MXPA97002475A - Analogues of the fibroblast acid growth factor that have stability and biological acticity improves - Google Patents

Abstract

The present invention describes analogs of proteins in the FGF family. These analogs are more stable than the corresponding proteins that exist naturally. The improved stability can be achieved by substituting at least one amino acid having a higher loop-forming potential for an amino acid residue having a lower loop-forming potential in the region of loop formation identified in the amino acid sequence of the loop. protein. The analogs of the present invention are especially useful in therapeutic applications

Description

ANALOGUES OF THE FIBROBLAST ACID GROWTH FACTOR THAT HAVE IMPROVED STABILITY AND BIOLOGICAL ACTIVITY BACKGROUND The complex healing process that follows injury to a tissue, such as by wound or burn, is mediated by a number of protein factors sometimes called soft tissue growth factors. These factors are required for the growth and differentiation of new cells, to replace the destroyed tissue. Included within this group of soft tissue growth factors is a family of proteins of fibroblast growth factors (FGFs). FGFs are mitogenic, and chemotactic for a variety of cells of epithelial, -mesenchymal, and neural origin. In addition, FGFs are angiogenic, that is, they are able to stimulate the formation of blood vessels. Members of the FGF family include acidic FGF, basic FGF, KGF, lnt-2, HST, FGF-5, and FGF-6. The acid FGF (aFGF) and the basic FGF (bFGF) are considered to be two "original" members of the FGF family. It is believed that both aFGF and bFGF are derived from the same ancestral gene, and both molecules have approximately 55% sequence identity, in addition to the same intron / exon structure. It is also known that acid FGF and bFGF bind to the same receptor, although the existence of specific receptors for aFGF and bFGF has not been ruled out. Several molecular weight forms of aFGF and REF: 24298 bFGF are found in different tissues. However, experiments by Southern blotting suggest that there is only one gene for aFGF and bFGF, with differences between these molecules probably due to post-translational processing. Both acidic and basic FGF are mitogenic for a wide variety of cell types of mesodermal and neuroectodermal origin, and are capable of inducing angiogenesis both in vitro and in vivo (see, for example, Gospodarowics et al (1979), Exp. Eye Res., 28: 501-514). The range of biological activities of the two classes is almost identical, although bFGF is about ten times more potent than aFGF in most bioassay systems. KGF exhibits a potent mitogenic activity for a variety of cells, and binds to the cell surface receptors that are on the Balb / MK keratinocytes, to which the aFGF and bFGF can also bind (Bottaro, et al. 1990), J. Biol. Chem., 265: 12767-12770). However, KGF is distinct from known FGFs (eg, aFGF and bFGF) in that it is not mitogenic for fibroblasts or endothelial cells (Rubin et al., (1989), Proc. Nati. Acad. Sci. USA , 86: 802-806). KGF also has different receptors on NIH / 3T3 fibroblasts of receptors for aFGF and bFGF, which do not interact with KGF. Bottaro et al. A shared distinctive feature of aFGF and bFGF is the propensity of these factors to bind tightly to heparin. The affinity of heparin for aFGF seems to be weaker than for bFGF, and aFGF has an anionic isoelectric point. (Thomas et al (1984), Proc. Nati, Acad. Sci. USA, 82: 6409-6413). The unique property of heparin binding of aFGF and bFGF has greatly enhanced the purification of these factors. The discovery that FGFs have a strong affinity for immobilized heparin has also spurred research into the regulatory role of heparin-like molecules in the in vivo biology of FGFs. Although the full spectrum of functions for heparin has yet to be determined, it is known that heparin can regulate the function of FGF in several ways (Lobb (1988), Eur. J. Clin. Invest., 18: 321-326). . For example, heparin-like molecules can play a direct role in the function of FGF, including the activation, or potentiation, of aFGFs (Uhllrich et al., (1986), Biochem. Biophys. Res. Comm., 137: 1205-1213). There is, however, no direct correlation between the affinity of FGF for immobilized heparin and its ability to be enhanced by soluble heparin. In this respect, the potentiating power of heparin appears to be selective for aFGF. For example, Uhllrich et al., (1986), supra, found that the degree of potentiatof pure aFGF was about ten times greater than that of pure bFGF, raising the potency of aFGF to approximately the same level as that of bFGF. . However, it was found that in the presence of fetal calf serum, the potentiating effect of heparin decreased significantly (Uhllrich et al., (1986), supra).

It is believed that the use of FGF proteins is effective in promoting the healing of tissue that has been subject to trauma. The unique angiogenic property of FGFs makes these factors especially valuable in the healing of deep wou It has been claimed that the natural proteins of bFGF are useful in the treatment of myocardial infarction (U.S. Patent Nos. 4,296,100 and 4,378,347). In addition, it has been found that human bFGF increases neuronal survival and neurite extension in fetal rat hippocampal neurons, suggesting that this factor may also be useful in the treatment of degenerative neurological disorders, such as Alzheimer's disease and of Parkinson (Wallicke et al., (1986), Proc. Nati, Acad. Sci. USA, 83: 3012-3016). A major obstacle to the effective use of aFGF in therapeutic applications seems to be related to its significantly lower biological activity, compared to that of bFGF. Although studies with heparin suggest that the potential observed difference between aFGF and bFGF can be substantially decreased by using heparin to increase the activity of aFGF at a level comparable to that of bFGF, the use of heparin in pharmaceutical preparations may not always be desirable. In this regard, it is important to note that heparin, a highly sulfated glycosaminoglycan of heterogeneous structure, is known as an anticoagulant, which works by accelerating the rate at which antithrombin III inactivates the proteases of homeostasis (Jacques (1980), Pharmacol. Rev, 31: 99-166). It is not known if it can be detrimental to use heparin in a pharmaceutical preparation for the treatment of deep wou where some degree of coagulation may be desired to achieve proper healing. In addition, practical considerations can be expected to arise when heparin is incorporated into a pharmaceutical preparation for wound healing. Concerns about the drug supply include the matter of controlling the composition of the pharmaceutical preparation (containing the combination of aFGF and heparin) at the time of entry into the patient's body. On the other hand, the negative effect of fetal calf serum on the enhancing effect of heparin on aFGF- (observed by Uhllrich et al) suggests that any advantage obtained by including heparin in the pharmaceutical preparation as an activating or enhancing factor for aFGF it could be completely denied or lost, once contact is made with the patient's own serum. It is an object of the present invention to provide a protein analog of the FGF family that has an increased stability, compared to the naturally occurring form of the protein. It is a further object of the present invention to provide an analog of aFGF that exhibits improved stability and biological activity in the absence of heparin. It is a further object of the present invention to provide an aFGF analog for therapeutic use.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides novel protein analogues in the FGF family. An analog is an analog of aFGF that is more stable and exhibits greater biological activity in the absence of heparin than aFGF that occurs naturally. Another analog is a KGF analog that has improved thermal stability, compared to naturally occurring KGF. Improved stability is achieved by substituting at least one amino acid having a higher loop-forming potential for an amino acid residue of lower loop-forming potential, at or near the Asn-His-Tyr loop-forming sequence -Asn-Thr-Tyr of the protein that exists naturally. In the case of aFGF, this loop formation sequence occurs in the area near the amino acids 92 to 96. In the case of KGF, this region of loop formation occurs in the vicinity of the amino acids 115- 119 A preferred analogue of the present invention incorporates the substitution of an amino acid having a higher looping potential for the histidine residue in the loop formation sequence. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are the adducts of the pr-lForp and arai acid acids of the [Ala47, Gly ^ JaFGFbovina recombinante. Figures 2A and 2B depict the acid sealant of the recombinant human [Gly ^ aFGF.

Figure 3 demonstrates the elution profiles for the [Ala47] and [Ala47, G! And 93] aFGF bovine analogs, using hydrophobic interaction chromatography. Figures 4A and 4B show the spectrum of circular dichroism for the [Ala47] and [Ala47, Gl 8JaFGF bovine analogs. Figure 5 shows the second derivative of the IRTF spectrum of bovine analogues of [Ala47] and [Ala47, Gly93] aFGF in the region of amide I '(narrowing of C = 0 in deuterated proteins). Figure 6 is a graph showing a log plot of the concentration of the [Ala47] and [Ala47, Gly ^ JaFGF bovine and [Ser70, _. Ser ^ JbFGF human versus the percentage of maximum stimulation. Figure 7 is a graph showing the loss of activity over time of the [Ala47] and [Ala47, Gly93] aFGF bovine analogues in the absence of heparin, compared to human [Ser70, SerM] bFGF. Figure 8 shows the structure of the [Ala47, Bovine Gly ^ aFGF analog of the present invention, determined by X-ray crystallography. Figures 9A and 9B show the sequences of the nucleic acid and amides of naturally occurring KGF. Figures 10A and 10B show the nucleic acid and acid sequences of the recombinant [Gly116] KGF.

