Chimeric fibroblast growth factor proteins, nucleic acid molecules, and uses thereof Number:6,800,286 from the United States Patent and Trademark Office (PTO) owispatent A chimeric fibroblast growth factor protein and recombinant nucleic acid molecule encoding the same are disclosed. The chimeric fibroblast growth factor protein is characterized by: fibroblast growth factor biological activity in the absence of heparan sulfate and, entry into a living cell in the absence of a receptor that binds to FGF. Also disclosed are a method of making the chimeric fibroblast growth factor protein and methods of using the chimeric fibroblast growth factor protein to promote fibroblast growth factor activity in a cell and to enhance a biological process associated with fibroblast growth factor activity.<H fibroblast, molecules, Chimeric, fibrob, 6800286.html, Patent, pto, 6800286

Title: Chimeric fibroblast growth factor proteins, nucleic acid molecules, and uses thereof

Abstract: A chimeric fibroblast growth factor protein and recombinant nucleic acid molecule encoding the same are disclosed. The chimeric fibroblast growth factor protein is characterized by: fibroblast growth factor biological activity in the absence of heparan sulfate and, entry into a living cell in the absence of a receptor that binds to FGF. Also disclosed are a method of making the chimeric fibroblast growth factor protein and methods of using the chimeric fibroblast growth factor protein to promote fibroblast growth factor activity in a cell and to enhance a biological process associated with fibroblast growth factor activity.

Patent Number: 6,800,286 Issued on 10/05/2004 to Olwin, et al.

Inventors: Olwin; Bradley B. (Boulder, CO), Rosenthal; Richard Scott (Raleigh, NC)
Assignee: The Regents of the University of Colorado (Boulder, CO)

Appl. No.: 09/377,675

Filed: August 19, 1999

Current U.S. Class: 424/185.1 ; 424/192.1; 514/12; 514/2; 530/350; 530/399

Field of Search: 530/350,399 514/2,12,44 424/192.1,185.1

References Cited [Referenced By]

U.S. Patent Documents


5604293	February 1997	Fiddes et al.
5804604	September 1998	Frankel et al.
5888762	March 1999	Joliot et al.

Foreign Patent Documents


WO 91/18981	Dec., 1991	WO
WO 97/12912	Apr., 1997	WO

Other References

Derossi et al., J. Biol. Chem., 269:10444-10450 (1994). .
Femig et al., Progress in Growth Factor Research, 5:353-377 (1994). .
Perez et al., J. Cell Sci., 102:717-722 (1992)..

Primary Examiner: Ulm; John
Assistant Examiner: Chernyshev; Olga N.
Attorney, Agent or Firm: Sheridan Ross, P.C.

Government Interests

GOVERNMENT SUPPORT

This invention was made in part with government support under NIH Grant AR39467 and NIH Grant HL07851, each awarded by the National Institutes of Health. The government has certain rights to this invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. .sctn. 119(e) from U.S. Provisional Application Ser. No. 60/097,160, filed Aug. 19, 1998, entitled "Sugar Free FGF". The entire disclosure of U.S. Provisional Application Ser. No. 60/097,160 is incorporated herein by reference.

Claims

What is claimed is:

1. A chimeric fibroblast growth factor-2 (FGF-2), comprising: a) a biologically active fibroblast growth factor-2 (FGF-2) protein having a first amino acid sequence that is encoded by a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence encoding a fibroblast growth factor-2 (FGF-2) protein represented by SEQ ID NO:5 or SEQ ID NO:6, wherein the FGF-2 protein has an FGF-2 biological activity selected from the group consisting of: promotion of cell proliferation, repression of terminal differentiation in a cell, promotion of angiogenesis, promotion of wound healing, promotion of osteogenesis, and promotion of nerve outgrowth; and, b) a penetratin peptide having a second amino acid sequence, wherein the penetratin peptide is selected from the group consisting of: i) a first peptide comprising an amino acid sequence selected from the group consisting of: 1) X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 -X.sub.8 -X9-X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 ; and, 2) X.sub.16 -X.sub.15 -X.sub.14 -X.sub.13 -X.sub.12 -X.sub.11 -X.sub.10 -X.sub.9 -X.sub.8 -X.sub.7 -X.sub.6 -X.sub.5 -X.sub.4 -X.sub.3 -X.sub.2 -X.sub.1 ; wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, X.sub.15, and X.sub.16 each represent an .alpha.-amino acid, between 6 and 10 of which are hydrophobic amino acids; and wherein X.sub.6 represents Trp; and, ii) a second peptide comprising amino acid residues 49-57 of HIV Tat protein (SEQ ID NO:17). wherein the biological activity of said penetratin peptide is to transport said chimeric fibroblast growth factor-2 (FGF-2) across a lipid bilayer of a cell independently of the presence of an FGF-2 receptor;

wherein said second amino acid sequence is linked to said first amino acid sequence; and

wherein said chimeric fibroblast growth factor-2 (FGF-2) is characterized by: i) said FGF-2 biological activity of (a) in the absence of heparan sulfate; and, ii) entry into a living cell in the absence of a receptor that binds to FGF-2.

2. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said chimeric FGF-2 has biological activity that is characterized by: a) repression of terminal differentiation in the absence of heparan sulfate; and, b) promotion of cell proliferation in the absence of heparan sulfate.

3. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said second amino acid sequence is linked to the N-terminus of said first amino acid sequence.

4. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first amino acid sequence is selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.

5. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said FGF-2 protein has an amino acid sequence comprising from position 18 through position 172 of SEQ ID NO:2 or from position 17 through 171 of SEQ ID NO:4.

6. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said second peptide does not comprise amino acid residues 22-36 or 73-86 of HIV Tat protein (SEQ ID NO:]17).

7. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first peptide is selected from the group consisting of a peptide comprising helix 3 of a homeobox domain and a homeobox domain.

8. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:9, amino acid residues 42 through 58 of SEQ ID NO:9, amino acid residues 43 through 59 of SEQ ID NO:9, amino acid residues 43 through 58 of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

9. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first peptide comprises amino acid residues 2-17 of SEQ ID NO:2.

10. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said second peptide comprises an amino acid sequence from an HIV Tat protein selected from the group consisting of amino acid residues 37-72 of SEQ ID NO:17, amino acid residues 38-72 of SEQ ID NO:17, amino acid residues 47-72 of SEQ ID NO:17, amino acid residues 37-58 of SEQ ID NO:17, amino acid residues 38-58 of SEQ ID NO:17, amino acid residues 47-58 of SEQ ID NO:17, amino acid residues 1-21 and 38-72 of SEQ ID NO:17, amino acid residues 47-62 of SEQ ID NO:17, amino acid residues 38-62 of SEQ ID NO:17, amino acid residues 1-72 of SEQ ID NO:17, amino acid residues 1-58 of SEQ ID NO:17, and amino acid residues 48-60 of SEQ ID NO:17.

11. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said second peptide comprises amino acid residues 48-60 of SEQ ID NO:17 or amino acid residues 2-14 of SEQ ID NO:4.

12. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said chimeric fibroblast growth factor-2 (FGF-2) comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 (HLX-FGF) and SEQ ID NO:4 (TAT-FGF).

13. A the rapeutic composition comprising the chimeric fibroblast growth factor-2 (FCF-2) of claim 1 and a pharmaceutically acceptable excipient.

14. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first amino acid sequence is SEQ ID NO:5.

15. The chimeric fibroblast growth factor-2 (FGF-2) of claim 1, wherein said first amino acid sequence is SEQ ID NO:6.

Description

FIELD OF THE INVENTION

The present invention generally relates to a chimeric fibroblast growth factor protein, and in particular, to a chimeric fibroblast growth factor protein which does not have an absolute requirement for heparan sulfate for biological activity. The present invention also relates to nucleic acid molecules encoding such a protein, and to therapeutic methods of using such a protein.

BACKGROUND OF THE INVENTION

Fibroblast growth factors (FGFs) comprise a growing family of proteins found throughout various organs and tissues of both developing and adult mammals. FGFs have been shown to mediate or influence numerous biological processes including mitogenesis, angiogenesis, wound healing, and neurogenesis, as well as limb patterning and outgrowth. Two particularly well known members of the FGF family include FGF-1 and FGF-2, also referred to as acidic FGF and basic FGF, respectively.

FGF-2, also referred to as basic fibroblast growth factor (bFGF), was one of the first FGFs to be identified and has been extensively studied. FGF-2 has been shown to be able to elicit various biological responses by binding to and activating specific cell-surface receptors called FGF receptor tyrosine kinases. In addition to the FGF receptor tyrosine kinase, it is generally agreed that heparan sulfate proteoglycans (or its soluble analog heparin) are necessary for both the FGF/FGF receptor interaction and the resulting biological activity. A relatively small number of studies have implicated the FGF ligand to have a role in mediating the biological activity of these factors, yet the mechanism by which this occurs remains poorly understood.

The commonly accepted paradigm for growth factor mediated activation of receptor tyrosine kinases depicts ligand-facilitated multimerization and trans-phosphorylation of the cognate receptor resulting in the recruitment of intracellular adapter and signal-transducing molecules. A complex cascade of intracellular signaling events terminating in the nucleus is thought to dictate the resulting biological response(s) (Fantl, et al., (1993) Ann. Rev. Biochem., 62:453-81; Klint, et al., (1999) Frontiers in Bioscience4: D165-77). Concomitantly, the ligand is internalized and subjected to degradation or other alternative fates (Cuatrecasas, (1982) Epidermal growth factor: uptake and fate. Ciba Foundation Symposium, 96-108; Lewis, et al., (1996)Exp. Eye Res., 62:309-24; Massagu, etal., (1986) J. Cell. Phys., 128:216-22; Naka, et al., (1993) Febs Letters, 329:147-52; Sorkin, et al., (1988) Exp. Cell Res., 175:192-205). However, mounting evidence for a number of growth factors and cytokines (FGF, nerve growth factor, PDGF, Schwannoma-derived growth factor, insulin, angiotensin 11 and growth hormone) suggest that they may act intracellularly and in many cases support a site of action for these factors in the nucleus (Jans, et al., (1998) Bioessays, 20:400-11; Prochiantz, et al., (1995) Bioessays, 17:39-44; Imamura, et al., (1990) Science, 249:1567-1570; Kimura, H. (1993) Proc. Natl Acad. Sci. USA, 90:2165-9). This has been extensively documented for the FGF family (Imamura, et al., (1990) Science, 249:1567-1570; Baldin, et al., (1990) EMBO J., 9:1511-1517; Imamura, et al., (1994) Exp. Cell Res., 215:363-372). However, the only specific activity described for FGF in the nucleus is enhancement of ribosomal RNA synthesis (Bouche, et al., (1987) Proc. Natl. Acad. Sci. USA, 84:6770-6774). This activity was also correlated with the ability of FGF-2 to bind to and regulate the activity of protein kinase CK2 which has been shown to act directly on nucleolin, a nucleolar protein involved in the control of rDNA transcription (Bonnet, et al., (1996) J. Biol. Chem., 271:24781-7). Additionally, a number of studies have shown that translocation of FGF-2 or FGF-1 to the nucleus either in the absence or presence of their cognate receptors is involved in DNA synthesis, but specific FGF targets have not been identified (Hawker, et al., (1994) Am. J. Phys., 266:H107-20; Hawker, et al., (1994) In Vitro Cellular And Developmental Biology. Animal30A:653-63; Wiedlocha, et al. (1996) Mol. Cell. Biol, 16:270-280; Wiedlocha, et al., (1994) Cell, 76:1039-1051). FGF-1 and FGF-2 ligands have been detected in intracellular compartments. Both ligands have been proposed to have specific intracellular sites of action that include stimulation of DNA synthesis for FGF-1 and stimulation of ribosomal gene transcription for FGF-2. A receptor-independent role for FGF-1 has been proposed using an FGF-1-Diphtheria toxin conjugate, which allowed receptor-independent, cytoplasmic entry of FGF-1.

