WO1994000570A1

WO1994000570A1 - Mammalian growth factor

Info

Publication number: WO1994000570A1
Application number: PCT/US1993/005962
Authority: WO
Inventors: Claudio Basillico; Daniela Talarico
Original assignee: New York University
Priority date: 1992-06-22
Filing date: 1993-06-22
Publication date: 1994-01-06
Also published as: AU4645993A

Abstract

A truncated mammalian growth factor, displaying homology to both basic and acidic fibroblast growth factor in a single polypeptide, is disclosed herein. The growth factor is substantially smaller (i.e. has fewer amino acid residues) than the full-length mammalian growth factor, has a higher affinity for fibroblast growth factor receptors than full-length K-FGF and basic fibroblast growth factor and increased mitogenic activity. Also disclosed herein are DNA sequences encoding the truncated growth factor, pharmaceutical formulations containing the truncated growth factor and methods to heal burns and wounds in a mammal by administering the pharmaceutical formulations.

Description

MAMMALIAN GROWTH FACTOR

This application is a continuation-in-part of co- pending U.S. Patent Application Serial No. 07/806,791, filed December 6, 1991 which is a continuation of U.S. Patent Application Serial No. 07/177,506 filed April 4, 1988 (abandoned) which is a continuation-in-part of U.S. Patent Application Serial No. 07/062,925, filed June 16, 1987 (abandoned) .

The United States Government has rights to this invention by virtue of grant No. CA42568 from The National Cancer^{^}Institute.

FIELD OF THE INVENTION

This invention pertains to a mammalian growth factor_, pharmaceutical formulations comprising said factor and methods for healing wounds or burns in mammals comprising administering said formulations.

BACKGROUND OF THE INVENTION

This invention pertains to a novel polypeptide having mammalian growth factor activity and to methods for using it. A variety of diffusible factors which stimulate the growth of cells in a hormone-like manner are generally called

"growth factors". Growth factors are often present in serum an have also been isolated from a variety of organs. They are protein molecules (or groups of such molecules) and in all know cases they interact with specific cell surface receptors to promote cellular growth and/or differentiation. Growth factors vary in their tissue specificity, i.e. some interact only with specific cell types, while others are active on a wider cell type range.

Among the best known groups of mammalian growth factors are: (1) platelet derived growth factor (PDGF) , release from platelets; (2) epidermal growth factor (EGF) ; (3) hematopoietic growth factors (including interleukins 1, 2, and 3) , required for growth and differentiation of lymphocytes, and colony stimulating factors (CSF) , promoting growth and differentiation of hematopoietic stem cells; (4) angiogenic

(literally "blood-vessel-forming") growth factors, such as the fibroblast growth factors (FGF) believed to promote growth and organization of endothelial cells into new blood vessels; (5) miscellaneous growth factors released by tumor cells. Two well-characterized angiogenic factors are basic and acidic fibroblast growth factors (FGF) , believed to be most important in vivo for endothelial cell growth. However, neithe basic FGF nor acidic FGF has proven useful as pharmaceutical agents for promotion of wound healing. Several factors may contribute to the unsuitability of basic FGF and acidic FGF as pharmaceutical agents. Neither factor is sufficiently stable for effective pharmaceutical formulation. Basic FGF demonstrates restricted interaction with FGF receptors in vitro and thus cannot be expected to interact with all FGF receptors in vivo. Finally, basic FGF and acidic FGF have thus far prove ineffective in animal models.

Co-pending U.S. Patent Application Serial No. 07/806,791 filed December 6, 1991 discloses an angiogenic mammalian growth factor isolated from Kaposi's Sarcoma cells an having substantial homology to each of acidic and basic fibroblast growth factor in a single polypeptide. The growth factor protein comprises 176 amino acid residues and is a matur (secreted) glycoprotein. This growth factor has variously been called K-FGF or FGF-4, and it has shown promising results as a wound healing agent in preclinical studies in an ischemic rabbi ear model. In such a model, K-FGF promoted wound healing bette than basic or acidic FGF.

Growth factors are believed to promote wound healing. For example, EGF present in saliva is believed to accelerate wound healing in mice. Schultz G.S et al. (Science 232_.:350-352 1986) report that transforming growth factor (TGF) -alpha and vaccinia virus growth factor (VGF) , both of which are substantially homologous to EGF, accelerated epidermal wound healing in pigs when topically applied to second degree burns and were significantly more active than EGF.

Of the above-mentioned growth factors, the angiogenic growth factors would be particularly useful as wound healing agents because of their ability to promote the formation and growth of new blood vessels. OBJECTS OF THE INVENTION

It is an object of the present invention to provide a novel growth factor useful as a wound healing agent in mammals.

Another object of the present invention is to provide a mammalian growth factor with increased biologic activities. Yet another object of the present invention is to provide novel pharmaceutical formulations and methods for promoting wound healing in mammals.

