The making of bispecific antibodies

Abstract

During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addition, different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.

Keywords: Appended IgG; Fab; Fc heterodimerization; bispecific antibodies; fusion proteins; immunoglobulin; scFv.

Figure 1.

Building blocks and generation of bispecific antibodies Examples of antigen-binding modules as well as homo- and heterodimierzation modules are shown, which can be used to generate bispecific molecules combining different binding sites within one molecule.

Figure 2.

The zoo of bispecific antibody formats Overview of bispecific antibody formats reduced to practice, grouped into molecules with symmetric or asymmetric architecture.

Figure 3.

Combinatorial diversity of bispecific IgGs Overview of possible combinations to arrange heavy and light chains from two different antibodies, including strategies to overcome incorrect heavy chain and heavy-light chain pairing.

Figure 4.

Strategies to generate bispecific IgGs Overview of strategies to generate bispecific IgG molecules including strategies to force correct assembly of heavy chains and / or heavy-light chains, or utilizing postproduction purification or assembly strategies.