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The DuoBody® technology is inspired by a naturally occurring process for generating bispecific antibodies, ‘Fab-arm exchange’

To understand the process of Fab-arm exchange, which has been an inspiration for Genmab’s DuoBody technology, it is important to recap the structural components of human immunoglobulin G (IgG)antibodies. Human IgGs exist in four subclasses, IgG1, -2, -3 and -4, that have distinct structural and functional properties. Figure 1 shows a schematic representation of the general IgG protein structure. IgGs are composed of four polypeptide chains: two heavy and two light chains. Each heavy chain consists of a variable domain (VH), a constant domain (CH1), the hinge region and two additional constant domains (CH2 and CH3). The light chains both consist of a variable domain (VL) and a constant domain (CL) and are paired to heavy chains by disulphide bonds and non-covalent interactions. The VL and VH domains form the antigen-binding sites of the IgG molecule. Each heavy-light chain pair represents an IgG Fab-arm (or half-molecule), which is connected to its counterpart by inter-heavy chain disulphide bonds in the hinge region, as well as by non-covalent interactions in the CH3 domain (see below figure).


Human IgG protein structure

Most IgG subclasses are symmetric and contain two identical antigen-binding sites. These antibodies are therefore monospecific (i.e. can bind to one particular epitope). Human IgG4 molecules are also produced as monospecific antibodies by B cells. Yet, once they are secreted by the B cell into the blood, they engage in a unique process, called Fab-arm exchange, in which they become bispecific (Van der Neut Kolfschoten et al, 2007). This dynamic and stochastic process involves the joining of Fab-arms (consisting of one heavy chain bound to one light chain), from one IgG4 antibody with Fab-arms from another. IgG4 antibodies consisting of Fab-arms with different antigen-specificities are therefore formed (see below figure). All IgG4 molecules in the body participate in this naturally occurring and continuously ongoing process of Fab-arm exchange (Van der Neut Kolfschoten et al, 2007). In humans, the ability to engage in Fab-arm exchange is an inherent and unique feature of IgG4.


Human IgG4 engages in Fab-arm exchange and dynamically forms bispecific antibodies in vivo

Intriguingly, bispecific antibodies are therefore naturally produced in human immunity. We have studied the mechanism of IgG4 Fab-arm exchange extensively and discovered that both the hinge region and the CH3 domain play essential roles in Fab-arm exchange (Van der Neut Kolfschoten et al, 2007; Labrijn et al, 2009; Labrijn et al, 2011). More specifically, the amino acid residues serine at position 228 (S228) in the hinge (Van der Neut Kolfschoten et al, 2007; Labrijn et al, 2009) and arginine at position 409 (R409) in the CH3 domain (Labrijn et al, 2011) turned out to be the critical residues that allow IgG4 antibodies to engage in Fab-arm exchange. Building on our in-depth understanding of the mechanism of IgG4 Fab-arm exchange, we developed a powerful platform for the generation of stable bispecific IgG1 antibodies: the DuoBody platform.

REFERENCES

Van der Neut Kolfschoten et al., Science 2007, 317: 1554-7.
Burton and Wilson, Science 2007, 317: 1507-8.
Labrijn et al., Nat Biotech 2009, 27: 767-71.
Labrijn et al., J Immunol 2011, 187: 3238-46.

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The DuoBody technology platform results in the efficient generation of stable bispecific antibody therapeutics 

Our extensive knowledge of the naturally occurring process of IgG4 Fab-arm exchange inspired us to develop a simple post-production process to generate bispecific antibodies, named “controlled Fab-arm exchange” (Figure 3; Labrijn et al, 2013).

In the first step of controlled Fab-arm exchange, the two parental IgG1 antibodies that will be combined into a bispecific are designed to contain a single, matched mutation in the CH3 domain: lysine at position 409 to arginine (K409R) in one parental antibody, phenylalanine at position 405 to leucine (F405L) in the other. The parental antibodies are produced and purified separately. Subsequently, they are mixed together under tailored reaction conditions. The reaction conditions, together with the mutations in the CH3 domains, allow the parental antibodies to dissociate into IgG1 Fab-arms and promote the formation of bispecific IgG1 molecules in a unidirectional manner which results in a very efficient generation of bispecific antibodies (Figure 3). Importantly, DuoBody molecules have a wild-type IgG1 hinge that is resistant to reduction under physiological conditions, which further ensures the in vivo stability of the bispecific end-product. With this simple post-production process we are able to generate bispecific antibodies with a remarkably high yield: typically about 95% is already obtained in the DuoBody reaction mixture. If required, a simple polishing step can be employed to obtain an essentially pure DuoBody product.



Figure 3. DuoBody technology: controlled Fab-arm exchange.

Bispecific antibodies generated by the DuoBody technology fully retain IgG1 structure and function (Labrijn et al, 2013). Fc-mediated effector functions are preserved, and DuoBody molecules have regular IgG1 pharmacokinetic properties.

REFERENCES
Labrijn et al, PNAS 2013;110(13):5145-5150.

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