Application of a Plug-and-Play Immunogenicity Assay in Cynomolgus Monkey Serum for ADCs at Early Stages of Drug Development

Abstract

Immunogenicity assessment during early stages of nonclinical biotherapeutic development is not always warranted. It is rarely predictive for clinical studies and evidence for the presence of anti-drug antibodies (ADAs) may be inferred from the pharmacokinetic (PK) profile. However, collecting and banking samples during the course of the study are prudent for confirmation and a deeper understanding of the impact on PK and safety. Biotherapeutic-specific ADA assays commonly developed can require considerable time and resources. In addition, the ADA assay may not be ready when needed if the study of PK and safety data triggers assay development. During early stages of drug development for antibody-drug conjugates (ADCs), there is the added complication of the potential inclusion of several molecular variants in a study, differing in the linker and/or drug components. To simplify analysis of ADAs at this stage, we developed plug-and-play generic approaches for both the assay format and the data analysis steps. Firstly, the assay format uses generic reagents to detect ADAs. Secondly, we propose a cut point methodology based on animal specific baseline variability instead of a population data approach. This assay showed good sensitivity, drug tolerance, and reproducibility across a variety of antibody-derived biotherapeutics without the need for optimization across molecules.

Single cell-produced and in vitro-assembled anti-FcRH5/CD3 T-cell dependent bispecific antibodies have similar in vitro and in vivo properties

“…A generic immune-complex immunoassay was used to screen cyno serum samples for anti-drug antibodies as described elsewhere. 38 In this assay, baseline and post-baseline samples from each animal were diluted and incubated with anti-FcRH5/CD3 variants, to allow formation of any ADA-drug complex. A mouse anti-human Fc-antibody (R10Z8E9, Genentech) immobilized on 96-well microtiter plates captured the ADA/ therapeutic immune-complexes.…”

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

“…Samples with an optical density higher than two times that of the control serum pool signal were reported as ADA positive. Serum samples from the monkey toxicology study were analyzed by the BioAnalytical Sciences Department at Genentech, using a qualified generic immuno-complex ADA immunoassay in cynomolgus monkey serum 65 . In this assay, serum samples were first incubated with the drug to form immune complexes with potential ADA in the sample.…”

Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

“…By preincubating the sample with excess drug, the immune-complex assay measures total ADA levels. Since the first report of the assay in 2010, the monkey ADA assay has been modified and successfully used by Carrasco-Triguero et al, for not only monovalent human IgG therapeutic antibodies, but also antibody-drug conjugates (see this special issue) [ 38 ]. The modified version uses a commercially available anti-human IgG Fc specific monoclonal antibody as coat and an anti-monkey IgG polyclonal antibody for detection.…”