Development of novel ultra-sensitive IL-11 target engagement assays to support mechanistic PK/PD modeling for an anti-IL-11 antibody therapeutic.

An in-house antibody generation campaign identified a diverse, high affinity set of anti-interleukin-11 (IL-11) monoclonal antibodies (mAbs) to enable successful development of novel, custom ultra-sensitive target engagement assays for detection of "free" (unbound to the dosed anti-IL-11 therapeutic mAb) and "total" (free and mAb-IL-11 complexed form) IL-11 in preclinical species and human. Antibody hits from distinct epitope communities were screened on various platforms, including enzyme-linked immunosorbent assay, Meso Scale Discovery, Simoa HD-1 and Simoa Planar Array (SP-X), and used for assay development and sensitivity optimization. The ultra-sensitive SP-X format achieved a lower limit of quantitation of 0.006 pg/mL, enabling the first reported baseline levels of IL-11 in healthy control plasma determined by custom bioanalytical assays. These newly established baseline levels supported mechanistic pharmacokinetic/pharmacodynamic modeling in mouse, cynomolgus monkey, and human for a greater understanding of preclinical study design and in vivo dynamic interaction of soluble IL-11 with an anti-IL-11 antibody therapeutic candidate. Modeling and simulation also helped refine the utility of assays with respect to their potential use as target engagement biomarkers in the clinic.Abbreviations IL-11: Interleukin-11, TE: Target engagement, PK/PD: Pharmacokinetic/pharmacodynamic, mAb: Monoclonal antibody, NHP: Non-human primate, IgG: Immunoglobulin G, Cyno: Cynomolgulus monkey, GFR: Glomerular filtration rate, BQL: Below quantitation levels, DRM: Disease relevant model, kDa: kilodaltons, SPR: Surface plasmon resonance, pSTAT3: phosphorylated STAT3, IL-11R: Interleukin-11 receptor, TPP: Target product protein, LLOQ: Lower limit of quantitation, RLU: Relative light units.

Whole blood stability evaluation of monoclonal antibody therapeutics using volumetric absorptive microsampling.

Volumetric absorptive microsampling (VAMS) is increasingly utilized for both nonclinical and clinical pharmacokinetic studies. Currently, VAMS is employed as the sampling method for the detection of antibodies for coronavirus disease 2019. Biotherapeutics whole blood stability on VAMS presents as a critical concern for the health and pharmaceutical industries. In this follow-up to our previous publication, we evaluated daclizumab and trastuzumab whole blood sample stability on VAMS. The drug recovery data we observed at room temperature for short term and -80°C for long term was very encouraging. The knowledge could help us better understand and plan important investigation timelines, especially pandemic situations where human whole blood samples from a large population are collected and in urgent need of data analysis.

Retrospective analysis of model-based predictivity of human pharmacokinetics for anti-IL-36R monoclonal antibody MAB92 using a rat anti-mouse IL-36R monoclonal antibody and RNA expression data (FANTOM5).

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1 κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.

Application of Mitra® microsampling for pharmacokinetic bioanalysis of monoclonal antibodies in rats.

AIM: Volumetric absorptive microsampling (VAM) is being increasingly applied in nonclinical pharmacokinetic studies. Although there are published results for VAM use in small molecule pharmacokinetics (PK) studies, there is limited data on the utility of VAM for protein therapeutics. RESULTS: We describe the use of Mitra® microsampler for blood sampling, ELISA quantitation and PK analysis of two marketed therapeutic monoclonal antibodies administered to rat. Results generated for these monoclonal antibodies using Mitra® were compared with both serum and whole blood sampling methods in the same study. CONCLUSION: The low relative standard deviation among the three sets of PK data suggest that Mitra® microsampler could be useful in early nonclinical PK studies for protein therapeutics where reduction and refinement of animal use is desirable.

GDF11 Decreases Pressure Overload-Induced Hypertrophy, but Can Cause Severe Cachexia and Premature Death.

