Bispecific Antibodies with Native Human IgG Structure As Mimetics for FVIII Activity

Background: Non-factor therapies for hemophilia A are being evaluated to overcome the need for frequent i.v. administration and address therapeutic issues caused by anti-FVIII inhibitors. One possibility to substitute the lack of FVIII activity is the development of a bispecific antibody (biAb) that targets the two coagulation factors FIXa and FX. The two arms' binding characteristics and relative orientation are thought to be important to optimally bridge FIXa and FX and to at least partially mimic the function of FVIIIa, and facilitate FXa generation.

Aim: To identify biAbs targeting FIXa and FX that have FVIII-mimetic activity using the κλ body platform, generating biAbs that are indistinguishable from native human IgG. As hemophilia A patients require life-long treatment, a native human IgG structure represents an advantage for the chronic treatment of this patient population.

Methods: We used the κλ body platform to identify novel biAbs with FVIII-mimetic activity. First, antibodies against different forms of FIX(a) and FX(a) were selected using phage display. Then, κλ bodies were produced by co-expressing a common heavy chain and two light chains. As the κλ body is the major form secreted in the supernatant of the transfected cells, conveniently, cell culture supernatants were used directly for functional screening and multiple arms optimized in parallel.

Results: Over 1000 unique antibodies were identified that target different epitopes on each factor, allowing for the generation of thousands of different biAbs combinations. To date, over 10,000 biAbs have been functionally screened. Unlike previous studies which only rarely identified FVIII-mimetic biAbs (Sampei et al., PLoS One 2013), the present approach resulted in isolation of ~350 unique arm combinations showing considerable FVIII mimetic activity. These results suggest that multiple geometries of the FIXa/FX biAb complex can lead to FVIII-mimetic activity.

Conclusion: Theκλ body technology is optimal for generating native human and non-engineered biAbs of high epitope diversity, and exploring a wide range of geometries which is crucial to obtain biAbs for efficient FX activation. Efficient expression supernatant screening allowed multiple arms to be optimized in parallel and significantly improved FVIII-mimetic activity. The native human IgG structure of the FVIII-mimetic κλ bodies make them ideal candidates for long-term treatment of hemophilia A.