Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


(19) 



J 



Europaisches Patentamt 
European Patent Office 
Office europeen dee brevets 



(12) 



(11) EP 0 433 225 B1 

EUROPEAN PATENT SPECIFICATION 



(45) Date ot publication and mention 
of the grant of the patent: 
07.04.1999 Bulletin 1999/14 

(21) Application number: 90810922.6 

(22) Date of filing: 27.11.1990 



(51) Intel 6: C07K 14/495, C07K 1/113, 
A61K 38/18, C12N 15/16 



(54) Process for the production of biologically active protein (e.g. TGF) 
Verfahren zur Herstellung von biologisch aktivem Protein (z.B. TGF) 
Procede de preparation d'une prot^ine (e.g. TGF) a activite biologique 



(84) Designated Contracting States: 

AT BE CH DE DK ES PR GB GR IT LI LU NL SE 

(30) Priority: 06.12.1989 GB 8927546 

(43) Date of publication of application: 
19.06.1991 Bulletin 1991/25 

(60) Divisional application: 981 1 3487.7 / 0 891 985 

(73) Proprietors: 

• Novartis AG 
4058 Basel (CH) 
Designated Contracting States: 

BE CH DE DK ES FR GB GR IT U LU NL SE 

• Novartis-Erfindungen Verwaltungsgesellschaft 
m.b.H. 

1235 V\fien (AT) 

Designated Contracting States: 
AT 

(72) Inventors: 

• CerlettI, Nice, Dr. 
CH-4103 Bottmingen (CH) 

• McMaster, Gary Kent, Dr. 
CH-4303 Kalseraugst (CH) 

• Cox, David, Dr. 
CH-4204 HImmelried (CH) 



m 

m 

CM 
CM 

CO 
CO 

o 

Q. 
LU 



• Schmltz, Albert, Dr. 
CH-4056 Basel (CH) 

• Meyhack, Bernd, Dr. 
CH-4312 Magden (CH) 

(56) References cited: 

• BIOCHEMISTRY, vol. 28, no. 7, 1989, pages 
2956-2960, Washington, US; J. HOPPE et al.: 
"Preparation of biologically acrtlve 
platelet-derived growth factor type BB from a 
fusion protein expressed In Escherichia coll" 

• JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 
250, no. 21, 1975, pages 8477-8482, Baltimore, 
US; A. KARIM AHMED et ai.: "Nonenzymic 
reactivation of reduced bovine pancreatic 
ribonuclease by air oxidation and by glutathione 
oxldoreduction buffers" 

• JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 
262, no. 10, 1987, pages 4486-4491, Baltimore, 
US; S. TANDON et al.: "Detergent-assisted 
refolding of guanidinlum chloride-denatured 
rhodanese" 

• BIOCHEMISTRY, vol. 7, no. 12, 1968, pages 
4247-4254, Washington, US; W. CHAN: "A 
method for the complete S-sulfonation of 
cysteine residues in proteins" 



Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give 
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in 
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 
99(1) European Patent Convention). . 



Printed by Jouve. 76001 PARIS (FR) 



EP 0 433 225 B1 



Description 

[0001] The present invention relates to a process for the preparation of biologically active, dtmeric, TGF-p (Trans- 
forming Growth Factor type P). to novel TGF-ps, and pharmaceutical compositions comprising it. TGF-p produced 
5 according to the invention can be used for the promotion and acceleration of wound healing and bone and tissue repair, 
the treatment of cancer, as a bone marrow protective agent, mediator of cardioprotection, anti inflammatory or immu- 
nosuppressive agent or as a growth regulator in mammalian cell cultures. 

Background of the invention 

TO 

[0002] Two growth modulating proteins have originally been characterized by their ability to reversibly induce phe- 
notyplc transformation of mammalian cells in vitro and have therefore been designated as Transforming Growth Factors 
type a and type ^ (Anzano, M. A. et al. (1 983) PNAS 80, 6264-6268). Despite their common nomenclature TGF-a and 
TGF-p have shown to be both structurally as well as functionally entirely distinct proteins each acting through its own 

^5 unique receptor system. TGF-a which competes with epidermal growth factor (EGF) for binding to the same cell surface 
receptor (Todaro, G.J. et al. (1 980) PNAS 77, 5258-5262) and which shares sequence homologies and similar activity 
with EGF (Marquardt, H. et al. (1984) Science 223, 1079-1082) is synthesized as a transmembraneous precursor of 
159 amino acids and is proteolytically processed into a peptide of 50 amino acid residues (Derynck, R. et al. (1984) 
Cell 38, 287-297). As a potent mitogen for mesenchymal cells, TGF-a is produced and released by numerous trans- 

20 formed cell lines and human cancers, but is also expressed in activated macrophages and in other normal tissues, 
thus making its role In neoplasia still unclear. 

[0003] TGF-p was originally purified to homogeneity from human platelets (Assoian, R.K. et al. (1 983) J. Biol. Chem. 
258, 7155-7160), human placenta (Frollk, C.A. et al. (1983) PNAS 80, 3676-3680) and bovine kidney (Roberts, A.B. 
et at. (1983) Biochemistry 22, 5692-5698) and identified as a homodimeric protein with a molecular mass of 25.000 D. 

2S First characterized by its ability to act synergistically with EGF or TGF-a to induce anchorage-Independent growth of 
untransformed NRK cells, recently, TGF-p has been shown to exhibit numerous regulatory effects on a wide variety of 
both normal and neoplastic celts indicating the importance of this protein as a multifunctional regulator of cellular activity. 
TGF-p may either stimulate mitogenesis, cell proliferation and growth, or may effectively inhibit said processes, or may 
exhibit other actions like e.g. control of adipogenesis, myogenesis, chondrogenesis, osteogenesis und immune cell 

30 function, stimulation of chemotaxis, or induction or inhibition of differentiation depending upon the cell or tissue type, 
and the presence or absence of other growth factors. Many of the actions of TGF-p are related to the response of cells 
or tissues to stress or injury, and to the repair of resultant damage. After Inflammation, TGF-p plays the major role In 
the formation of granulation tissue, increases the expression of genes associated with extracellular matrix formation 
such as fibronectin, collagen and several protease inhibitors and stimulates collagen-matrix contraction by fibroblasts. 

3S suggesting its possible role In connective tissue contraction (Roberts, A. and Sporn, M.B. (1 988) Adv. Cancer Res. 51 , 
107-145; Sporn, M B. and Roberts, A. (1989) J. Amer. Med. Assoc. 262, 938-941). 

[0004] Until now, three distinct types of TGF-ps designated as TGF-pi , TGF-P2 and TGF-P3 which are functionally 
closely related and share a high degree of receptor cross-reactivity have been cloned and characterized by sequence 
analysis. All TGF-ps are synthesized as 390 to 41 2 amino acid precursors that undergo proteolytic cleavage to produce 

40 the monomeric forms, which consist of the C-terminal 112 amino acids. In their mature, biologically active forms, TGF- 
Ps are acid- and heat-stable disulfide-linked homodimers of two polypeptide chains of 112 amino acids each. The 
complete amino acid sequences of human (Derynck, R. et al. (1985) Nature 316, 701-705), murine (Derynck, R. et al. 
(1986) J. Biol. Chem. 261 , 4377-4379) and simian TGF-pi (Sharpies, K. et al. (1987) DNA6, 239-244) show remarkable 
sequence conservation, differing only in a single amino acid residue. Comparison of the amino acid sequence of human 

45 TGF-pi , human TGF-p2 (de Martin, R. et al. (1987) EMBO J. 6, 3673-3677; Marquardt, H. et al. (1987) J. Bk>l. Chem. 
262. 12127-12131) and human TGF-P3 (Ten Dijke, P et al. (1988) PNAS 85, 4715-4719) has demonstrated that the 
three proteins exhibit in their mature forms about 70-80% sequence identity A heterodlmeric TGF-pi .2 has been 
isolated from porcine platelets and consists of one subunit of TGF-pi disulfide-linked to one subunit of TGF-P2 
(Cheifetz. S. et al. (1987) Cell 48, 409-415). 

so [0005] Recently, attempts have been undertaken aiming to produce TGF-ps by means of recombinant techniques 
rather than isolating these factors from natural sources (e.g. platelets) in order to obtain sufficient amounts for testing 
in various therapeutic modalities. However, it has proven to be extremely difficult to synthesize recombinant T<3F-p 
while retaining its biological activity. As can be seen from the sequences depicted in the sequence listing under SEQ 
ID No. 1, 2, and 3. the 112 amino acids containing mature forms of TOF-pl, TGF-P2 and TGF-P3 contain 9 cysteine 

55 residues each, at least some of which are involved in intrachain and interchain disulfide bond formation which results 
in the complex tertiary structure of the biologically active, dimeric molecules. Heterologous expression of TGF-p may 
lead to a product which, although having the correct primary structure, fails to fold properly to produce the correct 
secondary or tertiary structures and which, therefore, lacks the biological activity. 



2 



EP 0 433 225 B1 



To date, the secondary and tertiary structures of TGF-ps are unknown. 

[0006] Taking the complexity of the native TGF-p molecules into account, ft has generally been considered expedient 
to express the respective TGF-p genes in cells derived from higher organisms. The expression of simian and human 
TGF-pi in Chinese hamster ovary (CHO) cells under the control of the SV40 promoter is described in European Patent 

5 Applications 293785 and 200.341, respectively. Recombinant TGF-P2 could be expressed in the same cell line as 
disclosed in European Patent Application 268.561 in which process TGF-P2 activity was found without refolding, ob- 
viously due to a very low spontaneous refolding activity which does not lead to technically usable biologically active 
amounts of TGF-P2, and in German Oftenlegungsschrift 38 33897. Eukaryotic expression of a fusion protein of TGF- 
P3 (with TGF-pi) is disclosed in European Patent Application 267.463. 

10 [0007] Although expression of recombinant TGF-Ps can be achieved In eukaryotic systems, the yields of biologically 
active, correctly folded material obtained are still far from being satisfactory. On the other hand, it seemed unlikely that 
biologically active TGF-p could be obtained when the respective gene was expressed in a microbial host, since in e, 
g. bacteria the intracellular conditions are not conducive to refolding, disulfide bond formation and disulfide-stabilized 
dimerization which is apparently essential for activity. Thus, only very little biologically active TGF-P2 could be obtained 

'5 after expression of the respective gene in E. coli under the control of the lambda promoter as described in European 
Patent Application 268.561 . This lack of activity is considered to be due to the fact, that the biologically active, dimeric 
form of TGF-p2 fails to form spontaneously from the monomeric primary translation product when exposed to the 
reducing environment inside the bacterial cells. Another report describes the expression of TGF-p cDNA in E. coli 
under the control o1 the trp promoter yielding a radioactively labelled protein band with an apparent molecular weight 

20 of 1 3'000 D in an autoradiogram of a SDS polyacrylamide gel, but no activity was measured (Urushizaki, Y et al. (1 987) 
Tumor Res. 22, 41-55). 

[0008] When recombinant proteins are produced at high levels in bacterial (such as E. coli ) expression systems, 
they often appear in the form of highly insoluble intracellular precipitates referred to as inclusion bodies or retractile 
bodies (Brems, D.N. et al. (19B5) Biochemistry 24, 7662) which can be recognized as bright spots visible within the 
2S enclosure of the cells under a phase contrast microscope at magnifications down to 1 000 fold. These inclusion bodies, 
which can readily be separated from the soluble bacterial proteins, contain the recombinant protein in a mostly dena- 
tured and reduced form which does not exhibit the functional activity of its natural counterpart and which therefore is 
useless as a commercial product. 

It is therefore generally agreed, that the recombinant retractile protein has to be solubilized under conditions which are 

30 suitable in maintaining it in its denatured form and subsequently has to be refolded in order to undergo the transition 
from the denatured unfolded form to the proper, functionally active three-dimensional structure, the conformation of 
which is stabilized by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge 
interactions. In the case of cysteine containing proteins this process may also involve formation of disulphide bonds. 
When the formation of disulfide bonds is chemically promoted, the formation o1 incorrect intramolecular and. in the 

35 case of dimeric or multimeric proteins, intermolecular bridges should be prevented or at least minimized, since the 
formation of undesired, incorrectly folded isomers may yield non-homogenous material, thus complicating the further 
purification of the protein having the desired structure, or may generate a protein with reduced activity 
[0009] A number of publications have appeared which report refolding attempts for individual proteins produced in 
bacterial hosts, or which are otherwise in a denatured or non-native form. Formation of a dimeric, biologically active 

40 human colony stimulating factor-1 (CSF-1) after expression in E. coli is described in PCT Application No. 88/8003 and 
by Halenbeck. R. et al. (1989) Biotechnology 7. 710-715. The procedures described involve the steps of initial solubi- 
lization of CSF-1 monomers isolated from inclusion bodies under reducing conditions in a chaotropic environment 
comprising urea or guanidine hydrochloride, refolding which is achieved by stepwise dilution of the chaotropic agents, 
and final oxidation of the refolded molecules in the presence of air or a redox -system. In PCT Application No. 88/8849 

45 a process for recovering recombinant interleukin-2 (IL-2) is disclosed, characterized in that IL-2 isolated from retractile 
bodies is denatured under reducing conditions with 6 M guanidine hydrochloride, the soluble IL-2 is oxidized by a 
controlled oxidization in the presence of Cu2+ ions, and the oxidized IL-2 is refolded by reducing the concentration of 
the denaturant in the solution. Interleukin-2 and interferon-p (IFN-p) have been refolded using SDS for solubilization 
and Cu2+ ions as oxidation promoters of the fully reduced proteins (US Patent No. 4.572.798). The process for isolating 

50 recombinant retractile proteins as described in US Patent No. 4.620.948 involves strong denaturing agents to solubilize 
the proteins, reducing conditions to facilitate correct folding and denaturant replacement in the presence of air or other 
oxidizing agents to reform the disulfide bonds. The proteins to which said process can be applied include urokinase, 
human, bovine and porcine growth hormone, interferon, tissue-type plasminogen activator, FMDcoat protein, prorennin 
and the src protein. A method for renaturing unfolded proteins including cytochrome c, ovalbumin and trypsin inhibitor 

55 by reversibly binding the denatured protein to a solid matrix and stepwise renaturing it by diluting the denaturant is 
disclosed in PCT Application No. 86/5809. A modified monomeric form of human platelet-derived growth factor (PDGF) 
expressed in E. coli is S-sulfonated during purification in order to protect thiol moities and Is dimerized in the presence 
of oxidizing agents to yield the active protein (Hoppe. J. et al. (1989) Biochemistry 28, 2956). 



3 



EP 0 433 225 B1 

[0010] The foregoing references are merely representatives of a huge amount of literature dealing with the refolding 
of non-native proteins derived from different sources. The man skilled in the art on the other hand knows that the 
success of refolding experiments cannot be predicted. Unsuccessful experiments are usually not reported. There is 
no certainty that anyone of the reported refolding conditions would work at all with a given denatured protein such as 
5 TGF-p. Considering the fact, that TGF-P is a dimeric protein containing 9 cysteine residues per chain and a number 
of intramolecular as well as intermolecular disulfide bonds, which are required for activity, it is a particularly difficult 
challenge to produce biologically active TGF-p from its monomeric, denatured or othenwise non-native form. Nowhere 
in the literature is a specific process described for the preparation of biologically active dimeric TGF-p from its non- 
native form. 

10 

Obiect of the invention 

[0011] It is the object of the present invention to provide a process for the production of biologically active, dimeric 
TGF-p-tike protein from its denatured or otherwise non-native form. This object is achieved by the unexpected finding 
IS that considerable amounts of the desired dimeric product can be obtained when the monomeric form of said protein 
is subjected to refolding conditions. Surprisingly, the production of the active dime r is achieved under various conditions 
in a one step procedure which is superior over the multi step procedures described in the prior art for the refolding of 
other proteins. 

^0 Detailed description of the invention 

[0012] The present invention relates to a process for the production of a dimeric, biologically active Transforming 
Growth Factor type p (TGF-P)-like protein, comprising subjecting the denatured, monomeric form of said TGF-p-like 
protein to refolding conditions. 

[0013] The term "TGF-p-like protein" is intended to embrace TGF-pi,TGF-p2 and TGF-P3 of mammalian such as 
human or animal origin, e.g. simian, murine, porcine, equine or bovine, as well as heterodimeric TGF-ps consisting of 
two different subunits of 112 amino acids each. Further included within the definition are growth-regulating proteins of 
the TGF-p superfamily sharing a sequence homology of at least about 25 % with TGF-pi, TGF-p2 or TGF-p3, such 
as a T cell suppressor factor from human glioblastoma cells (G-TsF; Wrann, M. et al. (1987) EMBO J. 6,1633-1636), 
a growth inhibitor isolated from conditioned medium of BSC-1 monkey kidney cells (polyergin; Holley, R.W. et al. (1 980) 
PNAS 77, 5989-5992; Ristow. H.J. (1986) PNAS 83, 5531-5533) a cartilage-inducing peptide isolated from bovine 
bone (CIF-B; Seyedin, S.M. et al. (1987) J. Biol. Chem. 262, 1946-1949), TGF-P4 from chicken embryo chondrocytes 
(Jakowlew. S.B. et al. (1988) Molecular Endocrinology 2. 1186-1195) and TGF-P5 from Xenopus-Laevis (Kondaiah, P 
et al. (1990) J. Biol. Chem. 265, 1089- 1093), as well as fragments and mutants of the above mentioned proteins 
retaining the biological activity. Further included within the definition of "TGF-p-like protein" are two forms of inhibin 
and three forms of activin (gonadal proteins that regulate pituitary secretion of follicle stimulating hormone), Mullerian 
inhibiting substance (MIS, which inhibits the development of the Mullerian duct in mammalian male embryos), bone 
morphogenic proteins (BMP, a group of polypeptides involved In the induction of cartilage and bone formation), the 
transcript from the decapentaplegic gene complex of Drosophila (dpp, which acts to control morphogenesis in the fly 
embryo), Vg-1 (the product of the Xenopus transcript which is present in the vegetal pole of oocytes), and Vgr-1, a 
related mammalian gene (Mason, A. et al. (1986) Biochem. Biophys. Res. Commun. 135, 957-964; Cate, R. et al. 
(1986) Cell 45, 685-698; Wozney, J.M. et al. (1988) Science 242, 1528-1534; Padgett, R. et al. (1986) Nature 325, 
81-84; Weeks, D.L and Melton, D.A. (1987) Cell 51, 861-868; Lyons, K. et al. (1989) PNAS 86, 4554-4558). 
[0014] Preferred TGF-p-like proteins are human TGF-p1 (Derynck. R. et al. (1985) Nature 316, 701-705), human 
TGF-P2 (Marquardt, H. et al. (1987) J. Biol. Chem. 262, 12127-12131) and human TGF-P3 (Ten Dijke, P et al. (1988) 
PNAS 85, 4715-4719) with the amino acid sequences depicted in the sequertce listing under SEQ ID No. 1, 2 and 3, 
respectively. 

