Jose Pardinas

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.

Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding

ABSTRACT The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.

Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or “TLQ”) in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.

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.