A still further object of the present invention is to provide a truncated mammalian growth factor protein having substantial homology to each of acidic and basic fibroblast growth factor protein in a single polypeptide and having substantially higher specific activity than K-FGF protein. SUMMARY OF THE INVENTION

The present invention pertains to a previously unkno form of truncated mammalian growth factor protein having substantial homology to each of basic and acidic fibroblast growth factor proteins in a single polypeptide chain, said truncated mammalian growth factor being substantially smaller than the full-length mammalian growth factor (the truncated protein is hereinafter referred to as truncated K-FGF or K-FGF-

140) .

In another aspect, the present invention provides a polypeptide having the amino acid sequence (SEQ. ID. NO. 1) :

Ala Ala Val Gin Ser Gly Ala Gly Asp Tyr Leu Leu Gly He Lys Arg 1 5 10 15

Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu Gin Ala 20 25 30

Leu Pro Asp Gly Arg He Gly Gly Ala His Ala Asp Thr Arg Asp Ser 35 40 45 Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser He Phe Gly 50 55 60

Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys Leu Tyr 65 70 75 80

Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu He Leu Lea

85 90 95

Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe 100 105 110

He Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Ser 115 120 125 Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu. 130 135 140

In yet another aspect, the present invention provides a pharmaceutical formulation for treating a mammal suffering from wounds or burns comprising truncated K-FGF and a pharmaceutically acceptable carrier or diluent.

A still further aspect of the present invention involves a method for healing wounds or burns in a mammal in need of such treatment by administration of an effective amount for wound or burn healing of truncated K-FGF. A still further aspect of the present invention provides an isolated DNA having the sequence (SEQ. ID. NO. 2) : GCG GCC GTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC

ATC AAG CGG CTG CGG CGG CTC TAC TGC AAC GTG GGC ATC GGC TTC CAC CTC CAG GCG CTC CCC GAC GGC CGC ATC GGC

GGC GCG CAC GCG GAC ACC CGC GAC AGC CTG CTG GAG CTC 1

TCG CCC GTG GAG CGG GGC GTG GTG AGC ATC TTC GGC GTG 1

GCC AGC CGG TTC TTC GTG GCC ATG AGC AGC AAG GGC AAG 2

CTC TAT GGC TCG CCC TTC TTC ACC GAT GAG TGC ACG TTC 2 AAG GAG ATT CTC CTT CCC AAC AAC TAC AAC GCC TAC GAG 3

TCC TAC AAG TAC CCC GGC ATG TTC ATC GCC CTG AGC AAG 3

AAT GGG AAG ACC AAG AAG GGG AAC CGA GTG TCG CCC ACC 3

ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG TGA 4

A still further aspect of the present invention provides a truncated K-FGF protein characterized by (i) a molecular, weight of about 14,000 Daltons; and (ii) an average FGF-receptor binding affinity of about 9.5X10"^aM.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram depicting the amino acid sequence of the full-length K-FGF protein and the amino acid sequence of the truncated protein of the present invention, K- FGF-140.

Figure 2 is an autoadiograph of a sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of immunoprecipitated K-FGF forms produced in COS cells transfected with either wild type K-FGF or K-FGF-140 DNA or wit a mutated K-FGF cDNA which expresses an unglycosylated form of K-FGF that is processed to produce K-FGF-140.

Figure 3 is an autoradiograph of an SDS-PAGE analysis of the elution of K-FGF and K-FGF-140 from heparin affinity columns. Figure 4 is a graph showing the stimulation of DNA synthesis in quiescent BALB/c-3T3 cells by recombinant K-FGF a K-FGF-140.

Figure 5 (A and B) are graphs depicting a competitio assay of the ability of K-FGF and K-FGF-140 to displace labele basic fibroblast growth factor (bFGF) binding to Chinese Hamst Ovary (CHO) cells expressing the FGF receptor 1 (fig) or 2 (bek) .

Figure 6 (A-D) are a series of graphs depicting competition assays between K-FGF and K-FGF-140 for receptors o CHO cells expressing the FGF receptor 1 (fig) or 2 (bek) .

Figure 7 is a graph depicting a Scatchard analysis o K-FGF and K-FGF-140 binding to CHO cells expressing the FGF receptor 1 (bek) . DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references mentioned in the specification are incorporated by reference in their entirety. In the case of inconsistencies, the present disclosure, including definitions, shall prevail. The present inventors have surprisingly found a truncated form of the K-FGF protein which demonstrates substantially increased activity over that of either full-leng K-FGF, basic FGF, or acidic FGF. The truncated K-FGF protein not glycosylated and is substantially smaller (i.e. has fewer amino acid residues) than the full-length K-FGF protein. The truncated protein has a higher affinity for fibroblast growth factor receptors than either the mature, full-length K-FGF protein or bFGF. In addition, the truncated protein has a higher affinity for heparin than full-length K-FGF and increas mitogenic (i.e. growth promoting) activity. It is expected that, due to these increased biological activities, the truncated protein of the present invention will also have increased wound healing activity.

