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WO1994000570A9 - Facteur de croissance de mammiferes - Google Patents

Facteur de croissance de mammiferes

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Publication number
WO1994000570A9
WO1994000570A9 PCT/US1993/005962 US9305962W WO9400570A9 WO 1994000570 A9 WO1994000570 A9 WO 1994000570A9 US 9305962 W US9305962 W US 9305962W WO 9400570 A9 WO9400570 A9 WO 9400570A9
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WO
WIPO (PCT)
Prior art keywords
growth factor
fgf
gly
ggc
leu
Prior art date
Application number
PCT/US1993/005962
Other languages
English (en)
Other versions
WO1994000570A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU46459/93A priority Critical patent/AU4645993A/en
Publication of WO1994000570A1 publication Critical patent/WO1994000570A1/fr
Publication of WO1994000570A9 publication Critical patent/WO1994000570A9/fr

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Definitions

  • This invention pertains to a mammalian growth factor pharmaceutical formulations comprising said factor and methods for healing wounds or burns in mammals comprising administerin said formulations.
  • This invention pertains to a novel polypeptide havin 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 are often present in serum a have also been isolated from a variety of organs. They are protein molecules (or groups of such molecules) and in all kno cases they interact with specific cell surface receptors to promote cellular growth and/or differentiation. Growth facto vary in their tissue specificity, i.e. some interact only with specific cell types, while others are active on a wider cell type range.
  • platelet derived growth factor PDGF
  • EGF epidermal growth factor
  • CSF colony stimulating factors
  • fibroblast 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.
  • FGF fibroblast growth factors
  • Two well-characterized angiogenic factors are basic and acidic fibroblast growth factors (FGF) , believed to be most important in vivo for endothelial cell growth.
  • FGF acidic fibroblast growth factors
  • 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 vitr and thus cannot be expected to interact with all FGF receptors in vivo.
  • 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 a 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 matu (secreted) glycoprotein.
  • This growth factor has variously bee called K-FGF or FGF-4, and it has shown promising results as a wound healing agent in preclinical studies in an ischemic rabb ear model. In such a model, K-FGF promoted wound healing bett than basic or acidic FGF.
  • TGF transforming growth factor
  • VGF vaccinia virus growth factor
  • angiogeni growth factors would be particularly useful as wound healing agents because of their ability to promote the formation and growth of new blood vessels.
  • a still further object of the present invention is t 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.
  • 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-
  • the present invention provides a polypeptide having the amino acid sequence (SEQ. ID. NO. 1) :
  • 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 amoun 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
  • 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 "n M.
  • Figure l 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 wi a mutated K-FGF cDNA which expresses an unglycosylated form of K-FGF that is processed to produce K-FGF-140.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoretic
  • Figure 3 is an autoradiograph of an SDS-PAGE analysi 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 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 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) .
  • 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.
  • 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 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 disclosed in U.S. Patent Application Serial No. 07/806,791 fil 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.
  • K-FGF-140 was discovered during studies on the effec of glycosylation on the secretion of full-length K-FGF. Simia 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).
  • 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.
  • the N-terminal amino acid (alanine) of K FGF-140 is residue 67 of the full-length K-FGF protein.
  • Two d 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 (SEQ. ID. NO. 4) , and one dot indicates that there has been a conservative substitution, e.g. substitution of the same type amino acid such as phenyl-alanine substituted for tyrosine.
  • amino acid sequence of the truncated growth fact of the present invention are number 67' - 206'
  • FGF sequences are presented as 1" - 146" and 1" - 141" for basic a acidic FGF, respectively. That is to say residues 1" - 146" comprise the sequence of basic FGF, while residues 1" - 141" comprise acidic FGF.
  • the truncated protein h mitogenic activity that is 4-5 times greater (i.e. increased D synthesis and cell proliferation activity) than the full-lengt K-FGF protein as shown by its ability to induce proliferation 3T3 cells at concentrations 4-5 times lower than those of K-FG
  • 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 b the "wild type" or full-length K-FGF gene that have been cultivated in the presence of glycosylation inhibitors, such a tunicamycin.
  • 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.
  • the DNA sequence can be used to chemically synthesize the K-FGF-140 ge 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 an eukaryotic or prokaryotic system known in the art.
  • suitable eukaryotic expression systems include yea expression vectors (described by Brake, A. et al. , Proc. Nat. Acad. Sci. USA 81.: 4642-4646, 1984), Polyoma virus based expression vectors (described in Kern, F.G. et al. Gene 43: 23 245, 1986) Simian virus 40 (SV40) -based expression vectors in COS-1 Simian cells (as described in Gething, M.J. et al.
  • 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.
  • a procaryotic expression system e.g. I _ coli
  • a eukaryotic expression system e.g. COS cells
  • Particularly preferred expression vectors include E ⁇ _ coli, simian COS cells and baculovirus (insect) cells.
  • the DNA encoding the truncated mammalian growth fact of the present invention may be modified without changing the primary sequence of the encoded polypeptide in order to increa the efficiency of its production.
  • One such example is present in Example 2 below where AT nucleotides were incorporated into the 5' end of the molecule for cloning into I . coli.
  • an ATG encoding methionine was also added to the 5' end of the DNA.
  • Other modifications for cloning and expressio in other systems are known in the art and are within the scope the present invention.
  • the DNA sequence (SEQ. ID. NO. 5) of K-FGF-140 is as follows: GCG GCC GTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC
  • 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 EL . 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.
  • 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.
  • parenteral dosage forms may comprise a physiologic, sterile saline solution.
  • Topical dosag forms may comprise for example, lanolin, hydroxymethyl cellulos or propylene glycol.
  • 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.
  • antibiotic creams such as Silvadene, Marion Laboratories, Kans City, MI, Achromycin, Lederle Laboratories, Pearl River, N.Y., Terramycin, Pfipharmecs, New York, New York
  • 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.
  • 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 t 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.
  • K-FGF and K-FGF-140 are shown in Fig. l.
  • 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
  • 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 Biolog 8 . : 2933-41) . The sequence of this truncated protein is illustrated in Fig. 1.
  • a variety of different expression vectors may be use 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 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 Enzymol. , 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.
  • K-FGF-140 protein 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
  • FGF-140 was found in the soluble fraction. This fraction was loaded onto a heparin Toyopearl (Tosohaas) column, and the buff exchanged with 0.5M NaCl, 50 mM Tris pH 7.5 followed by 0.5M
  • 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.
  • 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 cD ⁇ A encoding a protein lacking the ⁇ -linked glycosylation signal (glyc(-) cD ⁇ A) . 40 hours later the cells were labelled with 35 S-methionine for 8 hours, in the presence ( or absence (-) of tunicamycin, a drug that inhibits ⁇ -linked glycosylation.
  • the 91203B expression plasmid described in Delli-Bovi et al. (1987) Cell. Vol. 50, pages 729-737
  • the cells were labelled with 35 S-methionine for 8 hours, in the presence ( or absence (-) of tunicamycin, a drug that inhibits ⁇ -linked glycosylation.
  • EXAMPLE 4 ELUTIO ⁇ OF K-FGF-140 FROM HEPARI ⁇ AFFINITY COLU Conditioned Medium labeled with 35 S-methionine produce 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. T results are shown in Fig. 3.
  • EXAMPLE 5 STIMULATION OF DNA SYNTHESIS IN QUIESCENT BALB/C-3T3 CELLS BY HUMAN RECOMBINANT
  • BALB/c-3T3 cells were incubated for two days in medi containing 0.5% serum, at which point cells were treated with different concentrations of K-FGF or K-FGF-140. 18 hours late the cells were labeled with 3 H-thymidine (1 ⁇ Ci/ml) for 6 hours Radioactivity incorporated into cellular DNA was counted after trichloroacetic acid (TCA) precipitation. The results are sho in Fig. 4.
  • K-FGF-140 For study the affinity of K-FGF-140 for FGF receptors the ability of K-FGF and K-FGF-140 to compete with 125 I-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 t CHO 3-7.5 cells expressing the FGF receptor-2 (Mansukhani, A. al. (1990) Proc. Natl. Acad. Sci. USA. Vol. 87, pages 4378-438 (B) was performed.
  • bFGF basic fibroblast growth factor
  • DMEM Dulbecco's modified EAGLE'S medium
  • 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
  • the data ar expressed as % of inhibition of Iodine labeled growth-factor binding by the indicated amount of unlabeled growth factor.
  • 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 125 I-label K-FGF or K-FGF-140 bound to high affinity receptors was remove 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 F receptors. The results are shown in Fig. 7.
  • K-FGF-140 will be assayed in an ischemic wound heali system.
  • the rabbit ear ischemic model of der ulcers in which healing of these ulcers is retarded because o induced ischemia (reduced blood flow) is used.
  • K-FGF-140 is applied either in an isotonic buffer o in a gel, applied in a single dose (1-5 ⁇ g) , and compared to untreated controls, or wounds treated with K-FGF or bFGF.
  • 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.
  • 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) tha K-FGF or bKFGF.

