JPH0240399A - Complex or composition of fibroblast cell growth factor mutein - Google Patents

Abstract

NEW MATERIAL:A complex of fibroblast cell growth factor(GOF) and glycosaminoglycan. USE:A healing promoter of burn, wound and postoperative tissue and remedy for thrombosis and arterioscherosis by regeneration action of blood-vessel. PREPARATION:For example, a plasmid containing DNA coding a human basic fibroblast cell growth factor (human bFGF) is digested with a restriction enzyme and bonded with a proper phage bector and then inserted into Escherichia coli bacterium to transform the bacterium and the bacterium is sown on a plate and plaque containing recombinant phage is picked up and a basic sequence of recombinant part is decided by dideoxynucleotide synthesis chain stopping method and a clone having human bFGFDNA is selected. Then the clone is cultured and a human bFGF mutein is collected and brought into contact with glycosaminoglycan (e.g., heparin) in an aqueous medium to provide the aimed complex.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to fibroblast growth factor (Fibroblast growth factor).
growth ractor, hereinafter abbreviated as FGF. ) A method for stabilizing mutin, a stable FGF mutin-containing aggregate or composition, and a method for producing these.

Conventional FGF was initially used to treat line Ra such as l3ALB/C3T3 cells.
It was isolated as a factor that exhibits a strong proliferation-promoting effect on T-blasts [Gosbodaguchiwitz, Nature (NaL
ure) + vol. 249, p. 123 (Mon. 1974).

It is now known that FGF has a proliferation-promoting effect on almost all mesodermal cells.

Based on its isoelectric point, FGF is divided into basic FGF (hereinafter abbreviated as bFGF) and acidic FGF (hereinafter abbreviated as a).
It is abbreviated as PGF. ) are broadly divided into two types. These F
Since GF has a strong proliferation-promoting effect and plasminogen activator-inducing effect on vascular endothelial cells, it is used as a prophylactic agent such as an angiogenesis promoter, wound treatment agent, antithrombotic agent, and antiarteriosclerotic agent. It is expected.

Traditionally, FGF has been purified to a single level from animal organs such as the bovine pituitary gland, but its supply is limited and there are concerns about antigenicity because it is derived from a different species of animal. It had problems such as. Recently, recombinant D
A method for producing large quantities of FGF has been developed by expressing the cloned human FGF gene in microorganisms and animal cells using NA technology (FEI3S Letters, Vol. 213) ,
+89-194 pages (1987): European Patent Application Publication No. 237.966).

Furthermore, a method for producing mutins with enhanced activity or improved stability was developed by modifying the amino acid sequence of FGF [Japanese Patent Application No. 63-5
Specification No. 0249, European Patent Application No. 881030
Specification No. 47.2, Biochemical and Biophysical Research Communications (Bio
chemical and biophysical
Research Communications) Volume 151, No. 70! -708 pages (1988)].

However, although FGF mutin has improved stability, it can be rapidly deactivated in an aqueous solution, and its biological activity can be easily reduced by freeze-drying. . Furthermore, when FGF mutin is administered over a long period of time, such as by intravenous drip, a decrease in the titer during this period may be unavoidable.

It has been reported that the inactivation of FGF can be prevented by the addition of certain glycosaminoglycans such as heparin [Journal of Cellular Finology (J.
Ce1lular Physiology) 128
Vol. 475 (1986)].

Means for Solving the Problems In view of the above circumstances, the present inventors conducted intensive research and surprisingly found that by adding glycosaminoglycan to an aqueous solution of FGF mutin, the stability of FGF mutin was significantly improved. As a result of further research based on this finding, the present invention was completed.

The present invention provides (1) a complex of F'GF mutin and glycosaminoglycan or a composition containing these;
2) A method for producing a complex of FGF mutin and glycosaminoglycan or a composition containing these by contacting PGF mutin and glycosaminoglycan in an aqueous medium; and (3) FGF mutin and glycosaminoglycan. This is a method for stabilizing FGF mutin by bringing the mutin and glycosaminoglycan into contact in an aqueous medium.

The above-mentioned FGF may be basic (hereinafter sometimes abbreviated as bFGF) or acidic (hereinafter sometimes referred to as bFGF).
It is sometimes abbreviated as aFGF. ) is also fine.

Examples of the above-mentioned FGF include those derived from mammals. The mammals include humans, monkeys, pigs, and cows.

Examples include sheep and horses.

Examples of the FGF include those extracted from various organs whose existence has already been clarified, such as the brain and pituitary gland.

Examples of the mutin of the present invention include the above-mentioned FGF mutin.

Examples of the FGF mutin used in the present invention include Japanese Patent Application No. 63-50249, European Patent Application No. 88103047.2, and Biochemical and Biophysical Research Communications.
Biophysical Research Com
communications) Volume 151, Nos. 701-708
(1988).

For example, the FGF mutin in the present invention is originally a mutated amino acid sequence of the original peptide or protein, and therefore, such mutations include addition of amino acids, deletion of constituent amino acids, and substitution with other amino acids. It will be done.

Examples of the addition of amino acids include those in which at least 1 amino acid is added.

The deletion of the constituent amino acids includes at least one FG
Name the F-constituent amino acids that are missing.

As a substitution to the native amino acid, at least one F
Examples include those in which a GF constituent amino acid is substituted with another amino acid.