"Substantially smaller" refers to the fact that the truncated K-FGF protein of the invention contains about 140 amino acid residues as contrasted with the 176 amino acid residues that are present in the full-length mature, secreted K FGF protein.

"K-FGF-140" is defined herein as the unglycosylated, truncated mammalian growth factor protein of the present invention.

"K-FGF" is defined herein as the full-length mature human growth factor having a molecular weight of about 18,000 Daltons (non-glycosylated) comprising 176 amino acid residues a disclosed in U.S. Patent Application Serial No. 07/806,791 file December 6, 1991.

"Mitogenic activity" in reference to the biological activity of the truncated protein of the present invention is defined herein as the ability of the protein to induce DNA synthesis and proliferation of cells in culture.

"Substantial homology to each of acidic and basic fibroblast growth factors" is defined herein as having regions of identity (either exact or by conservative substitution) to said growth factors as shown in Table 1 below. K-FGF-140 was discovered during studies on the effect of glycosylation on the secretion of full-length K-FGF. Simian COS cells that were transfected with a plasmid encoding the full-length human K-FGF protein and incubated with tunicamycin (an inhibitor of N-linked glycosylation) , accumulated an unglycosylated K-FGF protein within the cells of approximately 18,000 Daltons (the expected size of the unglycosylated full- length K-FGF protein). Surprisingly, only proteins of 12,000- 14,000 Daltons were detected in the culture medium (i.e. were secreted) . This was more clearly shown using a K-FGF cDNA mutated in such a way to express a protein in which amino acid 38 (Threonine) of the full length K-FGF precursor protein was replaced by Alanine. This protein cannot be glycosylated. COS cells transfected with a plasmid encoding this mutated form of K-FGF also accumulate within the cells an unglycosylated K-FGF protein of approximately 18,000 daltons, but produce in the medium only forms of 12,000-14,000 daltons. Apparently the removal of the sugar residues exposes sites on the K-FGF molecule that are very susceptible to cleavage by cellular proteases located on the cell surface. Thus the protein is cleaved to produce these smaller forms as soon as it becomes externalized. It has been determined that the 14,000 Dalton species is K-FGF-140, a truncated form of the full-length K-FG protein.

K-FGF-140 retains the same regions of homology to acidic and basic FGF as the full-length K-FGF protein (as show in Table 1 below) but has increased biological activity.

TABLE 1

BOVINE BASIC FIBROBLAST GROWTH FACTOR

67' AAQPKE AAVQSGAGDYLLG-IKRLRRLYCJ GIGFHLQALPDGRIGGAHADTRDSL- LEL

1" PA LPEDGGSGAFPPGHFKDPKRLYCKNG- GFFLRIHPDGRVDGVREKSDPHIKLQL

119' SPVERGWSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYE- SYKYPGMF

56" QAEERGVVSIKGVCANRYLAMKEDGRLIJ^KCVTDECFFFERLESNNyNTYR- SRKYSSWY

179' IALSKΝGKTKKGNRVSPTMKVTHFLPRL

• • • • • • • • • •

116 " VALKRTGQYKLGPKTGPGQKAILFLPMSAKS

BOVINE ACIDIC FIBROBLAST GROWTH FACTOR

67 ' AAQPKEAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSL ^■ LELS

1 " FΝLPLGΝYKKPKLLYCSΝG-

GYFLRILPDGTVDGTKDRSDQHIQLQLC

120 ' PVERGWSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPΝΝYΝAYESYKYPGM

48 " AESIGEVYIKSTETGQFLAMDTDGLLYGSQTPΝEECLFLERLEEΝHYΝTYISKKHAEK

W

178 ' FIALSKΝGKTKKGΝRVSPTMKVTHFLPRL 108 " FVGLKKΝGRSKLGPRTHFGQKAILFLPLPVSSD

A = Ala ; R = Arg ; Ν = Asn; D = Asp ; C = Cys ; Q = Gin; E = Glu ; G = Gly ; H = His ; I = He ; L = Leu ; K = Lys ; M = Met F = Phe ; P = Pro ; S = Ser; T = Thr ; W = Trp ; Y = Tyr; V = Val .

In Table 1, the N-terminal amino acid (alanine) of K- FGF-140 is residue 67 of the full-length K-FGF protein. Two do between a particular set of amino acid residues indicate exact identity between the truncated growth factor of the present invention and either one of basic (SEQ. ID. NO. 3) and acidic F (SEQ. ID. NO. 4) , and one dot indicates that there has been a conservative substitution, e.g. substitution of the same type o amino acid such as phenyl-alanine substituted for tyrosine. In addition, the amino acid sequence of the truncated growth facto of the present invention are number 67' - 206', while the FGF sequences are presented as 1" - 146" and 1" - 141" for basic an acidic FGF, respectively. That is to say residues 1" - 146" comprise the sequence of basic FGF, while residues 1" - 141" comprise acidic FGF.

