Published OnlineFirst January 7, 2020; DOI: 10.1158/1535-7163.MCT-19-0515 MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS 10D1F, an Anti-HER3 Antibody that Uniquely Blocks the Receptor Heterodimerization Interface, Potently Inhibits Tumor Growth Across a Broad Panel of Tumor Models Dipti Thakkar1, Vicente Sancenon1, Marvin M. Taguiam1, Siyu Guan1, Zhihao Wu1, Eric Ng1, Konrad H. Paszkiewicz2, Piers J. Ingram1,2, and Jerome D. Boyd-Kirkup1,2 ABSTRACT ◥ In recent years, HER3 has increasingly been implicated in the activation of the PI3K pathway in a broad panel of tumor models. progression of a variety of tumor types and in acquired resistance to Even as a monotherapy, 10D1F shows superior inhibition of EGFR and HER2 therapies. Whereas EGFR and HER2 primarily tumor growth in the same cell lines both in vitro and in mouse signal through the MAPK pathway, HER3, as a heterodimer with xenograft experiments, when compared with other classes of EGFR or HER2, potently activates the PI3K pathway. Despite its anti-HER3 antibodies. This includes models demonstrating critical role, previous attempts to target HER3 with neutralizing ligand-independent activation of heterodimerization as well as antibodies have shown disappointing efficacy in the clinic, most constitutively activating mutations in the MAPK pathway. Posses- likely due to suboptimal and indirect mechanisms of action that fail sing favorable pharmacokinetic and toxicologic profiles, 10D1F to completely block heterodimerization; for example, tumors can uniquely represents a new class of anti-HER3 neutralizing anti- escape inhibition of ligand binding by upregulating ligand- bodies with a novel mechanism of action that offers significant independent mechanisms of HER3 activation. We therefore potential for broad clinical benefit. developed 10D1F, a picomolar affinity, highly specific anti-HER3 10D1F is a novel anti-HER3 antibody that uniquely binds the neutralizing antibody that binds the HER3 heterodimerization receptor dimerization interface to block ligand-dependent and interface, a region that was hitherto challenging to raise antibodies independent heterodimerization with EGFR/HER2 and thus against. We demonstrate that 10D1F potently inhibits both EGFR: more potently inhibits tumor growth than existing anti-HER3 HER3 and HER2:HER3 heterodimerization to durably suppress antibodies. Introduction shown to inhibit proliferation and reduce tumor growth, even in cells that are resistant to TKIs (5). Members of the EGFR family (EGFR/HER family) are com- The HER family signal through the PI3K/AKT/mTOR and the monly implicated in the formation and progression of many tumor MAPK/ERKK pathways to promote cell survival and prolifera- types and therefore represent attractive targets for therapeutic tion (1, 6, 7). However, whereas HER2 and EGFR primarily activate intervention (1). Despite the relative clinical success of small- the MAPK/ERKK pathway, HER3 potently activates the PI3K/ molecule tyrosine kinase inhibitors (TKI), the anti-EGFR antibody AKT/mTOR pathway (8–10),asitsintracellulardomaincontains cetuximab, and the anti-HER2 antibodies pertuzumab and trastu- multiple docking sites for the regulatory p85 subunit of PI3K (11). zumab, tumors frequently develop resistance and patients relapse. HER3 lacks kinase activity and does not form stable homodimers; HER3 has emerged as a central player in both tumor progression therefore, HER3 must be transphosphorylated by binding to a and acquired resistance to EGFR and HER2-targeted therapies, as kinase-active heterodimer partner (commonly EGFR or HER2) for aberrant expression and/or activation of HER3 and its ligand signal transduction to take place (12, 13). The HER3 extracellular NRG1 is associated with poor responses and low survival rates in domain exists in a reversible equilibrium between a “closed” multiple indications (2, 3). Although critical in early development, inactive conformation and an “open” active conformation, in which HER3 is expressed at only low levels in adult tissues such as skin the dimerization arm within domain II is exposed to allow dimer- and colon, yet is commonly activated in many cancers, most ization along the domain II dimerization interface, and in particular notably breast, colorectal, and gastric cancer (4). Furthermore, through the cysteine-rich CR1 region (Fig. 1;refs.14–21). HER3 is knockdown of HER3 in representative cancer models has been “activated” when the equilibrium is shifted in favor of the open conformation, increasing the probability of forming active hetero- dimers. The conventional model for activation is ligand-dependent, 1Hummingbird Bioscience, 1 Research Link, Singapore. 