The evidence for the activity of FGF proteins in a variety of beneficial biological processes, combined with the evidence indicating an intracellular site of action and a potential direct role for FGF proteins in signal transduction affecting cell proliferation and differentiation, make FGF proteins a desirable candidate molecule for the development of modified proteins as regulators of cell growth and differentiation, for the use in applications such as promoting wound healing, treating myocardial infarction (Svet-Moldavsky, G. J., et al, Lancet (Apr. 23, 1977) 913; U.S. Pat. Nos. 4,296,100 and 4,378,347), treating degenerative neurological disorders, such as Alzheimer's disease and Parkinson's disease (Walicke, P., et al, Proc Natl Acad Sci (USA) (1986) 83:3012-3016), promoting angiogenesis, promoting bone healing, and promoting muscle healing. Therefore, there is a need in the art for modified FGF proteins having FGF biological activity and novel attributes which improve their suitability for use in therapeutic protocols.

SUMMARY OF THE INVENTION

The present invention generally relates to a chimeric fibroblast growth factor (FGF) protein characterized by: (a) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (b) an ability to enter a living cell in the absence of a receptor that binds to FGF. The present invention also relates to recombinant nucleic acid molecules encoding such a chimeric FGF protein, to therapeutic compositions including such a chimeric FGF protein, and to methods of making and using such a chimeric FGF protein.

One embodiment of the present invention is a chimeric fibroblast growth factor (FGF) which includes: (a) a biologically active fibroblast growth factor (FGF) protein having a first amino acid sequence; and, (b) a penetratin peptide having a second amino acid sequence. The penetratin peptide transports the chimeric fibroblast growth factor (FGF) across a lipid bilayer of a cell independently of the presence of an FGF receptor, and the second amino acid sequence is linked to the first amino acid sequence. The chimeric fibroblast growth factor (FGF) is characterized by: (i) fibroblast growth factor (FGF) biological activity in the absence of heparan sulfate; and, (ii) entry into a living cell in the absence of a receptor that binds to FGF. In one embodiment, the FGF biological activity of(i) is characterized by: (a) repression of terminal differentiation in the absence of heparan sulfate; and/or, (b) promotion of cell proliferation in the absence of heparan sulfate. In a preferred embodiment, the second amino acid sequence is linked to the N-terminus of the first amino acid sequence.

In the chimeric FGF of the present invention, the FGF protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding any naturally occurring FGF protein, with an FGF protein selected from the group consisting of fibroblast growth factor-1 (FGF-1) protein and fibroblast growth factor-2 (FGF-2) protein being preferred. The FGF protein encoded by the nucleic acid molecule has FGF biological activity. In a preferred embodiment, the FGF protein is selected from the group consisting of a fibroblast growth factor-1 (FGF-1) protein and a fibroblast growth factor-2 (FGF-2) protein. Other preferred FGF proteins include, but are not limited to: FGF proteins having an amino acid sequence selected from the group of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A preferred FGF protein for use in the chimera of the present invention is a fibroblast growth factor-2 protein. In one embodiment, the FGF protein has an amino acid sequence comprising from position 18 through position 172 of SEQ ID NO:2 (HLX-FGF-2) or from position 17 through 171 of SEQ ID NO:4 (TAT-FGF-2). Preferably, a biologically active FGF protein useful in a chimera of the present invention is encoded by a nucleic acid sequence comprising from nucleotide 59 to 523 of SEQ ID NO:1 (HLX-FGF-2) or from nucleotide 59 to 523 of SEQ ID NO:3.

In one embodiment, the penetratin peptide portion of a chimeric FGF of the present invention can include: (a) a first peptide having an amino acid sequence selected from the group consisting of: (i) X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 -X.sub.8 -X9-X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 ; and, (ii) X.sub.16 -X.sub.15 -X.sub.14 -X.sub.13 -X.sub.12 -X.sub.11 -X.sub.10 -X.sub.9 -X.sub.8 -X.sub.7 -X.sub.6 -X.sub.5 -X.sub.4 -X.sub.3 -X.sub.2 -X.sub.1 ; wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, X.sub.15, and X.sub.16 each represent an .alpha.-amino acid, between 6 and 10 of which are hydrophobic amino acids; and wherein X.sub.6 represents Trp; and,

(b) a second peptide comprising amino acid residues 49-57 of HIV Tat protein (SEQ ID NO:17). In a preferred embodiment, the second peptide of (b) does not comprise amino acid residues 22-36 or 73-86 of HIV Tat protein (SEQ ID NO:17).

The first penetratin peptide can include a peptide comprising helix 3 of a homeobox domain and a homeobox domain, and fragments and homologues thereof Such peptides comprise an amino acid sequence including, but are not limited to: SEQ ID NO:9, amino acid residues 42 through 58 of SEQ ID NO:9, amino acid residues 43 through 59 of SEQ ID NO:9, amino acid residues 43 through 58 of SEQ ID NO:9, amino acid residues 58 through 43 of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and/or SEQ ID NO:16. In one embodiment, such a peptide comprises amino acid residues 2-17 of SEQ ID NO:2. Preferably, such a peptide is encoded by a nucleic acid sequence comprising nucleotides 11 to 58 of SEQ ID NO:1.

The second penetratin peptide can include an HIV Tat protein or fragments or homologues thereof. Preferred peptides comprise an amino acid sequence that includes, but is not limited to: amino acid residues 37-72 of SEQ ID NO:17, amino acid residues 38-72 of SEQ ID NO:17, amino acid residues 47-72 of SEQ ID NO:17, amino acid residues 37-58 of SEQ ID NO:17, amino acid residues 38-58 of SEQ ID NO:17, amino acid residues 47-58 of SEQ ID NO:17, amino acid residues 1-21 and 38-72 of SEQ ID NO:17, amino acid residues 47-62 of SEQ ID NO:17, amino acid residues 38-62 of SEQ ID NO:17, amino acid residues 1-72 of SEQ ID NO:17, amino acid residues 1-58 of SEQ ID NO:17, and/or amino acid residues 48-60 of SEQ ID NO:17. In one embodiment, such a peptide comprises amino acid residues 48-60 of SEQ ID NO:17 or amino acid residues 2-14 of SEQ ID NO:4. Preferably, such a peptide is encoded by a nucleic acid sequence comprising residues 14 to 52 of SEQ ID NO:3.

