Examination of HER3 targeting in cancer using monoclonal antibodies

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.Growth factors and their plasma-membrane–embedded receptors regulate cellular proliferation and migration during both embryogenesis and oncogenesis (1). One example entails the epidermal growth factor (EGF) family and the corresponding human EGF receptor (HER)/ERBB family of four receptor tyrosine kinases: the EGF receptor (EGFR or ERBB1), HER2 (c-Neu or ERBB2), HER3 (ERBB3), and HER4 (ERBB4) (2). The intracellular parts of ERBB/HER receptors harbor a catalytic tyrosine kinase domain (3). After ligand binding to an ectodomain, these receptors form active homodimers or heterodimers (4–7). Unlike EGFR, HER3/ERBB3 presents very low tyrosine kinase activity (8). Nevertheless, it binds neuregulins (NRGs) and exerts profound influence on signaling pathways, primarily through dimerization with EGFR and HER2. In line with roles in cancer progression, and similarly to EGFR and HER2, mutant forms of HER3 have recently been reported in colon and gastric cancer (9).Cancer therapies that use monoclonal antibodies (mAbs) to target ERBB/HER family members are becoming a mainstay in oncology. For example, trastuzumab, which targets HER2, is currently used to treat gastric and breast cancer (10, 11). However, the majority of patients with metastatic HER2-positive breast cancer will become trastuzumab-resistant after prolonged treatment, a development significantly less common in an adjuvant or neo-adjuvant setting. It has been reported that trastuzumab-resistant tumors show elevated expression of HER3 (12), and, similarly, inhibition of HER2 using a kinase inhibitor also up-regulates HER3 (13). Likewise, HER3 has been implicated in the development of resistance to treatment with other ERBB/HER-targeted therapies (14, 15), agents blocking insulin-like growth factor receptors (16), and chemotherapeutic agents (17). For these reasons, anti-HER3 antibodies (Abs) are being developed by several laboratories, and some have reached initial clinical trials.Importantly, anticancer mechanisms of therapeutic Abs are multifactorial and incompletely understood. The involvement of Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity, as well as various cellular processes such as triggering of apoptosis, blocking angiogenesis, inhibiting tumor cell proliferation, interfering with signaling cascades, and accelerating receptor internalization, have been described (18). Our own studies proposed critical involvement of the endocytic system. In particular, combinations of two mutually noncompetitive Abs (Abs against different epitopes that do not cause steric hindrance) to EGFR (19) or to HER2 (20, 21) have been shown to accelerate receptor endocytosis and inhibit tumor growth, better than either Ab alone. Furthermore, a recently completed clinical trial that combined with chemotherapy two mutually noncompetitive anti-HER2 mAbs, for the treatment of HER2-positive breast cancer (22), confirmed the clinical benefit of combining noncompetitive mAbs.Anti-HER3 agents that are being developed or are in clinical trials show promising results, yet their efficacy might be viewed, in general, as variable and rather modest (23). Hence, it is imperative to develop new agents and resolve their molecular mechanisms of action. In this study, we generated in mice a relatively broad series of anti-HER3 mAbs and examined them for the ability to both degrade HER3 and decrease downstream signaling. The new mAbs displayed a variety of biochemical and biological attributes, such as binding affinity to HER3, ability to displace NRG and block downstream signals, sort HER3 for degradation, and recruit natural killer cells. Ultimately, the Abs were tested in vitro and in tumor-bearing animals for their ability to inhibit growth of cancer cells. We focus on an especially potent tumor-inhibitory Ab that can displace NRG, robustly sort HER3 for intracellular degradation, and also recruit immune effector cells.