10D1F, an Anti-HER3 Antibody That Uniquely Blocks the Receptor

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 heterodimers. The conventional model for activation is ligand-dependent, 1Hummingbird Bioscience, 1 Research Link, Singapore. 2Hummingbird Biosci- that is, the equilibrium shifts when HER3 in the open conformation 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;19:490–501 (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, 2020 American Association for Cancer Research. oncogenicfusions ofSLC3A2, CD74, orVAMP2 to NRG1isoforms have

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Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization

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).

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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)rateconstantsandtheequi- 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 well as exhibited only limited clinical efficacy. First-generation HER3- mouse, rat and cynomologus homologues of HER3 and human EGFR targeted antibodies (e.g., seribantumab), developed to block the and human HER2. ELISAs were carried out according to standard NRG1-binding region (class 1), did not address ligand-independent protocols. Ninety-six–well Maxisorp plates (Nunc) were coated with heterodimer formation (Fig. 1B) (38). Furthermore, second- 1 mg/mL of target antigen in PBS for 16 hours at 4C. After blocking generation antibodies (e.g., elgemtumab), developed to lock HER3 for 1 hour with 1% BSA in TBS at room temperature, anti-HER3 in the closed conformation (class 2; ref. 39), were hampered by antibodies were serially diluted and added to the plate. After 1-hour conformation-dependent binding that may have limited their incubation at room temperature, plates were washed three times potency. with TBS containing 0.05% Tween 20 (TBS-T) and were then incu- To overcome these limitations, we developed 10D1F, a human- bated with goat anti-human IgG Fc-HRP (Abcam, #ab97225) for ized IgG1 antibody selected for its ability to inhibit HER3 by 1 hour at room temperature. After washing, plates were developed binding an epitope on the heterodimerization interface, accessible with colorimetric detection substrate 3,30,5,50-tetramethylbenzidine irrespective of receptor conformation (Fig. 1B). We demonstrate (Turbo-TMB; Pierce) for 10 minutes. The reaction was stopped that 10D1F harnesses this novel mechanism of action to inhibit with 2 mol/L H2SO4, and OD was measured at 450 nm on a BioTek ligand-dependent and independent HER3-driven tumor growth Synergy HT. more potently than other HER3 therapies, suggesting significant potential for clinical benefit. Inhibition of dimerization All proteins were from Sino Biological. Maxisorp plates (Nunc) were coated with 1 mg/mL of HER2-Fc or EGFR-Fc antigen diluted Materials and Methods in PBS for 16 hours at 4C. Following overnight incubation, plates Antibody isolation were washed three times with washing buffer and blocked with 1% Six- to 8-week-old female BALB/c mice were repeatedly immunized BSA in TBS at room temperature for 1 hour. In a separate plate, with antigenic peptide, recombinant target protein, or cells expressing samples were prepared by preincubating for 1 hour at room the target protein. Twenty-four hours after the final immunization, temperature serially diluted anti-HER3 antibodies with 3 mg/mL total splenocytes were isolated and fused with the myeloma cell of human HER3-His antigen (EC50 of HER3-His binding to HER2- line P3X63.Ag8.65 (ATCC) using ClonaCell-HY Hybridoma Cloning Fc). NRG1 (0.1 mg/mL) was also added to the samples (EC80 of Kit, in accordance with the manufacturer's instructions (Stemcell HER3-His binding with NRG1). After blocking, plates were washed Technologies). After 7 to 10 days, single hybridoma clones were twice with washing buffer and incubated with samples for 1 hour at isolated and antibody-producing hybridomas were selected by screen- room temperature; plates were washed three times and further ing supernatants for antigen binding using ELISA and flow cytometry. incubated with anti-his HRP antibody (Abcam, #ab97225) for Variable regions of selected clones were sequenced and expressed 1 hour at room temperature and developed using standard ELISA in CHO cells for testing (Supplementary Methods). One clone, protocol. 10D1P, was selected for development and subsequently humanized and further affinity matured (Supplementary Methods) into the final Flow cytometry antibody 10D1F (Clone 10D1_c89, PCT/EP2019/058035; WO/2019/ Wild-type HEK293T cells (Supplementary Methods), which do not 185878; ref. 40). express HER3, were transiently transfected with HER3 cDNA expression plasmid (Sino Biological #HG10201-UT) using lipofectamine Antibody production 2000 (Thermo Fisher Scientific) following the manufacturer's protocol. For production of all antibodies, see supplemental methods. Twenty-four hours posttransfection, cells were harvested and used for analysis. Cells were incubated with 10 mg/mL of antibodies at 4C for Antibody affinity 1.5 hours. Cells were washed three times with FACS buffer (PBS with All proteins were from Sino Biological. Antibody affinity was 5 mmol/L EDTA and 0.5% BSA) and resuspended in FITC-conjugated calculated using Bio-Layer Interferometry on an Octet QK384 anti-Fc antibody (Thermo Fisher Scientific) for 40 minutes at 2Cto (ForteBio). First, anti-human IgG capture (AHC) sensors (Forte- 8C. Cells were washed again and resuspended in 200 mL of FACS flow Bio) were loaded with anti-HER3 IgG antibodies (25 nmol/L). buffer (PBS with 5 mmol/L EDTA) for flow cytometric analysis using Kinetic measurements were performed in the absence or presence MACSQuant 10 (Miltenyi Biotec). After acquisition, all raw data were of NRG1. NRG1 was used at 1:1 molar ratio with HER3 wherein the analyzed using Flowlogic software. Cells were gated using forward and complex was allowed to form at room temperature for 2 hours. side scatter, and the profile on percentage of positive cells was His-tagged human HER3 or HER3–NRG1 complexes were loaded determined.

