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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

Combination of C3E10 with either trastuzumab or pertuzumab synergistically inhibits ErbB2 heterodimerization and signaling. (a) Competitive binding assay. 3E10, trastuzumab and pertuzumab were evaluated for their ability to compete with Alexa Fluor 488-labeled 3E10, Alexa Fluor 488-labeled trastuzumab or Alexa Fluor 488-labeled pertuzumab for binding to ErbB2-overexpressing BT-474 cells. (b) Coimmunoprecipitation assay comparing the ability of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) to disrupt ligand-independent ErbB2/ErbB3 heterodimer formation in BT-474 cells. (c) Coimmunoprecipitation assay detecting EGF-induced ErbB2/EGFR and HRG-induced ErbB2/ErbB3 heterodimerization in BT-474 cells pretreated with control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each). (d) Immunoblots examining ErbB2 signaling in BT-474 cells upon treatment with control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) or trastuzumab plus C3E10 (5 μg/ml each) in the absence of ErbB ligand. (e) Immunoblots assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each) pretreatment on EGF- or HRG-activated ErbB2 signaling in BT-474 cells. (f) MTS assay comparing the effects of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the absence of ErbB ligand. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05, **P<0.001. (g) MTS assay assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) and pertuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the presence of HRG or EGF. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05.
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fig3: Combination of C3E10 with either trastuzumab or pertuzumab synergistically inhibits ErbB2 heterodimerization and signaling. (a) Competitive binding assay. 3E10, trastuzumab and pertuzumab were evaluated for their ability to compete with Alexa Fluor 488-labeled 3E10, Alexa Fluor 488-labeled trastuzumab or Alexa Fluor 488-labeled pertuzumab for binding to ErbB2-overexpressing BT-474 cells. (b) Coimmunoprecipitation assay comparing the ability of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) to disrupt ligand-independent ErbB2/ErbB3 heterodimer formation in BT-474 cells. (c) Coimmunoprecipitation assay detecting EGF-induced ErbB2/EGFR and HRG-induced ErbB2/ErbB3 heterodimerization in BT-474 cells pretreated with control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each). (d) Immunoblots examining ErbB2 signaling in BT-474 cells upon treatment with control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) or trastuzumab plus C3E10 (5 μg/ml each) in the absence of ErbB ligand. (e) Immunoblots assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each) pretreatment on EGF- or HRG-activated ErbB2 signaling in BT-474 cells. (f) MTS assay comparing the effects of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the absence of ErbB ligand. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05, **P<0.001. (g) MTS assay assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) and pertuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the presence of HRG or EGF. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05.

Mentions: Next, we investigated if the 3E10 antibody could compete with trastuzumab or pertuzumab for binding to ErbB2-overexpressing BT-474 cells. Our data showed that 3E10 did not compete with either trastuzumab or pertuzumab (Figure 3a). The 3E10 antibody and trastuzumab (or pertuzumab) bind to distinct regions of ErbB2 and inhibit receptor activation by different mechanisms. This raises a question of possible synergistic inhibition of ErbB2 signaling by 3E10 and trastuzumab (or pertuzumab). Our data showed that the combination of C3E10 (a mouse/human chimeric IgG1/κ antibody derived from 3E10) and trastuzumab inhibited ErbB2 heterodimerization significantly more efficiently than either mAb alone (Figure 3b). Similar results were observed for C3E10 plus pertuzumab (Figure 3c). Consistently, C3E10 in combination with either trastuzumab or pertuzumab synergistically inhibited the phosphorylation of ErbB receptors and their downstream signaling molecules MAPK and AKT (Figures 3d and e), and the proliferation of BT-474 cells (Figures 3f and g).