DETAILED DESCRIPTION OF THE INVENTION Novel analogs of the FGF family are provided in accordance with the present invention. These analogs exhibit improved stability, compared to that with the corresponding naturally occurring protein. In the case of the aFGF analog of the present invention, the analog exhibits improved stability and biological activity, in the absence of heparin. In the case of the KGF analog of the present invention, the analog exhibits improved thermal stability. The analogs of the present invention have at least one amino acid residue different from the corresponding naturally occurring protein, at or near the Asn-His-Tyr-Asn-Thr-Tyr loop formation sequence found in the form that exists naturally. In the case of aFGF, the loop formation sequence occurs in the area around amino acid residues 92 to 96 (based on the numbering of the known amino acid sequence for bovine aFGF, shown in Figure 1) . In the case of KGF, the loop formation sequence occurs in the area around the amino acid residues 115-119, as shown in Figures 9 and 10. The different amino acid (s) (s) ) is selected (n) for its highest loop forming potential, to stabilize this area of the analogue. Amino acids that have a relatively high loop-forming potential include glycine, proline, tyrosine, aspartic acid, asparagine, and serine (Leszcynski et al., (1986), Science, 234: 849-855) (the values relative of loop formation potentials were assigned based on the frequency of occurrence in loop structures of naturally occurring molecules)). Preferably, a different amino acid, which has a higher loop-forming potential, replaces the histidine residue in the loop-forming sequence. Still more preferably, the histidine in the loop formation sequence is replaced with a glycine residue. Other additions, substitutions, and / or deletions may be made to the analogs of the present invention. For example, the analog may also optionally include an amino acid substitution for non-conserved cysteine residues (eg, the cysteine residue at position 47 of the bovine aFGF molecule, and the cysteine residue at position 16 of the -molecule of human sFGF). In addition, analogs of the present invention that are expressed from E. coli host cells may include an initial methionine amino acid residue (i.e., at position -1, as shown in Figure 1). Alternatively, one or more of the terminal amino acid residues may be deleted from the DNA sequence, as is known to those skilled in the art, while substantially retaining the enhanced biological aity of the corresponding naturally occurring protein. DNA sequences encoding all or part of the analogs of the present invention are also provided. Such sequences may preferably include the incorporation of "preferred" codons for expression by selected E.coli host strains ("E.coli expression codons"), the provision of hydrolysis sites by endonuclease restrin enzymes, and or the provision of additional, initial, terminal, or intermediate DNA sequences that facilitate the construn of easily expressed vectors These novel DNA sequences include sequences useful for ensuring the expression of the analogs of the present invention in both eukaryotic and prokaryotes, such as from E. coli More specifically, the DNA sequences of the present invention may comprise the DNA sequence shown in Figure 1, wherein at least one codon encodes an amino acid residue in the surrounding area of the amino acids 92 to 96 is replaced by a codon encoding a different amino acid residue that has a pot Higher loop formation element (hereinafter "aFGF analog sequence (s)" or "analogous sequence (s)"), as well as a DNA sequence hybridizing to one of the analogous sequences or fragments of it, and a DNA sequence that, except for the degeneration of the genetic code, would hybridize to one of the analogous sequences. Correspondingly, the DNA sequences of the present invention may comprise the DNA sequence shown in Figure 10, wherein at least one codon encoding an amino acid residue in the area around amino acids 115-119 is replaced by a codon encoding a different amino acid residue having a higher loop-forming potential (hereinafter "KGF analog sequence (s)" or "analog sequence (s)"), as well as a DNA sequence that hybridizes to one of the analog sequences, or fragments thereof, and a DNA sequence which, except for the degeneracy of the genetic code, would hybridize to one of the analog sequences. The analogs of the present invention can be encoded, expressed, and purified by any of a number of recombinant technology methods known to those skilled in the art. The preferred produn method will vary depending on many factors and considerations, including the cost and availability of materials, and other economic considerations. The optimal produn procedure for a given situation will be apparent to those skilled in the art through minimal experimentation. The analogs of the present invention can be expressed at particularly high levels using E. coli host cells, and the resulting expression product is subsequently purified to near homogeneity using procedures known in the art. A typical purification procedure involves first solubilizing the inclusion bodies containing the analogues, followed by ion exchange chromatography, then re-folding the protein, and finally, hydrophobic interan chromatography.

The analogs of the present invention exhibit a surprising degree of improved stability. Contrary to naturally occurring aFGF, the aFGF analogs of the present invention demonstrate improved stability and biological activity in the absence of heparin. While it is known that more stable aFGF analogs can be obtained through the substitution of serine or other neutral amino acids instead of certain cysteine residues (eg, as described in published PCT Patent Application No. 88 / 04189, the single substitution for the unconserved cysteine residue at position 47 of the naturally occurring bovine aFGF is not believed to be significant for improving the biological activity and / or stability of an aFGF analogue. activity -exhibited by a bovine [Ala47] aFGF analogue (substituted with cysteine) compared to a bovine [Ala47, Gly93] aFGF analogue (having the substitution of the desired amino acid in the region of residue 92 to 96 of the aFGF), as shown in the following examples: Specifically, the analogue of [Ala47, bovine Gly ^ aFGF, although still less potent compared to bFGF, was found to be approximately ten times more potent than the analogue of [Ala47] bovine aFGF. With the addition of 45 μg / ml heparin, the bioactivity of the three forms of FGF was improved, and the bovine [Ala47, Gly93] analogue, the bovine [Ala47, Gly93] analog and the [Ser70, Ser ^ bFGF human had a substantially identical potency.

