Ihsan Nijem

Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

Phase I Dose-Escalation Study of Onartuzumab as a Single Agent and in Combination with Bevacizumab in Patients with Advanced Solid Malignancies

Purpose: This first-in-human study evaluated the safety, immunogenicity, pharmacokinetics, and antitumor activity of onartuzumab, a monovalent antibody against the receptor tyrosine kinase MET. Experimental Design: This 3+3 dose-escalation study comprised three stages: (i) phase Ia dose escalation of onartuzumab at doses of 1, 4, 10, 20, and 30 mg/kg intravenously every 3 weeks; (ii) phase Ia cohort expansion at the recommended phase II dose (RP2D) of 15 mg/kg; and (iii) phase Ib dose escalation of onartuzumab at 10 and 15 mg/kg in combination with bevacizumab (15 mg/kg intravenously every 3 weeks). Serum samples were collected for evaluation of pharmacokinetics, potential pharmacodynamic markers, and antitherapeutic antibodies. Results: Thirty-four patients with solid tumors were treated in phase Ia and 9 in phase Ib. Onartuzumab was generally well tolerated at all dose levels evaluated; the maximum tolerated dose was not reached. The most frequent drug-related adverse events included fatigue, peripheral edema, nausea, and hypoalbuminemia. In the phase Ib cohort, onartuzumab at the RP2D was combined with bevacizumab and no dose-limiting toxicities were seen. Onartuzumab showed linear pharmacokinetics in the dose range from 4 to 30 mg/kg. The half-life was approximately 8 to 12 days. There were no apparent pharmacokinetic interactions between onartuzumab and bevacizumab, and antitherapeutic antibodies did not seem to affect the safety or pharmacokinetics of onartuzumab. A patient with gastric carcinoma in the 20-mg/kg dose cohort achieved a durable complete response for nearly 2 years. Conclusions: Onartuzumab was generally well tolerated as a single agent and in combination with bevacizumab in patients with solid tumors. Clin Cancer Res; 20(6); 1666–75. ©2014 AACR.

Population pharmacokinetic analysis from phase I and phase II studies of the humanized monovalent antibody, onartuzumab (MetMAb), in patients with advanced solid tumors

Onartuzumab is a unique, humanized, monovalent (one‐armed) monoclonal antibody (mAb) against the MET receptor. The intravenous (IV) pharmacokinetics (PK) of onartuzumab were investigated in a phase I study and a phase II study in recurrent non‐small cell lung cancer (NSCLC) patients. The potential for drug–drug interaction (DDI) was assessed during co‐administration of IV onartuzumab with oral erlotinib, by measuring the PK of both drugs. The concentration–time profiles of onartuzumab were adequately described using a two‐compartment model with linear clearance (CL) at doses between 4 and 30 mg/kg. The estimates for CL, central compartment volume (V1), and median terminal half‐life were 0.439 L/day, 2.77 L, and 13.4 days, respectively. Statistically significant covariates included creatinine clearance (CrCL) on clearance, weight and gender on V1, and weight on peripheral compartment volume (V2), but the clinical relevance of these covariates needs to be further evaluated. The current analysis did not indicate obvious DDI between onartuzumab and erlotinib. MET diagnostic status did not impact the exposure of either agent. Despite the slightly faster clearance compared with typical bivalent mAbs, the PK of onartuzumab support dosing regimens of 15 mg/kg every 3 weeks or doses equivalent to achieve the target minimum tumoristatic concentration in patients.

Onartuzumab (MetMAb): Using Nonclinical Pharmacokinetic and Concentration–Effect Data to Support Clinical Development

Purpose: We characterized the pharmacokinetics of onartuzumab (MetMAb) in animals and determined a concentration–effect relationship in tumor-bearing mice to enable estimation of clinical pharmacokinetics and target doses. Experimental Design: A tumor growth inhibition model was used to estimate tumoristatic concentrations (TSC) in mice. Human pharmacokinetic parameters were projected from pharmacokinetics in cynomolgus monkeys by the species-invariant time method. Monte Carlo simulations predicted the percentage of patients achieving steady-state trough serum concentrations (Ctrough ss) ≥TSC for every 3-week (Q3W) dosing. Results: Onartuzumab clearance (CL) in the linear dose range was 21.1 and 12.2 mL/d/kg in mice and cynomolgus monkeys with elimination half-life at 6.10 and 3.37 days, respectively. The estimated TSC in KP4 pancreatic xenograft tumor-bearing mice was 15 μg/mL. Projected CL for humans in the linear dose range was 5.74 to 9.36 mL/d/kg with scaling exponents of CL at 0.75 to 0.9. Monte Carlo simulations projected a Q3W dose of 10 to 30 mg/kg to achieve Ctrough ss of 15 μg/mL in 95% or more of patients. Conclusions: Onartuzumab pharmacokinetics differed from typical bivalent glycosylated monoclonal antibodies with approximately 2-times faster CL in the linear dose range. Despite this higher CL, xenograft efficacy data supported dose flexibility with Q1W to Q3W dose regimens in the clinical setting with a TSC of 15 μg/mL as the Ctrough ss target. The projected human efficacious dose of 10 to 30 mg/kg Q3W should achieve the target TSC of 15 μg/mL. These data show effective pharmacokinetic/pharmacodynamic modeling to project doses to be tested in the clinic. Clin Cancer Res; 19(18); 5068–78. ©2013 AACR.

Nonclinical Evaluation of the Serum Pharmacodynamic Biomarkers HGF and Shed MET following Dosing with the Anti-MET Monovalent Monoclonal Antibody Onartuzumab

Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab. Mol Cancer Ther; 13(2); 540–52. ©2013 AACR.

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

ABSTRACT Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.

