# Patent

## Title
FGF-18 truncated variants having increased receptor specificity and uses thereof

## Abstract

The present invention relates to fibroblast growth factor 18 (FGF-18) variants having various truncations beyond the signal peptide domain of the N-terminus, which activate FGFR3 with increased specificity. The invention further relates to polynucleotides encoding the variants, pharmaceutical compositions comprising same and methods for use thereof in treating cartilage and skeletal disorders.

## Background
FIELD OF THE INVENTION
The present invention relates to fibroblast growth factor 18 (FGF-18) variants, which activate FGFR3 with increased specificity. In particular, the invention relates to FGF-18 variants having various truncations beyond the signal peptide domain of the N-terminus, nucleic acids encoding the variants, pharmaceutical compositions comprising same and methods of use thereof.
BACKGROUND OF THE INVENTION
Fibroblast Growth Factors and their Receptors
Fibroblast growth factors (FGFs) comprise a large family of evolutionarily conserved polypeptides involved in a variety of biological processes including morphogenesis, angiogenesis, and tissue remodeling as well as in the pathogenesis of numerous diseases. The various members of this family stimulate the proliferation of a wide spectrum of cells, including those deriving from mesenchymal, endothelial, epithelial and neuroectodermal origin. FGFs are expressed in a strict temporal and spatial pattern during development and have important roles in patterning and limb formation (Ornitz, Bioessays, 2000, Vol. 22, p. 108). All members of the FGF family share a homology core domain of about 120 amino acids, 28 aa residues are highly conserved and four are identical. The adjacent N- and C-termini are of variable length and share limited homology. The core domain comprises both the primary receptor binding sites and a heparin-binding domain, which are distinct from each other (Ornitz and Itoh, Gen. Biol., 2001, Vol. 2, No. 3, p. 30005).
Fibroblast growth factor 18 (FGF-18), is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 is primarily expressed in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. The first 27 amino acids of the human and mouse FGF-18 protein are hydrophobic residues that are predicted to be signal peptides for secretion. FGF-18 further contains two potential N-linked glycosylation sites and it was glycosylated and secreted when it was transfected into 293-EBNA cells. Glycosylation was determined not to be critical for FGF-18 activity as the glycosylated (mammalian cells expressed) and non-glycosylated (E. coli expressed) FGF-18 showed similar activity on target cells (Hu et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 10, p. 6063). WO 2006/063362 discloses expression vectors and methods using an E. coli expression system for the large scale production of FGF-18. Initially, FGF-18 was disclosed as being a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine (Hu et al., Molecular and Cellular Biology, 1998, Vol. 18, No. 10, p. 6063). US 2008/0193425 discloses a composition for intra-articular delivery of chondrogenic polypeptides comprising a pharmaceutically acceptable admixture comprising FGF-18 and hyaluronic acid. US 2010/0016223 discloses the treatment of cartilage disorder and osteoarthritis in particular, using FGF-18 and truncated variants which comprise or consist of residues 28 to 175-207.
The biological response of cells to FGF is mediated through specific, high affinity (Kd 20-500 pM) cell surface receptors that possess intrinsic tyrosine kinase activity and are phosphorylated upon binding to FGF. Five distinct Fibroblast Growth Factor Receptors (FGFRs) have been identified, FGFR1-4 are transmembrane-protein kinases while FGFR5 lacks a tyrosine kinase domain. The FGFR extracellular domain consists of three immunoglobulin-like (Ig-like) domains (D1, D2 and D3), a heparin binding domain and an acidic box. Alternative splicing of D3 in FGFR1-3 mRNAs generates six different receptor subtypes, each having unique ligand specificity and tissue distribution pattern. Most naturally occurring FGF ligands share the capacity to bind multiple FGF receptors. Fibroblast growth factor-18 (FGF-18) binds to FGF receptors-2 and -3. Compositions comprising an FGF-18 component can be used to target cells including tumor cells that express constitutively activated forms of FGF receptors-2 and -3 (WO 01/39788).
FGF Variants and Receptor Specificity
Attempts have been made to achieve altered FGF receptor specificity by inducing mutations at certain locations within the gene encoding the proteins and/or by inducing truncations in the FGF ligands. WO 02/36732 and WO 03/094835 disclose FGF-9 N- and/or C-terminus truncated variants having enhanced specificity for one receptor subtype compared to the corresponding wild type FGF. WO 03/094835 further discloses an FGF-4 variant having both an N-terminal truncation (55 amino acids) and an amino acid substitution in the β8-β9 loop, the variant exhibiting enhanced receptor specificity towards FGFR3 with substantially unchanged activity towards FGFR1 and FGFR2. WO 08/038,287 discloses FGF-2 and FGF-4 variants having N-terminus truncations which activate only one or two of the FGFR subtypes, preferably FGFR1. Kuroda et al., (Bone, 1999, Vol. 25, p. 431) demonstrated that a full-length FGF4 polypeptide (191 aa) and an N-terminal truncated version containing 134 amino acid residues exhibit comparable cellular proliferation on NIH3T3 cells and increase of bone density. The shortest form of FGF4 tested, containing only 111 amino acid residues, exhibited limited growth stimulatory activity. U.S. Pat. No. 5,998,170 discloses a biologically active FGF-16 molecule having from one to thirty-four amino acids deleted from the N-terminus or from one to eighteen amino acids deleted from the C-terminus. The truncated ligands were shown to retain biological activity including hepatocellular proliferation and increased production of triglycerides and serum proteins, when administered to animals.
X-ray crystallography has been used in an attempt to study the basis of specificity of FGFs to their receptors (Plotnikov et al., Cell, 2000, Vol. 101, p. 413; Olsen, et al., PNAS US, 2004, Vol. 101, p. 935; Mohammadi et al., Cyto. Growth Factor Rev., 2005, Vol. 16, No. 2, p. 107). The role of the N-terminal domain of the FGFs was partially resolved in a few of the abovementioned crystal structures. Olsen et al., (PNAS US, 2004, Vol. 101, p. 935) compared receptor binding of a full length FGF-1 (155 aa) to an N-terminal truncated form (21-155) and showed that the N-terminus of FGF-1 may be relevant to binding and activation of the FGFR3c isoform. The (21-155) form also exhibits reduced FGFR2 and FGFR3 phosphorylation. Plotnikov et al., (Plotnikov et al., Cell, 2000, Vol. 101, p. 413) determined the crystal structures of FGF-1 and FGF-2 complexed with the ligand binding domains (Ig-like domains 2 and 3) of FGFR1 and FGFR2, respectively and showed that certain N-terminal residues of FGF, in particular Phe17 and Lys18 of FGF-2 (Lys27 of 155 aa form), could be in contact with the D3 domain of FGFR2. The authors speculated that amino acids 7-13 of FGF-1 play a role in receptor binding. The deletion of that specific sequence, which had originally been proposed to be a nuclear localization sequence, reduces the ability of FGF-1 to induce cell proliferation in endothelial cell lines by about 250-fold (Imamura et al., Science, 1990, Vol. 249, p. 1567). Seno et al., (Eur. J. Biochem. 1990, Vol. 188, p. 239) disclosed certain FGF-2 variants having N- and C-terminus truncations and have characterized their ability to bind heparin. The mitogenic activity of those variants in BALB/c3T3 cells was determined and the N14 variant (corresponding to a 22 amino acid truncation of the 155 aa bFGF species) showed an activity of 68% that of the mature form of bFGF. A much larger truncation, N41, which corresponds to a 49 amino acid truncation of the 155 aa species, exhibits only about 2% mitogenic activity.
Attempts have been made to alter FGF receptor specificity and heparin binding by means of site directed mutagenesis within the FGF genes. U.S. Pat. No. 5,512,460 discloses a biologically active FGF-9 (glia activating factor, GAF) molecule comprising N-terminus and C-terminus truncations of 53 aa and 13 aa, respectively. U.S. Pat. No. 5,571,895 discloses an N-terminus 54 aa deletion yielding a 154 aa protein retaining its biological activity, as measured by glial cell growth activity. Bellosta et al., (Mol. Cell. Biol., 2001, Vol. 21, No. 17, p. 5946) disclosed mutated and truncated FGF-4 variants having reduced receptor binding and a low mitogenic potential.
Wild type FGF-18 was found to bind to FGF receptors (FGFRs)-3c and -2c but not to FGFR-1c (Hoshikawa, Brain Res. Mol. Brain Res., 2002, Vol. 105, No. 1-2, p. 60). Yet another paper (Hamidouche et al J Cell Physiol. 2010 Vol. 224(2), p. 509) demonstrates that FGF-18 signaling in mesenchymal stem cells is mediated via FGFR1 and FGFR2. Another study identified a novel mechanism for FGF-18 signaling. It involves an accessory receptor (Cfr) that binds to FGF-18 and increases its affinity towards FGFR3c (Miyaoka et al, Development. 2010 Vol. 137(1), p. 159). Zhang et al. (J. Bio. Chem. 2006, Vol. 281(23), p. 15694) discloses that FGF-18 specifically activates FGFRs 1c, 2c, and 3c and the two Ig-like domain forms of FGFR4. WO 01/39788 discloses FGF-18 peptide that lacks the signal peptide (amino acids 1-27) also known as the mature form of FGF-18, and FGF-18 functional fragments comprising C-terminal truncations as targeting compositions for inhibiting the proliferation of cells including tumor cells that express constitutively activated forms of FGF receptors-2 and -3.
Certain FGFRs have been implicated in certain malignancies and proliferative diseases. FGFR3 is the most frequently mutated oncogene in transitional cell carcinoma (TCC) of the bladder where it is mutated in about 50% of the cases; the FGFR3IIIc isoform is ectopically expressed in 15-20% of patients with multiple myeloma and is over expressed in the white blood cells of chronic myeloid leukemia (CML) patients. FGFR3IIIc isoform is also implicated in mediation of colorectal cancer growth and migration (Sonvilla et al, British Journal of Cancer 2010, Vol. 102, p. 1145). Single nucleotide polymorphisms (SNPs) of FGFR2 are associated with increased risk of breast cancer (Katoh, Inter. J. Oncol., 2008, Vol. 33, No. 2, p. 233). Mutations in FGFR2 and FGFR3 genes are associated with human gastric and colorectal cancers (Jang et al., Cancer Res., 2001, Vol. 61, p. 3541). A mutation in FGFR3 is linked to cervical carcinoma. FGFR4 was shown to be associated with pituitary tumors and breast cancer progression. FGF-18 is known to stimulate the proliferation of chondrocytes resulting in an increased cartilage formation (Davidson et al., The Journal of Biological Chemistry, 2005, Vol. 280, No. 21, p. 20509).
There remains an unmet need for highly selective FGF variants, useful in selective stimulation or inhibition of FGF receptor subtypes, thus being useful in treating cartilage and skeletal disorders.
SUMMARY OF THE INVENTION
The present invention provides Fibroblast Growth Factor 18 (FGF-18) N-terminal truncated polypeptides that show an increase in receptor specificity for FGF receptor 3 when compared to the corresponding wild type polypeptide. In particular, the invention provides FGF-18 polypeptides having an N-terminal truncation beyond the signal peptide domain, polynucleotides encoding same, pharmaceutical compositions comprising the polypeptides or polynucleotide encoding same, and use thereof in treating cartilage and skeletal disorders.
The present invention is based in part on the unexpected finding that certain N-terminal truncated variants of the mature form of FGF-18 have enhanced receptor specificity for FGFR3 thus affording selective activation of FGFR3. The selective activation of FGFR3 has now been shown to inhibit proliferation of chondrocytes while affording chondrocyte differentiation. The FGF-18 truncated polypeptides of the present invention provide chondrogenic differentiation for specific treatment of cartilage disorders.