The reason for improved mitogenic activity and stability of the bovine [Ala47, Gly93] aFGF analog relative to bovine [Ala47] aFGF in the absence of heparin was not immediately clear. The substitution of glycine by the histidine residue at position 93 seemed to make the aFGF molecule somewhat more hydrophobic, but did not appear to dramatically alter its tertiary structure, as determined by circular dichroism and IRTF spectroscopy. However, the relative differences in activities observed in the in vitro vitrq bioassays for the [Ala47, Gly ^ JaFGF bovine analog and bovine [Ala 7] aFGF analogue (with substitution only at position 47) suggested that the position 93 of the amino acid substituted by glycine of the [Ala47, Gly93] bovine aFGF analog could be in or near the region responsible for receptor binding. Although the receptor binding region in aFGF has not been determined, position 93 in aFGF corresponds to a region in bFGF that is reported to be in or near the receptor binding domain (Baird et al., (1988 ), Proc. Nat. Acad. Sci. USA, 85: 2324-2328). In addition, the [Ala47, Gly ^ JaFGF bovine analogue of the present invention, contrary to the bovine [Ala 7] aFGF analog, exhibited improved stability, maintaining its original mitogenic activity in the absence of heparin during the course of 250 hours, while that the bovine [Ala47] analogue rapidly lost its activity. The [Ala47, bovine Gly ^ aFGF analog was crystallized, and the resulting crystals were examined by X-ray crystallography. The crystallographic X-ray data obtained from the examination of these crystals supports the suggestion of the hydrophobic interaction chromatography data. that the residue 93 is exposed to solvent, that is, that the glycine for the substitution of histidine at position 93 makes the molecule less hydrophilic. Detailed examination of the sequence of bovine [Ala47, Gly93] aFGF analogue around residue 93 revealed a pool of approximately 8 amino acids with high loop forming potentials in the region from near the glutamic acid residue at position 90 to near the tyrosine residue at position 97. The relative potentials of amino acid loop formation have been reported, and glycine is identified as the amino acid residue that has the highest potential for loop formation among all amino acids acids (Leszcynski et al., supra). Thus, it is believed that the substitution of glycine by histidine stabilizes the putative loop, due to the much higher loop forming potential of the glycine residue compared to histidine. The KGF analogs of the present invention also demonstrate that the corresponding region in KGF is a loop exposed to solvent, which may be involved in binding to the receptor. Specifically, the [Gly 11 β] KGF analog of the present invention was found to exhibit altered, decreased mitogenic activity compared to naturally occurring KGF, as shown in the examples that follow. It was also found that the analog of [Gly116] KGF has a thermal stability of 5-7 ° C higher relative to naturally occurring KGF. Other analogs, in addition to the analogues of [Gly93] aFGF and [Gly116] KGF shown specifically herein, are contemplated by the present invention. These other analogs could easily be made by one skilled in the art, following the techniques provided herein. For example, there are no less than fifteen amino acids that are reported to have a higher potential for loop formation than histidine (Leszcynski et al., (1986), supra). These amino acids are (in descending order of loop formation potential) glycine, proline or tyrosine, aspartic acid or asparagine, serine, cysteine, glutamic acid, threonine, lysine, cystine, glutamine, arginine, phenylalanine, and tryptophan. Substitution of any of these residues by the histidine residue in the loop formation sequence of the naturally occurring protein could be expected to result in an analog of the present invention having improved stability. Of course, it will be preferred to replace the histidine residue in the loop formation sequence with amino acids having the highest possible loop-forming potential, without creating any potential negative effects, such as the formation of undesirable disulfide bonds through the insertion of additional cysteine or cystine residues. Thus, other preferred amino acid substitutions in the histidine residue in the loop formation sequence (ie, in addition to glycine) appear to include proline, tyrosine, aspartic acid, asparagine, serine, glutamic acid, threonine, lysine, glutamine, arginine, phenylalanine, and tryptophan. The present invention also contemplates the substitution of an amino acid having a high potential for loop formation by other amino acid residues within the region of amino acids 92 to 96 of naturally occurring aFGF (ie, amino acids). and 94-96), and the region of amino acids 115 to 119 of naturally occurring aFGF (ie, amino acids 92 and 115-117-119). The aFGF analogs of the present invention include, for example, analogues of aFGF having the threonine residue at position 96 of the naturally occurring aFGF replaced with glycine, proline or tyrosine, aspartic acid or asparagine, serine, or glutamic acid , in order of preference, although minimal improvement in stability and / or biological activity would not be expected with the substitution of glutamic acid by threonine, due to the similarity of loop forming potential of these two amino acids. The amino acid residues at positions 92, 94, 95, and 97 (asparagine, tyrosine, asparagine, and tyrosine, respectively) of the naturally occurring aFGF have a sufficiently high loop formation potential, so that they are expected to result minimum benefits of substitution for these particular residues. The analogs of the present invention are considered to include analogs of both human and animal (eg, bovine) origins, as well as all forms of a protein having the following loop-forming amino acid sequence: 92 93 94 95 96 (aFGF) -Asn-His-Tyr-Asn-Thr- 115 116 117 118 119 (KGF) For example, both forms of aFGF, human and bovine are known, and have been identified as possessing the identical sequence of amino acids (shown above) at positions 92 to 96. On the other hand, there is approximately a sequence identity of 92. % between human aFGF and bovine, and 97% "similar" (ie, 5% of the total 8% changes between the two forms of aFGF are "conservative"). Both forms, human and bovine of aFGF that exist naturally, exhibit substantially the same mitogenic activity in vitro. Because of their improved stability and biological activity in the absence of heparin, the novel biologically active aFGF analogs of the present invention are particularly suitable for use in pharmaceutical formulations for the treatment by physicians and / or veterinarians of many types of wounds of mammalian species. . The KGF analogs of the present invention, because of their improved thermal stability, may also be suitable for use in pharmaceutical preparations. The amount of biologically active analogue used in such treatments will, of course, depend on the severity of the wound being treated, the route of administration selected, and the specific activity or purity of the analog, and will be determined by the veterinarian or physician. service. The term "therapeutically effective amount of analogue" refers to the amount of analogue determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are easily determined by one ordinarily skilled in the art. The analogs of the present invention can be administered by any appropriate route to the wound or condition being treated. Conditions that can be profitably treated with therapeutic application (s) of the analog of the present invention include, but are not limited to, the healing of superficial wounds, bone healing, -angiogenesis, nerve regeneration, and generation and / or organ regeneration. The formulations of the present invention, for both veterinary and human uses, comprise a therapeutically effective amount of the analog, together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic ingredients. The carrier (s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, and not be harmful to the recipient thereof. The formulations can be conveniently presented in unit dosage form, and can be prepared by any of the methods well known in the art. All methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately associating the analog with liquid carriers, or finely divided solid carriers, or both. The following examples are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the disclosed procedures without departing from the spirit of the invention. Example 1 Analog Production of [Ala47, Gly93] aFGF Synthesis A [Ala47, Gly ^ JaFGF and a [Gly116] KGF analog was made, using site-directed mutagenesis. It will be appreciated, however, that these and other analogs of the present invention can be made by other methods, including chemical synthesis. In the case of the analog of [Ala47, Gly ^ JaFGF, a bovine sequence was used. Specifically, the analogue of [Ala47, Gly93] aFGF was prepared as follows: A bovine aFGF analogue was prepared and examined according to the present invention in the following examples. This analogue, [Ala47, Gly93] bovine aFGF, was constructed to contain both a desired substitution of amino acid (glycine for histidine at position 93) in the loop formation sequence of residue 92 to 96 of the aFGF molecule and an additional substitution of amino acid, of alanine by the non-conserved cysteine residue at position 47, as shown in Figure 1. A bovine [Ala47] aFGF analog was also prepared, which had only the amino acid substitution of alanine by cysteine, to be used as a control for the desired [Ala47, Gly93] bovine aFGF analogue. Although these examples demonstrate a bovine aFGF analog of the present invention, the same results can be achieved for highly homologous human aFGF analogs. For example, the amino acid sequence of the corresponding human [Gly93] aFGF analog of the present invention is shown in Figure 2. A synthetic gene, which codes for the [Ala47, Gly93] analogue of the bovine aFGF was assembled in two. Oligonucleotide sections with a total of 28 components. The amino acid sequence of Giménez-Gallego et al. (1985), Science, 230: 1385-1388 was used as the basis for this gene, with selected codon alternatives to optimize the expression of the analogue in E. coli (Giménez-Gallego et al., (1985), supra). Section I was assembled from 16 oligonucleotides, to provide a fragment of 287 nucleotides that could be inserted into a plasmid vector at the restriction endonuclease sites Xba I and Xho I. Section II was assembled from 12 oligonucleotides, to give a fragment of 170 nucleotides joined by the compatible ends of Xho I and Bam Hl. The two sections were inserted into the expression plasmid pCFM1156, which had been previously digested with Xba I and Bam Hl in a 3-component binding, to provide the complete aFGF gene under the control of the lambda pL promoter. The plasmid pCFM1156 is prepared from the known plasmid pCFM836. The preparation of plasmid pCFM836 is described in the patent of E.U.A. No. 4,710,473; Relevant portions of the specification, particularly Examples 1 to 7, are incorporated herein by reference. To prepare pCFM1156 from pCFM836, the two endogenous Nde I restriction sites are cut, the exposed ends are covered with T4 polymerase, and the coated ends are linked by the sticky end. The resulting plasmid is then digested with Cia I and Kpn I, and the fragment-cut DNA is replaced with a DNA oligonucleotide of the following sequence: -CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC-3'3'-TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC-5 'E cells Coli transformed with this plasmid were cultured in a 16 liter fermentation reactor (as described in Fox et al (1988), J. Biol. Chem., 263: 18452-18458). The gene encoding the [Gly93 Ala47] bovine aFGF was converted to the [Ala47] form using oligo site-directed mutagenesis. The aFGF gene was first transferred to the phage vector M13mp18, and single-stranded DNA was prepared to serve as a template for the mutagenesis reaction.

