Active variants of FGF with improved specificity

• US 20040014658A1
• Filed: 04/29/2003
• Published: 01/22/2004
• Est. Priority Date: 10/31/2000
• Status: Active Grant

• Current Assignee: ProChon Biotech Ltd. (Ocugen, Inc.)
• Original Assignee: ProChon Biotech Ltd. (Ocugen, Inc.)
• Inventors: Adar, Rivka, Bogin, Oren, Yayon, Avner
• Granted Patent: US 7,288,406 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/50 Fibroblast growth factor [FGF]

Abstract

The present invention provides active fibroblast growth factor variants demonstrating enhanced receptor subtype specificity. The preferred novel variants retain binding to FGF Receptor Type 3 (FGFR3) triggering intracellular downstream mechanisms leading to activation of a biological response. Methods of utilizing preferred FGF mutants in preparation of medicaments for the treatment of malignancies and skeletal disorders including osteoporosis and enhancing fracture healing and wound healing processes are provided.

Claims

1. An active variant of a fibroblast growth factor having at least one mutation in the beta 8 beta 9 loop, wherein said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

2. The active FGF variant of claim 1 wherein at least one substitution is replacement of tryptophan at position 14 of FGF-9, tryptophan143 of FGF-16, or tryptophanl 47 of FGF-20.

3. The active FGF variant of claim 1 wherein FGF is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-17, FGF-18, FGF-19, FGF-21, FGF-22, FGF-23 or FGF-CX;

and further wherein at least one substitution is;

replacement of the histidine 108 of FGF-1;

replacement of the asparagine 111 of FGF-2;

replacement of the glycine 125 of FGF-3;

replacement of the asparagine 165 of FGF-4;

replacement of the serine 170 of FGF-5;

replacement of the asparagine 167 of FGF-6;

replacement of the histidine 147 of FGF-7;

replacement of the asparagine 155 of FGF-8;

replacement of the glycine 160 of FGF-10;

replacement of the tyrosine 153 of FGF-11;

replacement of the tyrosine 155 of FGF-12;

replacement of the tyrosine 151 of FGF-13;

replacement of the tyrosine 153 of FGF-14;

replacement of the glycine 137 of FGF-15;

replacement of the asparagine 137 of FGF-17;

replacement of the asparagine 137 of FGF-18;

replacement of the glycine 130 of FGF-19;

replacement of the glycine 131 of FGF-21;

replacement of the glycine 123 of FGF-22;

replacement of the glycine 123 of FGF-23;

replacement of the tryptophan 147 of FGF-CX.

4. The active FGF variant of claim 1 wherein the FGF is FGF-9.

5. The active FGF-9 variant of claim 4 wherein the FGF-9 is selected from the group consisting of a human, mouse and chicken.

6. The active FGF-9 variant of claim 4 having SEQ ID NO:

1 wherein at least one substitution is replacement of tryptophan at position 144.

7. The active FGF-9 variant of claim 6 wherein the tryptophan at position 144 is replaced by an amino acid selected from the group consisting of glycine, arginine, valine and and glutamate.

8. The active FGF-9 variant of claim 4 having SEQ ID NO:

3 wherein at least one substitution is replacement of asparagine at position 143.

9. The active FGF-9 variant of claim 8 wherein the asparagine at position 143 is replaced by serine.

10. The active FGF variant of claim 1 wherein FGF is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23 or FGF-CX;

wherein at least one substitution is;

replacement of the asparagine 107 of FGF-1;

replacement of the asparagine 110 of FGF-2;

replacement of the leucine 124 of FGF-3;

replacement of the asparagine 164 of FGF-4;

replacement of the asparagine 169 of FGF-5;

replacement of the asparagine 166 of FGF-6;

replacement of the asparagine 146 of FGF-7;

replacement of the asparagine 154 of FGF-8;

replacement of the asparagine 159 of FGF-10;

replacement of the asparagine 152 of FGF-11;

replacement of the asparagine 154 of FGF-12;

replacement of the asparagine 150 of FGF-13;

replacement of the asparagine 152 of FGF-14;

replacement of the leucine 136 of FGF-15;

replacement of the asparagine 142 of FGF-16;

replacement of the asparagine 136 of FGF-17;

replacement of the asparagine 136 of FGF-18;

replacement of the aspartate 129 of FGF-19;

replacement of the asparagine 146 of FGF-20;

replacement of the aspartate 130 of FGF-21;

replacement of the asparagine 122 of FGF-22;

replacement of the asparagine 122 of FGF-23;

replacement of the asparagine 146 of FGF-CX.

11. The active FGF variant of claim 1 having fewer than 30 amino acid residues at the N-terminus or fewer than 15 residues at the C terminus extending beyond the core structure.