[0015] Biologically active TGF-P-like proteins are originally defined as being capable of inducing anchorage-inde- 
pendent growth of untranstormed eel! lines (Tucker R.F. et al. (1983) Cancer Research 43, 1581-1586) or inhibiting 
growth of neoplastic target cells (Roberts, A.B. et al. (1985) PNAS 82, 119-123). "Biological activity" for the purpose 
herein is defined as either 

(a) the cell migration promoting activity on normal Balb/c 3T3 fibroblasts, whjch can be measured by counting the 
number of celts that migrate into a "wounded" monolayer culture of said cells, in the presence of a serum-free 
medium containing the TGF-p-like protein, as compared to the number of cells that migrate in the absence of the 
TGF-p-like protein, or 

(b) the growth promoting activity on normal Balb/c 3T3 fibroblasts determined by the stimulatory effect of the TGF- 
P-like protein on cellular DNA synthesis and cell division. 



30 



3S 



40 



45 



4 



EP 0 433 225 B1 



(c) the growth inhibition of A375 melanoma cells determined by a colorimetrical assay which reflects the number 
of cells treated with the TGF-p-like protein for a given culture period as compared to the number of non-treated cells, 

(d) the accelerated healing of partial-thickness burn wounds, by a process of re-epithelialization, in old mice fol- 
lowing multiple topical applications of the TGF-p-like protein as compared to untreated control wounds, 

5 (e) the accelerated healing of full-thtckness incisional wounds, as determined by tensile strength measurements 

and the histological analyses of biopsies, in adult rats following single topical applications of the TGF-p-like protein 
as compared to untreated control wounds, or 

(f) the increase in formation of fibrous granulation tissue, together with a marked increase in vascularity of the said 
tissue, both in and around porous wound-chamber implants in adult rats following multiple local injections of the 
10 TGF-p-like protein into the chamber as compared to untreated control chambers. 

[0016] The monomeric form of the TGF-p-like protein can be produced by means of recombinant DNA technology 
or synthetically by methods well known in the art. The dimeric form is the mature, biologically active molecule consisting 
of two disulfide-linked polypeptide chains. 
IS [0017] the monomer is subjected to refolding conditions which allow the recovery of the biologically active dimer. 
This process does not involve any change in the primary structure (i.e. the amino acid sequence) of the monomer, but 
relates to the formation of the three-dimensional conformation of the dimeric product which is associated with the 
biological activity. This process includes the formation of disulfide bonds and the association of monomers into dimeric 
structures. 

20 [0018] Before being subjected to refolding conditions, the monomeric TGF-p-like protein has to be present in a de- 
natured (i.e. unfolded) form. Capable of effectively denaturing proteins are so-called chaotropic agents well known in 
the art, which, in aqueous solution and In suitable concentrations change the spatial configuration of the respective 
protein through alterations at the surface thereof, either through altering the state of hydration, the solvent environment, 
or the solvent-surface interaction. Examples of such chaotropic agents or denaturants include urea, guanidine hydro- 
ps chloride, sodium thiocyanate at concentrations in the range of about 4 to about 9 M, and detergents such as SDS, 
which are supplied in concentrations in the order of 0.01 to 2 percent. Also, acidification of the aqueous solution con- 
taining the TGF-p-like protein to a pH of about 2 to about 4 as well as basic conditions of e.g. pH 10 and above and 
elevated temperatures will result in denaturation of the monomer. 

[0019] The term "refolding conditions' refers to buffer conditions wherein the denatured monomer Is permitted to 
30 assume a conformation associated with the biological activity Conventional buffer systems such as Tris, phosphate 
or citrate buffers can be used at a pH of about 6 to about 10. Under refolding conditions Intra- and interchain disulfide 
bond formation is promoted. Such conditions include the presence of a solubillzing agent and a redox system which 
permits the continuous oxidation and reduction of the thiol/disulfide pairs. The buffer system may additionally contain 
suitable salts. 

35 [0020] Suitable solubillzing agents are detergents, preferably mild detergents, organic, water-miscible solvents, or 
phospholipids or a mixture of two or more such agents. 

[0021] Detergents are surface active compounds used in a concentration permitting folding of the TGF-p-like protein. 
Preferred are mild detergents which permit folding of the monomeric TGF-p-like protein into the spatial conformation 
which after dimerization is associated with the biological activity, while retaining said monomer in a soluble form. Mild 

40 detergents, which solubilize TGF-p-like proteins without inactivating them can be 3-(3-chlolamtdopropyl)dimethylam- 
monio-1 -propane-sulfonate (Chaps), 3-< 3-chlolamidop ropy l)dimethylammonio-2 -hydroxy- 1 -propanesulfonate (Chap- 
so). They are present in the refolding buffer at a concentration of about 1 to 100 mM, especially in the range of 30 to 
60 mM. Preferred detergents are the zwitterionic detergents 3-(3-chloIamidopropyl)dimethylammonlo-1 -propanesul- 
fonate and 3-(3<hlolamidopropyl)dlmethylammonio-2-hydroxy-1 -propanesulfonate. Most preferred is 3-(3-chlolami- 

45 dopropyl)dlmethylammonlo-1 -propanesulfonate. 

[0022] Organic, water-miscible solvents can replace the detergent in the refolding buffer. Such solvents are, for 
example, lower alkanols, especially C2-C4 alkanols such as ethanol or Isopropanol, or lower alkandiols, especially Cg- 
C4 alkandiols such as ethyleneglycol, at a concentration range of 10 to 50 percent per volume. 
[0023] Alternatively, phospholipids can replace the detergent or the organic, waternnisclble solvent in the refolding 

50 buffer. Such phospholipids are, for example, phosphatldylethanolamine, phosphatidykiholine, phosphatidylserine and 
phosphatldylinositole at a concentration range of 0.1 to 5 mg/ml as well as synthetic phospholipid derivatives or variants 
such as dihexanoylphosphatidylcholine or diheptanoylphosphatldytehollne in the same concentration range. 
[0024] Suitable redox systems which encourage the formation of disulfides are e.g. low molecular weight sulfhydryl/ 
disulfide reagent combinations such as glutathione in its oxidized and reduced form, dithiothreltol in its oxidized and 

55 reduced form, p-mercaptoethanol or p-mercaptomethanol in its oxidized and reduced form, cystine and its reduced 
form, and cystamine and its reduced form at a concentration of about 1 to 100 mM, especially of about 1 to 10 mM, 
wherein the molar ratio of the oxidized and the reduced form Is between 100:1 and 1 :100, especially between 6:1 and 
1:6. 



5 



EP 0 433 225 B1 



[0025] The preferred sulfhydryl/disulfide redox system is glutathione in its oxidized and reduced form. 
[0026] Alternatively, thioredoxin or dtsulfideisomerase at a concentration range of about 10 to 1 000 ^ig/mi, especially 
of about 50 to 200 ^g/ml can be used instead of the low molecular weight sutfhydryl/disulfide reagent combinations. 
[0027] Salts which can be used in the refolding buffer include salts of Na+, Li+, K+. NH4+, Mg2+. Ca2+, or Mn2+ with 

5 Cl-, F", Br. J-, HCOs", 5042-. phosphate, acetate, cyanate or rhodanid, or other alkali metal- or alkaline earthmetal - 
halogen or pseudohalogen compounds at a concentration of up to 3 M. Preferred is NaCI at a concentration of 1 to 2 M. 
[0028] The invention particularly relates to a process for the production of a dimerlc, biologically active Transforming 
Growth Factor type p-like protein, comprising subjecting the denatured, monomeric form of said TGF-p-like protein to 
buffer conditions comprising a low molecular weight sulfhydryl/disulfide redox system in the presence of a solubllizing 

70 agent at a pH of about 6 to about 10 and a temperature of about (fO to about 37°C. Preferably, the pH is about 8.0 
and the temperature is about 4'*C. 

[0029] In a preferred embodiment the sulfhydryl/disulfide redox system is glutathione in its oxidized and reduced 
form at a concentration of about 1 to 10 mM, wherein the molar ratio of the oxidized and the reduced form is 1 :1 to 1 : 
2, and the weak detergent is 3-(3-chlolamidopropyl)dimethylammonio-1 -propanesutfonate at a concentration of about 

IS 30 mM to about 60 mM. 

[0030] Particularly, the production of a dimeric. biologically active TGF-p-like protein is performed in a one step 
procedure, wherein the monomer of said protein is dissolved in the refolding buffer and the reaction mixture is incubated 
for 2 to 400 hours at 4°C while refolding and dimerization continuously take place. The protein concentration during 
the refolding reaction is of considerable importance since when being too high, the monomers might undergo substantial 

20 aggregation leading to the formation of undesired higher-order oligomers. Final yields of dimeric product are increased, 
if the protein concentration is less than about 2 mg/ml, a concentration range of 0.01 to 0.5 mg/ml is preferred. 
[0031] Optionally, to further promote disulfide formation, an effective amount of an oxidation promoting agent con- 
taining Cu2+ ions (such as CUCI2, Cu(N03)2 or o-phenanthroline/Cu2+ complexes) or Fe^ ions (such as FeCl3 or Feg 
(804)3) "T^iQbt be added to the refolding buffer. An effective amount is the amount which at minimum will be necessary 

2S to conduct the oxidation of sulfhydryl groups within a convenient time period and which is approximately equivalent to 
the concentration of free sulfhydryl groups In the TGF-p-like protein which are destined to be involved in forming the 
desired disulfide bonds. Preferable amounts range between 0.01 to 1 00 |iM. 

[0032] Furthermore, O2 or air may optionally be bubbled through the refolding buffer either In the presence or absence 
of oxidation promoting agents. Oxidation may also be performed using Ig (Kamber, B. et a!., 1980, Helv. 63. 899-915) 

30 or Benzochinon derivatives (Kamber. B. PCT appl. WO 89/01484). 

[0033] Sulf onation of proteins can be used to cleave disulfide bonds and to block the resulting thiol groups. Monomeric 
TGF-p-like proteins can optionally be S-sulfonated and thereby be prevented to become oxidized before being exposed 
to the refolding conditions. S-sulfonation Is performed using sodium sulfite In the presence of a reducing agent such 
as cysteine, resulting in the reversible protection of thiol residues as S-sulfonates. Under refolding conditions, the 

55 protection groups are removed by the excess of the sulfhydryl/disulfide redox system and dimerization occurs spon- 
taneously. 

[0034] The invention relates further to a process for the production of a dimeric. biologically active TGF-p-!ike protein, 
in which the monomeric form of said TGF-p-like protein is produced by the steps of: 

40 (a) culturing a microbial host comprising a nucleotide sequence encoding the TGF-p-like protein linked in the proper 

reading frame to an expression control sequence such that said protein is expressed, 
(b) recovering the TGF-p-like protein in a denatured, monomeric. soluble form, 

[0035] Suitable microbial hosts are yeast strains as Saccharomyces cerevisiae or bacteria such as Escherichia coll 
4S or Bacillus subtilis . 

Microbial hosts comprising a nucleotide sequence encoding the TGF-p-like protein linked in the proper reading frame 
to an expression control sequence can be prepared by recombinant DNA techniques which are well known in the art 
and which comprise the steps of 

so . preparing a hybrid vector comprising a DNA sequence encoding the TGF-p-like protein under the expression con- 
trol of a suitable expression control sequence, 
transforming said microbial host with said hybrid vector, and 
selecting transformed microbial host cells from untransformed host cells. 

55 [0036] The nucleotide sequence coding for TGF-p-like proteins such as mature human TGF-pi , TGF-P2 or TGF-P3 
are known (Derynck, R. etal. (1985) Nature 316, 701-705; Marquardt, H. etaL (1987) J. Biol. Chem. 262, 12127-12131; 
Ten Dijke, P et al. (1988) PNAS 85, 4715-4719) and can e.g. be chemically synthesized by methods known in the art. 
Altematively, cDNAs encoding TGF-p-like proteins can be prepared after Isolation of the respective mRNA from TGF- 



6 



EP 0 433 225 B1 



P-like proteins producing mammalian cells. Expression control sequences are promoter sequences which ensure the 
effective expression of the TGF-p-like proteins. 

[0037] The selection of a suitable vector is determined by the microbial host cell provided tor the transformation. 

[0038] Examples of vectors that are suitable for the expression of the TGF-p-like protein in an E. coli strain are 
5 bacteriophages, for example derivatives of the bacteriophage X, or plasmids, such as the plasmid pBR322 and its 

derivative pPLMu. Suitable vectors contain a complete replicon and a marker gene, which renders possible the selection 

and identification of the microorganisms transformed by the expression plasmids by means of a phenotype feature. 

Suitable marker genes impart to the microorganism, for example, resistance to heavy metals, antibiotics such as amp- 

Icillin or tetracyclln, and the like. 
10 [0039] Several promoters can be used for regulating the expression of TGF-P-like proteins in E. coli . Especially 

promoters of strongly expressed genes are used. Suitable promoters are the E. coli lac, tac, trp and Ipp promoters, 

furthermore the phage or the phage XpL promoter, and others. 

[0040] Vectors suitable for replication and expression in S.cerevisiae contain a yeast-replication origin and a selective 
genetic marker tor yeast. Hybrid vectors that contain a yeast replication origin, for example the chromosomal autono- 

is mously replicating segment (ars), are retained extrachromosomally within the yeast cell after transformation and are 
replicated autonomously during mitosis. Also, hybrid vectors that contain sequences homologous to the yeast 2 \x 
plasmid DNA can be used. Such hybrid vectors are integrated by recombination in 2 n plasmids already present within 
the cell, or replicate autonomously. Suitable marker genes for yeast are especially those that impart antibiotic resistance 
to the host or, in the case of auxotrophic yeast mutants, genes that complenrient the host lesions. Corresponding genes 

20 impart, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast nriutant, 
for example the URA3 . LEU2 , HISS or the TRPI gene. 

[0041] Promoters suitable for expression in yeast are, for example, those of the ADHI , ADHII , or PH05 gene, and 
also promoters involved in glycolysis, for example the PGK or the GAP promoter. 

[0042] Optionally, signal sequences which allow the secretion of the TGF-p-like protein can be included in the ex- 
2S pression vector. Suitable signal sequences are e.g. derived from the yeast acid phosphatase (PH05) or the yeast 
invertase gene. 

[0043] The transformed microbial hosts are cultured in a liquid medium containing assimilatable sources of carbon, 
nitrogen and inorganic salts, applying methods known in the art. 

[0044] Various carbon sources are usable. Example of preferred carbon sources are assimilable carbohydrates, 
50 such as glucose, maltose, mannitol, fructose or lactose, or an acetate such as sodium acetate, which can be used 
either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as casamino 
acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore 
yeast extract, malt extract, corn steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or 
nitrate which can be used either alone or in suitable mixtures. Inorganic salts which may be used include, for example, 
35 sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally the 
nutrient medium may also contain growth promoting substances. Substances which promote growth include, for ex- 
ample, trace elements, such as iron, zinc, manganese and the like, or individual amino acids. 
[0045] The monomeric TGF-p-like protein is recovered from the microbial host cells by methods well known in the 
art. These methods include lysis or mechanical disruption of the cells in order to release the desired protein, followed 
40 by the separation of the TGF-p-like protein from the host cell proteins, e.g. by precipitation and/or chromatographic 
means. 

[0046] In cases where the monomeric TGF-p-like protein is produced in the microbial host cells as an insoluble 
aggregate (inclusion body) it has to be solubilized before being exposed to the refolding conditions. Accordingly, the 
present invention further relates to a process wherein the monomeric TGF-p-like protein is produced by the steps of: 

45 

(a) isolating the water-insoluble protein fraction containing the TGF-p-like protein from the host cells and 

(b) solubilizing the TGF-p-like protein. 

[0047] Solubilization and denaturation of the monomer is achieved by acidification of the crude protein suspension 
50 containing the monomeric TGF-p-like protein in the non-soluble form to a pH of about 1 to about 4, preferably to about 
2.5, optionally in the presence of a reducing agent, such as DTT, or by the addition of chaotropic agents, preferably 
guanidine HCl or most preferably urea, in a concentration of about 4 to 9 M, basic pH or elevated temperatures as 
described before. The solubilized monomer can be purified from solubilizingchaotropes by dialysis and, if a precipitate 
occurs during dialysis, by additional centrifugation. The solubilized monomer is chromatographically purified and used 
55 tor refolding to get the biologically active, dimeric product. 

[0048] After refolding, the biologically active dimer is purified in order to remove impurities, in particular, pyf^ogens 
or other endotoxins which might be present in the preparation after production of the recombinant protein in microbial 
host cells. Separation of the dimer is performed by chromatography such as sizing get chromatography, hydrophobic 



7 



EP 0 433 225 B1 



inleraction chromatography or ion exchange chromatography, e.g. on a Mono S column and reverse phase HPLC. 
[0049] The present invention further relates to dimeric biologically active TGF-p-like proteins when produced accord- 
ing to the process of the invention. These TGF-p-like proteins can be used in a variety of therapeutic modalities. 
[0050] The invention relates further to a monomertc, S-sulfonated TGF-p-like protein, which can be produced by S-. 
5 sulfonating the monomeric TGF-p-like protein. Monomeric, S-sulfonated TGF-p-like proteins are novel compounds, 
which can be used for the production of biologically active, dimeric TGF-p-like proteins. 

[0051] The present invention concerns further a pharmaceutical composition comprising an effective amount of a 
dimeric, biologically active TGF-p-like protein produced according to the invention, or a pharmaceutically acceptable 
salt thereof in dosage unit form. 

10 [0052] Such composition is in the form of infusion solutions or preparations for parenteral, for example intramuscular 
or intravenous, oral, or especially for local, i.e. topical, administration, respectively. The solutions are preferably isotonic 
aqueous solutions or suspensions which can be prepared before use, for example from lyophilised preparations which 
contain the active ingredient alone or together with a pharmaceutically acceptable carrier Solutions for parenteral use 
are usually aqueous solutions. They are prepared in conventional manner and may contain in addition to the active 

IS ingredient physiological saline, a stabilizer, such as human serum albumin, amino acids, such as arginine or glycine, 
and a carbohydrate, such as glucose, man nose, dextran or hydroxyethy! starch. The pH may be adjusted with a buffer, 
e. g. a phosphate, succinate or an amino acid to about 4.5 to 7. Usually the vials are filled with the solution and lyophilized 
for longer storage. 

