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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 specific antibodies identify EphB4, cause EphB4 protein phosphorylation and degradation(A) Antibodies specifically recognizing Peptide 7 were isolated from the H200 polyclonal preparation. Western blot analysis shows that these antibodies (H7) specifically identify EphB4 with less non-specific cross-reactivity than the H200 antibody. β-actin was used as a loading control. (B) Western blot and graph showing that in a similar manner to H200, the H7 antibody, tested at two different dilutions, reduces total EphB4 protein level with a significant reduction seen at 72 h (p = 0.0001). (C) Immunoprecipitation of cell lysates from MCF-7 breast cancer cells showing treatment with the H7 antibody causes phosphorylation of the EphB4 protein, seen at 5 min after addition and maintained at 30 min (yellow box). Cells were stimulated with soluble clustered ephrin-B2-Fc ligand (eB2) as a positive control for phosphorylation with clustered Fc fragment only (Fc) as the negative control for this. (D) Alignment of the EphB4 sequence of peptides 1 and 2, which includes peptide 7 sequence AGSCVVDA (red box) with the corresponding sequence from EphA3. Asterisks identify the amino acids identified as contributing to the third ligand binding domain. (E) Comparison of the peptide sequence used to raise EphB4 monoclonal antibodies (green) with other members of the human Eph family. Amino acids identical to the EphB4 sequence are shaded in grey, homologous amino acids are shaded pink. The antigenic aspartic acid reside is within a red box. (F) Comparison of the peptide sequence used to raise human EphB4 monoclonal antibodies (green) with EphB4 sequences from other species. Amino acids identical to the human EphB4 sequence are shaded in grey. (G) Western blot analysis using the H200 and 13A7 antibodies with recombinant human EphB4 extracellular domain (rhEphB4ecd) (H) and recombinant mouse EphB4 extracellular domain Fc fusion protein (rmEphB4ecd-Fc) (M). The arrows indicate the position of the proteins compared to the molecular weight marker for which sizes are indicated on the left.
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Figure 4: Peptide specific antibodies identify EphB4, cause EphB4 protein phosphorylation and degradation(A) Antibodies specifically recognizing Peptide 7 were isolated from the H200 polyclonal preparation. Western blot analysis shows that these antibodies (H7) specifically identify EphB4 with less non-specific cross-reactivity than the H200 antibody. β-actin was used as a loading control. (B) Western blot and graph showing that in a similar manner to H200, the H7 antibody, tested at two different dilutions, reduces total EphB4 protein level with a significant reduction seen at 72 h (p = 0.0001). (C) Immunoprecipitation of cell lysates from MCF-7 breast cancer cells showing treatment with the H7 antibody causes phosphorylation of the EphB4 protein, seen at 5 min after addition and maintained at 30 min (yellow box). Cells were stimulated with soluble clustered ephrin-B2-Fc ligand (eB2) as a positive control for phosphorylation with clustered Fc fragment only (Fc) as the negative control for this. (D) Alignment of the EphB4 sequence of peptides 1 and 2, which includes peptide 7 sequence AGSCVVDA (red box) with the corresponding sequence from EphA3. Asterisks identify the amino acids identified as contributing to the third ligand binding domain. (E) Comparison of the peptide sequence used to raise EphB4 monoclonal antibodies (green) with other members of the human Eph family. Amino acids identical to the EphB4 sequence are shaded in grey, homologous amino acids are shaded pink. The antigenic aspartic acid reside is within a red box. (F) Comparison of the peptide sequence used to raise human EphB4 monoclonal antibodies (green) with EphB4 sequences from other species. Amino acids identical to the human EphB4 sequence are shaded in grey. (G) Western blot analysis using the H200 and 13A7 antibodies with recombinant human EphB4 extracellular domain (rhEphB4ecd) (H) and recombinant mouse EphB4 extracellular domain Fc fusion protein (rmEphB4ecd-Fc) (M). The arrows indicate the position of the proteins compared to the molecular weight marker for which sizes are indicated on the left.

