Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides
Inventors
HERSHKOVITZ, Oren • BAR-ILAN, Ahuva
Assignees
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Publication Number
US-9808534-B2
Publication Date
2017-11-07
Expiration Date
Abstract
This invention is directed to the use of a chorionic gonadotrophin carboxy terminal peptide (CTP) or fragments thereof for modifying a polypeptide or a fragment thereof in order to increase the hydrodynamic volume of the polypeptide or fragment thereof.
Core Innovation
The invention relates to recombinant coagulation factor polypeptides comprising chorionic gonadotrophin carboxy terminal peptide (CTP) units. The engineered recombinant constructs include three CTP units fused to the carboxy terminus of coagulation factor IX (FIX) or coagulation factor VIIa (FVIIa), with no CTPs attached to the N-terminus.
The recombinant CTP-modified polypeptides are expressed in Chinese hamster ovary (CHO) host cells, where expression comprises glycosylating the CTP units. The glycosylation comprises O-glycosylation, and the glycosylated CTP units incrementally increase hydrodynamic size.
For FIX and FVIIa embodiments, the glycosylated CTP units incrementally increase hydrodynamic size by about 48–53 kDa per glycosylated CTP unit for FIX and about 43–50 kDa per glycosylated CTP unit for FVIIa.
Claims Coverage
The independent claim coverage includes three inventive features and two alternative paths for FIX or FVIIa. The claims focus on carboxy-terminal CTP fusion, CHO expression with O-glycosylation of the CTP units, and defined hydrodynamic size increments per glycosylated CTP unit.
C-terminus CTP fusion without N-terminal CTPs for FIX or FVIIa
Fusing three chorionic gonadotrophin carboxy terminal peptide (CTP) units to the carboxy terminus of coagulation factor IX (FIX) or coagulation factor VIIa (FVIIa), with no CTPs attached to the N-terminus.
CHO expression to generate O-glycosylated CTP units
Expressing the recombinant CTP-modified polypeptides in a Chinese hamster ovary (CHO) host cell, wherein expression comprises glycosylating the CTP units and the glycosylation comprises O-glycosylation.
Hydrodynamic size increment per glycosylated CTP unit for FIX and FVIIa
The glycosylated CTP units incrementally increase the hydrodynamic size of the FIX by about 48–53 kDa per glycosylated CTP unit, and the hydrodynamic size of the FVIIa by about 43–50 kDa per glycosylated CTP unit.
The claims require carboxy-terminal fusion of three CTP units without N-terminal CTPs, CHO-cell expression that produces O-glycosylated CTP units, and hydrodynamic size increments specified for FIX and FVIIa per glycosylated CTP unit.
Stated Advantages
Incrementally increasing the hydrodynamic size of the recombinant polypeptide of interest.
Increased hydrodynamic volume is linked to improved serum half-life, bioavailability, potency, and reduced dosing frequency.
Increasing the hydrodynamic size increases bioavailability of the polypeptide.
Documented Applications
Methods of incrementally increasing the hydrodynamic size of recombinant coagulation factor IX (FIX) and recombinant coagulation factor VIIa (FVIIa) polypeptides.
Increasing bioavailability associated with increased hydrodynamic size of the recombinant polypeptide.
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