Abstract

Facteur de croissance tronqué présentant une homologie avec les facteur de croissance basique et acide dans un polypeptide unique. Le facteur de croissance est sensiblement plus petit (c'est-à-dire qu'il comporte moins de restes d'acides aminés) que le facteur de croissance de longueur normale de Mammiféres, et il présente une affinité plus importante pour les récepteurs de facteurs de croisance fibroblastiques que pour le facteur de croissance fibroblastique K de longueur normale et le facteur de croissance fibroblastique basique, ainsi qu'une activité nitrogène accrue. Cette invention concerne également des séquences d'ADN codant le facteur de croissance tronqué et des procédés de guérison et de cicatrisation de brûlures et de blessures dans lesquels on administre à un mammifère brûlé ou bléssé lesdites compositions pharmaceutiques.
PCT/US1993/005962 1992-06-22 1993-06-22 Facteur de croissance de mammiferes WO1994000570A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46459/93A AU4645993A (en) 1992-06-22 1993-06-22 Mammalian growth factor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90170592A 1992-06-22 1992-06-22
US901,705 1992-06-22

Publications (2)

Publication Number Publication Date
WO1994000570A1 WO1994000570A1 (fr) 1994-01-06
WO1994000570A9 true WO1994000570A9 (fr) 1994-05-26

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7432243B2 (en) 2002-04-08 2008-10-07 The Scripps Research Institute Truncated 24kDa basic fibroblast growth factor

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
US5126323A (en) * 1989-11-16 1992-06-30 Genetics Institute, Inc. Homogeneous purified k-fgf and compositions containing the same
US5206354A (en) * 1990-11-23 1993-04-27 American Cyanamid Company Dna sequence encoding active fragment of fibroblast growth factor, hbf-2

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