DK164283B - POLYPEPTIDE WITH CYSTATIN C ACTIVITY, DNA SEQUENCE FOR EXPRESSION OF 3-DES-OH CYSTATIN C OR A MODIFICATION THEREOF, PROCEDURES FOR THE PREPARATION OF 3-DES-HYDROXY-CYSTATINE MELDOMMANE MADRIDME OR MADIFICANE MADRIDME USE OF 3-DES-OH CYSTATIN C OR ITS MODIFICATION FOR THE PREPARATION OF A THERAPEUTIC PREPARATION - Google Patents

Description

DK 164283 B

The present invention relates to a polypeptide having cystatin C activity and a DNA sequence to express 3-des-OH-cystatin C or a modification thereof. Furthermore, the invention relates to a process for the preparation of 3-des-hydroxy-cystatin C or a modification thereof, and to a plasmid and a microorganism for carrying out the process. Finally, the invention relates to the use of 3-des-OH-cystatin C or a modification thereof for the preparation of a therapeutic composition.

10

The present 3-des-OH-cystatin C is a protein with 120 amino acids.

The amino acid sequence is as follows:

Ser-Ser-Pro-Gly-Lys-Pro-Pro-Arg-Leu-Val-Gly-Gly-Pro-Met-Asp-Ala-Ser-Val-Glu-Glu-Glu-Gly-Val-Arg-Arg Ala-Leu-Asp-Phe-Ala-Val-Gly-Glu-Tyr-Asn-Lys-Ala-Ser-Asn-Asp-Met-Tyr-Mis-Ser-Arg-Ala-Leu-Gln-Val-Val- Arg-Ala-Arg-Lys-Gln-Ile-20 Val-Ala-Gly-Val-Asn-Tyr-Phe-Leu-Asp-Val-Glu-Leu-Gly-Arg

Thr-Thr-Cys-Thr-Lys-Thr-Gln-Pro-Asn-Leu-Asp-Asn-Cys-Pro-Phe-His-Asp-Gln-Pro-Mis-Leu-Lys-Arg-Lys-Ala- Phe-Cys-Ser-Phe-Gln-ile-Tyr-Ala-Val-Pro-Trp-Gln-Gly-Thr-Met-Thr-Leu-Ser-Lys-Ser-Thr-Cys-Gln-Asp-Ala 25

Native human cystatin C isolated from urine and purified is known and the amino acid sequence is determined (1) after fragmentation of the polypeptide chain by both cyanogen-bromide treatment and trypsin cleavage. In determination 30, it was found that the amino acid at position 3 (Pro) is hydroxylated to a degree of about 50%, but somewhat varying. Thus, cystatin C consists of two nearly equal and very close components, one of which is hydroxylated at position 3 (Pro) while the other (3-des-OH-35 cystatin) is not. This relationship has so far been of no importance and therefore cystatin C has been used and studied in the known form.

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2

According to the present invention, the two components have been separated and, surprisingly, it has been found that the biological effect is associated solely with the component 3-des-OH-cystatin C.

5

As can be seen in the literature, cystatin C is an effective inhibitor of cysteine proteinases, i.a. papain and cath-epsin B.

Thus, cystatin C in the form of human cystatin C, also called human r-trace, is described by Grubb et al. (1), which indicates the amino acid sequence and describes some biological properties.

Cysteine proteinases participate in the intracellular catabolism of proteins and peptides, in the proteolytic reaction of prohormones, in the extracellular degradation of collagen and in the penetration of normal tissue by malignant cells.

20

Since cystatin C and thus also 3-des-OH-cystatin C inhibits cysteine proteinases that promote cancer growth or metastasis formation, it is a potential anticancer agent.

25

Furthermore, cystatin C and thus also 3-des-OH-cystatin C have been shown to possess antiviral and possibly antibacterial activity. 1 2 3 4 5 6

For the treatment of disc prolapse, cysteine pro 2 teinases, e.g. chymopapain. If these cysteine protein 3 axes diffuse into the cerebrospinal fluid, brain 4 bleeding occurs as a serious side effect associated with the 5

therapeutic treatment of disc prolapse. When cystatin C

6 and thus also 3-des-OH-cystatin C prevents such a side effect, it has therapeutic use for this purpose.

