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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

Antibody C2 is effective against MDA-MB-231 cells with surface EphB4 expression(A) Each antibody was tested for their ability to prevent tubular network formation of MDA-MB-231 cells grown on Matrigel. C2 was the most effective in duplicate wells. (B) Tumour growth data for MDA-MB231 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Doxorubicin™ (green triangles) or untreated (PBS) control group (blue diamonds). The C2 monoclonal antibody significantly reduces the size of tumours by 18 days (p < 0.001) and even performs better than Doxorubicin™ over this time course. (C) Tumour growth data for PC3 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Taxol (green triangles) or untreated (PBS) control group (blue diamonds). The C2 antibody does not affect PC3 tumour growth. (D) Immunofluorescence localization of EphB4 (green) in MDA-MB-231 cells using the C2 antibody. Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Bar = 10 μm. (E) Immunofluorescence localization of EphB4, using the C2 antibody (green), and the endoplasmic reticulum marker calnexin (red) in PC3 cells. DAPI (blue) stains the nucleus. Bar = 10 μm.
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Figure 6: Antibody C2 is effective against MDA-MB-231 cells with surface EphB4 expression(A) Each antibody was tested for their ability to prevent tubular network formation of MDA-MB-231 cells grown on Matrigel. C2 was the most effective in duplicate wells. (B) Tumour growth data for MDA-MB231 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Doxorubicin™ (green triangles) or untreated (PBS) control group (blue diamonds). The C2 monoclonal antibody significantly reduces the size of tumours by 18 days (p < 0.001) and even performs better than Doxorubicin™ over this time course. (C) Tumour growth data for PC3 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Taxol (green triangles) or untreated (PBS) control group (blue diamonds). The C2 antibody does not affect PC3 tumour growth. (D) Immunofluorescence localization of EphB4 (green) in MDA-MB-231 cells using the C2 antibody. Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Bar = 10 μm. (E) Immunofluorescence localization of EphB4, using the C2 antibody (green), and the endoplasmic reticulum marker calnexin (red) in PC3 cells. DAPI (blue) stains the nucleus. Bar = 10 μm.

Mentions: Cell based assays testing antibodies that recognise a native epitope of the target protein where the effect of the antibody on the growth of the cells can be determined are an important prelude to in vivo testing. Because EphB4 has been shown to be important to cancer cell migration and invasion, we screened our antibodies for their effect on the formation of cancer cell networks on Matrigel, a cell-based assay that recapitulates in vivo cell growth and particularly the ability of cancer cells to migrate and invade the extracellular matrix. In this assay, the C2 antibody best prevented network formation of MDA-MB-231 breast cancer cells in both duplicate wells with 1H4 showing a web-like network similar to that of the no antibody or mIgG controls (Figure 6A and data not shown). For this reason C2 was tested for its effect on the growth of established MDA-MB-231 murine xenografts. Based on acute tolerated dose (ATD) and maximum tolerated dose (MTD) studies performed by VivoPharm (Adelaide, Australia), the maximum dose of 50 mg/kg C2 antibody was chosen. After 18 days (with C2 treatment given every three days), MDA-MB-231 tumours showed a significant decrease in size when compared with control untreated tumours and this was comparable to the response of the tumours to the chemotherapeutic agent, Doxorubicin (Figure 6B). We also tested the PC3 prostate cancer cell line, which has been reported to over-express EphB4 endogenously and for which knockdown experiments have shown EphB4 expression is required for cancer cell viability, migration and invasion [6]. The PC3 tumours did not respond to the C2 antibody (Figure 6C). Immunofluorescence using the antibody C2 to visualize EphB4 in MDA-MB-231 and PC3 cells shows that the C2-responsive MDA-MB-231 cells express EphB4 on the surface and in the cytoplasm (Figure 6D) but the PC3 cells show little surface expression with most of the C2-recognising EphB4 co-localising with the endoplasmic reticulum marker calnexin (Figure 6E). This highlights the need for complementary development of a screening technique that can accurately identify patients with high level surface expression of EphB4 and therefore those most likely to benefit from a monoclonal antibody therapy targeting EphB4.