According to one aspect, the present invention provides an isolated FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes. In one embodiment, the N-terminal truncated FGF-18 polypeptides are specific to FGFR3.
The sequence of native FGF-18 including the signal peptide is set forth in SEQ ID NO: 1 and shown in FIG. 1. The signal peptide, the R34-H36 basic cluster 1 (BC1), the R42-R44 basic cluster 2 (BC2), and the R49-K50 basic cluster 3 (BC3) are underlined. The sequence of the polynucleotide encoding native FGF-18 which includes the signal peptide is set forth in SEQ ID NO: 71. The sequence of the recombinant wild-type mature FGF-18 (without the signal peptide) is set forth in SEQ ID NO: 2. The corresponding polynucleotide sequence is set forth in SEQ ID NO: 59.
In some embodiments, the N-terminus of the isolated FGF-18 polypeptide of the present invention retains between 0 and 28 amino acid residues extending beyond the core domain.
In one embodiment, the isolated FGF-18 polypeptide having an N-terminal truncation comprises an amino acid sequence as set forth in SEQ ID NO: 11. In other embodiments, the isolated FGF-18 polypeptide having an N-terminal truncation comprises an amino acid sequence as set forth in SEQ ID NO: 19. In other embodiments, the isolated FGF-18 polypeptide having an N-terminal truncation comprises an amino acid sequence as set forth in SEQ ID NO: 35. In yet other embodiments, the isolated FGF-18 polypeptide having an N-terminal truncation comprises an amino acid sequence as set forth in SEQ ID NO: 47. In certain embodiments, the isolated FGF-18 polypeptide having an N-terminal truncation consists of an amino acid sequence as set forth in any one of SEQ ID NOS: 11, 19, 35 or 47. Each possibility represents a separate embodiment of the present invention.
A description of the FGF-18 N-terminal truncated polypeptides of the present invention is set forth hereinbelow. It is to be noted that the N-terminal methionine residue (Met) is required for expression in the bacterial expression system, yet it is generally post-translationally cleaved and a polypeptide lacking the first Met is obtained. However, it is to be understood that FGF-18 polypeptides having the N-terminal methionine are encompassed within the scope of the present application.
The FGF-18 N-terminal truncated polypeptides are characterized as follows:
SEQ ID NO: 3, represents FGF-18Δ29, having a 28 amino acid N-terminal truncation with the Glu29 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 4. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 5, represents FGF-18Δ30, having a 29 amino acid N-terminal truncation with the Asn30 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 6. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 7, represents FGF-18Δ31, having a 30 amino acid N-terminal truncation with the Val31 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 8. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 9, represents FGF-18Δ32, having a 31 amino acid N-terminal truncation with the Asp32 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 10. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 11, represents FGF-18Δ33, having a 32 amino acid N-terminal truncation with the Phe33 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 12. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 13, represents FGF-18Δ34, having a 33 amino acid N-terminal truncation with the Arg34 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 14. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 15, represents FGF-18Δ35, having a 34 amino acid N-terminal truncation with the Ile35 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 16. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 17, represents FGF-18Δ36, having a 35 amino acid N-terminal truncation with the His36 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 18. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 19, represents FGF-18Δ37, having a 36 amino acid N-terminal truncation with the Val37 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 20. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 21, represents FGF-18Δ38, having a 37 amino acid N-terminal truncation with the Glu38 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 22. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 23, represents FGF-18Δ39, having a 38 amino acid N-terminal truncation with the Asn39 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 24. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 25, represents FGF-18Δ40, having a 39 amino acid N-terminal truncation with the Gln40 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 26. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 27, represents FGF-18Δ41, having a 40 amino acid N-terminal truncation with the Thr41 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 28. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 29, represents FGF-18Δ42, having a 41 amino acid N-terminal truncation with the Arg42 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 30. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 31, represents FGF-18Δ43, having a 42 amino acid N-terminal truncation with the Ala43 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 32. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 33, represents FGF-18Δ44, having a 43 amino acid N-terminal truncation with the Arg44 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 34. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 35, represents FGF-18Δ45, having a 44 amino acid N-terminal truncation with the Asp45 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 36. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 37, represents FGF-18Δ46, having a 45 amino acid N-terminal truncation with the Asp46 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 38. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 39, represents FGF-18Δ47, having a 46 amino acid N-terminal truncation with the Val47 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 40. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 41, represents FGF-18Δ48, having a 47 amino acid N-terminal truncation with the Ser48 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 42. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 43, represents FGF-18Δ49, having a 48 amino acid N-terminal truncation with the Arg49 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 44. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 45, represents FGF-18Δ50, having a 49 amino acid N-terminal truncation with the Lys50 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 46. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 47, represents FGF-18Δ51, having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 48. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 49, represents FGF-18Δ52, having a 51 amino acid N-terminal truncation with the Leu52 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 50. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 51, represents FGF-18Δ53, having a 52 amino acid N-terminal truncation with the Arg53 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 52. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 53, represents FGF-18Δ54, having a 53 amino acid N-terminal truncation with the Leu54 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 54. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 55, represents FGF-18Δ55, having a 54 amino acid N-terminal truncation with the Tyr55 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 56. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
SEQ ID NO: 57, represents FGF-18Δ56, having a 55 amino acid N-terminal truncation with the Gln56 replaced by a Met residue. This polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 58. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27.
In some embodiments, the isolated FGF-18 polypeptide of the present invention further comprises at least one additional modification in its polypeptide sequence, wherein the modification is selected from an amino acid deletion, an amino acid substitution and an amino acid insertion. Each possibility represents a separate embodiment of the invention. In one embodiment, the additional modification is an amino acid residue substitution in the retained N-terminus sequence. In another embodiment, the amino acid substitution is a conservative substitution. In further embodiments, the isolated FGF-18 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 67 representing FGF-18Δ51L52X, having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue and further having the Leu52 replaced by an amino acid residue other than leucine. In specific embodiments, the Leu52 is replaced by an amino acid residue selected from isoleucine, valine and methionine. Each possibility represents a separate embodiment of the present invention. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27. The polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 68. In additional embodiments, the isolated FGF-18 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 69 representing FGF-18Δ51L52I, having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue and further having the Leu52 replaced by an Ile residue. It is to be understood that the amino acid numbering includes the signal peptide located at amino acids positions 1-27. The polypeptide is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 70.
It is to be understood explicitly that the isolated FGF-18 polypeptide having an N-terminal truncation as disclosed herein may comprise extensions at the N-terminus other than reinstating those specific N-terminal residues that have been truncated. In other words, the isolated FGF-18 polypeptide of the present invention comprises at least one amino acid deletion at the N-terminus beyond the signal peptide. The isolated FGF-18 N-terminal truncated polypeptides of the present invention may further comprise at least one additional modification in the polypeptide sequence as described herein.
According to another aspect, the present invention provides an isolated polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes.
In various embodiments, the isolated polynucleotide comprises a sequence as set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58. Each possibility represents a separate embodiment of the invention. In additional embodiments, the isolated polynucleotide consists of a sequence as set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58. Each possibility represents a separate embodiment of the invention.
In certain embodiments, the present invention provides an isolated polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and further having at least one additional modification in its polypeptide sequence, wherein the modification is selected from an amino acid deletion, an amino acid substitution and an amino acid insertion, wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes. In one embodiment, the isolated polynucleotide encodes an FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and further having at least one amino acid residue substitution in the retained N-terminus sequence.
In other embodiments, the present invention provides a vector comprising a polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation as disclosed herein.
In other embodiments, the present invention provides a host cell comprising a vector, the vector comprising a polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation as disclosed herein.
In another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes; and a pharmaceutically acceptable diluent or carrier.
In one embodiment, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 11, and a pharmaceutically acceptable diluent or carrier. In another embodiment, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 19; and a pharmaceutically acceptable diluent or carrier. In another embodiment, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 35; and a pharmaceutically acceptable diluent or carrier. In yet another embodiment, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 47; and a pharmaceutically acceptable diluent or carrier.