As shown in Example 5 below, the truncated protein ha mitogenic activity that is 4-5 times greater (i.e. increased DN synthesis and cell proliferation activity) than the full-length K-FGF protein as shown by its ability to induce proliferation o 3T3 cells at concentrations 4-5 times lower than those of K-FGF The truncated K-FGF protein also has a higher affinity for two the FGF receptors than either the full-length K-FGF protein or basic fibroblast growth factor (bFGF) .

The growth factor of the present invention can be obtained from the medium of cells transfected or transformed by the "wild type" or full-length K-FGF gene that have been cultivated in the presence of glycosylation inhibitors, such as tunicamycin. Alternatively, K-FGF-140 can be obtained from the medium of cells transfected or transformed by a mutated K-FGF cDNA that produces a protein incapable of being glycosylated, a the one described above, or preferably by using a suitable DNA construct to transform or transfect a eukaryotic, plant, or bacterial cell (e.g. E . coli) , the latter described in Example below. The wild type full-length gene can be obtained as described in co-pending U.S. Patent Application Serial No. 07/806,791 filed December 6, 1991. Alternatively the DNA sequence can be used to chemically synthesize the K-FGF-140 gen using techniques well known in the art.

The DNA encoding the growth factor of the present invention can be cloned and the protein can be expressed in any eukaryotic or prokaryotic system known in the art. Non-limitin examples of suitable eukaryotic expression systems include yeas expression vectors (described by Brake, A. et al., Proc. Nat. Acad. Sci. USA £1: 4642-4646, 1984), Polyoma virus based expression vectors (described in Kern, F.G. et al. Gene 43: 237 245, 1986) Simian virus 40 (SV40) -based expression vectors in COS-1 Simian cells (as described in Gething, M.J. et al. Nature 293: 620-625, 1981) and baculovirus (insect) -based expression vectors (described in U.S. Patent No. 4,745,051, issued May 17, 1988 and U.S. Patent No. 4,879,232, issued November 7, 1989). - example of a procaryotic expression system (e.g. E_^. coli) is presented below in Example 2 and an example of a eukaryotic expression system (e.g. COS cells) is presented below in Exampl 3. Particularly preferred expression vectors include E _. coli. simian COS cells and baculovirus (insect) cells.

The DNA encoding the truncated mammalian growth facto of the present invention may be modified without changing the primary sequence of the encoded polypeptide in order to increas the efficiency of its production. One such example is presente in Example 2 below where AT nucleotides were incorporated into the 5' end of the molecule for cloning into J _ coli. In addition, an ATG encoding methionine, was also added to the 5' end of the DNA. Other modifications for cloning and expression in other systems are known in the art and are within the scope the present invention.

The DNA sequence ^" (SEQ. ID. MO. 5) of K-FGF-140 is as follows: GCG GCC GTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC

Ala Ala Val Gin Ser Gly Ala Gly Asp Tyr Leu Leu Gly 1 5 10 ATC AAG CGG CTG CGG CGG CTC TAC TGC AAC GTG GGC ATC

He Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly He 15 20 25

GGC TTC CAC CTC CAG GCG CTC CCC GAC GGC CGC ATC GGC Gly Phe His Leu Gin Ala Leu Pro Asp Gly Arg He Gly

30 35

GGC GCG CAC GCG GAC ACC CGC GAC AGC CTG CTG GAG CTC 1

Gly Ala His Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu 40 45 50 TCG CCC GTG GAG CGG GGC GTG GTG AGC ATC TTC GGC GTG 1

Ser Pro Val Glu Arg Gly Val Val Ser He Phe Gly Val 55 60 65

GCC AGC CGG TTC TTC GTG GCC ATG AGC AGC AAG GGC AAG 2 Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys

70 75

CTC TAT GGC TCG CCC TTC TTC ACC GAT GAG TGC ACG TTC 2

Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe 80 85 90

AAG GAG ATT CTC CTT CCC AAC AAC TAC AAC GCC TAC GAG 3

Lys Glu He Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu 95 100

TCC TAC AAG TAC CCC GGC ATG TTC ATC GCC CTG AGC AAG 3

Ser Tyr Lys Tyr Pro Gly Met Phe He Ala Leu Ser Lys ^~ 105 110 115 AAT GGG AAG ACC AAG AAG GGG AAC CGA GTG TCG CCC ACC 3

Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Ser Pro Thr 120 125 130

ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG TGA 4 Met Lys Val Thr His Phe Leu Pro Arg Leu

135 140

The polypeptide of the present invention can be purified by any one of the many techniques that are well known the art for use in conjunction with the expression system to produce the polypeptide. For example, when expressing the protein in J _. coli, a purification procedure such as that disclosed in Example 2 below may be used.

