SH30200

SH30200.01), with 1% penicillin/streptomycin and 10% FCS, respectively. antibodies in combination with the epidermal growth element receptor (EGFR)/HER2 specific TKI, lapatinib. In the 1st approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 relationships with additional possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination 10-Oxo Docetaxel with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody offers 10-Oxo Docetaxel improved inhibitory activity. Collectively, these observations provide support for the restorative use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert. Keywords: bispecific antibody, receptor internalization, HER3, HER2, antibody executive Introduction Multiple cancers are driven by aberrant signaling through the human being epidermal growth element receptor (HER), also known as ErbB, family members. Recent studies possess indicated that HER2-HER3 heterodimers can perform a central part in tumorigenesis.1-5 HER3 is a preferred dimerization partner for HER2, which has no known ligand and is constitutively active.5-7 Although HER3 has very low intrinsic kinase activity, there are six phosphorylation-dependent binding sites for PI3K within the cytosolic tail of this receptor.8 Consequently, HER2-HER3 heterodimers are the most effective activators identified to date of the PI3K/Akt pathway through both ligand-independent and ligand-dependent signaling.5,9 Ligand-dependent activation of HER3 involves the binding of heregulin or other ligands to induce a conformational switch in the dimerization arm, traveling heterodimer formation with kinase competent partners such as HER2 or EGFR.5,6 Consistent with HER3 like a driver of tumorigenesis, loss of HER3 expression in breast cancer cells results in reductions in both PI3K/Akt signaling and proliferation.1,2 Further, modeling studies demonstrate that HER3 represents a central node 10-Oxo Docetaxel in PI3K/Akt signaling.4 In conjunction with the limited effectiveness of solely targeting HER2 with monotherapies such as trastuzumab,10-12 these observations have motivated the development of therapeutics targeting HER3 or HER2-HER3 heterodimers.3,13-15 Recent data indicate the targeting of this axis with antibodies is less effective in the presence of heregulin,14 which is expressed in either autocrine or paracrine fashion in many tumor types.16-18 Thus, there is a need for the generation of improved therapeutics directed toward ligand-dependent activation of HER3. The current study involves a comparison of two unique methods using bispecific HER2/HER3 specific antibodies or multivalent HER3-specific antibodies to target HER3 in the presence of the HER3 ligand, heregulin. As an KLK7 antibody alternative to targeting HER family members with antibodies, the use of small molecule inhibitors of the tyrosine kinase activity of EGFR or HER2, or of the downstream kinase, PI3K, offers attracted much interest.19-25 However, these inhibitors can 10-Oxo Docetaxel lead to tumor escape due to upregulation of compensatory signaling pathways and complex cross-regulatory networks involving negative feedback loops. For example, the delivery of lapatinib, a tyrosine kinase inhibitor (TKI) that focuses on both EGFR and HER2, results in upregulation of HER2 and HER3 manifestation and PI3K/Akt signaling.26-29 Lapatinib resistance pathways can also include activating mutations of PI3K,30 and the inhibitory effects of lapatinib can be dampened by the presence of the HER3 ligand, heregulin.14,31-33 This suggests that lapatinib in combination with antibodies or bispecifics that bind to HER3 might provide an effective route for therapy, particularly for tumors involving autocrine or paracrine heregulin loops that can result in 10-Oxo Docetaxel limited efficacy of monotherapies. Related to the limited restorative effectiveness of TKIs such as lapatinib that inhibit EGFR and HER2 activation, HER3 can associate with additional activating receptors such as cMET and insulin-like growth element type I receptor I/insulin receptor substrate-1.5,7,34,35 A possible strategy to extinguish HER3-PI3K signaling would therefore be to inhibit multiple potential HER3 partners. This provides the impetus for directly focusing on HER3, which would avoid the difficulty of identifying and globally inhibiting all HER3 partners. Here, we have taken the approach of investigating the ability of a bispecific anti-HER2/HER3 antibody to drive HER3 into HER2-HER3 heterodimers that, through combination treatment with lapatinib, are.