The at least one amino acid in a mutin in which at least one amino acid is added to FGF does not include methionine, which is based on the initiation codon used when expressing the peptide, or a signal peptide.

The number of added amino acids is at least one, but any number may be used as long as the characteristics of FGF are not lost.

More preferably, it includes part or all of the amino acid sequence of a protein that is homologous to FGF and exhibits similar activity.

In a mutin that is deficient in at least one FGF-constituent amino acid of FGF, any number of missing constituent amino acids may be used as long as the characteristics of FGF are not lost.

Examples of the deficient constituent amino acids include human bFG
10 residues on the amino terminal side of F: Ver-Pro-Ala
-L eu -Pro -G lu -A sp -
Gly-Gly-Ser.

14 amino-terminal residues of human bFGF: MeL-Pro
-Ala-Leu-Pro-Glu-ΔspGly
-Gly-Ser-Gly-A! a-
P he -P ro.

41 amino-terminal residues of human bFGF.

Examples include the carboxyl-terminal 61 residues of human bFGF.

At least one FGF +M constituent amino acid of FGF In a mutin in which at least one FGF constituent amino acid is substituted with another amino acid, the number of at least one FGF constituent amino acid before substitution may be any number as long as the characteristics of FGF are not lost. .

An example of a constituent amino acid that is substituted is cysteine. Examples include things other than cysteine.

Cystine is particularly preferred. Examples of constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, and valine.

When the constituent amino acid before substitution is cysteine, the substituted amino acid is preferably, for example, a neutral amino acid. Specific examples of the neutral amino acid include glycine, valine, alanine, loinine, isoleucine, and thyronone. Examples include phenylalanine, histidine, tryptophan, serine-threonine, and methionine. Particularly preferred is serine-threonine.

When the constituent amino acid before substitution is other than cysteine, the substituted amino acid may be, for example, in terms of hydrophilicity, hydrophobicity, or charge of the amino acid.
Choose six properties that are different from the amino acid before being substituted. Specifically, if the moe amino acid to be substituted is aspartic acid, the substituted amino acid is asparagine.

Examples include threonine, valine, phenylalanine, and arginine, with asparagine and arginine being particularly preferred.

If the amino acid in the song to be replaced is arginine, the amino acid after being replaced is glutamine.

Examples include threonine, loinine, phenylalanine, and aspartic acid, with glutamine being particularly preferred.

When the constituent amino acid of 1rj to be substituted is glycine, the amino acids after substitution are threonine, loinine, phenylalanine, and serine.

Examples include glutamic acid and arginine, with threonine being particularly preferred.

If the constituent amino acid before substitution is serine,
The amino acids after substitution are methionine alanine, leucine 1. Examples include cysteine, glutamine, arginine, aspartic acid, and methionine is particularly preferred.

When the constituent amino acid of M to be substituted is valine,
Examples of the amino acid after substitution include serine, leucine, proline, glycine, lysine, and aspartic acid, with serine being particularly preferred.

The original constituent amino acids before substitution are preferably aspartic acid, arginine, glycine, serine, and valine.

Preferred amino acids after substitution are asparagine, glutamine, arginine, threonine, methionine, serine, and leucine.

The most preferred substituted mutin is one in which the constituent amino acid cysteine is replaced with serine.

In the above substitutions, two or more substitutions may be performed simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted.

The mutin may be a combination of two or three of the above additions, deletions, and substitutions.

To produce the mutin, a site-directed liquid film I
Site-directed muta
genesis) will be adopted. The technology is well known;
R,F,
) and J.P. Lecoq (I, ecoq, J
, P, ), Genetic Engineering (G
Genetic Engineering), Academic Press (1983), pp. 31-50. Oligonucleotide-directed mutagenesis is described in Smith, M. and Gillam, S., Noenetic Engineering Principles and Methods, Plenum Press, 1981.
2004), Volume 3, pages 1-32.

In order to produce the structural gene encoding the mutin, for example, (a) a single-stranded DNA consisting of a single strand of the FOP structural gene;
A is hybridized with a mutant oligonucleotide primer (the primers are complementary to the region containing the codon for cysteine to be replaced with this single strand, or optionally the antisense triplet that pairs with this codon). However, other amino acid encoding codons of the codon in question, or in some cases antisense
The discrepancy with triplets is not limited to this. ), (b)
The primer is extended by a DNA polymerase to form a mutant heterodimer (hcteroduplex), and (c) the mutant heterodimer is replicated.

Next, a phage DN carrying the mutated gene
A is isolated and incorporated into a plasmid.

Mutins can be produced by transforming a suitable host with the plasmid thus obtained and culturing the resulting transformant in a medium.

Glycosaminoglycan is a type of polysaccharide also called mucopolysaccharide, and is composed of unbranched polysaccharide chains in which disaccharides are repeatedly bonded.

It is characterized in that one of the two sugar residues is always an amino sugar (N-acetyl-D-glycosamine or N-acetyl D-galactosamine).

Glycosaminoglycans usually have a sulfate group or a carboxyl group, or both, in many of the sugar residues.

The glycosaminoglycan used in the present invention may be any glycosaminoglycan having the above characteristics, but one of the repeating trisaccharides is D-glucuronic acid, 7-iduronic acid, or D-galactose. and the other one is preferably N-acetyl-D-glycosamine or N-acetyl-D-galactosamine. In addition to these sugars, a trace of D-galactose,
It may contain D-quinlose, D-mannose, L-fucose, D-galactosamine, etc. Further, it is desirable that the trisaccharide unit has about 0.1 to 3.0 sulfate groups, and the molecular weight is about 1,000 to +00.000. It is preferably within the range of about 2,000 to 50°000.