The truncated K-FGF mammalian growth factor of the present invention can be employed as a wound-healing agent for various mammalian wounds, such as decubitus ulcers or burns. When employed as a wound or burn healing agent, the growth fact of the present invention may be administered to a mammal in nee of such treatment orally, parenterally, or preferably, topicall directly to the affected area in amounts broadly ranging betwee about 10 nanograms and about 10 micrograms per dose. The numbe of treatments required to treat a particular wound or burn and the duration of treatment can vary from individual to individua depending upon the severity of the wound or burn. A typical treatment would comprise 1 or 2 topical applications per day, that are applied directly to the surface of the wound or burn.

The growth factor of the present invention can be prepared in pharmaceutical formulations or dosage forms to be used as a wound or burn healing agent. Pharmaceutical formulations containing the mammalian growth factor of the present invention (or physiologically acceptable salts thereof) as at least one of the active ingredients may also contain pharmaceutically-acceptable carriers, diluents, fillers, salts and other materials well-known in the art depending upon the dosage form utilized. For example, parenteral dosage forms may comprise a physiologic, sterile saline solution. Topical dosag forms may comprise for example, lanolin, hydroxymethyl cellulos or propylene glycol. In an alternative embodiment, the mammali growth factor of the present invention may be mixed with antibiotic creams (such as Silvadene, Marion Laboratories, Kans City, MI, Achromycin, Lederle Laboratories, Pearl River, N.Y. , Terramycin, Pfipharmecs, New York, New York) well-known in the art.

As will be understood by those of ordinary skill in t art, the pharmaceutical formulations or dosage forms of the present invention need not contain an effective amount of the truncated protein of the present invention as such effective amounts can be achieved by administering a plurality of formulations or dosage forms. Although the truncated K-FGF growth factor of the present invention is particularly useful as a wound or burn healing agent it also can be employed as an agent to promote th growth of cells in tissue culture and/or as a partial serum substitute. The growth-promoting properties of truncated K-FGF are illustrated in Example 5 below. The invention is described further below in specific working examples which are intended to illustrate the present invention without limiting its scope.

EXAMPLE 1: AMINO ACID SEQUENCE OF THE HUMAN K-FGF PRECURSOR PROTEIN

The amino acid sequence of K-FGF and K-FGF-140 are shown in Fig. 1. In Fig. 1, arrows under the sequence indicate the sites of cleavage of the mature, secreted form of K-FGF.

Asterisks indicate the glycosylation signal. The result of the mutation introduced in the cDNA to eliminate glycosylation is indicated above the asterisks (Threonine to Alanine) . The [ si indicates the site of cleavage which generates K-FGF-140. EXAMPLE 2: CONSTRUCTION AND EXPRESSION VECTOR FOR K-FGF-140 The K-FGF-140 cDNA (which was mutated at the glycosylation site) was expressed in COS cells using media conditions that allowed tritiated leucine to be incorporated in the expressed protein. The leucine-labeled protein was purifie by precipitation with a polyclonal antibody raised against full- length K-FGF. The amino terminus of the purified K-FGF-140 protein was sequenced using a protein sequencer (Applied

Biosystems model 470A) . Tritium was found in several cycles an these cycles were assigned as leucine residues. There was a major sequence and a minor sequence. By a process of eliminati the major sequence was identified as starting at residue 67 of the K-FGF full-length sequence (Delli-Bovi et al. Cell 50: 729- 37 1987, Delli-Bovi et al. 1988 Molecular and Cellular Biology 8_.: 2933-41) . The sequence of this truncated protein is illustrated in Fig. 1.

A variety of different expression vectors may be used to produce the K-FGF-140 protein in E. coli. A bacterial expression vector was designed and constructed encoding the K- FGF-140 protein under the control of the bacteriophage lambda p promoter and the ell ribosome binding site.

The full-length cDNA sequence of K-FGF was altered using site directed mutagenesis (T.A. Kunkel et al. (1987) Methods in Enz mol. , Vol. 154, pages 367-382) to delete the sequence for the first 66 amino acids and place an initiator methionine in front of residue 67. It was also found desirable to change the codon usage pattern (using site directed mutagenesis) at the start of the truncated sequence to codons containing more Adenine or Thymidine. The sequence changes tha were made are illustrated in Table 2 below.

TABLE 2 Original Sequence GCG GCC GTC CAG AGC GGC GCC GGC GAC ... New Sequence ATG GCA GCA GTT CAA TCA GGA GCA GGC GAC ... Amino Acid Met Ala Ala Val Gin Ser Gly Ala Gly Asp ...

