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Updated: Mar 13, 2026
Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium
Published on: May 18, 2021
Physiologically relevant binding affinity quantification of monoclonal antibody PF-00547659 to mucosal addressin cell
Mengmeng Wang1, Amanda K Kussrow2, Mireia Fernandez Ocana1
1Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc, Andover, MA, USA.
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 KD between in vitro (Biacore) and clinically derived (KD,in-vivo ) values. Back-scattering interferometry (BSI) was applied to acquire physiologically relevant KD values which were used to establish in vitro and in vivo correlation (IVIVC).
Experimental Approach:
BSI was applied to obtain KD 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 KD,in-vivo and target concentrations; MAdCAM concentrations were also measured using LC-MS/MS.
Key Results:
BSI measurements revealed low KD values (higher affinity) for sMAdCAM in buffer and serum, yet a 20-fold higher KD value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted KD,in-vivo in serum was similar to clinically derived KD,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 KD values can be used to establish IVIVC, for PF-00547659 to MAdCAM despite the lack of correlation when using Biacore measured KD and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development.
