Fc-erythropoietin fused protein with improved pharmacokinetics

FIELD: medicine.

SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.

EFFECT: improved pharmacokinetic properties of erythropoietin.

23 cl, 14 ex, 6 tbl, 11 dwg

 

The text descriptions are given in facsimile form.

1. Highly purified sililirovany dimeric protein, which essentially consists of a dimeric Fc portion containing the CH2 and CH3 domain of human IgG2, hinge area human IgG, and human erythropoietin (EPO), where each chain dimeric Fc part is linked through its C-terminal end directly or through a link is REGO peptide with N-terminal end of the molecule of EPO, this protein has the following properties:
(i) the EPO molecule comprises residues 15-28 sialic acid;
(ii) plot Gln-Phe-Asn-Ser sequence replaced by Gln-Ala-Gln-Ser within the CH2 domain of the specified IgG2, and
(iii) amino acid sequence plot Leu-Ser-Leu-Ser near the C-terminal end of the CH3 domain of the specified IgG2 replaced by Ala-Thr-Ala-Thr.

2. Dimeric Fc-EPO protein according to claim 1, which further C-terminal Lys residue CH3 domain is replaced by Ala.

3. Dimeric Fc-EPO protein according to claim 1, in which the hinged section is derived from human IgG1.

4. Dimeric Fc-EPO protein according to claim 3, wherein said hinge section IgG1 is modified by replacing the amino acid Cys residue within the sequence Pro-Lys-Ser-Cys-Asp-Lys articulated plot the Ser residue, forming, thus, the sequence Pro-Lys-Ser-Ser-Asp-Lys within the hinge area.

5. Dimeric Fc-EPO protein according to claim 1, in which eritropoyetina part includes at least one of the following amino acid substitutions:
(i) a residue other than cysteine, at position 29 of the molecule of EPO,
(ii) a residue other than cysteine, at position 33 of the molecule of EPO,
(iii) a cysteine residue at position 88 of the molecule EPO and
(iv) the cysteine residue at position 139 of the EPO molecule.

6. Dimeric Fc-EPO protein according to claim 5, in which amino acid residue, Otley is hydrated from Cys, is in position 33 of the EPO molecule instead of the original residue Cys, and Cys residue is located at position 88 of the EPO molecule instead of the original Trp residue, allowing, thus, EPO part within the fused protein to form Cys29-Cys88disulfide bonds.

7. Dimeric Fc-EPO protein according to claim 6, in which amino acid residue other than Cys, located at position 33 is a Pro.

8. Dimeric Fc-EPO protein according to claim 1, in which the EPO portion includes one or more mutations selected from the group:
(i) Arg131→Glu131
(ii) Arg139→Glu139
(iii) His32→Gly32
(iv) Ser34→Arg34
(v) Pro90→Ala90.

9. Dimeric Fc-EPO protein of claim 1, wherein the linker peptide comprises a glycosylation site.

10. Dimeric Fc-EPO protein according to claim 9, in which the glycosylation site comprises the amino acid sequence Asn-Ala-Thr.

11. Dimeric Fc-EPO protein according to claim 1, in which the full IgG molecule, including a hinged section, derived from IgG2.

12. Dimeric Fc-EPO protein according to claim 1 which further includes CN domain.

13. Dimeric Fc-EPO protein according to claim 1, in which the protein contains 20-22 residue sialic acid.

14. Dimeric Fc-EPO protein according to claim 1, comprising the sequence:
EPKSSDKTHTCPPCPAPPVAGPSVFLPPPKPKDTLMISRTPEVTCVWDVSHEDPE
VQFNWYVDGVEVHNAKTKPREEQAQSTPRWSVltwhqdwlngkeykckvsnbr/> KGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPE3STNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSATATPGAAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENI
TVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPL
QLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYS
NFLRGKLKLYTGEACRTGDR (SEQ ID NO: 14).

15. Dimeric Fc-EPO protein according to claim 1, comprising the sequence:
EPKSSDKTHTCPPCPAPPVAGPSVFLPPPKPKDTLMISRTPEVTCVWDVSHEDPE
VQFNWYVDGVEVHNAKTKPREEQAQSTFRWSVLTWHQDWLNGKEYKCKVSN
KGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSATATPGAAPPRLICDSRVLERYLLEAKEAENITTGCAEGPSLNENITV
PDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLWSSQPCEALQL
HVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNF
LRGKLKLYTGEACRTGDR (SEQ ID NO: 15).

16. A DNA molecule encoding a protein according to claim 1.

17. Pharmaceutical composition suitable for the treatment of disorders hematopoiesis or hematopoietic failure in a mammal, comprising an effective amount of Fc-EPO fused protein described in claim 1, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.

18. The population of highly purified sililirovany Fc-EPO fused protein according to claim 1, which is acceptable for administration to a mammal, which is obtained by introducing a DNA molecule encoding the corresponding Fc-EPO protein in the cell KSS, expression, isolation and purification of a population of the corresponding Fc-EPO fused proteins, where this population has a longer half-life in serum compared with the population of the corresponding Fc-EPO if the s protein, synthesized in cells, NS/0, PerC6, or 293.

19. The population of purified Fc-EPO fused proteins on p where a specified population of fused proteins contains an average of 20-22 residues of sialic acid in purified Fc-EPO protein.

20. The population of purified Fc-EPO fused proteins on p or 19, where KSS cell is adapted for growth in an environment that does not contain protein, or in suspension.

21. A method of obtaining a population of highly sililirovany purified recombinant Fc-EPO fused protein according to claim 1, whereby the method includes the following steps:
(i) constructing a DNA molecule that encodes the corresponding Fc-EPO protein;
(ii) the transformation of the KSS cells using the selected DNA molecules in an environment that does not contain protein, or in suspension;
(iii) the expression of populations Fc-fused proteins encoded by the specified DNA molecule,
(iv) the collection, isolation and purification of a specified population of Fc-EPO fused proteins.

22. The method according to item 21, in which the specified synthesized population of fused proteins contains an average of 15 to 28 residues of sialic acid in purified Fc-EPO protein.

23. The method according to item 21, in which the specified synthesized population of fused proteins contains an average of 20-22 residues of sialic acid in purified Fc-EPO protein.



 

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FIELD: medicine.

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37 cl, 6 ex, 1 dwg

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The invention relates to the field of biotechnology and can be used to obtain a fused protein with increased eritropoetinovmi activity

The invention relates to a conjugate of a glycoprotein erythropoietin, which has at least one free amino group and has biological activity in vivo, leading to increase production of reticulocytes and red blood cells by the bone marrow cells, and which are selected from the group comprising human erythropoietin and its analogues, which have the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site

The invention relates to a fused protein with in vivo increased activity of erythropoietin

The invention relates to biotechnology and is used for the preparation of recombinant human erythropoietin (EPO)
The invention relates to a combined pharmaceutical preparations containing drugs erythropoietin and iron supplements

The invention relates to biotechnology and related analogue of erythropoietin

The invention relates to biotechnology, in particular the production of hybridomas

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EFFECT: high stability of new crystalline forms.

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