Approximately 0.5 μg of this DNA was mixed with 5 picomoles of each of the first mutagenic (5'-GAAGAAAACCATTACAACAC-3 ') and the first universal M13 used for the determination of the DNA sequence, heated at 65 ° C for 3 minutes, and allowed to cool slowly. The ringed template-first was mixed with ATP, a mixture of dNTP, the large fragment of DNA polymerase I, and T4 DNA ligase, then incubated at 15 ° C for 4 hours. Aliquots of this reaction mixture were added to competent E. coli JM101 cells, and plated on 0.7% L-agar. The resulting plates were replicated on nitrocellulose filters, and the filters were hybridized with first mutagenic labeled with 32P. DNA was prepared from phages that hybridized, the sequence was determined to verify the successful termination of the desired mutagenesis event. The resulting gene was then transferred back to the vector pCFM1156, for the expression of the recombinant protein. Purification of the aFGF analogue Both aFGF analogs, bovine [Gly93 Ala47] and [Ala47] were purified from the insoluble fraction obtained from the centrifugation of mechanically used E. coli cells expressing the recombinant protein. The pellet fraction was solubilized in 8 M urea, 0.1 M in glycine, pH 2.5, and subjected to centrifugation to remove the insoluble materials. The supernatant was loaded onto a column of S-Sepharose ™ (Pharmacia, Uppsala, Switzerland), equilibrated with 6 M urea, 10 mM in glycine, pH 3.0, and washed with 6 M urea, 20 mM in sodium citrate, pH 6.5 . The proteins that bound to the column were eluted with a linear gradient of sodium chloride from 0 to 0.5 M in 20 mM sodium citrate, pH 6.5. The fractions containing the aFGF were pooled, diluted 20 times with 20 mM sodium citrate, 0.1 M ammonium sulfate, and subjected to centrifugation to remove any precipitate. The supernatant was mixed with a volume of 20 mM sodium citrate, 2 M ammonium sulfate, and loaded onto a column of Fepil-Sepharose ™ (Pharmacia, Uppsala, Switzerland), equilibrated with 20 mM sodium citrate, ammonium sulfate 1 M, pH 6.5. The bound proteins were eluted from the column with a descending linear gradient (1 M to 0 M) of sodium sulfate. Fractions containing the aFGF were pooled and dialyzed against 20-mM sodium citrate, pH 6.5. This product was essentially homogeneous, as demonstrated by the fact that no other bands appeared in Coomassie blue in the SDS gel, as shown in Figure 4. Example 2 Gel Filtration Chromatography of the aFGF Analog gel filtration at room temperature, using a SuperoseM -12 column on a Pharmacia FPLC system (Pharmacia, Uppsala, Switzerland). The column was run at 0.5 ml / minute, in 20 mM sodium citrate, 0.2 M sodium chloride, pH 6.5. Gel filtration chromatography showed that the aFGF analogues, the purified [Gly93 Ala47] and [Ala47] bovine eluted as individual maxima in an elution position identical to that of ribonuclease A (Mr = 13,700). This indicated that both proteins are monomeric, and have the same hydrodynamic radius, although there is a possibility that both protein forms interact with the matrix of the column and give a delayed elution of the column. EXAMPLE 3 Hydrophobic Interaction Chromatography of aFGF Analogue Hydrophobic interaction chromatography was performed at room temperature, using a phenyl-Superose ™ -12 column on a Pharmacia FPLC system. The sample, in 2 M ammonium sulfate, 20 mM sodium citrate, pH 6.5, was loaded onto the column which had been equilibrated with 2 M ammonium sulfate. After washing with 2 M ammonium sulfate, the remaining protein was added to the column. eluted with a descending gradient of ammonium sulfate from 2 M to 0 M, followed by a final wash with 20 mM sodium citrate, pH 6.5. Because the position of elution of a protein in hydrophobic interaction chromatography (HIC) is strongly dependent on the exposure of hydrophobic regions in the folded state, this technique provides a sensitive test of the conformational homogeneity of similar proteins. Figure 3 presents the elution profiles for the aFGF, the [Gly93 Ala47] and the [Ala47] bovine analogues. The [Ala47] aFGF showed a major peak that eluted in 0.25 M ammonium sulfate, while the analog of [Ala47, Gly93] aFGF showed an individual maximum in 0.13 M ammonium sulfate, suggesting that both proteins exist primarily in a different individual conformation . Elution at a lower salt concentration by [Ala47, Gly93] aFGF indicates that it is slightly more hydrophobic than the [Ala47] form. This observation is consistent with the replacement of the histidine residue at position 93 by glycine, if the conformation of the protein is such that this residue is exposed to the solvent. Alternatively, the change in this residue could induce a total change in the conformation of the molecule, to produce a more hydrophobic structure. Example 4 Spectroscopy of the aFGF analogue Circular Dichroism The circular dichroism spectrum was determined at room temperature on a Jasco Model J-500C spectro-polarimeter (Jasco, Tokyo, Japan), equipped with an Oki If 800 Model 30 computer (Oki, Tokyo, Japan). ). The measurements were made at a bandwidth of 1 nm, using cells of 1 and 0.02 cm for the near and far ultraviolet ranges, respectively. The data were expressed as the average residual ellipticity, [?], Calculated using the average residual weight of 113 for both forms of aFGF. The circular dichroism (DC) spectra of the aFGF analogues of [Ala47, Gly93] and [Ala47] bovines were almost identical in both the far and near ultraviolet regions, as shown in Figures 4A and 4B, respectively. The DCs of the analogs were also very similar to the spectrum reported for human bFGF (Arakawa et al., (1989), BBRC, 161: 335-341). The simiiarity of the spectra in the near ultraviolet region is consistent with similar tertiary structures for the FGFs. Thermal Transition The thermal transition of the proteins was determined in a Response II spectrophotometer (Gilford, Medfield, Massachusetts), equipped with thermal programming and thermal support for cells. The samples were heated at an increase of 0.1 ° C / minute, or 0.5 ° C / minute, and their absorbance was monitored at 287 nm. Protein concentrations were determined spectrophotometrically, using an extinction coefficient of -0.98 for bFGF and 1.04 for both bovine aFGF analogs, at 280 nm for the 0.1% protein. The thermal denaturation of aFGF analogues was examined in the presence and absence of heparin, in both, 20 mM sodium citrate, pH 6.5 and 7.0. In all cases, the proteins precipitated when the temperature was increased. The temperature at which the abrupt increase in absorbance occurred was taken as the denaturation temperature. In the absence of heparin, this temperature was about 10 ° higher for the bovine [Ala47, Gly93] aFGF analog than for the bovine [Ala47] aFGF analogue. The addition of either an excess of 1.4 times or 8 times (w / w) of heparin increased the denaturation temperature for both forms by 14-20 ° C, depending on the rate of increase in temperature used. The difference between the denaturation temperature of the two forms remained at about 10 ° C. There was no apparent effect of the excess of 1.4 times or 8 times (w / w) of heparin on the DC spectrum of any of the proteins in the range of 240 to 340 nm, although in the case of the 8-fold excess of heparin, the spectrum of aFGF in the 240-260 nm region was masked by the absorbance of heparin itself. Infrared Spectroscopy with Fourier Transforms (I RTF) infrared spectra with Fourier Transforms (IRTF) were determined to further examine the conformational similarity of both aFGFs. For IRTF spectroscopy, the proteins were completely dialyzed against water. Each protein was prepared as a 2% solution in a 20 mM imidazole buffer solution, made in D20 (Sigma Chemical Co., 99.9% isotopic purity). The solutions were placed in IR cells with CaF2 windows and 100 μm spacers. For each spectrum, 1500 interferograms were collected and coded in a Nicolet 800 FTIR system, equipped with a KBr beam splitter coated with germanium and a DTGS detector. The optical bench was continuously purged with dry gaseous nitrogen. The spectrum of the second derivative was calculated (as described in Susi et al (1988), Biochem Biophys, Res. Comm., 115: 391-397). A 9-point smoothing function was applied to the subtracted spectrum of water vapor. Figure 5 shows the spectrum of the second derivative of the aFGF analogs of [Ala47, Gly93] and [Ala47] bovine in the amide I 'region (stretch of C = 0 in deuterated proteins). For polypeptides and proteins, the frequencies of the component bands in this region are related to the content of the secondary structure. Surewicz et al., (1988), Biochem. Biophys. Minutes 952: 115-130. The spectrum shows strong bands at 1630 and 1685 cm'1, which are indicative of a significant amount of β-structures in the two proteins. A strong band near 1647 cm "1 is indicative of the presence of irregular or disordered structures, the weakest maxima near 1666 and 1673 cm'1 come from bent structures, a small maximum is present near 1651 cm'1 in the spectrum of both proteins The amide components I 'near this frequency are typically assigned to a-helices., it was recently shown that this band can come from structures with a loop. Wilder et al., (1990), Abstracts of the Fourth Symposium of the Protein Society, San Diego. As shown in Figure 5, the highly resolved IRTF spectrum, contrary to DC, clearly demonstrates the presence of β-structures and folds, and the spectrum for the bovine [Ala47] aFGF analogue and the analog of [Ala47, Gly93 ] bovine aFGF are almost superimposable, again suggesting that these two proteins have a similar conformation.