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3

Of particular importance is the activity of cystatin C in the treatment of cerebral prone brain amyloidosis. In certain types of this disease, deposits of a so-called HCHWA amyloid protein (Proc.

5 Natl. Acad. Sci. Vol 83. pp 2974-2978, May 1986 and Biochem. Biophys. Res. Commun. 1986, 136,548-554). This protein has been found to be a variant of cystatin C in that it lacks the first 10 amino acids of the cystatin C sequence while cystatin C s amino acid Leu at position 68, 10 of the HCHWA protein's corresponding position 58 is replaced by the amino acid Gin. It is believed that this variant of cystatin C is associated with and perhaps responsible for the disease. In that case, it is to be expected that 3-des-OH cysteine T will be useful in the treatment of this disease.

15

In the breakdown of collagen in connective tissue, i.a. cysteine proteinases, and therefore 3-des-OH-cystatin C may probably inhibit such degradation of connective tissue.

The amino acid sequence of cystatin C, isolated from human urine, has been determined and has been shown to have a certain homologue! with c-Ha-ras oncogene products.

The invention is based on the use of a novel DNA sequence containing codons encoding 3-des-OH-cystatin C or modifications thereof. The DNA sequence can be a cDNA sequence formed from placental human cells and having the structure of claim 2.

30

Furthermore, the invention relates to modified 3-des-OH-cystatin C, wherein one or more amino acids at positions 5-17 and / or 55-59 are replaced by another amino acid. An example of such a modified 3-des-OH-cystatin C is a derivative wherein Gly at position 11 is changed to Arg.

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4

In the process of the invention for the preparation of 3-des-hydroxy-cystatin C or a modified 3-des-OH-cystatin C, a host organism such as a bacterium, a yeast cell or a mammalian cell line in which a vector is used is used. , such as a plasmid, having the structure suitable for expression of 3-des-OH-cystatin C or a modified 3-des-OH-cystatin C.

The DNA sequence in question is inserted together with signal sequences, promoters and more. into a plasmid that is incorporated into the microorganism, which is then cultured in a manner known per se.

In the method of the invention, a host organism containing a DNA sequence encoding 3-des-OH-cystatin C is grown in a substrate, after which the polypeptide formed is recovered by the culture medium. The preferred host organism is E. coli, but in principle any microorganism can be used, including yeast cells, or the culture can be done by mammalian cells.

The resulting 3-des-OH-cystatin C is recovered by the culture medium in a pure state in a manner known per se, such as by extraction with solvents and purification by chromatography. Affinity chromatography with monoclonal or polyclonal antibodies has proven particularly suitable for isolation of 3-des-OH-cystatin C in a pure state.

Isolation and analysis of cDNA clones was done by screening 30 of recombinant phages. Thus, xgt11 cDNA library from human placenta and liver mRNA is used. Screening has been performed at a density of 50,000 plaques per 130 mm Petri dishes with affinity purified antibody by a method of Young and Davis. Bound antibody was detected with alkaline phosphatase-conjugated antibody (Promega Biotechnology).

5

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λ phages, prepared by the plate lysate method, were isolated by centrifugation in a CsCl gradient. DNA was extracted by standard methodology. A mixed oligonucleotide probe specific for 3-des-OH-cystatin C was hybridized to the EcoRI-reacted product of phage DNA in a Southern blot experiment. Hybridizing cDNA insertion was ligated into EcoRI-linearized pUCC18 plasmid vectors, which were then used to transform E. coli JM83 cells. Plasmids were prepared by alkaline lysis method. DNA sequencing of inserts, subcloned into M13mp8, was performed using a modified dideoxy chain terminator technique.

The invention will now be described by way of example, in which: FIG. Figure 1 illustrates a sequencing strategy for human 3-des-OH cysteine C cDNA inserts; 2 shows the plasmid pUC18 / C6a; FIG. 3 shows the plasmids pHD162 and pHD262; 4 shows the plasmid pH0313, and FIG. Figure 5 is a diagram showing growth curves of type 1 herpes simplex wine in the presence of recombinant 3-des-OH cysteine C compared to known antiviral agents. 1 35

Furthermore, the invention is illustrated below by means of some exemplary embodiments.