2Hummingbird Biosci- that is, the equilibrium shifts whenHER3intheopenconformation ence, South San Francisco, California. is stabilized by binding of the NRG1 ligand (Fig. 1A, left). However, any dimerization partner at sufficient concentration will also shift Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). the equilibrium as it binds to and stabilizes HER3 that is transiently in the open conformation, known as ligand-independent activation Corresponding Author: Jerome D. Boyd-Kirkup, Hummingbird Bioscience, 1 – Research Link, Singapore, 117604. Phone: 65-62662617; E-mail: (Fig. 1A,right;refs.19 21). [email protected] Multiple mechanisms contribute to the increased HER3 activation associated with resistance to EGFR or HER2 directed therapy, including Mol Cancer Ther 2020;XX:XX–XX (i) transcriptional upregulation of HER3 (22, 23); (ii) increased levels of doi: 10.1158/1535-7163.MCT-19-0515 NRG1 (24, 25); and (iii) HER2 amplification (26, 27). More recently, Ó2020 American Association for Cancer Research. oncogenicfusions ofSLC3A2, CD74, orVAMP2 to NRG1isoforms have AACRJournals.org | OF1 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst January 7, 2020; DOI: 10.1158/1535-7163.MCT-19-0515 Thakkar et al. A Ligand-dependent activation Ligand-independent activation Presence of ligand shifts equilibrium state to favor Abundant EGFR/HER2 shifts equilibrium state to favor HER3 heterodimers open HER3 conformation and heterodimer formation ~10% ~90% ~90% ~10% ~10% ~90% ~90% ~10% PI3K PI3K PI3K PI3K Blocking ligand-dependent activation selects for cells capable of-ligand independent activation through upregulation of EGFR/HER2 Open EGFR/ Open Closed HER3 HER2 HER3 HER3 Heterodimer B HER3 Ectodomain - open conformaon HER3 Ectodomain - closed conformaon Exposed NRG1-binding site dimerizaon interface NRG1-binding site (class 1 binding (class 1 binding region) region) Exposed dimerizaon interface Closed conformaon interacon Membrane (class 2 binding region) Membrane Figure 1. Proposed model for ligand-dependent and ligand-independent activation of HER3 and therapeutic strategies for inhibition. A, Diagram of a proposed model for how the equilibrium between inactive conformation and active conformation of HER3 can be shifted toward the active conformation in the presence and absence of ligand. B, Structural models of HER3 conformations showing the target regions (red circles) for anti-HER3 therapeutic antibodies of different classes, representing the proposed mechanisms (MOA) for inhibiting HER3 heterodimerization. been identified in patients with lung-invasive mucinous adenocarcino- cascades, such as BRAF V600E, which confer TKI resistance in thyroid ma (28, 29). These fusions promote paracrine secretion of the EGF-like and colon carcinomas (30–32), may fail through HER3 activation. domain of NRG1 and increased HER3 activation. Furthermore, efforts Notably,activationof HER3 byNRG1promotes resistance to the specific to target constitutively activating mutations in the downstream signaling BRAF V600E inhibitor vemurafenib (33, 34). OF2 Mol Cancer Ther; 2020 MOLECULAR CANCER THERAPEUTICS Downloaded from mct.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst January 7, 2020; DOI: 10.1158/1535-7163.MCT-19-0515 Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization In support of the role of HER3 in drug resistance, anti-HER3 to antibody coated AHC sensors at different concentrations for antibodies restore sensitivity to vemurafenib in BRAF-V600E– 120 seconds, followed by a 120-second dissociation time. All mutant colon cancer (34) and blockade of HER2:HER3 signaling with measurements were performed at 25C with agitation at 1,000 rpm. combination therapy overcomes trastuzumab resistance in HER2- Sensorgrams were referenced for buffer effects and then analyzed positive breast cancer (35). HER3, therefore, represents a promising using the Octet QK384 -software (ForteBio). Kinetic responses were therapeutic target for the treatment of HER3-driven tumors and HER globally fitted using a one-site binding model to obtain values for – K K therapy resistant tumors. association ( on), dissociation ( off) rate constants and the equi- Although embryonic lethal in knockout mice (36), HER3 inhi- librium dissociation constant (KD). bition has been shown to be broadly safe in the clinic, with only low-grade toxicity observed, such as skin rash and gastric compli- ELISA cations, consistent with HER3's low expression in normal adult All proteins were from Sino Biological. Antibodies were analyzed tissues (37). Unfortunately, previous anti-HER3 antibodies have for binding to recombinant human HER3 ectodomain, as