Crystal structures of immunoglobulin Fc heterodimers reveal the molecular basis for heterodimer formation

• X-ray crystal structures of two heterodimeric Fc variants were determined.
• The X-ray structures provide the molecular bases for Fc heterodimerization.
• Addition of inter-CH3 disulfide bond increases the Fc heterodimer yield.
• The inter-CH3 disulfide-bonded Fc heterodimer shows improved thermal stability.

We determined the X-ray crystal structure of an immunoglobulin fragment crystallizable (Fc) heterodimer, EW-RVT, at a resolution of 2.5 Å and found that the designed asymmetric interaction residues located in the heterodimeric CH3 interface favor Fc heterodimer formation. We further generated an inter-CH3 disulfide-bonded heterodimeric Fc variant, EW-RVTS–S, which exhibited improved heterodimer formation and thermodynamic stability compared with the parent EW-RVT variant. The crystal structure of EW-RVTS–S superimposed very closely with the wild-type Fc structure. Our results provide the detailed structure of heterodimeric Fc scaffolds, which will be useful for the generation of immunoglobulin G (IgG)-like bispecific antibodies.

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