A FULLY Human Bispecific Antibody Functionally Rescues Factor VIII Deficiency EX VIVO
Authors: Wang Wei , Blackwood John , Magliozzi Roberto , Moraes Leonardo , Hollins Jeffrey , Sinopoli Alessandro , Chi Bertie , Mitchell Laura , Sellick Chris , Pearce Claire , Theurl Igor , Germaschewski Volker , Galson Jacob , Badiali Luca , Dickson Karen , Bradley Allan , Lee E-Chiang 
Published: November 2019

Background:

Hemophilia A is caused by deficiency of factor VIII (F.VIII), an essential blood-clotting cofactor. The current standard of care involves prophylactic or on-demand use of F.VIII. However, approximately 30% of severe hemophilia A patients develop inhibitory antibodies against the replacement factor VIII. Additionally, venous infusion of F.VIII is burdensome especially in pediatric patients. Hemlibra®, a humanized bispecific antibody (BiAb) that mimics the action of factor VIII, has been developed and approved for treating hemophilia A patients with or without inhibitors. Using Kymab's IntelliSelect® Bispecific antibody discovery platform, a fully human common light chain (CLC) bispecific antibody was developed and optimized to mimic the function of F.VIII. The biological activity of the CLC bispecific antibody was characterized using clinically relevant hemostatic assays.

Methods:

Kymab IntelliSelect® Transgenic mice were immunized with human factor IX (F.IX) or factor X (F.X). Isolated F.IX and F.X arms were combinatorially expressed as 2-heavy-2-light (2H2L) BiAbs. Light chain of a biologically active F.IX arm was chosen to generate CLC transgenic mice. CLC transgenic mice were immunized with F.X to identify F.X heavy chains pairing with CLC. The isolated F.X heavy chains were co-expressed with the heavy and light chains of the selected F.IX arm as CLC BiAbs and re-screened by functional assays. Biologically active BiAbs were further optimized to generate a lead BiAb, KY1049. KY1049 heterodimer was purified by a two-step method using Protein A and cation ion exchange chromatography (cIEX). The purified BiAb was analyzed by analytical HPLC and mass spectrometry (MS). The purified BiAb was also characterized using a combination of in vitro and ex vivo hemostatic assays including chromogenic FXase (FXase), activated partial thromboplastin time (aPTT) and thrombin generation assay (TGA). Binding of KY1049 to F.IX and F.X was studied using surface plasmon resonance (SPR).

Results:

More than 8,000 2H2L BiAbs were first screened by high-throughput chromogenic FXase assay. Functionally active 2H2L BiAbs were identified, consisting of one F.IX arm and different F.X arms. The light chain of this promiscuous F.IX arm was chosen to generate CLC transgenic mice expressing the light chain exclusively. More than 400 F.X heavy chains isolated from the CLC transgenic mice were screened to identify functionally active CLC BiAbs. Further work to iteratively and combinatorially optimize the variable domains resulted in KY1049, a lead BiAb which demonstrates robust hemostatic activity. KY1049 demonstrates a dose-dependent reduction in clotting time and a dose-dependent increase in thrombin burst, thereby functionally restoring the hemostatic activity of F.VIII-depleted plasma. Compared with a sequence identical analog (SIA) of Hemlibra®, KY1049 shows comparable efficacy in a range of concentration of IgG (0.1 to 300 nM). Importantly, KY1049 can be purified using a routine purification process. The purity and the identity of KY1049 was confirmed by mass spectrometry. KY1049 was shown to simultaneously bind F.IX and F.X using SPR in confirmation of its mode of action.

Summary/conclusions:

KY1049, developed using Kymab's IntelliSelect® Bispecific platform, is a potent F.VIII mimetic bispecific antibody with activities comparable to a SIA of Hemlibra®. This fully human CLC bispecific antibody can potentially provide a new treatment option for hemophilia A patients.

Disclosures

Wang: Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Blackwood:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Magliozzi:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Moraes:Kymab Ltd.: Employment, Equity Ownership. Hollins:Kymab Ltd.: Employment, Equity Ownership. Sinopoli:Kymab Ltd.: Employment, Equity Ownership. Chi:Kymab Ltd.: Employment, Equity Ownership. Mitchell:Kymab Ltd.: Employment, Equity Ownership. Sellick:Kymab Ltd.: Employment, Equity Ownership. Pearce:Kymab Ltd.: Employment, Equity Ownership. Theurl:Kymab Ltd.: Consultancy, Equity Ownership; Sierra Oncoloy: Research Funding. Germaschewski:Kymab Ltd.: Employment, Equity Ownership. Galson:Kymab Ltd.: Employment, Equity Ownership. Badiali:Kymab Ltd.: Employment, Equity Ownership. Dickson:Kymab Ltd.: Employment, Equity Ownership. Bradley:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties. Lee:Kymab Ltd.: Employment, Equity Ownership, Patents & Royalties.