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)

Combination of C3E10 with either trastuzumab or pertuzumab synergistically inhibits ErbB2 heterodimerization and signaling. (a) Competitive binding assay. 3E10, trastuzumab and pertuzumab were evaluated for their ability to compete with Alexa Fluor 488-labeled 3E10, Alexa Fluor 488-labeled trastuzumab or Alexa Fluor 488-labeled pertuzumab for binding to ErbB2-overexpressing BT-474 cells. (b) Coimmunoprecipitation assay comparing the ability of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) to disrupt ligand-independent ErbB2/ErbB3 heterodimer formation in BT-474 cells. (c) Coimmunoprecipitation assay detecting EGF-induced ErbB2/EGFR and HRG-induced ErbB2/ErbB3 heterodimerization in BT-474 cells pretreated with control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each). (d) Immunoblots examining ErbB2 signaling in BT-474 cells upon treatment with control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) or trastuzumab plus C3E10 (5 μg/ml each) in the absence of ErbB ligand. (e) Immunoblots assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each) pretreatment on EGF- or HRG-activated ErbB2 signaling in BT-474 cells. (f) MTS assay comparing the effects of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the absence of ErbB ligand. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05, **P<0.001. (g) MTS assay assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) and pertuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the presence of HRG or EGF. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05.
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fig3: Combination of C3E10 with either trastuzumab or pertuzumab synergistically inhibits ErbB2 heterodimerization and signaling. (a) Competitive binding assay. 3E10, trastuzumab and pertuzumab were evaluated for their ability to compete with Alexa Fluor 488-labeled 3E10, Alexa Fluor 488-labeled trastuzumab or Alexa Fluor 488-labeled pertuzumab for binding to ErbB2-overexpressing BT-474 cells. (b) Coimmunoprecipitation assay comparing the ability of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) to disrupt ligand-independent ErbB2/ErbB3 heterodimer formation in BT-474 cells. (c) Coimmunoprecipitation assay detecting EGF-induced ErbB2/EGFR and HRG-induced ErbB2/ErbB3 heterodimerization in BT-474 cells pretreated with control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each). (d) Immunoblots examining ErbB2 signaling in BT-474 cells upon treatment with control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) or trastuzumab plus C3E10 (5 μg/ml each) in the absence of ErbB ligand. (e) Immunoblots assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) or pertuzumab plus C3E10 (5 μg/ml each) pretreatment on EGF- or HRG-activated ErbB2 signaling in BT-474 cells. (f) MTS assay comparing the effects of control IgG (10 μg/ml), trastuzumab (10 μg/ml), C3E10 (10 μg/ml) and trastuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the absence of ErbB ligand. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05, **P<0.001. (g) MTS assay assessing the effects of control IgG (10 μg/ml), pertuzumab (10 μg/ml), C3E10 (10 μg/ml) and pertuzumab plus C3E10 (5 μg/ml each) on BT-474 cell proliferation in the presence of HRG or EGF. Results are shown as percentage of control cell proliferation. Error bars, s.d. *P<0.05.
Mentions: Next, we investigated if the 3E10 antibody could compete with trastuzumab or pertuzumab for binding to ErbB2-overexpressing BT-474 cells. Our data showed that 3E10 did not compete with either trastuzumab or pertuzumab (Figure 3a). The 3E10 antibody and trastuzumab (or pertuzumab) bind to distinct regions of ErbB2 and inhibit receptor activation by different mechanisms. This raises a question of possible synergistic inhibition of ErbB2 signaling by 3E10 and trastuzumab (or pertuzumab). Our data showed that the combination of C3E10 (a mouse/human chimeric IgG1/κ antibody derived from 3E10) and trastuzumab inhibited ErbB2 heterodimerization significantly more efficiently than either mAb alone (Figure 3b). Similar results were observed for C3E10 plus pertuzumab (Figure 3c). Consistently, C3E10 in combination with either trastuzumab or pertuzumab synergistically inhibited the phosphorylation of ErbB receptors and their downstream signaling molecules MAPK and AKT (Figures 3d and e), and the proliferation of BT-474 cells (Figures 3f and g).

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