In further embodiments, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and further having at least one additional modification in its polypeptide sequence, wherein the modification is selected from an amino acid deletion, an amino acid substitution and an amino acid insertion, wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes; and a pharmaceutically acceptable diluent or carrier. In some embodiments, the pharmaceutical composition comprises an FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and further having at least one amino acid residue substitution in the retained N-terminus sequence; and a pharmaceutically acceptable diluent or carrier. In specific embodiments, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 67; and a pharmaceutically acceptable diluent or carrier. In other embodiments, the pharmaceutical composition comprises at least one isolated FGF-18 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 69; and a pharmaceutically acceptable diluent or carrier.
In certain embodiments, the FGF-18 N-terminal truncated polypeptides of the present invention are admixed with at least one bioactive agent. In some embodiments, the at least one bioactive agent is a carboxylated polysaccharide. In particular embodiments, the carboxylated polysaccharide is hyaluronic acid. In additional embodiments, the at least one bioactive agent is a natriuretic peptide. In an exemplary embodiment, the at least one bioactive agent is a C-type natriuretic peptide.
According to another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one isolated polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes; and a pharmaceutically acceptable diluent or carrier. In additional embodiments, the pharmaceutical composition comprises as an active ingredient at least one isolated polynucleotide encoding an FGF-18 polypeptide having an N-terminal truncation and further having at least one additional modification in its polypeptide sequence, wherein the modification is selected from an amino acid deletion, an amino acid substitution and an amino acid insertion, for example an amino acid substitution in the retained N-terminus sequence. In specific embodiments, the pharmaceutical composition comprises at least one isolated polynucleotide comprising a sequence as set forth in SEQ ID NO: 68; and a pharmaceutically acceptable diluent or carrier. In other embodiments, the pharmaceutical composition comprises at least one isolated polynucleotide comprising a sequence as set forth in SEQ ID NO: 70; and a pharmaceutically acceptable diluent or carrier.
In one embodiment, the pharmaceutical composition comprises at least one polynucleotide comprising a sequence as set forth in SEQ ID NO: 12; and a pharmaceutically acceptable diluent or carrier. In another embodiment, the pharmaceutical composition comprises at least one polynucleotide comprising a sequence as set forth in SEQ ID NO: 20; and a pharmaceutically acceptable diluent or carrier. In another embodiment, the pharmaceutical composition comprises at least one polynucleotide comprising a sequence as set forth in SEQ ID NO: 36; and a pharmaceutically acceptable diluent or carrier. In yet another embodiment, the pharmaceutical composition comprises at least one polynucleotide comprising a sequence as set forth in SEQ ID NO: 48; and a pharmaceutically acceptable diluent or carrier.
In some embodiments, the pharmaceutical compositions of the present invention are formulated for administration via a route selected from intra-articular, intravenous, intramuscular, subcutaneous, intradermal, and intrathecal route. Each possibility represents a separate embodiment of the invention. In certain embodiments, the pharmaceutical compositions are formulated for administration to the site of a cartilage lesion.
In additional embodiments, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one isolated FGF-18 polypeptide having an N-terminal truncation, wherein the N-terminal truncation is extending beyond the signal peptide, and wherein the polypeptide has increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes or a polynucleotide encoding same for inducing cartilage repair, particularly joint cartilage repair. In other embodiments, the compositions of the present invention are useful for treating degenerative joint diseases. In one particular embodiment, the compositions of the present invention are useful in treating osteoarthritis.
In further embodiments, the present invention provides a method of inducing cartilage repair comprising the step of administering to an individual in need thereof a pharmaceutical composition comprising at least one isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide or a polynucleotide encoding same. The present invention further provides a method of treating a degenerative joint disease, particularly osteoarthritis, comprising the step of administering to an individual in need thereof a pharmaceutical composition comprising at least one isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide or a polynucleotide encoding same. In other embodiments, the present invention provides an isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide or a polynucleotide encoding same or a pharmaceutical composition comprising at least one isolated FGF-18 polypeptide as disclosed herein or a pharmaceutical composition comprising at least one polynucleotide encoding same for use in inducing cartilage repair or for use in treating a degenerative joint disease, particularly osteoarthritis. These methods of use of the present invention comprise the administration of a therapeutically effective amount of a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57 or a polynucleotide having a nucleotide sequence as set forth in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58. Each possibility represents a separate embodiment of the invention.
In additional embodiments, the present invention provides a method of inducing cartilage repair or treating a degenerative joint disease, particularly osteoarthritis comprising the step of administering to an individual in need thereof a pharmaceutical composition comprising at least one isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide and further having at least one additional modification in its polypeptide sequence, wherein the modification is selected from an amino acid deletion, an amino acid substitution and an amino acid insertion, for example an amino acid substitution in the retained N-terminus sequence, or a polynucleotide encoding same. In exemplary embodiments, the method comprises the administration of a therapeutically effective amount of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 67 or 69, or a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 68 or 70. Each possibility represents a separate embodiment of the invention.
In yet other embodiments, the present invention provides a method of inducing primary chondrocyte differentiation, comprising the steps of:
a. isolating a population of primary chondrocytes to be differentiated; and
b. exposing said chondrocytes to at least one isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide as disclosed herein.
In yet another aspect, the present invention provides methods for the use of an isolated FGF-18 polypeptide having an N-terminal truncation extending beyond the signal peptide as disclosed herein for inducing primary chondrocyte differentiation.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. The amino acid sequence of human FGF-18 (SEQ ID NO:1) and recombinant mature form of FGF-18 (SEQ ID NO:2).
FIGS. 2A-2D. Immobilized receptor binding assay analysis of FGF-18 variants demonstrating FGF receptor specificity. (2A) FGFR1; (2B) FGFR2; (2C) FGFR3 and (2D) FGFR4. Biotinylated FGF-18 (♦) and three FGF-18 variants (Δ37, v1, (●); Δ45, v2, (▪); and Δ51, v3, (▪)) were added at increasing concentrations to FGFR1-4 anchored to a MaxiSorp ELISA plate. Bound ligands were detected by HRP-streptavidin. Data are the average of duplicate wells.
FIG. 3. Effect of FGF-18Δ51 on cells proliferation of FDCPR3. FGF-18Δ51 (▴) effect on FDCPFGFR3 cell proliferation was compared to that of FGF-2 (♦) or FGF-18 wild type ligands (▪). Each of the three ligands was added to FDCPFGFR3 cells at different concentrations (100 ng/ml→0.39 ng/ml). After two days incubation at 37° C., cell proliferation was measured by the XXT reagent kit.
FIG. 4. Expression of FGFRs in primary chondrocytes. Primary chondrocytes were lyzed and probed by Western with anti-FGFRs antibodies. The receptors were detected by ECL.
FIG. 5. Effect of FGF-2 (F2) and FGF-18 (F18) on PAC proliferation. Human primary articular chondrocytes (PAC), passage 1, were seeded in 12 well plate (10,000 cells/well) in DMEM-F12+10% FBS. Ligands (FGFs) were added with 5 μg/ml heparin (where indicated) and cells proliferation was determined using an automated cell counter.
FIG. 6. Effect of low doses of FGF-18 and FGF-18 variants on PAC Proliferation. Human primary articular chondrocytes (PAC), passage 1, were seeded in 12 well plate (10,000 cells/well) in DMEM-F12+10% FBS. Ligands (FGFs) were added the next day and the cells were grown for four days without further medium exchanges. Heparin was used at 5 μg/ml final concentration. Cells were harvested by trypsinization and counted using an automated cell counter.
FIG. 7. Effect of high doses of FGF-18 and FGF-18 variants on PAC Proliferation. Human primary articular chondrocytes (PAC), passage 3, were seeded in 24 well plate (7,000 cells/well) in DMEM-F12+10% FBS. Ligands (FGFs) were added the next day to cells and cells were grown for one week without further medium change. Heparin was used at 5 μg/ml final concentration. Cells were harvested by trypsinization and counted using an automated cell counter.
FIG. 8. FGF-18 N-terminal variants inhibit RCS cell proliferation in a receptor specific manner. RCS cells were treated with wild-type or mutant FGFs with or without the anti-FGFR3 PRO-001 (WO 07/144,893) antibody for 3 days. Then, cell proliferation was measured by the Cy Quant analysis. Untreated cells were included as control.
FIGS. 9A-9C. Effect of FGF-18 wild type or FGF-18Δ51 treatment on chondrocytes' pellet culture formation. (9A) control; (9B) FGF-18 wild type; and (9C) FGF-18Δ51. Human articular chondrocytes (passage 1, d11, 0.5*106/pellet) were grown in chondrocyte differentiation medium for 2 weeks. Some pellets were supplemented with 15 ng/ml of FGF-18 wild type (wt) or FGF-18Δ51. Media was changed every 3 days. At the end of the experiment, pellets were fixed for histology. H&E stain of X40 magnification of representative pellets is presented.
FIG. 10. Effect of FGF-18 N-terminal variants on chondrocyte proliferation. Primary porcine chondrocytes were harvested from articular cartilage of adult pig. 300,000 cells were seeded per well. The ligands were added the next day at 100 ng/ml, followed by media replacement 3 days later. The cells were harvested at day 5 and counted.
FIG. 11. FGF-18Δ51L52I is expressed at higher levels than FGF-18Δ51. FGF-18Δ51 and FGF-18Δ51L52I transformed BL21(DE3) bacteria were cultured overnight. Overnight lysates were analyzed by Western blotting with anti-FGF-18 antibody.
FIGS. 12A-12D. FGF-18Δ51L52I receptor specificity. (12A) FGFR1; (12B) FGFR2; (12C) FGFR3; and (12D) FGFR4. FGFRs were anchored to a MaxiSorp plate. Biotinylated FGF ligands (FGF-18 wild type (♦); FGF-18Δ51 (▪); and FGF-18Δ51L52I (▴)) were added at increasing concentrations and bound ligands were detected by HRP-streptavidin.
FIGS. 13A-13B. FGF-18Δ51L52I is a potent activator of FGFR3. (13A) FGF-18 wild type (♦) vs. FGF-18Δ51 (▪); and (13B) FGF-18 wild type (♦) vs. FGF-18Δ51L52I (▴). FDCP-FGFR3 cells were cultured with FGF-18, FGF-18Δ51 and FGF-18Δ51L52I and cell proliferation was measured 2 days later by the XTT assay.
FIG. 14. FGF-induced GAG synthesis in chondrocytes. Cartilage taken from osteoarthritis patients was dissected and pre-incubated for 3 days in control medium (day 0) followed by 4 days in the presence of 50 ng/ml of the indicated FGFs. Cell supernatant was collected and levels of secreted glycosaminoglycans (GAGs) were determined.