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EXAMPLE 1

Construction of a cDNA encoding 3-des-OH-cystatin C

Five cDNA library from human cells of liver, prostate and placenta were screened for 3-des-OH-cystatin C coding clones. Ca. 1.2 x 10 5 recombinants of liver cells and 35 x 10 5 recombinants of prostate cells were screened with antibodies and a mixed oligonucleotide probe. The result of this screening was negative. In contrast, screening of 6 x 10 6 placental recombinants yielded nine clones that reacted with the antibody.

Three of these clones, designated C5, C6a and C12, respectively, were used below. Their insertions of 800, 800, and 700 base pairs, respectively, were hybridized specifically by Southern blot experiments to a mixed oligonucleotide constructed from protein sequence data.

By sequencing insertions from clones C6a and C12, subcloned into EcoRI cut M13mp8, it was revealed that the inserts shared the same 3 'sequence containing the polyadenylation signal AATAAA.

The entire sequence of both strands of the clone C6a cDNA insert was determined by sequencing randomly overlapping fragments, subcloned into M13mp8. Data obtained by sequencing cDNA inserts of clones C6a and C12 from the ends revealed that the sequence of C12 inserts 30 starts at position 91 of that of C6a, see FIG. First

The C6a insert contains 777 base pairs, including 77 base pairs of 5 'non-coding sequence, and 262 base pairs of 3' non-coding sequence. The polyadenylation signal AATAAA at position 756-761 is followed by 15 nucleotides. A possible translation initiation in accordance with the sequence found by Kozak is found 6 bp downstream of a stop codon.

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7

Initiation at this position and translation stops at the TAG code at positions 516-518 will result in a 120 amino acid long 3-des-OH-cystatin C with a signal sequence of 26 amino acid residues. The polypeptide formed was found to have higher activity than a corresponding human cysteine C isolated from urine.

As shown in FIG. 1, in human 3-des-OH-cystatin C cDNA inserts of clones C12 and C6a were sequenced from the ends by the chain terminator method. A "shotgun" sequencing analysis technique was used for sequencing both DNA strands of C6a inserts. The horizontal arrows indicate the direction and extent of each sequence analysis. The protein coding region of inserted C6a is framed and the shaded portion shows the region encoding mature protein.

The DNA structure encoding 3-des-OH cystatin C comprises nucleotides derived from amino acid sequence for clone 20 C6a and cDNA encoding human precystatin C. The numbering of the nucleotide sequence begins at the first nucleotide and extending in the direction of 5 'to 3'. The amino acid numbering begins with the first amino acid in the mature protein. The Kozak initiation and polyadenylation signal are emphasized.

EXAMPLE 2

Cytoplasmic expression of methionine extended 3-30 des-OH-cystatin C in E. coli

The plasmid pUC18 / C6a containing the cDNA sequence of 3-des-OH-cystatin C was cut with the restriction enzymes NcoI / HindIII and the plasmid fragment was purified.

A synthetic DNA linker containing the coding sequence for the amino acid methionine as well as the first ca. 40 bases of mature cystatin C were then introduced between NcoI and 8

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Hind the sites (Fig. 2). Instead of the codons present in the 3-des-OH-cystatin C s cDNA, codons were used in the linker which yield high yields of proteins in E. coli. This plasmid was cut with the restriction enzymes 5 ClaI / EcoRI, after which a fragment of ca. 600 bp containing the coding sequence for methionine-extended 3-des-OH-cystatin C was purified.

The plasmid pHD162 containing a synthetic promoter and 10 Shine and Dalgarno sequence was cut with the restriction enzymes Clal / EcoRI and the plasmid fragment was purified. This plasmid fragment was then assembled with the above 600 bp fragment to generate the 3-des-OH-cystatin C expression plasmid pHD262 (encoding methionine cystatin C) (Fig. 3). E. coli MC1061 containing the above expression plasmid was grown as described below.