In Table 2, the nucleotides which were changed are underlined. None of the changes resulted in a change m the amino acids sequence of the protein.

Other changes to more favorable codons or changes further into the sequence could also have been made. This AT rich sequence at the start of the gene was found to optimize th amount of K-FGF-140 protein expressed in E. coli B4. The K-FGF-140 gene was expressed in E. coli.

Expression of the gene was accomplished by growing cells at 30° (the permissive temperature for the temperature sensitive lambd repressor) . The culture was then shifted to 40° C where the lambda repressor fails to repress the pL lambda promoter and maintained at this temperature for 3 hours. Cells were harvest by centrifugation and stored at -80° C. The bacterial cells we broken in the presence of break buffer (6.0 g Tris adjusted to 7.0 with HC1, 1.9 g EDTA, 1.7 g PMSF, 1.0 g pABA all in 1 L water) in a homogenizer (Gaulin model 15) . The cells were pass three times through the homogenizer at a pressure differential 8000-9000 pounds per square inch (PSI) . The broken cell paste was frozen in liquid nitrogen and stored at -80°C.

Most of the K-FGF-140 protein was found in the insoluble fraction in the cell lysate and was harvested by centrifugation. The growth factor was extracted from the centrifugation pellet by suspension in extraction buffer (50 mM

Tris pH 7.5, 200 mM MgCl₂) - The extract was centrifuged and K-

FGF-140 was found in the soluble fraction. This fraction was loaded onto a heparin Toyopearl >, ^"osohaas) column, and the buff exchanged with 0.5M NaCl, 50 mM Tris pH 7.5 followed by 0.5M

NaCl, 20 mM Na phosphate pH 7.5. Finally, the K-FGF-140 protei was eluted with a gradient of 0.5-1.75 NaCl in 20 mM Na phospha pH 7.5. The protein was found to elute at a salt concentration of about 1.55 M NaCl whereas full-length K-FGF elutes at about 1.15 M NaCl.

EXAMPLE 3: IMMUNOPRECIPITATION ANALYSIS OF THE

K-FGF FORMS PRODUCED IN COS CELLS TRANSFECTED WITH THE GLYC-K-FGF-140 CDNA

COS cells were transfected with the 91203B expression plasmid (described in Delli-Bovi et al. (1987) Cell. Vol. 50, pages 729-737) containing either the full-length human K-FGF cD or a mutated cDNA encoding a protein lacking the N-linked glycosylation signal (glyc(-)cDNA) . 40 hours later the cells were labelled with ³⁵S-methionine for 8 hours, in the presence ( or absence (-) of tunicamycin, a drug that inhibits N-linked glycosylation. Labelled proteins from either the cell lysate (indicated as L) or medium (indicated as M) were immunoprecipitated with anti-K-FGF rabbit antibodies and electrophoresed on SDS-PAGE. The gel was then subjected to autoradiography. The results are shown in Fig. 2.

In Fig. 2, M.W. markers are indicated on the right. can be seen that the cell transfected with the glyc(-) cDNA expressed in the cell lysate a protein of apparent M.W. of 18,0 Daltons, identical to the one produced by the wild-type K-FGF in the presence of tunicamycin. This protein cannot however, detected in the culture medium, where only two bands of MW 12,000-14,000 were seen.

EXAMPLE 4: ELUTION OF K-FGF-140 FROM HEPARIN AFFINITY COLU Conditioned Medium labeled with ³⁵S-methionine produc from COS cells transfected with either of K-FGF or glyc-cDNAs (i.e. K-FGF-140) was absorbed to Heparin-Sepharose columns and eluted with increasing salt concentrations. Fractions were immunoprecipitated with anti-K-FGF antibodies, and electrophoresed on SDS-PAGE to identify the K-FGF proteins. Th results are shown in Fig. 3.

It can be seen that all or most of K-FGF eluted at 1.

M NaCl, while the truncated K-FGF forms eluted with a peak at

1.3-1.6 M NaCl.

EXAMPLE 5: STIMULATION OF DNA SYNTHESIS IN QUIESCENT BALB/C-3T3 CELLS BY HUMAN RECOMBINANT

K-FGF OR BY RECOMBINANT K-FGF-140

BALB/c-3T3 cells were incubated for two days in mediu containing 0.5% serum, at which point cells were treated with different concentrations of K-FGF or K-FGF-140. 18 hours later the cells were labeled with ³H-thymidine (1 μCi/ml) for 6 hours. Radioactivity incorporated into cellular DNA was counted after trichloroacetic acid (TCA) precipitation. The results are show in Fig. 4.

