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NIH Research Festival

September 23 – 25, 2024

Thiol-based Conjugation for the Generation of ADA Bridging Assay Reagents to Detect Antibodies to PTH-IA in Human Plasma

Authors

  • AJ Nduwumwami
  • E Wagner
  • AQ Wang
  • Y Fang
  • D Tao
  • X Xu

Abstract

Biotin-and sulfotag-conjugated biotherapeutic drugs are routinely used as reagents for the detection of anti-drug antibodies (ADA). The biotin and sulfotag labels contain N-hydroxysuccinimide (NHS) ester that allows covalent conjugation to primary amine groups on lysine or N-terminal residues of peptide or protein drugs. In cases where the lysine or N-terminal residues are part of epitopes recognized by ADA, those covalent modifications can mask the epitopes and impair ADA binding and assessment of immunogenicity. We devised a two-step approach to generate reagents that can detect antibodies against parathyroid hormone inverse agonist (PTH-IA) in human plasma. PTH-IA is a small peptide being developed as a therapeutic for Jansen’s Metaphyseal Chondrodysplasia (JMC). In the first step, sulfo-NHS biotin and the heterobifunctional sulfo-SMCC reacted with primary amines of BSA. In the second step, a 1:1 mixture of a cysteine extended-PTH-IA at either the N-terminus or C-terminus was conjugated to biotin-BSA-SMCC complex through the maleimide group of SMCC. A similar approach was used to synthesize the warhead wherein sulfotag-NHS ester was used instead of the sulfo-NHS-biotin. The generated Biotin-BSA-PTH-IA and Sulfotag-BSA-PTH-IA reagents were used to develop a bridging assay to detect anti-PTH-IA antibodies. The reagents allowed development of an ADA assay with sufficient sensitivity, low background, and good positive control performance. This approach allows conjugation of multiple drug molecules to each molecule of albumin tag and should increase epitope access and improve assay sensitivity.

Scientific Focus Area: Molecular Pharmacology

This page was last updated on Tuesday, August 6, 2024

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Current Research Festival

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