Kyra J. Cowan

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

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.

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.

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

ABStract 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.

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. Mol Cancer Ther; 12(6); 1122–30. ©2013 AACR.

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.

Fit-for-purpose biomarker immunoassay qualification and validation: three case studies

AIM To improve on the efficiency of biomarker assay readiness, and for reliable biomarker data to support three drug programs, we implemented a fit-for-purpose approach, qualifying two biomarker assays and validating a third. Results/methodology: The qualification strategy and selection of experiments for two exploratory biomarkers (CXCL1, CCL19) was determined by the intended use of the biomarker data. The third biomarker, IL-6, was validated as the data would be used in monitoring patient safety during dose-escalation studies in a Phase I trial. All three assays passed a priori acceptance criteria. CONCLUSION These assays highlight strategies and methodologies for a fit-for-purpose approach. Minimum qualification, full qualification and validation were chosen and supported programs at different stages of drug development.

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.

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

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.