The spectrum of the second derivative apparently showed no difference in conformation between the two aFGF analogs. However, it was evident that the deuteration of the interchangeable protons occurred faster for the bovine [Ala47] aFGF analog than for the [Ala47, Gly93] analog during equilibration of the lyophilized protein with the D20 solution. Since the two proteins have a similar conformation, the difference observed in the exchange rate H-D can not be explained by differences in the degree of exposure of the interchangeable protons between them. It is more likely that the [Ala47] aFGF analog has a more flexible structure, which makes the amide protons more accessible to the solvent. Example 5 Heparin Chromatography of the Analog of [Ala47, Gly93] aFGF Heparin-Sepharose ™ (Pharmacia) was packed in a 1 x 8 cm column, and equilibrated with 10 mM Tris.HCl, pH 7.2. The column was loaded, washed with Tris. 10 mM HCl, pH 7.2, and eluted with a linear gradient of sodium chloride from 0 to 2.8 M in the same buffer, at a flow rate of 0.5 ml / minute, using a Pharmacia FPLC system. Acidic and basic FGFs are distinguished by their avid binding to heparin and heparin-like molecules. Both analogs, the [Ala47, Gly93] and the [Ala47] bovine showed a single maximum that eluted in 1.54 M sodium chloride in Tris. 10 mM HCl, pH 7.2.

Example 6 Biological Activity of aFGF Analogs Bioassays in vitro The mitogenic activity on NIH 3T3 cells of the aFGF analogs of the previous examples was determined as described below. In addition, an analogue of [Ser70, Se bFGF human, prepared as described in Published PCT Patent Application No. 88/04189, was also examined in the bioactivity assay, together with aFGF analogues. NIH 3T3 cells were obtained from ATCC. The cells were cultured in DME supplemented with 10% calf serum, 10 units / ml of. penicillin, 2 mM glutamine and 10 units / ml of streptomycin. The cells were subcultured in a ratio of 1: 40 twice a week. On day 1 of the assay, subconfluent cultures were dispersed with trypsin, and plated in 24-well plates at a concentration of 20,000 cells / ml, 1 ml per well in the above medium. On day 5, the medium was replaced with 1 ml / well of DMEM without serum, but containing penicillin, streptomycin, and glutamine in the concentrations above. On day 6, experimental samples were added to the medium in volumes no greater than 100 μl. Eighteen hours later, the cells were pulsed for 1 hour with 1 ml of the above medium, which contained 2-10 μCi of tritiated thymidine at 37 ° C. After the pulse, the cells were washed once with medium, then 250 mM sucrose, 10 mM sodium phosphate, 1 mM EDTA, pH 8 was added, and the plates were incubated at 37 ° C for 10 minutes to release the cells. The cells were harvested in a Skatron harvester (Skatron, Inc., Sterling, Virginia). The filters were dried, placed in scintillation fluid, and counted in a Beckman scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.) The mitogenic activity of the aFGF analogs [Ala47, Gly93] and [Ala47] bovine on NIH 3T3 cells was examined as shown in Figure 6. In the absence of heparin, the analogue of [Ala47] aFGF produced a dose-dependent stimulation of 3H-thymidine consumption in the range of 1 to 100 ng / ml, with semi-maximal stimulation of 25 ng / ml Under the same test conditions, the analog of [Ala47, Gly93] aFGF was able to produce the same mitogenic effect at a much lower protein concentration, -the semi-maximal dose being of about 1 ng / ml The recombinant bFGF was 4-5 times more potent than [Ala47, Gly ^ aFGF, with a semi-maximal dose of 220 pg / ml when 4.5 μg / ml of heparin was added to both analogs, their in vitro activity increased, the analogue being more powerful of [Ala47, Gly ^ aFGF. In the presence of 45 μg / ml of heparin, the activities were improved in such a way that the dose response of the three molecules was almost identical, with a semi-maximum dose of 90 pg / ml. The stability of the aFGF analogues, determined by the retention of their respective mitogenic activity, was examined by incubation of a 0.1 mg ml solution of each FGF analog in 20 mM sodium citrate, pH 7 at 37 ° C, both in presence and in the absence of 1 mg / ml heparin. In the absence of heparin, the bovine [Ala47] aFGF analogue rapidly lost its activity, with a half-life of about 13 hours, as shown in Figure 7. However, in the presence of heparin, the [Ala47] bovine aFGF it did not lose its biological activity during the course of 250 hours of the experiment. In contrast, neither the bovine [Ala47, Gly93] aFGF analogue nor the human [Ser70, SerM] bFGF analog exhibited any loss of activity during the 250 hours, whether heparin was present or not. Example 7 Crystallography of the Analog of [Ala47, Gly93] aFGF [AU47, Gly ^ aFGF bovine-analogue crystals were grown by vapor diffusion against 0.2 M NH4S04, 2 M NaCl, 0.099 M sodium citrate, and sodium phosphate and 0.02 M potassium, pH 5.6. The protein drop contained equal volumes of the reservoir solution and a protein solution of 10 mg / ml. The crystals were trigonal (spatial group P3Í21, a = 78.6 A, c = 115.9 A) and diffracted at a resolution of 2.5 A. The intensity data were collected with a Siemens area detector (Madison, Wisconsin) of multiple wires, mounted on a rotating anode generator of 18 kw. The series of Siemens processing programs for data reduction was used. The phases of multiple isomorphic replacement (mir) were calculated at a resolution of 3 A of two derivatives, with a figure of merit of 0.68. After the solvent flattening, regions corresponding to two independent molecules of aFGF were identified in the asymmetric unit. The general non-crystallographic symmetry relationships between these molecules were determined from the rotation function, the real space translation function, and from density correlation studies. A molecular envelope was defined around an aFGF molecule averaged with a modified algorithm from B.C. Wang. The phases were iteratively refined by molecular averaging and solvent flattening. The initial maps revealed extended regions of β-sheet structures that were truncated in the loops, due to a small molecular envelope, as shown in Figure 8. The final map for the construction of the model was calculated with mir phases (of parameters of heavy atoms re-refined against averaged phases, as described in Rould et al, (1989), Science, 246: 1135-1142) and iteratively averaged with a molecular envelope generated by placing 6 A spheres near the atomic positions in the initial model. Averaging at a resolution of 3 A converged to a final R-factor of 17.8% between the factors of the observed structure and the calculated structure factors of the averaged map and solvent flattening. The TOM / FRODO graphic program, implemented for a Silicon Graphics 4D80 by C. Cambillau, was used to construct residues 10 to 136 of the aFGF sequence in an averaged electronic density map. The results of crystallography supported the hypothesis that the 90-97 region is involved in a loop structure. If this region is, in fact, involved in receptor binding (as suggested by Baird et al, (1988), supra), any substitution of amino acid that stabilizes the loop can stabilize and / or improve the biological activity of the receptor. molecule. This is presumably the mechanism for the improvement observed in the activity achieved with bovine [Ala47, Gly93] aFGF. Example 8 Production of the KGF Analog [Gly116] Synthesis To make the analog of [Gly116] KGF, a coding sequence for naturally occurring KGF was first obtained, and then altered in the. codon for the amino acid 116, to achieve a coding sequence for the analogue. The coding sequence for naturally occurring KGF was obtained using RNA isolated from human fibroblast cells (cell line AG1523), as a starting material from which to make cDNA for KGF, using standard techniques known in the art. The KGF cDNA was then used as a template in polymerase chain reactions (PCR) to amplify the KGF gene. Due to the presence of an internal Ndel site in the KGF gene, the PCR DNA was made as two fragments that were then joined to each other in a single Bsml site. Oligonucleotides 238-21 and 238-24 (shown below) were used to make a DNA product that was subsequently cut with BamHI and then isolated to provide a 188 base pair fragment of KGF. Oligonucleotides 238-22 and 238-24 were used to make a DNA product that was subsequently cut with Ndel and Bsml to provide a 311 base pair fragment of KGF. The two DNA fragments, when ligated together, created the gene for naturally occurring KGF, shown in Figure 9. To obtain a coding sequence for the desired [Gly116] KGF analogue, it was necessary to substitute a codon of glycine by the His116 codon in the KGF gene. This was achieved using oligonucleotide mutagenesis by PCR overlap with the soligonucleotides 315-17 and -31-5-18, which code for the KGF DNA sequence corresponding to the Gly116 region with the appropriate changes in base pairs to encode the Analog of [Gly116] KGF. The KGF DNA template for PCR was the same as shown in Figure 9, except that the DNA sequence between the Kpnl and EcoRI sites was replaced by chemically synthesized DNA as shown in Figure 10. They could be used any convenient oligonucleotides that correlated to the KGF 5'- and 3'- DNA regions of the site-directed mutational change, such as oligonucleotides 238-22 and 238-24 to provide the first exteriors for overlapping mutagenesis PCR. An EcoRI to BamHI DNA fragment containing the sequence coding for the [Gly116] KGF analog was then ligated into the expression plasmid pCFM1156 previously described, which already contained the KGF gene, in order to replace the corresponding region of the KGF gene with a region of the coding sequence containing the changes necessary to encode the analog of [Gly116] KGF (Figure 10). The ligand DNA was then transformed into an FM5 host (ATCC # 53911), and colonies containing the plasmid of the [Gly116] KGF analog pCFM1156 were isolated. The [Gly116] KGF FM5 / pCFM1156 analogue strain was then fermented, and cell paste was harvested using standard fermentation techniques or go 238-21 5 -ACAACGCGTGCAATGACATGACTCCA-3 'or go 238-22 5'-ACACATATGTGCAATGACATGACTCCA-3 'ohgo 238-24 5'-ACAGGATCCTATTAAGTTATTGCCATAGGAA-3, oligo 238-17 5'-GGAAAACGGTTACAACACATATGCA-3' ohgo 238-18 5'-GTGTTGTAACCGTTTTCCAGAATTAG-3 'Purification of the [Gly11ß] KGF analogue The cells containing the [ Gly116] KGF from the fermentation described above were first broken by suspending 665 g of the E. coli paste in about 4 I of 20 mM sodium phosphate, pH 6.8, 0.2 M NaCl, and then passing the suspension 3 times through a Gaulin homogenizer at 632.7 kg / cm2 (9,000 psi). The suspension was then subjected to centrifugation in a Beckman JA-10 rotor (Beckman Instruments, Fullerton, California) at 10,000 rpm, for 30 minutes at 4 ° C.