[0053] The compositions contain conventional adjuncts, for example preservatives, stabilisers, wetting agents and/ 
20 or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The present pharmaceutical com- 

positionS: which may, if desired, contain further pharmacologically valuable substances, are produced in a manner 

known per se. for example by means of conventional mixing, dissolving, lyophilising and/or sterilising processes, and 

contain from approximately 1 ng to ICQ iig/g, especially from approximately 10 ng to 10 ^g/g of preparation, and in the 

case of lyophilisates up to 100%, of the active Ingredient. 
25 [0054] The TGF-p-like proteins are dual in character in that they on the one hand stimulate the proliferation of certain 

cell types, namely fibroblasts, and on the other hand inhibit the proliferation of other cell types, namely tumor cells and 

cells of the immune system. 

[0055] The dimeric, biologically active TGF-p-like proteins produced according to the invention, optionally in the form 
of their salts, such as in particular non-toxic pharmaceutical acid addition salts, optionally in form of pharmaceutical ~ 

30 formulations, are applied in an effective amount. By the form "effective amount" is Intended an amount which exerts a 
significant healing, e.g. an amount which stimulates the desired cells to grow and which is not toxic to normal cells. 
This amount can be determined e.g. by in vitro growth experiments. Due to the dual character of TGF-p-like proteins, 
an "effective amount" is also such which to a significant extent inhibits the growth and proliferation of tumour cells and 
cells of the immune system. If human or veterinary use is intended, the amount has to be adjusted to the particular 

35 tissue to be treated, the mode of application, the severity of the disease, and the age and general condition of the 
patient to be treated. In general, the either single or daily dosages for adult humans will be in the range of about 0.01 
to 20 |ig for both the growth stimulating and the inhibiting effect. 

[0056] The pharmaceutical composition of this invention have a clinical use In the treatment of animals, particularly 
mammals, more particularly human beings, and, in the case of wound healing, most particularly of old human beings. 
40 [0057] The compositions of this invention promote cell migration and proliferation. Since wound healing involves 
both cell migration and cell proliferation patterns these in vitro findings become directly relevant to the in vivo wound 
healing process. 

[0058] Prevention or treatment of bed sores (decubitus ulcers) is a preferred use since they frequently occur in 
hospital patients, particularly geriatric and wheel chair patients. In elderly people the wound healing process is slower 
45 and this group of patients tends to show a higher incidence of wounds (not only decubitus and diabetic ulcers, but 
trauma, burns and the like) that either heal slowly or do not heal at all. 

[0059] Two types of application of the compositions of this invention are proposed for both veterinary and, in particular, 
human medicine. 

[0060] The first, and preferred application is a topical one for the promotion of surface wound healing, particularly in 
50 elderly human beings where the wound healing processes are noticeably slower. There are no limitations as to the 
type of wound that may be treated, and these include (but are not limited to): Surface ulcers including decubital (bed 
sore), diabetic, dental, oral, varicose and haemophiliac surface ulcers; bums (especially second and third degree); 
surgical incisions (including those of dental and cosmetic surgery); accidental wounds (including incisions, penetra- 
tions, lacerations and other traumata) and therapeutically induced wounds (including those induced during radbther- 
55 apy). When applied topically, the compositions may be combined with other ingredients, such as adjuvants, carriers, 
solubilizing agents and any other known, or as yet unknown, secondary growth factor(s). There are no limitations as 
to the nature of these ingredients except that they must be pharmaceutically and physiologically acceptable for admin- 
istration and must not degrade the activity or render harmfully toxic, the active ingredients of the compositions. When 



8 



EP 0 433 225 B1 



the compositions of this invention are applied to surface ulcers, burns, surgical or accidental wounds, the connpositions 
are preferably in the form of a powder, gel, ointment, salve or trrigant, or they may be impregnated into transdermal 
patches, plasters and bandages, preferably in a liquid or semi-liquid form, or they may be incorporated into a tooth 
paste or a gum or resin for chewing. 

5 [0061] The second application is a systemic one for the healing of internal wounds either following surgery, or damage 
to the tissues of the inner organs where surgery is either impossible or is not required. Again, there are no limitations 
as to the type of tissue or wound to be treated and these include (but are not limited to) deep surgical incisions to the 
inner organs and tissues; bone and cartilage (after fracture); gastric, duodenal and other intestinal ulcers. When applied 
systemically, the compositions of the invention may be formulated as liquids, pills, tablets, lozenges for enteral admin- 

^o istration, or in liquid form for parenteral injection. For the treatment of internal incisions following surgery, they may be 
in the form of an irrigant, preferably in combination with a physiologically acceptable saline solution. Again, the active 
ingredients of the compositions may be combined with other ingredients such as adjuvants, carriers, solubilizing agents; 
and any other known, or as yet unknown, secondary growth factor(s). There are no limitations as to the nature of these 
ingredients except that they must be pharmaceutically and physiologically acceptable for administration and must not 

IS degrade the activity, or render harmfully toxic, the active ingredients of these compositions. 

[0062] For healing the wounds, the amount of active ingredient to be applied has to be adjusted to the type, severity 
and location of the wound, and also to the age and general condition of the patient to be treated. In general a single 
or daily amount of from about 1 ^g to 20 |ig of TGF-p-like protein per 1 cm^ of wound has already a significant healing 
effect. For internal use a higher amount should be applied depending on the mode of administration due to the dilution 

20 of the TGF-p-like protein in the body fluids. 

[0063] Further uses of the TGF-p-like proteins produced according to the invention are in bone and tissue repair, 
treatment of cancer in mammals, as an anti-inflammatory or Immunosuppressive agent, as a growth regulator in mam- 
malian cell cultures or as a bone marrow protective agent or mediator of cardioprotection. 

26 Examples " 

[0064] The following examples illustrate the invention without being meant to be limitative. 

Example 1 : Cloning and sequencing of TGF-&1 . TGF-g2 and TGF-P3 cDNA " 

30 

A. Culturing of cells 

[0065] Human glioma cells from the CI-21 5 line (de Muralt, B. et al. (1985) Eur. J. Cancer Clin. OncoL 21 , 207) are 
grown in tissue culture flasks (Falcon T75) containing Dulbecco's Modified Eagle Medium (DMEM, Gibco) and 10 % 
35 foetal calf serum. 

B. RNA extraction 

[0066] 1x10^ cells from the CI-21 5 human glioma cell line are harvested and Dounce homogenized in 30 ml 5% 
40 citric acid with 0.2 % (w/v) NP40 detergent at 4°C. The nuclei are separated from the -cytoplasm by cent rifugat ion at 
2.500 rpm for 10 minutes at 4°C in a Sorvall RT 6000-B table-centrifuge. The supernatant is centrifuged at 1 5.000 rpm 
for 30 minutes at 4°C in a Sorvall RC 5-B centrifuge fitted with a SS-34 rotor. The resulting supernatant is discarded 
and the pellet is resuspended in 30 ml 0.2 M TRIS/HCI (pH 7.5), 5 mM EDTA, 2% SDS, 25.000 units/I Heparin (Sigma), 
and then extracted 3 times with phenol/chloroform (1:1, v/v), the chloroform consisting of 24 parts chloroform and 1 
45 part isoamylalcohot (v/v). To the final aqueous phase 1 volume 3 M sodium acetate (pH 5.0) and 2.5 volumes ethanol 
are added. The ethanol precipitate is washed twice with 70 % ethanol. The RNA pellet is resuspended in 2 ml 10 mM 
TRIS/HCI (pH 7.5), 1 mM EDTA, 0.05 % SDS. Polyadenylated RNA is isolated by oligo-dT cellulose chromatography 
as described by T Maniatis in "Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York 
(1982)". 

so 

C. Synthesis of cDNA 

[0067] The first strand cDNA is synthesized from 10 \ig of poty A+ RNA in 100 \i\ of a solution containing 50 mM 
TRIS (pH 8.3), 50 mM KCt, 10 mM MgClg, 1 mM DTT 30 pg/ml oligo-dT 12-18, 1 mM each dATP, dCTP. dGTP and 
66 dTTP, 50 units RNase inhibitor (Promega) and 1000 units of Moloney Leukaemia Virus reverse transcriptase (Gibco- 
BRL). The reaction is incubated for 1 hour at 37*^C. The reaction is then diluted to 400 \i\ with a second strand buffer 
containing 20 mM TRIS/HCI (pH 7.5), 5 mM MgCl2,-100 mM KCI. 12.5 units RNase H (Gibco-BRL) are added and the 
reaction mixture is incubated for 10 minutes at 37*C. The reaction mixture is cooled on ice for 5 minutes and 125 units 



9 



EP 0 433 225 B1 



of E. coli DNA polymerase I (Promegaj are added. The reaction mixture is then incubated for a further 2 hours at 16**C. 
40 \i\ 0.5 M EDTA are added followed by a phenol/chloroform (1:1, v/v) extraction. To the aqueous phase, 1/1 0 volume 
of 3 M sodium acetate (pH 6.0) and 4 volumes ethanol are added whereafter the reaction mixture is precipitated for 
30 minutes at -70'*C. 

5 [0068] The ethanol precipitation is centrtfuged for 1 0 minutes at 1 7.000 g, the pellet is washed twice with 70 % ethanol 
and dried in a Speed- Vac. The double stranded cDNA is dissolved in sterile water and electrophoresed in an agarose 
gel in TRIS-borate buffer (pH 8.8) to evaluate the size and quantity of the cDNA. 

[0069] 5 ^g of the cDNA are then methylated at the EcoRI sites by incubating for 1 hour at 37°C in 100 ^1 50 mM 
TRIS/HCI (pH 8.0), 0.1 mM EDTA, 80 ^iM adenosyl-methionine and 40 units EcoRI methylase (New England Biolabs). 
10 The reaction mixture is extracted with phenol/chloroform and the cDNA is ethanol precipitated and dissolved in sterile 
water as described above. 

[0070] 5 ^g of the cDNA are then prepared for linker ligation by incubating with 20 units of T4 DNA polymerase for 
10 minutes at 37°C in 200 ^1 33 mM TRIS-acetate (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 
0.5 mM DTT and 0.1 mM each dATP, dGTR dCTP and dTTP. The reaction is cooled to room temperature and then 20 
15 units of Klenow Polymerase (Gibco-BRL) are added and incubated for 5 minutes at room temperature and 5 minutes 
on ice. After adding 10 ^^ of 0.5 M EDTA the reaction mixture is phenol/chloroform extracted and the cDNA is ethanol 
precipitated and dissolved in sterile water as described above. 

[0071] 12-mer synthetic 5'-phosphorylated linkers (New England Biolabs No. 1070) are then ligated to 5 ^g cDNA 
in 100 |il of a solution containing 10 ^g linker in 50 mM TRIS/HCI (pH 7.8): 10 mM MgClg, 20 mM DTT, 1 mM ATP at 
20 1 6'*C using 4000 units T4 DNA ligase (New England Biolabs). The ligase is then heat inactivated at 70° C for 10 minutes, 
the reaction is diluted to 500 |il in 10 mM TRIS/HCI (pH 7.5), 6 mM MgCl2, 100 mM NaC! and is digested with 1000 
units of EcoR! (Boehringer) for 6 hours at 37''C. 50 |il 0.5 M EDTA are added and the reaction mixture is heated at 
70" C for 10 minutes. The heated reaction mixture is directly added to a Bio-gel A15M column (200-400 mesh, Bio- 
Rad) to remove the monomer linker fragments. cDNA of more than 300 bp elutes in the exclusion volume. 

25 

D. Cloning into lambda gtll 

[0072] Lambda gtl 1 arms are prepared by digesting 1 00 ^g of lambda vector DNA with EcoRI (New England Biolabs) 
according to the supplier. The digested DNA is dephosphorylated using 1 unit of Calf Intestine Alkaline Phosphatase 

30 from Boehringer Mannheim as described. 20-30 ng of cDNA f rom the Bio-gel A1 5M column are coprecipitated in ethanol 
with 1 ^g of gtni dephosphorylated arms and resuspended in 10 jil of a solution containing 50 mM TRIS/HCI (pH 7.8), 
10 mM MgClg, 20 mM DTT, 1 mM ATP, 15 % polyethylene glycol (MW 6000) and 200 units of T4 DNA ligase. The 
ligation mixture is incubated for 2 hours at 16°C. The reaction mixture is centrifuged for 10 minutes and the pellet is 
resuspended in ^Q \i\ of sterile water and then in vitro packaged for 3 hours at room temperature according to the 

55 supplier (Promega). 0.5 Ml of SM phage dilution buffer containing 50 mM TRIS/HCI (pH 7.5), 100 mM NaCI, 100 mM 
MgS04 and 0.01 % gelatine are added and stabilized with 25 ^il of chloroform. A total of 500.000 phages are amplified 
on 10 YT-plates (15 cm diamter) using 0.7 % agarose-YT (Sigma) with E. coli Y 1090 cells as described (Young, R. 
and Davis, R. (1 983) PNAS 80, 11 94). 

40 E . Screening and selection of clones containing TGF-&1 , TGF-B2 and TGF-&3 inserts 

[0073] Six replica nylon filters (Cuno) are made from each of the 10 YT-plates and the phages on the filters are 
denatured with 0.5 M NaOH, 1.5 M NaCI and neutralized as described in "Molecular Cloning: A Laboratory Manual" 
(T. Maniatis, Cold Spring Harbour Laboratory, New York, 1 982). The filters are placed in 0.2 x SSC, 0.2 % SDS at 90'C 
45 for 15 minutes and then prehybridized for 4 hours at 45**C in 2 x SSC, 1 % SDS, 0. 1% ficoll, 0.1% polyvinylpyrrolidone, 
0.1% bovine serum albumin, 50 mM NaP04 (pH 6.8), 50 jig/ml denatured salmon sperm DNA, 0.1 tig/m! oligo A 12-18 
and 100 ^ig/ml poly A+ RNA. Each of the 6 replicas is hybridized overnight at 45**C in the prehybridization buffer to 
which one out of six different ^^p-iabelled 39 bp oligomers (see below) to a concentration of 2 x 10^ cpm/ml had been 
added. 

£0 [0074] The six oligomers used for hybridization are synthesized on an Applied Biosystem DNA Synthesizer and 
correspond to the nucleotide sequence encoding either the first amino acids (oligomers 1 , 3 and 5) or the last amino 
acids (oligomers 2, 4 and 6) of the mature forms (112 amino acids) of TGF-pi (seeSEQ ID No. 1), TGF-P2 (see SEQ 
ID No. 2) and TGF-P3 (see SEQ ID No. 3), respectively 
[0075] The two oligomers used for the detection of T-GF-pl sequences are: 

£5 



10 



EP 0 433 225 B1 



1) 5' GCC CTG GAC ACC AAC TAT TGC TTC AGC TCC ACG GAG AAG 3' 

2) 5' TCA GCT GCA CTT GGA GGA GCG CAC GAT CAT GTT GGA GAG 3* 

5 

[0076] The two oligomers used for the detection of TGF-P2 sequences are; 

3) 5* GCT TTG GAT GCG GCC TAT TGC TTT AGA AAT GTG CAG GAT 3' 
10 4) 5* ^rj,j^ QQrj, Q^p^ ^rpY QCA AGA CTT TAC AAT CAT ATT AGA AAG 3* 

[0077] The two oligomers used tor the detection of TGF-p3 sequences are: 

75 

5) 5' GCT TTG GAC ACC AAT TAC TGC TTC CGC AAC TTG GAG GAG 3* 

6) 5* TCA GCT ACA TTT ACA AGA CTT CAC CAC CAT GTT GGA GAG 3* 

20 

[0078] 40 ng each oligomer is labelled at its 3' end using ^^p-dATP and 20 units of terminal transferase (Gibco-BRL) 
in a 20 ^1 reaction buffer containing 100 mM potassium cacodylate (pH 7.2), 2 mM C0CI2 and 0.2 mM DTT for 1 hour 
at 37°C. The reaction mixture is gel filtrated over a Sephadex G-50 column. The eluted labelled oligomers are heated 
at 95°C for 5 minutes and added to the prehybridization buffer as described above. 

25 [0079] The replicas 1 to 6 are hybridized with oligomers 1 to 6. respectively After hybridization, the filters are washed 
twice each with 2 x SSC, 1 x SSC and 0.1 x SSC at room temperature for 15 minutes. Positive plaques are identified 
by autoradiography and are rescreened by repeating the procedure given above until all of the plaques on the plate 
are positive. A single plaque is eluted in 1 ml of SM phage dilution buffer (see section 1.D), 100 ^1 are added to 1 ml 
E. coli Y 1090 cells and the mixture is kept for 20 minutes at room temperature. The E. coli Y 1090 cells and phages 

30 are added to 100 ml YT-medium containing 0.2 % maltose and incubated at 37°C for 7 hours. After adding 1 ml chlo- 
roform to the lysed cells, the phage DNA is purified according to the method described in "Molecular Cloning: A Lab- 
oratory Manual" (T Maniatis, Cold Spring Harbour Laboratory, New York, 1982). The purified DNA is dissolved in 1 ml 
10 mM TRIS/HCI (pH 7.5), 1 mM EDTA and 100 fil are digested to completion in a total volume of 1 ml with EcoRI 
following the recommendations of the supplier (Boehringer). The enzyme reaction is phenol/chloroform extracted and 

35 ethanol precipitated. The EcoRI cDNA inserts are purified by gel electrophoresis (Ultrapure BRL) using NA-45 DEAE 
paper (Schleicher and Schuell). The DNA is eluted in 50 mM TRIS/HCI (pH 7.5), 5 mM EDTA, 1 M NaCI, phenol/ 
chloroform extracted and ethanol precipitated. The resulting pellet is washed twice with 70 % ethanol and resuspended 
in 10 mM TRIS/HCI (pH 7.5), 1 mM EDTA. 

^0 F. Sequencing of cDNA inserts 

[0080] The EcoRI cDNA inserts are subcloned into Bluescript KS+ Vector (Stratagene). The cDNA identity is con- 
firmed by double-stranded sequencing according to the method described by F. Sanger et at. (1977) PNAS 74, 5463 
using the above oligomers (see section 1 E) and a Sequenase kit (U.S. Biochemicals). The nucleotide sequence cov- 
45 ering the 112 amino acids of the mature TGF-pi, TGF-P2 and TGF-P3 are depicted under SEQ ID No. 1, 2 and 3, 
respectively. 