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Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization

Epitope binning and mapping injection in SD rats and NCr nude mice, respectively. Vehicle (PBS) For epitope binning, human HER3-His protein (Sino Biological) in was administered as a negative control. Blood was obtained at baseline PBS was immobilized to Anti-Penta His sensor (HIS1K, Fortebio) on (24 hours), 6, 24, 96, 168, and 336 hours after administration. an Octet QK384 (Fortebio), for 5 minutes. Baseline signals were Antibody in the serum was quantified by ELISA using HER3-His measured for 30 seconds before loading 400 nmol/L saturating anti- (Sino Biological) coated plates and anti-human IgG Fc-HRP (Abcam body in PBS for 10 minutes at a shake speed of 1,000 rpm. Subse- #ab97225). The parameters for the pharmacokinetic analysis were quently, biosensors were immersed in 300 nmol/L competing antibody derived from a noncompartmental model: maximum concentration C – – fi t in PBS for 5 minutes at a shake speed of 1,000 rpm. Association of ( max), AUC (0 336 hours), AUC (0 in nity), half-life ( 1/2), clearance V antibodies was monitored on the sensorgram at each step. (CL), volume of distribution at steady state ( ss). A peptide-based epitope mapping study was also conducted to identify the potential binding region of the antibody (Supplementary Safety and toxicity Methods). Six- to 8-week-old female BALB/c mice and SD rats were injected intraperitoneally or via slow (2 minutes) intravenous injection, respec- In vitro tumor growth assays tively, with a single dose of 10D1F at the indicated concentrations. Cell lines (Supplementary Methods) were treated with 10-point Blood samples were obtained at 24 and 96 hours postinjection for serially diluted concentrations of therapeutic antibodies as indicated. mice and at 24, 6, 24, 96, 168, and 336 hours for the rats. Biochemistry Cell viability was measured using CCK-8 assay (Dojindo) at 3 to 5 days parameters were analyzed using Vetscan VS2 chemistry analyzer using posttreatment. The percentage of cell inhibition is shown relative to Abaxis Comprehensive Diagnostic Profile rotor and hematologic cells treated with buffer only (PBS). Data points represent the average analysis was performed using Vetscan HM5 analyzer. Gross necropsy of three replicates. IC50s were calculated by plotting percent inhibition (lesions, organ abnormalities, ascites, and hemorrhage) was performed as a function of antibody concentration and fitting the data points to a at the end of the study. four-parameter logistic model. IHC HER3 phosphorylation and pathway activation Arrays of frozen normal and malignant human tissue cryosections Cell lines (Supplementary Methods) were seeded in 6-well plates were from US Biomax (FMC282d). Slides were dried in a desiccator for fi with 10% serum O/N at 37 C, 5% CO2. Cells were starved with 0.2% 10 minutes and xed in 100% acetone for 10 minutes at room FBS culture medium for 16 hours, followed by treatment with different temperature. Endogenous peroxidase was blocked with 3% (v/v) in vitro antibodies at the IC50 determined by tumor growth assays for H2O2 for 15 minutes at room temperature. Slides were then blocked 0.5 or 4 hours. Before harvesting, cells were stimulated with 100 ng/mL with 10% goat serum for 30 minutes at room temperature, and of NRG1 (Sino Biological). Protein was harvested in RIPA lysis buffer incubated with 0.124 mg/mL (827 nmol/L) primary antibody (Thermo Fisher Scientific) according to the manufacturer's protocol (10D1F in a mouse IgG2a backbone) overnight at 4C and HRP and quantified by Bradford assay. Protein samples (50 mg) were polymer–conjugated goat anti-mouse secondary antibody for 30 fractionated by SDS-PAGE and transferred to nitrocellulose mem- minutes at room temperature. Slides were developed using Bond brane using Bio-Rad semidry transfer cell. Membranes were blocked Mixed DAB Refine for 5 minutes at room temperature, followed by with 5% BSA in TBS and immunoblotted with the indicated antibodies, rinse in DI H2O to stop the reaction. Following IHC, slides were with either anti-mouse IgG-HRP secondary antibody (Lifetech counterstained with hematoxylin for 5 minutes at room temperature, #A24512) or anti-rabbit IgG-HRP secondary antibody (Cell Signaling rinsed, dehydrated, and mounted in synthetic mounting media. Sec- Technology #7074S). Additional details in are provided in Supple- tions were scanned using a Leica SCN scanner at 20 magnification. mentary Methods. Blots were visualized with Bio-Rad Clarity Western ECL substrate and a Syngene Gel Doc (Thermo Fisher Scientific). Blots were quantified using densiometric analysis and normalized to b-actin. Results Existing anti-HER3 neutralizing antibodies attempt to inhibit Animal experiments ligand binding (class 1, e.g., seribantumab) or trap the receptor in an All animals were purchased from InVivos, housed under specific inactive conformation (class 2, e.g., elgemtumab) to prevent hetero- pathogen-free conditions, and treated in strict compliance with the dimerization of HER3 with its binding partners (Supplementary Institutional Animal Care and Use Committee guidelines. Table S1). However, these fail to completely suppress HER3 activation as both modes of HER3 activation, ligand-dependent and ligand In vivo tumor growth assays independent, are not fully blocked (Fig. 1A). Furthermore, although Tumor xenografts were established by subcutaneous injection in the many of these are IgG1 antibodies and thus capable of inducing right flank of NCr nude or NPG mice, approximately 6 weeks old with depletion of tumor cells through antibody-dependent cellular cyto- 1 106 tumor cells (except A549 cells, where 5 106 cells were used). toxicity (ADCC), this has been insufficient to differentiate their clinical Tumor volume was measured using calipers (Supplementary Meth- efficacy. We reasoned that a more effective strategy to abrogate ods). Once mean tumor volume reached 100 to 150 mm3, mice were pathway activation would be the prevention of all HER3 heterodimer- treated intraperitoneally with 25 mg/kg of either 10D1, elgemtumab, ization using a novel molecular mechanism of action that would cetuximab, trastuzumab, or vehicle control (PBS) as indicated. directly block the dimerization interface of HER3, irrespective of conformational state. Pharmacokinetics Single-dose pharmacokinetic profiles were analyzed in 6- to 8-week- Development of an anti-HER3 antibody against the HER3 old female NCr nude mice or female Sprague–Dawley rats (SD rats). dimerization interface 10D1F was administered in a single dose at the indicated concentration Raising antibodies that bind to the heterodimerization interface of via tail vein slow intravenous injection (2 minutes) or intraperitoneal HER3 is challenging due to the restricted surface area and the low

