IQ Survey Results on Current Industry Practices: Part 2-Quantitative Evaluations of Immunogenicity Assessment.

All biotherapeutics have the potential to induce an immunogenic response and generate anti-drug antibodies (ADAs), especially when administered as multiple doses over prolonged periods. However, a clinically meaningful effect of ADAs can be difficult to identify to communicate the impact of immunogenicity on drug exposure, safety and efficacy outcomes in product labels in a way that is useful for health care providers. The immunogenicity working Group, IQ Consortium (Clinical Pharmacology Leadership Group) has conducted a survey to understand the current practices in analyzing immunogenicity data generated during clinical development and its impact on pharmacokinetics, clinically relevant pharmacodynamic biomarkers, safety, and efficacy outcome measures.

Pharmacokinetic and Exposure-Response Modeling Support Body Surface Area-Based Dosing of Farletuzumab Ecteribulin in Japanese Patients with Solid Tumors.

The first-in-human, Phase 1 Study 101 showed antitumor activity and a tolerable safety profile of farletuzumab ecteribulin in Japanese patients with platinum-resistant ovarian and non-small cell lung cancer. A pharmacometric assessment evaluated farletuzumab ecteribulin pharmacokinetics and exposure-response (E-R) relationships for efficacy and safety to support dose optimization. Patients received 0.

Phase I study of BMS-986299, an NLRP3 agonist, as monotherapy and in combination with nivolumab and ipilimumab in patients with advanced solid tumors.

Purpose: BMS-986299 is a first-in-class, NOD-, LRR-, and pyrin-domain containing-3 (NLRP3) inflammasome agonist enhancing adaptive immune and T-cell memory responses.

Materials And Methods: This was a phase-I (NCT03444753) study that assessed the safety and tolerability of intra-tumoral BMS-986299 monotherapy (part 1A) and in combination (part 1B) with nivolumab, and ipilimumab in advanced solid tumors. Reported here are single-center results.

Nivolumab and ipilimumab population pharmacokinetics in support of pediatric dose recommendations-Going beyond the body-size effect.

Body size has historically been considered the primary source of difference in the pharmacokinetics (PKs) of monoclonal antibodies (mAbs) between children aged greater than or equal to 2 years and adults. The contribution of age-associated differences (e.g.

Pediatric model-based dose optimization using a pooled exposure-response safety analysis for nivolumab and nivolumab plus ipilimumab combination in melanoma.

An exposure-response (E-R) safety analysis was conducted across adult and pediatric (<18 years) studies to evaluate the potential impact of higher nivolumab and/or ipilimumab exposures in adolescents (≥12 to <18 years) versus adults with melanoma using the approved adult dosing regimens for nivolumab alone or in combination with ipilimumab. Data from 3507 patients across 15 studies were used to examine the relationship between nivolumab-ipilimumab daily average exposure and time to grade 2+ immune-mediated adverse events (gr2+ IMAEs). Results from the E-R safety model showed ipilimumab, but not nivolumab, exposure to be a statistically significant predictor of gr2+ IMAEs.

Changing the drug development and therapeutic paradigm with biologic drug combinations and bispecifics: How to choose between these two approaches?

Biologics are increasingly being co-developed in combination or as novel constructs like bispecific antibodies (BsAbs) with the goal of targeting multiple, non-redundant mechanisms of action. Rational design of combinations and dual-targeting approaches that consider disease complexities have the potential to improve efficacy and safety, to increase duration of clinical benefit, and to minimize clinical resistance mechanisms. Here we summarize examples of BsAbs and biologic combinations that have been approved by health authorities and present drug development considerations when deciding between these two strategies.

Exposure-Response Analysis to Support Nivolumab Once Every 4 Weeks Dosing in Combination with Cabozantinib in Renal Cell Carcinoma.

Purpose: A benefit:risk assessment for a less-frequent nivolumab 480 mg every 4 weeks + cabozantinib 40 mg every day dosing regimen was predicted using modeling and simulation of clinical trial data from nivolumab monotherapy studies and from the nivolumab 240 mg every 2 weeks + cabozantinib 40 mg every day dosing regimen, which demonstrated clinical benefit versus sunitinib in previously untreated advanced renal cell carcinoma (aRCC) in the phase III CheckMate 9ER trial (NCT03141177).

Patients And Methods: Multivariable Cox proportional hazards analyses were conducted using nivolumab monotherapy data in previously treated aRCC and data from CheckMate 9ER to evaluate progression-free survival (PFS), overall survival (OS), and grade ≥2 immune-mediated adverse events (IMAE).

Results: Nivolumab 240 mg every 2 weeks + cabozantinib versus nivolumab monotherapy showed improvement in PFS (HR, 0.

Nivolumab exposure-response analysis for adjuvant treatment of melanoma supporting a change in posology.

