Published OnlineFirst November 17, 2020; DOI: 10.1158/1078-0432.CCR-20-2845 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer A C Michael J. Giffin1, Keegan Cooke1, Edward K. Lobenhofer2, Juan Estrada1, Jinghui Zhan1, Petra Deegen3, Melissa Thomas4, Christopher M. Murawsky5, Jonathan Werner2, Siyuan Liu1, Fei Lee6, Oliver Homann7, Matthias Friedrich3, Joshua T. Pearson8, Tobias Raum9, Yajing Yang1, Sean Caenepeel1, Jennitte Stevens10, Pedro J. Beltran1, Jude Canon1, Angela Coxon1, Julie M. Bailis6, and Paul E. Hughes1 ABSTRACT ◥ Purpose: Small-cell lung cancer (SCLC) is an aggressive neuro- Results: AMG 757 showed potent and specific killing of even endocrine tumor with a high relapse rate, limited therapeutic those SCLC cell lines with very low DLL3 expression (<1,000 options, and poor prognosis. We investigated the antitumor activity molecules per cell). AMG 757 effectively engaged systemically of AMG 757, a half-life extended bispecific T-cell engager molecule administered human T cells, induced T-cell activation, and redir- targeting delta-like ligand 3 (DLL3)—a target that is selectively ected T cells to lyse tumor cells to promote significant tumor expressed in SCLC tumors, but with minimal normal tissue regression and complete responses in PDX models of SCLC and expression. in orthotopic models of established primary lung SCLC and met- Experimental Design: AMG 757 efficacy was evaluated in SCLC astatic liver lesions. AMG 757 was well tolerated with no AMG 757- cell lines and in orthotopic and patient-derived xenograft (PDX) related adverse findings up to the highest tested dose (4.5 mg/kg mouse SCLC models. Following AMG 757 administration, changes weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, in tumor volume, pharmacodynamic changes in tumor-infiltrating which is projected to enable intermittent administration in patients. T cells (TILs), and the spatial relationship between the appearance Conclusions: AMG 757 has a compelling safety and efficacy of TILs and tumor histology were examined. Tolerability was profile in preclinical studies making it a viable option for targeting assessed in nonhuman primates (NHPs). DLL3-expressing SCLC tumors in the clinical setting. Introduction Small-cell lung cancer (SCLC) is an aggressive neuroendocrine 1Oncology Research, Amgen Research, Thousand Oaks, California. 2Translational tumor prone to early metastasis, accounting for 10%–15% of all lung Safety & Bioanalytical Sciences, Amgen Research, Thousand Oaks, California. cancers (1–3) and associated with poor 5-year survival (4). Disease 3Translational Safety & Bioanalytical Sciences, Amgen Research (Munich) 4 relapse and resistance to therapy are common following an initial GmbH, Munich, Germany. Therapeutic Discovery, Amgen Research, South San response to etoposide and platinum-based chemotherapy with or Francisco, California. 5Therapeutic Discovery, Amgen Research, Burnaby, British Columbia, Canada. 6Oncology Research, Amgen Research, South San Francisco, without radiotherapy (5). Immune checkpoint blockade has increased California. 7Genome Analysis Unit, Amgen Research, South San Francisco, overall survival (OS) in SCLC despite relatively modest response California. 8Pharmacokinetics & Drug Metabolism, Amgen Research, South San rates (6–9). The anti-programmed cell death-1 (anti–PD-1) antibodies Francisco, California. 9Therapeutic Discovery, Amgen Research (Munich) GmbH, nivolumab and pembrolizumab received accelerated approval in the 10 Munich, Germany. Therapeutic Discovery, Amgen Research, Thousand Oaks, United States for the treatment of metastatic SCLC with progression California. on or after platinum-based chemotherapy and at least one other line of Note: Supplementary data for this article are available at Clinical Cancer therapy (10, 11); however, subsequent studies did not confirm Research Online (http://clincancerres.aacrjournals.org/). increased OS (12, 13). The promise of targeted therapies has also not M.J. Giffin and K. Cooke contributed equally to this article. yet been realized in SCLC; a DLL3-targeted antibody–drug conjugate Current address for Michael J. Giffin, Process Development, Amgen Inc., Thou- with early evidence of clinical activity demonstrated no benefitina sand Oaks, California; current address for Joshua T. Pearson, Pharmacodynam- subsequent phase III trial (14). Therapeutic agents with different ics, Pharmacokinetics, and Drug, Merck Research Labs, South San Francisco, mechanisms of action are still urgently needed for patients with SCLC. California; and current address for Pedro J. Beltran, Discovery Biology, UNITY Bispecific T-cell engager (BiTE) molecules are a clinically validated Biotechnology, South San Francisco, California. therapeutic modality that redirect a patient’s T cells to kill tumor Corresponding Authors: Paul E. Hughes, Amgen Research, One Amgen Center cells (15). Blinatumomab, the first BiTE molecule in clinical develop- Drive, Thousand Oaks, CA 91320–1799. Phone: 805–447–1137; Fax: 805-498- ment, is approved for the treatment of relapsed/refractory B-cell 5666; E-mail: [email protected]; and Julie M. Bailis, Amgen Research, 1120 – – – precursor acute lymphoblastic leukemia (16 18) and also demon- Veterans Blvd, South San Francisco, CA 94080. Phone: 650 244 2361; E-mail: fi – [email protected] strates ef cacy in non-Hodgkin lymphoma (19 21). The BiTE mol- ecule AMG 420, which targets BCMA, demonstrated a 70% response Clin Cancer Res 2020;XX:XX–XX rate in multiple myeloma with 50% minimal residual disease–negative doi: 10.1158/1078-0432.CCR-20-2845 complete responses at the maximum tolerated dose in a phase I study Ó2020 American Association for Cancer Research. (22). In solid tumors, therapeutic index has been a major challenge for AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst November 17, 2020; DOI: 10.1158/1078-0432.CCR-20-2845 Giffin et al. Translational Relevance Methods Cell lines Small-cell lung cancer (SCLC) is an aggressive neuroendo- DMS 79, NCI-H2171, NCI-H889, SHP-77, NCI-H211, NCI-H460, crine malignancy that is associated with a high relapse rate and HEK-293, and Chinese hamster ovary (CHO) cell lines were purchas- dismal prognosis. Recent immunotherapeutic approaches using ed from the American Type Culture Collection, NCI-H82 from the immune checkpoint inhibitors have only modestly improved DSMZ-German Collection of Microorganisms and Cell Cultures, and clinical outcomes. AMG 757 is a first-in-class, half-life-extended COR-L279 from Sigma-Aldrich. Cell lines were analyzed for authen- bispecific T-cell engager molecule that redirects T cells to ticity using DNA-fingerprinting techniques such as short tandem specifically kill DLL3-expressing tumor cells. In biologically repeat profiling. All cell lines tested negative for mycoplasma con- relevant orthotopic and patient-derived xenograft SCLC disease tamination. Research Resource Identifiers for cell lines are listed in models, AMG 757 promoted significant tumor regression and Supplementary Tables (Supplementary Table S1). complete antitumor responses against established tumors. The antitumor effect of AMG 757 is linked to its ability to promote intratumoral infiltration of activated T cells and facilitate T-cell– Cell-based assays fi fl mediated killing of DLL3-expressing SCLC tumors. This, together DLL3 expression levels on cell lines were quanti ed in ow cyto- with its acceptable nonclinical safety profile, suggest that AMG 757 metry experiments using standard receptor quantitation methods may be a promising novel option for SCLC therapy. AMG 757 is (QIFIKIT assay [Dako] or Quantum Simply Cellular assay [Bangs currently under evaluation in a phase I clinical study Laboratories]) and analyzed using BD FACSDiva and GraphPad Prism (NCT03319940) for patients with SCLC. or QuickCals software (see Supplementary Methods). Transfected HEK-293 cells were plated at 5,000 cells/well and treated with activated T cells at 50,000 cells/well (10:1 effector to target [E:T] cell ratio) and BiTE molecules at 50 ng/mL final concen- the successful development of T-cell bispecific antibodies (23, 24). tration, titrated 4-fold across the plate (12-point titration curve) in Development of AMG 110 (MT110), which targets EpCAM, was duplicate. After incubation for 20 hours, cell viability was measured discontinued due to on-target, dose-limiting toxicity in the liver and with the Steady-Glo Luciferase assay (Promega). Effector cells (2 Â 105 human or NHP PBMC or isolated human gastrointestinal tract (24). The clinical activity of the CEA-targeting T- þ cell bispecific antibody, cibisatamab, is also associated with dose- CD3 T cells) were cocultured with Vybrant (Thermo Fisher Scien- fi – limiting on-target toxicity in gastrointestinal tissues (23). These data ti c) DiO/Dil labeled target cell lines at an E:T cell ratio of 2:1 or 5:1 fl highlight the need to identify therapeutic targets with tumor-specific and serial dilutions of AMG 757 in 96-well, at-bottom plates. fi or tumor-selective expression profiles (23, 24). Cells were cultured for 48 hours at 37 C in a 5% CO2 humidi ed To identify potential BiTE molecule targets in SCLC, we profiled incubator and washed with FACS buffer. PBMCs were stained with a fluorochrome-labeled anti-CD14 antibody (BD Biosciences) to define