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The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TP L , retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TP L showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TP L potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TP L was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TP L treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TP L to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic. Cancer Res; 73(21);

Human epidermal growth factor receptors (HERs or ErbBs) play crucial roles in numerous cellular processes. ErbB2 is a key member of ErbB family, and its overexpression is recognized as a frequent molecular abnormality. In cancer, this overexpression correlates with aggressive disease and poor patient outcomes. Dimer-dependent phosphorylation is a key event for the signal transduction of ErbBs. However, the molecular mechanism of the dimerization of ErbB2 remains elusive. In the present work, we report the homodimer architecture of the ErbB2 extracellular domain (ECD) which is unique compared with other dimer-models of ErbBs. The structure of the ErbB2 ECD homodimer represents a “back to head” interaction, in which a protruding β-hairpin arm in domain II of one ErbB2 protomer is inserted into a C-shaped pocket created by domains I–III of the adjacent ErbB2 protomer. This dimerized architecture and its impact on the phosphorylation of ErbB2 intracellular domain were further verified by a mutagenesis study. We also elucidated the different impacts of two clinically administered therapeutic antibodies, trastuzumab and pertuzumab, on ErbB2 dimerization. This information not only provides an understanding of the molecular mechanism of ErbBs dimerization but also elucidates ErbB2-targeted therapy at the molecular level.

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in ErbB2-overexpressing breast and gastric cancer, but resistance to the drug is common. Here, we investigated the antitumor activity of the combination of trastuzumab and the SRC inhibitor saracatinib in ErbB2-overexpressing trastuzumab-resistant gastric cancer. The ErbB2-overexpressing human gastric cancer cell line NCI-N87 was treated with trastuzumab to obtain the trastuzumab-resistant cell line NCI-N87R. The NCI-N87R cell line showed a marked increase in SRC activity and ErbB signaling compared with the NCI-N87 cell line. Our data demonstrated that trastuzumab plus saracatinib was much more potent than either agent alone in reducing the phosphorylation of ErbB3 and AKT in both NCI-N87 and NCI-N87R gastric cancer cell lines. Trastuzumab and saracatinib synergistically inhibited the in vitro growth of NCI-N87 and NCI-N87R cell lines. Further data showed that combination therapy of trastuzumab with saracatinib resulted in a significant benefit over either agent alone in both NCI-N87 and NCI-N87R xenograft models, suggesting its potential use for treating ErbB2-overexpressing gastric cancer.

Trastuzumab, an anti-HER2/ErbB2 humanized antibody, has shown great clinical benefits in ErbB2-positive breast cancer treatment. Despite of its effectiveness, response rate to trastuzumab is limited and resistance is common. Here, we developed a new anti-ErbB2 antibody, denoted as H2-18, which was isolated from a phage display human antibody library. Previous studies have demonstrated that trastuzumab recognizes the juxtamembrane region of domain IV, and pertuzumab, another humanized ErbB2-specific antibody, binds to ErbB2 near the center of domain II. Our crystallographic analysis showed that the epitope recognized by H2-18 is within domain I of the ErbB2 molecule. H2-18 potently induced programmed cell death (PCD) in both trastuzumab-sensitive and -resistant breast cancer cell lines, while trastuzumab and pertuzumab, either used alone or in combination, only exhibits very weak PCD-inducing activity. More importantly, H2-18 could inhibit the growth of trastuzumab-resistant breast cancer cells far more effectively than trastuzumab plus pertuzumab, both in vitro and in vivo. In conclusion, H2-18 shows a unique ability to overcome trastuzumab resistance, suggesting that it has the great potential to be translated to the clinic.

MicroRNAs (miRs) have shown tremendous potential to act as therapeutic targets for cancer treatment. In this context, the present study was designed to investigate the potential of miR-143 in the treatment of breast cancer. Results showed that miR-143 to be significantly (P < 0.05) downregulated in breast cancer tissues and cell lines. The miR-143 has inhibitory effect on CAMA-1cell growth which was manifested as significant (P < 0.05) decline in loss of viability of cancer cells. The loss of cell viability was revealed to be due to the induction of apoptotic cell death as evident from acridine orange/ethidium bromide (AO/EB) and 4′,6-diamidino-2-phenylindole (DAPI) staining assays. The apoptotic cell percentage was found to be 35.7% in miR-143 mimics transfected in comparison to 6.4% in miR-NC transfected cells. The western blot analysis showed that miR-143 caused enhancement in Bax and suppression in Bcl-2 expression in CAMA-1 cells. The miR-143 also suppressed the bone metastasis of the CAMA-1 cells by suppressing the expression of Jag1 and deactivation of the Rho-signalling pathway. The transwell assays also showed considerable anti-metastatic effects of miR-143 on CAMA-1 cells. Taken together, miR-143 has growth inhibitory anti-metastatic effect on breast cancer and thus may prove beneficial in breast cancer treatment.

(2013) A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity, mAbs, 5:6, 946-955,