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A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors.

Meng Y, Zheng L, Yang Y, Wang H, Dong J, Wang C, Zhang Y, Yu X, Wang L, Xia T, Zhang D, Guo Y, Li B - Oncogenesis (2016)

Bottom Line: The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer.Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling.Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Nankai University, Tianjin, People's Republic of China.

ABSTRACT
The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

No MeSH data available.


Related in: MedlinePlus

Mapping of 3E10-specific epitope on the extracellular domain of ErbB2. (a) Amino-acid sequences of the insert from 3E10-positive phage clones. Sequences were aligned for the consensus motif, which is indicated by underlined letters. (b) Effect of alanine substitutions on 3E10 binding to ErbB2. Data are expressed as means±s.d. (c) Surface representation of the extracellular domain of ErbB2 (PDB accession code 1N8Z). Domains I, II, III and IV are green, dark cyan, magenta and gray, respectively. The ErbB2 residues within 5 Å of trastuzumab (PDB accession code 1N8Z) and pertuzumab (PDB accession code 1S78) are colored red and yellow, respectively. The 3E10 epitope residues, T474, R477 and P478, are colored blue. (d) 3E10 did not cross-react with ErbB3. Different concentrations of the 3E10 antibody were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein (R&D Systems), followed by incubation at 37 °C for 1 h. After washing, horseradish peroxidase-labeled goat anti-mouse IgG H&L (Abcan) was added and the plates were further incubated for 1 h at 37 °C. Finally, 3,3′,5,5′-tetramethylbenzidine (TMB) was added as a substrate and the absorbance was read at 450 nm. As a positive control, different concentrations of horseradish peroxidase-labeled ErbB-3 antibody (C-17) (sc-285; Santa Cruz Biotechnology) were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein, followed by incubation at 37 °C for 1 h. After washing, TMB was added as a substrate and the absorbance was read at 450 nm.
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fig2: Mapping of 3E10-specific epitope on the extracellular domain of ErbB2. (a) Amino-acid sequences of the insert from 3E10-positive phage clones. Sequences were aligned for the consensus motif, which is indicated by underlined letters. (b) Effect of alanine substitutions on 3E10 binding to ErbB2. Data are expressed as means±s.d. (c) Surface representation of the extracellular domain of ErbB2 (PDB accession code 1N8Z). Domains I, II, III and IV are green, dark cyan, magenta and gray, respectively. The ErbB2 residues within 5 Å of trastuzumab (PDB accession code 1N8Z) and pertuzumab (PDB accession code 1S78) are colored red and yellow, respectively. The 3E10 epitope residues, T474, R477 and P478, are colored blue. (d) 3E10 did not cross-react with ErbB3. Different concentrations of the 3E10 antibody were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein (R&D Systems), followed by incubation at 37 °C for 1 h. After washing, horseradish peroxidase-labeled goat anti-mouse IgG H&L (Abcan) was added and the plates were further incubated for 1 h at 37 °C. Finally, 3,3′,5,5′-tetramethylbenzidine (TMB) was added as a substrate and the absorbance was read at 450 nm. As a positive control, different concentrations of horseradish peroxidase-labeled ErbB-3 antibody (C-17) (sc-285; Santa Cruz Biotechnology) were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein, followed by incubation at 37 °C for 1 h. After washing, TMB was added as a substrate and the absorbance was read at 450 nm.

Mentions: To identify the peptides recognized by 3E10, phage clones were isolated by panning the PhD-7 phage display peptide library with 3E10. Three rounds of selection were performed and at each round, the library was precleared on a control mouse IgG2a,κ antibody. After the third round of panning, the binding of the isolated phage clones to 3E10 was determined in an enzyme-linked immunosorbent assay (ELISA). Sequence analysis of 3E10-positive phage clones identified five distinct amino-acid sequences (Figure 2a). Alignment of these sequences resulted in the consensus motif THKRP, which could be aligned with the 474T—R477P478 sequence located at the extracellular domain III of ErbB2 (Figure 2a). To prove that the 474T—R477P478 sequence within ErbB2 is the epitope recognized by 3E10, alanine substitutions were introduced into ErbB2-ECD at residues T474, R477 and P478, and the binding of 3E10 to these ErbB2-ECD mutants was measured by ELISA. The results showed that alanine substitution in any one of residues T474, R477 and P478 significantly reduced 3E10 binding activity for ErbB2 (Figure 2b). Double alanine substitutions at positions 477 and 478 further reduced the binding activity, and triple alanine substitutions at positions 474, 477 and 478 almost totally abolished 3E10 binding to ErbB2 (Figure 2b). In contrast, the binding of 9F12, another mouse anti-ErbB2 mAb that does not compete with 3E10, to these ErbB2-ECD mutants was approximately the same as to wild-type ErbB2-ECD (Figure 2b). These data demonstrate that the 474T—R477P478 sequence within ErbB2 is the 3E10 epitope. The 3E10 epitope appeared to be located opposite to the dimerization interfaces in domains II and IV (Figure 2c), implying that 3E10 did not directly disrupt the interaction of ErbB2 with other ErbBs. Therefore, we speculate that the 3E10 antibody may exert the ErbB2 heterodimerization-blocking activity through causing major conformational changes of the ErbB2 molecule.


A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors.

