A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Zhi Liu,Esther C Leng,Kannan Gunasekaran,Martin Pentony,Min Shen,Monique Howard,Janelle Stoops,Kathy Manchulenko,Vladimir Razinkov,Hua Liu,William Fanslow,Zhonghua Hu,Nancy Sun,Haruki Hasegawa,Rutilio Clark,Ian N Foltz,Wei Yan

doi:10.1074/jbc.m114.620260

Zhi Liu, Esther C Leng + Show 15 more

Open Access

https://doi.org/10.1074/jbc.m114.620260

Copy DOI

Abstract

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.

Highlights

Bispecific heterodimeric antibody consisting of two different heavy chains and two different light chains requires heterodimerization of heavy chains and cognate light-heavy chain pairings
We attempted to achieve this by engineering the VH-VL and CH1-CL interfaces as they are both involved in the HC-LC recognition and engagement process [27,28,29,30,31]
The ADCC-enhanced anti-HER2 ϫ EGF receptor (EGFR) hetero-IgG1 V23_W165 significantly reduced the tumor size at the end of study when compared with the combination of two parent IgG1 antibodies (p ϭ 0.0473), whereas the ADCC-norm anti-HER2 ϫ EGFR heteroIgG1 V23 did not show any significant difference (p ϭ 0.2701). These results showed that anti-HER2 ϫ EGFR hetero-IgG1s strongly inhibited the tumor growth, and ADCC enhancement helps antibody to overcome K-RAS mutation, which is in line with the report by Schlaeth et al [46]

Summary

Introduction

Bispecific heterodimeric antibody consisting of two different heavy chains and two different light chains requires heterodimerization of heavy chains and cognate light-heavy chain pairings. Results: Cognate light-heavy chain pairing can be achieved by an antibody engineering approach. Conclusion: Bispecific hetero-IgG antibodies can be made in mammalian cells. We describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies

Objectives

Results

Conclusion