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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-specific antibody H200 and its effect on cancer cell growth in vitro(A) The H200 antibody recognizes a 200 amino acid sequence in the extracellular domain of EphB4 spanning the cysteine rich domain (CYS) and the first fibronectin type III repeat (FIII). LBD—globular ligand binding domain, SAM—sterile alpha motif, PDZ—PDZ domain. (B) Flow cytometry comparing parental MCF10A cells with EphB4 over-expressing MCF10A-B4 cells using the H200 antibody showing an increase in fluorescence of the EphB4 over-expressing cells. (C) Western blot analysis with the H200 antibody identifying EphB4 over-expressed in the MCF10A-B4 cells (B4) compared with the parental empty vector only MCF10A-VO cells. β-actin was used to demonstrate equal loading. (D) Immunofluorescence using the H200 antibody to identify EphB4 (green) over-expressed in the MCF10A-B4 cells compared to the parental cells transfected with the empty vector (VO). Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Co-localisation of EphB4 and F-actin (yellow). Bar = 10 μm. (E) Photomicrographs of cells treated with 0.4 μg/ml H200 antibody added to confluent monolayers of SW480 colon cancer cells for different lengths of time. (F) Trypan blue assay of SW480 cells treated with four different EphB4-specific antibodies for 48 h. Antibodies included the Ziemiecki lab rabbit polyclonal antibody (S), EphB4 (N-19) (N), EphB4 (H-200) (H) and EphB4 (C-16) (C) all from Santa Cruz Biotechnology. The H200 antibody showed a reduction in the number of viable cells compared to the control untreated (Con) and the other three antibodies irrespective of the presence (FCS) or absence (CLM) of complement. (G) Trypan blue assay of SW480 cells treated with either 0.2 μg/ml or 1 μg/ml H200 for 48 or 72 h. A significant reduction in the number of viable cells was seen at 72 h with the low concentration (p < 0.05) and at 48 h with the higher concentration (p < 0.001), with no viable cells remaining after 72 h treatment with 1 μg/ml H200. (H) Caspase-3 assay of SW480 cells treated with different concentrations of H200 (as per Figure 1G). The reduction in cell viability correlates with an increase in caspase-3 activity, statistically significant at 72 h with 0.2 μg/ml (p = 0.002) and at 48 h with 1 μg/ml (p = 0.003).
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Figure 1: Validation of the EphB4-specific antibody H200 and its effect on cancer cell growth in vitro(A) The H200 antibody recognizes a 200 amino acid sequence in the extracellular domain of EphB4 spanning the cysteine rich domain (CYS) and the first fibronectin type III repeat (FIII). LBD—globular ligand binding domain, SAM—sterile alpha motif, PDZ—PDZ domain. (B) Flow cytometry comparing parental MCF10A cells with EphB4 over-expressing MCF10A-B4 cells using the H200 antibody showing an increase in fluorescence of the EphB4 over-expressing cells. (C) Western blot analysis with the H200 antibody identifying EphB4 over-expressed in the MCF10A-B4 cells (B4) compared with the parental empty vector only MCF10A-VO cells. β-actin was used to demonstrate equal loading. (D) Immunofluorescence using the H200 antibody to identify EphB4 (green) over-expressed in the MCF10A-B4 cells compared to the parental cells transfected with the empty vector (VO). Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Co-localisation of EphB4 and F-actin (yellow). Bar = 10 μm. (E) Photomicrographs of cells treated with 0.4 μg/ml H200 antibody added to confluent monolayers of SW480 colon cancer cells for different lengths of time. (F) Trypan blue assay of SW480 cells treated with four different EphB4-specific antibodies for 48 h. Antibodies included the Ziemiecki lab rabbit polyclonal antibody (S), EphB4 (N-19) (N), EphB4 (H-200) (H) and EphB4 (C-16) (C) all from Santa Cruz Biotechnology. The H200 antibody showed a reduction in the number of viable cells compared to the control untreated (Con) and the other three antibodies irrespective of the presence (FCS) or absence (CLM) of complement. (G) Trypan blue assay of SW480 cells treated with either 0.2 μg/ml or 1 μg/ml H200 for 48 or 72 h. A significant reduction in the number of viable cells was seen at 72 h with the low concentration (p < 0.05) and at 48 h with the higher concentration (p < 0.001), with no viable cells remaining after 72 h treatment with 1 μg/ml H200. (H) Caspase-3 assay of SW480 cells treated with different concentrations of H200 (as per Figure 1G). The reduction in cell viability correlates with an increase in caspase-3 activity, statistically significant at 72 h with 0.2 μg/ml (p = 0.002) and at 48 h with 1 μg/ml (p = 0.003).