Mentions: Peptide 7 was immobilized onto a MicroLink (gel matrix used to isolate interacting antibodies (designated H7) from the H200 polyclonal antibody preparation. To confirm that isolated antibodies could specifically identify EphB4 we again compared the MCF10A and MCF10A-B4 cells using Western blot analysis (Figure 4A). When compared with the H200 pAb, the H7 antibody detected only a single strongly immunoreactive band at the predicted 120 kDa, seen in the MCF10A-B4 sample and less strongly in MCF10A, consistent with detection of the over-expressed EphB4 in MCF10A-B4 and the endogenous EphB4 in MCF10A. Extended stimulation (for 72 h) led to a loss of EphB4 protein from the MCF-7 cells in a similar manner to that of the parental H200 antibody (Figure 4B and Figure 2F). Addition of the purified H7 antibody to MCF-7 cells caused an increase in EphB4 phosphorylation as soon as 5 min after addition which was still maintained at 30 min suggesting that the antibody activates EphB4 signaling in treated cells (Figure 4C). This would suggest that the antibody is behaving as an ephrin-B2 mimetic as ligand stimulation of EphB4 normally leads to phosphorylation of the kinase domain, then internalization and degradation of the receptor. In keeping with this hypothesis, the AGSCVVDA sequence contains three amino acids that align with those of a defined third ligand interface region on the EphA3 receptor (Figure 4D). Comparison with sequences of other Eph receptors shows variability at key residues suggesting high epitope specificity of the H7 antibody (Figure 4E). For example, analysis of the sequence using the EMBOSS antigenic prediction program (http://emboss.sourceforge.net/apps/release/6.0/emboss/apps/antigenic.html) identifies the aspartic acid residue in the EphB4 sequence (red box in Figure 4E) as the key amino acid within the most antigenic sequence in the 200 amino acid region targeted by the H200 antibody. Comparison with sequences from other species shows few differences in the amino acid sequence of the corresponding regions but the most differences to mouse EphB4 (Figure 4F) and Western blot analysis shows that although the H200 antibody can detect both human and mouse EphB4 proteins, the 13A7 antibody that targets the peptide 7 sequence specifically, only identified human EphB4 protein (rhEphB4ecd) and not murine EphB4 protein (rmEphB4ecd-Fc) (Figure 4G).


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 specific antibodies identify EphB4, cause EphB4 protein phosphorylation and degradation(A) Antibodies specifically recognizing Peptide 7 were isolated from the H200 polyclonal preparation. Western blot analysis shows that these antibodies (H7) specifically identify EphB4 with less non-specific cross-reactivity than the H200 antibody. β-actin was used as a loading control. (B) Western blot and graph showing that in a similar manner to H200, the H7 antibody, tested at two different dilutions, reduces total EphB4 protein level with a significant reduction seen at 72 h (p = 0.0001). (C) Immunoprecipitation of cell lysates from MCF-7 breast cancer cells showing treatment with the H7 antibody causes phosphorylation of the EphB4 protein, seen at 5 min after addition and maintained at 30 min (yellow box). Cells were stimulated with soluble clustered ephrin-B2-Fc ligand (eB2) as a positive control for phosphorylation with clustered Fc fragment only (Fc) as the negative control for this. (D) Alignment of the EphB4 sequence of peptides 1 and 2, which includes peptide 7 sequence AGSCVVDA (red box) with the corresponding sequence from EphA3. Asterisks identify the amino acids identified as contributing to the third ligand binding domain. (E) Comparison of the peptide sequence used to raise EphB4 monoclonal antibodies (green) with other members of the human Eph family. Amino acids identical to the EphB4 sequence are shaded in grey, homologous amino acids are shaded pink. The antigenic aspartic acid reside is within a red box. (F) Comparison of the peptide sequence used to raise human EphB4 monoclonal antibodies (green) with EphB4 sequences from other species. Amino acids identical to the human EphB4 sequence are shaded in grey. (G) Western blot analysis using the H200 and 13A7 antibodies with recombinant human EphB4 extracellular domain (rhEphB4ecd) (H) and recombinant mouse EphB4 extracellular domain Fc fusion protein (rmEphB4ecd-Fc) (M). The arrows indicate the position of the proteins compared to the molecular weight marker for which sizes are indicated on the left.
