Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/ receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC.Cancer Res; 76(13); 3942-53. Ó2016 AACR.

BACKGROUND & PURPOSELoperamide is a selective m opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating m opioid receptor agonism with d opioid receptor antagonism, by combining reference compounds or using a novel compound ('MuDelta'), could normalize GI motility without constipation. EXPERIMENTAL APPROACHMuDelta was characterized in vitro as a potent m opioid receptor agonist and high-affinity d opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic d opioid receptor immunoreactivity was quantified. KEY RESULTSd Opioid receptor antagonism opposed m opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated d opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model. CONCLUSIONS AND IMPLICATIONSMuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.

Small molecule inhibitors targeting mutant epidermal growth factor receptor (EGFR) are standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo anti-tumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR and this activity is associated with receptor downregulation. Despite these robust anti-tumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro. Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo anti-tumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophagemediated anti-tumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

energy storage, membrane components, and signaling. Extracellular hydrolysis of dietary TG in circulating lipoproteins yields FFAs and sn -2 MG, which are then taken up by cells ( 1,2 ). MGs are also produced intracellularly from membrane phospholipids and the consecutive action of phospholipase C and diacylglycerol lipase, or from the hydrolysis of stored TG by adipose TG lipase (ATGL) and hormone sensitive lipase (HSL) ( 2-5 ). The ultimate fate of intracellular MGs is hydrolysis to FFAs and glycerol or reesterifi cation by acyltransferases into diacylglycerol and TG ( 6, 7 ).MG lipase (MGL) is considered the rate-determining enzyme in MG catabolism. MGL accounts for roughly 85% of MG hydrolysis in the brain, with the remainder being catalyzed by the enzymes ABHD6 and ABHD12 ( 8,9 ). MGL is expressed in many other tissues as well, including brain, liver, skeletal muscle, adipose, and intestine ( 10-13 ). Within cells, MGL localizes to both the cytosolic and membrane fractions and hydrolyzes sn -1 and sn -2 MGs of varying acyl chain lengths and degrees of unsaturation, with almost no activity toward other lipids, such as TG and lyso-phospholipids ( 10,(14)(15)(16)(17)(18).MGL is involved in energy balance through two important functions. Abbreviations: AA, arachidonic acid; AEA, arachidonoyl ethanolamide; 2-AG, 2-arachidonoyl glycerol; AUC, area under the curve; CB, cannabinoid; EC, endocannabinoid; HFD, high-fat diet; HOMA-IR, homeostatic model assessment of insulin resistance; iMGL, mice that overexpress monoacylglycerol lipase specifi cally in the intestinal mucosa; LFD, low-fat diet; MG, monoacylglycerol; MGL, monoacylglycerol lipase; OFTT, oral fat tolerance test; OGTT, oral glucose tolerance test; RER, respiratory exchange ratio .

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.