Nivolumab monotherapy is approved as adjuvant treatment for melanoma based on results from the pivotal CheckMate 238 trial. We present a model-based, benefit-risk assessment of nivolumab in adjuvant melanoma supporting a posology change from a weight-based to a less frequent, flat-dosing regimen. The exposure-response (E-R) relationship for efficacy was evaluated using recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) end points from the CheckMate 238 trial.

Evaluating a Multiscale Mechanistic Model of the Immune System to Predict Human Immunogenicity for a Biotherapeutic in Phase 1.

A mechanistic model of the immune response was evaluated for its ability to predict anti-drug antibody (ADA) and their impact on pharmacokinetics (PK) and pharmacodynamics (PD) for a biotherapeutic in a phase 1 clinical trial. Observed ADA incidence ranged from 33 to 67% after single doses and 27-50% after multiple doses. The model captured the single dose incidence well; however, there was overprediction after multiple dosing.

Nivolumab Clearance Is Stationary in Patients With Resected Melanoma on Adjuvant Therapy: Implications of Disease Status on Time-Varying Clearance.

Nivolumab clearance (CL) in patients with advanced melanoma (MEL) decreases over the treatment duration, with change in CL associated with improved disease status, measured by reduced tumor burden. Here, we characterize the pharmacokinetics of nivolumab administered as adjuvant therapy for patients with MEL (AdjMEL) whose tumors were removed by surgical resection. A population pharmacokinetic model was developed using data from 1,773 patients with AdjMEL, MEL, non-small cell lung cancer, and other solid tumors who received nivolumab over a dose range of 0.

Surface plasmon resonance as a tool for ligand-binding assay reagent characterization in bioanalysis of biotherapeutics.

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process.

Developing a Natural History Progression Model for Duchenne Muscular Dystrophy Using the Six-Minute Walk Test.

The 6-minute walk test (6MWT) is used as a clinical endpoint to evaluate drug efficacy in Duchenne Muscular Dystrophy (DMD) trials. A model was developed using digitized 6MWT data that estimated two slopes and two intercepts to characterize 6MWT improvement during development and 6MWT decline. Mean baseline 6MWT was 362 (±87) meters.

Perspectives on Subcutaneous Route of Administration as an Immunogenicity Risk Factor for Therapeutic Proteins.

An increasing number of therapeutic proteins are being developed for delivery through the subcutaneous (SC) route of administration. Relative to intravenous (IV) administration, the SC route offers more convenience to patients, flexibility in dosing, and potential to reduce health care costs. There is a perception that SC administration can pose a higher immunogenicity risk than IV administration for a given protein.

Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform.

The Gyrolab™ xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation.

A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin.

The effect of the neonatal Fc receptor on human IgG biodistribution in mice.

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-β ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice.

The impact of glycosylation on the pharmacokinetics of a TNFR2:Fc fusion protein expressed in Glycoengineered Pichia Pastoris.

Purpose: P. pastoris has previously been genetically engineered to generate strains that are capable of producing mammalian-like glycoforms. Our objective was to investigate the correlation between sialic acid content and pharmacokinetic properties of recombinant TNFR2:Fc fusion proteins generated in glycoengineered P.

Lymphatic transport and catabolism of therapeutic proteins after subcutaneous administration to rats and dogs.

The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ∼70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph.

Metabolism of biologics: biotherapeutic proteins.

Recombinant therapeutic protein drugs have now been in clinical use for nearly three decades and have advanced considerably in complexity over this time period. Regulatory approvals of some early pioneering protein drugs did not require characterization of metabolism, but more recently regulatory expectations and guidance have appropriately evolved. Sponsors may now be expected to investigate metabolism of newer biologics as the structural complexity of proteins has increased markedly, particularly with the introduction of conjugated and modified proteins.

Pharmacokinetics of IgG1 monoclonal antibodies produced in humanized Pichia pastoris with specific glycoforms: a comparative study with CHO produced materials.

A glycoengineered Pichia pastoris host was used to produce an IgG1 with either afucosylated N-glycosylation (afucosylated biantennary complex) or without N-glycosylation (N297A) while a wild type P. pastoris host was used to produce an IgG1 containing fungal-type N- and O-linked glycosylation. The PK properties of these antibodies were compared to a commercial IgG1 produced in CHO cells following intravenous administration in wild type C57B6, FcγR-/- or hFcRn transgenic mice.

Monoclonal antibodies with identical Fc sequences can bind to FcRn differentially with pharmacokinetic consequences.

The neonatal Fc receptor (FcRn) is a key determinant of IgG homeostasis. It binds to the Fc domain of IgG in a strictly pH-dependent manner and protects IgG from lysosomal degradation. The impact of FcRn salvage pathway on IgG monoclonal antibody (mAb) pharmacokinetics (PK) has been well established.

Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis.

Introduction: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays.