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The DuoBody® technology platform results in the efficient generation of stable bispecific antibody therapeutics, and is easily applied to both drug discovery and large-scale development

Our in-depth knowledge of the naturally occurring process of IgG4 Fab-arm exchange inspired us to develop a novel platform for the discovery and development of bispecific antibodies: the DuoBody platform.

The DuoBody technology involves three basic steps to generate stable bispecific human IgG1antibodies in a post-production exchange reaction. In a first step, two IgG1s, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines (Figure 4, step 1). Subsequently, these IgG1 antibodies are purified according to standard processes for recovery and purification (Figure 4, step 2). After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield (typically >95%) (see figure, step 3; Labrijn et al, 2013).

DuoBody production process

The DuoBody production process: 3 steps to generate bispecific antibodies

REFERENCES

Labrijn et al, PNAS 2013;110(13):5145-5150.
Gramer et al. mAbs 2013;5(6): 962–973.
Labrijn et al. Nature Protocols 2014;9(10):2450-63.

 

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By leveraging the natural Fab-arm exchange process, DuoBody® technology allows for regular IgG1 structure and function to be retained

Bispecific antibodies generated with the DuoBody platform fully retain IgG1 structure and function (Labrijn et al, 2013). Fc-mediated effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), are preserved (Figure 5), and DuoBody molecules have regular IgG1 pharmacokinetic properties (Labrijn et al, 2013). DuoBody characteristics


Regular IgG1 Fc-effector functions and pharmacokinetic properties of DuoBody molecules. Bispecific IgG1 molecules have a similar ability to induce complement-dependent cytotoxicity (CDC; left panel) and antibody-dependent cellular cytotoxicity (ADCC; middle panel) as wild-type IgG1. The clearance rate of bispecific IgG1 in mice is similar to the clearance rate of regular IgG1 (right panel).

REFERENCES

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

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The DuoBody® technology platform allows for efficient screening and discovery of bispecific antibodies in the final format

It is often difficult to predict the behavior of bispecific antibodies based on the activity of the separate parental antibodies or the combination thereof (Labrijn et al, 2013). Thus, there is a need to screen directly for the optimal antibody combination in a bispecific format. The simple post-production exchange reaction developed makes the DuoBody technology platform ideal for high-throughput bispecific antibody library generation and screening in the final format. After separate production of antibody libraries carrying matched DuoBody mutations (Figure, step 1), the antibodies are purified (Figure, step 2), combined in a grid set up and subjected to the post-production exchange reaction (Figure, step 3). Finally, a library of bispecific antibodies that can be screened for desired functionalities is generated (Figure, step 4). Our platform therefore allows for selection of the best bispecific antibodies based on functional data. Thus, the DuoBody platform enables discovery and selection of the lead bispecific molecule in final bispecific antibody format.

The DuoBody platform is compatible with large-scale manufacturing

We have shown that the DuoBody platform is compatible with large-scale manufacturing (Labrijn et al, 2013). Remarkably, simple upscaling of the bench scale protocol with only limited optimization resulted in excellent bispecific antibody yields at large-scale. In a proof-of-concept study, a bispecific product, generated at 0.5 kg/25 L scale, was of outstanding quality: a bispecific antibody yield of 94.7% was obtained and proper and efficient assembly of the bispecific antibody was observed (Labrijn et al, 2013). Stability studies performed with materials produced at large scale showed that it was stable for at least 18 months at +5°C. The DuoBody platform therefore builds on a highly robust process which is easily scalable and compatible with standard large scale manufacturing of IgG.

DuoBody mixing grid


The simple post-production exchange reaction employed by the DuoBody platform allows for generation of bispecific antibody libraries. X and Y represent antibody libraries. X+Y is the number of productions to be performed, and X*Y is the number of different bispecific combinations that is generated during the discovery process.

REFERENCES

Labrijn et al, PNAS 2013;110(13):5145-5150.
Gramer et al. mAbs 2013;5(6): 962–973.
Labrijn et al. Nature Protocols 2014;9(10):2450-63.

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