Physiologically relevant binding affinity quantification of monoclonal antibody PF‐00547659 to mucosal addressin cell adhesion molecule for in vitro in vivo correlation

BACKGROUND AND PURPOSE: A monoclonal antibody (PF‐00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans‐membrane (mMAdCAM) target forms, showed over 30‐fold difference in antibody‐target K(D) between in vitro (Biacore) and clinically derived (K(D,in‐vivo)) values. Back‐scattering interferometry (BSI) was applied to acquire physiologically relevant K(D) values which were used to establish in vitro and in vivo correlation (IVIVC). EXPERIMENTAL APPROACH: BSI was applied to obtain K(D) values between PF‐00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue. CHO cells and tissue were minimally processed to yield homogenate containing membrane vesicles and soluble proteins. A series of binding affinities in serum with various dilution factors was used to estimate both K(D,in‐vivo) and target concentrations; MAdCAM concentrations were also measured using LC–MS/MS. KEY RESULTS: BSI measurements revealed low K(D) values (higher affinity) for sMAdCAM in buffer and serum, yet a 20‐fold higher K(D) value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted K(D,in‐vivo) in serum was similar to clinically derived K(D,in‐vivo), and the BSI‐estimated serum sMAdCAM concentration also matched the measured concentration by LC–MS/MS. CONCLUSIONS AND IMPLICATIONS: Our results successfully demonstrated that BSI measurements of physiologically relevant K(D) values can be used to establish IVIVC, for PF‐00547659 to MAdCAM despite the lack of correlation when using Biacore measured K(D) and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development.