Overview of Diabody-Fc

Bispecific antibodies (BsAbs) are a class of antibodies or antibody constructs with two different binding sites, which can simultaneously recognize and bind to two different antigens or two different epitopes on the same antigen. BsAbs have more advantages than MoAbs, such as achieving multiple targeting, enhancing cytotoxic effects, reducing drug resistance, reducing dosage and toxic side effects. However, BsAbs also face some challenges, such as structural complexity, poor stability, production difficulty, safety issues, etc. To solve these challenges, various platforms and strategies have been designed to generate BsAbs, forming different types and formats of BsAbs. Among them, Diabody-Fc is a novel BsAbs structure, consisting of two single-chain Fv (scFv) fragments forming a bivalent Fv fragment (diabody), fused with an Fc fragment. Diabody-Fc has several characteristics and advantages. First, diabody-Fc has a size of about 100 kDa, smaller than the conventional IgG-type BsAbs (about 150 kDa), which is conducive to tumor tissue penetration and distribution. Second, diabody-Fc provides a stable dimerization interface through the Fc fragment, avoiding nonspecific dimerization and multimerization, and improving the stability and solubility of the molecule. Third, diabody-Fc only requires one gene encoding one polypeptide chain, which can be efficiently expressed and purified in eukaryotic systems. In addition, diabody-Fc can bind to immune effector cells or complement system through the Fc fragment, enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). What's more, diabody-Fc can bind to neonatal Fc receptor (FcRn) on neovascular endothelial cells through the Fc fragment, prolonging the half-life and clearance rate, reducing dosage and toxic side effects.

Structure Features of Diabody-Fc

Diabody-Fc is a BsAbs structure consisting of two single-chain Fv (scFv) fragments forming a bivalent Fv fragment (diabody), fused with an Fc fragment. scFv is the smallest antibody binding fragment consisting of a variable heavy chain (VH) and a variable light chain (VL) domain connected by a flexible peptide. It has a molecular weight of about 25 kDa. diabody is a bivalent Fv fragment formed by two scFv connected by a short peptide linker, which prevents the pairing of the VH and VL domains within one chain and forces them to pair between different chains. It has a molecular weight of about 50 kDa. Fc fragment is an antibody structural domain composed of constant heavy chain (CH) domains. It has a molecular weight of about 50 kDa. Therefore, the total molecular weight of Diabody-Fc is about 100 kDa.

Fig.1 Schematic diagram of Diabody-Fc (Creative Biolabs)

Each scFv fragment consists of a VH and a VL domain, with the VH domain at the N-terminus and the VL domain at the C-terminus. Two scFv fragments are connected by a short peptide linker, which prevents the pairing of the VH and VL domains within one chain and forces them to pair between different chains, forming a diabody. diabody has a compact structure, with two antigen binding sites separated by about 30 Å and facing approximately 90° apart. diabody is fused with an Fc fragment by a long peptide linker, which allows the Fc fragment to stably dimerize and provide immune effector functions and extended half-life.

Clinical Data of Diabody-Fc

Currently, only one Diabody-Fc has been approved for marketing, namely Emicizumab (trade name Hemlibra). Emicizumab is a Diabody-Fc targeting coagulation factor IXa and coagulation factor X, which can mimic the function of missing coagulation factor VIII, for the treatment of hemophilia A. It has been approved for marketing in the United States, the European Union, Japan, Canada, Australia, Switzerland, China and other countries and regions. Emicizumab is suitable for patients of all ages with congenital hemophilia A, regardless of whether they have anti-coagulation factor VIII antibodies. Clinical trial data of Emicizumab showed that compared with prophylactic infusion of coagulation factor VIII, Emicizumab can significantly reduce the incidence and severity of bleeding events.

In additon, there are several Diabody-Fc in different stages of clinical trials, mainly targeting tumor-associated antigens or immune checkpoints, for the treatment of malignant tumors. These Diabody-Fc are mainly developed and led by pharmaceutical companies or biotechnology companies, such as Pfizer, Genentech/Roche, MacroGenics, Merus, etc. Clinical trial data of these Diabody-Fc show that they can effectively activate or inhibit immune cells, induce tumor cell death or enhance the effect of other treatment methods.

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