12. The active FGF variant of claims 11 wherein the FGF is FGF-9.

13. The active FGF-9 variant of claim 12 having SEQ ID NO:

2.

14. The active FGF-9 variant of claim 13 wherein the tryptophan at position 144 is replaced by an amino acid selected from the group consisting of arginine, glycine, valine and glutamate.

15. The active FGF-9 variant of claim 12 having SEQ ID NO:

4.

16. The active FGF-9 variant of claim 15 wherein the asparagine at position 143 is replaced by a serine.

17. The active FGF variant of claim 3 wherein the FGF variant has fewer than 30 amino acid residues at the N-terminus or fewer than 15 residues at the C terminus extending beyond the core structure.

18. An active variant of a fibroblast growth factor having fewer than 30 amino acid residues at the N-terminal or fewer than 15 residues at the C terminus extending beyond the core structure of said FGF, wherein said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

19. The active FGF variant of claim 18 wherein the FGF is FGF-9.

20. The active FGF-9 variant of claim 19 having SEQ ID NO:

5.

21. The active FGF-9 variant of claim 18 wherein said variant is selected from the polypeptides having SEQ ID NO:

6 or SEQ ID NO;

7.

22. The active FGF-9 variant of claim 19 wherein said variant includes heterologous sequences at the N-terminus or C-terminus.

23. The active FGF-9 variant of claim 22 having SEQ ID NO:

8.

24. A polynucleotide sequence encoding an active variant of a fibroblast growth factor having at least one mutation in the beta 8 beta 9 loop, wherein said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

25. The polynucleotide sequence of claim 24 wherein the FGF is FGF-9.

26. The polynucleotide sequence of claim 25 wherein the sequence is selected from the polypeptides having SEQ ID NO:

14 or SEQ ID NO;

16

27. The polynucleotide sequence of claim 24 having fewer than 30 amino acid residues at the N-terminus or fewer than 15 residues at the C terminus extending beyond the core structure.

28. The polynucleotide sequence of claim 27 wherein the sequence is selected from the polypeptides having SEQ ID NO:

15 or SEQ ID NO;

17.

29. A polynucleotide sequence encoding an active variant of a fibroblast growth factor having fewer than 30 amino acid residues at the N-terminal or fewer than 15 residues at the C terminus extending beyond the core structure of said FGF, said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

30. The polynucleotide sequence of claim 29 wherein the sequence is selected from the group consisting of polynucleotides having SEQ ID NO:

18, polynucleotides having SEQ ID NO;

19, polynucleotides having SEQ ID NO;

20, and polynucleotides having SEQ ID NO;

21.

31. A vector comprising a polynucleotide sequence encoding an active variant of a fibroblast growth factor having at least one mutation in the beta 8 beta 9 loop, said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

32. A vector comprising a polynucleotide sequence encoding an active variant of a fibroblast growth factor having fewer than 30 amino acid residues at the N-terminal or fewer than 15 residues at the C terminus extending beyond the core structure of said FGF, said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype.

33. A pharmaceutical composition comprising as an active ingredient a variant of a fibroblast growth factor having at least one substitution in the beta 8 beta 9 loop of an FGF, wherein said substitution producing variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype, and a pharmaceutically acceptable diluent or carrier.

34. A pharmaceutical composition comprising as an active ingredient an active variant of a fibroblast growth factor having fewer than 30 amino acid residues at the N-terminal or fewer than 15 residues at the C terminal extending beyond the core structure of said FGF, and wherein said FGF variant having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF, by decreasing the biological activity mediated by at least one receptor subtype while retaining the activity mediated through another receptor subtype, and a pharmaceutically acceptable diluent or carrier.

35. A method for the treatment of an individual, the method comprising the step of administering an active FGF variant of a fibroblast growth factor having at least one substitution in the beta 9 loop of an FGF, wherein the substitution produces enhanced specificity for one receptor subtype compared to a corresponding wild type FGF, by decreasing the biological activity mediated through another receptor subtype.

36. The method of claim 35, wherein the treatment is for a disease or disorder related to fibroblast growth factor receptors.

37. The method of claim 35, wherein the treatment is for a skeletal disorder, wound healing, cancer or bone growth.

38. The method of claim 35, further comprisng the step of formulating a pharmaceutical composition for parenteral administration via intra-articular, intravenous, intramuscular, subcutaneous, intradermal, or intrathecal routes.

39. The method 35, wherein the method further comprises the step of administering a pharmaceutical compositoin comprising the FGF variant.

40. The method of claim 39, wherein the pharmaceutical composition is formulated by combining the active FGF variant with a pharmaceutically acceptable carrier;

and administering an appropriate form of the composition to the individual via intra-articular, intravenous, intramuscular, subcutaneous, intradermal, or intrathecal routes.