Modified coagulation factors with prolonged in vivo half-life

Abstract

The present invention relates to nucleic acid sequences coding for modified coagulation factors, preferably coagulation factor VIII, and their derivatives; recombinant expression vectors containing such nucleic acid sequences; host cells transformed with such recombinant expression vectors; and recombinant polypeptides and derivatives coded for by said nucleic acid sequences, whereby said recombinant polypeptides and derivatives have biological activities and prolonged in vivo half-lives compared to the unmodified wild-type proteins. The invention also relates to corresponding sequences that result in improved in vitro stability. The present invention further relates to processes for the manufacture of such recombinant proteins and their derivatives. The invention also relates to a transfer vector for use in human gene therapy, which comprises such nucleic acid sequences.

1 - 32 . (canceled) 
     
     
         33 . A polynucleotide or a group of polynucleotides encoding a modified factor VIII (FVIII) polypeptide, comprising a FVIII polypeptide having an N-terminal amino acid and a C-terminal amino acid, and a half-life enhancing polypeptide (HLEP) inserted within the B-domain between the N-terminal amino acid and the C-terminal amino acid of the FVIII polypeptide,
 wherein the FVIII polypeptide is capable of being cleaved from the HLEP moiety during activation in vivo, wherein the modified FVIII polypeptide exhibits a prolonged half-life prior to activation during a bleeding event and a half-life substantially the same as that of an unmodified FVIII peptide following activation, and   wherein the HLEP comprises albumin or an immunoglobulin constant region polypeptide.   
     
     
         34 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the modified FVIII polypeptide has a prolonged functional or antigenic half-life as compared to a FVIII polypeptide lacking an inserted HLEP. 
     
     
         35 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the modified FVIII polypeptide has an improved in vivo recovery as compared to the FVIII polypeptide lacking an inserted HLEP. 
     
     
         36 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the modified FVIII polypeptide has increased stability in serum-free culture media and/or in animal protein-free culture media as compared to the FVIII polypeptide lacking an inserted HLEP. 
     
     
         37 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the B-domain of FVIII or a part thereof is replaced with the HLEP. 
     
     
         38 . The polynucleotide or group of polynucleotides according to  claim 37 , wherein more than 75% of the B-domain is deleted, or more than 75% of the B-domain is replaced by linker sequences. 
     
     
         39 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the modified FVIII polypeptide has at least 10% of the biological activity of the FVIII polypeptide lacking an inserted HLEP. 
     
     
         40 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the half-life enhancing polypeptide is albumin. 
     
     
         41 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the B-domain of FVIII has been replaced partly or completely with human albumin. 
     
     
         42 . The polynucleotide or group of polynucleotides according to  claim 33 , wherein the immunoglobulin constant region polypeptide is an immunoglobulin G Fc domain. 
     
     
         43 . A plasmid or vector comprising the polynucleotide according to  claim 33 , or a group of plasmids or vectors comprising the group of polynucleotides according to  claim 33 . 
     
     
         44 . The plasmid or vector, or the group of plasmids or vectors, according to  claim 43 , wherein the plasmid(s) or vector(s) are expression vector(s). 
     
     
         45 . The vector, or the group of vectors, according to  claim 43 , wherein the vector(s) are transfer vector(s) for use in human gene therapy. 
     
     
         46 . A host cell comprising the polynucleotide or group of polynucleotides according to  claim 33 . 
     
     
         47 . A method of producing a modified FVIII polypeptide, comprising culturing the host cell according to  claim 46  under conditions such that the modified FVIII polypeptide is expressed.