Abstract A25: Novel anti-HER3 strategy for pancreatic cancer therapy. (2012)

The purpose is to identify novel antibodies that inhibit proliferation of pancreatic cancer cells for therapeutic development. A large scale, random screening of hybridoma libraries was performed against pancreatic cancer cell lines (S2013, S2VP-10) resistant to anti-DR5 antibody, and several candidates including clone 1A5, enhanced anti-DR5 mediated apoptosis in the resistant cells. Mass spectroscopy analysis of the cell surface protein immunoprecipitated by 1A5 identified the protein as HER3. A second antibody, 3D4 anti-HER3, was generated by immunizing mice with SP2/0 cells transfected with full-length cDNA encoding HER3. The binding specificity of both anti-HER3 antibodies was confirmed by ELISA and flow cytometry using either soluble receptor or cell surface expressing recombinant cell lines. Both antibodies specifically bind to HER3 with no cross-reactivity against EGFR and HER2. Although both antibodies had a similar binding profile to recombinant soluble HER3, their ability to inhibit the binding of NRG1, a ligand for HER3, was different. 1A5 did not inhibit NRG1 binding to HER3 while 3D4 inhibited NRG1 binding in a dose-dependent fashion. The expression and phosphorylation profiles of EGFR, HER2 and HER3 were analyzed in a panel of pancreatic cancer cell lines. All cell lines expressed high levels of EGFR. Five and 7 out of 8 lines expressed variable levels of HER2 and HER3, respectively. TGF-α induced a differential phosphorylation response among cell lines although they all expressed high levels of EGFR, suggesting that the activation of EGFR by TGF-α does not correlate with the expression levels of EGFR. In addition, NRG1 did not induce EGFR activation. All HER2 positive cell lines had low levels of autophosphorylation of HER2. Stimulation with NRG1 significantly induced HER2 phosphorylation in 4 out of 6 HER2 positive lines, which correlated with NRG1-induced HER3 phosphorylation. This novel and important observation suggests that HER3 plays a critical role in activation of HER2 since no ligand has been identified for HER2 activation. Compared to NRG1, TGF-α had no effect on either HER2 or HER3 activation. All HER3 positive lines had levels of HER3 autophosphorylation, suggesting that these pancreatic cancer cells had a level of ligand-independent activation. Treatment with 1A5 led to de-phosphorylation of HER3 in all cell lines which ranged from 30% to 60% reduction from the basal levels. No effect of erlotinib or lapatinib on ligand-independent activation of HER3 was observed. Erlotinib moderately reduced EGFR phosphorylation at a low dose. While 1A5 alone had a minimum effect on EGFR activity, the combination of erlotinib and 1A5 led to a significant reduction of EGFR phosphorylation, suggesting that the ligand-independent activation of HER3 might be a key to the resistance of pancreatic cells to erlotinib. While 1A5 or lapatinib alone partially inhibited the degree of HER2 autophosphorylation, 1A5 and lapatinib in combination synergistically inhibited HER2 activity. 3D4 effectively blocked NRG1induced HER3 activation. Lapatinib alone inhibited NRG1-induced activation of HER3. 3D4 or lapatinib inhibited NRG1-induced HER2 activation, further confirming the role of HER3 ligand-dependent activation in HER2 activation. In the absence of NRG1 stimulation, the two anti-HER3 antibodies combined with either erlotinib or lapatinib yielded a profound inhibition of proliferation against all cell lines except HER3 negative Panc 1 cells. The combination of anti-HER3 with lapatinib was superior to the erlotinib combination in most cell lines, presumably due to co-activation of HER2 with HER3. These results suggest a novel mechanism by which pancreatic cancer develops resistance to EGFR targeted therapies, and a target, the HER3 ligand-independent activation pathway, for overcoming the resistance. Citation Format: Donald J. Buchsbaum, Xian Chen, Enyun Shen, Tong Zhou. Novel anti-HER3 strategy for pancreatic cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A25.