G. Amplification of cDNA inserts and subcloning intoplasmid PGem-5 

so [0081] The above oligomers (see section 1 .E) for identifying TGF-pi , TGF-P2 and TGF-P3 sequences are used to 
amplify the cDNA inserts encoding the mature 112 amino acids. forms (including the stop codon). 
[0082] The EcoRI cDNA inserts of the Bluescript KS+ plasmids (see section 1 .F) are gel purified as described above 
(see section 1 .E). 50 ng of each cDNA insert are amplified in the presence of 2 x 2^g of the respective two oligonoers 
by a polymerase chain reaction in a 100 |il reaction mixture containing 10 mM TRIS/HCI (pH 8.35), 50 mM KCI, 1.6 

55 mM MgCl2. 0.05 % (w/v) NP-40, 0.05 % (w/v) Tween 20 and 200 ^im of each dATP, dGTP, dCTP and dTTP using 5 
units Taq Polymerase (Perkin-Elmer Cetus). 30 rounds of amplification are performed under the following temperatures 
using a Perkin-Elmer Cetus Heating Block: 93*'C/0.1 minutes, 65''C/0.2 minutes, 71 "C/l .5 nninutes. The resulting 339 
bp fragments covering the coding sequences of TGF-pi, TGF-P2 and TGF-P3, respectively, are gel purified and sub- 



11 



EP 0 433 225 B1 



cloned into plasmid PGem-52F(+) (Promega) digested with Ncol, dephosphorylated with Calf Intestinal Alkaline Phos- 
phatase (Boehringer) and filled in with Klenow polynnerase (Gibco-BRL). The resulting constructs are designated as 
pGKM 125 (TGF-pi), pGKM 740 (TGF-p2) and pGKM 126 (TGF-p3) and are used to transform competent E. coli Y 
1090 cells (see example 2). Clones carrying the correct inserts encoding TGF-pi , TGF-P2 and TGF-p3 are designated 
5 as E. coli Y1090/pGKM 125 (TGF-pi), E. coli Y1090/pGKM 740 (TGF-p2) and E. coli Y1090/pGKM 126 (TGF-p3), 
respectively. 

Example 2: Expression of TGF-Bl. TGF-B2 and TGF-&3 in E. coli 
10 A. General methods 

Bacterial strain (E.coli K12): 

[0083] LC 137: htpR^^JonRg, \ac^^, mal^^, trpg^, pho^^, rspL, tsx::Tn10, supCig (Goff, S.A. et aL (1984) PNAS 81. 
IS 6647-6651). 

Plasnnlds: 

[0084] 

20 

pPLMu:. (Buell, G. et al. (1985) Nucleic Acids Res. 13, 1923-1938). This plasmid carries the bacteriophage X Pl 
promoter with the phage Mu ner gene ribosome binding site (Van Leerdam, E. et al. (1982) Virology 123, 
19-28). 

25 pcle57: Plasmid encoding a thermolabile XCIgsy repressor and conferring resistance to kanamycin (Remault, E. et 
al. (1 983) Gene 22, 1 03-1 1 3). 

SDS gel-electrophoresis: 

30 [0085] SDS polyacrylamide gel-electrophoresis (SDS-PAGE) and protein staining Is done as described previously 
(Laemmli, U.K. (1 970) Nature 227, 680-685) using the Miniprotean II cell from BIORAD and 1 mm thick 18 % polyacr- 
ylamide gels. 

Heat induction: 

35 

[0086] 7 ml of LB-Medium (Maniatis et al. (1982), Molecular Cloning, Cold Spring Harbor Laboratory, New York) in 
a 20 ml culture tube containing 40 |ig of each ampiciHin and kanamycin (LB/amp/kan) are inoculated with a single 
colony and incubated with shaking overnight at 30°C. 5 ml of this overnight culture are added to 15 ml of LB/amp/kan 
in a 100 ml Erienmeyer flask. This flask is transferred to a 42*'C waterbath shaker A 2 ml sample is taken before 
^0 transfer (non-inducing conditions) and 1 ml samples at 1 hour intervals after the transfer (inducing conditions). Cells 
are pelleted by centrifugation (5 min, 10.000 rpm in an Eppendorf centrifuge) and the supernatant is discarded. The 
pellet Is resuspended in 100 \i\ of sample buffer for SDS-PAGE and heated for 10 min at 95''C. 5 \i\ aliquots are loaded 
for SDS-PAGE. 

4S Preparation of competent cells: 

[0087] Competent E. coli cells are prepared by the calcium chloride procedure as described in Maniatis et al. (1 982), 
Molecular Cloning, Cold Spring Harbor Laboratory, New York. Cells carrying plasmid pclgsy are grown at 30**C. 

so B. Construction of expression vectors pPLMu.hTGF-BI. pPLMu.hTGF-32 and pPLMu.hTGF-03 and expression of TGF- 
p1,TGF-&2 and TGF-B3 

[0088] E. coli Y1090/pGKM 125, E. coli Y1090/pGKM 740 and E. coli Yl090/pGKM 126 (see example 1 .G) cells are 
grown in LB medium and plasmid DNA is prepared by the method of Birnboim, H.C. and Doly, H. (1979) Nucleic Acids 
ss Research 7, 1513. 5 |ig of plasmid DNA are cut to completion in 50 |il restriction buffer with either Ncol and Sail 
(pGKM125), Ncol and EcoRV (pGKM740) or Ncol alone (pGKM126) following the recommendations of the supplier 
(Boehringer). The DNA is precipitated by addition of 5 ^il 3 M sodium acetate, 100 mM MgCl2, 5 mM EDTA and 150 ^1 
ethanol. After incubation at -70*'C for 15 min the DNA is pelleted by centrifugation at 13,000 g for 15 min in a SS34 



12 



EP 0 433 225 B1 



rotor in a Sorvall centrifuge. The supernatant is discarded and the pellet is resuspended in 80^1 0.089 M TRIS borate, 
0.089 M boric acid and 0.002 M EDTA (TBE buffer) containing 0.25 % bromphenol blue and 0.25 % xylene cyanol. 4 
times 20 ^1 samples are electrophoresed through a 1 % agarose gel in TBE buffer containing 0.5 ^g/ml ethidium bromide 
at 50 volts till the bromphenol blue marker reaches the bottom of the 10 cm long and 0.8 cm thick gel. The DNA 

s fragments coding for mature TGF-pi , TGF-P2 and TGF-p3, respectively, are visualized under short wave UV light, cut 
out with a razor blade and etectroeluted from the gel piece in a Schleicher & Schull Biotrap apparatus applying 200 
mamp for 1.5 hours. The eluted DNA fragments are precipitated (see above) and resuspended in 20 ^1 TE. 
[0089] 5 ^l of plasmid pPLMu are linearized by digestion with either Ncol and Sail, Ncol and EcoRV or Ncol atone 
and gel purified as described above for the fragment DNAs. 100 ng of the linearized and puriffed pPLMu vector DNA 

10 and 3 times the molar equivalent of the respective purified fragment DNA are incubated at A^C for 15 hours in 20 ^l of 
ligation buffer (70 mM TRIS/HCI, pH 7.5, 10 mM MgCl2, 5 mf^ DTT. 0.1 mM adenosine-triphosphate) containing 1 unit 
of DNA ligase (Boehringer). 

[0090] 10 |il of the ligation mixture are added to 200 pi of cold (4°C) competent E. coli LC 137 cells carrying plasmid 
pclgsy. After 30 min the cells are heat shocked by incubation for 1 .5 min in a 42°C water bath. 2 ml of LB medium are 

75 added and the culture is shaken for 60 min at 30°C. 200 |il aliquots are plated on LB plates containing ampicillin and 
kanamycin and incubated for 22 hours at 30*0. Single colonies are cultivated and plasmid DNA is analysed. Subcloning 
of the DNA fragments coding for TGF-pl , TGF-p2 and TGF-p3 in pPLMu results in plasmids pPLMu.hTGF-pi . pPLMu. 
hTGF-p2 and pPLMu.hTGF-p3, respectively Clones containing the above constructs are referred to as E. coli LC 
1 37/pPLMu.hTGF-pi . E. coli LC 1 37/pPLMu.hTGF-p2 and E. coli LC 1 37/pPLMu.hTGF-p3, respectively. 

20 [0091] E. coli LC 137/pPLMu.hTGF-3l. E. coli LC 137/pPLMu.hTGF-p2 and E. coli LC 137/pPLMu.hTGF-P3 cells 
are heat induced (see example 2. A) and the expressed proteins are analysed by SDS-PAGE. TGF-pi, TGF-p2 and 
TGF-P3 all appear as heat induced proteins 2 hours after heat induction migrating with an apparent molecular weight 
of approximately 12.000 D. 

25 c. Fermentation of transformants 

[0092] Overnight cultures of E. coli LC l37/pPLMu.h.TGF-pi. E, coH LC 137/pPLMu.h.TGF-p2 and E coy LC 
1 37/pPLMu.h.TGF-p3 in 21 Erienmeyer flasks containing 750 ml of LB medium with 40 mg/l of ampiclINn and kanamycin 
are grown at SO^C. 300 ml of the overnight cultures are added to 750 ml of LB medium containing antibiotics as men- 
30 tioned above in 2 I Erienmeyer ffasks and heated to 42*'C by shaking for approximately 3.5 minutes in a 65*'C water 
bath. The flasks are then transferred to a 42'*C shaker and incubated tor 3 hours. The flasks are cooled down to 1 2**C 
in an ice water bath and the cells are collected after centrifugation for 10 minutes at 8.000 rpm in a GSA rotor (Sorvall). 

Example 3: Expression of TGF-Bl, TGF-g2 and TGF-33 in Saccharomyces cerevisiae 

36 

[0093] The coding sequences of mature TGF-pi , TGF-P2 and TGF-P3 are expressed in Saccharomyces cerevisiae 
under the control of the inducible promoter of the yeast acid phosphatase (PH05). 
[0094] The expression vectors are constructed in two steps: 

40 A. construction of plasmid pJDB207/PH05-RIT 12, 

B. construction of plasmids pJDB207R/PH05-TGF-pi, pJDB207R/PH05-TGF-p2 and pJDB207R/PH05-TGF-p3, 
where A) provides the yeast vector and the PH05 transcriptional terminator and B) provides the expression cas- 
settes with an insert coding for mature TGF-pi , TGF-P2 and TGF-P3, respectively under the control of the PH05 
promoter. 

45 

A. Construction of plasmid pJDB207/PH05-RIT 12 

[0095] Plasmid p31 RIT 12 (European patent application EP 277.31 3) is linearized with restriction endonuclease Sail. 
Partial Hindlll digestion in the presence of ethidiumbromide results in a 1 kb Sail/Hindi 11 fragment comprising the 276 
50 bp Sall/BamHI pBR322 sequence, the 534 bp promoter of the yeast acid phosphatase PH05 . the yeast invertase signal 
sequence (coding for 19 amino acids) and the PH05 transcriptional terminator. 

The 1 kb Sall/Hindlll fragment of p3lRIT 12 is cloned in to the yeast -E.coli shuttle vector pJDB207 (Beggs, J.D. in: 
Molecular Genetics in yeast, Alfred Benzon Symposium 16, Copenhagen, 1981. pp. 383-389), which had been cut 
with Sail and Hindlll. The resulting plasmid containing the 1 kb insert is referred to as pJDB207/PH05-RIT 12. 

55 

B. Construction of plasmid pJDB207R/PH05-TGF-82 

[0096] Plasmid pGKM740 (TGF-P2) (see example 1 .G) is cut with Ncol. The sticky ends are filled in a reaction with 



13 



EP 0 433 225 B1 



Klenow DNA polymerase. EcoRI linker (5'-CCGGAATTCCGG; Biolabs) are added and the nnixture is ligated. The re- 
sulting circular plasmid is referred to as pGKMA668 (TGF-P2) and is cut with EcoRI and Sail. A 0.4 kb EcoRI/Sall 
fragment is isolated from an agarose gel, purified and resuspended in sterile water at a concentration of 25fig/ml. The 
fragment contains the mature coding sequence of TGF-P2 with an ATG in frame to codon GCT which defines amino 

5 acid Ala 1 of mature TGF-P2. 

[0097] The PH05 promoter is isolated from plasmid p31RIT 12 (see above) on a 534 bp BamHI/EcoRI fragment. 
Plasmid pJDB207/PH05-RIT 12 is cut with BamHI and Xhol. The large, 6.8 kb BamHI/Xhol fragment is isolated. The 
PH05 transcriptional terminator remains on the fragment. The BamHI/EcoRI PH05 promoter fragment, the EcoRI/Sall 
fragment coding for TGF-P2, and the BamHI/Xhol vector fragment are ligated. One correct clone with the TGF-P2 gene 

yo under the control of the PH05 promoter cloned in an anticlockwise orientation into pJDB207 is referred to as pJDB207R/ 
PH05-TGF-P2. 

[0098] I nan analogous manner, mature TGF-pl and TGF-P3 are expressed in S. cerevisiae . The plasmids containing 
the coding sequences of TGF-pl and TGF-P3 are pGKM125 and pGKM126, respectively (see example 1.G). After 
digestion of these plasmids with Ncol, addition of EcoRI linkers and ligation, the resulting circular plasmids are cut with 
^5 EcoRI and Sail. The EcoRI/Sall fragments are cloned into pJDB207 as described above. The resulting plasmids are 
referred to as pJDB207R/PH05-TGF-p1 and pJDB207R/PH05-TGF-p3 

C. Transformation of S. cerevisiae strain GRF18 

20 [0099] Saccharomvces cerevisiae strain GRF18 (MATa. his3-n. his3-15, leu2-3, Ieu2-112. can^ . DSM 3665 ) is 
transformed with plasmids 

pJDB207R/PH05-TGF-pi 
p JDB207R/PH05-TG F-p2 
25 pJDB207R/PH05-TGF-p3 

using the transformation protocol described by Hinnen, A. et al. (1978) PNAS75, 1929: 
Transformed yeast cells are selected on yeast minimal medium plates deficient in leucine. 
Single transformed yeast colonies are isolated and referred to as 

30 

Saccharomvces cerevisiae GRF18/pJDB207R/PH05-TGF-p1 
Saccharomvces cerevisiae GRF18/pJDB207R/PH05-TGF-p2 and 
Saccharomvces cerevisiae GRF18/pJDB207R/PH05-TGF-P3. 

35 D. Fermentation of S. cerevisiae transformants and preparation of cell extracts 

[0100] The yeast transformants, as mentioned above, contain plasmids with PH05 pronrioter-controlled expression 
cassettes and therefore require derepression of the promoter for the expression of TGF-pi , TGF-P2 or TGF-p3. Trans- 
formants are each grown in two successive precultures (10 ml and 50 ml) in yeast high Pj minimal medium prepared 

40 according to the recipe of the Difco Yeast Nitrogen Base without amino acids but containing 10 g/I L-asparagine instead 
of (NH4)2S04, 1 g/I L-hlstidine and 20 g/I glucose. The cells of the second preculture are washed in 0.9 % NaCt and 
all the cells are used to inoculate 100 ml of low Pj minimal medium prepared according to the recipe of the Difco Yeast 
Nitrogen Base medium (without amino acids), but containing 0.03 g/I KH2PO4, 10 g/I L-asparagine, 1 g/I L-histidine 
and 20 g/I glucose. The cultures are agitated at 30®C at 180 rpm. 

^5 [0101] Cells from 1 0 ml of culture are collected at 5 h, 24 h and 48 h by centrifugation at 3000 rpm and washed once 
in 0.9 % NaCI. The cell pellet is resuspended in lysis buffer [66 mM potassium phosphate pH 7.4, 4 mM Zwittergent 
(Calbiochem)]. 8 g of glass beads (0.5-0.75 mm in diameter) are added and the suspension is shaken vigerously 4-5 
times lor 2 min each on a Vortex Mixer in the cold. The cell extract is decanted to get rid of the glass beads. Cell debris 
in the extract are sedimented by centrifugation for 5 min at 3000 rpm at A°C. The supernatant and pellets are separated 

50 and stored at -20°C. 

Example 4: Production of dimeric. biologically active TGF-&1.TGF-&2 and TGF-B3 

[0102] The procedures given below for the production of dimeric, biologically active TGF-P2 can be applied in analogy 
55 for the recovery of dimeric, biologically active TGF-pi , TGF-P3, and other "TOF-p-like proteins", respectively. 



14 



EP 0 433 225 B1 



A. Recovery of non-soluble, monomeric TGF-B2 from E. coli 

[0103] E. coli LC 1 37/pPLMu.hTGF-p2 cells are fermented as described in example 2.C. Cell disruption and recovery 
of non-soluble TGF-P2 is performed at 4*C. About 18 g of wet cells are suspended in 60 ml of 0.1 M TRIS/HCI, 10 rr\M 

5 EDTA, 1 mM PMSF (Phenyl f^ethan Sulphonyl Fluoride), pH 8.3 (disruption buffer). The cells are passed two times 
through a Frenchpress (SLM Instruments, Inc.) according to the manufacturers instructions and the volume is brought 
to 200 ml with the disruption buffer. The suspension is centrifuged for 20 min at 15.000 g. The pellet obtained is sus- 
pended in 100 ml disruption buffer containing 1 M NaCi and centrifuged for 10 min as above. The pellet is suspended 
in 100 m! disruption buffer containing 1 % Triton X-100 (Pierce) and again centrifuged for 10 min as above. The washed 

10 pellet is then suspended in 50 ml of 20 mM Tris/HCI, 1 mM EDTA, 1 mM PMSF, 1 % DTT and homogenised in a Teflon 
tissue grinder. The resulting suspension contains crude monomeric TGF-P2 in a non-soluble form. 

B. Solubilization and purification of monomeric TGF-&2 

15 [0104] 10 ml of the TGF-P2 suspension obtained according to example 4.A or 4.C are acidified with 10 % acetic acid 
to pH 2.5 and centrifuged in an Eppendorf centrifuge for 10 min at room temperature. The supernatant is chromato- 
graphed on a Sephacryl S-100 column (Pharmacia, 2.6 x 78 cm) in 10 % acetic acid at a flow rate of 1.4 Ml/min. 
(Alternatively, the chromatography can be performed on Sephacryl S-100 HR (Pharmacia) and the column can be run 
in 1% acetic acid or 5 mM HCI, respectively.) Fractions containing monomeric, denatured TGF-P2 eluting between 190 

20 min and 220 min are pooled. This material is used for refolding to get biologically active, dimeric TGF-P2 (examples 
4.G.J.K.L) or for further purification for structural analysis (example 4.D.). 