DETAILED DESCRIPTION OF THE INVENTION
According to the principles of the present invention, it is now disclosed that isolated FGF-18 polypeptides having an N-terminal truncation which extends beyond the signal peptide show improved specificity for FGFR3. These variants show enhanced chondrogenic activity and are shown to afford differentiation of primary chondrocytes with no mitogenic activity.
The present invention provides an isolated FGF-18 polypeptide having an N-terminal truncation. The location of the N-terminal truncation, according to the principles of the present invention, extends beyond the signal peptide, i.e. an N-terminal truncation of the mature form of FGF-18. In one embodiment, the N-terminus retains between 0 and 28 amino acid residues at the N-terminus of the mature form extending beyond the core domain. When referring to an amino acid numbering which includes the signal peptide located at amino acids positions 1-27 (SEQ ID NO:1), the core domain starts at amino acid residue Leu57. The FGF-18 truncated polypeptides of the present invention include truncations of between 29 amino acids to 56 amino acids. These truncations therefore extend beyond the signal peptide, whereby the variants retain between 28 and 0 amino acid residues, respectively, at the N-terminus of the mature form extending beyond the core domain.
The amino acid sequences of the truncated polypeptides of the present invention are set forth herein below:







(FGF-18Δ29):


SEQ ID NO: 3


MNVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISA


 


RGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSK


 


ECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFM


 


KRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ30):


SEQ ID NO: 5


MVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISAR


 


GEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKE


 


CVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMK


 


RYPKGQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ31):


SEQ ID NO: 7


MDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARG


 


EDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKEC


 


VFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKR


 


YPKGQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ32):


SEQ ID NO: 9


MFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGE


 


DGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECV


 


FIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRY


 


PKGQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ33):


SEQ ID NO: 11


MRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGED


 


GDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVF


 


IEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYP


 


KGQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ34):


SEQ ID NO: 13


MIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDG


 


DKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFI


 


EKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPK


 


GQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ35):


SEQ ID NO: 15


MHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDG


 


DKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFI


 


EKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPK


 


GQPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ36):


SEQ ID NO: 17


MVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGD


 


KYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIE


 


KVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKG


 


QPELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ37):


SEQ ID NO: 19


MENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDK


 


YAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEK


 


VLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQ


 


PELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ38):


SEQ ID NO: 21


MNQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKY


 


AQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKV


 


LENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQP


 


ELQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ39):


SEQ ID NO: 23


MQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYA


 


QLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVL


 


ENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPE


 


LQKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ40):


SEQ ID NO: 25


MTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQ


 


LLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLE


 


NNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPEL


 


QKPFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ41):


SEQ ID NO: 27


MRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLL


 


VETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENN


 


YTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQK


 


PFKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ42):


SEQ ID NO: 29


MARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLV


 


ETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNY


 


TALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKP


 


FKYTTVTKRSRRIRPTHPA


 


(FGF-18Δ43):


SEQ ID NO: 31


MRDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVE


 


TDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYT


 


ALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPF


 


KYTTVTKRSRRIRPTHPA


 


(FGF-18Δ44):


SEQ ID NO: 33


MDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVET


 


DTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTA


 


LMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFK


 


YTTVTKRSRRIRPTHPA


 


(FGF-18Δ45):


SEQ ID NO: 35


MDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETD


 


TFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTAL


 


MSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKY


 


TTVTKRSRRIRPTHPA


 


(FGF-18Δ46):


SEQ ID NO: 37


MVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDT


 


FGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALM


 


SAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYT


 


TVTKRSRRIRPTHPA


 


(FGF-18Δ47):


SEQ ID NO: 39


MSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTF


 


GSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMS


 


AKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTT


 


VTKRSRRIRPTHPA


 


(FGF-18Δ48):


SEQ ID NO: 41


MRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFG


 


SQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSA


 


KYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTV


 


TKRSRRIRPTHPA


 


(FGF-18Δ49):


SEQ ID NO: 43


MKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGS


 


QVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAK


 


YSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVT


 


KRSRRIRPTHPA


 


(FGF-18Δ50):


SEQ ID NO: 45


MQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQ


 


VRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKY


 


SGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK


 


RSRRIRPTHPA


 


(FGF-18Δ51):


SEQ ID NO: 47


MLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQV


 


RIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYS


 


GWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKR


 


SRRIRPTHPA


 


(FGF-18Δ52):


SEQ ID NO: 49


MRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVR


 


IKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSG


 


WYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRS


 


RRIRPTHPA


 


(FGF-18Δ53):


SEQ ID NO: 51


MLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRI


 


KGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGW


 


YVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSR


 


RIRPTHPA


 


(FGF-18Δ54):


SEQ ID NO: 53


MYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIK


 


GKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWY


 


VGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRR


 


IRPTHPA


 


(FGF-18Δ55):


SEQ ID NO: 55


MQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKG


 


KETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYV


 


GFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRI


 


RPTHPA


 


(FGF-18Δ56):


SEQ ID NO: 57


MLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGK


 


ETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVG


 


FTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIR


 


PTHPA






Currently preferred are the N-terminal truncated FGF-18 polypeptides identified by SEQ ID NOS: 11, 19, 35 and 47. Each possibility represents a separate embodiment of the invention.
The present invention further provides an isolated FGF-18 polypeptide having an N-terminal truncation and further having at least one additional modification (e.g. an amino acid deletion, an amino acid substitution or an amino acid insertion), preferably in the retained N-terminus. Without being bound by any theory or mechanism of action, the at least one additional modification provides enhanced stability, solubility or yield of the polypeptide. In some embodiments, the isolated FGF-18 polypeptide has a 50 amino acid N-terminal truncation with Gln51 being replaced by a Met residue and a replacement of the penultimate leucine (L52) with an amino acid other than leucine. In particular embodiments, the at least one modification is the conservative replacement of the penultimate leucine (L52) with another non-charged, hydrophobic amino acid, for example isoleucine, valine or methionine. Each possibility represents a separate embodiment of the invention.
The amino acid sequences of the polypeptides are set forth herein below:







(FGF-18Δ51L52X):


SEQ ID NO: 67


MXRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQV


 


RIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYS


 


GWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKR


 


SRRIRPTHPA






X is an amino acid residue other than leucine, for example isoleucine, valine or methionine.







(FGF-18Δ51L52I):


SEQ ID NO: 69


MIRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQV


 


RIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYS


 


GWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKR


 


SRRIRPTHPA






According to the principles of the present invention, the polypeptides of the present invention have increased receptor selectivity when compared to the isolated wild-type FGF-18 polypeptide by a gain of activity or loss of activity by at least a factor of two toward at least one receptor subtype but not toward all FGFR subtypes. It has now been disclosed for the first time that these truncated variants specifically activate FGFR3 while essentially eliminating the activation of other receptor subtypes including, in particular FGFR1 and FGFR2.
As used herein and in the claims the term “FGFR” denotes a receptor specific for FGF molecule(s), which is necessary for transducing the signal, exerted by FGF to the cell interior, typically comprising an extracellular ligand-binding domain, a single transmembrane helix, and a cytoplasmic domain that contains a tyrosine kinase activity. The term “FGFR” includes various isotypes of the receptors including soluble versions comprising the extracellular domain and lacking the transmembrane and kinase domains. As used herein and in the claims the term “an isolated FGF-18 polypeptide having increased receptor selectivity” denotes an isolated FGF-18 polypeptide molecule, having either enhanced or reduced biological activity toward at least one but not all FGFR, compared to the corresponding wild type FGF-18. The biological activity toward at least one receptor, but not all FGF receptors, is reduced or increased by a factor of at least 2. In some embodiments, the biological activity toward at least one receptor, but not all FGF receptors, is reduced or increased by a factor of at least 4, at least 5, at least 7 or at least 10. Biological activity, according to the principles of the present invention, can be measured by methods known in the art. In some embodiments, biological activity is measured by a cell proliferation or cell differentiation assay or alternatively by substrate phosphorylation.
As contemplated herein, the variants of the present invention afford differentiation of primary chondrocytes. The differentiation can be determined as is known in the art by morphological and/or phenotypic changes and/or by biochemical or molecular changes. The variants of the present invention essentially do not show mitogenic activity. “Mitogenic activity” as used herein refers to the ability to induce an increase in the number of cells.
The N-terminal truncated variants of the present invention can be prepared by any manner known in the art. Exemplary method for producing the variants is through recombinant DNA technologies, for example by Polymerase Chain Reaction (PCR) using specific primers for each of the truncated forms as disclosed herein. The PCR fragments may be purified on an agarose gel and the purified DNA fragment may be cloned into an expression vector and transfected into host cells. The host cells may be cultured and the protein harvested according to methods known in the art. As used herein, the term “polymerase chain reaction” (“PCR”) refers to the methods of amplifying nucleic acids, as disclosed in e.g. U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188 hereby incorporated by reference.
According to the principles of the present invention the N-terminal truncated variants of the present invention may further comprise at least one modification including, but not limited to, a substitution of at least one amino acid, preferably in the retained N-terminus. The term “at least one modification” as used herein refers to an amino acid sequence that is altered by one or more amino acids including an amino acid deletion, an amino acid substitution or an amino acid insertion. An amino acid substitution is selected from a conservative substitution or a non-conservative substitution. “Conservative” substitutions refer to replacements wherein the amino acid that replaces the naturally occurring amino acid has similar structural or chemical properties, e.g., replacement of leucine with any one of isoleucine, valine or methionine. Each possibility represents a separate embodiment of the invention. Of these, leucine and isoleucine are the most closely related amino acids and are therefore considered interchangeable by those of skill in the art. “Non-conservative” substitutions refer to replacements wherein the amino acid that replaces the naturally occurring amino acid has different structural or chemical properties, e.g. replacement of leucine with histidine. Analogous minor modification may also include amino acid deletions or insertions, or both.
The present invention further provides the isolated polynucleotides encoding the N-terminal truncated variants of the present invention and a vector and a host cell comprising the polynucleotides. The polynucleotides encoding the variants of the present invention are identified by SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58. Each possibility represents a separate embodiment of the invention. Currently preferred are polynucleotides identified by SEQ ID NOS: 12, 20, 36 and 48. Each possibility represents a separate embodiment of the invention. In further embodiments, the present invention provides isolated polynucleotides encoding the N-terminal truncated variants which further comprise at least one modification as defined herein and a vector and a host cell comprising the polynucleotides. The polynucleotides are identified by SEQ ID NO: 68 or 70. Each possibility represents a separate embodiment of the present invention. Selected sequences are set forth herein below:







encoding FGF-18Δ37:


SEQ ID NO: 20


atggagaaccagacgcgggctcgggacgatgtgagccgtaagcagctgcg


 


gctgtaccagctctacagccggaccagtgggaaacacatccaggtcctgg


 


gccgcaggatcagtgcccgcggcgaggatggggacaagtatgcccagctc


 


ctagtggagacagacaccttcggtagtcaagtccggatcaagggcaagga


 


gacggaattctacctgtgcatgaaccgcaaaggcaagctcgtggggaagc


 


ccgatggcaccagcaaggagtgtgtgttcatcgagaaggttctggagaac


 


aactacacggccctgatgtcggctaagtactccggctggtacgtgggctt


 


caccaagaaggggcggccgcggaagggccccaagacccgggagaaccagc


 


aggacgtgcatttcatgaagcgctaccccaaggggcagccggagcttcag


 


aagcccttcaagtacacgacggtgaccaagaggtcccgtcggatccggcc


 


cacacaccctgcctag


 


encoding FGF-18Δ45:


SEQ ID NO: 36


atggatgtgagccgtaagcagctgcggctgtaccagctctacagccggac


 


cagtgggaaacacatccaggtcctgggccgcaggatcagtgcccgcggcg


 


aggatggggacaagtatgcccagctcctagtggagacagacaccttcggt


 


agtcaagtccggatcaagggcaaggagacggaattctacctgtgcatgaa


 


ccgcaaaggcaagctcgtggggaagcccgatggcaccagcaaggagtgtg


 


tgttcatcgagaaggttctggagaacaactacacggccctgatgtcggct


 


aagtactccggctggtacgtgggcttcaccaagaaggggcggccgcggaa


 


gggccccaagacccgggagaaccagcaggacgtgcatttcatgaagcgct


 


accccaaggggcagccggagcttcagaagcccttcaagtacacgacggtg


 


accaagaggtcccgtcggatccggcccacacaccctgcctag


 


encoding FGF-18Δ51:


SEQ ID NO: 48


atgctgcggctgtaccagctctacagccggaccagtgggaaacacatcca


 


ggtcctgggccgcaggatcagtgcccgcggcgaggatggggacaagtatg


 


cccagctcctagtggagacagacaccttcggtagtcaagtccggatcaag


 


ggcaaggagacggaattctacctgtgcatgaaccgcaaaggcaagctcgt


 


ggggaagcccgatggcaccagcaaggagtgtgtgttcatcgagaaggttc


 


tggagaacaactacacggccctgatgtcggctaagtactccggctggtac


 


gtgggcttcaccaagaaggggcggccgcggaagggccccaagacccggga


 


gaaccagcaggacgtgcatttcatgaagcgctaccccaaggggcagccgg


 


agcttcagaagcccttcaagtacacgacggtgaccaagaggtcccgtcgg


 


atccggcccacacaccctgcctag


 


encoding (FGF-18Δ51L52X)


SEQ ID NO: 68


ATGNNNCGGCTGTACCAGCTCTACAGCCGGACCAGTGGGAAACA


 


CATCCAGGTCCTGGGCCGCAGGATCAGTGCCCGCGGCGAGGATGGGG


 


ACAAGTATGCCCAGCTCCTAGTGGAGACAGACACCTTCGGTAGTCAAGT


 


CCGGATCAAGGGCAAGGAGACGGAATTCTACCTGTGCATGAACCGCAAA


 


GGCAAGCTCGTGGGGAAGCCCGATGGCACCAGCAAGGAGTGTGTGTTC


 


ATCGAGAAGGTTCTGGAGAACAACTACACGGCCCTGATGTCGGCTAAGT


 


ACTCCGGCTGGTACGTGGGCTTCACCAAGAAGGGGCGGCCGCGGAAGG


 


GCCCCAAGACCCGGGAGAACCAGCAGGACGTGCATTTCATGAAGCGCT


 


ACCCCAAGGGGCAGCCGGAGCTTCAGAAGCCCTTCAAGTACACGACGG


 


TGACCAAGAGGTCCCGTCGGATCAGGCCCACACACCCTGCCTAG






NNN is a codon coding for an amino acid other than leucine, for example isoleucine, valine or methionine.







encoding (FGF-18Δ51L52I)


SEQ ID NO: 70


ATGATACGGCTGTACCAGCTCTACAGCCGGACCAGTGGGAAACA


 


CATCCAGGTCCTGGGCCGCAGGATCAGTGCCCGCGGCGAGGATGGGG


 


ACAAGTATGCCCAGCTCCTAGTGGAGACAGACACCTTCGGTAGTCAAGT


 


CCGGATCAAGGGCAAGGAGACGGAATTCTACCTGTGCATGAACCGCAAA


 


GGCAAGCTCGTGGGGAAGCCCGATGGCACCAGCAAGGAGTGTGTGTTC


 


ATCGAGAAGGTTCTGGAGAACAACTACACGGCCCTGATGTCGGCTAAGT


 


ACTCCGGCTGGTACGTGGGCTTCACCAAGAAGGGGCGGCCGCGGAAGG


 


GCCCCAAGACCCGGGAGAACCAGCAGGACGTGCATTTCATGAAGCGCT


 


ACCCCAAGGGGCAGCCGGAGCTTCAGAAGCCCTTCAAGTACACGACGG


 


TGACCAAGAGGTCCCGTCGGATCAGGCCCACACACCCTGCCTAG






Within the scope of the present invention are nucleic acid molecules containing polynucleotide sequences having at least 90% sequence identity, preferably about 95%, and more preferably about 97% identity to the above encoding nucleotide sequences as understood by those of skill in the art.
The invention also provides isolated nucleic acid molecules that hybridize under high stringency conditions to polynucleotides having SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 68 and 70 or the complement thereof. As used herein, highly stringent conditions are those which are tolerant of up to about 5-20% sequence divergence, preferably about 5-10%. Without limitation, examples of highly stringent (−10° C. below the calculated Tm of the hybrid) conditions use a wash solution of 0.1×SSC (standard saline citrate) and 0.5% SDS at the appropriate Ti below the calculated Tm of the hybrid. The ultimate stringency of the conditions is primarily due to the wash conditions, particularly if the hybridization conditions used are those which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency remove the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above may be performed by hybridizing in a solution of 6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA at an appropriate incubation temperature Ti. See generally Sambrook et al. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Press) for suitable high stringency conditions.
Stringency conditions are a function of the temperature used in the hybridization experiment and washes, the molarity of the monovalent cations in the hybridization solution and in the wash solution(s) and the percentage of formamide in the hybridization solution. In general, sensitivity by hybridization with a probe is affected by the amount and specific activity of the probe, the amount of the target nucleic acid, the detectability of the label, the rate of hybridization and hybridization duration. The hybridization rate is maximized at a Ti (incubation temperature) of 20-25° C. below Tm for DNA:DNA hybrids and 10-15° C. below Tm for DNA:RNA hybrids. It is also maximized by an ionic strength of about 1.5M Na+. The rate is directly proportional to duplex length and inversely proportional to the degree of mismatching. Specificity in hybridization, however, is a function of the difference in stability between the desired hybrid and “background” hybrids. Hybrid stability is a function of duplex length, base composition, ionic strength, mismatching, and destabilizing agents (if any).
The Tm of a perfect hybrid may be estimated for DNA:DNA hybrids using the equation of Meinkoth et al. (Anal. Biochem. 1984, Vol. 138(2), p. 267), as

Tm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L 

and for DNA:RNA hybrids, as

Tm=79.8° C.+18.5(log M)+0.58(% GC)−11.8(% GC)2−0.56(% form)−820/L 

where

    
    
        M, molarity of monovalent cations, 0.01-0.4 M NaCl,
        % GC, percentage of G and C nucleotides in DNA, 30%-75%,
        % form, percentage formamide in hybridization solution, and
        L, length hybrid in base pairs.
    
    


