Short communicationCrystal structures of immunoglobulin Fc heterodimers reveal the molecular basis for heterodimer formation
Graphical abstract
Introduction
The fragment crystallizable (Fc) region of immunoglobulin G (IgG), comprising the hinge region, CH2, and CH3 domains of the heavy chain, forms a homodimer, which is initially driven by non-covalent inter-CH3 interactions and subsequently by covalent disulfide linkages in the hinge region (Labrijn et al., 2011). This Fc homodimerization is the basis of the homodimeric assembly of heavy chains with light chains to form the conventional IgG antibody with monospecific, bivalent antigen binding activity. Bispecific antibodies (bsAbs), which are single molecules that simultaneously bind to two different targets, often provide improved clinical benefits in the treatment of complicated diseases, such as tumors and immune disorders (Kontermann, 2012). IgG-like formats based on heterodimeric Fc scaffolds, which are designed by asymmetric mutations in the CH3 homodimeric interface of IgG1 to favor the heterodimeric Fc (Spreter Von Kreudenstein et al., 2014), are useful for industrial applications because they retain natural IgG properties, including physicochemical stability, long serum half-life, and immune system recruiting ability (Klein et al., 2012, Kontermann, 2012).
Heterodimeric Fc technology has been pioneered by Carter and co-workers at Genentech Inc. (San Francisco, CA, USA) with the so-called “knobs-into-holes (KiH)” Fc variant (Ridgway et al., 1996), which has a T366WCH3A “knob” mutation (EU numbering (Edelman et al., 1969)) in one CH3 domain (CH3A) and T366S/L368A/Y407VCH3B “hole” mutations in the other CH3 domain (CH3B) (Atwell et al., 1997). These mutations replace the symmetric hydrophobic interactions at the core CH3 interface with asymmetric hydrophobic interactions, and favor heterodimer over homodimer formation. Thereafter, a similar strategy has been applied for other heterodimeric Fc variants, including so-called HA-TF (Moore et al., 2011), ZW1 (Von Kreudenstein et al., 2013), and SEEDBody (Davis et al., 2010). Other approaches for Fc heterodimer generation include DD-KK (Gunasekaran et al., 2010) and EEE-RRR (Strop et al., 2012), in which the residues involved in the symmetric electrostatic interactions at the CH3 interface are replaced with those for asymmetric electrostatic interactions.
We recently reported a heterodimer Fc variant, EW-RVT, with the interaction pairs K409WCH3A–D399V/F405TCH3B (dubbed the W-VT pair) and K360ECH3A–Q347RCH3B (dubbed the E-R pair), which were designed to replace the conserved electrostatic interactions with asymmetric hydrophobic interactions and to add asymmetric long-range electrostatic interactions at the rim of the heterodimeric CH3 interface, respectively (Choi et al., 2013). To improve the heterodimer yield and stability of heterodimeric Fc variants, it is necessary to determine three-dimensional structures to better understand the heterodimeric CH3 interface. Although many heterodimeric Fc variants have been reported, X-ray crystal structures are only currently available for two Fc variants, KiH (PDB ID: 4NQS) (Elliott et al., 2014, Mimoto et al., 2014) and ZW1 (PDB ID: 4BSW) (Von Kreudenstein et al., 2013).
In this study, we determined the X-ray crystal structure of the EW-RVT Fc heterodimer at a resolution of 2.5 Å to elucidate the molecular interactions favoring Fc heterodimer formation over the homodimer. Based on the tertiary structure of the EW-RVT Fc heterodimer, we further introduced a disulfide bond at the CH3A–CH3B interface, generating the EW-RVTS–S variant, which exhibited improved Fc heterodimer yield and thermodynamic stability compared with EW-RVT. We also determined the crystal structure of the EW-RVTS–S Fc heterodimer at 2.5 Å resolution, and the overall structure of EW-RVTS–S was similar to those of the wild-type Fc homodimer and EW-RVT Fc heterodimer. Our results provide deep insight into the structural basis favoring heterodimer formation of the two designed heterodimeric Fc variants.
Section snippets
Construction, expression, and purification of heterodimeric Fc variants
The plasmids encoding the single-chain variable fragment (scFv)-Fc and Fc proteins were constructed as described previously (Choi et al., 2013). The cloned Fc of human IgG1 includes residues 225–447 (EU numbering) spanning the hinge-CH2–CH3 region (Edelman et al., 1969). The two plasmids encoding the scFv-FcCH3A/FcCH3B or FcCH3A/FcCH3B proteins were transiently co-transfected in pairs into 100-ml to 400-ml HEK293F cell cultures in FreeStyle 293 media following the standard protocol (Choi et
Crystal structure of the EW-RVT heterodimeric Fc variant
The heterodimer yield of the EW-RVT Fc variant was approximately 91% under co-expression conditions (Choi et al., 2013). To elucidate the structural properties of the heterodimeric CH3A–CH3B interactions driving heterodimer formation, we sought to solve the crystal structure. The EW-RVT Fc heterodimer (Fc-EW-RVT) was purified from mammalian HEK293F cell cultures after transient co-transfection of two respective plasmids carrying two Fc fragments, i.e., hinge-CH2–CH3A and hinge-CH2–CH3B (Choi et
Conclusion
Here we determined the X-ray crystal structure of the EW-RVT Fc heterodimer at 2.5 Å resolution to understand the molecular interactions at the CH3A–CH3B interface favoring heterodimer over homodimer formation. As designed, the W-VT pair residues (K409WCH3A–D399VCH3B/F405TCH3B) and E-R pair residues (K360ECH3A–Q347RCH3B) are located in the heterodimeric CH3 interface to form asymmetric, complementary hydrophobic interactions and electrostatic interactions at 3.45 Å, respectively, which
Conflict of interest
The authors declare no conflict of interest for this study.
Acknowledgements
This work was supported by grants from the Basic Science Research Program (2012R1A1A2039306), the Global Frontier Project (2013M3A6A4043874), the Pioneer Research Center Program (2014M3C1A3051470), and the Converging Research Center Program (2009-0093653) of the National Research Foundation, funded by the Korean government.
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These two authors contributed equally to this work.