C. Recovery of monomeric TGF-32 from Saccharomyces cerevlsiae 

2S [0105] The pellet of broken cells obtained from a 500 ml fermentation performed as described in example 3.D is 
suspended in 20 ml 4M urea, 0. M TRIS, 1% 1 DTT, pH 8.0. The mixture is kept at room temperature for 30 minutes 
with intermittant vortexing every 5 minutes. Insoluble material is removed by centrifugation at 30*000g for 30 minutes 
at 4°C and the supernatant is adjusted to pH 2.5 with acetic acid and dialysed extensively against 5% acetic acid 
overnight at 4**C. The solution is centrifuged as above and the clear supernatant is concentrated by ultrafiltration on a 

30 YM 10 membrane (Amicon) to a final volume of 4 ml. The sample is then chromatographed on Sephacryl S-100 HR 
(Pharmacia) in 5% acetic acid as described in example 4.B yielding monomeric TGF-P2. 

D. Further purification of monomeric TGF-&2 by RP-HPLC 

35 [0106] Aliquots of the pooled fractions from the Sephacryl S-100 column (example 4.8) are purified on a Vydac 
214TP5415 HPLC reverse phase column (4.6 x 150 mm. The Separations Group, Hesperia, CA, USA). The column 
is equilibrated in a mixture of 70 % TFA 0.1% in water and 30 % TFA 0.08 % in acetonitrile, and the product is eluted 
by a linear gradient over 30 min ending with a mixture of 55 % TFA 0.1% in water and 45 % TFA 0.08 % in acetonitrile 
at a flow rate of 1 ml/min. The eluate is monitored for absorbance at 216 nm and individual peaks are collected manually 

40 according to the UV absorbance. Denatured, monomeric TGF-P2 is eluted at 21 .5 min. Depending on the individual 
reverse phase column used for the separation the same preparation of TGF-p2 is eluted around 16 min and 18 min, 
respectively. 

[0107] TGF-P2 fractions are analysed by RP-HPLC using the same column and solvent system as above. TGF-P2 
is eluted by a linear gradient over 42 min starting from 100 % TFA 0.1 % in water and ending with a mixture of 30 % 
45 TFA in water and 70 % TFA 0.08 % in acetonitrile. TGF-p2 is eluted as a single peak after 30.4 min. Depending on the 
individual column used retention times of 29 min and 29.9 min, respectively, are obtained. 

[0108] TGF-p2 is analyzed after admixture of chemically reduced natural porcine TGF-P2 (British Biotechnology 
Limited, Oxford, UK) which has an identical primary structure as human TGF-p2 (Marquardt, H. et al. (1987) J. Biol. 
Chem. 262, 12127-12131). The mixture elutes as a single peak confirming the identity of the material. 

so 

E. Analysis of monomeric TGF-B2 by SDS-PAGE 

[0109] Individual aliquots of the Sephacryl S-100 column (example 4.B) or the reverse phase column (example 4.D) 
are dried in vacuo and analysed by SDS-PAGE (Lammli, U.K. (1970) Nature 227, 680) on 15 % polyacrylamide slab 
55 gels stained with Coomassie Blue R-250. A single band of an apparant molecular weight of about 12.000 D is obtained 
which is indistinguishable from reduced natural porcine TGF-p2. 



15 



EP 0 433 225 B1 

F. N-terminal amino acid sequence determination of monomeric TGF-P2 



[0110] TGF-P2 from example 4.B Is evaporated in vacuo, dissolved in 25 ^1 acetic acid and subjected to amino acid 
sequence determination on a gas phase protein sequencer model 470A (Applied Biosystems). 
5 [0111] The N-terminal amino acid sequence is: 

5 10 15 

Ala-Leu-Asp-Ala-Ala-Tyr-X-Phe-Arg-Asn-Val-Gln-Asp-Asn-X- 

X-Leu-Arg-Pro 

IS wherein X denotes an amino acid not positively identified. 

[0112] Similarly, the N-terminal amino acid sequence is determined for the 4-vinylpyridine derivative of TGF-p2 pre- 
pared as described by Marquardt, H. et al. (1987) J. Biol. Chem. 262, 12127-12131. 
[0113] The N-terminal amino acid sequence is: 

20 

5 10 15 

Ala-Leu-Asp-Ala-Ala-Tyr-Cys-Phe-Arg-Asn-Val-Gln-Asp-Asn-X- 
20 25 

25 

Cys-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-X-Arg-Asp-Leu- 

wherein X denotes an amino acid not positively identified. Cysteine was determined as S-pyridylethyicysteine. 

30 G. Generation of dimeric, biologically active TGF-32 

[0114] 3 mg of monomeric denatured TGF-P2 from example 4.B is dissolved in 140 ml 50 mM Tris/HCI pH 8.0, 1 M 
NaCI, 5 mM EDTA, 2 mt\A reduced glutathione, 1 mM oxidised glutathione and 33 mM Chaps (Calbiochem). After 72 
hours at 4°C the pH of the solution is adjusted to pH 2.5 with HCl and the mixture is concentrated 10 times by ultrafil- 

55 tration on a YM 10 membrane (Amicon, Danvers, MA, USA) in an Amicon stirred cell. The concentrated solution is 
diluted to the original volume with 10 mM HCl and concentrated to a final volume of 10 ml by the same method. The 
precipitate formed is removed by centrifugation at 5000 g for 30 minutes. The supernatant contains disulfide linked 
dimeric TGF-(i2 as judged by SDS-PAGE under non-reducing conditions. The biological activity of the preparation is 
measured by the cell migration and growth assay (example 5.A) and the cell growth inhibition assay (example 5.B). 

40 [0115] Alternatively, instead of using monomeric TGF-p2, the S-sulfonated TGF-P2 derivative (example 4.M) is used 
for the generation of dimeric active TGF-p2 by applying essentially the procedure described in this example with the 
exception of the sodium chloride concentration which is 2M. Purification and Isolation of the dimeric TGF-p2 Is per- 
formed with the same methods as dimeric TGF-P2 generated from the underivatised monomeric protein (example 4.H 
and 4.1). 

45 

H. Isolation of dimeric TGF-&2 by cation exchange chromatography on a Mono S column 

[0116] The concentrated solution from example 4.G is applied at a flow rate of 1 ml/min onto a Mono S HR 5/5 column 
(Pharmacia) equilibrated In a mixture of 85 % buffer A (20 mM sodium acetate, 30 % isopropanol, pH 4.0) and 15 % 

50 buffer B (buffer A containing 1 M sodium chloride). The column is then washed at the same flow rate keeping the buffer 
mixture composition constant until the absorbance reading at 280 nm has reached baseline level, followed toy a linear 
gradient over 20 minutes starting upon injection at the equilibration conditions and ending with a mixture of 50 % buffer 
A/50% buffer B. Dimeric biologically active TGF-P2 is eluted 9 minutes after the start of the gradient and collected 
manually. As judged by biological activity determination, SDS-PAGE under non-reducing conditions and RP-HPLC no 

55 dimeric TGF-P2 was found in the flow through fraction. Additionally, no monomeric TGF-P2 was detected by SDS- 
PAGE in the dimeric TGF-P2 peak eluted from the column by the salt gradient. 



16 



EP 0 433 225 B1 



I. Further purification of dimeric TGF-B2 

[0117] Dimeric TGF-P2 from example 4.G is diluted with the same volume of 0.1 % TFA in water and subjected to 
RP-HPLC on a Vydac 214TP5415 column(4.6 x 150 mm, The Separations Group, USA) equilibrated In a mixture of 
5 80 % TFA 0.1% in water and 20 % TFA 0.08 % in acetonitrile. The column is eluted by a linear gradient over 40 min 
starting upon injection at the equilibration conditions and ending with a mixture of 60 % TFA 0.1% in water and 40 % 
TFA 0.08 % in acetonitrile at a flow rate of 1 ml/mln. The eluate is monitored for absorbance at 216 nm. TGF-P2 is 
eluted with a retention time of 32.7 min and collected manually. SDS-PAGE analysis under non reducing conditions 
revealed a single sharp band of apparent molecular weight of about 25 kD. The obtained dimeric TGF-P2 isof high purity. 

10 

J. Alternative method I for the generation of dimeric, biologically active TGF'&2 

[01 1 8] Monomeric TGF-P2 from example 4. B is dissolved at a concentration of 0. 1 mg/ml in 50 mM sodium phosphate, 
pH 8.0, 2M NaCI, 5 mM EDTA, 2.5 mM cysteine, 1 mM cystine and 50 mM Chaps (Calbiochem). After 300 hours at 

IS 4°CthepH is adjusted to pH 2.5 with 10% TFA. Then 30mg/mlof SepralyteC-1 (preparative grade, 40^m, Analytichem 
International, Harbor City, CA, USA), pretreated sequentially with 0.1 % TFA in acetonitrile and 0.1% TFA in water, is 
added and the mixture is gently stirred for 30 min at room temperature. The gel is filtered over a glass frit covered with 
fresh prewashed Sepralyte C-1 (20% the amount added to the refolding solution). The gel is washed first with (5 times 
the gel volume) buffer A (0.2 M NaCI/0.1%TFA/water), then with a mixture of 80 % buffer A and 20 % buffer B (0.08% 

20 TFA in acetonitrile). TGF-P2 is eluted with a mixture of 70 % buffer A and 30 % buffer B. The eluate is applied directly 
onto a Mono S column HR 5/5 (Pharmacia). Purification and isolation of TGF-p2 is performed as in examples 4.H and 
4.1, respectively. 

[0119] Alternatively, acetonitrile in buffer A and buffer B, respectively, used for washing the Sepralyte C-1 gel and 
elution of TGF-p2 is replaced by isopropanol. Washing is then performed with a mixture of 90 % buffer A and 10 % 
25 buffer B and elution of TGF-P2 is achieved with a stepwise gradient (steps of 2% buffer B) starting with a mixture of 
80 % buffer A and 20 % buffer B and ending with a mixture of 70 % buffer A and 30 % buffer B. Further procedure is 
as in examples 4. H and 4.1, respectively. 

K. Alternative method II for the generation of dimeric, biologically active TGF-&2 

30 

[0120] Monomeric TGF-p2 from example 4-B is dissolved at a concentration of 0.5 mg/mt in 100 mM Tris/HCI, pH 
8.5, 1M NaCI, 5 mM EDTA, 1 mM reduced glutathione, 1 mM oxidised glutathione and 50 mM Chaps (Calbiochem). 
After 450 hours at 4°C the mixture is adjusted to pH 4.0 with acetic acid, diluted by addition of 7 volumes of 20 mM 
sodium acetate, pH 4.0 and pumped onto a Mono S column HR 5/5 (Pharmacia). Further procedure as in example 
35 4.H and 4.1, respectively. 

L. Alternative method III for the generation of dimeric, biologically active TGF-32 using thioredoxin as a disulfide 
promoting agent 

40 [0121] Monomeric TGF-p2 from example 4.B is dissolved at a concentration of 0.025 mg/ml in 100 mM Tris/HCI, pH 
8.0, 50 mM Chaps, 0.05 mg/ml thioredoxin. The mixture is incubated at 4'*C for 24 hours. As determined by the cell 
migration and growth assay (example 5. A) the yield of refolded dimeric active TGF-P2 is similar to that of the method 
described in example 4.G. Purification and isolation of dimeric TGF-p2 is as in examples 4.H and 4.1. TGF-p2 is sep- 
arated from thioredoxin by the Mono S column of example 4.H. 

45 

M. Preparation of S>sulfonated TGF-&2 and its use for the generation of dimeric. biologically active TGF'B2 

[0122] Monomeric TGF-p2 from example 4.B is dissolved at room temperature in'6M urea, 100 mM Tris/HCI, pH 8.0, 
50 mM sodium sulfite and 0.2 mM cysteine. Formation of S-sulfonated TGF-p2 is monitored by RP-HPLC using the 
so conditions of example 4.D. The retention time of S-sulfonated TGF-P2 is 31 .8 min. After completion of the reaction, 
the pH of the solution is adjusted to pH 2.0 with 1 N HCI. S-sulfonated TGF-P2 is desalted on a FPLC 'Fast Desalting 
Column' HR1 0/10 (Pharmacia) in 10 mM HCI. Refolding of S-sulfonated TGF-p2 to give dimeric active TGF-P2 is done 
essentially according to the procedure of example 4.G. 

ss N. Recyclisation of incorrectly folded TGF-&2 

[0123] Solid guanidinium hydrochloride and DTT is added to the material not bound to the Mono S column of example 
4.H to give a concentration of 6 M and 5 mM. respectively, and the pH is adjusted to pH 8.5 with solid Tris. After 1 hour 



17 



EP 0 433 225 B1 



at room temperature the mixture is subjected to RP-HPLC using the same column and solvent system as in example 
4.D. Reduced monomeric TGF-P2 is collected, and acetonitrile is removed in the vacuum. This preparation is then 
subjected to the refolding procedure of example 4.G either directly or together with newly isolated monomeric TGF-P2 
from example 4.B or 4.C, thus Improving the total yield of refolded active dimeric TGF-(}2. 

5 

O. Generation of heterodimeric, biologically active TGF-& 

[0124] Heterodimeric TGF-ps consisting of two different disulfide-linked polypeptide chains of 112 amino acids each 
can be prepared by subjecting equimolar amounts of the two respective monomers to refolding conditions as described 
fo in example 4.G. Purification and isolation of the dimers is performed according to examples 4.H and 4.1 allowing the 
separation of the heterodimeric form from the homodimers. 

R Peptide mapping and sequence determination of monomeric TGF-&1. TGF-&2 and TGF-33. respectively 
IS TGF-P2: 

[0125] 92 ^g (6.7 nmoles) S-pyridylethylated recombinant TGF-P2 described in example 4.R are dried In an vaccum 
centrifuge and redissolved in 200 pi 5 mM HCI. 200 |il 0.2 M Tris-acetate buffer. pH 7.8, containing 10 mM Zwittergent 
3-12 detergent (Calbiochem Corporation, La Jolla. CA) is added and mixed with the protein solution. The cleavage Is 

20 carried out with 2 pg (dissolved in 50 |il water) endoproteinase Asp-N (from Pseudomonas tragi mutant, Sequence 
Grade, Boeh ringer Mannheim Biochemica. FRG) at 37^*0. After 13 hours, 50 pi 10 % (v/v) TFA are added and the 
mixture is separated by RP-HPLC on a C4 narrow-bore column (Vydac 214TP52, 2.1 x 250 mm) with a linear gradient 
of 6 to 40 % (v/v) acetonitrile in 0.1% TFA/water in 35 min at a flow rate of 0.1 ml/min and UV detection at 216 nm. 
The collected peaks are analysed by plasma desorption mass spectroscopy as described in example 4.S. 

2S [0126] The comparison of the measured molecular mass (in Dattons, D) of the peptides in their protonated form 
(M+H+) with the calculated molecular mass allows the following identification: 



Retention Time (min) 


M+H+ (D) 


Calculated Mass M (D) 


Peptide Sequence 


16.1 


566.1 


564.6 


DFKR 


23.9 


1832.3 


1831.1 


NTINPEASASPCCVSQ 


25.9 


1292.5 


1291.5 


DAAYCFRNVQ 


29.0 


1307.7 


1306.6 


DNCCLRPLY 


31.2 


1320.0 


1318.5 


DTQHSRVLSLY 


32.1 


1421.1 


1419.7 


DNCCLRPLYI 


33.0 


3132.3 


3131.5 


DTQHSRVLSLYNTINPEASASPCCVSQ 


36.9 


3425.3 


3424.9 


DLGWKWHIEPKGYNANFCAGACPYL WSS 


44.5 


3739.5 


3739.5 


DLEPLTILYYIGKTPKIEQLSNMIVKSC KCS 



TGF-pl: 

[0127] 32 ^ig (2.5 nmoles) S-pyridylethylated recombinant TGF-pi (prepared similarly as S-pyridylethylated recom- 
binant TGF-P2) are cleaved with 1 .5 \ig endoproteinase Lys-C using the same procedure as for the digestion of TGF- 
P2 except that the incubation time is 9h and a linear gradient of 12 to 27 % acetonitrile in 90 min is used on a CI 8 
column (Vydac 218TP5205. 2.1 x 50 mm). 



so 



55 



Retention Time (min) 


M+H^ (D) 


Calculated Mass M (D) 


Peptide Sequence 


9.4 


810.6 


808.9 


WIHEPK 


13.2 


619.2 


617.7 


DLGWK 


20.4 


1584.6 


1583.7 


ALDTNYCFSSTEK 


34.9 


1613.3 


1611.9 


VEQLSNMIVRSCK 


50.6 


1869.8 


1868.3 


NCCVRQLYIDFRK 


79.8 


2875.2 


2874.3 


<3YHANFCLGPCPYIWSLDTQYSK 


87.1 


4189.1 


4189.0 


VLALYNQHNPGASAAPCCVPQALEPL 








PIVYYVGRKPK 



18 



EP 0 433 225 B1 



(continued) 



Retention Time (min) 


M+H-^ (D) 


Calculated Mass M (D) 


Peptide Sequence 


89.8 


3965.0 


39637 


VLALYNQHNPGASAAPCCVPQALEPL 
PIVYYVGRK 



TGF-P3: 



[0128] 20 ^ig (1 .46 nmoles) S-pyridylethylated recombinant TGF-p3 (prepared similarly as S-pyridylethoxylated re- 
combinant TGF-P2) are digested with 0.4 ^g endoproteinase Asp-N as described tor TGF-P2 except that the incubation 
time is 22.5 hours and the separation is carried out on a CI 8 column (Vydac 218TP5205, 2.1x50 mm) with a linear 
gradient of 16 to 32 % acetonitrile in 80 min. 



IS 



20 



25 



30 



Retention Time (min) 


M+H+ (D) 


Calculated Mass M (D) 


Peptide Sequence 


7.0 


1308.0 


1306.6 


ENCCVRPLY 


8.8 


1381.0 


1379.5 


DTNYCFRNLE 


11.6 


1205.5 


1206.3 


DTTHSTVLGLY 




1252.4 


1250.4 


DTNYCFRNL 


19.4 


1421.5 


1421.5 


DTTHSTVLGLYNT 




1551.2 


1551.9 


DLEPLTILYYVGR 


36.5 


3030.4 


3029.4 


DTTHSTVLGLYNTLNPEASASPC CVPQ 


39.8 


2782.6 


2781.2 


DTNYCFRNLEENCCVRPLYI 


45.6 


3457.3 


3456.0 


DLGWKWVHEPKGYYANFCSGPCPYL RSA 


77.9 


3726.5 


3725.5 


DLEPLTILYYVGRTPKVEQLSNMV VKSCKCS 


82.6 


6736.9 


6736.9 


DTTHSTVLGLYNTLNPEASASPCC 

VPQDLEPLTILYYVGRTPKVEQL 

SNMWKSCKCS 



Q. Structural characterisation ot monomeric TGF-B2 expressed in Saccharamyces cerevisiae 



[0129] An aliquot of the material from example 4.C is further purified by RP-HPLC as described in example 4.D and 
35 the N-termlnal amino acid sequence is determined as described in example 4.F. 
[0130] The amino acid sequence isf^ 



5 10 15 

Ala-Leu-Asp-Ala-Ala-Tyr-X-Phe-Arg-Asn-Val-Gln-Asp-Asn-X- 



20 25 



X-Leu-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Lys-Arg-Asp-Leu-Gly 



50 wherein X denotes an amino acid not positively identified. 