Abstract 4717: Final results from the Phase I study of MetMAb, a monovalent antagonist antibody to the receptor Met, dosed as single agent and in combination with bevacizumab in patients with advanced solid malignancies

Background: The receptor tyrosine kinase Met and/or its ligand, hepatocyte growth factor (HGF), are frequently over-expressed in cancers. Aberrant Met activation can enhance invasion, proliferation, and survival and may promote angiogenesis. MetMAb was uniquely engineered as a recombinant, humanized, monovalent monoclonal antibody to act as an antagonist of HGF-induced Met signaling. Materials and Methods: This 3+3 dose-escalation study consisted of three phases: 1) dose-escalation evaluating 1, 4, 10, 15, 20 and 30 mg/kg IV Q3W; 2) expansion at 15mg/kg IV Q3W; and 3) combination testing MetMAb, at 10 and 15mg/kg IV Q3W, plus bevacizumab (15mg/kg Q3W). Pre- and post- dose serum was collected for evaluation of pharmacodynamic biomarkers that could be affected by inhibition of Met and/or VEGF signaling. Results: 43 patients were treated in this study (21 in escalation, 13 in expansion, and 9 in combination). MetMAb has a half-life of approximately 11 days, and there were no apparent PK interactions with bevacizumab. MetMAb was generally well tolerated, both alone and in combination. The most frequent treatment-related adverse events included: fatigue, peripheral edema and hypoalbuminemia. In patients treated with the combination, no Grade 3-5 treatment-related adverse events were reported; a Grade 1, and dose-limiting adverse event of hemoptysis was reported in a patient who had central-necrosis of pulmonary metastases. A patient with gastric carcinoma achieved a complete response after 4 cycles of single-agent MetMAb; this patient came off study after 10 cycles with a sustained complete response. Conclusions: MetMAb, when administered as a single-agent, or in combination with bevacizumab was generally safe and well tolerated. A Phase II trial testing MetMAb in combination with bevacizumab and paclitaxel in patients with triple negative breast cancer is currently ongoing, while a Phase III trial testing MetMAb in combination with erlotinib in advanced NSCLC patients is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4717. doi:10.1158/1538-7445.AM2011-4717

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys

Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%-2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures.

Optimizing hybrid LC–MS/MS binding conditions is critical: impact of biotransformation on quantification of trastuzumab

BACKGROUND Hybrid ligand-binding (LB) LC-MS/MS protein quantitative assays involve a LB step for analyte enrichment that has less stringent requirements than the conventional LB assays. RESULTS Herceptin™(trastuzumab) binding to HER2 extracellular domain was evaluated using on-bead and off-bead capture formats. The two formats yielded significantly different trastuzumab concentrations in human and monkey serum pharmacokinetic samples. Biotransformations, including deamidation of asparagine and isomerization of aspartic acid near the complementarity-determining regions of trastuzumab, had a profound impact on the LB step for analyte enrichment and trastuzumab quantification. CONCLUSION Quantitative measurements were profoundly impacted by LB conditions in a hybrid LB LC-MS/MS protein assay due to biotransformations. Therefore, similar to conventional LB assays, binding conditions should be carefully evaluated during assay development.

Abstract 1670: Circulating biomarkers of MetMAb activity in a phase Ia dose escalating clinical trial

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The HGF/Met pathway has attracted interest as a promising target for cancer therapy both due to direct pathway activation in solid tumors as well as to activation as a mechanism of resistance to EGFR pathway inhibition. MetMAb is a recombinant, humanized, monovalent monoclonal antibody that antagonizes HGF driven Met signaling and is currently being evaluated in a clinical trial for NSCLC in combination with Erlotinib, and in phase 1 (in all solid tumors) in combination with Avastin. In this study we explored the utility of candidate pharmacodynamic biomarkers (PDB) in monitoring responses to MetMAb in the Phase Ia clinical trial, including a downstream marker of Met signaling, IL-8, and two proximal markers, serum HGF and shed Met (sMet) extracellular domain (ECD). A panel of four distinct secreted proteins including VEGF, PAI1, IL-8, uPAR were evaluated as potential biomarkers of Met pathway inhibition in preclinical models of efficacy. Of these, IL-8 demonstrated a desirable dynamic range that prompted its evaluation as a potential biomarker of drug activity in a Phase Ia dose ranging study with MetMAb using an MSD based ELISA assay. Circulating IL-8 levels, decreased as early as 24 hours after drug administration in patients that had higher than normal physiologic levels at baseline, irrespective of the MetMAb dose administered. In addition, a dose dependent increase in sMet was observed in all patients. These results are consistent with those obtained in preclinical studies that show that MetMAb can prevent normal clearance of hu-Met-ECD in mice. A trend towards increased circulating HGF levels post MetMAb administration was observed in most patients. However, an exception was noted in one gastric cancer patient that showed an objective complete response to MetMAb treatment. This patient demonstrated significantly higher levels of serum HGF and upon treatment showed an immediate and sustained decrease in circulating serum HGF. These data along with the observation that this patients tumor co-expressed Met and HGF suggests that MetMAb decreased serum HGF by disrupting an autocrine loop that was driving high serum HGF levels and sustaining tumor growth. The data show that IL-8, HGF and sMet levels can be modulated by treatment with MetMAb. As modulation of IL-8 was observed in most dose groups the utility of this marker to guide dose response is minimal. MetMAb can bind to and prevent the clearance of the Met ECD and the results presented here confirm this phenomenon in patients. Correspondingly, there appears to be no utility of sMet as a biomarker for response to MetMAb. The reduction in serum HGF observed in one patient with an objective CR suggests utility HGF as a MetMAb biomarker may depend on tumor biology. Together, these data have allowed the translation of preclinical biology in humans and demonstrate Met pathway inhibition using MetMAb. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1670.