Mentions: The commercial H200 polyclonal antibody (Santa Cruz) was raised to a 200 amino acid sequence in the extracellular domain of human EphB4 that spans the cysteine-rich region and the first fibronectin type III repeat (Figure 1A). To confirm that this antibody can identify EphB4 we compared the non-transformed breast cell line MCF10A which expresses a low endogenous level of EphB4 with MCF10A cells engineered to over-express full length EphB4 (MCF10A-B4) using three different techniques. Initially cell surface expression of EphB4 in both the parental and derivative was compared by flow cytometry. A clear increase in fluorescence of the MCF10A-B4 when compared with the MCF10A cells (green peak shifted to right) shows that the H200 polyclonal antibody (Ab) is binding to surface expressed EphB4 in the MCF10A–B4 cells (Figure 1B). The H200 pAb was then used in Western blot analysis using total protein isolates extracted from both cell lines. A strongly immunoreactive band at the predicted molecular weight of EphB4 (120 kDa) was detected with the H200 antibody in the sample from the MCF10A-B4 cells compared to low expressing control empty vector only MCF10A-VO cells (VO) (Figure 1C). Finally, EphB4 was detected in MCF10A-VO and MCF10A-B4 cells using immunofluorescence (Figure 1D). The increased green fluorescence in the MCF10A-B4 cells when compared with MCF10A-VO cells indicates that the H200 pAb recognizes over-expressed EphB4 protein. Together these results show that the H200 pAb recognizes 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)

Validation of the EphB4-specific antibody H200 and its effect on cancer cell growth in vitro(A) The H200 antibody recognizes a 200 amino acid sequence in the extracellular domain of EphB4 spanning the cysteine rich domain (CYS) and the first fibronectin type III repeat (FIII). LBD—globular ligand binding domain, SAM—sterile alpha motif, PDZ—PDZ domain. (B) Flow cytometry comparing parental MCF10A cells with EphB4 over-expressing MCF10A-B4 cells using the H200 antibody showing an increase in fluorescence of the EphB4 over-expressing cells. (C) Western blot analysis with the H200 antibody identifying EphB4 over-expressed in the MCF10A-B4 cells (B4) compared with the parental empty vector only MCF10A-VO cells. β-actin was used to demonstrate equal loading. (D) Immunofluorescence using the H200 antibody to identify EphB4 (green) over-expressed in the MCF10A-B4 cells compared to the parental cells transfected with the empty vector (VO). Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Co-localisation of EphB4 and F-actin (yellow). Bar = 10 μm. (E) Photomicrographs of cells treated with 0.4 μg/ml H200 antibody added to confluent monolayers of SW480 colon cancer cells for different lengths of time. (F) Trypan blue assay of SW480 cells treated with four different EphB4-specific antibodies for 48 h. Antibodies included the Ziemiecki lab rabbit polyclonal antibody (S), EphB4 (N-19) (N), EphB4 (H-200) (H) and EphB4 (C-16) (C) all from Santa Cruz Biotechnology. The H200 antibody showed a reduction in the number of viable cells compared to the control untreated (Con) and the other three antibodies irrespective of the presence (FCS) or absence (CLM) of complement. (G) Trypan blue assay of SW480 cells treated with either 0.2 μg/ml or 1 μg/ml H200 for 48 or 72 h. A significant reduction in the number of viable cells was seen at 72 h with the low concentration (p < 0.05) and at 48 h with the higher concentration (p < 0.001), with no viable cells remaining after 72 h treatment with 1 μg/ml H200. (H) Caspase-3 assay of SW480 cells treated with different concentrations of H200 (as per Figure 1G). The reduction in cell viability correlates with an increase in caspase-3 activity, statistically significant at 72 h with 0.2 μg/ml (p = 0.002) and at 48 h with 1 μg/ml (p = 0.003).