The ion exchange chromatography was performed by applying the supernatant of the centrifuged suspension to a column of S-Sepharose Rapid Flow (Pharmacia, Uppsala, Switzerland) (5 x 23 cm, 450 ml of total volume), equilibrated with sodium phosphate. mM, pH 7.5, 0.2 M NaCl, at a flow rate of 25 ml / minute. The column was then washed with 2 liters of 20 mM sodium phosphate, pH 7.5, 0.4 M NaCl, and the [Gly116] KGF analog was eluted with a linear gradient of 0.4 M NaCI at 0.6 M in 20 mM sodium phosphate, pH 7.5. The total volume of the gradient was 7 liters (about 16 times the volume of the column). Fractions containing KGF were pooled and concentrated about 22 times on a YMMR-10 membrane (Amicon) in a stirred cell of 400 ml. Size exclusion chromatography (TSC) was performed by applying half of the volume of concentrated KGF (total volume of 80 ml) obtained from ion exchange chromatography to a column of Sephadex "^ G-75 (Pharmacia) (4.4 x 85 cm, total volume of 1300 ml), equilibrated with 20 mM potassium phosphate, pH 6.8, 0.5 M NaCl, and developing the column with this buffer solution.The process was then repeated with the second half of the preparation of the concentrated KGF. then performed a second ion exchange chromatography procedure, first yielding the CET fractions that corresponded to the monomeric form of the [Gly 1ß] KGF analog, then diluting the pooled fractions with five volumes of 20 mM sodium phosphate, pH 6.8 , NaCl 0.2 M, and applying the diluted fractions to a column of S-Sepharose Rapid Flow (Pharmacia) (5 x 23 cm, 450 ml of total volume), equilibrated with 20 mM sodium phosphate, pH 6.8, NaCl 0.4 M. This column was then washed with about 1.5 liters of 20 mM sodium phosphate, pH 6.8, 0.4 M NaCl. The purified [Gly116] KGF analog was eluted with a linear gradient of 0.4 M NaCl at 0.6 M in phosphate of 20 mM sodium, pH 6.8. The total volume of the gradient was 10 liters (about 22 times the volume of the column). Samples containing the [Gly116] KGF analogue, determined by SDS-PAGE, were pooled, and the KGF content was determined by UV absorption. EXAMPLE 9 KGF Analog Spectroscopy Infrared Spectroscopy with Fourier Transforms (IRTF) Samples were prepared for infrared spectroscopy by diafiltering protein solutions in 20 mM sodium phosphate, 0.5 M NaCl, pH = 6.8 in a 20 mM sodium phosphate buffer solution. , NaCl 0.15 M, pD = 6.8 prepared in D20 (Sigma, 99.9% + of isotopic purity). The pD values were determined by adding 0.45 to the pH reading of a glass electrode pH meter, according to Covington et al., (1968), Anal. Chem. 40: 700-706. The final protein concentration was 30 mg / ml. The protein solutions were placed in IR cells with CaF2 windows and 100 μM Teflon spacers.

For structural characterizations, 256 double-sided interferograms were co-added and subjected to Fourier transforms after the application of a Happ-genzel apodization function, using a Nicolet 800 FTIR system. The resolution was set at 2 cm "1. The derivative spectrum and the Fourier self-deconvolutions were performed according to Susi and Byler (1983), Biochim, Biophys, Res. Comm., 115: 391-397, using the Nicolet programming elements The curve fitting was performed using the PeakfitMR program (Jandel Scientific Co.) The infrared spectrum of KGF showed a strong similarity to that of bFGF and aFGF, indicating similar structures for these three proteins. Thermal stability studies of naturally occurring KGF and the analog of [Gly1 ß] KGF were performed by placing the IR cells in an electric heating jacket controlled by an automatic temperature controller (Specac Inc., Fairfield, Connecticut). was increased at a speed of 0.5 ° C / minute IR spectra were collected at a resolution of 8 cm "1. At temperatures below 50 ° C, the spectra seemed to change little of the spectrum at room temperature. At temperatures above 50 ° C, the spectrum indicated that naturally occurring KGF undergoes a thermotropic transition at this point. The maximums near 1616 and 1685 cm "1 were evident in the spectrum and, by 65 ° C, these maximums dominated the spectrum with a corresponding loss of intensity at 1643 cm'1.This spectral transition represents the cooperative splitting of the KGF that exists Naturally, the observed thermal transition was not reversible, most likely due to the aggregation of the split protein.The melting temperature, Tm, for naturally occurring KGF was estimated to be 60 ° C, while the Tm for the Analog of [Gly116] KGF was estimated to be 65 ° C, 5 ° C higher than that of naturally occurring KGF, indicating that the [Gly116] KGF analogue has a higher relative thermal stability than naturally occurring KGF Ultraviolet Spectroscopy The thermal denaturation of both, the analogue of [Gly116] KGF and the Naturally occurring KGF was studied using a Response II UV spectrophotometer (Gilford, Medfield, Mass.) With a Peltier temperature controller and a thermal programmer. Solutions of KGF in 20 mM sodium phosphate, pH 6.8, were mixed with 8 M guanidine HCl or 20 mM sodium phosphate, for a final protein concentration of about 0.5 mg / ml, and a final concentration of guanidine HCl of 0 at 2 M. The thermal scan speed was set at 0.5 ° C / minute, and the monitored wavelength was 286 nm. The addition of a small amount of guanidine HCl eliminated the precipitation of the proteins during the thermal denaturation, but did not make the denaturation reversible. Therefore, the samples to be compared were run simultaneously.

