Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain

Sebastian Fenn,Karl-Peter Hopfner,Christian Gassner,Hubert Kettenberger,Wolfgang Schaefer,Julia J Griese,Sabine Imhof-Jung,Christian Klein,Christian B Schiller,Joerg Thomas Regula,Harald Duerr,Markus Thomas,Joerg Moelleken

doi:10.1371/journal.pone.0061953

Sebastian Fenn, Karl-Peter Hopfner + Show 11 more

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https://doi.org/10.1371/journal.pone.0061953

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Abstract

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the “CrossMab” format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the “knobs-and-holes” approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Highlights

Therapeutic antibodies are used to treat a multitude of human diseases
To reveal potential structural effects of the crossover procedure on the resulting bispecific antibodies, we report the crystal structure of a human Angiopoietin 2 binding ‘‘CrossFab’’, i.e. a Fab derivative in which the VL domain is fused to the CH1 domain, while the VH domain is fused to the CL domain (‘‘CH1CL crossover’’ in [16])
CrossMabCH1CL was shown to be expressed in eukaryotic cells with high fidelity concerning the correct heavy chains (HCs)-light chains (LCs) association [16]

Summary

Introduction

Therapeutic antibodies are used to treat a multitude of human diseases. They can routinely be obtained by mature technologies such as immunization or in-vitro display approaches. The natural variability of complimentarity-determining regions (CDRs) allows for the discovery of specific, high-affinity antibodies. Most of these therapeutic antibodies have the Immunoglobulin G (IgG) format which confers long serum half-life due to an FcRn-mediated recycling mechanism. A typical IgG antibody consists of two identical heavy chains (HCs) and two identical light chains (LCs) [3]. Typical IgG-type antibodies comprise two identical antigen-binding arms (Fabs), and an effector domain, Fc. Each Fab contains one light chain and heavy chain (reviewed by [5])

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