Fibroblast growth factor receptor extracellular domain acidic region muteins

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

 

The text descriptions are given in facsimile form.

1. Polypeptide, essentially corresponding to mutein acidic areas of the extracellular domain (ECD) of the receptor FGFR4, for the treatment of a disease associated with one or more ligands of the FGFR family, where mutein acidic areas FGFR4 ECD selected from chimeras acid is ons FGFR4 ECD options and long acid box FGFR4, and where mutein acidic areas FGFR4 ECD has a greater number of acidic residues in the linker area D1-D2 than FGFR4 ECD wild type, and where mutein acidic areas FGFR4 ECD optionally contains at least one point mutation that inhibits glycosylation.

2. The polypeptide according to claim 1, where the acid Chimera zone FGFR4 ECD selected from the linker chimeras D1-D2 domain of FGFR4 ECD, chimeras exon 4 FGFR4 ECD, chimeras acid Boxing FGFR4 ECD, chimeras long acid box FGFR4 ECD and chimeras short acid box FGFR4 ECD.

3. The polypeptide according to claim 2, where:
a) Chimera linker D1-D2 domain of FGFR4 ECD contains D1-D2 linker selected from the D1-D2 linker FGFR1, D1-D2 linker FGFR2 and D1-D2 linker FGFR3, instead of the D1-D2 linker FGFR4;
b) the Chimera of exon 4 FGFR4 ECD contains exon 4, selected from exon 4 FGFR1, exon 4 FGFR2 and exon 4 FGFR3, instead of exon 4 FGFR4;
c) Chimera acid Boxing FGFR4 ECD contains:
i) acid box selected from acid Boxing FGFR1 acid box FGFR2 acid and Boxing FGFR3, instead of acid Boxing FGFR4;
ii) the area of acid box selected from areas of acid Boxing FGFR1, zones of acid Boxing FGFR2 and areas of acid Boxing FGFR3, instead of acid Boxing FGFR4;
iii) the area of acid box selected from areas of acid Boxing FGFR1, zones of acid Boxing FGFR2 and areas of acid Boxing FGFR3, instead of areas of acid Boxing FGFR4;
iv) acid box selected from acid Boxing FGFR1 acid box is FGFR2 acid and Boxing FGFR3, instead of a zone of acid Boxing FGFR4;
d) Chimera long acid box FGFR4 ECD contains a long acid box selected from the long acid box FGFR1, long acid box FGFR2 and long acid box FGFR3, instead of the long acid box FGFR4; and
e) Chimera short acid box FGFR4 ECD contains a short acid box selected from the short acid box FGFR1, short acid box FGFR2 and short acid box FGFR3, instead of the short acid box FGFR4.

4. The polypeptide according to claim 3, where:
a) Chimera linker D1-D2 domain of FGFR4 ECD contains:
i) an amino acid sequence selected from SEQ ID NO:22, 26, 28 and 32, instead of the D1-D2 linker FGFR4 selected from SEQ ID nos:16 and 17; or
ii) an amino acid sequence selected from SEQ ID NO: 35 to 38;
b) the Chimera of exon 4 FGFR4 ECD contains:
i) an amino acid sequence selected from SEQ ID NO:23, 92, 29 and 33, instead of exon 4 FGFR4 selected from SEQ ID nos:18 and 19; or
ii) an amino acid sequence selected from SEQ ID NO: 39 to 42;
c) Chimera acid Boxing FGFR4 ECD contains:
i) an amino acid sequence selected from SEQ ID NO:24, 30 and 34 instead of acid Boxing FGFR4 having the amino acid sequence of SEQ ID NO:20;
ii) an amino acid sequence selected from SEQ ID NO: 56 to 65, instead of acid Boxing FGFR4 having the amino acid sequence of SEQ ID NO:20; or
iii) consequently is any amino acid, selected from SEQ ID NO: 56 to 65, instead of the zone of acid Boxing FGFR4 having an amino acid sequence selected from SEQ ID NO: 46 to 55;
iv) an amino acid sequence selected from SEQ ID NO:24, 30 and 34 instead of the zone of acid Boxing FGFR4 having an amino acid sequence selected from SEQ ID NO: 46 to 55; or
v) an amino acid sequence selected from SEQ ID NO: 43 to 45 and 157;
d) Chimera long acid box FGFR4 ECD contains:
i) an amino acid sequence selected from SEQ ID NO: 98 to 100, instead of the long acid box FGFR4 selected from SEQ ID NO:96 and 97; or
ii) an amino acid sequence selected from SEQ ID NO: 105 to 107; and
e) Chimera short acid box FGFR4 ECD contains:
i) an amino acid sequence selected from SEQ ID NO: 102 to 104, instead of the short acid box FGFR4 having the amino acid sequence of SEQ ID NO:101; or
ii) an amino acid sequence selected from SEQ ID nos: 108 to 110.

5. The polypeptide according to claim 1, where the option of long acid box FGFR4 ECD contains a variant FGFR4 ECD with a larger number of acidic amino acid residues in the long acid box compared to the long acid box of the wild type FGFR4.

6. The polypeptide according to claim 5, where:
a) at least two non-acidic residue in a long acid box FGFR4 ECD independently from each other substituted acid of astatke is, selected from Glu (E) and Asp (D);
b) at least three non-acidic residue in a long acid box FGFR4 ECD independently from each other substituted acid residue selected from Glu (E) and Asp (D); or
c) at least four non-acidic residue in a long acid box FGFR4 ECD independently from each other substituted acid residue selected from Glu (E) and Asp (D).

