A full-length IgG contains two identical heavy chains and light chains. Each heavy chain associates with a light chain through a disulfide bond and non-covalent interactions to form a heterodimer, and both heterodimers associate to forming a complex Y-shaped antibody. Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously bind to two different antigens or epitopes. Bispecific antibodies are emerging as the next wave of antibody-based therapies. Advances in genetic engineering technology has resulted in a range of recombinant bispecific antibody formats. Generally, bispecific antibody can be divided into two major categories, those with an Fc region and those without an Fc region. Many of these formats involve linking antibody fragments in tandem. Nevertheless, most of these formats have been limited by some of their liabilities, such as instability, short half-life, poor manufacturability, and immunogenicity.

Bispecific antibody with IgG-like architecture, which is based on the heterodimerization of two different IgG molecules, is a promising format because it maintains the overall size and natural structure of human IgG with good stability, half-life, and pharmacokinetics profile. To generate IgG-like bispecific antibody, two challenges have to overcome. One is to facilitate heterodimerization of two distinct heavy chains and prevent homodimerization of two identical heavy chains. The second is to have correct pairing of cognate heavy and light chain. Based on knobs into holes (KIH) technology for heavy chain heterodimerization, we have developed a new technology to facilitate correct pairing of cognate heavy and light chain. Our approach replaces the native interchain disulfide bond between the heavy and light chain in one Fab arm with an engineered interchain disulfide bond. The native interchain disulfide bond between the heavy and light chain in another Fab arm is not modified (wild-type). We have identified several different engineered interchain disulfide bonds, which can be chosen to use for the production of IgG-like bispecific antibody.

Our approach can be used to efficiently produce an IgG-like bispecific antibody from two pre-existent antibodies (IgG1, IgG2, IgG3, IgG4, Kappa and Lambda light chains). This approach is easy to operate without using any artificial linkers and common light chains. The resulting IgG-like bispecific antibody is stable and amenable to commercial manufacturing.