Specific examples of the glycosaminoglycan include those extracted and purified from various organs, such as heparin, heparan sulfate, and dermatan sulfate. Further examples include low-molecular heparin, low-molecular heparan sulfate, etc. that have been reduced in molecular weight using hydrogen peroxide or the like.

The glycosaminoglycan used in the present invention may be free or in salt form.

Examples of the salt include sodium salt, potassium salt,
Examples include ammonium salts and trimedelammonium salts.

Furthermore, when contacting FGF mutin in an aqueous medium, a free glycosaminoglycan may be added and then an appropriate amount of alkali or acid may be added to adjust the desired pH. By adding alkali, glycosaminoglycans are
It may be in the form of a salt in an aqueous medium6, and free and salt forms may be present together.

When the FGF mutin of the present invention is contacted with a glycosaminoglycan in an aqueous medium, it is advantageous if the contact is further carried out in the presence of a di- or tribasic carboxylic acid, since the FGF mutin is further stabilized.

Examples of the dibasic carboxylic acid include tartaric acid, maleic acid, malic acid, and fumaric acid.

Examples of the tribasic carboxylic acid include citric acid and isocitric acid.

The above-mentioned carboxylic acid may be free or may be in the form of a salt. Examples of such salts include sodium salts, potassium salts, and ammonium salts.

Alternatively, a free carboxylic acid may be added to an aqueous medium, and then an appropriate amount of alkali or acid may be added to obtain the desired 1)H. In an aqueous medium by adding an alkali,
The carboxylic acid may be in the form of a salt, or a mixture of free and salt forms.

When FGF mutin is brought into contact with glycosaminoglycan in an aqueous medium, the amount of glycosaminoglycan used is
Approximately 0 glycosaminoglycan per mole of F mutin
．． It is preferred to add in a range of 1 to 100 moles, more preferably about 0.5 to 4 moles.

The concentration of glycosaminoglycans in the aqueous medium is approximately 0.
．． 0005 to 51/V% is preferable, and furthermore, about 0.0
1 to IW/V% is preferred.

The concentration of FGF mutin in the aqueous medium is approximately 0.00
05 to 51/V% is preferable, and about 0°0l-II
/V% is preferred.

The amount of the carboxylic acid used is such that the concentration in the aqueous medium is approximately 1
mM to IM is preferred, and more preferably about 10 mM to 500 mM
is preferred.

For contacting in an aqueous medium, FGF mutin.

The goal can be achieved by simply mixing the glycosaminoglycans and also the carboxylic acids in an aqueous medium.

As the aqueous medium, it is preferable to use distilled water for injection.

The conditions for mixing are FGF mutin.

The glycosaminoglycan and the carboxylic acid may be mixed together as an aqueous solution, or may be added as a solid to form a solution in an aqueous medium. The temperature during mixing is approximately 0
~40°C, and the pH is preferably in the range of about 3 to IO, more preferably in the range of about 5 to 9. The time required for mixing is usually about 1 to 30 minutes.

By the above method, a composition of stabilized FGF mutin is obtained.

In the stabilized FGF mutin composition, it is thought that PGF mutin and glycosaminoglycan are bonded to each other at a certain ratio to form a complex. Therefore, it is possible to isolate a stabilized FGF mutin complex if desired.

Isolation and collection methods include, for example, Sephadex and T.
Examples include a gel filtration method using SK gel, ion exchange chromatography using DFAE- or CM-) Jovar, and isoelectric point fractionation. Therefore, depending on the purpose, the FGF mutin complex may be isolated and collected, or it may be used as a composition without being isolated or collected.

The complex is formed by FGF mutin and glycosaminoglycan in a ratio of about 1.0.5 to about 1:5. Furthermore, since the complex dissociates in the presence of a high concentration of salts, such as 1M NaCI2, it is necessary to avoid using solvents with high ionic strength during the isolation procedure.

In this way, a stabilized FGF mutin complex or composition is obtained. That is, as shown in the Examples below, the complex or composition of the present invention is stable against heat, pi-1, and protease.

In particular, the complex or composition of the present invention is stable not only under neutral conditions but also under acidic and alkaline conditions.

The stabilized FGF mutin complex or stabilized FOP mutin composition may be used as such or in the presence of other pharmacologically acceptable carriers and excipients.

For use in warm-blooded animals (e.g. humans) as pharmaceutical compositions (e.g. injection tablets, capsules, solutions, ointments) together with diluents.

It can be safely administered parenterally or orally to mice, rats, hamsters, rabbits, dogs, and cats.

The preparation is preferably in the form of, for example, an injection, a solution for injection, a frozen product, or a lyophilized product.

When formulating a pharmaceutical composition, pharmaceutically acceptable additives, diluents, excipients, etc. are used as desired, according to known pharmaceutical manufacturing methods.

For example, when preparing an aqueous solution for injection, an aqueous solvent (e.g., distilled water) or a water-soluble solvent (e.g., physiological saline) may be used.