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immunogenicity of this region. Therefore, computational structural 10D1F inhibits HER3 heterodimerization analysis and modeling were used to predict HER3 specific epitopes To assess the ability of 10D1F to prevent HER3 heterodimerization within the dimerization interface that would be accessible in both with EGFR and HER2, we conducted plate-based in vitro dimerization conformations and conserved across mammalian orthologs. The assays. 10D1F inhibited HER3 dimerization with EGFR (Fig. 3C) and selected target region was located on the dimerization interface of HER2 (Fig. 3D) in a dose-dependent manner, with significantly higher HER3 subdomain II and spatially separated from the ligand-binding potency than the anti-HER3 class 1 and class 2 antibodies. Notably, the site located between subdomains I and III (Fig. 1B). anti-HER3 class 2 antibody did not cause any significant inhibition of mAbs against the target region were raised by immunization of mice HER3 heterodimerization. and subsequent isolation of hybridoma clones using Hummingbird's Rational Antibody Discovery Platform. One clone, 10D1P, was select- 10D1F demonstrates superior inhibition of proliferation in a fi K ed for further development based on preliminary binding af nity ( d panel of cancer cell lines 52.6 nmol/L), specificity within the EGFR family and species cross- Given the unique binding region and resulting superior inhibition of reactivity to HER3 orthologs (Supplementary Fig. S1). 10D1P was heterodimerization, it was hypothesized that 10D1F would show more subsequently humanized and affinity matured (Supplementary Meth- potent inhibition of HER3-mediated cell growth and survival path- ods). The final antibody, 10D1F, was selected from among the ways than other anti-HER3 antibodies. To this end, we measured the optimized variants based on its in vitro physicochemical and func- effect of 10D1F on the proliferation of a panel of cancer cell lines tional properties, including increased binding affinity, thermal stability (gastric: N87, SNU16; SCCHN: FaDu; lung: A549, HCC95; kidney: (>70C), and low aggregation potential after freeze-thaw (below ACHN; ovarian: OvCar8; thyroid: BCPAP). Although the genetic and Fig. 2). background and expression profiles of these cell lines are unique, they were selected as examples of cancer cell lines where HER3 activation of 10D1F binds HER3 with high affinity and specificity the PI3K pathway is likely to be important to growth and proliferation. fi K 10D1F demonstrated high af nity to human HER3 ( d of Furthermore, based on mRNA and protein expression data, these cell 1pmol/L),a>1,000-fold improvement over the parental antibody, lines should reflect the main classes of alternative HER3 activation. We and no cross-reactivity with EGFR or HER2 (Fig. 2A and B). There included cell lines with high NRG1, expected to show predominantly wasnodifferenceobservedforthebindingaffinity in the presence ligand-driven HER3 activation (FaDu, A549, HCC95), those with low or absence of NRG1, that is, to a more open or closed conformation NRG1 and high HER2/EGFR for which HER3 activation is expected to of HER3. This contrasted to that observed with examples of ligand be driven by HER2/EGFR (N87, SNU16), and those where both HER2/ blockers (class 1) and conformational change blockers (class 2) EGFR and NRG1 are high and where HER3 activation could be driven where binding for both was decreased in the presence of NRG1 by both mechanisms (OvCAR8, ACHN). In addition, one cell line (Supplementary Fig. S1E–S1F). Consistently, 10D1F bound with harboring the BRAF mutation V600E and known to be resistant to high specificity to HER3-overexpressing HEK293T cells, but not BRAF inhibitors was selected (BCPAP), which also showed the highest wild-type cells (Fig. 2C). Moreover, although there was a 10-fold EGFR, HER2, and HER3 protein expression (Supplementary difference in affinity between human/cyno and rodent HER3 for the Table S2). As a monotherapy, 10D1F effectively inhibited the prolif- parental antibody, 10D1F exhibited identical binding to all HER3 eration of all cell line models in a dose-dependent manner. Further- orthologs (Fig. 2D). more, 10D1F inhibited proliferation more potently than anti-HER3 class 1 or class 2 antibodies as well as selected anti-EGFR or anti-HER2 10D1F binds to a unique epitope in the dimerization interface of antibodies (Fig. 4A; Supplementary Fig. S3A). Remarkably, 10D1F HER3, distinct from other anti-HER3 antibodies also potently inhibited the proliferation of the BRAF V600E–mutant To verify the binding site of 10D1F, we conducted an epitope line BCPAP (Fig. 4A; ref. 41). binning experiment using class 1 (seribantumab) and class 2 (elgemtumab) anti-HER3 antibodies. The epitope of the seribantu- 10D1F inhibits HER3 phosphorylation and pathway activation mab has previously been mapped to domain I of HER3, within the Given its effects on in vitro proliferation of cancer cell lines, we ligand-binding site, whereas the epitope of elgemtumab has been hypothesized that 10D1F would effectively inhibit dimerization- mapped to a discontinuous epitope across the interaction surface of induced receptor phosphorylation and downstream signaling. To test HER3 subdomains II and IV, hypothesized to lock the HER3 in an this, we measured the levels of total and phosphorylated HER family inactive conformation (Figs. 1B and 3). 10D1F did not compete receptors as well as downstream PI3K/AKT/mTOR pathway and with class 1 or class 2 antibodies for binding to human HER3 MAPK/ERKK pathway intermediates by Western blot analysis in a ectodomain (Fig. 3A). subset of the cancer cell lines that responded to 10D1F previously To further elucidate the 10D1F-binding region, a peptide-based (A549, N87, FaDu, and OvCar8). Cells were treated with 10D1F (or epitope mapping study was conducted (Supplementary Methods). HER3 class 1 and class 2 antibodies) at the IC50 for each cell line, as This identified the consensus sequence CFGPNPNQCC to have the determined during the proliferation inhibition assay, for 30 minutes strongest interaction with 10D1F, and thus likely to contain multiple (N87, FaDu, and OvCar8) or 4 hours (A459) prior to stimulating residues that constitute the 10D1F epitope (Supplementary Fig. S2). HER3 with NRG1. Consistent with the observed blockade of receptor This sequence maps to the desired target region in the dimerization heterodimerization, 10D1F completely abrogated phosphorylation of interface (Fig. 3C). It should be noted, however, that the observed HER3 in FaDu and OvCar8, and substantially attenuated the phos- interaction with the linear peptides was weak, suggesting that 10D1F phorylation in A549 and N87 (Fig. 4B; Supplementary Fig. S3B). In binds a discontinuous epitope involving additional residues outside addition, 10D1F decreased phosphorylation of HER2 in A549, N87, (yet topologically close) of this sequence. and OvCar8, did not alter the levels of pan-HER3 in N87 and FaDu, Together, these results demonstrate that 10D1F binds a novel and only slightly decreased pan-HER3 levels in A549 and OvCar8. epitope on the domain II dimerization interface, topologically distinct Furthermore, 10D1F markedly reduced the levels of phosphorylated from the epitopes of other anti-HER3 antibodies. AKT in A549, N87, FaDu, and OvCar8, and phosphorylated mTOR in