Meng Y, Zheng L, Yang Y, Wang H, Dong J, Wang C, Zhang Y, Yu X, Wang L, Xia T, Zhang D, Guo Y, Li B - Oncogenesis (2016)

Mapping of 3E10-specific epitope on the extracellular domain of ErbB2. (a) Amino-acid sequences of the insert from 3E10-positive phage clones. Sequences were aligned for the consensus motif, which is indicated by underlined letters. (b) Effect of alanine substitutions on 3E10 binding to ErbB2. Data are expressed as means±s.d. (c) Surface representation of the extracellular domain of ErbB2 (PDB accession code 1N8Z). Domains I, II, III and IV are green, dark cyan, magenta and gray, respectively. The ErbB2 residues within 5 Å of trastuzumab (PDB accession code 1N8Z) and pertuzumab (PDB accession code 1S78) are colored red and yellow, respectively. The 3E10 epitope residues, T474, R477 and P478, are colored blue. (d) 3E10 did not cross-react with ErbB3. Different concentrations of the 3E10 antibody were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein (R&D Systems), followed by incubation at 37 °C for 1 h. After washing, horseradish peroxidase-labeled goat anti-mouse IgG H&L (Abcan) was added and the plates were further incubated for 1 h at 37 °C. Finally, 3,3′,5,5′-tetramethylbenzidine (TMB) was added as a substrate and the absorbance was read at 450 nm. As a positive control, different concentrations of horseradish peroxidase-labeled ErbB-3 antibody (C-17) (sc-285; Santa Cruz Biotechnology) were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein, followed by incubation at 37 °C for 1 h. After washing, TMB was added as a substrate and the absorbance was read at 450 nm.
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fig2: Mapping of 3E10-specific epitope on the extracellular domain of ErbB2. (a) Amino-acid sequences of the insert from 3E10-positive phage clones. Sequences were aligned for the consensus motif, which is indicated by underlined letters. (b) Effect of alanine substitutions on 3E10 binding to ErbB2. Data are expressed as means±s.d. (c) Surface representation of the extracellular domain of ErbB2 (PDB accession code 1N8Z). Domains I, II, III and IV are green, dark cyan, magenta and gray, respectively. The ErbB2 residues within 5 Å of trastuzumab (PDB accession code 1N8Z) and pertuzumab (PDB accession code 1S78) are colored red and yellow, respectively. The 3E10 epitope residues, T474, R477 and P478, are colored blue. (d) 3E10 did not cross-react with ErbB3. Different concentrations of the 3E10 antibody were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein (R&D Systems), followed by incubation at 37 °C for 1 h. After washing, horseradish peroxidase-labeled goat anti-mouse IgG H&L (Abcan) was added and the plates were further incubated for 1 h at 37 °C. Finally, 3,3′,5,5′-tetramethylbenzidine (TMB) was added as a substrate and the absorbance was read at 450 nm. As a positive control, different concentrations of horseradish peroxidase-labeled ErbB-3 antibody (C-17) (sc-285; Santa Cruz Biotechnology) were added to 96-well plates precoated with 3 μg/ml of recombinant human ErbB3/Her3 Fc chimera Protein, followed by incubation at 37 °C for 1 h. After washing, TMB was added as a substrate and the absorbance was read at 450 nm.
Mentions: To identify the peptides recognized by 3E10, phage clones were isolated by panning the PhD-7 phage display peptide library with 3E10. Three rounds of selection were performed and at each round, the library was precleared on a control mouse IgG2a,κ antibody. After the third round of panning, the binding of the isolated phage clones to 3E10 was determined in an enzyme-linked immunosorbent assay (ELISA). Sequence analysis of 3E10-positive phage clones identified five distinct amino-acid sequences (Figure 2a). Alignment of these sequences resulted in the consensus motif THKRP, which could be aligned with the 474T—R477P478 sequence located at the extracellular domain III of ErbB2 (Figure 2a). To prove that the 474T—R477P478 sequence within ErbB2 is the epitope recognized by 3E10, alanine substitutions were introduced into ErbB2-ECD at residues T474, R477 and P478, and the binding of 3E10 to these ErbB2-ECD mutants was measured by ELISA. The results showed that alanine substitution in any one of residues T474, R477 and P478 significantly reduced 3E10 binding activity for ErbB2 (Figure 2b). Double alanine substitutions at positions 477 and 478 further reduced the binding activity, and triple alanine substitutions at positions 474, 477 and 478 almost totally abolished 3E10 binding to ErbB2 (Figure 2b). In contrast, the binding of 9F12, another mouse anti-ErbB2 mAb that does not compete with 3E10, to these ErbB2-ECD mutants was approximately the same as to wild-type ErbB2-ECD (Figure 2b). These data demonstrate that the 474T—R477P478 sequence within ErbB2 is the 3E10 epitope. The 3E10 epitope appeared to be located opposite to the dimerization interfaces in domains II and IV (Figure 2c), implying that 3E10 did not directly disrupt the interaction of ErbB2 with other ErbBs. Therefore, we speculate that the 3E10 antibody may exert the ErbB2 heterodimerization-blocking activity through causing major conformational changes of the ErbB2 molecule.

Bottom Line: The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer.Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling.Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Nankai University, Tianjin, People's Republic of China.

ABSTRACT
The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

No MeSH data available.


Related in: MedlinePlus