Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

Athéna C Patterson-Orazem,Ahlam N Qerqez,Laura R Azouz,Minh Thu Ma,Shannon E Hill,Yemo Ku,Lisa A Schildmeyer,Jennifer A Maynard,Raquel L Lieberman

doi:10.1016/j.jbc.2021.101067

Abstract

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Highlights

Antibodies are indispensable and ubiquitous tools for targeting a specific antigen that are widely used across biomedical research fields
Myocilin is best known for its high expression in the trabecular meshwork (TM) [14, 15], an ocular tissue in the anterior segment involved in the filtration of aqueous humor and the regulation of intraocular pressure (IOP) [16]
Boosters alternating between human CCLZ (hCCLZ) and mouse leucine zipper (LZ) until strong titers to both immunogens were detected in mouse sera (Fig. S1A)

Summary

Introduction

Antibodies are indispensable and ubiquitous tools for targeting a specific antigen that are widely used across biomedical research fields. Recombinant antibodies avoid the batch-to-batch variability of polyclonal antibodies [4] and the genetic drift of monoclonal antibodies [5], while allowing for facile conversion to other formats (human, mouse, or rabbit constant regions, Fab fragments) Such antibodies can be designed with the structure, function, and dysfunction of their targets in mind and can be combined with constant domains that allow for use of the same reagent on samples from human and animal tissues. Sensitivity of the LZ conformational state, defined by comparing immunoreactivity in ELISA, dot blot, and western blot, identified one highly conformationally selective and one less selective antibody Both antibodies immunoprecipitate full-length myocilin from spent media of primary human TM cells. Additional maturation steps applied to the conformationally selective antibody yielded a final antibody suitable for fluorescence imaging of myocilin in fixed human TM cells These new antibodies should find broad application in glaucoma and other research areas where myocilin plays a role

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