R. Refolding of monomeric TGF-P2 expressed in Saccharamyces cerevisiae and isolation and characterisation of 
dimeric TGF>B2 

55 [0131] Refolding of the monomeric TGF-p2 expressed in Saccharomyces cerevisiae and isolation of dimeric biolog- 
ically active TGF-P2 is performed as described in example 4.G, 4.H and 4.1 respectively 



19 



EP 0 433 225 B1 



S. Molecular mass of dimeric TGF-B2 

[0132] An aliquot of 6 \ig monomeric TGF-P2 and of 20 dimeric biologically active TGF-P2 obtained in example 
4.D and 4. 1, 'respectively is dissolved in 25 % acetic acid, adsorbed on nitrocellulose and analysed on a BIO ION 20 
5 Plasma Desorption Mass Spectrometer (Applied Biosystems, Uppsala, Sweden). The molecular masses determined 
are 

M= 12738.0 for monomeric TGF-P2 (calculated Mass M= 12719.7) 
10 M= 25'422.0 for dimeric TGF-P2 (calculated Mass M= 25'421 .2 assuming all cysteins as disulfides) 
T. Molecular mass of dimeric TGF-&3 

[0133] Dimeric biologically active TGF-ps is prepared similarly to TGF-P2 described in examples 4.A, 4.B, 4.G, 4.H 
IS and 4.1. The molecular mass of dimeric biologically active TGF-P3 is determined as described in example 4.S. The 
molecular mass found is: 

M = 26'434.0 (calculated Mass M = 25'427.2 assuming all cysteins as disulfides) 
20 Example 5 : In vitro activity test for TGF-B1 . TGF-02 and TGF-63 

A. Cell migration and growth assav 

[0134] The assay is based on the chemotactic activity of TGF-p on fibroblasts (Postlethwaite, A.E. et al. (1987) J. 

25 Exp. Med. 165, 251) and is performed as described by Burk, R. (1973) PNAS 70, 369. 

[0135] The cell migration promoting activity of TGF-pl , TGF-P2 and TGF-ps is assayed by measuring the number 
of normal Balb/c 3T3 fibroblasts which migrate over a culture period of 22 hours into a wounded monolayer culture of 
said cells in serum-free medium (Dulbecco's Modified Eagle Medium, Gibco) containing TGF-pl , TGF-P2 or TGF-P3, 
respectively, as compared to the number of fibroblasts which migrate into a wounded monolayer culture in the absence 

30 of TGF-p. 

[0136] The growth promoting activity of TGF-pi, TGF-p2 and TGF-ps is determined by the stimulatory effect on 
cellular DNA synthesis and cell division. This activity is apparent in said monolayer cultures observed under the light 
microscope after a culture period of 44 hours and is quantified by either 

3S (a) counting the number of cell nuclei, in any given field of view, in cultures of said cells grown in serum-free medium 

containing TGF-pl, TGF-p2 or TGF-p3, respectively, as compared to the number of cell nuclei counted, in any 
given field of view, in cultures grown in the absence of TGF-p, or 

(b) measuring the amount of radio-labelled ^H-thymidine uptake in cultures of said cells grown in serum-free me- 
dium containing TGF-pl, TGF-p2 or TGF-p3, respectively, as compared to the amount of ^H-thymidine uptake in 
40 cultures grown in the absence of TGF-p. 

[0137] In these dose response experiments concentrations of the completely purified TGF-p 1, TGF-P2 and TGF-ps 
proteins (see example 4.K) in the range of 0.1 to 1 000 pg per milliliter of culture medium are sufficient to elicit 50 % of 
the maximal migration and growth promoting response. 

45 

B. Cell growth inhibition assav 

[0138] The colorimetrical assay is based on the inhibitory effect of TGF-p on the growth of human A 375 melanoma 
cells (Brown, T.J. et al. (1987) J. Immunol. 139, 2977). TGF-pi, TGF-p2 and TGF-ps samples are serially diluted (1: 

50 3) in flat bottomed 96-well tissue culture plates (Falcon) containing RPMI-1640 medium (<3ibco) and 5% foetal calf 
serum. Control wells receive medium alone. 1 .5 x 10^ A375 melanoma cells are added to each well. After a 72 hours 
Incubation period at 37°C in 5% COg, the A375 cell monolayers are washed once, fixed and stained with crystalviolet 
for 1 5 minutes. Unbound stain is washed out intensively. The stained cells are lysed with 33 % acetic acid to release 
the stain (which Is confined to the cell nuclei) and the OD was measured at 590 nm with a multiskan-8 Channel Pho- 

55 tometer equipped with an Olivetti M 24 PC to calculate the activity of the test compounds. Since the intensity of staining 
in each well is directly related to the number of nuclei (and therefore to the number of cells), this technique provides 
a colorimetrical assay for measuring the ant i-prol iterative effects of TGF-pi, TGF-p2 and TGF-P3 molecules. 
[0139] Treatment with purified TGF-pi , TGF-P2 and TGF-p3 over a concentration range of 0,001 to 10 nM inhibits 



20 



EP0 433 225 B1 

the growth of A375 melanoma cells. 

Example 6: In vivo activity tests for refolded TGF-B1. TGF-&2 and TGF-&3 

s A. Healing of Partial-Thickness Wounds in Old Mice 

[0140] It is recognised that wound heating processes become impaired with advancing age (Grove, G.L. (1982) Arch. 
Dermatol. Res. 272 : 381) and therefore represent major problems in the field of geriatric medicine. Therefore, the in 
vivo biological effects of the refolded active dimeric TGF-[is on the healing of partial-thickness wounds (formed by 

10 second degree burning) are investigated in a partially deficient or impaired wound repair situation, namely in old ani- 
mals, using the following protocol similar to the one described by Schultz, G.S. etal. (1987) Science 235: 350. 
[0141] Single middermal themial injuries are made on the dorsal thorax of anaesthetized old C57/BL6 mice (aged 
450 days or more), whose backs have been previously shaved and depilitated with a commercial cream-type hair 
remover, by a single 10 second application of a brass template (1 x 1 cm. 8 gm) which has been equilibrated at 80°C 

IS in a water bath. The resulting blister is surgically removed and the burns are treated daily, for 5 days, with a topical 
application of 25 |il sterile vehicle buffer solution (consisting of 0.8 % wA/ Hydroxypropyl cellulose in a solution of 10 
mM Histidine, 140 mM NaCI, pH 7.4) containing various amounts (500 ng, 100 ng or 10 ng) of the refolded active 
dimeric TGF-p form, or with buffer solution alone, or are left untreated. All topically applied materials are sterile, endo- 
toxin-f ree and pyrogen-free, and all mice are individually caged for the duration of the experiment. Each experimental 

20 group consists of 5 animals. 

[0142] After 5 days of treatment with TGF-p, the mice are anaesthetized, the blisters (if present) are surgically re- 
moved from the burns, and the burns are photographed. Areas of burns that have regenerated epithelium are outlined 
onto uniform thickness transparent overhead projector film and the percentage of each original burn area that has 
healed is calculated by planimetry. Results are also compared with the epithelial regeneration process in young (56-84 

25 day old) C57/BL6 mice with identical middermal burns which are left untreated for the duration of the experiment. 

[01 43] An example of such an experiment using refolded dimeric active TGF-p2 is shown in the following table where 
values shown represent the mean and range of group evaluations. 



Group 


Animals 


TGF-P2 dose per incision (ng) 


%age of original burn area healed on day 6 


1 


Old 


500 


59 ±8 


2 


Old 


100 


55 ±6 


3 


Old 


10 


46±7 


4 


Old 


Buffer Only 


10±9 


5 


Old 


Untreated 


16±6 


6 


Young 


Untreated 


66 ±9 



[0144] The results of the planimetrical analyses shown in the above table demonstrate that topical applicatbn of 
refolded active dimeric TGF-P2 daily for 5 days in a suitable vehicle buffer stimulates and accelerates epithelial regen- 
eration in partial-thickness wounds on old mice in a dose dependant fashion (Groups 1 -3) when compared with vehicle 
buffer only or untreated wounds (Groups 4 & 5 respectively). Young mice are apparently competent enough to suc- 
cessfully re-epithelialize their wounds in the absence of any topically applied TGF-p <Group 6). Histological analyses 
reveal the extent of the enhanced re-epithelialization process together with a hyperkeratosis of the regenerated epi- 
dermis on Day 6 in the TGF-p-treated wounds. 

B. Healing of Full-Thickness Wounds in Adult Rats 

[0145] The biological effects of refolded active dimeric TGF-ps are also investigated in a second in vivo model of 
wound repair, namely on the healing of full-thickness wounds (formed by surgk:al incisioning) in adult rats, using the 
following protocol similar to the one described by Mustoe. T A. et al. (1 987) Science 237: 1 333. 
[0146] Single, full-thickness 5 cm long linear incisions are made with surgical scissors 1 .5 cm on both sides of the 
dorsal midline of pentobarbitone anaesthetized male Wistar rats (300-350 g) whose backs have been previously shaved 
and depilitated with a commercial cream-type hair remover. In the experimental groups, edges of the left side incisions 
(as viewed with the dorsal side uppermost) receive single topical applications(100 ^J) of a sterile vehicle buffer (con- 
sisting of 0.8 % w/v Hydroxypropyl cellulose in a solution of 1 0 mM Histidine, 1 40 mM NaCI, pH 7.4) containing various 
amounts (2 ^g, 1 pg, 0.1 m9 or 0.01 ng) of a refolded active dimeric TGF-p form. Edges of the contralateral right side 
incisions receive corresponding equal amounts of a placebo control (Bovine Serum Albumin) in the said vehicle buffer 



21 



EP 0 433 225 B1 



and edges of incisions in control animals receive vehicle buffer alone in the left side incisions and no treatment in the 
right side incisions following surgical incisioning. All topically applied materials are sterile, endotoxin^ree. and pyrogen- 
free. Edges of each wound are then coapted with 5 evenly placed, interrupted horizontal mattress sutures of 5-0 Ethilon. 
All animals are caged separately and the wounds are left to heal for varying periods up to and including 21 days post 

5 treatment. After sacrifice the entire dorsal skin is removed from each animal and all subcutaneous fat is carefully 
dissected from the underside of each of the skins using a surgical scalpel. A template consisting of two parallel surgical 
blades (8 mm distance between blades) is then used to excise strips of skin (between sutures on each incision) for 
tensile strength measurements. Samples are taken from one end of each incision for histological analysis. The maxi- 
mum load tolerated by each excised skin sample is measured with a Universal Tensile Strength Machine Model 144501 

10 (Zwick, Ulm, FRG). Measurements are made on 30 mm x 8 mm strips which are secured between hydraulic clamps 
and then stretched to breaking point at a rate 10 mm per minute, with the maximum load recorded on a chart recorder 
Measurements are made on triplicate samples from each wound and experimental groups consisted of 4 animals. 
Breaking strength is not measured on wounds showing evidence of infection or excessive haemorrhaging (less than 
3% of ail wounds). 

IS [0147] An example of such an experiment using refolded dimeric active TGF-P2 is shown in the following table where 
values shown represent the average ratios of tensile strength between TGF-p2>treated wounds and placebo-treated 
wounds at 3 equally spaced ttmepoints over a 21 day day time period. 



Group 


TFG-p2 dose per incision (|ig) 


Ratio of Tensile Strength TGF-p: Placebo Treatment at 






Day 7 


Day 14 


Day 21 


1 


2.00 


1.9:1 


1.7:1 


1.4:1 


2 


1.00 


1.8:1 


1.4:1 


1.3:1 


3 


0.10 


1.4:1 


1.3:1 


1.2:1 


4 


0.01 


1.2:1 


1.1:1 


1.0:1 


5 


None* 


1.0:1 


1.0:1 


1.0:1 



(• ratio of vehicle buffer only v no treatment) 



[0148] The results of the tensile strength measurements shown in the above table demonstrate that a single topical 
application refolded active dimeric TGF-P2 in a suitable vehicle buffer enhances the breaking strength up to 2 fold, and 
accelerates the healing, of full-thickness incisional wounds in adult rats in a dose dependent fashion over a 21 day 
time period (Groups 1 -4) when compared against the control group (Group 5). Histological analyses reveal the marked 
increase influx of mononuclear cells, fibroblasts and collagen production in TGF-p-treated wounds over the 21 day 
period as compared to control wounds. A transient hyperkeratosis is also evident in T-GF-p-treated wounds up to 14 
days after the treatment. 



C. Wound Chamber Implant Model in Adult Rats 



[0149] The biological effects of refolded active dimeric TGF-ps are also investigated in a third in vivo model of wound 
repair, namely on the cellular ingrowth, vascularization and formation of fibrous granulation tissue in and around porous 
chamber implants in adult rats, based on a protocol similar to the one described by Sporn, M.B. etaL, (1983) Science 
219: 1329. 

[0150] Empty rigid polytetrafluoroethylene tubes (Internal and external diameters, 10 and 1 2 mm respectively; length 
32 mm), each perforated by apporixmately 250 regularly spaced holes (diameter 1 mm) and sealed at each end with 
a removable cap of identical material, are gas sterilized and surgically insertedsub-cutaneously, in symmetrrcal fashion, 
through small incisions into the dorsal flanks of pentobarbitone anaesthetized adult Wistar rats (350-400 g). One gas- 
sterilized tissue cage is implanted into each flank and the incisions are closed with single surgical clips (Clay-Adams 
Auto-Clips. 9 mm) which are removed 5 days after surgery. Following surgical insertion the chambers become errcap- 
sulated with fibrous connective tissue although there is a relative absence of cells within the chambers themselves. 
This model provides a sterile, defined and enclosed space within each chamber where various parameters of a wound 
healing response can be quantitated. Animals are used for experimentation 1 4 days after implantation of the chambers, 
after full healing of the surgical incision. 

[0151] At this time daily injections of 100 |j.l sterile vehicle buffer solution (consisting of 0.5 % wA/ Hydroxypropyl 
cellulose in a solution of 10 mM Histidine, 140 mM NaCl, pH 7.4) containing various amounts (1 ^ig, 0.1 ^ig or 0.01 ^g) 
of a refolded, active dimeric TGF-P form are given directly into the left side chambers (as viewed with the dorsal side 
uppermost). Right side chambers receive corresponding equal amounts of a placebo control (Bovine Serum Albumin) 
in the said vehicle buffer. Control animals receive vehicle buffer alone in the lieft side chambers whereas right side 



22 



EP 0 433 225 B1 



chambers remain untreated for the duration of the experiment. Experimental groups consist of 5 animals. Injections 
are made once daily for 5 days and all injected materials are sterile, endotox in-free and pyrogen-free. All animals are 
individually caged for the duration of the experiment and are sacrificed 24 hr after the last series of injections. Chambers 
are then removed from each animal by aseptic technique, and the fibrous tissue from inside each chamber 

s is'wet'weighed- The total serous protein in the chamber fluid is estimated using the method of Lowry et al. , (1951) J. 
Biol. Chem. 193 : 265. Samples of fibrous tissue removed from inside and outside each chamber are prepared for 
histological analysis. Sterility of the chamber contents is checked by incubation of chamber fluid samples on brain/ 
heart infusion plates for 72 hr at 37°C. Measurements are not made on chambers showing evidence of infection or 
rejection (less than 3% of alt chambers). 

10 [0162] An example of such an experiment using refolded dimeric active TGF-P2 is shown in the following table where 
values shown represent the average ratios of measurements obtained for protein in 5 matched pairs of chambers (left 
V right) from each group of animals. 



20 



Group 


TGF-P2 dose per left chamber (jig) 


Ratio of protein in matched chambers (left : right) 


Fibrous Tissue 


Serous Protein 


1 


1.00 


3.0:1 


1.5:1 


2 


0.10 


2.5:1 


1.4:1 


3 


0.01 


2.1:1 


1.31 


4 


None* 


1.0:1 


1.0:1 



* ratio of vehicle buffer only v no treatment 



[01 53] The results of the protein measurements shown in the above table demonstrate that local injection of refolded 
active dimeric TGF-P2 daily for 5 days in a suitable vehicle buffer enhances, up to 3 fold, the accumulation of total 
fibrous tissue, in a dose-dependant manner, in left-sided chambers as compared to the right-sided contralateral cham- 
bers which have received corresponding equal amounts of a placebo protein. A small dose-dependent increase in the 
amount of serous protein in left-sided chambers is also observed following multiple injection with TGF-p2 (Groups 1 -3). 
No differences are apparent between left-sided and right-sided chambers in the Control group (Group 5). 
[0154] On post-mortem biopsy of animals in Groups 1-3 it is consitently observed that the left-sided TGF-P-treated 
chambers are more firmly attached to the surrounding connective tissue of the body wall than the contralateral right- 
sided chambers that have received placebo injections. Furthermore, histological analyses show that the thickness and 
vascularity of the fibrous tissue surrounding the TGF-p-treated chambers is markedly greater than that of the tissue 
surrounding the placebo-treated chambers. Sheets of migrating fibroblasts and rriononuclear cells are also evident 
within the fibrous tissue inside TGF-p-treated chambers. No apparent differences are obsen/ed in either the thickness 
or vascularity of the fibrous tissue surrounding the chambers, nor in the degree of attachment of chambers to the 
connective tissue of the body wall in the control group (Group 4). These results suggest that the diffusion of TGF-p 
from the chamber is responsible for the observed differences in effect. A sterile infiltrate of inflammatory cells, consisting 
predominantly of macrophages, is found in the serous fluid of TGF-p-treated chambers, whereas contralateral placebo- 
treated chamber fluid shows a predominance of polymorphonuclear leukocytes. The contents of all 40 chambers In 
Groups 1-4 shown in the Example are found to be sterile after incubating samples of the chamber contents on brain/ 
heart infusion for 72 hr at 37**C. 



Example 7: Pharmaceutical composition 

4S 

[0155] 



A. Cream 


Ingredients: 


% (v/v) 


Sorbitan monostearate 


2.0 


Polyoxyethylene sorbitan monostearate 


3.0 


Cetyl alcohol 


5.0 


Light liquid paraffin 


8.0 


Isopropyl myristate 


2.0 


Active substance, TGF-p-tike protein 


1.0-1 0-5 



23 



EP 0 433 225 B1 



(continued) 



A. Cream 


Ingredients: 


% (v/v) 


Propylene glycol 


2.0 


Glycerin 


2.0 


Deionised water 


76.0 


Preservatives and other stabilizers 


q.s. 