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Figure 4: Peptide specific antibodies identify EphB4, cause EphB4 protein phosphorylation and degradation(A) Antibodies specifically recognizing Peptide 7 were isolated from the H200 polyclonal preparation. Western blot analysis shows that these antibodies (H7) specifically identify EphB4 with less non-specific cross-reactivity than the H200 antibody. β-actin was used as a loading control. (B) Western blot and graph showing that in a similar manner to H200, the H7 antibody, tested at two different dilutions, reduces total EphB4 protein level with a significant reduction seen at 72 h (p = 0.0001). (C) Immunoprecipitation of cell lysates from MCF-7 breast cancer cells showing treatment with the H7 antibody causes phosphorylation of the EphB4 protein, seen at 5 min after addition and maintained at 30 min (yellow box). Cells were stimulated with soluble clustered ephrin-B2-Fc ligand (eB2) as a positive control for phosphorylation with clustered Fc fragment only (Fc) as the negative control for this. (D) Alignment of the EphB4 sequence of peptides 1 and 2, which includes peptide 7 sequence AGSCVVDA (red box) with the corresponding sequence from EphA3. Asterisks identify the amino acids identified as contributing to the third ligand binding domain. (E) Comparison of the peptide sequence used to raise EphB4 monoclonal antibodies (green) with other members of the human Eph family. Amino acids identical to the EphB4 sequence are shaded in grey, homologous amino acids are shaded pink. The antigenic aspartic acid reside is within a red box. (F) Comparison of the peptide sequence used to raise human EphB4 monoclonal antibodies (green) with EphB4 sequences from other species. Amino acids identical to the human EphB4 sequence are shaded in grey. (G) Western blot analysis using the H200 and 13A7 antibodies with recombinant human EphB4 extracellular domain (rhEphB4ecd) (H) and recombinant mouse EphB4 extracellular domain Fc fusion protein (rmEphB4ecd-Fc) (M). The arrows indicate the position of the proteins compared to the molecular weight marker for which sizes are indicated on the left.
Mentions: Peptide 7 was immobilized onto a MicroLink (gel matrix used to isolate interacting antibodies (designated H7) from the H200 polyclonal antibody preparation. To confirm that isolated antibodies could specifically identify EphB4 we again compared the MCF10A and MCF10A-B4 cells using Western blot analysis (Figure 4A). When compared with the H200 pAb, the H7 antibody detected only a single strongly immunoreactive band at the predicted 120 kDa, seen in the MCF10A-B4 sample and less strongly in MCF10A, consistent with detection of the over-expressed EphB4 in MCF10A-B4 and the endogenous EphB4 in MCF10A. Extended stimulation (for 72 h) led to a loss of EphB4 protein from the MCF-7 cells in a similar manner to that of the parental H200 antibody (Figure 4B and Figure 2F). Addition of the purified H7 antibody to MCF-7 cells caused an increase in EphB4 phosphorylation as soon as 5 min after addition which was still maintained at 30 min suggesting that the antibody activates EphB4 signaling in treated cells (Figure 4C). This would suggest that the antibody is behaving as an ephrin-B2 mimetic as ligand stimulation of EphB4 normally leads to phosphorylation of the kinase domain, then internalization and degradation of the receptor. In keeping with this hypothesis, the AGSCVVDA sequence contains three amino acids that align with those of a defined third ligand interface region on the EphA3 receptor (Figure 4D). Comparison with sequences of other Eph receptors shows variability at key residues suggesting high epitope specificity of the H7 antibody (Figure 4E). For example, analysis of the sequence using the EMBOSS antigenic prediction program (http://emboss.sourceforge.net/apps/release/6.0/emboss/apps/antigenic.html) identifies the aspartic acid residue in the EphB4 sequence (red box in Figure 4E) as the key amino acid within the most antigenic sequence in the 200 amino acid region targeted by the H200 antibody. Comparison with sequences from other species shows few differences in the amino acid sequence of the corresponding regions but the most differences to mouse EphB4 (Figure 4F) and Western blot analysis shows that although the H200 antibody can detect both human and mouse EphB4 proteins, the 13A7 antibody that targets the peptide 7 sequence specifically, only identified human EphB4 protein (rhEphB4ecd) and not murine EphB4 protein (rmEphB4ecd-Fc) (Figure 4G).

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