EXAMPLE 3 20

Periplasmic expression of 3-des-OH-cystatin C in E. coli

The plasmid pUC18 / C6a mentioned in Example 2 was cut with the restriction enzymes NcoI / HindIII and the plasmid fragment was purified. A synthetic DNA linker containing the coding sequence for Outer membrane protein A (OmpA) from E. coli as well as the first ca. 40 bases of mature 3-des-OH-cystatin C were then introduced between the NcoI and 30 HindIII sites. Optimal E. coli codons were used. This plasmid was then cut with the restriction enzymes Clal / EcoRI, after which a fragment of ca. 700 bp containing the coding sequence for the OmpA signal peptide and 3-des-OH-cystatin C were purified.

The above fragment was then introduced into an expression plasmid containing the λ PR promoter, an optimal Shine 35 9

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and Dalgarno sequence, the polylinker region of the plasmid pUCC18, and the temperature-sensitive Cl repressor gene from λ.

The resulting plasmid pHD313 (Fig. 4) was then introduced into E. coli MC1061. The expression of 3-des-OH-cystatin C and the purification of the polypeptide were performed as described above.

Example 4 Periplasmic Expression of 3-Des-OH-Cystatin C in E. coli

Plasmid pUC18 was cut with Apal / EcoRI and a fragment of ca. 680 base pairs were purified and applied to a synthetic Apal / Clal linker containing the coding sequence of the ca. 3 first amino acids of pre-3-des-OH-cystatin C. The fragment was then introduced into the expression plasmid mentioned in Example 3 containing the temperature-inducible λPR promoter.

EXAMPLE 5

Expression of periplasmically modified 3-des-OH cysteine T in E. coli 25

From the expression plasmid pHD313 (Example 3), a HindIII / EcoRI fragment of ca. 700 bp. The fragment was introduced into M13 MP18 and subjected to in vitro mutagenization, so that the coding sequence for cystatin C was changed to contain one or more of the following modifications, Table 1. After in vitro mutagenization, a number of clones were cultured and sequenced. Clones of correct sequence were then cut with Clal / EcoRI, followed by a fragment of ca. 700 bp were isolated and introduced into the expression plasmid pHD313. The resulting plasmids were grown and the product purified as described above.

10

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Table 1 3-des-OH-cystatin C mutants with modified DNA sequence 5 1. Gly11 - * pos. charged, e.g. Arg 2. Gly11 - * neg. charged, e.g. Glu 11 3. Gly -► strongly hydrophobic, e.g. Trp 10 11 11 11 11 4. 4.Gly -> Ala, 5Gly -> Ser 5. Substitution by single amino acids, e.g.

ISLAND

a.) Arg - ♦ Glu g 15 b.) Leu - ♦ Arg, Glu c.) Val10 - Arg, Glu d.) Gly12 - »Arg, Glu 13 e.) Pro - * Arg, Glu, Trp 14 f. Arg - * Arg, Glu 20 6. Substitution of the whole region e.g.

Arg ^ -With "* · ^ - ♦ Gly-Gly-Gly-Gly-Gly-Gly-Gly EXAMPLE 6 25

Preparation of 3-des-OH-cystatin C on a technical scale by portion fermentation E. coli MC1061 pHD 313-1-1, which expresses 3-des-OH-cysteine C with correct N-terminal, is grown on LB petri plate containing ampicillin. A single colony is transferred to a 100 ml shake flask containing LB medium as well as 50 m g / l ampicillin. This is incubated at 30 ° C, 200 rpm for 20 hours.

The fermentation is carried out in 10 L of laboratory fermentor under the following conditions: pH = 7.2, aeration = 1 WM, stirring

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ring = 1000 rpm and temperature = 30 ° C in the first phase.

The medium used consists of yeast extract, casamino acids and various salts.

5 The fermenter is seeded with 100 ml culture from the shake flask and the glucose dosing starts when the biomass concentration is OD525 = 5. The dosage rate should be 3.5 g glucose / l and maintained during the remainder of the process. The formation of 3-des-OH-cystatin C is induced by raising the temperature of the fermentor to 40 ° C in the late growth phase (OD -, - = 50). 3-des-OH-Cystatin C forms 525 nm rapidly and - 2 hours after raising the temperature, the fermentation is terminated by lowering the temperature to 20 ° C and raising the pH to 9.0. The whole fermentation process takes 15 approx. 12 hours.

In the process, final concentrations of 3-des-OH-cystatin C of approx. 1000 mg / l (3-des-OH-cystatin C constitutes about 10% of the total protein in the cell).