RATIONALE: Possible beneficial effects of GDF11 (growth differentiation factor 11) on the normal, diseased, and aging heart have been reported, including reversing aging-induced hypertrophy. These effects have not been well validated. High levels of GDF11 have also been shown to cause cardiac and skeletal muscle wasting. These controversies could be resolved if dose-dependent effects of GDF11 were defined in normal and aged animals as well as in pressure overload-induced pathological hypertrophy. OBJECTIVE: To determine dose-dependent effects of GDF11 on normal hearts and those with pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS: Twelve- to 13-week-old C57BL/6 mice underwent transverse aortic constriction (TAC) surgery. One-week post-TAC, these mice received rGDF11 (recombinant GDF11) at 1 of 3 doses: 0.5, 1.0, or 5.0 mg/kg for up to 14 days. Treatment with GDF11 increased plasma concentrations of GDF11 and p-SMAD2 in the heart. There were no significant differences in the peak pressure gradients across the aortic constriction between treatment groups at 1 week post-TAC. Two weeks of GDF11 treatment caused dose-dependent decreases in cardiac hypertrophy as measured by heart weight/tibia length ratio, myocyte cross-sectional area, and left ventricular mass. GDF11 improved cardiac pump function while preventing TAC-induced ventricular dilation and caused a dose-dependent decrease in interstitial fibrosis (in vivo), despite increasing markers of fibroblast activation and myofibroblast transdifferentiation (in vitro). Treatment with the highest dose (5.0 mg/kg) of GDF11 caused severe body weight loss, with significant decreases in both muscle and organ weights and death in both sham and TAC mice. CONCLUSIONS: Although GDF11 treatment can reduce pathological cardiac hypertrophy and associated fibrosis while improving cardiac pump function in pressure overload, high doses of GDF11 cause severe cachexia and death. Use of GDF11 as a therapy could have potentially devastating actions on the heart and other tissues.

Optimizing NBE PK/PD assays using the Gyrolab Affinity Software; conveniently within the bioanalyst's existing workflow.

AIM: The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. RESULTS: Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after <100% target coverage was seen in a cynomolgus pharmacokinetic/PD study dosed with anti-CTGF antibodies. The results uncovered magnitude differences in binding affinities between the dosed antibody, target and assay reagents. CONCLUSION: The binding affinity values were used to investigate reduced target coverage and results highlight potential of the affinity software for incorporation into the bioanalyst's existing Gyrolab workflow for characterizing reagents and optimizing pharmacokinetic/PD bioanalytical assays.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

GDF11 Treatment Attenuates the Recovery of Skeletal Muscle Function After Injury in Older Rats.

Loss of skeletal muscle mass and function results in loss of mobility for elderly patients. Novel therapies that can protect and/or restore muscle function during aging would have profound effects on the quality of life for this population. Growth differentiation factor 11 (GDF11) has been proposed as a "youthful" circulating factor that can restore cardiac, neural, and skeletal muscle functions in aging animals. However, conflicting data has been recently published that casts doubt on these assertions. We used a complex rat model of skeletal muscle injury that physiologically mimics injuries seen in patients; to investigate the ability of GDF11 and to enhance skeletal muscle regeneration after injury in older rats. Our data showed that GDF11 treatment resulted in a significant increase in tissue fibrosis, accompanied by attenuated functional recovery, as compared to animals treated with vehicle alone. GDF11 impaired the recovery of skeletal muscle function in older rats after injury.

Utility of immunodeficient mouse models for characterizing the preclinical pharmacokinetics of immunogenic antibody therapeutics.

Prior to clinical studies, the pharmacokinetics (PK) of antibody-based therapeutics are characterized in preclinical species; however, those species can elicit immunogenic responses that can lead to an inaccurate estimation of PK parameters. Immunodeficient (SCID) transgenic hFcRn and C57BL/6 mice were used to characterize the PK of three antibodies that were previously shown to be immunogenic in mice and cynomolgus monkeys. Four mouse strains, Tg32 hFcRn SCID, Tg32 hFcRn, SCID and C57BL/6, were administered adalimumab (Humira®), mAbX and mAbX-YTE at 1 mg/kg, and in SCID strains there was no incidence of immunogenicity. In non-SCID strains, drug-clearing ADAs appeared after 4-7 days, which affected the ability to accurately calculate PK parameters. Single species allometric scaling of PK data for Humira® in SCID and hFcRn SCID mice resulted in improved human PK predictions compared to C57BL/6 mice. Thus, the SCID mouse model was demonstrated to be a useful tool for assessing the preclinical PK of immunogenic therapeutics.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

BACKGROUND: Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). DISCUSSION & CONCLUSION: This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

GDF11 does not rescue aging-related pathological hypertrophy.

RATIONALE: Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE: To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS: Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS: Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.