Thermal denaturation experiments using UV spectroscopy were unsuccessful in the presence of guanidine HCl or 1 M, due to the development of turbidity with heating. However, the turbidity developed at a lower temperature for the naturally occurring KGF than for the analogue of [Gly116] KGF, suggesting that the first protein is denatured and, consequently, added at a lower temperature. In 1.5 M guanidine HCl, the absorbance at 287 nm decreased when the protein was denatured, and then increased due to aggregation. The temperatures at which the decrease occurred and the increase in absorbance were respectively 25 and 44 ° C for the analogue of [Gly116] KGF. The addition of 2 M guanidine HCl resulted in partial denaturation for both the naturally occurring KGF and the analogue of [Gly116] KGF at 25 ° C. The increase in temperature resulted in complete denaturation for the proteins, and the denaturation ended around 37 ° C for naturally occurring KGF and 46 ° C for the analogue of [Gly116] KGF. These results indicate that the analogue of [Gly116] KGF is thermally more stable by several degrees at the melting temperature, and are in accordance with infrared analysis, indicating an increase of about 5 ° C in the Tm of the analogue of [Gly1ß] ] KGF relative to the KGF that exists naturally.

Example 10 Mutagenic Activity of the [Gly116] KGF Analog The mitogenic activity of the [Gly116] KGF analog was analyzed using the mitogenesis assay by Rubin et al., (1989), Proc. Nati Acad. Sci. USA, 86: 802-806. The analog of [Gly116] KGF showed less 3 H-thymidine incorporation than that observed for naturally occurring KGF, indicating a lower specific activity for the analogue of [Gly116] KGF.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Amgen Inc. (¡i) TITLE OF THE INVENTION: Growth Factor Analogs Fibroblast Acid Having Improved Stability and Biological Activity (iii) NUMBER OF SEQUENCES: 17 ( iv) MAILING ADDRESS: (A) RECIPIENT: Amgen Inc (B) ADDRESS: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: USA (F) ZIP CODE: 91320-1789 ( v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS. Patentln Reléase # 1.0, Version # 1.25 (vi) DATA OF THE APPLICATION PRESENT: (A) NUMBER OF THE APPLICATION: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (2) INFORMATION FOR SEQ ID NO: 1: (i) ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 463 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: TCT? G ????? ACC ?? GGAGG TAATAAATAA TGTTC? ACCT GCCGCTGGGT ?? CT ?? C ???? (0 AACCTAAGCT TCTGTACTGC TCT ?? CGGCG GTT? CTTCCT GCGC? TTCTC CCGGATGGCA 120 CTGTAGACGG TACC ?? AG? T CGTTCCGACC AGC? C? TTC? GCTCCAßCTC GCTGCAG ?? T 180 CT? TCGGTG? AGTTT? C? TC A? ATCC? CCG AAACTGGTCA GTTCCTGGCT ATGGAT? CTG 240 ATGGTCTCCT CT? CGGTTCT CAGACTCCG? ACG ?? GAGTG CCTGTTCCTC G? ßCGTCTGG 300 AAGA ??? CGG TTACAACACC T? C? TCTCCA ????? C? CGC TG ????? CAC TGGTTCGTTG 360 GTCTG ????? AAACGGTCGT TCTAAACTGG GTCCGCGC? C TCACTTCGGT C? O ??? GCT? 420 TCCTGTTCCT CCCTCTGCCG GTTTCTTCCG ATT? ATAGG? TCC 463 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 141 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: MßC Phß? Sn L * u Pro L «u Gly Asn Tyc Lys Lys Pro Lys Leu Leu Tyc 1 5 10 15 Cys S «c Asn Gly Gly Tyr Ph * L * u Ar? II * L * u Pro Asp Gly The Val 20 25 30 Asp Gly The Lys Asp Aeg S * c Asp G n His II * Gln L * u Gln L * u Ala 35 40 45 Ala Glu Ser I * Gly Glu Val Tyc XI * Lys S * r The Glu The Gly Gln 50 55 60 Ph * L * u Ala Mßt Asp The? Sp Gly Leu Leu Tyc Gly S * r Gln The Peo. 65 7fr 75 80 Asn Glu Glu Cys Leu Ph * L * u Glu Arg L * u Glu Glu? Sn Gly Tyr? Sn 85 90 95 The Tyc XI * S * e Lys Lys His Wing Glu Lys HÜ Trp Ph * Val Gly L * u 100 IOS 110 Lys Lys? Sn Gly Aeg S * e Lys L * u Gly Peo Aeg The His Ph * Gly Gln 115 120 125 Lys Wing XI * Lew Phß- L * u Pro Leu Pro- Val. $? S * e? Sp 130 135 140 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 155 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECUL protinin (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: M * t? Glu Gly Glu XI * The The Ph * The? The Leu The Glu Lys Ph? 1 5 10 15? Sn Leu Peo Peo Gly? Sn Tyc Lys Lys Peo Ly? Leu Leu Tyc? The S? C 20 25 30 Asn Gly Gly His Ph? Leu Aeg Xl? Leu Peo Asp Gly The Val? Sp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His lie Gln Leu Gln Leu Ser? La Glu 50 55 60 Sà £ o Val Gly Glu Val Tyr lie Lys Sec The Glu The Gly Gln Tyc Leu 65 70 75 80 Wing Met? Ap Thr? Sp Gly Leu Leu Tyr Gly Sec Gln The Peo? Sn Glu 85 90 95 Glu Cys Leu Phà Le Leu Glu Arg Leu Glu Glu Asn Gly Tyr Kan The Tyc 100 105 110 lie Sà £ o Lys Lys HisÃ, la Glu Lys ? sn Tep Ph? Val Gly Leu Lys Lys 115 120 125? sn Gly S? e Cys Lys? rg Gly Pro Arg The His Tyc Gly Gl? Lys? 130 135 140 lie Leu Phß Leu Pro Leu Pro to Sec See? Sp 145 150 155 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 502 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: T? TGTGC? TG? C? TG? CTC C? G? GC ??? T GGCTAC ??? GTGAACTGTT CCAGCCCTGA 60 GCG? C? C? C? AG ?? GTTATG ATTACATGG? AGGAGGGG? T ATAAG? GTG? G ?? G? CTCTC 120 TGTCG ?? C? C? GTGGTACCT GAGGATCGAT AA? AGAGGC? GT ???? GG G? CCC ?? G? G 180 ATG ?? GAAT? ? TT? C? T? T C? TGG ??? TC AGGACAGTG? C? ßTTGGAAT TGTGGC ?? TC 240 ??? GGGGTGG A? AGTG ?? TT CT? TCTTGC? TG ?? C ?? GG ?? GG ?? CT CT? TGC ??? G 300 ??? G ?? TGC? ? TG ?? G? TTG T ?? CTTC ??? G ?? CT ?? TTC TGG ???? CC? TT? C ?? C? C? 360 TATGCATCAG CT ??? TGG? C? C? CAACGGA GGGG ??? TGT TTGTTGCCTT ??? TC ???? G 420 GGG? TTCCTG T ?? G? GGAAA A ???? CG ?? C A ?? G ?? C ??? A ?? C? GCCC? CTTTCTTCCT 480 ATGGC ?? T ?? CTTAATAGGA TC 502 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 497 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (ix) ASPECTS: (A) NAME / KEY: - (B) POSITION: complement (1 .497) (xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 5: CTATTAAGTT ATTGCCATAG GAAGAAAGTG GGCTGTTTTT TGTTCTTTCT TCGTTTTTTT 60 TCCTCTTACA GGAATCCCCT TTTGATTTAA GGCAAC ??? C ATTTCCCCTC CGTTGTGTGT 120 CCATTTAGCT GATGCATATG TGTTGT? ATG GTTTTCCAGA ATTAGTTCTT TGAAGTTACA 180 ATCTTCATTG CATTCTTTCT TTGCATAGAG TTTTCCTTCC TTGTTCATTG CAAGATAGAA 240 TTCACTTTCC ACCCCTTTGA TTGCC? CA? T TCCA? CTGCC? CTGTCCTG? TTTCC? TG? T 300 ATTGTA? TT? TTCTTC? TCT CTTGGGTCCC TTTT? CTTTG CCTCTTTT? T CG? TCCTC? G 360 GTACCACTGT GTTCGACAGA AG? GTCTTCT C? CTCTT? T? TCCCCTCCTT CC? TGT? TC 423? TAACTTCTT GTGTGTCGCT C? GGGCTGG? ? C? GTTC? C? TTTGT? GCC? TTTGCTCTGG 480 AGTCATGTC? TTGCAC? 497 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY, unknown (ii) ) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: Mßt Cys Asn Asp Mßt The Pro Glu Gln Mßt Ala The Asn Val Asn Cys 1 5 10 15 Ser See Pro Glu Aeg His Thr Arg See Tyr Asp Tyc Met Glu Gly Gly 20 25 30? Sp lie? Eg Val? Eg? Rg Leu Ph? Cys? Eg The Gln Trp Tyr Leu Arg 35 40 45 Xl? Asp Lys Aeg Gly Lys Val Lys Gly The Gln Glu Mßt Lys? Sn? Sn 50 55 60 Tyr Asn Xlß Mßt Glu lie Arg Thr Val Wing Val Gly Xlß Val Ala lie 65 70 75 ao Lys Gly to Glu Ser Glu Phß Tyr Leu Ala Mßt Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phß Lys Glu Leu 100 105 no lie Leu Glu? Sn His Tyr Kan Thr Tyr Ala Ser Ala Lys Trp Thr His 115 120 15 Asn Gly Gly Glu Mßt Phß al? La Leu? Sn ßln Lya Gly lie Pro Val 130 135 140 Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr? His Phß Leu Pro 145 150 155 160 Met? La lie Thr (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 503 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown - - (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: TATGTGCA? TG? CATGACTC CAG? GCAA? T GGCTAC ??? T GTG ?? CTGTT CC? GCCCTG ? 60 GCG? C? C? C? AG? GTT? TG ATT? C? TGG? AGGAGGGG? T AT ?? G? GTG? GA? G? CTCTT 120 CTGTCG ?? C? C? GTGGT? CC TGCGT? TCG? C ??? CGCGGC ??? GTC ??? GG GC? CCC ?? G? 180 G? TG ????? C AACT? CA? T? TT? TGG ??? T CCGT? CTGTT GCTGTTGGT? TCGTTGC ?? T 2 0 CAAAGGTGTT GAATCTG ?? T TCT? TCTTGC A? TGAAC? AG G ?? GG ???? C TCTATGC ??? 300 G ??? G ?? TGC ?? TG? AGATT GT ?? CTTC ?? AG ?? CT ?? TT CTGGAAAACG GTTAC ?? C 360 AT? TGCATCA GCTAAATGGA CAC? CAACGG AGGGG ??? TG TTTGTTGCCT T ??? TC ???? 420 GGGGATTCCT GTAAGAGGAA A ????? CG ?? G ??? G ?? C ?? ???? C? GCCC? CTTTCTTCC 480 TATGGC ?? T? ACTTAATAGG? TC S0J (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 497 base pairs (B) TYPE: nucleic acid (C) UNKNOWN TYPE OF FLEECE (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: cDNA (ix) ASPECTS: (A) NAME / KEY - (B) POSITION: complement (1. 497) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CTATTAAGTT ATTGCCATAG GAAGAAAGTG GGCTGTTTTT TGTTCTTTCT TCGTTTTTTT 60 TCCTCTTACA GGAATCCCCT TTTGATTTAA GGCAACAAAC ATTTCCCCTC CGTTGTGTGT 120 CCATTTAGCT GATGCATATG TGTTGTAACC GTTTTCCAG? ATTAGTTCTT TGAAGTT? CA 130 ATCTTC? TTG C? TTCTTTCT TTGCATAGAG TTTTCCTTCC TTGTTCATTG CAAGAT? G ?? 240 TTC? G? TTC? ACACCTTTG? TTGCAACGAT ACCAACAGCA ACAGT? CGG? TTTCCAT ?? T 300 ATTGTAGTTG TTTTTCATCT CTTGGGTGCC CTTG? CTTTG CCGCGTTTGT CG? T? CGC? G 360 GT? CC? CTGT GTTCGACAG? AGAGTCTTCT CACTCTTAT? TCCCCTCCTT CCATGT ?? TC 420 ATA? CTTCTT GTGTGTCGCT CAGGGCTGG? ACAGTTCAC? TTTGTAGCC? TTTGCTCTGG 480 AGTCATGTC? TTGC? C? 7 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) ) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Met Cya? Sn? Sp Mßt The Peo Glu Gln Met? The? Sn? Val Kan Cys 1 5 10 15 Sßc Söße Peo Glu? Eg His The? Eg Ser Tyr? Sp Tyr Mßt ßlu Gly Gly 20 25 30? Sp Il?? Eg Val? Eg? Rs Leu Ph? Cys? Eg Thr ßln Tcp Tyr Leu? Eg 35 « 0 45 Xlß? Sp Lys? Eg Gly Lys Val Lyß Gly Thr Gln Glu Mßt and *? Sn? Sn 50 55 60 Tyr? Sn ll? Mßt ßlu Il?? Eg Thr val? The Val ßly ll? Val? La Il? 65 70 75 80 Lys Gly Val Glu Ser Glu Phß Tyr Leu Ala Mßt Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu 100 105 no lie Leu Glu Asn Gly Tyr Asn Thr Tyr? The Ser ? Lys Trp Thr His 115 120 125? sn Gly Gly Glu Mßt Phß Val? Leu Asn Gln Lys Gly He Pro Val N 130 135 140 Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro 145 150 155 160 ' Met Ala He Thr (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid ' (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown '(ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55TION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH : 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: GAAGAAAACC ATTACAACAC twenty (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA - - (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: ACAACGCGTG CAATGACATG ACTCCA 26 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: ACACATATGT GCAATGACAT GACTCCA 27 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: ACAGGATCCT ATTAAGTTAT TGCCATAGGA 31 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: GGAAAACGGT TACAACACAT ATGCA 25 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unknown (D) TOPOLOGY: unknown ( ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: GTGTTGTAAC CGTTTTCCAG AATTAG_26_It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention , is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (16)