20

Extraction I of 3-des-OH-cystatin C from E. coli

Cell suspension from the fermentation is centrifuged. The cells are resuspended in 20-25% w / w sucrose, 0.1M EDTA, 0.2M Tris pH 9.0 and left under stirring for 20 minutes.

The supernatant thereof contains 3-des-OH-cystatin C from the periplasmic phase (70% of total extraction by mechanical homogenization).

30

Extraction II of 3-des-OH-cystatin C from E. coli

Cell suspension from fermentation is adjusted to pH = 10.5 with 5M NaOH. After stirring for 20 minutes, the suspension is centrifuged. The supernatant thereof contains 3-des-OH cysteine C.

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12

Extraction III of 3-des-OH-cystatin C from E. coli

Cell suspension from fermentation is centrifuged and resuspended in 0.5M Tris pH = 10.5. After stirring for 20 minutes, the supernatant is centrifuged. The supernatant thereof contains 3-des-OH-cystatin C.

The extract is dialyzed against 20 mM ethanolamine, pH = 10.0 with a Spectrapor® type dialysis membrane (cut-off 10 3500) and then applied to a Q-Sepharose ion equilibrated in 20 mM ethanolamine, pH = 10.0. Fractions containing 3-des-OH-cystatin C are combined and concentrated on a Diaflo®YM2 type filter. Next, the material is applied to a column of the Bio-Gel® P60 type which is equilibrated in 0.05 M ammonium hydrogen carbonate. Fractions containing 3-des-OH-cystatin C are collected. The yield is typically 50-60% in this isolation procedure.

EXAMPLE 7 20

Construction of a synthetic gene encoding 3-des-OH-cystatin C using optimal E. coli codons

From the literature, it is known that highly expressed E. coli 25 proteins require the use of certain codons for the individual amino acids. Simultaneously with the preparation of cDNA for 3-des-OH-cystatin C, a 3-des-OH-cystatin C gene was synthesized based on the published amino acid sequence containing the codons preferred for highly expressed E. coli proteins. The gene was simultaneously modified in such a way that it allowed a fusion with the signal sequence from either outer membrane protein A from E. coli or the signal sequence from fibria protein K88 / 99 from E. coli. After synthesis, the above gene was cloned stepwise into plasmid pUC18. The gene was then isolated and introduced into the expression plasmid mentioned in Example 3 in combination with one of the above signal sequences. plasmid

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13 was introduced into E. coli MC1061 and grown as described above.

The ompA-3-des-OH cystatin c gene with optimal codons for 5 E. coli has the following structure: CG-ATG-AAA-AAA-ACT-GCT-ATC-GCT-ATC-GCT-GTT-GCT-CTG-GCT -GGT-TTC-GCT-ACT-GTT-GCT-CAG-GCT-TCT-TCT-CCT-GGT-AAA-CCG-10 CCT-CGT-CTG-GTT-GGT-GGT-CCG-ATG-GAC-GCT- TCT-GTT-GAA-GAA-GAA-GGT-GTT — CGT-CGT-GCT-CTG-GAC-TTC-GCT-GTT-GGT-GAA-TAO AAC-AAA-GCT-GTT-AAC-GAC-ATG-TAC -CAC-TCT CGT GCT CTG CAG GTT GTT CGT GCT CGT-AAA CAG ATC GTT GCT GGT GTT AAC TAC TTC CTG GAC GTT GAA -CTG-GGT-CGT-ACC-ACC-TGC-ACC-AAA-ACC-χ 5 CAG-CCG-AAC-CTG-GAC-AAC-TGC-CCG-TTC-CAC-GAC-CAG-CCG-CAC-

CTG-AAA CGT-AAA GCT TTC TGC TCT TTC CAG ATC TAC GCT-GTT CCG TGG CAG GGT ACC A'TG-ACC-CTG TCT AAA TCT ACC TGC CAG GAC GCT TAA TAG

EXAMPLE 8

Preparation and isolation of 3-des-OH-cystatin C 25

Bacterial media containing 3-des-OH cystatin c were centrifuged and bacterial-free medium was concentrated on a Diaflo ™ YM2 filter. The concentrate was dialyzed

TM

to 20 mM ethanolamine, pH = 10.0 in a Spectrapor 1 2 3 4 5 6 dialysis membrane (mole weight of cutting 3500) and then applied 2

TM

3 a Q-Sepharose column, equilibrated in 20 mM ethanolamine, pH = 10.0. Fractions containing 3-des-OH-cystatin C were pooled and concentrated on a Diaflo ™ YM2

TM

5 ter. The material was applied to a Bio-Gel column, equilibrated in 0.05 M ammonium bicarbonate. Fractions containing 3-des-OH-cystatin were combined. In this isolation procedure, the yield of 3-des-OH-cystatin C is typical

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14 50-60%.

