Patent EA-004317-B1 (EA)
Title: Production of recombinant blood clotting factors in human cell lines
Inventor: HAUSER CHARLOTTE
IPC: C12N5/08
Filed: 2001-03-21
Abstract: 1. A factor VIII mutein in which the B-domain between positions Arg740 and GIu1649 has been replaced by an Arg-rich linker peptide having at least 3 Arg residues and comprising 10 to 25 amino acid residues; wherein said factor VIII numbering is relative to the mature wild-type factor VIII sequence shown in SEQ ID NO:2. 2. The factor VIII mutein according to claim 1, wherein having at least one of the following mutations (a), (b) and (c): (a) Val at position 162 has been replaced by a neutral amino acid residue selected from Gly, Ala, Leu, Ile, Met and Pro; (b) Ser at position 2011 has been replaced by a hydrophilic amino acid residue selected from Asn, Thr and GIn; (c) Val at position 2223 has been replaced by an acidic amino acid residue selected from Glu and Asp. 3. The factor VIII mutein according to claim 2, wherein the factor VIII mutein has at least one of the mutations (a) and (b). 4. The factor VIII mutein according to 2, wherein the factor VIII mutein has all three mutations (a), (b) and (c). 5. The factor VIII mutein according to any one of claims 2 to 4, wherein in mutation (a) Val at position 162 has been replaced by Ala, in mutation (b) Ser at position 2011 has been replaced by Asn, and/or in mutation (c) Val at position 2223 has been replaced by Glu. 6. The factor VIII mutein according to any one of claims 1 to 5, wherein the Arg-rich linkerpeptide has 14 to 20 amino acid residues. 7. The factor VIII mutein according to any one of claims 1 to 6, wherein the linker comprises: the amino acid sequence SFSQNSRH, and/or the amino acid sequence QAYRYRRG. 8. The factor VIII mutein according to claim 7, wherein the linker comprises the amino acid sequence SFSQNSRHQAYRYRRG. 9. The factor VIII mutein according to claim 1, which comprises amino acids 1-1440 SEQ ID NO:4, 13 or 15. 10. A DNA sequence coding for the factor VIII mutein as defined in any one of claims 1 to 9. 11. The DNA sequence of claim 10, which has at least one of the mutations T485C, G6032A and T6668A relative to the DNA sequence of the mature wild-type factor VIII shown in SEQ ID NO:1, preferably the DNA sequence comprises all three of said mutations. 12. A vector comprising the DNA as defined in claim 10 or 11. 13. The vector according to 12 which is pTGFB-1, pTGTB-2hyg-s or pTGFB-3 as shown in SEQ ID NO:3, 12 and 14, respectively. 14. The vector according to 12 which is a gene transfer vector. 15. A host cell being transformed with a vector as defined in claim 12 and/or comprising a DNA sequence as defined in claim 10. 16. A method for the production of the factor VIII mutein of claim 1 which comprises: (a) culturing a transformed host cell as defined in claim 15 and (b) isolating the factor VIII mutein from the culture broth. 17. A pharmaceutical composition comprising the factor VIII mutein as defined in any one of claims 1 to 9 or a gene transfer vector as defined in claim 14. 18. Use of the factor VIII mutein as defined in any one of claims 1 to 9 or a gene transfer vector as defined in claim 14 for preparing a medicament for treating hemophilia, preferably for treating hemophilia A. 19. A method according to claim 16, comprising: (a) culturing an immortalized human cell line stably expressing Simian virus T antigen and carrying a vector having a promoter functionally linked to a DNA sequence coding for the (a) culturing an immortalized human cell line stably expressing Simian virus T antigen and carrying a vector having a promoter functionally linked to a DNA sequence coding for the factor VIII mutein. 20. The method according to claim 19, wherein the immortalized human cell line is an immortalized kidney, bladder, liver, lung, cardiac muscle, smooth muscle, ovary or gastrointestinal cell. 21. The method according to claim 20, wherein the immortalized human cell line is derived from an embryonic human kidney cell and preferably is cell line 293T (DSM ACC2494). 22. The method according to any one of claims 19 to 21, wherein the vector further comprises a selection marker and/or regulatory sequences. 23. The method according to any one of claims 19 to 22, wherein the culturing is performed in the presence of von Willebrand Factor (vWF). 24. The method according to any one of claims 16 and 19 to 23 which further comprises: (c) purifying the blood clotting factor isolated in step (b), and/or (d) subjecting the blood clotting factor isolated in step (b) or purified in step (c) to a virus inactivation treatment. 25. An immortalized human cell line stably expressing at least one viral transcription activator protein and carrying a vector coding for factor VIII mutein as defined in any one of claims 19 to 21.