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Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization

A 10D1F – HER3 binding in C 10D1F – HER3 binding in 10D1F – Nave binding absence of NRG presence of NRG

Color population HEK293T WT HEK293T O/E HER3

BD10D1F - Binding to HER3 orthologs 10D1F - Binding to HER family proteins

Human HER1 1.0 1.0 Human HER2 Human HER3 0.8 Human HER3 0.8 Mouse HER3 Human IgG isotype 0.6 0.6 Cyno HER3 Irrelevant antigen Rat HER3 0.4 0.4 Human IgG isotype 0.2 0.2 Irrelevant antigen 0.0 0.0 Absorbance 450 nm Absorbance 450 nm - -0.2 0.2 - - - -12 -10 -8 12 10 8 Concentration log (mol/L) Concentration log (mol/L) Human HER3 Mouse HER3 Rat HER3 Cyno HER3 Human EGFR Human HER2 Human HER3 1.437e-011 3.322e-011 3.032e-011 2.581e-011 EC50 (mol/L) EC50 (mol/L) N/A N/A 1.437e-011

E 10D1F - Thermal stability F 10D1F - Freeze-thaw stability

15,000 10D1F_1 SEC (Superdex 200 10/300) Cycle 1 10D1F_2 Cycle 2 10,000 60 Cycle 3 10D1F_3 Cycle 4 5,000 no_sample_1 40 Cycle 5 no_sample_2 Cycle 6 Cycle 7 20 0 no_sample_3 Cycle 8

First derivative Cycle 1-repeat fluorescence (RFU) Tm=70°C 0 -5,000 Absorbtion 280 nm (mAU) 20 30 40 50 60 70 80 90 100 5 1015202530 Temperature (°C) Retention volume (mL)