Tm is reduced by 0.5-1.5° C. (an average of 1° C. can be used for ease of calculation) for each 1% mismatching. The Tm may also be determined experimentally.
Filter hybridization is typically carried out at 68° C., and at high ionic strength (e.g., 5-6×SSC), which is non-stringent, and followed by one or more washes of increasing stringency, the last one being of the ultimately desired high stringency. The equations for Tm can be used to estimate the appropriate Ti for the final wash, or the Tm of the perfect duplex can be determined experimentally and Ti then adjusted accordingly.
The present invention also relates to a vector comprising the nucleic acid molecule of the present invention. The vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. The term “expression vector” and “recombinant expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. The expression vector may comprise sequences encoding heterologous domains including, but not limited to, protein detection, purification or cleavage sequences that may be fused at the N- or C-terminus to the desired coding sequence, to yield a fusion protein. The present invention encompasses expression vectors that are integrated into host cell genomes, as well as episomal vectors.
Within the scope of the present invention is the combination of the FGF-18 variants with at least one other bioactive agent. As used herein and in the claims a “bioactive agent” refers to a pharmaceutically active compound which provides enhanced effect when mixed with the variants of the present invention. This includes, but is not limited to, peptides and peptide analogs, peptidomimetics, oligopeptides, proteins, apoproteins, glycoproteins, antigens and antibodies or antibody fragments thereto, receptors and other membrane proteins, aptamers, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, hormones, lipids, phospholipids, liposomes; toxins; tyrosine kinase inhibitors, photoreactive agents, antibiotics; analgesics and anti-inflammatory substances; antimicrobial agents; antihypertensive agents; antiviral agents; antihistamines; anti-cancer drugs including chemotherapeutic agents; tranquilizers; neuroprotective agents; antispasmodics; anti-Parkinson agents, vitamins, nucleotides, oligonucleotides, polynucleotides, and their biologically functional analogs and derivatives, plasmids, cosmids, artificial chromosomes, other nucleic acid vectors, antisense polynucleotides including those substantially complementary to at least one endogenous nucleic acid, promoters, enhancers, inhibitors, other ligands for regulating gene transcription and translation and the like. Each possibility represents a separate embodiment of the invention.
In particular embodiments the present invention provides the combination of the FGF-18 variants with at least one other bioactive agent, wherein the at least one other active agent is a carboxylated polysaccharide. In currently preferred embodiments, the carboxylated polysaccharide is hyaluronic acid (HA) and its derivatives including, but not limited to, the partial esters of hyaluronic acid with aliphatic, aryliphatic, heterocyclic and cycloaliphatic alcohols. Suitable molecular weights of hyaluronic acid and its partial esters range from about 104 Daltons to about three million (3×106) Daltons.
Disclosed herein for the first time is a mixture of at least one FGF-18 variant of the present invention and at least one natriuretic peptide which provides a synergistic effect. The terms “synergistic”, “cooperative” and “super-additive” and their various grammatical variations as used herein refer to e.g. an observed effect which is higher in the presence of the components together than the sum of the individual effects of each component when administered separately. In one embodiment, the observed combined effect of the FGF-18 variant and the natriuretic peptide is significantly higher than the sum of the individual effects. The term significant means that the observed p<0.05. Currently preferred is the admixture of at least one N-terminal truncated variant of FGF-18 with C-type natriuretic peptide (CNP). CNP is a known regulator of longitudinal bone growth. Genetic ablation of CNP or its signaling pathway components results in severe skeletal dysplasias caused by reduced chondrocyte proliferation and differentiation (Chusho et al., PNAS US, 2001, Vol. 98, p. 4016-; Miyazawa et al., 2002, Endocrinology, Vol. 143, p. 3604; Tamura et al., PNAS US, 2004, Vol. 101, p. 17300). This phenotype is remarkably similar to that caused by mutant FGFR3, the causative agent of achondroplasia. These findings led, in recent years, to intense investigation of the mechanism of action of CNP and its interaction with the FGF signaling pathway in chondrocytes. FGFs exert their effect mainly through the MAPK, STAT and PI3K pathways. Krejci et al. (Journal of Cell Science, 2005, Vol. 118, p. 5089) demonstrated that CNP counteracts FGF's action through MAPK inhibition. Yet, in another study, it has been shown that STAT in not affected by CNP (Ozasa et al., Bone, 2005, Vol. 36, p. 1056). Modulation of PI3K by CNP is currently not well characterized. In addition, CNP increases extracellular matrix mass independent of its interference with FGF signaling (Krejci et al., Journal of Cell Science, 2005, Vol. 118, p. 5089). Thus, CNP may utilize both direct and indirect ways to antagonize FGF signaling in chondrocytes. CNP is known as a chondrogenic factor and is usually considered to be an antagonist to FGFs. It is believed to operate by counteracting FGFs. Unexpectedly, it is now disclosed for the first time that the FGF variants of the present invention when admixed with CNP provide a synergistic effect on FGFR3. According to the principles of the present invention, the terms admixture or combination of FGF-18 variants and CNP exclude covalent conjugate or chimeric recombinant of the two components.
The present invention further provides pharmaceutical compositions comprising at least one polypeptide or polynucleotide encoding same and at least one diluent or carrier. The carrier may be any of those conventionally used and is limited only by chemical-physical considerations, such as solubility and lack of reactivity with the active ingredient of the invention, and by the route of administration. The choice of carrier will be determined by the particular method used to administer the pharmaceutical composition. Some non-limiting examples of suitable carriers include lactose, glucose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, cellulose derivatives (e.g. methylcellulose), water, calcium carbonate, various types of starch, vegetable oils and polyethylene glycols. Each possibility represents a separate embodiment of the invention. The compositions may further comprises lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents, surfactants, emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; colorants, buffering agents (e.g., acetates, citrates or phosphates), disintegrating agents, moistening agents, antibacterial agents, antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), and agents for the adjustment of tonicity such as sodium chloride. Each possibility represents a separate embodiment of the invention. Other pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Each possibility represents a separate embodiment of the invention. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other compounds which are known to increase the resistance to proteolitic degradation might also be added.
The pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Each possibility represents a separate embodiment of the invention.
The pharmaceutical compositions of this invention may be administered by any suitable means, such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally. Each possibility represents a separate embodiment of the invention. Ordinarily, intra-articular administration is currently preferred. The dosage will be prescribed according to common regimes in the art, while taking into consideration variables such as: weight, age, extent of damage etc.
The N-terminal truncated FGF-18 ligands with increased specificity for FGFR3 are useful in the treatment of various pathological conditions of the cartilage and are capable of specifically targeting the cartilage for inducing cartilage repair. Thus, the compositions of the present invention are suitable for treating any cartilage defect or disorder. The terms “cartilage defect” or “cartilage disorder” as used herein refer to cartilage that has been damaged by disease, injury or trauma including indications such as, but not limited to, rheumatoid arthritis, osteoarthritis and joint injuries. Each possibility represents a separate embodiment. The treatment is suitable for an acute injury as well as for a chronic condition requiring prolonged treatment.
The N-terminal truncated FGF-18 ligands with increased specificity for FGFR3 are useful in the treatment of various skeletal disorders and in the treatment of degenerative joint diseases. An exemplary degenerative joint disease is osteoarthritis, a disease which involves the degradation of joints, including articular cartilage and subchondral bone.
The compositions of the present invention are suitable for treating a subject in need thereof, preferably a mammal, particularly a human.
FGF-18 variants activity is conveniently determined using biological assays performed in-vitro, ex-vivo and in vivo. The assays are used to demonstrate the activity elicited upon binding of an FGF-18 variant of the present invention to its receptors. The biological assays routinely used to test activities of variant FGFs include, but are not limited to, the following: binding of variant FGFs to cloned FGF receptors expressed on immortalized cell lines, thereby eliciting a biological response including cell proliferation or inhibition of cell proliferation; cell proliferation and differentiation in cell culture systems; phosphorylation assays; and enhancement of cartilage repair in animal models of cartilage disease and trauma.
The present invention further provides a method of inducing primary chondrocyte differentiation by exposing primary chondrocytes to at least one N-terminal truncated FGF-18 variant of the present invention to induce differentiation.
The principles of the invention are demonstrated by means of the following non-limiting examples.
EXAMPLES
Example 1
Preparation of FGF-18 Polynucleotide and Polypeptide Variants
The FGF-18 variants were prepared by standard PCR amplification and cloning into a pET9a expression vector. The vector has unique NdeI-BamHI restriction sites and the PCR-generated DNA fragments encompassing the coding region of a number of proteins and variants were produced having NdeI and BamHI sites at the 5′ and 3′ ends, respectively. The polynucleotide fragments encoding the proteins were ligated into digested pET9a and the plasmid was used to transform E. coli cells, such as BL21-DE3, using standard transformation protocols.
The primers used for construction of the variants are shown in Table 1, herein below.







TABLE 1







Forward and reverse primers used in preparation of


the FGF-18 variants of the present invention.











POLY-





NUCLEOTIDE





ENCODING




FGF-18
THE FGF-18




poly-
POLYPEPTIDE

Backward


peptide
SEQ ID NO:
FORWARD PRIMER
Primer













FGF-18Δ31
8
5′ GGAATTCCATATG
SEQ ID




GACTTCCGCATC CACG
NO: 61




SEQ ID NO: 65



 


FGF-18Δ33
12
5′ GGAATTC CATATG
SEQ ID




CGCATCCACGTGGAGAAC
NO: 61




SEQ ID NO: 66



 


FGF-18Δ37
20
5′ GGAATTC CATATG
5′ ACGT




GAGAACCAGACGCGGGC
GGATCC




SEQ ID NO: 60
CTAGGCAGGG





TGTGTGG





SEQ ID





NO: 61


 


FGF-18Δ45
36
5′ GGAATTC CATATG
SEQ ID




GATGTGAGCCGTAAGCAG
NO: 61




SEQ ID NO: 62



 


FGF-18Δ51
48
5′ GGAATTC CATATG
SEQ ID




CTGCGGCTGTACCAGCTC
NO: 61




SEQ ID NO: 63



 


FGF-18
59
5′ GGAATTC CATATG
SEQ ID


(recom-

GCCGAGGAGAACGTGG
NO: 61


binant

SEQ ID NO: 64



wild





type)









