Hybrid liquid chromatography high resolution and accuracy mass spectrometry approach for quantification of antibody-drug conjugates at the intact protein level in biological samples.

Preclinical in vivo and in vitro characterization of Antibody-Drug Conjugates (ADCs) involves the development of several bioanalytical methods to address many drug exposure questions. The current pharma industry approach requires at least three different assays that must be run, i.e., total antibody (mAb), conjugated payload or conjugated mAb, and free payload assays. Herein we present analytical performances of a quantitative hybrid Ligand Binding/Liquid Chromatography High Resolution and Accuracy Mass Spectrometry (LB/LCHRAM) method that can condense much of the necessary bioanalytical information in one method. The method includes an immuno-capture step, and it detects whole ADC molecules. It was applied to plasma mouse samples and showed reliable bioanalytical performance according to full method validation standards. Quantitation using extracted ion chromatograms and deconvoluted mass peaks was evaluated. The limit of quantitation resulted in 0.5ng of protein on column with a linear dynamic range spanning from 0.5 to 10µg/mL. Moreover, lower drug-to-antibody ratio (DAR) ADC species can be simultaneously detected, also enabling qualitative characterization of in vivo ADC conjugation.

R399E, A Mutated Form of Growth and Differentiation Factor 5, for Disease Modification of Osteoarthritis.

OBJECTIVE: To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug. METHODS: Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. RESULTS: Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1ß (IL-1ß), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 µg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-µg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. CONCLUSION: R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

Anti-drug Antibody Sample Testing and Reporting Harmonization.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

A strategic approach to nonclinical immunogenicity assessment: a recommendation from the European Bioanalysis Forum.

Immunogenicity assays are required to evaluate anti-drug antibody (ADA) responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAs are an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity; unexpected changes in exposure in the absence of a pharmacodynamic marker or evidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.

Update to the European Bioanalysis Forum recommendation on biomarkers assays; bringing context of use into practice.

In 2012, the European Bioanalysis Forum published a recommendation on biomarker method development and the bioanalysis of biomarkers in support of drug development. Since then, there has been significant discussion on how to bring the topic of context of use of biomarker assays to the forefront so that the purpose of the assay, the use of the data and the decisions being made with the data are well defined and clearly understood, not just by the bioanalytical scientist, but across all stakeholders. Therefore, it is imperative that discussions between the bioanalytical laboratory and the end users of the data happen early (and regularly) in the drug development process to enable the right assays to be developed and appropriately validated to generate the correct data and allow suitable decisions to be made. This updated refinement to the previous European Bioanalysis Forum recommendation will highlight the items to consider when discussing context of use for biomarker assay development and validation, thus enabling the correct conversations to occur and the move away from the misapplication of PK assay validation criteria to biomarker assays.

Translational strategies in drug development for knee osteoarthritis.

Osteoarthritis (OA) is a common disease worldwide with large unmet medical needs. To bring innovative treatments to OA patients, we at Merck have implemented a comprehensive strategy for drug candidate evaluation. We have a clear framework for decision-making in our preclinical pipeline, to design our clinical proof-of-concept trials for OA patients. We have qualified our strategy to define and refine dose and dosing regimen, for treatments administered either systemically or intra-articularly (IA). We do this through preclinical in vitro and in vivo studies, and by back-translating results from clinical studies in OA patients.

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material.

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

Effects of RG7652, a Monoclonal Antibody Against PCSK9, on LDL-C, LDL-C Subfractions, and Inflammatory Biomarkers in Patients at High Risk of or With Established Coronary Heart Disease (from the Phase 2 EQUATOR Study).

RG7652 (MPSK3169A), a fully human immunoglobulin G1 (IgG1) monoclonal antibody directed against proprotein convertase subtilisin/kexin type 9 (PCSK9), blocks the interaction between PCSK9 and low-density lipoprotein (LDL) receptor. EQUATOR (ClinicalTrials.govNCT01609140), a randomized, double-blind, and dose-ranging phase 2 study, evaluated RG7652 in patients (1) at high risk for or (2) with coronary heart disease (CHD). The primary end point was change in LDL cholesterol (LDL-C) from baseline to day 169. Patients (n = 248; median age, 64 years; 57% men; 52% with established CHD; 82% on statins) with baseline LDL-C levels of 90 to 250 mg/dl (mean, 126 mg/dl) continuing on standard-of-care therapy were randomized to receive 1 of 5 RG7652 doses or placebo, subcutaneously every 4, 8, or 12 weeks for 24 weeks. Significant dose-dependent reductions in LDL-C levels from baseline to nadir (56 to 74 mg/dl [48% to 60%]) were observed in all RG7652-dosed patients; effects persisted to day 169 with the highest doses (reduction vs placebo up to 62 mg/dl [51%]) with no unexpected safety signals. RG7652 reduced apolipoprotein B and lipoprotein(a) levels. LDL-C subfraction analysis by nuclear magnetic resonance spectroscopy revealed a prominent decrease in large LDL-C and some decrease in small LDL particles. There was significant reduction in mean particle size of LDL-C on day 169 but no significant reductions in systemic markers of inflammation (high-sensitivity C-reactive protein, interleukin-6, and tumor necrosis factor-alpha). RG7652 reduced LDL-C levels and was well tolerated in patients at high risk for or with CHD on standard-of-care therapy. In conclusion, RG7562 treatment affected large LDL-C and, to a lesser extent, small LDL-C particles; RG7562 did not affect systemic circulating pro-inflammatory cytokines or high-sensitivity C-reactive protein.