70 

[0156] Heat the aqueous phase to 55-60°C, dissolve the active substance in it, and disperse the melted lipid phase 
in it by vigorous stirring. Cool to toom temperature and homogenize. 

[0157] In a similar manner a cream comprising 0.01 to 20 ^g/ml, respectively, can be produced. 
[01 58] Of this cream 1 00 ^il/cm^ of wound is applied. 

75 



B. Ointment 


Ingredients: 


% (v/v) 


Sorbitan trioleate 


5.0 


Wax, microcrystalline 


3.0 


Light liquid paraffin 


9.0 


Isopropyl myristate 


10.0 


Lanolin alcohols 


3.0 


Active substance, TGF-p-like protein 


1.0-1 0-5 


Propylene glycol 


2.0 


Glycerin 


2.0 


Magnesium sulphate, hydrous 


0.7 


Deionised water 


65.3 


Preservatives 


q.s. 



[0159] Dissolve the active substance in the aqueous phase, with gentle heating, and disperse the solution in the 
melted lipid phase. Cool to room temperature and homogenize. 

[0160] In a similar manner an ointment comprising 0.01 to 20 ^ig/ml, respectively, can be produced. Of this ointment 
100 p-l/cm^ of wound is applied. 



40 


C. Parenteral Solution 


Ingredients: 






Active Substance. TGF-p-like protein 


0.05 mg/ml 




± Human Serum Albumin 


1 mg/ml 




Arginine or Glycine 


20 mg/ml 


45 


± Carbohydrate 


5-20 mg/ml 




PH 


7 



[0161] The carbohydrate is glucose, mannose, dextran, hydroxyethyl starch or a mixture thereof. 
[0162] The pH is adjusted with phosphate, succinate, amino acids or a mixture thereof. 
so [0163] Vials with 0.05 mg TGF-p-ltke protein/0.5 ml are made and lyophilised. 

Deposition of microorganisms 

[0164] The following microorganisms were deposited at the Deutsche Sammlung von f^ikroorganismen (DSM), 
55 Mascheroder Weg 1 b. D-3300 Braunschweig (FRG): 



24 



EP 0 433 225 B1 



microorganismus 


deposition date 


accession number 


E. coli LC 137/pPLMU.hTGF-pi 
E. coli LC 137/pPLMU.hTGF-p2 
E. coli LC 137/pPLMU.hTGF-p3 
Saccharomvces cerevisiae GRF 18 


November 28,1989 
November 28.1989 
November 28.1989 
March 4, 1986 


DSM 5656 
DSM 5657 
DSM 5658 
DSM 3665 



SEQ ID No. 1 

10 

Sequence Type: Nucleotide with corresponding polypeptide 
Sequence Length: 339 base pairs 
Strandedness: double 
Topology: linear 
'5 Source: human cDNA 

Immediate experimental source: E. coli LC 137/pPLMu.hTGF-pi (DSM 5656) 
Features: from 1 to 336 coding region for TGF-pi 



20 



25 



30 



35 



40 



45 



50 



25 



EP 0 433 225 B1 



GCC CTG GAC 
Ala Leu Asp 



AAC TGC TGC 
Asn Cys Cys 
15 

GAC CTG GGC 
Asp Leu Gly 



ACC AAC TAT 
Thr Asn Tyr 
5 

GTG CGG CAG 
Val Arg Gin 



TGG AAG TGG 
Trp Lys Trp 
30 



TGC TTC AGC 
Cys Phe Ser 



CTG TAC ATT 
Leu Tyr lie 
20 

ATC CAC GAG 
He His Glu 
35 



TCC ACG GAG 
Ser Thr Glu 
10 

GAC TTC CGC 
Asp Phe Arg 
25 

CCC AAG GGC 
Pro Lys Gly 



AAG 39 
Lys 



AAG 78 
Lys 



TAC 117 
Tyr 



CAT GCC AAC TTC TGC CTC GGG CCC TGC CCC TAC ATT TGG 156 
His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr He Trp . 
40 45 50 

AGC CTG GAC ACG CAG TAC AGC AAG GTC CTG GCC CTG TAC 195 
Ser Leu Asp Thr Gin Tyr Ser Lys Val Leu Ala Leu Tyr 
55 60 65 



25 



30 



AAC CAG CAT AAC CCG GGC GCC TCG GCG GCG CCG TGC TGC 234 
Asn Gin His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys 

70 - 75 

GTG CCG CAG GCG CTG GAG CCG CTG CCC ATC GTG TAC TAC 273 
Val Pro Gin Ala Leu Glu Pro Leu Pro He Val Tyr Tyr 
80 85 90 



35 



GTG GGC CGC AAG CCC AAG GTG GAG CAG CTG TCC AAC ATG 
Val Gly Arg Lys Pro Lys Val Glu Gin Leu Ser Asn Met 
95 100 



312 



ATC GTG CGC TCC TGC AAG TGC AGC TGA 339 
He Val Arg Ser Cys Lys Cys Ser 
105 110 



SEQ ID No. 2 

Sequence Type: Nucleotide with corresponding polypeptide 
Sequence Length: 339 base pairs 
Strandedness: double 
Topology: linear 
Source: human cDNA 

Imnnediate experimental source: E. coli LC 137/pPLMu.hTGF-p2 (DSM5657) 
Features: from 1 to 336 coding region for TGF-P2 



£5 



26 



EP 0 433 225 B1 



GCT TTG GAT GCG GCC TAT TGC TTT AGA AAT GTG CAG GAT 
Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gin Asp 

5 10 

AAT TGC TGC CTA CGT CCA CTT TAC ATT GAT TTC AAG AGG 
Asn Cys Cys Leu Arg Pro Leu Tyr He Asp Phe Lys Arg 
15 20 25 

GAT CTA GGG TGG AAA TGG ATA CAC GAA CCC AAA GGG TAC 
Asp Leu Gly Trp Lys Trp He His Glu Pro Lys Giy Tyr 
30 35 

AAT GCC AAC TTC TGT GCT GGA GCA TGC CCG TAT TTA TGG 
Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu Trp 
40 45 50 

AGT TCA GAC ACT CAG CAC AGC AGG GTC CTG AGC TTA TAT 
Ser Ser Asp Thr Gin His Ser Arg Val Leu Ser Leu Tyr 
55 60 65 



AAT ACC ATA AAT CCA GAA GCA TCT GCT TCT CCT TGC TGC 
Asn Thr He Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 

70 75 

GTG TCC CAA GAT TTA GAA CCT CTA ACC ATT CTC TAC TAC 
Val Ser Gin Asp Leu Glu Pro Leu Thr He Leu Tyr Tyr 
80 85 90 



ATT GGC AAA ACA CCC AAG ATT GAA CAG CTT TCT AAT ATG 
He Gly Lys Thr Pro Lys He Glu Gin Leu Ser Asn Met 
95 100 



ATT GTA AAG TCT TGC AAA T-GC AGC TAA 
He Val Lys Ser Cys Lys Cys Ser 
105 110 



SEQ ID No. 3 



Sequence Type: Nucleotide with corresponding polypeptide 
Sequence Length: 339 base pairs 
Strandedness: double 
Topology: linear 
Source: human cDNA 

Immediate experimental source: E. coli LC 137/pPLMu.hTGF-p3 (OSM'6658) 
Features: from 1 to 336 coding region for TGF-p3 



27 



EP 0 433 225 B1 



GCT TTG GAC ACC AAT TAG TGC TTC CGC AAC TTG GAG GAG 39 
Ala Leu Asp Thr Asn Tyr Cys Phe Arg Asn Leu Glu Glu 

5 10 

AAC TGC TGT GTG CGC CCC CTC TAC ATT GAC TTC CGA CAG 78 
Asn Cys Cys Val Arg Pro Leu Tyr lie Asp Phe Arg Gin 
15 20 25 



GAT CTG GGC TGG AAG TGG GTC CAT GAA CCT AAG GGC TAC 117 
Asp Leu Gly Trp Lys Trp Val His Glu Pro Lys Gly Tyr 
30 35 

TAT GCC AAC TTC TGC TCA GGC CCT TGC CCA TAC CTC CGC 156 
Tyr Ala Asn Phe Cys Ser Gly Pro Cys Pro Tyr Leu Arg 
40 45 50 

AGT GCA GAC ACA ACC CAC AGC ACG GTG CTG GGA CTG TAC 195 
Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu Tyr 
55 60 65 

AAC ACT CTG AAC CCT GAA GCA TCT GCC TCG CCT TGC TGC 234 
Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 

70 75 

GTG CCC CAG GAC CTG GAG CCC CTG ACC ATC CTG TAC TAT 27 3 

Val Pro Gin Asp Leu Glu Pro Leu Thr He Leu Tyr Tyr 
80 85 90 



GTT GGG AGG ACC CCC AAA GTG GAG CAG CTC TCC AAC ATG 
Val Gly Arg Thr Pro Lys Val Glu Gin Leu Ser Asn Met 
95 100 



312 



GTG GTG AAG TCT TGT AAA TGT AGC TGA 
Val Val Lys Ser Cys Lys Cys Ser 
105 110 



339 



Claims 



1 . Process for the production of a dimeric, biologically active Transforming Growth Factor type p (TFG-P)-like protein 
or a salt thereof, comprising refolding the denatured monomeric form of said TGF-p-like protein in the presence 
of a solubilizing agent selected from the group consisting of a mild detergent which permits folding of the monomeric 
TGF-beta-ltke protein into the spatial conformation which after dimerization is associated with the biological activity, 
while retaining said monomer in a soluble form, of a lower alkanol, of a lower alkandiol, of a phospholipid, and of 
a mixture ot two or more such agents. 

2. Process according to claim 1 comprising as additional step the production of the monomeric form of said TGF-p- 
like protein by the steps: 

(a) culturing a microbial host comprising a nucleotide sequence encoding the TGF-p-like protein linked in the 
proper reading frame to an expression control sequence such that said protein is expressed, and 



28 



EP0 43d 225 B1 



(b) recovering the TGF-p-like protein in a denatured, monomeric, soluble form. 

3. Process according to clainn 2 wherein the monomeric TGF-p-ltke protein is present as an insoluble aggregate in 
the microbial host cells and wherein the process further comprises the steps of: 

(a) isolating the water-insoluble protein fraction containing the TGF-p-like protein from the host cells and 

(b) solubilizing the insoluble TGF-p-like protein aggregate. 

4. Process according to claim 2 in which the microbial host is a yeast or a bacterium, 

5. Process according to claim 2 in which the nucleotide sequence encodes a protein selected from the group con- 
sisting of the bone morphogenetic proteins and the transforming growth factors p. 

6. Process according to claim 3 in which the insoluble aggregate is solubilized at a pH of about 1 to about 4. 

7. Process according to claim 3 in which the insoluble aggregate is solubilized at a pH of about 2.5. 

8. Process according to claim 3 in which the insoluble aggregate is solubilized with a chaotropic agent. 

9. Process according to claim 8 in which the chaotropic agent is urea or guanidine-HCl. 

10. Process according to claim 9 in which the chaotropic agent has a concentration of about 4 to about 9 M. 

11. Process according to claim 8 in which a detergent is used. 

12. Process according to claim 1 in which the TGF-p-like protein is selected from the group consisting of the bone 
morphogenetic proteins and the transforming growth factors p. 

13. Process according to claim 1 in which the TGF-p-like protein is selected from the group consisting of the bone 
morphogenetic proteins, TGF-P2 and TGF-P3. 

14. Process according to claim 1 in which the TGF-p-like protein is selected from the group consisting of TGF-P2 and 
TGF-P3. 

15. Process according to claim 1 , in which a low molecular weight sulfhydryl/disulfide redox system is present. 

16. Process according to claim 1 in which a low molecular weight sulfhydryl/disulfide redox system is present which 
is selected from the group consisting of glutathione in its oxidized and reduced form, dithiothreitol in its oxidized 
and reduced form, p-mercaptoethanol in its oxidized and reduced form, cystine and its reduced form, and cystamine 
and its reduced form. 

17. Process according to claim 16 in which the sulfhydryl/disulfide redox system is used at a concentration of about 
1 to 100 mM. 

18. Process according to claim 15 in which the sulfhydryl/disulfide redox system is glutathione in its oxidized and 
reduced form at a concentration of about 1 to 10 mM. 

19. Process according to claim 15 in which the sulfhydryl/disulfide redox system is glutathione in its oxidized and 
reduced form at a concentration of about 1 to 10 mM, wherein the molar ratio of the oxidized and the reduced form 
is between 1:1 and 1:2. 

20. Process according to claim 1 in which a protein selected from the group consisting of thioredoxin and disulfidei- 
somerase is present as redox system. 

21. Process according to claim 1 in which a protein selected from the group consisting of thioredoxin and disulfidei- 
somerase is present as redox system at a concentration of about 10 to 1000 pg/ml. 

22. Process according to claim 1 in which thioredoxin is present. 



29 



EP 0 433 225 B1 



23. Process according to claim 1 in which thioredoxin is present at a concentration of about 10 to 1000 \ig/m\. 

24. Process according to claim 1 in which the solubilizing agent is a mild detergent which permits folding of the mon- 
omeric TGF-beta-like protein into the spatial conformation which after dimerization is associated with the biological 

5 activity, while retaining said monomer in a soluble form. 

25. Process according to claim 24 in which the mild detergent is selected from the group consisting of a non-ionic 
detergent, an ionic detergent and a zwitterionlc detergent. 

10 26. Process according to claim 24 in which the detergent is selected from the group consisting of sulfobetaines, 3- 
(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate, 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy- 
1-propanesulfonate, digitonin, cholate and deoxycholate. 

27. Process according to claim 24 in which the detergent is selected from the group consisting of sulfobetaines, 3- 
^5 (3-chlolamidopropyl)dimethylammonio-1-propanesulfonate. 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy- 

1-propanesulf onate, digitonin, cholate and deoxycholate at a concentration of about 1 to 100 mM. 

28. Process according to claim 26 in which the detergent is selected from the group consisting of 3-(3-chlolamidopropyl) 
dimethylammonio-1-propanesulfonate and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propanesul- 

20 fonate. 

29. Process according to claim 26 in which the detergent is selected from the group consisting of 3-(3-chlolamidopropyl) 
dimethylammonio-1-propanesulfonate and 3-(3-chloiamidopropyl)dimethylammonio-2-hydroxy-1-propanesul- 
fonate at a concentration of about 30 mM to 60 mM. 

25 

30. Process according to claim 1 in which the lower alkanol or alkandiol or mixture thereof is used at a concentration 
of about 10 to 50 % per volume. 

31. Process according to claim 1 in which the solubilizing agent is a phospholipid. 

30 

32. Process according to claim 1 in which the pH is about pH to about 6 to about 10. 

33. Process according to claim 1 in which the pH is about 8,0 

35 34. Process according to claim 1 in which the temperature is about 0°C to about ST'^C. 

35. Process according to claim 1 in which the the temperature is about 4''C. 

36. Process according to claim 1 in which oxidation promoting agents containing Cu2+ or Fe^ metal ions are present. 

40 

37. Process according to claim 36 in which the Cu2+ or Fe^ metal ions are at a concentration of about 0.01 to 1 00 |iM. 

38. Process according to claim 1 in which additionally is bubbled through the reaction system. 

^5 39. Process according to claim 1 in which the dimeric protein obtained is purified by chromatography. 

40. Process according to claim 1 in which the monomeric TGF-P is S-sulfonated prior to refolding. 

41. Process according to claim 1 in which the solubilizing agent is 3-(3<hlolamidopropyl)dimethylammonio-l-pro- 
50 panesulfonate at a concentration of about 30 mM to 60 mM and in which glutathione is present in its oxidized and 

reduced form at a concentration of about 1 to 10 mM, wherein the molar ratio of the oxidized and the reduced form 
is 1:1 to 1:2 

42. Process according to claim 1 5 in which the sulfhydryl/disulfide redox system is used at a molar ratio of the oxidized 
S5 and the reduced form between 100:1 and 1:100. 

43. Process according to claim 14 in which the sulfhydryl/disulfide redox system is glutathione in its oxidized and 
reduced form at a molar ratio of the oxidized and the reduced form between 6:1 and 1 :6. 



30 



EP 0 433 225 B1 



Patentanspruche 

1. Verfahren zur Herstellung eines dimeren, biologisch aktiven dem transform ierenden Wachstumsfaktor vom Typ p 
(TGF-P) ahnlichen Proteins oder eines Salzes hiervon, gekennzelchnet durch Ruckfaltung der denaturierten nno- 

5 nomeren Form des TGF-p-ahnlichen Proteins in Gegenwart eines auflosenden Mittels, ausgewahit aus der Grup* 

pe, bestehend aus einem milden Detergenz, das die Faltung des monomeren TGF-p ahnlichen Proteins in die 
raumliche Konformation eriaubt, die nach der Dimerisierung mit der biologischen Aktivitat zusammenhangt, wobei 
dieses Monomer in einer loslichen Form eines niederen Alkanols, eines niederen Alkandiols, eines Phospholipids 
und eines Gemisches aus zwei Oder mehr dieser Mittel gehalten wird. 

10 

2. Verfahren nach Anspruch 1 , das den zusatzlichen Herstellungsschritt der monomeren Form dieses TGF-p ahnli- 
chen Proteins durch die folgenden Stufen umfa6t 

(a) Kultivierung eines mikrobiellen WIrts, der eine Nukleotidsequenz enthalt, welche fOr das TGF-p ahnliche 
Protein im richtigen Leserahmen verbunden mit einer Expressionskontrollsequenz kodiert, so dafJ dieses Pro- 
tein exprimiert wird, und 

(b) Gewinnung des TGF-p ahnlichen Proteins in einer denaturierten, monomeren, loslichen Form. 

3. Verfahren nach Anspruch 2. worin das monomere TGF-p ahnliche Protein als unldsliches Aggregat in den mikro- 
20 biellen Wirtszellen vorkommt und wobei das Verfahren ferner die folgenden Schritte umfaBt 

(a) Isolierung der das TGF-p ahnliche Protein enthaltenden wasserunloslichen Proteinfraktion aus den Wirts- 
zellen und 

(b) Auflosung des unloslichen TGF-p ahnlichen Proteinaggregats. 

25 

4. Verfahren nach Anspruch 2.. worin der mikrobielle Wirt eine Hefe oder ein Bakterium ist. 

5. Verfahren nach Anspruch 2, worin die Nukleotidsequenz fur ein Protein kodiert, das aus der Gruppe ausgewahit 
ist, die besteht aus den knochenmorphogenetischen Proteinen und den transformierenden V\fachstumsfaktoren p. 