1. _ CLAIMS 1. An analog of a naturally occurring protein in the FGF family, characterized in that at least one amino acid in the loop formation sequence of -Asn-His-Tyr-Asn-Thr- in the naturally occurring protein is it replaces by a residue of a different amino acid that has a higher loop-forming potential.
2. 2. The analog according to claim 1, characterized in that the naturally occurring protein is KGF.
3. 3. The analog according to claim 2, characterized in that the amino acid replaced is the amino acid 116.
4. 4. The analogue according to claim 3, characterized in that the amino acid having a higher loop forming potential is selects from the group consisting of glycine, proline, tyrosine, aspartic acid, asparagine, serine, glutamic acid, threonine, lysine, glutamine, arginine, phenylalanine, and tryptophan.
5. 5. The analog according to claim 4, characterized in that the amino acid having a higher loop-forming potential is glycine.
6. 6. The analogue according to claim 5, characterized in that it has the amino acid sequence shown in Figure 10.
7. 7. The analog according to claim 6, characterized in that at least one terminal amino acid residue is deleted, while the analog substantially retains the improved biological activity.
8. The analog according to claim 7, characterized in that at least one cysteine residue of the naturally occurring KGF is replaced by a residue of a neutral amino acid.
9. 9. A DNA sequence, characterized in that it encodes the prokaryotic or eukaryotic expression of an analogue according to claim 1.
10. 10. The DNA sequence according to claim 9, characterized in that the analogue is the analog according to the invention. claim 6.
11. A pharmaceutical composition characterized in that it comprises a therapeutically effective amount of an analog according to claim 1, and one or more pharmaceutically acceptable adjuvants.
12. 12. The pharmaceutical composition according to claim 11, characterized in that the analog is the analogue according to claim 6.
13. 13. A method for treating a wound, characterized in that it comprises administering to the wound a therapeutically effective amount of an analog of according to claim 1.
14. 14. The method according to claim 13, characterized in that the analogue is the analogue according to claim 6.
15. 15. The analogue according to claim 5, characterized in that it has the amino acid sequence shown in Figure 1.
16. 16. The analogue according to claim 5, characterized in that it has the amino acid sequence shown in Figure 2.