A chimeric fibroblast growth factor (FGF) of the present invention includes a chimera comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 (HLX-FGF-2) and SEQ ID NO:4 (TAT-FGF-2). Preferably, such a chimeric FGF is encoded by a recombinant nucleic acid molecule having a nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively.

Another embodiment of the present invention relates to a therapeutic composition comprising the chimeric fibroblast growth factor (FGF) of the present invention and a pharmaceutically acceptable excipient.

Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule encoding a chimeric fibroblast growth factor (FGF) of the present invention as described above. Such a recombinant nucleic acid molecule comprises: (a) a first isolated nucleic acid sequence encoding a biologically active fibroblast growth factor (FGF) protein; and, (b) a second isolated nucleic acid sequence encoding a penetratin peptide that transports the chimeric fibroblast growth factor (FGF) across a lipid bilayer of a cell independently of the presence of an FGF receptor, wherein the second nucleic acid sequence is linked to the first nucleic acid sequence. The first and second nucleic acid sequences are operatively linked to a transcription control sequence. Such a chimeric fibroblast growth factor (FGF) is characterized by: (i) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (ii) entry into a living cell in the absence of a receptor that binds to FGF. Preferred chimeric FGF proteins encoded by a recombinant nucleic acid molecule of the present invention are described above.

Another embodiment of the present invention relates to a recombinant cell that expresses the recombinant nucleic acid molecule of the present invention described above. Another embodiment of the present invention is a recombinant virus that comprises the recombinant nucleic acid molecule of the present invention.

Yet another embodiment of the present invention relates to a method to produce a chimeric fibroblast growth factor (FGF), comprising culturing in an effective medium a recombinant cell comprising a recombinant nucleic acid molecule encoding a chimeric fibroblast growth factor protein as described above.

Another embodiment of the present invention relates to a method to promote fibroblast growth factor biological activity in a cell and particularly, to repress terminal differentiation and promote proliferation in a cell. Such a method includes the steps of administering to a cell a chimeric fibroblast growth factor (FGF) protein of the present invention as described above. In one embodiment, the cell has reduced heparan sulfate proteoglycan production characterized by a reduction in both repression of terminal differentiation and promotion of proliferation in the presence of naturally occurring fibroblast growth factor. In another embodiment, the cell is a cell of patient that has a condition selected from the group consisting of stroke, nerve damage, bone damage, muscle damage, and a wound. Such a chimeric FGF can be administered by any route, including in vitro, in vivo, and ex vivo.

Another embodiment of the present invention relates to a method to enhance a biological process selected from the group consisting of mitogenesis, angiogenesis, wound healing, neurogenesis, limb patterning, limb outgrowth, comprising administering to cells associated with the biological process a chimeric fibroblast growth factor (FGF) of the present invention as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that chimeric FGF-2 molecules are capable of crossing the plasma membrane independent of receptor-mediation.

FIG. 2 is a graph showing that synchronized MM14 cells subjected to trypsin treatment to remove extracellular receptors proliferate in response to chimeric, but not wild type, FGF-2.

FIG. 3 is a graph showing that a mouse myoblast cell line (MM14) requires the s presence of heparan sulfates (or heparin) in order to respond to FGF and grow (as measured by the incorporation of radioactivity into DNA (CPM)).

FIG. 4A is a graph demonstrating that HLX-FGF-2 chimeric protein has an attenuated requirement for heparan sulfate.

FIG. 4B is a graph demonstrating that TAT-FGF-2 chimeric protein has an attenuated requirement for heparan sulfate.

FIG. 5A is a graph showing that chimeric FGF-2 requires an FGF receptor in order to function in L6AI (FR-) cells.

FIG. 5B is a graph showing that chimeric FGF-2 requires an FGF receptor in order to function in BaF3 lymphoid cells.

FIG. 5C is a graph showing that chimeric FGF-2 functions in BaF3 cells stably of transfected with an expression construct encoding FGF receptors.

FIG. 6 is a graph illustrating that MM14 cells expressing an FGF receptor lacking tyrosine kinase (dnFGFR) show repressed differentiation in the presence ofHLX-FGF-2, but not in the presence of wild type FGF-2.

FIG. 7A is a graph showing that in MM14 cells treated with Tyrphostin A25, growth is substantially inhibited in cells treated with FGF-2, but not in cells treated with HLX-FGF-2.

FIG. 7B is a graph showing that in MM14 cells treated with Tyrphostin B42, growth is substantially inhibited in cells treated with FGF-2, but not in cells treated with HLX-FGF-2.

FIG. 8A is a graph demonstrating that treatment of cells with tyrosine kinase inhibitor Tyrphostin AG1296 in the presence of wild type FGF-2, TAT-FGF-2, or HLX-FGF-2, effectively blocked DNA synthesis.

FIG. 8B is a graph demonstrating that treatment of cells with tyrosine kinase inhibitor Sugen SU4894 in the presence of wild type FGF-2, TAT-FGF-2, or HLX-FGF-2, effectively blocked DNA synthesis.

FIG. 8C is a graph demonstrating that treatment of MM14 cells with PD098059, a specific inhibitor of MKK1, blocked both wild type and penatratin-FGF-stimulated proliferation of the cells.

FIG. 8D is a graph demonstrating that treatment of MM14 cells with U0126, an inhibitor of both MKK1 and MKK2, blocked both wild type and penatratin-FGF-stimulated proliferation of the cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to a chimeric fibroblast growth factor (FGF) protein characterized by: (a) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (b) an ability to enter a living cell in the absence of a receptor that binds to FGF. The present invention also relates to recombinant nucleic acid molecules encoding such a chimeric FGF protein, to therapeutic compositions including such a chimeric FGF protein, and to methods of making and using such a chimeric FGF protein.

The present inventors describe herein the development of a novel cell-permeant FGF chimera, also referred to herein as a penetratin-FGF chimera or penetratin-FGF fusion protein (described in detail below), that is capable of acting intracellularly to mediate FGF biological activity, such as repression of differentiation and/or stimulation of proliferation in a cell. Surprisingly, the inventors have discovered that, although the penetratin-FGF chimera of the present invention has biological activity substantially similar to a naturally occurring FGF, the biological activity of the penetratin-FGF chimera does not demonstrate the absolute heparan sulfate-dependency seen for wild type FGFs. Furthermore, the chimeric penetratin-FGF molecule facilitated cell proliferation even when the ligand binding domain of the cell surface receptors was removed by proteolysis. Unexpectedly, the present inventors have also found that the intracellular activity of the penetratin-FGF chimera is completely dependent on the expression of FGF receptors.