EXAMPLE 9

5 Characterization of 3-des-OH-cystatin C

3-des-OH-cystatin C, isolated as described above from a culture of pHD 313-transformed E. coli, was physicochemically characterized for biological activity. The results of the characterization were compared with results from similar analyzes of human cystatin C.

A double-immuno diffusion technique based on Ouchterlony showed that all cystatin C epitopes recognized by a polyclonal anti-serum grown against human cystatin C are also present in 3-des-OH cystatin C. Agarose gel electrophoresis at pH-8.6 and SDS-PAGE under reducing or non-reducing conditions 20 showed that 3-des-OH-cystatin C and human cystatin C are identical in terms of charge and molecular weight. An amino acid analysis showed that the amino acid composition of 3-des-OH-cystatin C is almost identical to the composition of human cystatin C. Automatic sequence determination showed that 3-des-OH-cystatin C has the N-terminal sequence: SSPGKPPRLVGGPMDASVEEEGV-ALDFAVGEYNK , which is identical to the sequence in human cystatin C (cf. ref. 1), and in addition 100% of the proline residue at position 3 of 3-des-OH-cystatin C lacked a hydroxyl group. Amino acid analysis of disulfide-linked polypeptides showed that 3-des-OH-cystatin C has disulfide bridges between Cys ^ -Cys ^ and Cys ^ -Cys ^, i.e. the same as for human cystatin C (cf. ref. 15).

Biological activity of 3-des-OH-cystatin C is determined by titration against concentrated determined papain. 3-des-OH-cystatin C was 83% active, which is higher than the 15 corresponding figures for human cystatin C (55%). The strength of the binding 3-des-OH-cystatin C on the cysteine proteinases papain (EC 3.4.22.2), cathepsin B (EC 3.4.22.1) and dipeptidylpeptidase I (EC 3.4.14.1) was determined. The equilibrium constant for dissociation of 3-des-OH-cystatin C enzyme complex (<0.005 nM, 0.50 nM and 3.5 nM) was within the experimental error limit, identical to the equilibrium constant of human cystatin C enzyme complex (<0.005 nM, 0.27 nM and 3.5 nM). Thus, physicochemical characterization and assays performed by the action of 3-des-OH-cystatin with cysteine proteinases showed that 3-des-OH-cystatin C has full biological activity and is identical to human cystatin C except that the hydroxyl group in position 3 completely missing.

EXAMPLE 10

Comparison of the biological activity of 3-des-OH-cystatin C with cystatin C of human origin 20

The papain-inhibitory capacity of recombinant 3-des-OH-cystatin C was compared to the corresponding activity of human cystatin C. 1 2 3 4 5 6 7 8 9 10 11

Human cystatin C was isolated from urine from patients with 2 mixed glomerular-tubular proteinuria by ultrafilter 3 ring, ion exchange chromatography and gel filtration as described in literature (ref. 2.3) and lyophilized. A total of 5 different preparations of both human cystatin C and 6 3-des-OH-cystatin C were tested for their cysteine protein 7 inhibitory capacity. Cystatin C and 3-des-OH-Cy8 statin C preparations derived from human urine 9, respectively, and prepared by recombinant technique were separately 10

dissolved in 0.15 mol / liter ammonium bicarbonate. Cystatin C

Eleven concentrations of the resulting solutions were determined physically chemically and by immuno-diffusion and quantification of amino acids released by hydrolysis in 6 moles / liter 16

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The HCIJ cysteine proteinase inhibition capacity of these solutions was determined by adding a solution of isolated papain (ref. 4) which is a plant cysteine proteinase which is generally used as a model for cysteine proteinase and then determined residual proteolytic activity of the mixtures formed.

