The newest FDA Immunogenicity Testing Guidance mentioned that LBA could be developed for antagonistic mAb NAb assay. However, in the case of dual-receptors drug target or hard-to-express drug target/ligand, it is also very challenging to have LBA NAb assay. Here, we share a case study of fully automated NAb assay development and validation using a MSD-based cell binding assay.

Immunogenicity and Immunotoxicity are common challenges to address during drug development and can impact both the efficacy and safety of the drug. To assess these risks, a variety of tools can be applied during preclinical development. These include a range of different human primary cell assays to assess the innate and adaptive immune response to the drug as well as both target and off-target mediated immune reactions. This presentation will focus on an overview of these human primary cell assays and how studies can be designed and executed for the assessment of immunogenicity and immunotoxicity risk during preclinical development.

An ELISA method with Acid Dissociation was developed for the detection of ADA against a biosimilar therapeutic at PPD (2015). Pre-validation data indicated that the method was able to detect up to 100 ng/mL of the positive control in the presence of 43.3 mcg/mL drug. This degree of drug tolerance was predicted to be fit-for-purpose for sample analysis; measured serum concentrations of the innovator drug during Phase III trials ranged from 0.50 to 6.00 mcg/mL. An alternate dosing strategy for a biosimilar drug was also tested. As a result, serum drug concentrations surpassed the drug tolerance of the originally validated assay. An improved ECL-Bead ADA method with increased drug tolerance was developed at PPD (2019) and the impact of the improved drug tolerance was evaluated using a subset of study samples.

Compounds containing two or more structural domains with a distinct mode of action-relevant functionality have been defined as multi-domain biotherapeutics (MDB). Several modalities, including endogenous protein fusions with Fc fragment or another polypeptide, bispecific antibodies, antibody-drug conjugates, as well as polyethylene glycol conjugates, have been viewed as examples of MDB type. Similar to other biotherapeutics, MDB compounds have a potential to induce host immune response, commonly detected in a form of anti-drug antibodies (ADA). The need to characterize ADA specificity to a particular domain of MDB has been identified as a potential requirement based on the compound nature and associated immunogenicity risk factors. MDB-related immunogenicity risk factors and related strategy of ADA specificity evaluation will be discussed.

It is expected that immunogenicity will be monitored throughout the clinical development program of a biotherapeutic. The ADA assay used to monitor immunogenicity is needed to support multiple clinical studies through different development phases, over several years. Thus, it is important to have an assay life cycle management process in place, which monitors assay performance over time and in different clinical indications. In this presentation, case studies will be used to discuss and compare approaches for monitoring quality control (QC) performance of ADA assays, to study the factors that may potentially influence assay performance, and to address potential assay drifts during clinical bioanalysis.

The FDA has licensed two bispecific antibodies (BsAbs) and the number in clinical development has steadily increased in recent years. I will discuss the current expectation and guideline from the FDA for novel modalities with a focus on bi- and tri-specific antibodies.

Monitoring emerging immune responses to polyethylene glycol (PEG) is of critical interest to establish the safety and efficacy of pegylated therapeutics. In vitro drug tolerance assessments of an anti-PEG antibody assay suggested potential interference from high levels of pegylated therapeutic in serum samples. This presentation will discuss a Biotin-PEG Extraction Acid Dissociation (BEAD) strategy that reduced the concentration of pegylated protein and matrix components in samples before assay, and improved anti-PEG antibody assay drug tolerance.

Immunogenicity prediction through in silico and in vitro assays is widely employed for screening and selection of candidate biopharmaceuticals across our industry. During clinical development, these techniques, together with mathematical modeling and simulation, can help to understand the overall immunogenicity risk, pointing towards immunogenicity risk management strategies. I will review current practice and provide examples across the drug discovery and development pipeline.

Immunogenic response, such as generation of anti-drug antibodies (ADA), against biotherapeutic products can have detrimental effects on drug safety, efficacy, and pharmacokinetics. Therefore, prediction and quantification of the risk for immunogenicity of therapeutic protein drugs before clinical trials is a crucial need. This presentation will describe state-of-the-art in silico analyses and in vitro assays for characterization of the immunogenic potential of a panel of biotherapeutic proteins and their correlation to the clinically observed immunogenicity outcome. A novel T cell assay with shortened turnaround time, improved efficiency, and robustness will also be discussed.

Immunogenicity is one of the most complex issues to address in drug design and development and requires intelligent application of integrated platforms to mitigate the risk to your biologic. In this talk I will present case studies to illustrate the range of solutions that ProImmune provides including DC-T/T cell proliferation assays for lead selection/optimization, MAPPS assays for characterization of antigen presentation; HLA-peptide binding assays to characterize individual epitopes & undiluted whole blood cytokine storm assays.

Validation of immunogenicity assays results in cut points that may or may not be relevant when applied to the study population once the method is implemented for clinical study support. The potential for the validated cut point to be unsuitable during sample analysis should be considered when determining cut point strategies during method validation. This talk will address approaches for choosing the population used to establish cut points during pre-study validation, evaluating the suitability of validated cut points during sample analysis, and highlight strategies for in-study cut point determination and implementation when the validated cut point is deemed unsuitable.