More particularly, the present inventors have designed and constructed chimeric proteins which include FGF proteins fused to short peptide sequences which are referred to collectively herein as "penetratins", which have been shown to be capable of shuttling various cargo molecules across the plasma membrane (See for example U.S. Pat. No. 5,888,762, 1999 to Joliot et al., PCT Publication No. WO 91/18981 to Centre National de la Recherche Scientifique, PCT Publication No. WO 97/12912 to Centre National de la Recherche Scientifique, U.S. Pat. No. 5,804,604,1998) to Frankel et al., Perez et al., 1992, J. Cell Sci. 102;717-722, Derossi et al., 1994, J. Biol. Chem, 269:10444-10450, Bloch-Gallego, et al., (1993) J. Cell Biol. 120:485-92; Joliot, et al., (1991) Proc. Nat'l Acad. Sci. USA, 88:1864-8; Le Roux, et al., (1993) Proc. Nat'l Acad. Sci. USA, 90:9120-4; Mann, et al. (1991) Embo Journal, 10:1733-9; Vives, et al., (1997) J. Biol. Chem., 272:16010-7; Viv s, et al., (1994) J. Virol., 68:3343-53, each of which is incorporated herein by reference in its entirety). Specifically, two chimeric FGF-2 proteins containing either a portion of the Drosophila Antenapedia protein (HLX-FGF-2) or the HIV Tat protein (TAT-FGF-2) were constructed (see Examples, Table 2). The initial utility of these chimeric molecules lies in their ability to enter a cell independent of receptor-mediated or other common endocytic pathways as is evidenced by the capacity of the chimeric proteins to facilitate internalization at 4.degree. C. Indeed, both the TAT- and HLX-FGF-2 fusion proteins are capable of entering cells that do not possess detectable cell surface FGFRs and that fail to respond to FGF in an FGFR dependent manner. Moreover, cellular entry by these fusion molecules was efficient at 4.degree. C., consistent with penetratin-mediated internalization. It is noted that one advantage of a chimeric FGF protein of the present invention is that it is capable of penetrating not only the plasma membrane of a cell in the absence of binding to a receptor, but it is also capable of permeating any membrane within a cell (e.g., to enter the nucleus), and therefore, it's entry into an intracellular compartment is not constrained within a cell. The FGF-2 fusions were also indistinguishable from wild type FGF-2 in their biological activity and receptor binding. Both wild type and penetratin FGF-2 proteins required heparan sulfate to bind efficiently to fibroblast growth factor receptor 1 (FGFR1) present on MM14 cells.

Surprisingly, however, the present inventors discovered that the chimeric FGF-penetratin proteins had unexpected properties which distinguished them from naturally occurring FGF proteins and from any other previously described modified FGF proteins. More particularly, the present inventors discovered that the biological activity of HLX-FGF-2 and TAT-FGF-2 was not absolutely dependent on heparan sulfate. Both penatratin-FGF-2 fusion proteins were active in the absence of heparin in cells treated with chlorate. Additionally, FGFR1 expressing BaF3 cells, which do not express heparan sulfate proteoglycans, responded to HLX-FGF-2 treatment even in the absence of heparin. Control experiments with the isolated HLX penetratin peptide failed to elicit any detectable response in either MM14 or BaF3 cells (data not shown). As such, it is unlikely that the heparin-independent activity demonstrated by the FGF-2 fusion proteins is due to the presence of the penetratin sequence. Thus, these experiments indicated a disparity between the binding of penetratin-FGF-2 fusions to cell surface FGFR1, which requires heparan sulfate, and the ability of the fusions to promote proliferation in MM14 cells or survival in BaF3 cells. The present inventors additionally discovered that surprisingly, the intracellular activity of the penetratin-FGF chimera was completely dependent on the expression of FGF receptors, that inactivation of the FGFR1 tyrosine kinase is necessary for the biological activity of the penetratin-FGF chimeras of the present invention, and that activation of the raf/ERK pathway appears to be required for the biological activity of the penetratin-FGF chimeras.

In addition to a plethora of therapeutic uses for the penetratin-FGF chimeras that encompass current uses for wildtype FGF (e.g., wound healing, neurogenesis, etc.) the properties of the novel chimeric FGF protein described herein suggest potential therapeutic roles for penetratin-FGF chimeras in pathological conditions where normal FGF function does not occur due to biochemical defects in the normal FGF signaling pathways. For example, it is known that genetic defects in FGF receptors are responsible for most forms of dwarfism, and therefore, a chimeric FGF of the present invention, which does not require binding to an FGF receptor for entry into a cell, are believed to be extremely valuable as a therapeutic composition for the treatment of dwarfism. Additionally, it has been shown that the biological function of heparan sulfate with regard to FGF activity can be either inhibitory or stimulatory, depending on the cell type and the FGF receptor type. The relative heparan sulfate independence of a chimeric FGF of the present invention can therefore be used advantageously in that an FGF receptor could be activated in a cell where the FGFR is not normally activated due to inhibitory action of heparan sulfate, and vice versa. In other words, the ability to bypass the regulatory function of heparan sulfate on FGF receptors can be used advantageously in therapeutic protocols depending on the desired result. Other pathological conditions may also be responsive to FGF therapy (e.g., at the time of filing, wild-type FGF-2 is in clinical trials for the treatment of stroke) even though specific defects in FGF signaling do not exist. One current hypothesis regarding FGF receptors is that they are tightly regulated and not easily activated. Therefore, penetratin-FGF chimeras of the present invention are believed to be able to potentiate therapeutic effects by stimulating cells which are normally recalcitrant to FGF treatment and therefore be more effective than wildtype FGF.

