Mark L. Chiu

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.

Engineering antibody therapeutics

The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment.

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.

Early Engineering Approaches to Improve Peptide Developability and Manufacturability

Downstream success in Pharmaceutical Development requires thoughtful molecule design early in the lifetime of any potential therapeutic. Most therapeutic monoclonal antibodies are quite similar with respect to their developability properties. However, the properties of therapeutic peptides tend to be as diverse as the molecules themselves. Analysis of the primary sequence reveals sites of potential adverse posttranslational modifications including asparagine deamidation, aspartic acid isomerization, methionine, tryptophan, and cysteine oxidation and, potentially, chemical and proteolytic degradation liabilities that can impact the developability and manufacturability of a potential therapeutic peptide. Assessing these liabilities, both biophysically and functionally, early in a molecule’s lifetime can drive a more effective path forward in the drug discovery process. In addition to these potential liabilities, more complex peptides that contain multiple disulfide bonds can pose particular challenges with respect to production and manufacturability. Approaches to reducing the disulfide bond complexity of these peptides are often explored with mixed success. Proteolytic degradation is a major contributor to decreased half-life and efficacy. Addressing this aspect of peptide stability early in the discovery process increases downstream success. We will address aspects of peptide sequence analysis, molecule complexity, developability analysis, and manufacturing routes that drive the decision making processes during peptide therapeutic development.

Expression and purification of human TRPV1 in baculovirus-infected insect cells for structural studies

TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.

Fc Engineering Approaches to Enhance the Agonism and Effector Functions of an Anti-OX40 Antibody

Agonistic antibodies directed against immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising cancer immunotherapies. Several Fc engineering approaches discovered recently can augment the anti-tumor activities of TNFR antibodies by enhancing their agonistic activities and/or effector functions. In this study, we compared these approaches for their effects on an anti-OX40 antibody. Both S267E/L328F and V12 mutations facilitated enhanced binding to FcγRIIB and thus increased FcγRIIB cross-linking mediated agonist activity. However, both mutations abrogated the binding to FcγRIIIA and thereby decreasing the antibody-dependent cellular cytotoxicity activities. In contrast, the E345R mutation, which can promote antibody multimerization upon receptor binding, facilitated anti-OX40 antibody to have increased agonism by promoting the clustering of OX40 receptors without the dependence on FcγRIIB cross-linking. Nonetheless, cross-linking to FcγRIIB can lead to a further boost of the agonism of the anti-OX40 antibody with IgG1 Fc but not with the silent IgG2σ Fc. The antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities of the anti-OX40 antibody with the E345R mutation were affected by the choice of IgG subtypes. However, there was little change in the antibody-dependent cellular phagocytosis activity. In summary, different Fc engineering approaches can guide the design of engineered antibodies to OX40 and other TNFR with improved anti-tumor activity.

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.

An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants

Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models.

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.