A single colony was selected and grown overnight (ON) at 37° C. in 5 ml 2YT (8O g Trypton; 50 g yeast extract; 25 g NaCl for 5 Liters) supplemented with 200 μg/ml ampicillin. The next day, the culture was diluted 100 times. At O.D.600=0.6 0.1 mM IPTG was added and bacteria were further fermented over night. The bacterial suspension was centrifuged at 4000 rpm (4° C.) for 15 minutes, and the medium was discarded. The bacterial pellet was then suspended in 10 ml of 1×PBS+1 M NaCl buffer containing protease inhibitors, sonicated on ice, shaked over night at 4° C. and centrifuged at 10,000 rpm (4° C.) for 15 minutes. The protein supernatant was collected, filtered and diluted 5 times with 1×PBS. FGF polypeptides were purified on FPLC using a heparin Sepharose column (HiTrapTM Heparin, Amersham Pharmacia biotech) with a 0-2.5M NaCl (in PBS). The purified proteins were later stored at −70° C.
Example 2
FGF-18 Variant Binding to FGFR-Transfected FDCP Cell Lines
The FDCP cell line is a murine immortalized, interleukin 3 (IL-3)-dependent cell line of myelocytic bone marrow origin that does not express endogenous FGF Receptors (FGFR). Upon transfection with FGFR cDNA, the FDCP cell line exhibits a dose-dependent proliferative response to FGF that can replace the dependence on IL-3. FGFR transfected FDCP cells can therefore be used to screen variant FGFs for specific inhibitors, activators or for FGFR signaling. FDCP cells response to various ligands is quantitated by a cell proliferation assay with XTT reagent (Cell Proliferation Kit, Biological Industries Co.). The method is based on the capability of mitochondrial enzymes to reduce tetrazolium salts into a colorogenic compound, which can be quantitated and is indicative of cell viability.
Specifically, FDCP cells stably expressing FGFR4, FGFR3-IIIc, FGFR2IIIc or FGFR1IIIc were grown in “full medium” (Iscove's Medium containing 2 ml glutamine, 10% FCS, 100 μg/ml penicillin, 100 μg/ml streptomycin) supplemented with 5 μg/ml heparin and 10 ng/ml FGF. Cells were split every 3 days and kept in culture for up to one month. One day prior to the experiment the cells were split. Before the experiment, the cells were washed 3 times (1000 rpm, 6 min) with full medium. The cells were re-suspended and counted with the VI-cell counter (Beckman Coulter). Twenty thousand (2×104) cells were added to each well of 96-well plate in 50 μl full medium containing heparin. Conditioned medium containing FGF wild type parent or variants at varying concentrations with heparin was added in an additional volume of 50 μl full medium to bring the final volume to 100 μl. The plate was incubated for 48 hours at 37° C. To assay cell proliferation, 100 μl of PMS reagent was added to 5 ml of XTT reagent and mixed thoroughly (according to manufacturer's protocol). Fifty micro-liters (50 μl) of the latter solution were added into each well, and the plates incubated at 37° C. for 4 hours and the color was read by a spectro-ELISA reader at A490nm. In these experiments FDCP cells expressing FGFR4, FGFR3, FGFR2 or FGFR1 were grown in the presence of varying concentrations of the polypeptides of the invention.
Example 3
Effect of Variants on Growth Inhibition of RCS Chondrocytes
RCS is a rat chondrosarcoma derived cell line expressing preferentially high levels of FGFR2 and FGFR3 and low levels of FGFR1. In this cell line, FGFR functions as an inhibitor of cell proliferation similar to its expected role in the growth plate of the developing bone and in accord with the achondroplasia phenotype. In order to inhibit cell proliferation, the variants have to specifically induce FGFR signal transduction allowing the measuring of FGF affinity and specificity to the FGFRs reflected by the concentration dependence of induced growth inhibition.
The screening was performed on RCS cells in 96 well plates. Cells were seeded at a concentration of about 2,000 cells/well. The following day 10 ng/ml FGF or FGF variants and 5 μg/ml heparin were added to the cells. Positive and negative controls for cell proliferation were included in this assay at the same concentrations as the tested compounds. The Cy-Quant® assay kit was used to measure the amount of the cells. The results were measured in a fluoro ELISA reader.
Example 4
Chondrocyte Expansion
The effect of FGF-18 and FGF-18 variants on proliferation of articular chondrocytes was tested.
Articular Chondrocyte Culture:
Chondrocytes were isolated from human biopsies and cultured using the FGF variants of the present invention to identify the effect of the variants on proliferation and differentiation. The procedure employed for the isolation and propagation of articular chondrocytes is presented below.
Reagents:
Dulbecco's DMEM/F12 (Gibco BRL)
Fetal Bovine Serum (FBS) (Gibco BRL)
Streptomycin, Penicillin, Nystatin Solution (Biological Indus. Ltd.)
Trypsin-EDTA (Gibco BRL, cat. no. T8154) or Versene-Trypsin (Bio LAB Ltd.)
Isolation of Cells from Cartilage Biopsy:
A piece of cartilage tissue was minced into 2 to 4 mm pieces with a sterile scalpel. The collagenase solution was diluted 1:4 in FBS-DMEM, added to the tissue sample and left to incubate on a rotator at 37° C., overnight (ON). The cells were centrifuged (1200 rpm, 5-10 min). The medium was aspirated, the cells washed in 5 ml medium and re-centrifuged. The cells were re-suspended in culture medium and seeded in 25 cm2 or 75 cm2 flasks at a concentration of approximately 1×106 cells per flask. The cells were incubated in a 5% CO2 incubator at 37° C. The cell medium was replaced every 2-3 days.
Procedure for Passaging Cells (Trypsinization):
When the cell culture reached the desired confluency, the medium was removed and the cells trypsinized in the following manner: One milliliter (1 ml) of the trypsin solution was added to a 25 cm2 flask or 2.5 ml to a 75 cm2 flask. The flask was gently shaken by hand for two seconds and the trypsin solution was immediately removed. Another 1 ml of trypsin was added to a 25 cm2 flask or 2.5 ml to a 75 cm2 flask. The flask was gently shaken by hand for  ̃30 seconds and left to incubate at 37° C. for 3-5 minutes. Verification that cells were detached was performed under the microscope. The trypsin was neutralized by adding FBS-DMEM; followed by addition of 10 ml to a 25 cm2 flask and 25 ml to a 75 cm2 flask. The cells were split to 2-3 new flasks and 20 ml fresh pre-warmed medium was added. The expansion of cells and trypsinization was performed as necessary.
Furthermore, the cell population grown on the above matrices expresses several of the chondrocyte differentiation markers. One of several phenotypes expressed during chondrocyte differentiation is glycosaminoglycan (GAG) production. The production of GAGs is identified in histological staining using Alcian blue and quantitated using the DMB (3,3′-dimethoxybenzidine dihydrochloride) dye method.
Proliferation of the cartilage cells in the presence of the different variants can be quantitated by cell counting, CyQUANT® (Molecular Probes) or XTT reagent (Biological Industries, Co.).
Example 5
Human Articular Chondrocytes: Isolation and Proliferation
Human articular chondrocytes were isolated from pieces of cartilage using digestion enzymes. Isolated primary chondrocytes were seeded in 75 cm2 flasks (5×104 cells) containing DMEM/F12, 20% pooled human serum, 10 ng/ml of different FGF variants of the present invention and 5 μg/ml low molecular weight heparin (LMW). Cells were counted at different time points using the Vi-Cell XR.
Example 6
Human Articular Chondrocytes: In-Vitro Chondrogenesis Assay
The expanded chondrocytes were used for in-vitro chondrogenesis assay to produce high density pellet cultures. The pellet cultures were incubated for up to 21 days in chondrocytes differentiation medium containing DMEM, 10 ng/ml TGF-b, 100 nM dexamethasone, 0.28 mM ascorbic acid, 1 mM sodium pyruvate, 40 μg/ml proline, 10 μg/ml bovine insulin, 5.5 μg/ml human transferin, 5 μg/ml sodium selenite, 0.5 mg/ml bovine serum albumin and 1.7 μg/ml linoleic acid. Some pellet cultures were supplemented with FGF variant of the present invention or the FGF wt.
Example 7
Human Articular Chondrocytes: Histology Analysis of Pellet Cultures
Pellet cultures incubated for up to weeks in differentiation medium were fixed in PFA solution and paraffin blocks were prepared. Sections were used for analysis of proteoglycans by Alcian blue (AB) and Safranin O (SO) stains.
Example 8
Cell Proliferation of Primary Porcine Chondrocytes
Primary porcine chondrocytes were harvested from articular cartilage of adult pig using overnight collagenase:pronase digestion. The cells were seeded in 6-well plate (300,000/well) that was pre-coated with 200 ug/ml plasma fibrinogen in DMEM-F12 for 1 hr at 370 C. The cells were cultured in 2 ml/well DMEM-F12+2% FBS+100 ug/ml ascorbic acid+ITS-1 (1:100)+pen-strep. The variants (100 ng/ml) were added the next day. The media were replaced with fresh variants 3 days later. The cells were harvested at day 5 and counted.
Example 9
Chondrocyte Differentiation
Cartilage taken from osteoarthritis patients was dissected and pre-incubated for 3 days in control medium (day 0) followed by 4 days in the presence of 50 ng/ml of different FGFs. Cell supernatant was collected and levels of secreted glycosaminoglycans (sGAGs) were determined.
Example 10
In-Vivo Studies in a Mouse Model of Injury-Induced Osteoarthritis
The beneficial effects of the FGF-18 variants of the present invention are evaluated in a mouse model of injury-induced osteoarthritis using intra-articular injections. Specifically, the variants of the present invention are compared to wild type FGF-18 in terms of their efficacy. An evaluation of the possible side effects is also performed. Three groups, each containing 6 mice, are tested. Group 1. carrier (0.5% hyaluronic acid); Group 2. FGF-18 wild type (0.1 μg)+carrier; and Group 3. FGF-18 variant of the present invention (0.1 μg)+carrier. The injections are administered bi-weekly for a period of 3 weeks. The FGFs are administered in sterile PBS and a sterile solution of 2× concentrated carrier solution (1% hyaluronic acid (HA; MW=1.5×106 Da)).
Experimental osteoarthritis is induced by transsection of the medial anterior meniscotibial ligament which causes destabilization of the medial meniscus (DMM). The DMM model was developed by Glasson et al (Osteoarthritis Cartilage. 2007; 15:1061-9). Briefly, mice are anesthetized, hair is removed from the skin of the right knee joint and an incision is made in the skin medial from the patellar ligament. Subsequently, an incision is made in the joint capsule, again medial from the patellar ligament. The joint is opened and suppatellar fat is removed. The ligament is identified and transected. After verifying that all bleeding into the joint has stopped, the joint is flushed with excess saline and the joint capsule is closed with 3 sutures (resorbable). Finally, the skin is closed using 1 suture (resorbable) and tissue adhesive. The mice are housed in groups of 5-6 animals and 8 weeks after surgery full blown osteoarthritis pathology is present and knee joints are isolated for histology.
Treatment with various formulations is initiated 3-4 weeks post surgery. Immediately prior to injection, sterile solution of 2× carrier solution (1% hyaluronic acid) is mixed with an equal volume of sterile PBS containing the various FGF ligands at concentration that yields amounts of 0.1 μg or 0.5 μg of ligand per injection. Final dilution before injection is performed by diluting the ligands with equal volume of 2× carrier solution. The final volume of injection is 6-10 μl per animal. Animals receive bi-weekly intra-articular injections for 3 consecutive weeks (a total of 6 injections).
Evaluation is performed at 8 weeks post surgery using both H&E and proteoglycan staining (e.g. toluidine blue, saffranin-O).
Mouse strain: C57Bl/6 (12 weeks old, 25-30 g)
Group size: 6 animals
Osteoarthritis model: DMM
Housing: 6 mice per cage with wood chip bedding
Conditions: Mice are acclimatized after arrival for at least 2 weeks
Food/water: Food and acidified water ad libitum
Scoring of the osteoarthritis by histology is performed as follows: osteoarthritis joints are fixed in formalin for 3 days, followed by decalcification for 1 week and embedding in paraffin. All joints are sectioned semi-serial: coronal plane, 7 μm whole knee joint sections. Staining by Fast Green/Saffranin 0. Scoring of osteoarthritis cartilage damage is performed following an advanced version of the murine OARSI score, at 4 locations (med and lat femur; med and lat tibia). Extra scores which are further included are synovial inflammation, osteophyte formation and subchondral bone changes.
Results:
FGF-18 and the N-terminal truncated variants (FGF-18Δ37, FGF-18Δ45 and FGF-18Δ51) were tested for receptor specificity by binding to immobilized extracellular domain of FGFR1-4 (FIG. 2). Biotinylated FGF's were added at increasing concentrations to FGFR1-4 anchored to a MaxiSorp ELISA plate. Bound ligands were detected by HRP-streptavidin. While FGF-18 wild type activates FGFR1-4, the FGF-18 truncated variants of the present invention do not activate FGFR1-2 while maintaining the activation toward FGFR3-4 (Table 2). The variants of the present invention are thus shown to be specific for FGFR3-4.