One embodiment of the present invention relates to a chimeric fibroblast growth factor (FGF), comprising: (a) a biologically active fibroblast growth factor (FGF) protein having a first amino acid sequence; and, (b) a penetratin peptide having a second amino acid sequence, the second amino acid sequence being linked to the first amino acid sequence. The penetratin peptide transports the chimeric fibroblast growth factor (FGF) across a lipid bilayer of a cell independently of the presence of an FGF receptor. It is noted that the penetratin peptide can transport the chimeric fibroblast growth factor across any cellular membrane, and is not limited to transport across the plasma membrane. The chimeric fibroblast growth factor (FGF) is characterized by: (i) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (ii) an ability to enter a living cell in the absence of a receptor that binds to FGF. In one embodiment, fibroblast growth factor biological activity can include: (a) an ability to repress terminal differentiation in the absence of heparan sulfate; and (b) an ability to promote cell proliferation in the absence of heparan sulfate. Other biological activities of FGF are described below.

According to the present invention, the terms "chimera" or "chimeric" with regard to a protein refer to a protein that is composed of amino acid sequences derived from at least two distinct sources (i.e., at least two heterologous amino acid sequences). As used herein, a chimeric protein is not a single naturally occurring protein, but rather, has been synthesized or genetically engineered. One type of chimeric protein is known in the art as a fusion protein. According to the present invention, the phrases "chimeric fibroblast growth factor protein" and "penetratin-FGF chimera" or "penetratin-FGF protein" are used to refer to the same chimeric protein of the present invention, and thus can be used interchangeably.

According to the present invention, a chimeric fibroblast growth factor (FGF) protein is characterized as having fibroblast growth factor biological activity in the absence of heparan sulfate. Fibroblast growth factor biological activity is defined herein a measurable activity that is indicative of the biological activity of a naturally occurring fibroblast growth factor protein, as measured by an in vitro or in vivo assay. Such biological activities include, but are not limited to: an ability to promote cell proliferation (e.g., in a cell line such as MM14 as described in the Examples), an ability to repress terminal differentiation in a cell (e.g., in a cell line such as MM14 described in the Examples or bovine brain capillary endothelial cells), an ability to promote angiogenesis in vivo in a chicken chorioallantoic membrane assay, promotion of wound healing in vivo, promotion of osteogenesis on osteoblasts in an in vivo or in vitro assay, promotion of nerve outgrowth (primary neurons or neuronal cell lines). In vitro and in vivo assays for measuring FGF biological activity are well known in the art. For example, such assays are described in Gospodarowicz et al., 1985, J. Cell Physiol. 122:323-332, Gospodarowicz, 1983, J. Cell Physiol. 97:1677-1685, Esch et al., 1985, Proc. Nat. Acad. Sci. (USA) 82:6507-6511, Gospodarowicz et al., 1986, J. Cell Physiol. 127:121-136, Davidson et al., 1985, J.C.B. 100:1219-1227, and the Examples section. It is noted that modified forms of FGF, such as the chimeric penetratin-FGF proteins described herein, may have different quantitative activity and specificity than a naturally occurring FGF protein, and such variations are intended to be encompassed by the present invention.

According to the present invention, FGF biological activity of a protein is evaluated separately from the protein's requirement for entry into a cell (e.g., by receptor or by other means) as well as from the protein's requirement for heparan sulfate or exogenously added heparin (e.g., a protein can have FGF biological activity in the absence of heparan sulfate or other exogenously added heparin and/or in the absence of entering a cell via an FGF receptor). In other words, the ability to enter a cell via an FGF receptor or a dependence on heparan sulfate for activity is not used as a measure of FGF biological activity according to the present invention. A particular advantage of a chimeric FGF protein of the present invention is that the protein possesses the unique property of "heparan sulfate independence" (i.e., no absolute requirement for heparan sulfate for biological activity), a feature which has not been reported for any of the at least 20 known FGF family members or for any previously described modified FGF. FGFs are known to mediate numerous biological responses in many different tissues. The effects of FGFs require both a receptor tyrosine kinase specific for FGFs as well as heparan sulfate proteoglycans (HSPGs; or an analog compound, heparin) to bring about a biological response. It has become increasingly clear that HSPGs are critically important to the biological activity of naturally occurring FGF (e.g., as both inhibitory and stimulatory factors). Therefore, the chimeric FGF proteins of the present invention will have potential therapeutic value for use in pathologic conditions manifested by the inability of "normal" FGFs to generate necessary biological signals due to aberrant or abnormal heparan sulfate proteoglycan production. Furthermore, HSPGs/heparin have been postulated to stabilize FGFs and protect them from extracellular proteolysis. The "heparan sulfate independence" demonstrated by the chimeric FGF proteins of the present invention also suggest that these proteins may be more resistant to degradation than the native FGF protein.

A chimeric fibroblast growth factor protein of the present invention also has the ability to enter a living cell in the absence of a receptor that binds to FGF. In other words, although a chimeric FGF protein of the present invention may be capable of binding to an FGF receptor and of entering a cell via internalization of the chimeric FGF/FGF receptor complex, a chimeric FGF protein of the present invention can also enter a cell or cross any cellular membrane independently of receptor-mediated or other common endocytic pathways, and independently of the temperature and energy requirements of most biological processes (e.g., a chimeric FGF protein can enter a cell at 4.degree. C.).

One portion of a chimeric FGF protein of the present invention is a biologically active fibroblast growth factor protein. Accordingly, a biologically active FGF protein meets the requirements for FGF biological activity as discussed in detail above. Specifically, a biologically active FGF protein has a measurable activity or function that is indicative of the biological activity of a naturally occurring fibroblast growth factor protein, as measured by an in vitro or in vivo assay (described in detail above). According to the present invention, reference to a "fibroblast growth factor (FGF) protein" can include any full-length FGF protein, truncated FGF protein, or any homologue of such an FGF protein. According to the present invention, an FGF homologue includes proteins in which at least one or a few, but not limited to one or a few, amino acids of full-length FGF have been accidentally or deliberately deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the FGF homologue has FGF biological activity as described previously herein. An FGF protein homologue can be identified as a protein having at least one epitope which elicits an immune response against a naturally occurring FGF protein. In another embodiment, a homologue of an FGF protein is a protein having an amino acid sequence that is sufficiently similar to a naturally occurring FGF amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) a complement of a nucleic acid molecule encoding the naturally occurring FGF protein. A nucleic acid sequence complement of nucleic acid sequence encoding FGF refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand which encodes FGF. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand, such complementary strand having a sequence that is a complement to the single strand DNA. As such, nucleic acid molecules which encode an FGF protein of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence of an FGF protein, and/or with the complement of the nucleic acid that encodes amino acid sequence of an FGF protein. Methods to deduce a complementary sequence are known to those skilled in the art. It should be noted that since amino acid sequencing and nucleic acid sequencing technologies are not entirely error-free, the sequences presented herein, at best, represent apparent sequences of FGF proteins included in the present invention.

As used herein, stringent hybridization conditions refer to standard hybridization as conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138,267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

More particularly, stringent hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 75%, and most particularly at least about 80%. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10.degree. C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6.times. SSC (0.9 M Na.sup.+) at a temperature of between about 20.degree. C. and about 35.degree. C., more preferably, between about 280.degree. C. and about 40.degree. C., and even more preferably, between about 35.degree. C. and about 45.degree. C. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6.times. SSC (0.9 M Na.sup.+) at a temperature of between about 30.degree. C. and about 45.degree. C., more preferably, between about 38.degree. C. and about 50.degree. C., and even more preferably, between about 45.degree. C. and about 55.degree. C. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G+C. content of about 40%. Alternatively, T.sub.m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 TO 9.62.

Preferably, nucleic acid molecules encoding biologically active FGF proteins suitable for use in a chimeric FGF protein of the present invention have at least about 70%, more preferably, at least about 80% and most preferably, at least about 90% identity with a nucleic acid sequence encoding a naturally occurring FGF protein. As used herein, reference to a percent (%) identity refers to a BLAST homology search with the default parameters identified in Table 1.

TABLE 1 BLAST Search Parameters HISTOGRAM Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual). DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page). See also EXPECT and CUTOFF. ALIGNMENTS Restricts database sequences to the number specified for which high scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual). EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin end Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual). CUTOFF Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT. MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response. STRAND Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence. FILTER Mask oil segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993), or segments consisting of short-periodicity internal repeats, as determined by the SNU program of Claverie & States (Computers and Chemistry, 1993), or, for BLASTN, by the DUST program of Tetusov and Lipman (in preparation). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g.. hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences. Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in protein sequences (e.g., "XXXXXXXXX"). Users may turn off filtering by using the "Filter" option on the "Advanced options for the BLAST server" page. Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs. It is not unusual for nothing at all to be masked by SEG, SNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect. NCBl-gi Causes NCBl gi identifiers to be shown in the output, in addition to the accession and/or locus name.

Protein homologues of the present invention can be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.

In one embodiment of the present invention, FGF proteins suitable for use in the chimeric FGF protein of the present invention include biologically active FGF proteins that are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to the complementary strand of a nucleic acid molecule encoding FGF-1 or FGF-2. Preferred biologically active FGF proteins for use in a chimeric FGF protein of the present invention include FGF proteins comprising all or a biologically active fragment of an FGF protein including FGF-1 or FGF-2, with FGF-2 being particularly preferred. Other preferred biologically active proteins for use in a chimeric FGF protein of the present invention include FGF proteins comprising all or a biologically active fragment of a protein having an amino acid sequence selected from the group of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, which are amino acid sequences for bovine FGF-2, human FGF-2, bovine FGF-1 and human FGF-1, respectively. In yet another embodiment, an FGF protein suitable for use in a chimeric FGF of the present invention includes an FGF protein having an amino acid sequence corresponding to positions 18 through 172 of SEQ ID NO:2, or to positions 17 through 171 of SEQ ID NO:4. As discussed above, suitable FGF proteins include biologically active fragments and homologues of any of the above identified FGF amino acid sequences and of any other FGF amino acid sequences. Additionally, the nucleic acid and amino acid sequences of many FGF proteins are known in the art, such information being publicly available, for example, on a database such as GenBank. At the time of filing, at least 20 types of FGF have been identified. All known naturally occurring FGF proteins share similar requirements for FGF receptor binding and heparan sulfate dependence, and the use of any of such proteins in a chimeric FGF protein of the present invention results in chimeric FGF proteins having similar advantageous and unexpected properties as those described for the specific penetratin-FGF chimeras disclosed herein. It is noted that in the production of a chimeric FGF protein of the present invention, modifications to the sequences can be made to facilitate the production of the chimera For example, the amino acid sequence of an FGF protein portion of the chimera may be modified to substitute a non-methionine residue for the initial methionine residue to avoid problems with multiple translation start sites in the nucleic acid molecule encoding the chimera. Such modifications are well within the ability of one of skill in the art.

Another portion of a chimeric FGF of the present invention is a penetratin peptide, wherein the penetratin peptide transports the chimeric FGF across a lipid bilayer of a cell independently of the presence of an FGF receptor. According to the present invention, a "penetratin peptide" is an amino acid sequence which is capable of transporting itself and a heterologous protein linked to it across a lipid bilayer or any cellular membrane independent of receptor-mediated or other common endocytic pathways. Therefore, a penetratin peptide does not require a receptor for entry into a cell, nor does it have temperature and energy requirements associated with receptor-mediated or other endocytic biological processes (e.g., the transport can occur at 4.degree. C.). Such penetratin peptides are known in the art, but particularly preferred penetratin peptides for use in the present invention include peptides derived from the helix 3 of the homeobox domain and peptides derived from HIV Tat protein. Helix 3 homeobox peptides and homologues thereof suitable for use in a chimeric protein of the present invention are described in detail in U.S. Pat. No. 5,888,762, 1999 to Joliot et al., PCT Publication No. WO 91/18981 to Centre National de la Recherche Scientifique, PCT Publication No. WO 97/12912 to Centre National de la Recherche Scientifique, each of which is incorporated herein by reference in its entirety. HIV Tat peptides and homologues thereof suitable for use in a chimeric protein of the present invention are described in detail in U.S. Pat. No. 5,804,604, 1998, to Frankel et al., which is incorporated herein by reference in its entirety. Although these publications appreciated the use of penetratin peptides to enable the delivery of a heterologous peptide across a cell membrane independent of a receptor, none of the references disclose the production or use of a chimeric FGF protein of the present invention, and none of the references disclose the unexpected and surprising properties of the chimeric FGF protein of the present invention.