The cysteine proteinase activities of the papain solution and cystatin C-papain mixtures were determined using the substrate Z-Phe-Arg-NHMec (ref. 5). The molar concentration of active enzyme in the papain solutions was determined by the irreversible papain inhibitor E-64 (ref. 6).

The molar inhibitor capacities of the various preparations of cystatin C were calculated as the ratio of the papain inhibitor capacity of the cystatin C solutions, assuming 1 mol of cystatin C to inhibit 1 mol of papain, thereby increasing the molar concentration of cystatin C in the solution. The measurements were measured by physicochemical or immunochemical methods assuming a molecular weight of 13343 for cystatin C. Measured in this way, the molar inhibitory capacity of cystatin C isolated from human urine was obtained to 5-55%, while the inhibitory capacity of several samples. 25 of recombinant 3-des-OH-cystatin C ranged from 83-98%.

It can be seen from this that the biological potency of all investigated preparations of recombinant 3-des-OH-cystatin C substantially exceeds the biological potency of all cystatin C preparations isolated from human biological fluids.

EXAMPLE 11

Therapeutic use of recombinant 3-des-OH-cystatin C 35 as a CNS protective agent in chymopapine treatment of sciatica 17

Immediately prior to chymopapine treatment of patients with sciatica, a molar amount of 3-des-OH-cystatin C, 3 times higher than the molar amount of chymopapine used, was injected into the cerebrospinal fluid. For example, 8 mg of chy-5 mopapine (the active component of currently available drugs: Disease®; Travenol Laboratories Ltd.,

Thetford, UK and Chymodiactin®; Smith Laboratories, Inc., Rosemont, IL, U.S.A.) is to be used for intradiscal injection, 12 mg of 3-des-OH-cystatin C is injected into the cere-10 bridge spinal fluid. The injection preparation could be composed of 5 mg of 3-des-OH-cystatin C per day. ml of sterile physiological saline solution. This will protect against any side effect caused by proteolytic degradation of nerve tissue caused by incorrect injection of chymopapain into the cerebrospinal fluid.

If clinical or radiographic evidence of erroneous chymopapine injection into the cerebrospinal fluid is observed instead or at the time of the intended intradermal chymopapain injection, a molar amount of 3-des-OH cystatin C, which is three times higher than the molar amount of the chymopapain used and it is immediately injected into the cerebrospinal fluid to inhibit the tissue-damaging effect of chymopapain. Immediately after injection of 3-des-OH-cystatin C, the patient is subjected to some movement to ensure a rapid distribution of the injected solution of 3-des-OH-cystatin C with the cerebrospinal fluid on the patient.

EXAMPLE 12

Therapeutic use of 3-des-OH-cystatin C in the treatment of Herpes simplex 35 Compositions containing recombinant 3-des-OH-cystatin C, prepared according to the invention, were compared for antiviral activity with the known antiviral drugs 18

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Cyclovir® and Z-LVG-CNH

In the first series of experiments, paper slices were impregnated with Z-LVG-CNH2 (24 µg per slice) and placed on monolayers of green monkey kidney cells infected with poliovirus 1 or herpes simplex virus type 1 (HSV).

In this screening for antiviral activity, no plaque formation with poliovirus was observed. However, striking 10 inhibition zones occurred around these slices when cells were infected with HSV. It was then investigated whether adding Z-LVG-CNH2 to the culture medium could affect the growth of poliovirus or HSV in cell cultures.

In this case too, HSV replication was blocked (> 99.9% inhibition) while the effect on poliovirus replication was hardly noticeable (see Table 2).