Solvents such as Ringer's solution), oily solvents (e.g. sesame oil, olive oil), or, if desired, solubilizing agents (e.g. sodium salidylate, sodium acetate), buffers (e.g. sodium citrate, glycerin), isotonic agents ( For example, glucose,
Invert sugar), stabilizers (e.g., human serum albumin, polyethylene glycol), preservatives (e.g., benzyl alcohol,
phenol), soothing agents (e.g., benzalkonium chloride,
It is manufactured by conventional means using additives such as brocaine hydrochloride (brocaine hydrochloride).

In addition, for example, in order to prepare a solid injection preparation, a diluent (
e.g., distilled water, saline, glucose), excipients (e.g.,
Mix carboxymethyl cellulose (CMC) (sodium alginate), preservatives (e.g. benzyl alcohol, benzalkonium chloride, phenol), soothing agents (glucose, calcium gluconate, propylene hydrochloride), etc., and harden by conventional means. It can be manufactured into shaped injection preparations.

Furthermore, when formulating a formulation, monosaccharides such as glucose, amino acids, various salts, human serum albumin, etc. may be added, and in addition, tonicity agents, pl [regulators, soothing agents, preservatives, etc.] may be added. In addition, stable and effective FGF mutin preparations can be produced by FI.

The FGF mutin complex or composition obtained by the above method has the effect of promoting the proliferation of fibroblasts, is highly stable, and has low toxicity, so it can be used as a healing promoter for burns, wounds, postoperative tissues, etc. Alternatively, it can be used as a therapeutic agent for thrombosis, arterial fecal disease, etc. due to angiogenic action. Moreover, it can be used as a reagent for promoting cell culture.

When using the FGF mutin complex-like composition obtained by the method of the present invention as the above-mentioned medicine, for example, the amount of PGP mutin should be 1. An appropriate amount is selected and administered from about 1 ng to 100 μg/kg per day.

Further, when the FGF mutin-combined composition obtained by the method of the present invention is used as a reagent for promoting cell culture, the amount of FGF mutin is about 0.01-10 μg per medium IQ, and more preferably about 0.0 μg per medium IQ. 1
-10 μg is preferably added to the medium.

In this way, by contacting FGF mutin with glycosaminoglycan in an aqueous medium, FGF mutin can be stabilized and a stabilized FGF mutin complex can be obtained. can be formulated with a stable and sustained action.

Plasmid pTBe used in Reference Example 1 below
The transformant E. coli (Esc
herjchia coli) K 12 MM 2
94 /pTB669 (IFO14532, FERMB
P-1281) is European Patent Application Publication No. 237.
It was manufactured by the method described in Example 3 of Japanese Patent No. 966.

The combination human basic 1? used in the Examples below? GF mutein C923 (hereinafter sometimes abbreviated as rhbFGF mutein C23) is disclosed in European Patent Application No. 8, Patent Application No. 63-50249.
It is produced by the method described in No. 8103047.2, Biochemical and Biophysical Research Communications, Vol. 151, pp. 701-708 (1988). That is, the rhbP G F mutin cs23 is a transformant of Enoerychia coli M M
294/1) Tr3762 (IFO14613, FE
Reference Examples 1 to 3 described below using RM[3P-1645)
(This is the same method as described in the above-mentioned documents, etc.)
It was produced and purified by the method described in .

Transformant E described above, colt KI2 MM294
/1) TB669 (contains plasmid pTB669 used in Reference Example 1 described below), and E,
E. coli MM294/pTB 762 (see Example 3 below) has been deposited with the Fermentation Research Institute (IFO) and the Institute of Microbial Technology (FRl), Agency of Industrial Science and Technology, Ministry of International Trade and Industry. Their accession numbers and access points are shown in Table I below. Regarding deposits to FRI, initially domestic deposits were made and a deposit number indicated by the FERMP number was assigned, and the deposit was changed to a deposit based on the Budapest Treaty, and a deposit number indicated by the FErtM BP layer number was assigned. It is kept at the same research institute (FRI).

(Left below) Note that the amino acid numbers in the amino acid sequence of human bFGF in the following reference examples are those of human bFGF shown in Figure 1.
The N-terminal Met of the amino acid sequence shall be counted as the first.

Reference Example 1 (Production of recombinant DNA having a nucleotide sequence encoding mutin) (1) Cloning of MI3 vector of human bFGF gene: Obtained in Example 3 of European Patent Application Publication No. 237,966. The resulting plasmid pT8669 was digested with restriction enzymes EcoR1 and BamHl. Phage vector MI3mp8 (Clay Metznong (J, Me
ssing), Methods in Engineering, 1
01.20-78 (1983)) Reproduction (RF) DNA
was digested with restriction enzymes Econl and Bamf (I), and p which had been previously digested with EcoR [and Bam1-11]
It was mixed with a human bFGF DNA fragment derived from TB669. Next, the mixture was ligated with T4 DNA ligase, and the ligated DNA was transformed into E. coli JM105 strain capable of infection, and plated on a plate using Xgal as an indicator balance [Clay Messing et al., New York・
Acids Research (Nucleic Ac1ds)
Res, ) (1981) 9) J309-321], the plaque containing the recombinant phage (white plaque) was lifted up, and the base sequence of the recombinant portion was determined using the ditheochine nucleotide synthesis chain termination method [J, Messing et al. Nucleic Acids Research
Ac1ds Res,) 9, 309 (+98
1)] to confirm that it was correctly inserted into human bFGFDN.

Single-stranded phage DNA was purified from this M13-PO clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides.

