Abstract
Heterodimeric Fc designed by engineering the CH3 homodimeric interface of immunoglobulin G1 serves as an attractive scaffold for the generation of bispecific antibodies (bsAb) due to the favorable properties of the Fc region. In this study, we describe a heterodimeric Fc generated by substituting the conserved electrostatic interactions at the CH3 core interface with asymmetric hydrophobic interactions and introducing asymmetric, long-range electrostatic interactions at the rim of the CH3 interface. Coexpression of Fc proteins carrying the combined CH3 variant pairs in HEK293F cells produced the heterodimer, which was purified with more than 90% yield, and retained wild-type Fc biophysical properties. The heterodimeric Fc was exploited to generate a bsAb simultaneously targeting both the Met receptor tyrosine kinase and the VEGF receptor 2 (VEGFR-2), with two respective antigen-specific, single-chain variable fragments (scFv) into the N-terminus. The Met × VEGFR-2 bsAb bound concurrently to the two target antigens, efficiently inhibited the downstream signaling and tube formation stimulated by the two receptors in human endothelial cells, and exhibited more potent antitumor efficacy in MKN45 human gastric cancer xenograft models than both the parent monospecific antibody alone. Collectively, based on the newly designed heterodimeric Fc-based bsAb, our results provide the therapeutic potential of bsAb targeting both Met and VEGFR-2 simultaneously for the treatment of human cancers.
Highlights
Bispecific antibodies, which simultaneously bind to two different molecules or two epitopes within a single molecule, may provide improved clinical benefits in the treatment of complicated diseases, such as tumors and immune disorders, compared with conventional monospecific monoclonal antibodies because of the synergistic or additive effects [1]
Design of the heterodimeric Fc variant Homodimerization of the heavy chain of human IgG1 is driven by both hydrophobic interactions at the CH3 interface and electrostatic interactions around the rim of the hydrophobic core (Fig. 1A; refs. 10, 26)
Highly pure, stable heterodimeric Fc, we intended to add two kinds of mutations into the CH3 domain of human IgG1: (i) substitution of charged amino acids around the rim of the hydrophobic core with hydrophobic amino acids with larger or smaller side chains to replace the symmetric electrostatic interactions with asymmetric hydrophobic interactions and (ii) substitution of amino acids weakly involved in the CH3 interactions due to their distance with amino acids with charged, long side chains to form asymmetric longrange electrostatic interactions
Summary
Bispecific antibodies (bsAb), which simultaneously bind to two different molecules or two epitopes within a single molecule, may provide improved clinical benefits in the treatment of complicated diseases, such as tumors and immune disorders, compared with conventional monospecific monoclonal antibodies (mAb) because of the synergistic or additive effects [1]. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Tion), difficulties in large-scale manufacturing, and potential immunogenicity [1, 3]. To minimize these problems, bispecific heterodimeric IgG antibody formats that are as close as possible to human IgG [4] have been developed. Bispecific heterodimeric IgG antibody formats that are as close as possible to human IgG [4] have been developed These bispecific heterodimeric IgG antibodies use heterodimeric Fc technology enabling two different heavy chains to be assembled with a common light chain [5] or with the cognate light chain by CrossMab technology [6]
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