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P021 An electrochemiluminescence (ECL) immunoassay for the detection of antidrug antibodies against anti-mucosal addressin cell adhesion molecule (MAdCAM) monoclonal antibody SHP647

Q. Wang1, M. Goetsch*2

1Pfizer, Groton, CT, USA, 2Shire, Zug, Switzerland

Background

Immunogenicity assessment is a regulatory requirement for biotherapeutic product (BTP) approval since antibodies that develop in response to a BTP may directly impact product safety and efficacy. A well-designed anti-drug antibody (ADA) immunoassay is critical for monitoring the immunogenicity profile of a BTP during its development. SHP647 is a fully human IgG2қ monoclonal antibody that targets human MAdCAM to reduce lymphocyte homing to the gut and gastrointestinal inflammation, and is in development for the treatment of Crohn’s disease (CD) and ulcerative colitis (UC). A sensitive and specific ECL immunoassay for the detection of ADAs against SHP647 was developed and validated to support its use in clinical trials of SHP647.

Methods

SHP647 was either biotinylated as the capture agent, or labelled with ruthenium as the detection reagent. In the assay, human serum samples, positive controls and negative controls were diluted with assay buffer prior to co-incubation with both the capture and detection reagents overnight to form an antibody-drug complex. After incubation, each mixture was added to Streptavidin coated MSD plate to allow complexes to bind to the plate. In the presence of tripropylamine-containing read buffer, ruthenium produces a chemiluminescent signal that was triggered when voltage was applied. The resulting chemiluminescence was measured in relative units on a SECTOR Imager 6000™ instrument. Data are presented as endpoint log titers (log2) (the reciprocal of the serial dilution at which the sample response would be equal to the cut point of the assay).

Results

The assay precision (inter-run ≤4.0% and intra-run ≤3.4%) in normal human serum was demonstrated. Relative assay sensitivity was 3.25 ng/ml. The matrix specificity (recovery) ranged from 96.9% and 109.4% in 10 individual lots of normal, CD, or UC human serum. The assay achieved the detection of 300 ng/ml of ADA in the presence of 300 µg/ml of the drug. Interference was observed in the presence of 100 ng/ml soluble MAdCAM. The assay screening cut point factors and confirmatory assay cut points in normal, CD and UC populations were established.

Conclusion

The ECL immunoassay with sensitivity and high tolerance to both soluble MAdCAM and SHP647 for the detection of anti-SHP647 antibodies was successfully developed and validated in compliance with the regulatory requirements. The assay was used to support the Phase 2 OPERA II trial (NCT01298492) where the highest level of soluble MAdCAM in samples at Week 12 did not exceed 54 ng/ml and no samples had SHP647 level higher than 74.5 μg/ml. Therefore, the assay is considered suitable to support the OPERA II trial. However, the assay might not be able detect low levels of ADA when serum drug levels are high.