Sym015: a Highly Efficacious Antibody Mixture Against MET-Amplified Tumors
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Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-0782 Cancer Therapy: Preclinical Clinical Cancer Research Sym015: A Highly Efficacious Antibody Mixture against MET-Amplified Tumors Thomas Tuxen Poulsen, Michael Monrad Grandal, Niels Jørgen Østergaard Skartved, Rikke Hald, Lene Alifrangis, Klaus Koefoed, Trine Lindsted, Camilla Frohlich,€ Sofie Ellebæk Pollmann, Karsten Wessel Eriksen, Anna Dahlman, Helle Jane Jacobsen, Thomas Bouquin, Mikkel Wandahl Pedersen, Ivan David Horak, Johan Lantto, and Michael Kragh Abstract Purpose: Activation of the receptor tyrosine kinase MET is analogue of emibetuzumab, a monoclonal IgG4 antibody against associated with poor clinical outcome in certain cancers. To target MET currently in clinical development. Sym015 also induced MET more effectively, we developed an antagonistic antibody antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggest- mixture, Sym015, consisting of two humanized mAbs directed ing that secondary effector functions contribute to the efficacy of against nonoverlapping epitopes of MET. Sym015. Experimental Design/Results: We screened a large panel of Retrospectively, all responsive, high MET-expressing models well-annotated human cancer cell lines and identified a subset were scored as highly MET-amplified by in situ hybridization, with highly elevated MET expression. In particular, cell lines of pointing to MET amplification as a predictive biomarker for lung cancer and gastric cancer origin demonstrated high MET efficacy. Preclinical toxicology studies in monkeys showed that expression and activation, and Sym015 triggered degradation of Sym015 was well tolerated, with a pharmacokinetic profile sup- MET and significantly inhibited growth of these cell lines. Next, we porting administration of Sym015 every second or third week in tested Sym015 in patient- and cell line–derived xenograft models humans. with high MET expression and/or MET exon 14 skipping altera- Conclusions: The preclinical efficacy and safety data provide tions, and in models harboring MET amplification as a mecha- a clear rationale for the ongoing clinical studies of Sym015 nism of resistance to EGFR-targeting agents. Sym015 effectively in patients with MET-amplified tumors. Clin Cancer Res; 23(19); inhibited tumor growth in all these models and was superior to an 5923–35. Ó2017 AACR. Introduction (NSCLC; refs. 3, 4). In addition, genetic alterations leading to MET exon 14 skipping have recently gained interest. MET exon 14 The receptor tyrosine kinase (RTK) MET, also known as c-MET encodes the juxtamembrane receptor domain containing the or hepatocyte growth factor (HGF) receptor (HGFR), is essential binding site for the ubiquitin ligase CBL, responsible for triggering for embryonic and liver development, wound healing, and tissue MET internalization and degradation. Exon 14 skipping thus regeneration. Lately, evidence has accumulated for the role of induces stabilization of MET on the cell surface, leading to elevated MET activation as an oncogenic driver in a number of enhanced and constitutively active MET signaling (5, 6). A shift malignancies (1). In a context-dependent manner, high activation to MET dependency as a mechanism of resistance to tyrosine of MET induces increased cell proliferation, survival, motility, kinase inhibitors (TKI) targeting other RTKs is frequently scattering, and angiogenesis and drives epithelial–mesenchymal observed, and up to 20% of EGFR-mutated NSCLCs develop transition and cell invasion (2). resistance by MET gene amplification in response to treatment There are multiple mechanisms for aberrant MET activation in with EGFR-targeting TKIs (7, 8). tumorigenesis, including overexpression of its ligand HGF, as well A number of MET-targeted TKIs and therapeutic antibodies as amplification, activating mutations, and other modifications of have entered clinical development, but so far, none have been the MET gene. In particular, MET gene amplification is observed in approved as MET-targeting agents (9). TKIs generally have limited a distinct subset of patients and is associated with poor prognosis target specificity, which may lead to pleiotropic effects and in malignancies such as gastric and non–small cell lung cancer unwanted toxicity. Furthermore, after long-term therapy with MET-targeting TKIs, subsets of quiescent tumor cells survive treatment, resulting in a rebound effect with aggressive tumor Symphogen A/S, Ballerup, Denmark. regrowth upon release of treatment pressure (10, 11). MET-target- Note: Supplementary data for this article are available at Clinical Cancer ing antibodies in clinical development include the one-armed Research Online (http://clincancerres.aacrjournals.org/). engineered antibody onartuzumab (MetMAb), which demon- Corresponding Author: Thomas Tuxen Poulsen, Symphogen A/S, Pederstrup- strated activity in early-stage clinical trials (12–15), but failed to þ þ vej 93, Ballerup DK-2750, Denmark. Phone: 45 4526-5050; Fax: 45 4526- improve survival when used in combination with erlotinib to treat 5060; E-mail: [email protected] NSCLC in a phase III study (16). More recently, other antibodies doi: 10.1158/1078-0432.CCR-17-0782 targeting MET have entered the clinic. These include ABT-700, Ó2017 American Association for Cancer Research. which has demonstrated activity in patients with MET-amplified www.aacrjournals.org 5923 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 5, 2017; DOI: 10.1158/1078-0432.CCR-17-0782 Poulsen et al. mycoplasma-free, cultured per supplier recommendations, con- Translational Relevance tinuously evaluated for visual changes, and either used within In certain malignancies, such as gastric cancer and lung 6 months of purchase or profiled by cell authentication testing cancer, increased activation of the receptor tyrosine kinase (LGC Standards, Germany). Cells were discarded after 6 weeks of (RTK) MET is associated with poor clinical outcome and culture to minimize the risk of alterations. The cell line resistance to targeted therapies. A number of MET-targeting HCC827R1_Cet#3 was obtained by harvesting cells derived from mAbs are in clinical development, but so far, none have been cetuximab-resistant xenograft tumors of the erlotinib-resistant approved. In recent years, preclinical and early clinical studies cell line HCC827R1 (26). have demonstrated enhanced efficacy and antitumor activity of antibody mixtures directed against different RTK targets, Quantification of receptor protein levels in cell lines and leading to development of Sym015, an MET-targeting anti- xenografts body mixture comprising two antibodies directed against Samples were diluted to 0.2–1.0 mg/mL in a master mix contain- nonoverlapping epitopes. In the current study, we demon- ing fluorescent standards and reducing agent, and processed strate that Sym015 inhibits MET-dependent cancer cell and under standard conditions in a Sally/Sally Sue instrument in vitro in vivo tumor growth in multiple MET-dependent and (ProteinSimple, CA, USA). The following rabbit primary antibo- models, including models exhibiting resistance to EGFR- dies (from Cell Signaling Technology, MA, USA) diluted 1:50 were fi targeting agents. The promising preclinical ef cacy and safety used (clone number and phosphorylation site denoted in paren- data justify the ongoing clinical studies of Sym015 in patients theses): MET, pMET (D26; Y1234/Y1235), pMET (130H2; MET fi with -ampli ed tumors. Y1349), EGFR (C74B9), pEGFR (Y1068), and Pan-Actin. Sym015 and control antibodies Sym015 comprises a balanced (1:1) mixture of two MET- tumors (17, 18), and ARGX-111, which has been glycoengineered targeting monoclonal antibodies (Hu9006 and Hu9338) gener- to enhance secondary effector functions and also demonstrated ated and characterized as described in detail elsewhere (ref. 25, efficacy in a single patient with MET gene amplification (19, 20). Grandal and colleagues, manuscript submitted). Briefly, mice One of the most advanced MET-targeting antibody therapeutics in were immunized with soluble, recombinant MET protein and clinical development is emibetuzumab (LY2875358), a human- MET-overexpressing human cancer cells. Splenic B cells were ized IgG4 reported to block ligand binding and induce receptor harvested and sorted to single cells, antibodies were isolated and internalization (21). Emibetuzumab has demonstrated prelimi- cloned using Symplex technology (27), humanized, and nary evidence of antitumor activity in combination with erlotinib expressed as full-length IgG1 in mammalian cells. A negative in patients with MET-positive tumors and is currently being control antibody targeting a nonhuman antigen was cloned and evaluated in phase II studies (22). expressed using the same approach. The Sym015 concentration As an alternative to mAbs, mixtures comprising more than used for in vitro and in vivo experiments refers to the total antibody one antibody have demonstrated strong potential as RTK- concentration (e.g., for 50 mg/mL Sym015, the concentration of targeting anticancer agents. A major therapeutic advantage of each constituent antibody is 25 mg/mL). Cetuximab (Erbitux) was antibody mixtures is their ability to orchestrate receptor inter- purchased from Merck KGaA, Germany. An analogue of the MET- nalization and degradation in a manner superior to mAbs, as targeting antibody emibetuzumab (LY2875358; ref. 21) was previously demonstrated for different targets in the human generated on the basis