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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

Peptide exclusion assays to map the critical epitope recognized by EphB4 H200 antibody(A) Six 25 amino acid peptides were designed to the first 125 amino acids of the 200 amino acid region (201–400) recognised by the H200 antibody and encompassing most of the cysteine rich region (184–320). Peptide 7 spans the overlap between peptides 1 and 2. (B) Cell viability was determined in the presence of H200 pAb pre-incubated with a mixture of peptides 1–6 before addition to the cells (Ab+ all peptides, 0.1 mg/ml each) and cell viability was determined using a trypan blue assay after 65 h. Viability of cells treated with the peptide blocked H200 pAb was consistent with the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) for 65 h (***p < 0.001). This experiment was performed in duplicate. (C) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1–6 individually, as indicated, before addition to the cells. At 65 h, peptides 1 and 2 restored viability to 50% of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (*p < 0.01, **p < 0.05, ***p < 0.001). (D) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1 and 2, or 0.2 mg/ml Peptide 1, or 0.2 mg/ml Peptide 2 as indicated before addition to the cells. At 65 h, viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (***p < 0.001). (E) H200 pAb was pre-incubated with 0.2 mg/ml Peptide 7 (spans the overlapping sequence common to Peptides 1 and 2). Viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone). Because the peptide 7 solution was acidic and this may have negatively affected the antibody binding, antibody was also pre-treated with PBS adjusted to pH 4.7. Viable cells after antibody pH treatment was consistent with viability the antibody untreated (Ab alone) (***p < 0.001, NS = not significant).
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Figure 3: Peptide exclusion assays to map the critical epitope recognized by EphB4 H200 antibody(A) Six 25 amino acid peptides were designed to the first 125 amino acids of the 200 amino acid region (201–400) recognised by the H200 antibody and encompassing most of the cysteine rich region (184–320). Peptide 7 spans the overlap between peptides 1 and 2. (B) Cell viability was determined in the presence of H200 pAb pre-incubated with a mixture of peptides 1–6 before addition to the cells (Ab+ all peptides, 0.1 mg/ml each) and cell viability was determined using a trypan blue assay after 65 h. Viability of cells treated with the peptide blocked H200 pAb was consistent with the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) for 65 h (***p < 0.001). This experiment was performed in duplicate. (C) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1–6 individually, as indicated, before addition to the cells. At 65 h, peptides 1 and 2 restored viability to 50% of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (*p < 0.01, **p < 0.05, ***p < 0.001). (D) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1 and 2, or 0.2 mg/ml Peptide 1, or 0.2 mg/ml Peptide 2 as indicated before addition to the cells. At 65 h, viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (***p < 0.001). (E) H200 pAb was pre-incubated with 0.2 mg/ml Peptide 7 (spans the overlapping sequence common to Peptides 1 and 2). Viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone). Because the peptide 7 solution was acidic and this may have negatively affected the antibody binding, antibody was also pre-treated with PBS adjusted to pH 4.7. Viable cells after antibody pH treatment was consistent with viability the antibody untreated (Ab alone) (***p < 0.001, NS = not significant).

Mentions: The EphB4 polyclonal antibody was raised against a recombinant protein corresponding to amino acids 201–400 mapping within an extracellular domain region of human EphB4 (Figure 1A). The sequence includes the cysteine-rich domain and the first fibronectin type III repeat and accordingly it was possible that several different antigenic regions were recognized. We chose to explore the cysteine-rich region in the first instance and designed six, 25 amino acid peptides corresponding to this region for antibody competition studies (Figure 3A, peptides 1–6). Each peptide (0.1 mg/ml) was tested individually over 65 h in culture for potential toxic effects on cancer cell growth. None of the peptides caused morphological, cell growth or apoptotic changes (data not shown), indicating that the peptides themselves were not toxic to the cells.