A phase 1 study to evaluate the safety and LDL cholesterol-lowering effects of RG7652, a fully human monoclonal antibody against proprotein convertase subtilisin/kexin type 9.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) downregulates low-density lipoprotein (LDL) receptors, thereby leading to a rise in circulating LDL cholesterol (LDL-C). RG7652 is a fully human monoclonal antibody against PCSK9. This placebo-controlled, phase 1 ascending-dose study in healthy subjects evaluated the safety of RG7652 and its efficacy as a potential LDL-C-lowering drug. HYPOTHESIS: Anti-PCSK9 antibody therapy safely and effectively reduces LDL-C. METHODS: Subjects (N = 80) were randomized into 10 cohorts. Six sequential single-dose cohorts received 10, 40, 150, 300, 600, or 800 mg of RG7652 via subcutaneous injection. Four multiple-dose cohorts received 40 or 150 mg of RG7652 once weekly for 4 weeks, either with or without statin therapy (atorvastatin). RESULTS: Adverse events (AEs) were generally mild; the most common AEs were temporary injection-site reactions. No serious AEs, severe AEs, AEs leading to study-drug discontinuation, or dose-limiting toxicities were reported. RG7652 monotherapy reduced mean LDL-C levels by up to 64% and as much as 100 mg/dL at week 2; the effect magnitude and duration increased with dose (≥57 days following a single RG7652 dose ≥300 mg). Exploratory analyses showed reduced oxidized LDL, lipoprotein(a), and lipoprotein-associated phospholipase A2 with RG7652. Antidrug antibody against RG7652 tested positive in 2 of 60 (3.3%) RG7652-treated and in 4 of 20 (20.0%) placebo-treated subjects. Simultaneous atorvastatin administration did not appear to impact the pharmacokinetic profile or lipid-lowering effects of RG7652. CONCLUSIONS: Overall, RG7652 elicited substantial and sustained dose-related LDL-C reductions with an acceptable safety profile and minimal immunogenicity.

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

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.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Preclinical pharmacokinetics, pharmacodynamics, tissue distribution, and tumor penetration of anti-PD-L1 monoclonal antibody, an immune checkpoint inhibitor.

MPDL3280A is a human monoclonal antibody that targets programmed cell death-1 ligand 1 (PD-L1), and exerts anti-tumor activity mainly by blocking PD-L1 interaction with programmed cell death-1 (PD-1) and B7.1. It is being investigated as a potential therapy for locally advanced or metastatic malignancies. The purpose of the study reported here was to characterize the pharmacokinetics, pharmacodynamics, tissue distribution and tumor penetration of MPDL3280A and/or a chimeric anti-PD-L1 antibody PRO304397 to help further clinical development. The pharmacokinetics of MPDL3280A in monkeys at 0.5, 5 and 20 mg · kg(-1) and the pharmacokinetics / pharmacodynamics of PRO304397 in mice at 1, 3 10 mg · kg(-1) were determined after a single intravenous dose. Tissue distribution and tumor penetration for radiolabeled PRO304397 in tumor-bearing mouse models were determined. The pharmacokinetics of MPDL3280A and PRO304397 were nonlinear in monkeys and mice, respectively. Complete saturation of PD-L1 in blood in mice was achieved at serum concentrations of PRO304397 above ∼ 0.5 µg · mL(-1). Tissue distribution and tumor penetration studies of PRO304397 in tumor-bearing mice indicated that the minimum tumor interstitial to plasma radioactivity ratio was ∼ 0.3; saturation of target-mediated uptake in non-tumor tissues and desirable exposure in tumors were achieved at higher serum concentrations, and the distribution into tumors was dose-and time-dependent. The biodistribution data indicated that the efficacious dose is mostly likely higher than that estimated based on simple pharmacokinetics/pharmacodynamics in blood. These data also allowed for estimation of the target clinical dose for further development of MPDL3280A.

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

Targeting oxidized LDL improves insulin sensitivity and immune cell function in obese Rhesus macaques.

Oxidation of LDL (oxLDL) is a crucial step in the development of cardiovascular disease. Treatment with antibodies directed against oxLDL can reduce atherosclerosis in rodent models through unknown mechanisms. We demonstrate that through a novel mechanism of immune complex formation and Fc-γ receptor (FcγR) engagement, antibodies targeting oxLDL (MLDL1278a) are anti-inflammatory on innate immune cells via modulation of Syk, p38 MAPK phosphorylation and NFκB activity. Subsequent administration of MLDL1278a in diet-induced obese (DIO) nonhuman primates (NHP) resulted in a significant decrease in pro-inflammatory cytokines and improved overall immune cell function. Importantly, MLDL1278a treatment improved insulin sensitivity independent of body weight change. This study demonstrates a novel mechanism by which an anti-oxLDL antibody improves immune function and insulin sensitivity independent of internalization of oxLDL. This identifies MLDL1278a as a potential therapy for reducing vascular inflammation in diabetic conditions.

HGF as a circulating biomarker of onartuzumab treatment in patients with advanced solid tumors.

The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non-small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, on-treatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted.

Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys.

Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.

Pharmacokinetics and biodistribution of a human monoclonal antibody to oxidized LDL in cynomolgus monkey using PET imaging.

PURPOSE: Oxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging. METHODS: Anti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 ((64)Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging. RESULTS: Anti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to 12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow. CONCLUSIONS: The clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.