(2) Site-directed mutagenesis 0, 1 mM adenosine triphosphate (ATP), 50 mM
Hydroxymethylaminomethane hydrochloride (Tris-II
C+) ptr 8, Oll 0 mM MgCI
Synthetic oligonucleotide 5'>CGT TCT TGCTGT AGAG in 50μρ in the presence of 2.5mM dithiothreitol (DTT) and 9 units of T4 kinase.
CCGCT <3' CCys2 (primer for changing i to Ser (
The recognition sequence of the restriction enzyme Rsal disappears. )] was treated with T4 kinase for 1 hour at 37°C. 5
0mM NaCl,I, OmM) Lis-11CI.

pl-18,0, I OmM MgCIt and l0m
Mixture containing M β-mercaptoethanol 50
Single-stranded (ss) M13-P0DN was prepared by heating this kinased primer (12 pmoles) at 67°C for 5 min and at 42°C for 25 min in μQ.
Miscellaneous+i was formed on 5 μg of A. The annealed mixture was then cooled over water and treated with 0, 5 mM each deoquinonucleotide triphosphate (dNTP), 80 mM Tris1-IC.
1% pH 7, 4, 8mM MgCIt, 100mN
Add to 50 μQ of reaction mixture containing 9 units of aCl S DNA polymerase l K lenow fragment, 0, 5 mM ATP and 2 units of T4 DNA licase,
React at 7℃ for 3 hours and at 25℃ for 2 hours, 0.2mM
The reaction was stopped by adding 2 μQ of EDTA. It was used to transform JM105 cells capable of infection, allowed to grow overnight, and 5sDNAΔ was isolated from the culture medium supernatant. This 5
Using the sDNA as a template for the second cycle of brimer extension, gel-purified RF yMD DNA was transformed into competent JM105 cells, plated on agar plates, and incubated overnight to form phage plaques. was gotten.

(3) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used changes from one that encodes cysteine 70 to one that encodes serine. CGCTCA
Let CTCC<3'. (A recognition sequence for the restriction enzyme HaeII is generated.) (4) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used encodes cysteine 88. Encode serine from 5'>GTA ΔCA GACTTA G
Let AA GCT AGT <3'. (A recognition sequence for the restriction enzyme Alul is generated) (5) Specific site-directed mutagenesis; Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used encodes cysteine 93. 5'>TCG AAG AAG AAA G
Let ACTCA TCC<3'. (A recognition sequence for restriction enzyme order I nrl is generated) (6) Screening and identification of mutagenized plaques; Plates containing mutated M2S-PO plaques (item (+) above) and non-mutated M2S-PO
The two plates containing phage plaques were cooled to 4°C, and the plaques from each plate were layered onto two nitrocellulose round filters, in the case of the first filter, the dried filters were layered onto an agar plate for 5 min. For the second filter, the transfer was carried out for 15 minutes. Next, 0.2N Na
Oh.

1.5M NaCl and 0.2% Triton X-100
Place the filters on thick filter paper soaked in water for 5 minutes, then add 0,5 M Tris-tl CI, ptl
, and overlaid on filter paper soaked in 1.5 M NaCl for an additional 5 minutes to neutralize. 2XSS filters in the same way
C (standard citrate) soaked filter twice, dried and dried in a vacuum drying oven at 80° C. for 2 hours.

Duplicate filters were washed with 10 d of DNA hybridization buffer (5X SSC), pH 7.0, 4x per filter.
Denhard solution (polyvinylpyrrolidone.

Ficoll and bovine serum albumin, 1× = 0.02% each
), 0.1% sodium dodecyl sulfate (SDS).

50mM sodium phosphate buffer, pH 7,0 and 100
μg/rnfl of denatured salmon sperm DNA.
Prehybridization was performed for 4 hours at 5°C. Add oligonucleotide primer to lO5cpm/rnll, at 42°C.
Hybridization was performed for 24 hours. 30 min each in wash buffer containing 0.1% SDS and decreasing amounts of SSC.
Filters were washed at 0°C. Filters were first washed with buffer containing '2X SSC, and control filters containing non-mutated M2S-PO plaques were tested for the presence of radioactivity using a Geiger counter. Stepwise reduction of 5sca degree and unmutated M
The filters were washed until no detectable radioactivity remained on the control filter containing +3-PO plata. S
The lowest concentration of SC used was 0.1 x SSC. Filters were air dried and autoradiographed at -70°C for 2-3 days. 10,000 mutated M2S-PO plaques and 100 non-mutated control plaques were screened with a kinased oligonucleotide probe. Control plaques had little or no hybridization with the probe, while 3-10 mutated M2S-PO plaques hybridized with the probe.

One of the mutant MI3-PO plaques was picked up and JM
I05 culture medium was inoculated. 5sDNA was produced from the supernatant, and double-stranded (ds) DNA was produced from the bacterial pellet. Appropriate 11 nucleotide primer and 5sDNA
The base sequence was analyzed using

As a result, the TGC (Cys26) codon becomes TCT
(Sar) codon, TGT (Cys7
0) The codon was converted to an Acc (Set) codon, and the TGT (Cys88) codon was changed to T CT (S e
r) Converted to a codon, TG'r (Cys93)
:] It was confirmed that ton was converted to TCT (Set) codon.

Among the mutated M+3-PO farnos, the codon Cys-
The one with 26 changed to Ser is MI3-Pi.