Figure 2. 10D1F binds with high affinity and specificity to a species-conserved epitope on HER3 and shows high stability. A, Biolayer interferometry (octet) binding kinetics of 10D1F to human HER3 in the presence (open conformation) and absence (closed conformation) of the ligand, NRG1. Data were normalized to reference (blue/orange) and fitted with 1:1 global fitting (red). B, Binding specificity of 10D1F by ELISA using human HER1, HER2, and HER3 antigens. Data shown are mean n ¼ 3 measurements and error bars are SEM. C, Binding specificity of 10D1F to native HER3 was analyzed by flow cytometry using HEK293T cells stably transfected with human HER3 and parental HEK293T. D, ELISA binding of 10D1F to HER3 orthologs; human, mouse, rat, and cyno HER3. Data shown are mean of n ¼ 3 measurements. E, Thermostability of 10D1F analyzed by differential scanning fluorimetry. F, Aggregation/degradation propensity of 10D1F analyzed by freeze-thaw stability test. Data were acquired for 7 cycles of freeze-thaw. N/A, not applicable.

N87, FaDu, and OvCar8, indicating downstream inhibition of the domain with activatory Fcg receptors on natural killer (NK) cells (42), PI3K/AKT/mTOR pathway. In all cell lines, 10D1F showed the great- and can also inhibit signaling indirectly by triggering internalization est inhibition of cell pathways, in comparison with the HER3 class 1 or and degradation of the receptor, thus reducing the concentration of class 2 antibodies. activated receptors at the cell surface (40). Most anti-HER3 antibodies, such as elgemtumab, are capable of 10D1F induces ADCC but does not cause signiﬁcant HER3 inducing ADCC as they are IgG1 isotype antibodies similar to 10D1F internalization (Supplementary Table S1). Some, such as seribantumab, are IgG2 Some anti-HER family antibodies may have other mechanisms of antibodies and are not expected to induce ADCC. This can be action beyond neutralization. Trastuzumab, for example, can deplete demonstrated in vitro using isolated NK cells cocultured with HER2-expressing cells by ADCC due to interaction of the antibody Fc HER3-overexpressing cells. 10D1F and elgemtumab stimulated

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A HER3 Epitope binning All steps aligned by step antigen immobilization (sensor location) 3.0

2.5

2.0

1.5

1.0

Shift (nm) Shift 0.5

0 Competing Saturating antibody antibody −0.5

−1.0 0 200 400 600 800 1,000 1,200 1,400 Time (s)

B HER3 Heterodimer HER3 Ectodomain - open conformation 10D1F 10D1F Seribantumab

Membrane

HER3 Ectodomain - closed conformation

Seribantumab 10D1F

Membrane

Elgemtumab Membrane

C EGFR:HER3 heterodimerization D HER2:HER3 heterodimerization

120 120 10D1F 100 100 10D1F HER3 Class 1 Ab 80 80 HER3 Class 1 Ab HER3 Class 2 Ab 60 HER3 Class 2 Ab 60 Isotype control Isotype control

40 40 % Inhibition %

20 Inhibition % 20 0 0

-20 -20 -10 -9 -8 -7 -6 -9 -8 -7 -6 -5 -4 Antibody Conc.(LogM) Antibody Conc.(LogM)

10D1F HER3 Class 1 Ab HER3 Class 2 Ab Isotype control 10D1F HER3 Class 1 Ab HER3 Class 2 Ab Isotype control (mol/L) EGFR:HER3IC50 4.781e-009 5.419e-008 N/A N/A HER2:HER3 IC50 (mol/L) 7.88e-008 N/A N/A N/A

Figure 3. 10D1F binds to a unique epitope on the HER3 dimerization interface, topologically distant from other HER3 antibodies, to inhibit heterodimerization with EGFR and HER2. A, 10D1F epitope binning, using examples of class 1 (seribantumab) and class 2 (elgemtumab) anti-HER3 antibodies. An in-tandem method was used to test competitive binding of antibody pairs to HER3 by biolayer interferometry and signals were aligned to baseline. B, Structural models of HER3 showing the epitopes of 10D1F (yellow), an example of a class 1 anti-HER3 antibody (seribantumab, blue) and an example of a class 2 anti-HER3 antibody (elgemtumab, green) on different HER3 conformations. Left, a model of the HER3 heterodimer with EGFR including bound EGF ligand (pink) – NRG1 binding would be at a similar region on HER3. C, Inhibition of EGFR:HER3 dimerization, analyzed by competition ELISA. D, Inhibition of HER2:HER3 dimerization, analyzed by competition ELISA. Data shown are mean of triplicate measurements and error bars are SEM. N/A, not applicable.

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Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization

Figure 4. 10D1F demonstrates superior inhibition of cancer cell line proliferation by potently inhibiting downstream signaling through the PI3K pathway. A, In vitro proliferation experiments using A549 (lung) and N87 (gastric) cells treated with serially diluted anti-HER3 antibodies for 5 days with cell viability determined by CCK-8 assay. Cell proliferation values are relative to untreated cells and represent average of three replicates SEM. B, Western blots of A549 and N87 cells, treated with anti-HER3 antibodies for 4 and 24 hours, respectively, before stimulating with NRG1 (50 ng/mL), harvesting cells, and immunoblotting with the indicated antibodies.