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Related In: Results  -  Collection

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Figure 1: Validation of the EphB4-specific antibody H200 and its effect on cancer cell growth in vitro(A) The H200 antibody recognizes a 200 amino acid sequence in the extracellular domain of EphB4 spanning the cysteine rich domain (CYS) and the first fibronectin type III repeat (FIII). LBD—globular ligand binding domain, SAM—sterile alpha motif, PDZ—PDZ domain. (B) Flow cytometry comparing parental MCF10A cells with EphB4 over-expressing MCF10A-B4 cells using the H200 antibody showing an increase in fluorescence of the EphB4 over-expressing cells. (C) Western blot analysis with the H200 antibody identifying EphB4 over-expressed in the MCF10A-B4 cells (B4) compared with the parental empty vector only MCF10A-VO cells. β-actin was used to demonstrate equal loading. (D) Immunofluorescence using the H200 antibody to identify EphB4 (green) over-expressed in the MCF10A-B4 cells compared to the parental cells transfected with the empty vector (VO). Phalloidin-TRITC (red) stains F-actin, DAPI (blue) stains the nucleus. Co-localisation of EphB4 and F-actin (yellow). Bar = 10 μm. (E) Photomicrographs of cells treated with 0.4 μg/ml H200 antibody added to confluent monolayers of SW480 colon cancer cells for different lengths of time. (F) Trypan blue assay of SW480 cells treated with four different EphB4-specific antibodies for 48 h. Antibodies included the Ziemiecki lab rabbit polyclonal antibody (S), EphB4 (N-19) (N), EphB4 (H-200) (H) and EphB4 (C-16) (C) all from Santa Cruz Biotechnology. The H200 antibody showed a reduction in the number of viable cells compared to the control untreated (Con) and the other three antibodies irrespective of the presence (FCS) or absence (CLM) of complement. (G) Trypan blue assay of SW480 cells treated with either 0.2 μg/ml or 1 μg/ml H200 for 48 or 72 h. A significant reduction in the number of viable cells was seen at 72 h with the low concentration (p < 0.05) and at 48 h with the higher concentration (p < 0.001), with no viable cells remaining after 72 h treatment with 1 μg/ml H200. (H) Caspase-3 assay of SW480 cells treated with different concentrations of H200 (as per Figure 1G). The reduction in cell viability correlates with an increase in caspase-3 activity, statistically significant at 72 h with 0.2 μg/ml (p = 0.002) and at 48 h with 1 μg/ml (p = 0.003).
Mentions: The commercial H200 polyclonal antibody (Santa Cruz) was raised to a 200 amino acid sequence in the extracellular domain of human EphB4 that spans the cysteine-rich region and the first fibronectin type III repeat (Figure 1A). To confirm that this antibody can identify EphB4 we compared the non-transformed breast cell line MCF10A which expresses a low endogenous level of EphB4 with MCF10A cells engineered to over-express full length EphB4 (MCF10A-B4) using three different techniques. Initially cell surface expression of EphB4 in both the parental and derivative was compared by flow cytometry. A clear increase in fluorescence of the MCF10A-B4 when compared with the MCF10A cells (green peak shifted to right) shows that the H200 polyclonal antibody (Ab) is binding to surface expressed EphB4 in the MCF10A–B4 cells (Figure 1B). The H200 pAb was then used in Western blot analysis using total protein isolates extracted from both cell lines. A strongly immunoreactive band at the predicted molecular weight of EphB4 (120 kDa) was detected with the H200 antibody in the sample from the MCF10A-B4 cells compared to low expressing control empty vector only MCF10A-VO cells (VO) (Figure 1C). Finally, EphB4 was detected in MCF10A-VO and MCF10A-B4 cells using immunofluorescence (Figure 1D). The increased green fluorescence in the MCF10A-B4 cells when compared with MCF10A-VO cells indicates that the H200 pAb recognizes over-expressed EphB4 protein. Together these results show that the H200 pAb recognizes 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