In Fig. 4, 0.5% = negative control, 10% = cells stimulated with 10% serum. As can be seen from the data in Fig 5, 0.1 ng of K-FGF-140 was capable of producing the same stimulation of DNA synthesis as that of 0.5 ng of K-FGF. Furthermore, maximum stimulation .in ³H-thymidine uptake occurre using lng/ml of K-FGF-140 and 5ng/ml full-length K-FGF. Treatment with lng/ml of K-FGF-140 led to a greater amount of cell proliferation than all other additions, including 10% seru EXAMPLE 6: RECEPTOR BINDING

To study the affinity of K-FGF-140 for FGF receptors, the ability of K-FGF and K-FGF-140 to compete with ¹²⁵I-labeled basic fibroblast growth factor (bFGF) for binding to CHO 4-1 cells expressing FGF receptor-1 (Mansukhani, A. et al. (1992) Proc. Natl. Acad. Sci. USA. Vol. 89, pages 3305-3309) (A) or to CHO 3-7.5 cells expressing the FGF receptor-2 (Mansukhani, A. e al. (1990) Proc. Natl. Acad. Sci. USA. Vol. 87, pages 4378-4382 (B) was performed. Cells (lxlO⁶ cells/35 mm dish) were incubat at 4° C with Dulbecco's modified EAGLE'S medium (DMEM) containi 0.15% gelatin, 25 mM Hepes (pH 7.4), Heparin (10 g/ml) , ¹²⁵I- labeled bFGF (4 ng/ml s.a. 3.2 x 10¹⁷ cpm/mole, Collaborative Research) and the indicated concentration of unlabeled bFGF, K- FGF or K-FGF-140. After 2 hours the cells were washed with 2M NaCl buffered at pH 7.4 to remove growth factor bound to the matrix, and with 2M NaCl buffered at pH 4.0 to remove the ligan bound to high affinity receptors. The amount of ¹⁵I-labeled bFG bound to high affinity receptors was determined. The results a shown in Fig. 5 (A and B) .

In the CHO clone expressing the FGF receptor 1 (Fig 5A) the data show that about 8 times more K-FGF than bFGF or K- FGF-140 was needed to compete for the binding of ¹⁵I-labeled bFGF; in the CHO clone expressing the FGF receptor 2 (Fig. 5B) , K-FGF-140 was more efficient than K-FGF and bFGF in competing f the binding of ¹²⁵I-labeled bFGF. In this case the affinity of K

FGF-140 for the receptors was about three times higher than tha of bFGF or full-length K-FGF.

EXAMPLE 7: COMPETITION BETWEEN K-FGF AND K-FGF-140 FOR RECEPTOR BINDING

Clones CHO 4-1 expressing FGF receptor-1 (Fig. 6A and

Fig. 6C) or CHO 3-7.5 expressing FGF receptor-2 (Fig. 6B and Fi

6D) were incubated with ¹²⁵I-labeled K-FGF-140 (11 ng/ml, specifi activity 7.7 x 10¹⁶ cpm/mole) (Fig. 6A and B) or with ¹²⁵I-labeled K-FGF (8 ng/ml, specific activity 9.9 x 10¹⁶ cpm/mole) (Fig. 6C and D) , Heparin (10 ug/ml) and the indicated concentration of unlabeled K-FGF or K-FGF-140. After 2 hours at 4° C the medium was removed, the cells were washed with ice cold Tris and were lysed in 0.6% SDS/50mM.Tris/HCl pH 7.4, 0.15M NaCl, 5mM.EDTA, a the cell associated radioactivity was determined. The data are expressed as % of inhibition of Iodine labeled growth-factor binding by the indicated amount of unlabeled growth factor.

The data presented in Fig. 6 show that K-FGF-140 had higher affinity for both FGF (Fig. 6A and 6C and Fig. 6B and 6D receptors than full-length K-FGF protein. EXAMPLE 8: SCATCHARD ANALYSIS OF K-FGF AND

K-FGF-140 BINDING TO CHO CELLS EXPRESSING THE FLG RECEPTOR

Scatchard analysis of the binding of K-FGF and K-FGF- 140 was performed on CHO 4-1 cells expressing the FGF receptor- as follows. Cells at 4.8xl0⁵/35mm dish were incubated at 4° C with DMEM containing 0.15% gelatin, 25 mM. Hepes(pH 7.4), Hepa

(10 ug/ml) and various concentration of ¹²⁵I-labeled K-FGF or K-

FGF-140 from 0.15 to 20 ng/ml. After 2 hours the medium was removed, the cells were washed with ice cold Tris and ¹²⁵I-label K-FGF or K-FGF-140 bound to high affinity receptors was removed by extraction in 0.6%SDS/50mMTris/HCl pH 7.4, 0.15 mM NaCl, 5m EDTA. Non-specific binding was obtained using the same amount growth factor on parental CHO DG44 cells that do not express FG receptors. The results are shown in Fig. 7.

