Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody

Jiyeon Lee,Hye-Jin Kim,Jooho Roh,Youngsil Seo,Minjae Kim,Hye-Ryeong Jun,Chuong D Pham,Myung-Hee Kwon

doi:10.1074/jbc.m113.508499

Abstract

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering.

Highlights

A subset of anti-DNA antibodies have DNA-hydrolysis and/or cell-penetration activity
We reported previously the results of a mutational analysis based on the x-ray crystallographic structure of 3D8 scFv, which showed that the His residues in CDR1 of the heavy chain (HCDR1) and CDR3 of the light chain (LCDR3) are critical for DNA hydrolysis [15]. 3D8 scFv is endocytosed via the caveolae-mediated pathway and localizes to the cytosol without translocating to the nucleus [14, 16]
3D8-H1i denotes an scFv in which HCDR1 of 3D8 was replaced, and 3D8-L3i denotes an scFv in which LCDR3 was replaced

Summary

Introduction

A subset of anti-DNA antibodies have DNA-hydrolysis and/or cell-penetration activity. Conclusion: CDR1 of the light chain plays a crucial role in maintaining the properties of 3D8 scFv. Significance: Single complete CDR deactivation allows exploration of the molecular features of multifunctional anti-DNA antibodies. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering

Objectives

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Conclusion