Sheri Moores

A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor–Resistant Lung Tumors

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. ©2016 AACR.

Cross-arm binding efficiency of an EGFR x c-Met bispecific antibody

abstract Multispecific proteins, such as bispecific antibodies (BsAbs), that bind to two different ligands are becoming increasingly important therapeutic agents. Such BsAbs can exhibit markedly increased target binding and target residence time when both pharmacophores bind simultaneously to their targets. The cross-arm binding efficiency (χ) describes an increase in apparent affinity when a BsAb binds to the second target or receptor (R2) following its binding to the first target or receptor (R1) on the same cell. χ is an intrinsic characteristic of a BsAb mostly related to the binding epitopes on R1 and R2. χ can have significant impacts on the binding to R2 for BsAbs targeting two receptors on the same cell. JNJ-61186372, a BsAb that targets epidermal growth factor receptor (EGFR) and c-Met, was used as the model compound for establishing a method to characterize χ. The χ for JNJ-61186372 was successfully determined via fitting of in vitro cell binding data to a ligand binding model that incorporated χ. The model-derived χ value was used to predict the binding of JNJ-61186372 to individual EGFR and c-Met receptors on tumor cell lines, and the results agreed well with the observed IC50 for EGFR and c-Met phosphorylation inhibition by JNJ-61186372. Consistent with the model, JNJ-61186372 was shown to be more effective than the combination therapy of anti-EGFR and anti-c-Met monovalent antibodies at the same dose level in a mouse xenograft model. Our results showed that χ is an important characteristic of BsAbs, and should be considered for rationale design of BsAbs targeting two membrane bound targets on the same cell.

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

Background: Cancer cells express surface antigens at different levels from normal cells. Results: Differences in EGFR and c-MET receptor density levels influenced the in vitro activity of an EGFR × c-MET bispecific antibody. Conclusion: Consideration of target expression levels is important for bispecific design. Significance: In addition to multiple pathway targeting, the unique avidity of bispecific antibodies contributes to their promise for cancer therapy. The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.

Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

ABSTRACT Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients.

Abstract B241: Bispecific antibody targeting EGFR and cMet demonstrates superior activity compared to the combination of single pathway inhibitors.

Many tumors respond initially to targeted therapy, only to develop resistance over time thereby allowing the tumors to progress. In patients treated with EGFR small molecule inhibitors, the cMet pathway is often upregulated, either through MET gene amplification or an increase in the ligand HGF, to compensate and provide resistance to the EGFR monotherapy. Because both EGFR and cMet signal through some of the same survival and growth-promoting pathways, dual inhibition of these receptors may improve efficacy and prevent resistance through these mechanisms. We have designed a bispecific EGFR-cMet antibody (EM1-mAb) with multiple mechanisms of action resulting in superior activity compared to a combination of single EGFR and cMet inhibitors. Fab arm exchange was used to produce EM1-mAb, a technique that allows for efficient large-scale preparation of bispecific antibodies. EM1-mAb prevented binding of the ligands EGF and HGF to their respective receptors, EGFR (IC50 = 10 nM) and cMet (IC50 = 30 nM). Ligand-induced phosphorylation of each receptor was inhibited in cell-based assays. Blocking signaling from both EGFR and cMet with a combination of single monospecific antibodies resulted in an enhanced inhibition of pERK, a downstream effector of both receptors. The bispecific EM1-mAb further increased the potency of inhibition of pERK (55-65-fold) compared to the combination of single monospecific antibodies, suggesting an avidity effect on downstream signaling. EM1-mAb was evaluated in SCID-beige mice implanted with tumor cells engineered to express human HGF. Complete regression of 8/8 tumors was observed upon treatment with EM1-mAb dosed twice a week at 20 mg/kg. After the dosing period of four weeks, mice were monitored for an additional 10 weeks and no tumor regrowth was observed. Our data demonstrate that the bispecific antibody EM1-mAb, generated using Fab arm exchange, inhibited EGFR and cMet pathways simultaneously, resulting in superior activity in cellular downstream signaling compared to the combination of single pathway inhibitors. These attributes allow for a more efficient path toward clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B241. Citation Format: Sheri Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Diana Chin, Susan Chippari, Peter Haytko, Frank McCabe, Joost Neijssen, Paul Parren, Janine Schuurman, Sam Wu, Ricardo Attar, Mark Anderson. Bispecific antibody targeting EGFR and cMet demonstrates superior activity compared to the combination of single pathway inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B241.

Characterization of In Vivo Resistance to Osimertinib and JNJ-61186372, an EGFR/Met Bispecific Antibody, Reveals Unique and Consensus Mechanisms of Resistance

Approximately 10% of non–small cell lung cancer (NSCLC) patients in the United States and 40% of NSCLC patients in Asia have activating epidermal growth factor receptor (EGFR) mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be cotargeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry–based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy-resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third-generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase (SFK) substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as SFK signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro. This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages. Mol Cancer Ther; 16(11); 2572–85. ©2017 AACR.

Abstract A11: Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation

Non-small cell lung cancers (NSCLCs) with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but most acquire resistance over time through numerous mechanisms. In these patients, the cMet pathway is often activated to compensate and provide resistance to the EGFR targeted monotherapy; this activation can occur by MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. Controlled Fab-arm exchange was used to produce JNJ-61186372, a technique that allows for efficient large-scale preparation of bispecific antibodies with a regular IgG 1 structure. JNJ-61186372 was shown to bind EGFR and cMet and efficiently inhibited ligand-induced phosphorylation of both receptors. In addition to this important mechanism of action, we have engineered the antibody to contain lower than normal fucose levels to increase Fc-dependent effector mechanisms. JNJ-61186372 exhibited antibody dependent cellular cytotoxicity (ADCC) activity in vitro in a range of NSCLC cell lines with EGFR mutations, KRas mutation, and/or amplified MET gene. Furthermore, the low fucose form of JNJ-61186372 demonstrated more effective ADCC activity compared to its normal fucose counterpart. The bispecific JNJ-61186372 antibody showed increased potency (2-3 fold) compared to the combination of monovalent EGFR and monovalent cMet antibodies, demonstrating the beneficial effects of dual targeting in a single molecule. Antibody dependent cell-mediated phagocytosis (ADCP) activity of JNJ-61186372 was also confirmed in vitro. We have also demonstrated that Fc-dependent effector functions contributed to in vivo anti-tumor growth activity of JNJ-61186372, in a xenograft model with EGFR mutations and cMet activation. Our data demonstrate that the bispecific antibody JNJ-61186372, generated using controlled Fab-arm exchange, has in vitro ADCC and ADCP activity in EGFR mutant settings, either with or without cMet pathway activation, and with KRas mutation. In addition, the Fc-dependent effector mechanisms contributed to in vivo anti-tumor efficacy. The dual signaling inhibition of EGFR and cMet pathways by JNJ-61186372, combined with enhanced Fc effector function, may provide multiple mechanisms to combat resistance in EGFR mutant NSCLC patients. Citation Format: Keri L. Soring, Katharine D. Grugan, Randall J. Breszki, Jose Pardinas, Leopoldo Luistro, Barbara Bushey, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Matthew V. Lorenzi, Mark Chiu, Sheri Moores. Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A11.

Abstract DDT01-03: Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03