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Why assessing the pharmacokinetics of antibody-drug conjugates is so complex

Antibody-drug conjugates (ADCs) are drugs which combine very potent payloads with a highly selective antibody which guides the payload to its intended target. The first approval of an ADC treatment was in 2000 but the number of ADCs on the market remains low. Approximately 13 ADCs are in use, mainly in oncology. The complexity of developing this type of molecule for therapeutic purposes has limited the number of ADCs up to now. However, that is changing as a new generation of ADCs are being developed thanks to improvements in the structural design, primarily in the linker between the antibody and the payload. In this article we look at some of the challenges of typical bioanalytical ADC assays and some recent developments in the area. 

Finding the right bioanalytical methods

Assessing the pharmacokinetics (PK) and metabolism of ADCs requires robust and accurate bioanalytical methods. The nature of the drug makes this challenging, as several potential PK analytes may need to be quantified such as the conjugated and free antibody, a small molecule toxin, linker and several combinations of these. Further adding to the complexity, ADCs are heterogenous in structure, unstable by design and their individual components have very different concentrations in the circulation. 

Multiple bioanalytical assays are usually needed for ADCs, and for PK typically a series of methods have to be developed. In the simplest form at least three assays may be necessary; one assay for the ADC (antibody linked to at least one cytotoxic payload), one for total antibody (including those without payload) and an assay for free payload, which together provides information on the degree of payload release. Getting a full picture of the biological situation may be more complicated. Some payloads can undergo metabolism both after release and while still attached to the antibody, and many of the linker molecules can be cleaved in different ways under physiological conditions. This can result in several different analytes composed of free payload, or its metabolite, connected to linker fragments. In addition, determining the concentrations of ADC-containing metabolised payload may be required depending on the therapeutic activity of each of the ADC-related molecules. 

Drug-to-antibody ratio and ligand binding assays

Complexity further increases since the ADC usually is a heterogeneous group of molecules with different numbers of payloads per antibody rather than a single molecule. This number is often referred to as the drug-to-antibody ratio (DAR) and it may affect the binding capacity of an ADC and therefore its response in ligand binding assays (LBAs). Newer generation ADCs include molecules of even greater structural complexity, such as bispecific ADCs for dual antigen binding and ADCs containing multiple types of payloads.

Advances in bioanalytical approaches

The bioanalytical platforms required for the different PK assessments varies. Liquid chromatography mass spectrometry-based methods (LC-MS/MS) are often used for (small molecule) cytotoxin PK assays. LBAs typically measure the antibody PK. Recent major advances have made LC-MS/MS a reliable complementary technique for protein bioanalysis and nowadays LC-MS/MS-based approaches can also be used for most antibody analyses. An immunocapture step can be added to selectively extract the molecule(s) of interest from the biological sample. This is usually referred to as a hybrid LBA-LC-MS/MS assay.  

Assessing immunogenicity should be carried out by measuring anti-drug antibodies (ADAs). This sometimes means using a number of different assays with their own specificity against the relevant domains of the ADC. Critical reagents are required for most of these assays and the assays will only be as good as the quality of the available reagents.  LBAs are typically used for ADA assessment, but recently hybrid LBA-LC-MS/MS approaches have also been explored. These are a useful complementary technique as they can provide semi-quantitative concentrations of ADA isotypes. 

Conclusion

Given the complexity of performing bioanalysis for ADCs, designing a suitable analytical approach is complicated. The biology of the ADC in question, the goals of the drug development and the characteristics of the assays must all be considered as part of this crucial step. 

This blog is adapted from the article Finding your way in ADC bioanalytics.

ICON has extensive experience with ADC assays, including LC-MS/MS, LBA PK, and ADA methods for ADC drug development. Find out more or reach out directly. 

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