ADCC to a similar level, while seribantumab did not (Supplementary pathway observed with 10D1F was not the result of internalization Fig. S4). of HER3 from the cell surface. The ability of anti-HER3 antibodies to drive internalization of HER3 is less clear. To further investigate the mechanism of action 10D1F inhibits tumor growth in mouse xenograft models of 10D1F, we evaluated whether 10D1F is internalized upon binding to The results of the in vitro proliferation inhibition assay suggested cell-surface expressed HER3. We used a selection from our panel of that 10D1F would be effective as a single agent across HER3-driven cancer cell lines (HCC95, N87, OvCar8), and treated these cells with tumors. To test the monotherapy efficacy of 10D1F in vivo,we 10D1F, anti-HER2, or anti-HER3 class 1 and class 2 antibodies tagged conducted tumor growth inhibition studies in murine cell-derived with pH sensitive dye that only fluoresces after the antibody has been xenograft (CDX) models of tumors with high NRG1 (FaDu, A549), internalized and trafficked to the low-pH endosome (Supplementary high HER2/EGFR (N87), or high NRG1 and HER2/EGFR (OvCar8). Methods). Internalization was measured for 24 hours by live fluores- Female NCr nude mice were subcutaneously implanted and treated cence microscopy (Supplementary Fig. S4). Whereas the anti-HER2 either once a week (FaDu, OvCar8) or twice a week (N87, A549) with antibody was clearly internalized by N87 cells (high HER2 expression), vehicle control (PBS) or 25 mg/kg 10D1F and other anti-HER family only the class 2 anti-HER3 antibody was efficiently internalized, and by antibodies. 10D1F outperformed both class 1 and class 2 anti-HER3 only one of the HER3-expressing lines, OvCar8. 10D1F was not antibodies as well as other relevant anti-HER family antibodies in high internalized to detectable levels in any of the tested cancer cell lines. NRG1 CDX models, demonstrating complete inhibition of tumor Conversely, all anti-HER3 antibodies were significantly internalized by growth in A549, FaDu, and OvCar8 CDX models (Fig. 5). Remarkably, a recombinant HER3-overexpressing cell line. Alongside the minimal 10D1F also showed significant antitumor efficacy (64% TGI) in the effects on total HER3 protein observed by Western blot analysis, these high HER2/EGFR N87 model in contrast to the class I and class 2 anti- results indicate that the downregulation of the PI3K/AKT/mTOR HER3 antibodies, which failed to show any effect. Further support for

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Figure 5. 10D1F demonstrates superior in vivo tumor growth inhibition in multiple xenograft tumor models. Female NCr nude mice were subcutaneously implanted with N87 (A), A549 (B), FaDu (C), and OvCaR8 (D). Once tumors reached a volume of 100 to 200 mm3, mice were randomized and dosed with 25 mg/kg 10D1F at indicated time points. Tumor volumes were measured twice a week. Each data point represents the mean tumor volume SEM from n ¼ 8 mice.

the broad efficacy of 10D1F's mechanism of blocking HER3 dimer- and 250 mg/kg, i.v.) of 10D1F, respectively, and antibody concentra- ization was seen in additional tumor models treated with the parental tion in blood was measured at 0, 0.5, 6, 24, 96, 163, and 336 hours after antibody, 10D1P, where potent efficacy was observed in very high administration. 10D1F exhibited a half-life of more than 10 days in NRG1 (HCC95), HER2/EGFR high (SNU16), or high HER2/EGFR both mice and rats (Fig. 6). and NRG1 (ACHN) models (Supplementary Fig. S5A). Of note, To examine the potential for adverse effects, acute dose toxicity although the majority of CDX experiments used NCr Nude mice with studies were conducted. BALB/c mice were treated with increasing competent NK cells and active ADCC, 10D1P was also tested in an doses (25, 50, 100, and 250 mg/kg, i.p.) of 10D1F and hematology and NPG mouse background without NK cells and ADCC and demon- biochemistry profiles were obtained 96 hours posttreatment. There strated identical results (Supplementary Fig. S5A, bottom right). were no signals of toxicity observed in the hematologic or biochemical Furthermore, examining the tumors from treated mice (FaDu and parameters, nor behavioral or gross anatomic differences, at any of the OvCar8) confirmed that the PI3K pathway was indeed robustly and doses tested (Supplementary Tables S3). To further explore the safety durably suppressed (Supplementary Fig. S5B, c.f. Fig. 4B). profile of 10D1F, SD rats were treated with the same doses, and the hematology and biochemistry profiles were recorded at 0, 6, 24, 96, 10D1F exhibits favorable pharmacokinetic and safety profiles 168, and 336 hours posttreatment. With the exception of a mild To confirm the tumor specificity and evaluate the potential for off- increase in ALP at the highest doses, which was not observed in the target binding of 10D1F in healthy tissues, we examined the staining mice, there were again no behavioral or gross anatomic differences pattern of 10D1F to human malignant and normal tissue cryosections observed, and no significant toxicity signals noted for hematologic or by IHC (Fig. 6). In agreement with the specificity noted in previous biochemical parameters (Supplementary Table S3). ELISAs, 10D1F exhibited preferential binding to cancerous tissue, with marginal or no cross-reactivity to healthy tissue. Therapeutic antibodies must possess half-lives in plasma compat- Discussion ible with appropriate dosing regimens and must demonstrate minimal There has been significant interest in HER3 as a therapeutic target to toxicity to normal tissues. To determine the pharmacokinetic para- treat cancer progression and acquired resistance to MAPK inhibitors meters of 10D1F in rodents, NCr nude mice or SD rats were admin- including EGFR and HER2-targeted antibodies and TKIs. Several istered with a single dose (25 mg/kg, i.p.) or multiple doses (25, 50, 100, antibody-based strategies have been pursued to suppress HER3-

