Antibody–drug conjugates nonclinical support Bioanalytical Challenge
early to late stages of drug development. Based on the limited industry experience, the evolution of assays could result in differences in observed PK profile and calcula- tion of key PK parameters over the course of ADC devel- opment. However, by applying rational scientific under- standing of what each assay format and assay reagent is measuring, an appropriate interpretation of observed data can be made. Moreover, once enough understand- ing of the most relevant ADC analyte and DAR species is built, keeping the assay formats and assay reagents simi- lar in late stages of drug development such as between IND-enabling and first-in-human studies might help simplify interpretation of the observed results.
Future perspective The number of ADC programs across industry is grow- ing rapidly. The increased number of ADCs in the clinic may help better define what ADC analytes are most useful in understanding fate of ADCs in vivo. Novel site-specific ADCs may have less heterogeneity as well as greater in vivo stability than conventional conjugates. The use of novel small-molecule drug classes, linker types and conjugation chemistries will also require increased investment in appropriate bio- analytical strategies to support their progress through-
Executive summary
• Antibody–drug conjugates (ADCs) hold potential to deliver improved overall safety and reduced nonspecific off-target toxicity of chemotherapeutics agents.
• ADC bioanalysis at different stages of drug development may vary and so are the associated bioanalytical challenges.
• Novel bioanalytical approaches and strategies including combination of ligand-binding assays (LBA), LC–MS-based platforms are needed to overcome challenges unique to each stage of drug development.
• LBAs offer unique advantages for quantitation of ADCs: – High assay sensitivity and throughput; – Broad range of quantitation; – Minimal sample volume; – Ability to measure in biological matrix without additional sample extraction steps.
• The complex multicomponent structure of ADC also presents unique challenges in ADC bioanalysis using LBAs: – ADCs are heterogeneous mixtures of various drug–antibody ratio (DAR) species. The DAR heterogeneity comes from conjugation chemistry, linker lability and actual site of conjugation;
– ADC heterogeneity is dynamically changing in vivo due to spontaneous or environment-induced deconjugation and due to differences in the clearance rate of various DAR species;
– Multiple assays are needed for monitoring diverse ADC analytes; – LBAs may be sensitive to the amount of conjugated small molecule drug present on the ADC molecule.
• ADC-related analytes include the conjugated antibody, the total antibody, antibody-conjugated small molecule drug, released unconjugated small molecule drug and its metabolites, changes in DAR over time and ADAs against any component of the multicomponent ADC.
• In early drug discovery, the goal of ADC bioanalysis is to allow comparison of multiple ADC candidates with various antibodies, linker–small molecule drug combinations to select an optimal candidate.
• During pre-clinical regulated studies, the exposure–safety relationship of the lead candidate is defined using analytically validated assays.
• There is no single bioanalytical assay strategy that fits all ADCs, rather strategy needs to be adapted on a case-by-case basis depending on the physicochemical characteristics of the ADC, and the drug development stage.
out the drug development. A combination of LBA and LC–MS-based methods will be needed to support future ADC programs. But the balance between plat- form investments would be defined by the specifics of the ADC program and the need to measure all the relevant analytical species. Novel small-molecule drug classes may be challeng-
ing for generation of appropriate reagents against them and may need an early increased investment to define absorption, distribution, metabolism and elimination before specific LBA-based PK assays could be developed. The relationship between systemic exposure and
cellular response remains complex with a multiday transduction process involving extravasation into tissues, cellular binding, internalization, intracel- lular trafficking, small molecule release and finally intracellular target engagement. Thus, tissue PK and cellular biodistribution studies can be expected to play an important role in understanding this rela- tionship and would require additional bioanalytical investment.
Acknowledgements The authors would like to thank N Duriga, T Taylor and E Hamel for their contributions to the presented data.
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