30 



6. 


Verfahren 


nach 


Anspruch 


3, 


worin das unlosliche Aggregat bei einem pH von etwa 1 bis etwa 4 aufgelost wird. 


7. 


Verfahren 


nach 


Anspruch 


3, 


worin das unlosliche Aggregat bei einem pH von etwa 2,5 aufgelost wird. 


8. 


Verfahren 


nach 


Anspruch 


3, 


worin das unlosliche Aggregat mit einem chaotropen Mittel aufgelost wird. 


9. 


Verfahren 


nach 


Anspruch 


8, 


worin das chaotrope Mittel Harnstoff oder Guaninidin-HCI ist. 


10. 


Verfahren 


nach 


Anspruch 


9. 


worin das chaotrope Mittel eine Konzentration von etwa 4 M bis etwa 9 M aufweist. 


11. 


Verfahren 


nach 


Anspruch 


8, 


worin ein Detergenz verwendet wird. 


12. Verfahren 


nach 


Anspruch 


1, 


worin das TGF-p ahnliche Protein aus der Gruppe ausgewahtt wird, die besteht aus 



den knochenmorphogenetischen Proteinen und den transformierenden Wachstumsfaktoren p. 

45 

13. Verfahren nach Anspruch 1, worin das TGF-p ahnliche Protein aus der Gruppe ausgewahit ist, die besteht aus 
den knochenmorphogenetischen Proteinen, TGF-p2 und TGF-p3. 

14. Verfahren nach Anspruch 1, worin das TGF-p ahnliche Protein aus der Gruppe ausgewahit ist, die besteht aus 
50 TGF-P2 und TGF-B3. 

15. Verfahren nach Anspruch 1, worin ein niedermolekulares Sulfhydryl/Disulfidredoxsystem anwesend ist. 

16. Verfahren nach Anspruch 1, worin ein niedermolekulares Sulfhydryl/Disulfidredoxsystem anwesend ist, das aus 
55 der Gruppe ausgewahit ist, die besteht aus Glutathion in der oxidierten und reduzierten Form, Dithiothreit in der 

oxidierten und reduzierten Form, p-Mercaptoethanol in der oxidierten und reduzierten Form, Cystin und die redu- 
zierte Form und Cystamin und die reduzierte Form. 



31 



EP 0 433 225 B1 



17. Verfahren nach Anspruch 16, worin das Sulfhydryl/Disulfidredoxsystem bei einer Konzentration von etwa 1 bis 
100 mM verwendet wird. 

18. Verfahren nach Anspruch 1 5, worin das Sulfhydryl/Disulfidredoxsystem Glutathion in der oxidierten und reduzierten 
5 Form bei einer Konzentration von etwa 1 bis 10 mM ist. 

19. Verfahren nach Anspruch 15, worin das Sulfhydryl/Disulfidredoxsystem Glutathion in der oxidierten und reduzierten 
Form in einer Konzentration von etwa 1 bis 10 mM ist. worin das molare Verhattnis der oxidierten und der redu- 
zierten Formzwischen 1:1 und 1:2 liegt. 

20. Verfahren nach Anspruch 1, worin ein Protein als Redoxsystem anwesend ist, das aus der Gruppe ausgewahit 
ist, die besteht aus Thioredoxin und Disulfidisomerase, 

21. Verfahren nach Anspruch 1, worin ein Protein als Redoxsystem in einer Konzentration von 10 bis 1000 ^g/ml 
'5 anwesend ist, das aus der Gruppe ausgewahit ist, die besteht aus Thioredoxin und Disulfidisomerase. 

22. Verfahren nach Anspruch 1 , worin Thioredoxin vorkommt. 

23. Verfahren nach Anspruch 1, worin Thioredoxin in einer Konzentration von etwa 10 bis lOOO^g/ml vorkommt. 

20 

24. Verfahren nach Anspruch 1 , worin das auflosende Mittel ein mildes Delergenz ist. das die Faltung des monomeren 
TGF-p ahnlichen Proteins in die raumliche Konformation eriaubt, die nach der Dimerisierung mit der biologischen 
Aktivitat zusammenhangt, wobei das Monomer in einer loslichen Form gehalten wird. 

25 25. Verfahren nach Anspruch 24, worin das milde Detergenz aus derOruppe ausgewahit ist, die besteht aus einem 
nichtionischen Detergenz. einem ionischen Detergenz und einem zwitterionischen Detergenz. 

26. Verfahren nach Anspruch 24, worin das Detergenz aus der Gruppe ausgewahit Ist, die besteht aus Sulfobetainen, 
3-(3-Cholanamidopropyl)dimethylammonto-1-propansulfonat. 3-(3-Cholanamidopropyl)dimethylammonio-2-hy- 

30 droxy-1-propansulfonat, Digitonin, Cholat und Desoxycholat. 

27. Verfahren nach Anspruch 24, worin das Detergenz aus der Gruppe ausgewahit ist. die besteht aus Sulfobetainen, 
3-(3-Cholanamidopropyl)dimethylammonio-1-propansutfonat, 3-(3-Cholanamidopropyl)dimethylammonio-2-hy- 
droxy-1-propansulfonat, Digitonin, Cholat und Desoxycholat mit einer Konzentration von etwa 1 bis 100 mM. 

3S 

28. Verfahren nach Anspruch 26, worin das Detergenz aus der Gruppe ausgewahit ist, die besteht aus 3-(3-Cho- 
lanamidopropyl)dimethylammonio-1-propansulfonat und 3-(3-Cholanamidopropyl)dimethylammonio-2-hydroxy- 
Vpropansulfonat. 

40 29. Verfahren nach Anspruch 26. worin das Detergenz aus der Gruppe ausgewahit ist, die besteht aus 3-{3-Cho- 
lanamidopropyl)dimethylammonio-1-propansulfonat und 3-(3-Cholanamidopropyl)dimethylammonio-2-hydroxy- 
1-propansulfonat mit einer Konzentration von etwa 30 mM bis 60 mM. 

30. Verfahren nach Anspruch 1 , worin das niedere Alkano! oder Alkandiol oder Gemisch hiervon mit einer Konzentra- 
45 tion von etwa 1 0 bis 50 Volumenprozent verwendet wird. 

31. Verfahren nach Anspruch 1 , worin das auflosende Mittel ein Phospholipid ist. 

32. Verfahren nach Anspruch 1 , worin der pH etwa pH 6 bis etwa pH 10 ist. 

so 

33. Verfahren nach Anspruch 1 , worin der pH etwa 8,0 betragt. 

34. Verfahren nach Anspruch 1, worin die Temperatur etwa 0°C bis etwa 37°C betragt. 

55 35. Verfahren nach Anspruch 1 , worin die Temperatur etwa 4°C betragt. 

36. Verfahren nach Anspruch 1 , worin oxidationsfordernde Mittel. die Cu2+ oder Fe^ Metallionen enthalten anwesend 
sind. 



32 



EP 0 433 225 B1 



37. Verfahren nach Anspruch 36, worin die Cu^* o6er Fe^* Metallionen in einer Konzentration von etwa 0,01 bis 100 
|iM anwesend sind. 

38. Verfahren nach Anspruch 1 , worin zusatzlich Og durch das Reaktionssystem geblasen wird. 

39. Verfahren nach Anspruch 1, worin das erhaltene dimere Protein durch Chromatographie gereinigt wird. 

40. Verfahren nach Anspruch 1 . worin das monomere TGF-p vor dem Ruckfatten S-sulfoniert wird. 

41. Verfahren nach Anspruch 1 , worin das auflosende Mitte! 3-(3-Cholanamidopropyl)dimethylammonio-1-propansul- 
fonat mit einer Konzentration von etwa 30 mM bis 60 nnM ist und worin Glutathion in der oxidierten und reduzierten 
Form In einer Konzentration von etwa 1 bis 10 mM anwesend Ist, worin das molare Verhaltnls der oxidierten und 
reduzierten Form 1:1 bis 1:2 betragt. 

42. Verfahren nach Anspruch 15. worin das Sulfhydryl/Disulfidredoxsystem in einem molaren Verhaltnis der oxidierten 
und reduzierten Form zwischen 100:1 und 1:100 verwendet wird. 

43. Verfahren nach Anspruch 14, worin das Sulfhydryl/Disulfidredoxsystem Glutathion in der oxidierten und reduzierten 
Form In einem molaren Verhaltnls der oxidierten und reduzierten Form zwischen 6:1 und 1:6 ist. 



Revendications 

1. Proced6 de production d'une proteine dimere, bioiogiquement active, de type facteur de croissance transformant 
P (TGF p), ou d'un de ses sels, comprenant le repllage de ia forme monomere d6natur6e de ladltc protdine de 
type TGF-p en presence d'un agent solubilisant choisi dans le groupe constitu6 par un detergent doux qui permet 
le pliage de la prot6lne monomere de type TGF-p dans la conformation spatiale qui. apr6s dim6risation, est as- 
sociee a I'activite biologique, tout en maintenant ledit monomere sous forme soluble, un alcanol inf^rieur, un al- 
canediol infdrieur, un phospholiplde, et un melange de deux ou plusieurs de ces agents. 

2. Proced6 selon la revendication 1 , comprenant, comme etape supplementaire, ia production de la forme monomdre 
de tadlte proteine de type TGF-p par les 6tapes : 

(a) de mise en culture d'un hote microbien comprenant une sequence nucl6otidique codant pour la prot6lne 
de type TGF-p, Ii6e dans le cadre de lecture correct ^ une s6quence de contrfile de I'expression, de sorte que 
ladite prot6ine est exprim6e, et 

(b) de r6cup6ratlon de la prot6ine de type TGF-p sous forme monomere, d6natur6e, soluble. 

3. Proc6d6 selon la revendication 2, dans lequel la proteine monomere de type TGF-p est presents sous forme d'un 
agregat insoluble dans les cellules botes microbtennes et dans lequel le proc6d6 comprend en outre les etapes: 

(a) d'isolement de la fraction prot^ique insoluble dans I'eau contenant la prot6ine de type TGF-p ^ partir des 
cellules botes et 

(b) de solubilisation de ragr6gat insoluble de prot6ine de type TGF-p. 

4. Procede selon la revendication 2, dans lequel.t'hote microbien est une levure ou une bact6rie. 

5. Proced6 selon la revendication 2, dans lequel la sequence nucleotidique code pour une proteine choisie dans le 
groupe constitu6 par les prot6ines morphog6n6tiques de I'os et les tacteurs de croissance transformants p. 

6. Proc6de selon la revendication 3, dans lequel I'agr^gat insoluble est solubilis6 a un pH d'environ 1 ^ environ 4. 

7. Proc^d^ selon la revendication 3, dans lequel I'agr^gat insoluble est solubilisd ^ un pH d'environ 2,5. 

8. Proc6d6 selon la revendication 3, dans lequel I'agr^gat insoluble est solubilis^ avec un agent chaotrope. 

9. Proc6d6 selon la revendication 8, dans lequel I'agent chaotrope est rur6e ou le guanidine-HCI. 



33 



EP 0 433 225 B1 



10 



10. Proc6de selon ta revendication 9, dans lequel I'agent chaotrope a une concentration d'environ 4^ environ 9M. 

11. Proc6d6 selon ta revendication 8, dans tequel un detergent est utilise. 

12. Proc6d6 selon la revendication 1. dans lequel ta prot6ine de type TGF-p est choisie dans le groupe constitu6 par 
tes prot6ines morphog6netiques de I'os et las tacteurs de croissance transformants p. 

13. Proc6de selon la revendication 1, dans lequel ta prot6ine de type TGF-p est choisie dans le groupe constitu6 par 
les prot6ines morphog6n6tiques de I'os, TGF-P2 et TGF-p3. 

14. Proc6de selon la revendication 1, dans lequel ta prot6ine de type TGF-p est choisie dans le groupe constitu6 par 
TGF-p2etTGF-p3 

15. Proc6d6 selon la revendication 1, dans lequel un syst6me redox sutfhydryle/disutfure de laible masse mol6culaire 
^5 est present. 

16. Proc6d6 selon la revendication 1 , dans lequel est present un systdme redox sulthydryte/disulfure de taible masse 
moldculaire qui est choisi dans le groupe constitu^ par te glutathion dans sa forme oxyd^e et r^duite, le dfthiothrditol 
dans sa forme oxyd6e et r6duite, le p-mercapto6thanol dans sa forme oxyd6e et r6duite, la cystine et sa forme 

20 redutte, et la cystamine et sa forme r6duite. 

17. Proc6d6 selon la revendication 16, dans lequel le syst6me redox sulfhydryle/disulfure est utilise ^ une concentra- 
tion d'environ 1 a 100 ml^. 

2S 18. Proc6de selon la revendication 15, dans lequel le systeme redox sulfhydryle/disulfure est le glutathion dans sa 
forme oxyd6e et r^durte a une concentration d'environ 1^10 mM. 

19. Proc6de selon la revendication 15, dans lequel le systeme redox sulfhydryle/disulfure est le glutathion dans sa 
forme oxyd6e et reduite a une concentration d'environ 1 a 10 mM, le rapport molaire de la forme oxydee et de la 

30 forme r6duite 6tant compris entre 1 : 1 et 1 :2. 

20. Proc6d6 selon la revendication 1 , dans lequel une prot6ine choisie dans le groupe constitu6 par la thioredoxine 
et la disulfurisom^rase est pr^sente en tant que systeme redox. 

55 21. Proc6d6 selon la revendication 1, dans lequel une prot6ine choisie dans le groupe constitu6 par la thioredoxine 
et la dlsulfurisom6rase est pr6sente en tant que systeme redox k une concentration d'environ 10 h 1000 ^ig/mt. 

22. Proc6de selon la revendication 1 , dans lequel est presente ta thioredoxine. 

<o 23. Proc6de selon ta revendication 1, dans lequel la thioredoxine est presente a une concentration d'environ 10 a 
1000 fig/ml. 

24. Proc^de selon la revendication 1 , dans lequel I'agent solubitisant est un detergent doux qui permet le pliage de la 
protelne monomere de type TGF-p dans la conformation spatiale qui, apres dim6risation, est associ6e a t'activit6 

45 biologique, tout en maintenant ledit monomere sous forme soluble. 

25. Proc6d6 selon la revendication 24, dans lequel le ddtergent doux est choisi dans le groupe constitu6 par un d6- 
tergent non ionique, un detergent tonique et un detergent zwitt^rionlque. 

50 26. Proced6 selon la revendication 24, dans lequel le detergent est choisi dans le groupe constrtu6 par les sulfobe- 
taines. te 3-(3-chloramidopropyl)dlm6thylammonio-1-propanesulfonate, le 3-(3-chloramidopropyl)dim6thylammo- 
nio-2-hydroxy-1-propanesulfonate, la digitonine, le chotate et le d6soxycholate. 

27. Proc6de selon la revendication 24, dans lequel le detergent est choisi dans le groupe const itu6 par les sulfob6- 
55 tatnes, le 3-(3-ch!oramidopropyl)dlmethylammonio-1-propanesulfonate: le 3-(3-chloramidopropyl)dim6thylammo- 

nio-2-hydroxy-1-propanesulfonate, la digitonine, le cholate et le d6soxycholate a une concentration d'environ 1 a 
100ml\/l. 



34 



EP 0 433 225 B1 



28. Proc6d6 selon la revendication 26, dans lequel !e detergent est choisi dans le groupe const itu6 par le 3-(3-chlo- 
rannidopropyl)dim6thylammonio-1 -propanesulfonate et le 3-(3-chlorannldopropyl)dim6thylamnnonio-2-hydroxy- 
1-propanesulfonate. 

29. Precede selon la revendication 26. dans lequel !e detergent est choisi dans le groupe constitu6 par le 3-(3-chlo- 
ramidopropyl)dim6thylamnnonio-1 -propanesulfonate et le 3-(3-chloramidopropyl)dim6thylammonio-2-hydroxy- 
1 -propanesulfonate k une concentration d'environ 30 mf^ k 60 mM. 

30. Proc6d6 selon la revendication 1 , dans lequel I'alcanol ou I'alcanediol int^rieur ou un melange de ceux-cl est utilise 
a une concentration d'environ 10 a 50% par volume. 

31. Proc6d6 selon la revendication 1, dans lequel I'agent solubilisant est un phospholipide. 

32. Proc6d6 selon la revendication 1 , dans lequel le pH est d'environ 6 6 environ 10. 

33. Proced6 selon la revendication 1 , dans lequel le pH est d'environ 8,0. 

34. Procede selon la revendication 1, dans lequel la temperature est d'environ 0°C k environ 37°C, 

35. Proc6d6 selon la revendication 1, dans lequel la temperature est d'environ 4°C. 

36. Proc6d6 selon la revendication 1 , dans lequel des agents favorisant I'oxydation et contenant des ions m6talliques 
Cu2+ ou Fe3+ sont presents. 

37. Proced6 selon la revendication 36, dans lequel les ions mdtalliques Cu2+ ou Fe^ sont ^ une concentration d'environ 
0,01 ^lOOfiM. 

38. Proc^d^ selon la revendication 1, dans lequel en outre, on fait barboter de I'Og dans le systeme r^actionnel. 

39. Proc6de selon la revendication 1, dans lequel la prot6ine dim6re obtenue est purifi6e par chromatographie. 

40. Proc6d6 selon la revendication 1, dans lequel le TGF-p monomdre est S-sulfon6 avant le repliage. 

41. Proc6d6 selon la revendication 1, dans lequel I'agent solubilisant est le 3-(3-chloramidopropyl)dimethylammonto- 
1 -propanesulfonate k une concentration d'environ 30 k 60 mM et dans lequel le glutathion est pr6sent dans 
sa forme oxyd6e et rdduite a une concentration d'environ 1^10 mM, le rapport molaire de la forme oxyd6e et de 
la forme r6duite 6tant de 1 :1 ^ 1 :2. 

42. Proc6d6 selon la revendication 15, dans lequel le systeme redox sulfhydryle/disulfure est utillsd k un rapport mo- 
laire de la forme oxyd6e et de la forme reduite comprls entre 100:1 et 1 :100. 

43. Proc6d6 selon la revendication 14, dans lequel le syst6me redox sulfhydryle/disulfure est le glutathion dans sa 
forme oxydee et sa forme r6duite a un rapport molaire de la forme oxyd6e et de la forme r6duite compris entre 6: 
1 et 1 :6. 



35 



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

Defective images within tliis document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□TfADED text OR DRAWING 

2^ BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT 

□ REFERENCE(S) or EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.