Function-structure approach reveals novel insights on the interplay of Immunoglobulin G 1 proteoforms and Fc gamma receptor IIa allotypes

Frontiers in Immunology

Immunology

Alexis Dunkle¹  Karishma Mistry¹  Kevin Whang¹  Tilman Schlothauer²  Alexander Knaupp²  Dietmar Reusch³  Felix Kuhne³  Sebastian Pitschi³  Sunidhi Lenka⁴  Saeed Izadi⁴  Peilu Liu⁵  Lance Cadang⁵  Feng Yang⁵  Steffen Lippold⁵ 

[1] Biological Technologies, Genentech, A Member of the Roche Group, South San Francisco, CA, United States;Pharma Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany;Pharma Technical Development Europe, Roche Diagnostics GmbH, Penzberg, Germany;Pharmaceutical Development, Genentech, A Member of The Roche Group, South San Francisco, CA, United States;Protein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, United States;

关键词: affinity chromatograghy;    CD32 (FcgRII);    ADCP;    deamidation;    molecular dynamics;    mass spectrometry;    glycosylation;    critical quality attribute (CQA);   

DOI  :  10.3389/fimmu.2023.1260446

 received in 2023-07-17, accepted in 2023-08-30,  发布年份 2023

来源: Frontiers

【 摘 要 】

Human Fc gamma receptor IIa (FcγRIIa) or CD32a has two major allotypes with a single amino acid difference at position 131 (histidine or arginine). Differences in FcγRIIa allotypes are known to impact immunological responses such as the clinical outcome of therapeutic monoclonal antibodies (mAbs). FcγRIIa is involved in antibody-dependent cellular phagocytosis (ADCP), which is an important contributor to the mechanism-of-action of mAbs by driving phagocytic clearance of cancer cells. Hence, understanding the impact of individual mAb proteoforms on the binding to FcγRIIa, and its different allotypes, is crucial for defining meaningful critical quality attributes (CQAs). Here, we report a function-structure based approach guided by novel FcγRIIa affinity chromatography-mass spectrometry (AC-MS) assays to assess individual IgG1 proteoforms. This allowed to unravel allotype-specific differences of IgG1 proteoforms on FcγRIIa binding. FcγRIIa AC-MS confirmed and refined structure-function relationships of IgG1 glycoform interactions. For example, the positive impact of afucosylation was higher than galactosylation for FcγRIIa Arg compared to FcγRIIa His. Moreover, we observed FcγRIIa allotype-opposing and IgG1 proteoform integrity-dependent differences in the binding response of stress-induced IgG1 proteoforms comprising asparagine 325 deamidation. The FcγRIIa-allotype dependent binding differences resolved by AC-MS were in line with functional ADCP-surrogate bioassay models. The molecular basis of the observed allotype specificity and proteoform selectivity upon asparagine 325 deamidation was elucidated using molecular dynamics. The observed differences were attributed to the contributions of an inter-molecular salt bridge between IgG1 and FcγRIIa Arg and the contribution of an intra-molecular hydrophobic pocket in IgG1. Our work highlights the unprecedented structural and functional resolution of AC-MS approaches along with predictive biological significance of observed affinity differences within relevant cell-based methods. This makes FcγRIIa AC-MS an invaluable tool to streamline the CQA assessment of therapeutic mAbs.

【 授权许可】

Unknown   
Copyright © 2023 Lippold, Mistry, Lenka, Whang, Liu, Pitschi, Kuhne, Reusch, Cadang, Knaupp, Izadi, Dunkle, Yang and Schlothauer