TABLE 2







EC50 scores of biotinylated FGF ligands in immobilized


receptor assay of FDCPR1-4-Fc.













EC50 (ng/ml)
R1
R2
R3
R4

















F18
28.21
9.51
5.04
9.23



F18Δ37
—
—
5.94
5.86



F18Δ45
—
—
9.16
8.10



F18Δ51
—
—
10.21
9.93










To further explore the receptor binding specificity of these variants, their biological activity in a receptor dependent cell based FDCP assay was examined (FIG. 3). Table 3 summaries the EC50 scores of FGF-2 wild type, FGF-18 wild type, FGF-18Δ37, FGF-18Δ45 and FGF-18Δ51 ligands used. Primary chondrocytes harbor multiple FGFRs (FIG. 4) and respond to various FGFs by proliferation or differentiation depending on their differentiation state and expressed receptor profile. Activation of FGFR1 and FGFR2 usually leads to cell division and prolifreration while FGFR3 signaling typically results in chondrocyte differentiation, maturation and matrix deposition.







TABLE 3







EC50 scores of FGF ligands in cell proliferation of FDCPR3.










Ligand
EC50 (ng/ml)














F18 w.t.
15.40



F18Δ37
3.14



F18Δ45
5.44



F18Δ51
3.97



F2 w.t.
1.19










FGF-18 was further compared to FGF-2 wild type, a ligand that preferentially binds and activates FGFR1 and FGFR2, for its proliferative effect on young primary articular chondrocytes (PAC). FGF-18 was found to be approximately 5 folds less active than FGF-2 (both at 20 ng/ml, FIG. 5) in activating PAC. In a subsequent experiment, FGF-18 and the N-terminal variants of the present invention were tested for their proliferative effect on the same cells. While FGF-18 wild type treatment led to approximately 1.5 folds increase in PAC proliferation as compared to the control, FGF-18 truncated variants showed no detectable effect on chondrocytes cell proliferation (FIG. 6).
In order to further explore the proliferative capacity of FGF-18 and its FGFR3 specific variants, FGF-18 wild type and FGF-18Δ51 were tested at very high doses of up to 200 ng/ml. Remarkably, even at 200 ng/ml, FGF-18 was not saturable with approximately 2.5 folds increase in PAC as compared to un-treated cells, suggesting an extremely low affinity of this ligand to its receptors at the conditions tested. At all concentrations used, FGF-18Δ51 had no proliferative activity. Thus, the FGF N-terminal truncated variants of the present invention are highly selective to FGFR3 and show lack of a proliferative response via FGFR3 in primary chondrocytes (FIG. 7).
Rat chondrosarcoma (RCS) cell line is a unique chondrocyte derived cell type representing a mature prehypertrophic chondrocyte that responds to FGF signaling by growth arrest in the G1 phase of the cell cycle (Aikawa et al., J. Biol. Chem., 2001, Vol. 276, p. 29347). Addition of FGF-9 or FGF-18 to RCS cells resulted in a complete block of cell proliferation (FIG. 8). Addition of N-terminal truncated FGF-18 variants also inhibited RCS cell proliferation yet to a lesser extent. Incubation of RCS cells with PRO-001 (WO 07/144,893), a potent and highly specific anti-FGFR3 antibody partially reversed the inhibitory action of FGF-9 yet completely restored cell proliferation in the presence of the receptor specific FGF9W144G ligand (WO 02/36732). Likewise, PRO-001 moderately reversed the action of wild-type FGF-18 and completely reversed the action of FGF-18 N-terminal variants suggesting that the wild-type FGF-18 activates in addition to FGFR3 other FGFRs while the N-terminal variants are FGFR3 specific.
The effect of FGF-18 wild type and FGF-18Δ51 on chondrocytes' pellet culture formation is shown in FIG. 9. The ligands provided an increase in mass culture of PAC.
To further delineate the location of receptor specificity, FGF-18 variants lacking 31 or 33 amino acids from the N-terminus (FGF-18Δ31 and FGF-18Δ33, respectively) were tested using functional ELISA. While FGF-18Δ33 did not bind FGFR2 and FGFR1, FGF-18Δ31 recognized all 4 receptors similar to wild-type FGF-18. Table 4 indicates the EC50 of the different N-terminal FGF-18 variants to each FGF receptor.







TABLE 4







EC50 scores of the indicated variants to each receptor type


using functional ELISA.










EC50 (ng/ml)













FGFR1
FGFR2
FGFR3
FGFR4

















FGF18 WT
22.80
 8.76
6.59
10.21



FGF18Δ31
24.38
11.22
8.55
9.36



FGF18Δ33
ND
ND
8.27
7.11



FGF18Δ37
ND
ND
9.75
8.28



FGF18Δ51
41.94
43.47
9.99
10.62







ND—Not Detected






The mitogenic activity of FGF-18Δ31 and FGF-18Δ33 was further compared to the mitogenic activity of FGF-18Δ37, FGF-18Δ51 and wild-type FGF-18 in primary porcine chondrocytes. FGF-18Δ33 did not induce proliferation while FGF-18Δ31 demonstrated comparable mitogenic activity with that of the wild-type FGF-18 (FIG. 10). Without being bound by any theory or mechanism of action, the receptor binding activity as well as the mitogenic activity results imply that amino acids residues 32-37 of FGF-18 are important for binding and activation of FGFR1 and FGFR2 by FGF-18.
Another FGF-18 N-terminal truncated variant (451) which further comprises a conservative substitution at the penultimate leucine (L52) with isoleucine denoted FGF-18Δ51L52I (also denoted as FGF-18Δ51I) was produced. BL21 DE3 bacteria were transformed with pET-FGF-18Δ51L52I and the expression level was compared to that of pET-FGF-18Δ51. Western immunoblot analysis demonstrated a significant increase in protein level of FGF-18Δ51L52I (FIG. 11). The specificity of FGF-18Δ51L52I was then compared to that of FGF-18Δ51 by functional ELISA. FIG. 12 shows that FGF-18Δ51L52I retained the same receptor specificity as FGF-18Δ51. In order to evaluate the biological activity of FGF-18Δ51L52I, the variant was added to FDCP-FGFR3 cells and cell proliferation was compared to that induced by FGF-18Δ51 and wild-type FGF-18. FGF-18Δ51L52I and FGF-18Δ51 were both superior to the wild-type FGF-18 (FIG. 13).
Next, the effect of FGF-18Δ51L52I on chondrocyte differentiation was examined. FGFs induced GAG secretion and FGF-18Δ51L52I was at least as potent as wild-type FGF-18 (FIG. 14). FGF-18Δ37 induced GAG synthesis most effectively.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

## Claims

1. An isolated FGF-18 polypeptide having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue, the isolated FGF-18 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 67, and SEQ ID NO: 69, and having increased receptor selectivity when compared to an isolated wild-type FGF-18 polypeptide by a gain of activity by at least a factor of two toward FGFR3 but not toward all FGFR subtypes.

2. The isolated FGF-18 polypeptide according to claim 1, wherein the isolated FGF-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 47.

3. The isolated FGF-18 polypeptide according to claim 1, wherein the isolated FGF-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 67.

4. The isolated FGF-18 polypeptide according to claim 1, wherein the isolated FGF-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 69.

5. A pharmaceutical composition comprising as an active ingredient at least one isolated FGF-18 polypeptide having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue, the isolated FGF-18 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 67, and SEQ ID NO: 69, and having increased receptor selectivity when compared to an isolated wild-type FGF-18 polypeptide by a gain of activity by at least a factor of two toward FGFR3 but not toward all FGFR subtypes.

6. The pharmaceutical composition of claim 5, further comprising at least one bioactive agent.

7. The pharmaceutical composition according to claim 6, wherein the at least one bioactive agent is a carboxylated polysaccharide.

8. The pharmaceutical composition according to claim 7, wherein the carboxylated polysaccharide is hyaluronic acid.

9. The pharmaceutical composition according to claim 6, wherein the at least one bioactive agent is a C-type natriuretic peptide.

10. A method of inducing cartilage repair comprising the step of administering to an individual in need thereof a pharmaceutical composition according to claim 5.

11. A method of inducing cartilage repair in an individual with a degenerative joint disease comprising the step of administering to an individual in need thereof a pharmaceutical composition according to claim 5.

12. The method according to claim 11, wherein the degenerative joint disease is osteoarthritis.

13. The pharmaceutical composition of claim 5, wherein the isolated FGF-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 47.

14. The pharmaceutical composition of claim 5, wherein the isolated FGF-18 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 67 and SEQ ID NO: 69.

15. A pharmaceutical composition comprising as an active ingredient at least one isolated polynucleotide encoding an FGF-18 polypeptide having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue, the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 67, and SEQ ID NO: 69, and having increased receptor selectivity when compared to an isolated wild-type FGF-18 polypeptide by a gain of activity by at least a factor of two toward FGFR3 but not toward all FGFR subtypes.

16. A method of inducing cartilage repair comprising the step of administering to an individual in need thereof a pharmaceutical composition according to claim 15.

17. A method of inducing cartilage repair in an individual with a degenerative joint disease comprising the step of administering to an individual in need thereof a pharmaceutical composition according to claim 15.

18. An isolated FGF-18 polypeptide having a 50 amino acid N-terminal truncation with the Gln51 replaced by a Met residue and the Leu52 replaced by an amino acid residue other than leucine, the isolated FGF-18 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 67 and SEQ ID NO: 69, and having increased receptor selectivity when compared to an isolated wild-type FGF-18 polypeptide by a gain of activity by at least a factor of two toward FGFR3 but not toward all FGFR subtypes.

19. The polypeptide according to claim 18, wherein the Leu52 is replaced by an amino acid residue selected from isoleucine, valine and methionine and wherein said polypeptide has the amino acid sequence of SEQ ID NO: 67.

20. The polypeptide according to claim 19, wherein the Leu52 is replaced by isoleucine, and wherein said polypeptide has the amino acid sequence of SEQ ID NO: 69.