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Antitumor mAb 10D1F Uniquely Blocks HER3 Heterodimerization

A Malignant Normal Malignant Normal

Vulva Lung

Stomach Liver

Esophagus Kidney

Skin Colon

Testis Breast

Thyroid Rectum gland

BC10D1F - PK in NCr nude mice 10D1F - PK in SD 400 400 250 mg/kg 100 mg/kg 300 300 m g/mL)

m g/mL) 25 mg/kg 10 mg/kg 200 200

100 100 Concentation ( Concentation ( 0 0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Time (h) Time (h)

Cmax Tmax AUC0-336 AUC0-Infinity Elimination T1/2 CL Vss Cmax Tmax AUC0-336 AUC0-Infinity Elimination T1/2 CL Vss Dose Dose (mg/mL) (h) (mg/mL*h) (mg/mL*h) Rate KEL(1/h) (h) (L/h) (L) (mg/mL) (h) (mg/mL*h) (mg/mL*h) Rate KEL(1/h) (h) (L/h) (L) 25 mg/kg 257.297 6.000 64677.050 107359.970 0.003 252.753 4.66E-06 0.002 10 mg/kg 159.851 6 21286.213 32453.407 0.003 199.43 0.000077 0.023 25 mg/kg 217.686 6 28911.974 168078.210 0.001 639.67 0.000037 0.034 100 mg/kg 250.516 6 69078.168 169396.299 0.002 438.79 0.000148 0.094 250 mg/kg 297.919 24 81208.032 196765.343 0.002 440.24 0.000318 0.201

Figure 6. 10D1F exhibits preferential binding to cancerous tissue, with marginal or no cross-reactivity to healthy human tissue and has a favorable pharmacokinetic profile, with a serum half-life of more than 10 days in rodents. A, Normal and malignant tissue arrays were stained with 10D1F at 0.124 mg/mL. Scale bar, 100 mm. B, Serum concentration of 10D1F in NCr nude mice analyzed by ELISA at the indicated time points. 10D1F was administered in a single dose at 25 mg/kg via intraperitoneal injection. Values represent mean of n ¼ 3 mice SEM. C, Serum concentration of 10D1F in serum of Sprague–Dawley rats was analyzed by ELISA at the indicated time points. 10D1F was administered in a single dose at the indicated concentration via tail vein slow intravenous bolus injection. Values represent mean of n ¼ 3 rats SEM. mediated signaling, which include (i) inhibiting heterodimerization a heterodimer partner and promoting ligand-independent heterodi- between HER2 and HER3 (pertuzumab; ref. 43); (ii) blocking NRG1 merization, the leading hypothesis for why the ligand blocking (class 1) binding (e.g., seribantumab; ref. 38); and (iii) trapping HER3 in the anti-HER3 antibodies failed (26, 27). closed inactive conformation (e.g., elgemtumab; ref. 40). However, Here, we report an anti-HER3 antibody, 10D1F, that selectively none of these strategies have been completely effective in inhibiting binds with high affinity and specificity at a binding site on the domain HER3-mediated signaling; pertuzumab does not address the promis- II heterodimerization interface, which is unique and distant from cuity of HER3 to form heterodimers with EGFR, seribantumab fails to previously described anti-HER3 antibodies and available in all con- prevent ligand-independent dimerization, and elgemtumab does not formations. 10D1F was found to inhibit HER3 heterodimerization efficiently trap HER3 in the inactive conformation. In contrast to these with EGFR and HER2 more effectively than other anti-HER3 anti- previous approaches, we hypothesized that directly blocking the bodies and demonstrated superior efficacy over other HER-targeted heterodimerization interface of HER3 in all conformations would agents in inhibiting tumor growth of both in vitro and in vivo models of provide a broader and more efficacious mechanism for inhibiting cancers with ligand-dependent and ligand independent HER3 activa- HER3 activation, and prevent tumors from escaping by overexpressing tion. As little evidence of 10D1F internalization was observed, and

