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Antibody Technology Platforms
DuoBody®
DuoBody® is a biotechnology platform for discovering and developing bispecific antibodies, also called dual-targeting molecules.
DuoBody® is a versatile biotechnology platform for generating bispecific antibodies. The DuoBody technology platform is capable of generating bispecific antibodies which are dual targeting agents, leading to a variety of effects, including simultaneous recruitment of immune cells and tumor cell recognition.
ÐǺ£ÆåÅÆ DuoBody Collaborations
In July 2012, ÐǺ£ÆåÅÆ entered into a collaboration with Janssen to create and develop bispecific antibodies using our DuoBody® platform. ÐǺ£ÆåÅÆ created panels of bispecific antibodies to multiple disease target combinations identified by Janssen that they are developing further in both the research setting and in clinical trials.
Bispecific antibodies provide a flexible solution for several dual-targeting applications. They can be used for cell receptor blocking, activation or down modulation; used to recruit and target T cells; or to help deliver toxic payloads.
DuoBody® is a proven (Neijssen et al. 2021) technology platform for high-throughput bispecific antibody library generation and screening in the bispecific antibody format. (see Fig. 3)
The DuoBody® biotechnology involves three basic steps to generate stable bispecific human IgG1 antibodies. In a first step, two IgG1s, each containing single matched mutations in the third constant (CH3) domain (lysine at position 409 to arginine, K409R; phenylalanine at position 405 to leucine, F405L), are produced separately using recombinant mammalian expression systems (Figure 2, step 1). Subsequently, these IgG1 antibodies are purified according to standard processes for recovery and purification (Figure 2, step 2).
After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions, called controlled Fab-arm exchange, resulting in a bispecific antibody product with a very high yield (typically >95%) (Figure 2, step 3; Labrijn et al., 2014).
This DuoBody® process can be easily performed at discovery scale as well as commercial manufacturing scale.
If required, a simple polishing step can be employed to obtain an essentially pure DuoBody® product. Importantly, bispecific antibodies generated with the DuoBody® platform fully retain IgG1 structure and function (Labrijn et al., 2013). In addition, the DuoBody® platform is compatible with additional Fc-engineering mutations, in case such mutations are required for the specific application. The ease and versatility of the DuoBody® platform has also been translated to mouse (and rat) antibodies to enable the efficient generation of surrogate bispecific mouse IgG1, IgG2a and IgG2b (and rat IgG1, IgG2a, IgG2b and IgG2c molecules) via controlled Fab-arm exchange (Labrijn et al., 2017).
The DuoBody® platform builds on insights from the natural biology of antibodies with the goal of creating innovative treatments.
The inspiration for our DuoBody® platform comes from our extensive knowledge of the naturally occurring process of IgG4 Fab-arm exchange. To understand the process of Fab-arm exchange, 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. 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 cells, they can 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 recombination 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.
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. 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). More specifically, the amino acid residues serine at position 228 (S228) in the hinge (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 our powerful platform for the generation of stable bispecific IgG1 antibodies: the DuoBody® platform.
References
IgG4 biology and Fab-arm exchange:
Van der Neut Kolfschoten et al., Science 317(5844), 1554-7 (2007)
Labrijn et al., Nat Biotech 27(8), 767-71 (2009)
Labrijn et al., J Immunol 187(6), 3238-46 (2011)
DuoBody® platform:
Labrijn et al., PNAS 110(13): 5145-5150 (2013)
Gramer et al., mAbs 5(6): 962–973 (2013)
Labrijn et al., Nature Protocols 9(10): 2450-63 (2014)
Labrijn et al., Sci Report 7(1) (2017)
Van den Bremer et al., Anal. Chem. 89 (20), 10873-10882 (2017)
DuoBody® applications:
Moores et al., Canc Res 76 (13), 3942-53 (2016)
Jarantow et al., J Biol Chem 290 (41), 24689-704 (2015)
Zheng et al., mAbs 8 (3), 551-61 (2016)
Grugan et al., mAbs 9 (1), 114-126 (2017)
Goulet et al., J Biol Chem 293 (2), 651-661 (2018)
Evans et al., mAbs 11 (6), 1101-1112 (2019)
Neijssen et al. J Biol Chem 296:100641 (2021)
Østergaard et al. Blood 138(14):1258-68 (2021)
Review bispecific antibodies:
Labrijn et al., Nat Rev Drug Discov 18 (8), 585-608 (2019)
DuoBody Glossary
Antigen-Binding Sites
Synonym for paratope. The region of an antibody that binds to antigens, which is formed by determinants in the variable domains of the heavy and/or light chains.
Controlled Fab-arm exchange
The in vitro post-production process employed by the DuoBody® platform in which IgG1 half-molecules recombine with other IgG1 half-molecules to generate bispecific IgG1 antibodies. This unidirectional reaction only takes place under tailored reaction conditions in vitro with IgG1 antibodies containing single matched point-mutations in the CH3 domains and forms the basis of the DuoBody® technology. Although this process was originally developed as an IgG1-based platform, controlled Fab-arm exchange can be extended to other IgG subclasses to suit particular applications.
Epitope
Binding site of an antibody on an antigen.
Fc-engineering
Altering of certain functions of the Fc-domain of an antibody by the introduction of mutations that alter the amino acid sequence. The Fc-domain is involved in the employment of immune effector functions by interacting with Fc-receptors on effector cells or the complement factor C1q.
Hinge region
The region between the CH1 and CH2 domains of the heavy chain, which is highly flexible. Disulphide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule.
IgG antibodies
A certain subclass of immunoglobulin or antibody. IgG, and in particular the IgG1 subclass, is the antibody class that is most commonly used for antibody therapeutics.
Parental antibodies
Monospecific antibodies containing matched single mutations with different specificity from which a bispecific antibody is generated in the process of controlled Fab-arm exchange.
Post-production process
Refers to the controlled Fab-arm exchange process that takes place after the separate production of the two parental IgG1 monoclonal antibodies, each containing single matched mutations in the third constant (CH3) domain.
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