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)

Antibody C2 is effective against MDA-MB-231 cells with surface EphB4 expression(A) Each antibody was tested for their ability to prevent tubular network formation of MDA-MB-231 cells grown on Matrigel. C2 was the most effective in duplicate wells. (B) Tumour growth data for MDA-MB231 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Doxorubicin™ (green triangles) or untreated (PBS) control group (blue diamonds). The C2 monoclonal antibody significantly reduces the size of tumours by 18 days (p < 0.001) and even performs better than Doxorubicin™ over this time course. (C) Tumour growth data for PC3 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Taxol (green triangles) or untreated (PBS) control group (blue diamonds). The C2 antibody does not affect PC3 tumour growth. (D) Immunofluorescence localization of EphB4 (green) in MDA-MB-231 cells using the C2 antibody. Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Bar = 10 μm. (E) Immunofluorescence localization of EphB4, using the C2 antibody (green), and the endoplasmic reticulum marker calnexin (red) in PC3 cells. DAPI (blue) stains the nucleus. Bar = 10 μm.
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Figure 6: Antibody C2 is effective against MDA-MB-231 cells with surface EphB4 expression(A) Each antibody was tested for their ability to prevent tubular network formation of MDA-MB-231 cells grown on Matrigel. C2 was the most effective in duplicate wells. (B) Tumour growth data for MDA-MB231 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Doxorubicin™ (green triangles) or untreated (PBS) control group (blue diamonds). The C2 monoclonal antibody significantly reduces the size of tumours by 18 days (p < 0.001) and even performs better than Doxorubicin™ over this time course. (C) Tumour growth data for PC3 xenograft tumours treated daily for 18 d with 50 mg/kg anti-EphB4 antibody C2 (red squares), positive control chemotherapeutic Taxol (green triangles) or untreated (PBS) control group (blue diamonds). The C2 antibody does not affect PC3 tumour growth. (D) Immunofluorescence localization of EphB4 (green) in MDA-MB-231 cells using the C2 antibody. Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Bar = 10 μm. (E) Immunofluorescence localization of EphB4, using the C2 antibody (green), and the endoplasmic reticulum marker calnexin (red) in PC3 cells. DAPI (blue) stains the nucleus. Bar = 10 μm.
Mentions: Cell based assays testing antibodies that recognise a native epitope of the target protein where the effect of the antibody on the growth of the cells can be determined are an important prelude to in vivo testing. Because EphB4 has been shown to be important to cancer cell migration and invasion, we screened our antibodies for their effect on the formation of cancer cell networks on Matrigel, a cell-based assay that recapitulates in vivo cell growth and particularly the ability of cancer cells to migrate and invade the extracellular matrix. In this assay, the C2 antibody best prevented network formation of MDA-MB-231 breast cancer cells in both duplicate wells with 1H4 showing a web-like network similar to that of the no antibody or mIgG controls (Figure 6A and data not shown). For this reason C2 was tested for its effect on the growth of established MDA-MB-231 murine xenografts. Based on acute tolerated dose (ATD) and maximum tolerated dose (MTD) studies performed by VivoPharm (Adelaide, Australia), the maximum dose of 50 mg/kg C2 antibody was chosen. After 18 days (with C2 treatment given every three days), MDA-MB-231 tumours showed a significant decrease in size when compared with control untreated tumours and this was comparable to the response of the tumours to the chemotherapeutic agent, Doxorubicin (Figure 6B). We also tested the PC3 prostate cancer cell line, which has been reported to over-express EphB4 endogenously and for which knockdown experiments have shown EphB4 expression is required for cancer cell viability, migration and invasion [6]. The PC3 tumours did not respond to the C2 antibody (Figure 6C). Immunofluorescence using the antibody C2 to visualize EphB4 in MDA-MB-231 and PC3 cells shows that the C2-responsive MDA-MB-231 cells express EphB4 on the surface and in the cytoplasm (Figure 6D) but the PC3 cells show little surface expression with most of the C2-recognising EphB4 co-localising with the endoplasmic reticulum marker calnexin (Figure 6E). This highlights the need for complementary development of a screening technique that can accurately identify patients with high level surface expression of EphB4 and therefore those most likely to benefit from a monoclonal antibody therapy targeting EphB4.

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