Challenges in the Development of a Method for the Detection of Anti-PEGylated-aptamer Antibodies

Immunogenicity assays capable of properly determining the presence of anti-drug antibodies (ADA) and anti-PEG antibodies have been recognized of great importance due to the fact that pre-existing anti-PEG antibodies could have an impact on the efficacy of a drug coupled to polyethylene glycol (PEG).

AON-D21 is a clinical stage PEGylated L-configured oligonucleotide aptamer targeting complement factor C5a. In this study, the sponsor required a method to be developed for the determination of anti-AON-D21 and anti-PEG antibodies in human serum.

In the work we present to the 8th EBF Young Scientist Symposium, a number of challenges were faced during the development of the assay mainly around robust detection of positive control antibodies by using biotin and sulfo-tag labelled AON-D21 as capture and detection reagents. Several methods were evaluated with the most optimal assay design being the Affinity Capture Elution- AGL (ACE-AGL) format, which captures the ADA binding to immobilized biotin-AON-D21, followed by an acid elution step of ADA and finally the re-capture of these on a MSD plate coated with protein AGL. The ADA are then detected using sulfo-tag labelled AON-D21. In confirmatory assays, this is competed by unlabelled AON-D21 or PEG alone to identify the ADA binding site.

Special emphasis was put in identifying the best positive control by assessing different monoclonal anti-PEG antibodies in order to optimise assay sensitivity and reproducibility.

Finally, a suitable method capable of detecting both anti-PEG and anti-AON-D21 antibodies simultaneously was developed. The method has been validated and is being used to support a Phase I clinical trial in humans

You may find below the poster for your ready reference:


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