG alone. Combination therapy also reduced the weight of HeyA8MDR tumors by 47% (95% CI = 24% to 72%) compared with paclitaxel. Mice bearing SKOV3ip1 or HeyA8 tumors that were treated with combination therapy survived longer than those treated with paclitaxel alone (median survival = 144 versus 69 days and 46 versus 37 days, respectively). EA5-treated tumors had reduced microvascular density, proliferation, and VEGF protein and mRNA levels, with increased endothelial cell apoptosis. EphA2 was associated with Src, which was rapidly dephosphorylated after EA5 treatment.
EA5 in combination with paclitaxel decreased tumor growth in an orthotopic ovarian cancer mouse model through antiangiogenic mechanisms associated with reduced levels of VEGF and phosphorylated Src. Humanized antibody constructs against EphA2 are worthy of future study.
Full-text · Article · Dec 2006 · Journal of the National Cancer Institute