Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins

Abstract

The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgG-format bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the use of heterodimeric Fc as an alternative scaffold of wild-type Fc for naturally monomeric or heterodimeric proteins, to create Fc-fusion proteins with novel therapeutic modality.

Keywords: Fc engineering; Fc-fusion proteins; antibody engineering; bispecific antibody; heterodimeric Fc; immunocytokines.

Figure 1

Schematic diagram showing the assembly of conventional IgG and heterodimeric Fc-based IgG-format bsAbs. (A) The homodimeric interactions between the wild-type CH3 domains are the initial driving force for HC homodimerization and subsequently disulfide bonds in the hinge regions and between the HC and LC complete the assembly of conventional IgGs, which are bivalent and monospecific. (B) Heterodimer Fc technology introduces asymmetric mutations in each CH3 domain, which enforces two different HCs to be predominantly assembled together, while disfavoring homodimerization between the same HCs. Heterodimeric Fc fragments facilitate the generation of IgG-format bsAbs, which can simultaneously bind to two different antigens.

Figure 2

Schematics of the major interactions contributing to homodimeric CH3 interactions in the wild-type (A) and heterodimeric CH3A–CH3B interactions in the KiH (B), DD-KK (C), and EW-RVT (D) heterodimeric Fc variants. In (A), due to the twofold symmetry of the inter-CH3 interface, each pairwise interaction is represented twice in the structure. In (B–D), the upper panels highlight the main heterodimeric Fc-driving interactions with the indicated color codes. The lower panels show the crystal structures of the wild-type Fc [PDB 3AVE (19)] and the representative heterodimeric Fc variants of KiH [PDB 4NQS (28)], DD-KK [PDB 5DK2 (29)], and EW-RVT [PDB 4X98 (30)], highlighting the inter-CH3 domain interfaces. The images were generated using PyMol software (DeLano Scientific LLC).

Figure 3

Overview of heterodimeric Fc-based antibodies, which are subdivided into three classes: (A) monospecific and monovalent antibodies, (B) IgG-like formats with appendages of scFv and scFab, and (C) intact IgG formats with correct LC association. Each domain is color coded as indicated in the dotted box. Connecting peptide linkers in scFv and scFab fragments are shown by thin black lines. Details are described in the text.

Figure 4

Fc-fused monomeric or heterodimeric proteins. (A) Wild-type Fc-based Epo-Fc dimer vs. Epo-Fc monomer. (B) Aglycosylated Fc-fused GLP-1/GCG monomeric peptide, generated by the LAPScovery technology. (C) Wild-type Fc-based Fc-FSH tandem homodimer vs. Fc-FSH heterodimer. (D) Heterodimeric Fc KiH-based IgG-fused IL2v monomer, IgG-IL2v, developed by Roche. (E) Potential use of heterodimeric Fc for the generation of Fc-fused monomeric or heterodimeric proteins to present the fusion partner in its naturally occurring form. The Fc-fused monomer can easily be generated by the fusion of monomeric protein to the N- or C-terminus of one heterodimeric Fc chain. The Fc-fused heterodimer can be generated by separate fusion of the two subunits of heterodimeric proteins to each chain of the heterodimeric Fc at the N- or C-terminus.