M2S-P with codon Cys-70 set
2. M2S with codon Cys-88 set
-P3. M with codon Cys-93 set
It was set as 2S-P4.

Reference Example 2 (Screening and identification of mutagenized plaques) Plates containing mutated M13-P2 phage plaques obtained in Reference Example 1 and reference example! The two plates containing the non-mutated M13-P2 phage plaques obtained in were cooled to 4°C, and the plaques from each plate were transferred onto two nitrocellulose circular filters.
The dried filters were transferred onto an agar plate in a 5 minute overlay for the first filter and 15 minutes for the second filter. Then 0.2N Na0Il l , 5M
Place the filters on thick filter paper soaked for 5 minutes in NaCl and 0.2% Triton
Neutralization was achieved by placing the mixture on a filter paper soaked in NaCl for an additional 5 minutes. Filters were washed twice in a similar manner on 2X SSC (+ffi quasi-citrate filters), dried,
It was dried in a vacuum drying oven at 80°C for 2 hours.

Duplicate filters were prepared with IOd of DNA hybridization buffer (5x SSC), pH 7,0, 4x Denhard's solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin, lx = 0.02% each) per filter.

0.1% sodium dodecyl sulfate (SDS), 50mM
Sodium phosphate buffer, pH 7.0 and 100μg/
Planar hybridization was performed with denatured salmon sperm DNA of 7I7 for 4 hours at 55°C. Oligonucleotide primers were hybridized to 105 cpm/1 nl for 24 hours at 42°C. Filters were washed at 50° C. for 30 minutes each in wash buffer containing 0.1% SDS and decreasing amounts of SSC. Filters were first washed with buffer containing 2X SSC and control filters containing non-mutated M13-P2 plaques were tested for the presence of radioactivity using a Geiger counter. The SSC concentration was reduced stepwise and the filters were washed until no detectable radioactivity remained on the six unmutated MI3-P2 plaques on the control filters. The minimum concentration of SSC used was 0.1XSSC. Air dry the filter and −
Autoradiographs were taken after exposure at 70°C for 2-3 days. Too many mutated M13-P2 plaques and 100 non-mutated control plaques were screened with a kinased oligonucleotide probe. In the control plaque there was no hybridization with the probe, whereas in the mutated Ml3-P2 plaque 3
~10 hybridized with the probe.

One of the mutant MI3-r'2 plaques was picked up and J
M t O5 culture medium was inoculated. 5sDN from supernatant
Make A and extract two ff1(ds)DN from the bacterial pellet.
I made A. The base sequence was analyzed using appropriate oligonucleotide primers and sS DNA.

As a result, the TGC (Cys26) codon is changed to TCT (Se
r) converted to a codon, 'rc'r(cys88
) codon has been converted to a TCT (Set) codon,
It was confirmed that the TGT (Cys93) codon was converted to a TCT (Set) codon.

Among the mutated Ml3-P2 phages, the codon Cys-
26 and -70 are set as Ml 3-P
12. Ml3-P23.codon CyS-70 and -88 are set. The codon Cys-70 and -93 were set as Ml3-I) 24.

Reference Example 3 (Expression of gene encoding human bFGF mutin in E. coli) The replicative form (RF) of Ml3-P23 obtained in Reference Example 2 was treated with restriction enzymes EcoRI and psL.
+ to obtain an approximately 0.5 Kb DNA fragment containing the human bFGF mutin-encoding region.

On the other hand, the plasmid ptrp which carries the trp promoter
781 (Kurokawa, T. et al. Nuclic Acids Research)
1ds Res, ) 11, 3077-3085 (
(1983)) The DNA was cut with EcoRI-PstI to create approximately 3.2 Kb containing the trp promoter, tetracytalline resistance gene and plasmid replication initiation site.
DNA fragments were isolated. The 0.5Kb Eco containing the gene region encoding the mutin of human bF'GF
RI-PstI DNA fragment and this 3.2Kb
Plasmid pTB762 for human bFGF mutin expression by ligating DNA fragments by T4 DNA ligase reaction
was built.

Using this plasmid pTr3762, E. coli MM29
Plasmid containing the gene encoding mutin C23 by transforming 1) i'13762-containing K E 5charichia col 1M
M294/I) TE101 (IFO14613°FEf
IM UP-1645) was obtained.

(2) Preparation of bacterial cell extract Moeki transformants were each treated with 1% glucose.

The cells were cultured in M9 medium containing 0.4% casamino acids and 8 μg/d tetracycline, and when the K 1eft value was approximately 200, 3β indolyl acrylic acid was added at a concentration of 25 μg/day, and the cells were further cultured for 4 hours. .

After culturing, the bacterial cells were collected and added with 1/20 amount of 20mM Tris.
MCI, ptr 7.6. 1 Suspended in 0% sucrose solution. PMSF) was added to this suspension using phenylmethylsulfonyl fluoride.
TA was added at 1 OmM, NaCl at 0.1M, spermidine hydrochloride at 10mM, and lysozyme at 100 μg/d (all final concentrations) at 0°C.

After standing for 45 minutes, ultrasonication was applied for 30 seconds.

This solution was centrifuged at 1800 Orpm (Serval centrifuge, 5S
The mixture was centrifuged for 30 minutes to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract D containing 5% calf serum
0.2 d (2XlO' pieces) per hole in a tank 966 microtiter plate (flat bottom) suspended in MEM medium.
Seed each one, culture, and the next day.

