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Publication Number
US-11685771-B2
Publication Date
2023-06-27
Expiration Date
Abstract
Provided are recombinant Factor VIII proteins, e.g., human Factor VIII proteins with heterologous moieties inserted into flexible permissive loops located in the Factor VIII A domains, while retaining the procoagulant activity of Factor VIII.
Core Innovation
The disclosure provides an isolated nucleic acid encoding a recombinant Factor VIII (FVIII) protein with a specified domain architecture comprising a first polypeptide and a second polypeptide that are fused or exist as a heterodimer. Two half-life-extending moieties are inserted immediately downstream of selected amino acids within surface-exposed permissive loops or acidic spacer regions. The encoded recombinant FVIII protein retains procoagulant activity.
The half-life-extending moieties are selected from albumin, albumin-binding polypeptides, Fc, CTP, PAS, PEG, HES, clearance-receptor components, and XTEN. Candidate insertion sites tolerate these moieties while procoagulant activity and pharmacokinetic persistence vary by insertion position and insert composition. Selected constructs exhibit extended half-life in mice and increased expression, providing utility for bleeding disorders.
Claims Coverage
The claims define one independent claim directed to an isolated nucleic acid encoding a recombinant FVIII protein, comprising four inventive features.
Recombinant FVIII domain architecture
The encoded recombinant FVIII protein comprises a first polypeptide of Formula I and a second polypeptide of Formula II, with defined FVIII domains and acidic spacer regions, wherein the polypeptides are fused or exist as a heterodimer.
Two half-life-extending moieties at selected insertion locations
First and second half-life-extending moieties are inserted within the recombinant FVIII protein immediately downstream of selected amino acids or amino acid pairs corresponding to mature native human FVIII.
Half-life-extending moiety compositions
The half-life-extending moieties are independently selected from albumin, albumin-binding polypeptides, Fc, PAS, the C-terminal peptide of the beta subunit of human chorionic gonadotropin, PEG, HES, albumin-binding small molecules, clearance receptors or fragments including LRP1, and XTEN.
Procoagulant activity
The encoded recombinant FVIII protein exhibits procoagulant activity.
The independent claim combines a specified recombinant FVIII domain architecture with two half-life-extending moieties inserted at selected locations while requiring procoagulant activity.
Stated Advantages
Selected XTEN insertion constructs retain FVIII procoagulant activity, exhibit slower loss of activity over time compared to native FVIII, and demonstrate extended half-life in mice.
An a3-region insertion is associated with increased measured FVIII expression and detectable activity.
FVIII tolerates insertion of the described moieties at selected permissive loops and the a3 region, with activity varying based on insertion site and insert composition.
PEG conjugation at an inserted cysteine preserves specific activity, reported as 101% after conjugation compared with 100% before conjugation.
Multi-XTEN FVIII variants retain activity and exhibit extended persistence, with three XTEN insertions extending half-life to 16 hours in mice lacking VWF, reported as 64-fold over BDD-FVIII.
Documented Applications
Therapeutic use of recombinant FVIII variants for bleeding disorders, including hemophilia A treatment.
Vectors and host cells for producing recombinant FVIII variants, and methods for their production.
Visualization or localization of recombinant FVIII.
Evaluation of multi-XTEN FVIII variants for half-life extension in mice lacking VWF.
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