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)

Peptide exclusion assays to map the critical epitope recognized by EphB4 H200 antibody(A) Six 25 amino acid peptides were designed to the first 125 amino acids of the 200 amino acid region (201–400) recognised by the H200 antibody and encompassing most of the cysteine rich region (184–320). Peptide 7 spans the overlap between peptides 1 and 2. (B) Cell viability was determined in the presence of H200 pAb pre-incubated with a mixture of peptides 1–6 before addition to the cells (Ab+ all peptides, 0.1 mg/ml each) and cell viability was determined using a trypan blue assay after 65 h. Viability of cells treated with the peptide blocked H200 pAb was consistent with the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) for 65 h (***p < 0.001). This experiment was performed in duplicate. (C) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1–6 individually, as indicated, before addition to the cells. At 65 h, peptides 1 and 2 restored viability to 50% of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (*p < 0.01, **p < 0.05, ***p < 0.001). (D) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1 and 2, or 0.2 mg/ml Peptide 1, or 0.2 mg/ml Peptide 2 as indicated before addition to the cells. At 65 h, viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (***p < 0.001). (E) H200 pAb was pre-incubated with 0.2 mg/ml Peptide 7 (spans the overlapping sequence common to Peptides 1 and 2). Viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone). Because the peptide 7 solution was acidic and this may have negatively affected the antibody binding, antibody was also pre-treated with PBS adjusted to pH 4.7. Viable cells after antibody pH treatment was consistent with viability the antibody untreated (Ab alone) (***p < 0.001, NS = not significant).
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Figure 3: Peptide exclusion assays to map the critical epitope recognized by EphB4 H200 antibody(A) Six 25 amino acid peptides were designed to the first 125 amino acids of the 200 amino acid region (201–400) recognised by the H200 antibody and encompassing most of the cysteine rich region (184–320). Peptide 7 spans the overlap between peptides 1 and 2. (B) Cell viability was determined in the presence of H200 pAb pre-incubated with a mixture of peptides 1–6 before addition to the cells (Ab+ all peptides, 0.1 mg/ml each) and cell viability was determined using a trypan blue assay after 65 h. Viability of cells treated with the peptide blocked H200 pAb was consistent with the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) for 65 h (***p < 0.001). This experiment was performed in duplicate. (C) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1–6 individually, as indicated, before addition to the cells. At 65 h, peptides 1 and 2 restored viability to 50% of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (*p < 0.01, **p < 0.05, ***p < 0.001). (D) H200 pAb was pre-incubated with 0.1 mg/ml of each of the peptides 1 and 2, or 0.2 mg/ml Peptide 1, or 0.2 mg/ml Peptide 2 as indicated before addition to the cells. At 65 h, viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone) (***p < 0.001). (E) H200 pAb was pre-incubated with 0.2 mg/ml Peptide 7 (spans the overlapping sequence common to Peptides 1 and 2). Viability was restored to the level of the control PBS-treated cells (untreated). There were no viable cells after treatment with the antibody alone (Ab alone). Because the peptide 7 solution was acidic and this may have negatively affected the antibody binding, antibody was also pre-treated with PBS adjusted to pH 4.7. Viable cells after antibody pH treatment was consistent with viability the antibody untreated (Ab alone) (***p < 0.001, NS = not significant).
Mentions: The EphB4 polyclonal antibody was raised against a recombinant protein corresponding to amino acids 201–400 mapping within an extracellular domain region of human EphB4 (Figure 1A). The sequence includes the cysteine-rich domain and the first fibronectin type III repeat and accordingly it was possible that several different antigenic regions were recognized. We chose to explore the cysteine-rich region in the first instance and designed six, 25 amino acid peptides corresponding to this region for antibody competition studies (Figure 3A, peptides 1–6). Each peptide (0.1 mg/ml) was tested individually over 65 h in culture for potential toxic effects on cancer cell growth. None of the peptides caused morphological, cell growth or apoptotic changes (data not shown), indicating that the peptides themselves were not toxic to the cells.

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