The medium was replaced with DMEM medium containing 0.5% calf serum.

After culturing for 30 days, the bacterial cell extract was serially diluted 5 times with DME medium containing 05% 13SA, and 10 microorganisms were added per well.
After 20 hours, 'I-1-T
dr(5Ci/mmol, 0,5 mCi/rnI
.

RCCA Mersham) was added to each formula at 2ttQ. After 6 hours, cells were treated with 0.2% trypsin-○, 02%
Peel off with phosphate buffered saline (PBS) treatment containing EDTA,
Using a titer tick cell harvester, cells were collected on a glass filter, and the 'I(-T) incorporated into the cells was collected.
drfi was measured using a scintillation counter.

As a result, E. coli MM294/pTB 76
The bacterial cell extract of No. 2 showed FGF activity.

In this way, Cys at positions 70 and 88 of human bFGF were replaced with 5erf and rhb FG, respectively.
F Mutin C923 was obtained.

(4) Extract 25Pn1 obtained in (3) above. (prepared from 500d of culture solution) was added with 20mM Tris-HCI.
DEAE cellulose (DE52. Whatman, UK) equilibrated with p[-17,4,0,2M NaCl solution
The extract was passed through a column (diameter 2×10 cm) to remove nucleic acid components in the extract. Flow-through from column and 20mM T
The column washes with ris-HCl, pH 7, 4, 0, 2M NaCl solution were combined and collected (DEAE pass-through fraction 60d).

These two fractions were added to a heparin column Shodex AF-
It was applied to a high performance liquid chromatography apparatus (Gilson, France) equipped with pakHR-894 (8 mm [DX5 cm, manufactured by Showa Denko). column, 20111MT
ris-HCI pI (7,4 solution 1 then 20mM
Tris-11CI pl[7,4,0,5M Na
After washing with Cl solution, 20mM Tris-HCI
pH 7.4, 0.5M to 2M Na in buffer
Linear gradient elution of C1
telution, 6(L+4.Flow rate 1.0d
/m1n) was performed.

The peak fraction eluted between 15 and 25 minutes was r
Since it was found to contain hbF G F mutin C923, both portions were collected. The specific activity based on the PGF activity of bovine brain-derived F'CF (purity 95% or more) manufactured by Takara Shuzo Co., Ltd. is l, l mg-proteinF.
GF/mg protein.

Reference Example 4 (Method for Measuring Human bFGF Activity Using Endothelial Cells) FGF activity in the Examples described below was measured by the method shown below.

Samples were serially diluted 2-fold with DMEM medium containing +θ% calf serum, and 50μ/well was added to a Nunc 96-well microtiter plate (flat bottom), followed by American Type Culture Collection (ATCC).
) tallow cardiac endothelial cells (CRL 139) purchased from
5) was seeded in 50 μl2 (2XIO' pieces) per well and cultured for 3 days. Then, MTT solution (5 mg/m
20 μρ of P B S (Sigma, Inc.) was added to each formula. 4.5 hours later 10% 5DSO, 0IN1-1 (J!
After adding 100 μQ of the solution and leaving it in an incubator overnight, the absorbance at 590 μm was measured using a titer tick multi-scan [Tada et al., Journal or Immunological Methods].
unological methods): 93 volumes,
157 pages (1986)].

Example! 200% rhbFGF mutin C923 obtained in Reference Example 3 was added to 20mM phosphate buffer (ptl 7,4).
Heparin sodium (molecular weight 6,000 to 25,000) or low molecular weight heparin sodium (molecular weight fi 13.900 to 6
400) and incubated at 37°C for 48 hours. The final concentration of heparin sodium was 200 μg/d (molar ratio I:1) and 2 mg/d (molar ratio 1:10).
The final concentration of low molecular weight heparin sodium was set at 67 μg/d (molar ratio I:1). A non-additive group was set as a control group. Table 2 shows the residual activity after 48 hours. In the control group,
Although FGF activity was almost lost, FGF activity was stably maintained in the heparin sodium and low molecular weight heparin sodium addition groups.

Heparin sodium 1:10 100
Low molecular weight heparin sodium l:l 10
Example 2 Heparin sodium (molecular! .000), sodium heparan sulfate (molecules 910.000~+5.
000) or dermatan sulfur Fi! Glycosaminoglycans (molecular weight 11,000 to 25,000) were added to a final concentration of 500 μg/d or 25 μg/d and incubated at 37° C. for 48 hours.

A non-additive group was set as a control group. Table 3 shows the residual activity after 18:00 and 1 m. Although FGF activity was almost lost in the control group, FGF activity was stably maintained in the glycosaminoglycan added group.

Heparin sodium (average molecular weight 12.000) was added to the final concentration of 73 μg/− (molar ratio 1:1) and incubated at 56° C. for 4 hours. A group without heparin sodium was set as a control.

Table 4 shows the residual activity after 30 minutes and 4 hours.

As is clear from the results shown in Table 4, in the control group, F
Despite the decrease in GF activity, in the heparin sodium addition group, the decrease in FGF activity was prevented at high temperatures.

