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Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4.

Stephenson SA, Douglas EL, Mertens-Walker I, Lisle JE, Maharaj MS, Herington AC - Oncotarget (2015)

Bottom Line: An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth.Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface.A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Princess Alexandra Hospital, Queensland, Australia.

ABSTRACT
EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

No MeSH data available.


Related in: MedlinePlus

Validation of the EphB4-specificity of the epitope targeting monoclonal antibodies and their use for detecting EphB4 using different techniques(A) Monoclonal antibodies were screened using flow cytometry to detect EphB4 expression on MCF-7 cells. The shift in peak fluorescence to the right (black filled) is compared with the matched isotype control (mIgG1, mIgG2a or rIgG) (red line) and is due to antibody binding. The H200 was used for comparison and the BerEP4 antibody that detects the epithelial glycoprotein EpCam was used as the positive control. (B) Western blot analysis showing that denatured and reduced EphB4 (120 kDa) can be identified using antibodies 13A7, 13B11 and 2D9. The Life Technologies “Zymed” monoclonal antibody was used as the positive control. (C) EphB4 was immunoprecipitated from total protein lysates using several of the monoclonal antibodies. EphB4 was identified in the immunoprecipitated sample via Western blot analysis using the Zymed antibody. (D) Immunofluorescence comparing EphB4 detection (green) in the MCF10A and MCF10A-B4 cells using the monoclonal antibodies. Bar = 80 μm.
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Figure 5: Validation of the EphB4-specificity of the epitope targeting monoclonal antibodies and their use for detecting EphB4 using different techniques(A) Monoclonal antibodies were screened using flow cytometry to detect EphB4 expression on MCF-7 cells. The shift in peak fluorescence to the right (black filled) is compared with the matched isotype control (mIgG1, mIgG2a or rIgG) (red line) and is due to antibody binding. The H200 was used for comparison and the BerEP4 antibody that detects the epithelial glycoprotein EpCam was used as the positive control. (B) Western blot analysis showing that denatured and reduced EphB4 (120 kDa) can be identified using antibodies 13A7, 13B11 and 2D9. The Life Technologies “Zymed” monoclonal antibody was used as the positive control. (C) EphB4 was immunoprecipitated from total protein lysates using several of the monoclonal antibodies. EphB4 was identified in the immunoprecipitated sample via Western blot analysis using the Zymed antibody. (D) Immunofluorescence comparing EphB4 detection (green) in the MCF10A and MCF10A-B4 cells using the monoclonal antibodies. Bar = 80 μm.

Mentions: A panel of murine monoclonal antibodies was raised to the sequence AGSCVVDAVPAPGPSLY (Figure 4E). These antibodies were validated to the EphB4 protein using several methods. Firstly, each antibody was used to detect cell surface expression of EphB4 in MCF-7 cells by flow cytometry (Figure 5A). All antibodies caused an increase in fluorescence of the MCF-7 cells when compared with mIgG labelled cells (black peak shifted to right of clear peak) which shows that the antibodies are binding to the MCF-7 cells via the EphB4 expressed by these cells. Differences in the shape of the peaks, with some antibodies showing a single peak while others showed two peaks, may reflect heterogeneity of EphB4 expression levels and epitope presentation in the MCF-7 cell population, but also the affinities of the various antibodies. The BerEP4 antibody that recognises the epithelial EpCam glycoprotein was used as a positive control and mIgG1a, mIgG2a and rIgG antibodies were used as negative controls. Antibodies were then tested using Western blot analysis of MCF10A-B4 lysates. Three antibodies, 13B11, 2D9 and 13A7, were able to identify denatured and reduced EphB4 protein with 13A7 performing best in this application (Figure 5B). In a third validation experiment, antibodies were used to immunoprecipitate EphB4 from total MCF10A-B4 protein lysates and this was then detected by Western blot analysis using the Zymed antibody. Several antibodies successfully pulled down EphB4 confirming that maintenance of epitope folding is important for binding of some antibodies (Figure 5C). Finally, antibody recognition of EphB4 expressed on MCF10A and MCF10A-B4 cells was compared using immunofluorescence. A stronger green fluorescence signal was seen in MCF10A-B4 cells compared with MCF10A cells using several of the antibodies with 2D9, 6H4 and 11H4 performing particularly well (Figure 5D).


Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4.

Stephenson SA, Douglas EL, Mertens-Walker I, Lisle JE, Maharaj MS, Herington AC - Oncotarget (2015)

Validation of the EphB4-specificity of the epitope targeting monoclonal antibodies and their use for detecting EphB4 using different techniques(A) Monoclonal antibodies were screened using flow cytometry to detect EphB4 expression on MCF-7 cells. The shift in peak fluorescence to the right (black filled) is compared with the matched isotype control (mIgG1, mIgG2a or rIgG) (red line) and is due to antibody binding. The H200 was used for comparison and the BerEP4 antibody that detects the epithelial glycoprotein EpCam was used as the positive control. (B) Western blot analysis showing that denatured and reduced EphB4 (120 kDa) can be identified using antibodies 13A7, 13B11 and 2D9. The Life Technologies “Zymed” monoclonal antibody was used as the positive control. (C) EphB4 was immunoprecipitated from total protein lysates using several of the monoclonal antibodies. EphB4 was identified in the immunoprecipitated sample via Western blot analysis using the Zymed antibody. (D) Immunofluorescence comparing EphB4 detection (green) in the MCF10A and MCF10A-B4 cells using the monoclonal antibodies. Bar = 80 μm.
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Figure 5: Validation of the EphB4-specificity of the epitope targeting monoclonal antibodies and their use for detecting EphB4 using different techniques(A) Monoclonal antibodies were screened using flow cytometry to detect EphB4 expression on MCF-7 cells. The shift in peak fluorescence to the right (black filled) is compared with the matched isotype control (mIgG1, mIgG2a or rIgG) (red line) and is due to antibody binding. The H200 was used for comparison and the BerEP4 antibody that detects the epithelial glycoprotein EpCam was used as the positive control. (B) Western blot analysis showing that denatured and reduced EphB4 (120 kDa) can be identified using antibodies 13A7, 13B11 and 2D9. The Life Technologies “Zymed” monoclonal antibody was used as the positive control. (C) EphB4 was immunoprecipitated from total protein lysates using several of the monoclonal antibodies. EphB4 was identified in the immunoprecipitated sample via Western blot analysis using the Zymed antibody. (D) Immunofluorescence comparing EphB4 detection (green) in the MCF10A and MCF10A-B4 cells using the monoclonal antibodies. Bar = 80 μm.
Mentions: A panel of murine monoclonal antibodies was raised to the sequence AGSCVVDAVPAPGPSLY (Figure 4E). These antibodies were validated to the EphB4 protein using several methods. Firstly, each antibody was used to detect cell surface expression of EphB4 in MCF-7 cells by flow cytometry (Figure 5A). All antibodies caused an increase in fluorescence of the MCF-7 cells when compared with mIgG labelled cells (black peak shifted to right of clear peak) which shows that the antibodies are binding to the MCF-7 cells via the EphB4 expressed by these cells. Differences in the shape of the peaks, with some antibodies showing a single peak while others showed two peaks, may reflect heterogeneity of EphB4 expression levels and epitope presentation in the MCF-7 cell population, but also the affinities of the various antibodies. The BerEP4 antibody that recognises the epithelial EpCam glycoprotein was used as a positive control and mIgG1a, mIgG2a and rIgG antibodies were used as negative controls. Antibodies were then tested using Western blot analysis of MCF10A-B4 lysates. Three antibodies, 13B11, 2D9 and 13A7, were able to identify denatured and reduced EphB4 protein with 13A7 performing best in this application (Figure 5B). In a third validation experiment, antibodies were used to immunoprecipitate EphB4 from total MCF10A-B4 protein lysates and this was then detected by Western blot analysis using the Zymed antibody. Several antibodies successfully pulled down EphB4 confirming that maintenance of epitope folding is important for binding of some antibodies (Figure 5C). Finally, antibody recognition of EphB4 expressed on MCF10A and MCF10A-B4 cells was compared using immunofluorescence. A stronger green fluorescence signal was seen in MCF10A-B4 cells compared with MCF10A cells using several of the antibodies with 2D9, 6H4 and 11H4 performing particularly well (Figure 5D).

Bottom Line: An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth.Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface.A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Princess Alexandra Hospital, Queensland, Australia.

ABSTRACT
EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

No MeSH data available.


Related in: MedlinePlus