Development and implementation of a gyrolab-based generic anti-drug antibody assay for antibody-drug conjugates in cynomolgus monkey studies.

Introduction: Non-clinical immunogenicity yields valuable insights into pharmacokinetics, efficacy, and safety. Given the complex nature of ADC therapeutics, early detection of anti-drug antibodies (ADAs) is critical for elucidating exposure and toxicity issues that may be translated into the clinic. A universal ADA assay employing generic reagents and a standardized cut point in nonclinical studies can enhance cost efficiency, expedite development, and address limitations indrug tolerance associated with traditional bridging assays. Methods: Gyrolab-based generic ADA assay was carried out by initially spiking ADC into cynomolgus samples at a concentration of 300 mg/mL. Drug-ADA complexes were isolated using an anti-human Fc antibody, followed by detection with an anti-cynomolgus detection antibody. A total of 22 distinct ADCs were assessed across 50 cynomolgus subjects, each evaluated in six replicates to determine the cut point. Sensitivity and positive control (PC) assessments were performed by titrating an anti-human light chain generic surrogate positive control. Results and Discussion: A Gyrolab-based ADA assay with a universal cut point of 2.64 was developed, with 16 out of 22 ADCs acceptable for producing a standardized cut point. The average sensitivity of the positive control was 99.5 ng/ml and drug tolerant up to 1 mg/mL. Factors such as various linkers, drug antibody ratio (DAR), or backbone variations (variable region, Fc mutants and engineered cysteines) had minimal effect; however, one antibody variable region gave elevated signals. A workflow for assay used in Good Laboratory Practice (GLP) study was established, and its application demonstrated in a case study.