Heparin sodium Heparin sodium Sodium heparan sulfate Sodium heparan sulfate Sodium dermatan sulfate 25 +00 Example 3 The rhbr obtained in Reference Example 3 was added to a 50 mM sodium citrate (pl (7□4) solution;' G F Mutin C
923 was added to 100 μg/d, heparin sodium Heparin sodium No addition No addition 4 hours 8 16 hours 7 30 minutes 5 4 hours Example 4 2mM phosphate buffer (pH 7.0) rhbF G F Mutin C523 obtained in Reference Example 3 was added at 100 μg/
Heparin sodium (average molecular weight: 12.000) was added to the mixture to give a final concentration of 73 μg/molecular weight (molar ratio 1:1), and the mixture was incubated at 37°C with pt13. o,
It was incubated for 2 hours under conditions of 5.0, 7.4 and 9.7. Table 5 shows the residual FGF activity after 2 hours.

As is clear from the results shown in Table 5, both pH
Although the FGF activity was significantly decreased in the control group, heparin sodium added
F activity was maintained high.

Table 5 Heparin Sodium Heparin Sodium Heparin Sodium Heparin Sodium No Addition No Addition No Addition Addition 3.0 7 5.0 +0 7.4 9 3.0 3 5.0 4 7.4 6 Example 5 In Reference Example 3 Obtained rhbF G F mutin C823
was added to 8 mM OCQ at 100 μg/14, and 73 μg of heparin sodium with an average molecular weight of 12,000 (molar ratio 1:1) was added to 8 mM OCQ.
g/d (final pt+2.1). A group without heparin sodium addition was used as a control. The reaction solution was heated to 37°C.
The cells were incubated for 30 minutes and the residual FGF activity was measured (Table 6). Although FGF activity was almost lost in the control group, FGF activity in the heparin sodium addition group
The fact that the activity was stably maintained indicates that rhbFGF mutin CS23 was stabilized against pepsin digestion.

Table 6 Additives Residual FGF activity (%) 8, Sodium valine 100 Not added
0 Example 6 rhbF G F Mutin C823 obtained in Reference Example 3
(980 μg/d) and I volume of low molecular weight heparin sodium (3 mg/d) having an average molecular weight of 4,900 were mixed. 750μQ of the mixed solution was added to TSK-Gel 3000S.
W (manufactured by Tosoh Corporation) column (0,75 x 60 am) was attached with O, 1 containing 0, I M N at S Oa.
Elution was performed using M phosphate buffer (plTG, Q). Elution rate is l, OJ, /min, detection wavelength is 23
0 nm, the mixture gave a single peak (peak 1) (Figure 2).

As a result of fractionating and analyzing peak 1, rhbFGF mutin C! ;23 and low-molecular-weight m-heparin sodium at a molar ratio of 1.2. rhb
Elution 1 when FGF mutin C923 and low molecular weight heparin sodium were impregnated alone was compared to the second [f! Jr.

The complex was eluted on the higher molecular weight side than rhbFGF mutin 23 and low molecular weight eheparin sodium. Its molecular weight was calculated from FIG. 2 to be about 70,000.

Example 7 rhbF G F Mutin C523 obtained in Reference Example 3
,0.5+ng; Sodium heparin sulfate 0.37m
g, solution containing 15 mg of sodium citrate (1) I-
174) was prepared to obtain a stable injection solution.

Effects of the Invention Since FGF mutin can be stabilized by bringing glycosaminoglycan into contact with FOP mutin in an aqueous medium, it is possible to formulate FGF mutin while maintaining its stable action. can.

[Brief explanation of the drawing]

FIG. 1 shows the nucleotide sequence encoding human bFGF and the amino acid sequence deduced from it. FIG. 2 shows the gel filtration pattern of the composition of rhbFGF mutin C923 and low molecular weight heparin obtained in Example 6. Agent Patent Attorney Iwa 1) Hiroshi

Claims (12)

[Claims] (1) A complex of a fibroblast growth factor (FGF) mutein and a glycosaminoglycan, or a composition containing these. (2) The complex or composition according to claim 1, wherein the FGF mutein is a mutein in which at least one human basic FGF-constituting amino acid is substituted with another amino acid. (3) The complex or composition according to claim 1, wherein the glycosaminoglycan is heparin, heparan sulfate or dermatan sulfate. (4) The composition according to claim 1, further comprising a di- or tribasic carboxylic acid. (5) A complex of a fibroblast growth factor (FGF) mutein and a glycosaminoglycan, or a composition containing these, characterized in that the FGF mutein and a glycosaminoglycan are brought into contact with each other in an aqueous medium. Manufacturing method. (6) The production method according to claim 5, wherein the FGF mutein is a mutein in which at least one human basic FGF-constituting amino acid is substituted with another amino acid. (7) The production method according to claim 5, wherein the glycosaminoglycan is heparin, heparan sulfate or dermatan sulfate. (8) The manufacturing method according to claim 5, further comprising a di- or tribasic carboxylic acid. (9) A method for stabilizing a fibroblast growth factor (FGF) mutein, which comprises contacting a fibroblast growth factor (FGF) mutein with a glycosaminoglycan in an aqueous medium. (10) The stabilization method according to claim 9, wherein the FGF mutein is a mutein in which at least one human basic FGF-constituting amino acid is substituted with another amino acid. (11) The stabilization method according to claim 9, wherein the glycosaminoglycan is heparin, heparan sulfate or dermatan sulfate. (12) The stabilization method according to claim 9, wherein the FGF mutein is contacted with a glycosaminoglycan and a di- or tribasic carboxylic acid.