In Fig. 7, Scatchard analysis of binding to high affinity receptors gave a straight line, indicating a single class of binding sites. The data also indicate that the avera binding affinity of K-FGF-140 for the fig receptor is about 9.5X10^"UM or about three times higher than that of full-length K-FGF which has an average binding affinity of about 28.5X10^"UM EXAMPLE 9: WOUND HEALING ASSAY

K-FGF-140 will be assayed in an ischemic wound heali system. For this purpose the rabbit ear ischemic model of der ulcers, in which healing of these ulcers is retarded because of induced ischemia (reduced blood flow) is used. After wounding mm wounds) K-FGF-140 is applied either in an isotonic buffer o in a gel, applied in a single dose (1-5 μg) , and compared to untreated controls, or wounds treated with K-FGF or bFGF. At various days after the beginning of the experiment (up to day 10) the extent of wound healing is determined by measuring a) epithelium formed at the gap of epithelia tissue at the beginn and end of the experiment) by histological cross sections; b) gap between the two edges of the granulation tissues; and c) formation of new granulation tissue as measured by staining of immature vs. mature collagen. These techniques are described i Ann, S.T. and Mustoe, T.A. Annals Plastic Surgery 24: 17-23 (1990) and Mustoe, T.A. , Pierce G.F., Morishima, C. and Deuel, T.P. J. Clinical Invest. 87: 694-703 (1991). From the in vitro experiments present above showing that K-FGF-140 has higher potency and receptor affinity then K- FGF, it is expected that K-FGF-140 will prove effective at accelerating wound healing in the system, and will prove more potent (effective at lower concentration, faster response) than K-FGF or bKFGF.

Claims

WHAT IS CLAIMED IS; 1. A truncated mammalian growth factor protein hav substantial homology to each of basic and acidic fibroblast growth factor proteins in a single polypeptide, said truncated mammalian growth factor being substantially smaller than the full-length mammalian growth factor.

2. The truricated mammalian growth factor of claim said truncated mammalian growth factor being unglycosylated.

3. The truncated mammalian growth factor of claim having a molecular weight of about 14,000 Daltons.

,

4. A polypeptide having the amino acid sequence: Ala Ala Val Gin Ser Gly Ala Gly Asp Tyr Leu Leu Gly He Lys Ar 1 5 10 15 Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu Gin Al 20 25 30 Leu Pro Asp Gly Arg He Gly Gly Ala His Ala Asp Thr Arg Asp Se 35 40 45 Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser He Phe Gl 50 55 60 Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys Leu Ty 65 70 75 8 Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu He Leu Le 85 90 95 Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Ph 100 105 110 He Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Se 115 120 125 Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu. 130 135 140

5. A pharmaceutical formulation for treating a mam suffering from wounds or burns comprising a truncated mammalia growth factor protein having substantial homology to each of acidic and basic fibroblast growth factors in a single polypeptide and truncated mammalian growth factor being substantially smaller than the full-length mammalian growth factor and a pharmaceutically acceptable carrier or diluent.

6. The pharmaceutical formulation of claim 5 comprising a topical pharmaceutical formulation.

7. A method for healing wounds or burns in a mammal comprising administering to a mammal in need of such treatment a wound or burn healing effective amount of a truncated mammalian growth factor having a substantial homology to each of basic and acidic fibroblast growth factor in a single polypeptide, said truncated mammalian growth factor being substantially small tha the full-length mammalian growth factor.

8. An isolated DNA having the sequence: GCG GCC GTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC 3 ATC AAG CGG CTG CGG CGG CTC TAC TGC AAC GTG GGC ATC 7 GGC TTC CAC CTC CAG GCG CTC CCC GAC GGC CGC ATC GGC 11 GGC GCG CAC GCG GAC ACC CGC GAC AGC CTG CTG GAG CTC 15 TCG CCC GTG GAG CGG GGC GTG GTG AGC ATC TTC GGC GTG 19 GCC AGC CGG TTC TTC GTG GCC ATG AGC AGC AAG GGC AAG 23 CTC TAT GGC TCG CCC TTC TTC ACC GAT GAG TGC ACG TTC 27 AAG GAG ATT CTC CTT CCC AAC AAC TAC AAC GCC TAC GAG 31 TCC TAC AAG TAC CCC GGC ATG TTC ATC GCC CTG AGC AAG 35 AAT GGG AAG ACC AAG AAG GGG AAC CGA GTG TCG CCC ACC 39 ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG TGA 42

9. A truncated mammalian growth factor comprising a polypeptide chain having regions that are homologous to each of acidic and basic fibroblast growth factor, said polypeptide bei substantially smaller than the full-length mammalian growth factor.

10. A truncated K-FGF protein having a molecular weight of 14,000 Daltons, and an average FGF receptor binding affinity of about 9.5X10-^UM.

11. The truncated protein of claim 10 wherein said protein has about 4 to 5 times greater mitogenic activity than full-length K-FGF when assayed in the BALB/c-3T3 mitogenic ass