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identical efficacy of 10D1 was shown in a HER3 CDX model using a Alongside its superior efficacy, 10D1F demonstrated favorable NSG-like mouse (with no ADCC), our data strongly support the specificity, pharmacokinetic and toxicologic profiles, with no overt hypothesis that the primary mechanism of action of 10D1F is the toxicities detected in hematology or biochemistry parameters in rodent inhibition of HER3 heterodimerization, and that this is a more effective models. strategy than previous approaches to targeting HER3. Intriguingly, The data presented in this study positions 10D1F as a promising while some internalization of a class 2 HER3 antibody was seen in the candidate therapeutic to treat patients with solid tumors driven by OvCar8 cell line, other cell lines demonstrated minimal internaliza- HER3 heterodimers. Of note, our data strongly suggest that BRAF tion, independent of class/binding site. Indeed, this inconsistent V600E mutant, and tumors resistant to TKI, trastuzumab, and cetux- internalization has also been noted for the anti-HER3 antibody imab may be suitable indications for clinical assessment. patritumab (the antibody component of the antibody–drug conjugate, U3-1402; ref. 44). The much lower tumor overexpression observed for Disclosure of Potential Conflicts of Interest HER3 compared with HER2 (Supplementary Table S2), and lack of D. Thakkar is the principal scientist (Head of Discovery) at and has ownership significant and consistent internalization, casts doubt on HER3 as a interest (including patents) in Hummingbird Bioscience. Z. Wu has ownership reliable tumor target for delivery of a cytotoxic payload. interest (including patents) in Hummingbird Bioscience and is a coinventor of the patent for humanized anti-HER3 antibody. K.H. Paszkiewicz is the chief technology All previous attempts to inhibit HER3 have, at best, exerted only officer at and has ownership interest (including patents) in Hummingbird Bioscience. partial inhibition but failed to sustainably block downstream signaling. P.J. Ingram is the CEO at and has ownership interest (including patents) in In contrast, we show that 10D1F either fully or substantially reduces Hummingbird Bioscience. J.D. Boyd-Kirkup is the chief scientificofficer at and has phosphorylation of HER3 and AKT in NRG1, HER2/EGFR, and ownership interest (including patents) in Hummingbird Bioscience. No potential NRG1 þ HER2/EGFR–driven cell line models without significantly conflicts of interest were disclosed by the other authors. affecting the levels of pan-HER3. These results show that effective ’ inhibition of heterodimerization by 10D1F causes a more potent and Authors Contributions sustained downregulation of the PI3K/AKT/mTOR signaling path- Conception and design: D. Thakkar, M.M. Taguiam, P.J. Ingram, J.D. Boyd-Kirkup Development of methodology: D. Thakkar, M.M. Taguiam, P.J. Ingram, J.D. Boyd- way, which in turn leads to superior efficacy of this antibody. Notably, Kirkup 10D1F as a single agent potently inhibits the growth of BCPAP cells Acquisition of data (provided animals, acquired and managed patients, provided that carry the constitutively activating BRAF mutation V600E in the facilities, etc.): D. Thakkar, V. Sancenon, S. Guan MAPK/ERKK pathway and are known to be resistant to BRAF V600E Analysis and interpretation of data (e.g., statistical analysis, biostatistics, inhibition. These cells also express the highest levels of HER3, EGFR, computational analysis): D. Thakkar, V. Sancenon, M.M. Taguiam, S. Guan, and HER2. These findings suggest that activation of the PI3K/AKT/ E. Ng, K.H. Paszkiewicz, P.J. Ingram, J.D. Boyd-Kirkup Writing, review, and/or revision of the manuscript: D. Thakkar, V. Sancenon, mTOR pathway by HER3 is a critical mechanism for driving cell K.H. Paszkiewicz, P.J. Ingram, J.D. Boyd-Kirkup proliferation in these cells and has important therapeutic implications Administrative, technical, or material support (i.e., reporting or organizing data, as it suggests that 10D1F, either as monotherapy, or in combination constructing databases): D. Thakkar, M.M. Taguiam, K.H. Paszkiewicz with BRAF inhibitors, may be beneficial in patients with drug-resistant Study supervision: D. Thakkar, P.J. Ingram, J.D. Boyd-Kirkup BRAF mutations. Other (engineering of antibodies): Z. Wu Increased levels of the HER3 ligand NRG1 and genomic rearrange- ments involving NRG1 fusions are also associated with resistance to Acknowledgments trastuzumab in breast cancer and certain types of lung carcinomas (45). The authors would like to thank all members of the Hummingbird Bioscience fi team, especially Sabrina Ng, Shalini Paliwal, Shani Ajumal, Akila Sadasivam, Rahmat 10D1F demonstrated potent ef cacy in the NRG1 high models, Hidayat, Raihanah Ayob, and Michelle Su, for their assistance during the develop- including SCCHN (FaDu) and lung (A549, and HCC95). Notably, ment of the molecule and data acquisition for this study. The authors would also like HCC95 was found to express the highest levels of NRG1 mRNA among to thank Raymond Price for assistance during the preparation of this manuscript. All a panel of 67 lung cancer cell lines, due to a gene amplification (45). work was fully funded by Hummingbird Bioscience. Although HCC95 does not harbor an NRG1 fusion, it is thus a useful preclinical model to evaluate the efficacy of drugs in highly NRG1- The costs of publication of this article were defrayed in part by the payment of page advertisement driven cancers. Our in vitro tumor growth inhibition data clearly show charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that 10D1F is more effective than existing class 1 and class 2 anti-HER3 antibodies in HCC95 and other NRG1-driven models, suggesting fi Received May 15, 2019; revised August 15, 2019; accepted October 10, 2019; additional clinical bene t over existing therapies. published first January 8, 2020.

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10D1F, an Anti-HER3 Antibody That Uniquely Blocks the Receptor Heterodimerization Interface, Potently Inhibits Tumor Growth Across a Broad Panel of Tumor Models

Dipti Thakkar, Vicente Sancenon, Marvin M. Taguiam, et al.

Mol Cancer Ther 2020;19:490-501. Published OnlineFirst January 7, 2020.

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