Abstract
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G1 monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG1. Physicochemical characterization of these fractions revealed that a rarely seen crystallizable fragment (Fc) modification, N325 deamidation, exhibited a positive correlation with the loss of ADCC activity. A further surface plasmon resonance study showed that this modification disrupted the binding between the IgG1 Fc and Fcγ receptor IIIa, resulting in decreased ADCC activity of the IgG1 antibody. Mutants of N325/D and N325/Q were made to confirm the effect of N325 deamidation on ADCC. We hypothesize that N325 deamidation altered the local three-dimensional structure, which might interfere with the binding and interaction with the effector cell. Because of its impact on biological activity, N325 deamidation is a critical quality attribute for products whose mechanism of action includes ADCC. A thorough understanding of the criticality of N325 deamidation and appropriate monitoring can help ensure the safety and efficacy of IgG1 or Fc-fusion products.
Highlights
To the target while the Fc region of the antibody recognizes the Fc γ receptor (FcγR) IIIa expressed on natural killer (NK) cells
To understand the degradation pathway and establish a list of critical quality attributes (CQAs), a therapeutic mAb (IgG1-A) with Antibody-dependent cell-mediated cytotoxicity (ADCC) activity as one of the mechanisms of action was exposed to elevated temperature conditions at 25 °C for 10 months and to stressed conditions at 40 °C for 4 months
Similar results were observed for another IgG1 antibody targeting epidermal growth factor receptor (IgG1-C), and mutations of N325/D and N325/Q decreased both FcγRIIIa binding and ADCC activities. These results indicate the essential role of N325 for FcγRIIIa binding and effector function, which supports our conclusion that N325 deamidation caused ADCC
Summary
To the target while the Fc region of the antibody recognizes the Fc γ receptor (FcγR) IIIa expressed on natural killer (NK) cells. Removal of the fucose from the first N-acetylglucosamine of the N-linked oligosaccharide at this residue can substantially strengthen the binding affinity of engineered antibodies to FcγRIII and enhance ADCC in vitro and in vivo[16,17,18,19]. Reports are limited on how chemical degradations in the Fc region of mAbs could affect ADCC function. Methionine oxidation has been found to substantially affect the structure and stability of the human IgG1 Fc region, but this change has no effect on binding between IgG1 and FcγRs20,21. The aim of this study was to identify the cause of the biological activity loss after heat stress and understand the degradation pathway of the IgG1 antibody. We explore the clinical relevance of N325 deamidation, a control strategy to measure N325 deamidation, and correlation of N325 deamidation with stability study data using orthogonal methods
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