7. The polypeptide according to claim 5, where:
a) at least one acid residue is inserted between amino acids 103 and 104 of SEQ ID NO:1 and 2; or
d) two acid residue is inserted between amino acids 103 and 104 of SEQ ID NO:1 and 2.

8. The polypeptide according to claim 5, where the number of acidic residues in the long acid box FGFR4 is at least seven.

9. The polypeptide according to claim 5, where:
a) remains the domain of FGFR4 ECD from 104 to 114 (SEQ ID NO:145) substituted residues FGFR1 ECD from 106 to 117 (SEQ ID NO:149);
b) remains the domain of FGFR4 ECD from 104 to 114 (SEQ ID NO:145) substituted residues FGFR1 ECD from 107 to 117 (SEQ ID NO:150);
c) remains the domain of FGFR4 ECD from 104 to 110 (SEQ ID NO:146) substituted residues FGFR1 ECD from 105 to 113 (SEQ ID NO:151);
d) remains the domain of FGFR4 ECD from 113 to 116 (SEQ ID NO:147) substituted residues FGFR1 ECD 116-119 (SEQ ID NO:152);
e) remains the domain of FGFR4 ECD from 109 to 113 (SEQ ID NO:148) substituted residues FGFR1 ECD from 112 to 116 (SEQ ID NO: 153); or
f) a variant of the long acid box FGFR4 ECD contains an amino acid sequence selected from SEQ ID NO: 111 to 119.

10. The polypeptide according to claim 1, where mutein sour is Noah zone FGFR4 contains at least one point mutation, which inhibits glycosylation.

11. The polypeptide of claim 10, where at least one point mutation that inhibits glycosylation selected from the N91 AND N156A, N23 7A, N269A, N290A and N301A.

12. The polypeptide of claim 10, where mutein acidic areas FGFR4 contains an amino acid sequence selected from SEQ ID NO:120, 121 and 168.

13. Merged molecule FGFR4 ECD for the treatment of a disease associated with one or more ligands of the FGFR family, containing the polypeptide according to any one of claims 1 to 12, covalently linked with a merge partner.

14. Merged molecule FGFR4 ECD indicated in paragraph 13, in which the partner merge selected from an Fc, albumin, and polyethylene glycol.

15. Merged molecule FGFR4 ECD through 14, in which the merge partner is Fc.

16. Merged molecule FGFR4 ECD indicated in paragraph 15, where the fused molecule FGFR4 ECD contains an amino acid sequence selected from SEQ ID nos: 86 to 88, from 124 to 140, 143, 144 and 158.

17. Merged molecule FGFR4 ECD for the treatment of a disease associated with one or more ligands of the FGFR family, where the fused molecule FGFR4 ECD has the amino acid sequence of SEQ ID NO:86.

18. Polynucleotide that encodes a polypeptide for the treatment of a disease associated with one or more ligands of the FGFR family according to any one of claims 1 to 12 or fused molecule FGFR4 ECD according to any one of p-17.

19. Pharmaceutical composition for treating diseases associated the th with one or more ligands of the FGFR family, containing an effective amount of the polypeptide or fused molecules FGFR4 according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

20. A method of treating disorders of angiogenesis in a patient comprising the administration to a patient the pharmaceutical composition according to claim 19.

21. The method according to claim 20, where the violation of angiogenesis is selected from cancer and macular degeneration.

22. The method according to item 21, where the cancer is selected from colorectal cancer, liver, lung, breast and prostate.

23. Polypeptide, essentially corresponding to mutein acidic areas FGFR2 ECD for the treatment of a disease associated with one or more ligands of the FGFR family, where mutein acidic areas FGFR2 ECD has a greater number of acidic residues in the linker area D1-D2 than FGFR2 ECD wild-type.

24. The polypeptide according to item 23, where mutein acidic areas FGFR2 ECD is a Chimera short acid box FGFR2 ECD.

25. The polypeptide according to paragraph 24, where the Chimera short acid box FGFR2 ECD contains at least the short acid box instead of FGFR1 at least the short acid box FGFR2.

26. The polypeptide according to paragraph 24, where:
a) remains the domain of FGFR2 ECD from 111 to 118 (SEQ ID NO:155) substituted residues FGFR1 ECD from 105 to 112 (SEQ ID NO:154); or
b) Chimera short acid box FGFR2 ECD contains the amino acid sequence of SEQ ID NO:122.

27. Polypeptide, essentially corresponding to mutein acidic areas FGFR3 ECD to ensure C is the disease, associated with one or more ligands of the FGFR family, where mutein acidic areas FGFR3 ECD has a greater number of acidic residues in the linker area D1-D2 than FGFR3 ECD wild-type.

28. The polypeptide according to item 27, where mutein acidic areas FGFR3 ECD is a Chimera short acid box FGFR3 ECD.

29. The polypeptide according p where Chimera short acid box FGFR3 ECD contains at least the short acid box instead of FGFR1 at least the short acid box FGFR3.

30. The polypeptide according p where:
a) remains the domain of the FGFR3 ECD 110 to 117 (SEQ ID NO:156) substituted residues FGFR1 ECD from 105 to 112 (SEQ ID NO:154); or
b) Chimera short acid box FGFR3 ECD contains the amino acid sequence of SEQ ID NO:123.

31. The polypeptide according to claims 1, 23 or 27, where the disease is associated with one or more ligands of the FGFR family, is a cancer and/or violation of angiogenesis.

32. Merged molecule FGFR4 ECD according to item 13 or 17, where the disease is associated with one or more ligands of the FGFR family, is a cancer and/or violation of angiogenesis.



 

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33 cl, 18 dwg, 2 tbl, 4 ex

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