In one embodiment of the present invention, a penetratin portion of a chimeric FGF includes a peptide having an amino acid sequence selected from the group consisting of:

(i) X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 -X.sub.8 -X9-X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 ; and,

(ii) X.sub.16 -X.sub.15 -X.sub.14 -X.sub.13 -X.sub.12 -X.sub.11 -X.sub.10 -X.sub.9 -X.sub.8 -X.sub.7 -X.sub.6 -X.sub.5 -X.sub.4 -X.sub.3 -X.sub.2 -X.sub.1 ; wherein X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, X.sub.15, and X.sub.16 each represent an .alpha.-amino acid, between 6 and 10 of which are hydrophobic amino acids; and wherein X.sub.6 represents Trp.

Such a peptide represents a homologue of a helix 3 portion of a homeobox domain protein and is disclosed, for example, in PCT Publication No. WO 97/12912, ibid. In a preferred embodiment, the first peptide is selected from the group of a peptide comprising helix 3 of a homeobox domain and a homeobox domain. As discussed in U.S. Pat. No. 5,888,762, ibid., the term "homeobox peptide" denotes a family of related peptide sequences which occur in various animal species in the products of genes involved in embryogenesis. Genes encoding homeoproteins are expressed at various stages of embryo development and their products control the cell migration and differentiation phenomena involved in the morphogenesis of the organism. Homeobox sequences homologous to that of Drosophila have been found in all vertebrates including mammals. The homeobox sequence encodes a polypeptide sequence of 60 amino acids which corresponds to a structurally and functionally conserved region which is present in all homeoproteins, the homeodomain. The sequence of the homeodomain which is encoded by the homeobox sequence of the Antennapedia gene of Drosophila is represented herein by SEQ ID NO:9. All known homeodomains share the same helix/.beta.-turn/helix structure, despite some differences in their primary sequences. As used herein "helix 3" is defined as the portion of a homeobox peptide (domain), which is involved in the low-affinity binding with the wide groove of DNA. Helix 3 extends from amino-acid 43 to amino-acid 58 of the homeobox peptide (SEQ ID NO:9). Within the context of the present invention "helix 3" also refers to peptides which may slightly differ in their sequence from the helix 3 of naturally occurring homeodomains, provided that the differences do not affect the ability of the peptide to serve as a penetratin peptide in the chimeric protein of the present invention.

Particularly preferred penetratin peptides derived from the helix 3 sequence of a homeobox domain include, but are not limited to: SEQ ID NO:9, amino acid residues 42 through 58 of SEQ ID NO:9, amino acid residues 43 through 59 of SEQ ID NO:9, amino acid residues 43 through 58 of SEQ ID NO:9, amino acid residues 58 through 43 of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. SEQ ID NOs:10-16 are mutants of helix 3 peptides which have penetratin activity and are described in detail in PCT Publication No. WO 97/12912, ibid., and in U.S. Pat. No. 5,888,672, ibid. In a preferred embodiment of the present invention, the first penetratin peptide comprises amino acid residues 2-17 of SEQ ID NO:2.

In another embodiment of the present invention, a penetratin portion of a chimeric FGF can include a peptide comprising amino acid residues 49-57 of HIV Tat protein (SEQ ID NO:17). In a preferred embodiment, such a peptide does not comprise amino acid residues 22-36 or 73-86 of HIV Tat protein (SEQ ID NO:17). Penetratin peptides derived from HIV Tat protein are described in detail in U.S. Pat. No. 5,804,604, ibid., and all of such peptides are incorporated for use in the present invention by reference.

As described in U.S. Pat. No. 5,804,604, Tat protein trans-activates certain HIV genes and is essential for viral replication. The full-length Tat protein is characterized by a basic region which contains two lysines and six arginines (amino acids 49-57) and a cysteine-rich region which contains seven cysteine residues (amino acids 22-37). The basic region (i.e., amino acids 49-57 of SEQ ID NO:17) is thought to be important for nuclear localization (Ruben, S. et al., J. Virol. 63: 1-8 (1989); Hauber, J. et al., J. Virol. 63 1181-1187 (1989)). Residues 38-58 of SEQ ID NO:17 or protamine, enhance uptake of Tat. Therefore, as shown in U.S. Pat. No. 5,804,604, ibid., the entire 86 amino acids which make up the Tat protein are not required for the uptake activity of Tat. For example, a protein fragment or a peptide which has fewer than the 86 amino acids, but which exhibits uptake into cells and uptake into the cell nucleus, can be used (a functionally effective fragment or portion of Tat).

Preferred peptides derived from HIV Tat protein which are suitable for use in the penetratin portion of a chimeric FGF of the present invention include peptides comprising an amino acid sequence including, but not limited to amino acid residues 37-72 of SEQ ID NO:17, amino acid residues 38-72 of SEQ ID NO:17, amino acid residues 47-72 of SEQ ID NO:17, amino acid residues 37-58 of SEQ ID NO:17, amino acid residues 38-58 of SEQ ID NO:17, amino acid residues 47-58 of SEQ ID NO:17, amino acid residues 1-21 and 38-72 of SEQ ID NO:17, amino acid residues 47-62 of SEQ ID NO:17, amino acid residues 38-62 of SEQ ID NO:17, amino acid residues 1-72 of SEQ ID NO:17, amino acid residues 1-58 of SEQ ID NO:17, and/or amino acid residues 48-60 of SEQ ID NO:17. In a preferred embodiment, such a peptide comprises an amino acid sequence including amino acid residues 48-60 of SEQ ID NO:17 or amino acid residues 2-14 of SEQ ID NO:4.

To produce a chimeric FGF of the present invention, an amino acid sequence comprising a biologically active FGF protein as described above is linked to an amino acid sequence comprising a penetratin peptide as described above. The amino acid sequence of the penetratin portion of the chimera can be linked to either the N-terminal end or the C-terminal end of the FGF protein portion, and preferably, is linked to the N-terminal end of the FGF protein portion. The entire amino acid sequence of the chimeric FGF protein can contain additional amino acid residues other than those included in the FGF protein portion or the penetratin portion. For example, a methionine residue is typically added to the