TABLE 2

Anti-viral activities of 3-des-OH-cystatin C and Z-LVG-CNH «20 Δ

Virus Yield (pfu / ml) 3-des-OH- Medium Z-LVG-CNH2 Cysteine with 1% in 1%

Virus Medium tin C DMSO DMSO

Poliovirus a η n 7 type 1 1.2 x 10 ° 1.1 x 10 ° 1.5 x 10 ° 4.8 x 10 HVS type 1 8.8 x 107 2.4 x 103 6.4 x 106 1, 4 x 103 METHOD: Cells on green monkeys (strain GMK-AH1) were infected with poliovirus (Mahoney strain) or HSV-19 type 1 (F strain) with a multiplicity of 1 and 10, respectively). After 2 hours, serum-free medium (MEM, Flow Laboratories) was added to contain 0.10 mM cystatin C or 0.4 mM Z-LVG-CNH2. Z-LVG-CNH2 was dissolved in MEM medium containing 1% dimethyl sulfoxide (DMSO) to keep the peptide derivative in solution. Plaque titrations (pfu: plaque-forming units) were performed on GMK cells as previously described. In all 10 experiments, 3 Petri dishes (Falcon 6 cm diameter) were used in each dilution step. Values determined by counting at least 40 pfu and the values given in the table are from typical and reproducible experiments.

15

FIG. 5 illustrates the growth curves of herpes simplex virus type 1 in the presence of various concentrations of recombinant 3-des-OH-cystatin C, Z-LVG-CNH + and Acyclovir®. In the study, CMK-AH1 cells were seeded at 10 pfu 20 (plaque-forming units) per ml. cell corresponding to 5 x 10 ml for 1 hour at 37 ° C. After washing, a 3 residual infectivity of approx. 10 pfu, and this value thus represents background levels at the beginning of the growth curves. At this time, serum-free medium (MEM) containing various concentrations of the inhibitors was added to the HSV-infected cells followed by culture at 37 ° C for 48 hours. Finally, the cells were frozen and thawed and plaque titrations were performed. The concentration of 3-des-OH-cystatin C required for inhibition of viral replication to the background level was approx. 0.13 mM, while the corresponding concentrations of Z-LVG ~ CNH2 and Acyclovir were 0.44 mM and 0.22 mM, respectively. The anti-HSV potency of recombinant cystatin C and of the peptide derivative, Z-LVG-CNH2, corresponding to a portion of its proteinase binding center, is of the same order of magnitude as that of Acyclovir.

20

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In the experiments described above, 3-des-OH-cystatin C and Z-LVG-CNH2 were added to the medium after the cells were inoculated with virus and then thoroughly washed. Since the inhibitors were not present in the first step, they evidently affect the later stages of HSV replication. This was also shown when Z-LVG-CNH2 (0.40 mM) was mixed with a diluted HSV suspension and was present only during the 2 hour viral adsorption to the cells. In this case, the number of plagues was not reduced. Incidentally, inhibition of 10 HSV replication was not restricted to monkey cells.

15 20 "25 30 35 21

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3. Løfberg H. Grubb A & Brun A (1981) Biomed Res, 298-10306.

4. Lindahl P, ALriksson E, Jornvall H & Birk I (1988) Biochemistry 28: 5074-5082).

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8. Biochem. Biophys. Res. Commun. (1986), 136, 548-554. 1 35

Claims (8)

1. A polypeptide having cystatin C activity, characterized in that it consists of a 3-des-OH-cystatin C recombinant technique or a modification thereof. 2. DNA sequence for expression of 3-des-OH-cystatin C, characterized in that it is a DNA copy or cDNA of the structure: 26 -21 155 GCC COG CTG CTC CTG CTG GCC ATC CTG GCC GIG GCC CTG GCC GIG AGO CCC GCG GCC GGC Ala Pro Leu Leu Leu Leu Ala Ile Leu Ala Val Ala Leu Ala Val Ser Pro Ala Ala Gly -1 20 - 215 ICC AGT CCC GGC AAG CCG CCG CGC CTG GIG GGA GGC CCC ATG GAC GCC AGC GTG GAG GAG Ser Ser Pro Gly List Pro Pro Arg Leu Val Gly Gly Pro With Asp Ala Ser Val GLu Glu 1 20 275 GAG GGT GTG CGG CGT GCA CTG GAC TTT GCC GTC GGC GAG TAC AAC AAA GCC AGC AAC GAC 25 Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn List Ala Ser Asn Asp 40 335 ATG TAC CAC AGC CGC GCG CTG CAG GTG GTG CGC GCC CGC AAG CAG ATC GTA GCT GGG GTG With Tyr His Ser Arg Ala Leu Gin Val Val Arg Ala Arg List Gin Ile Val Ala Gly Val 60 30 395 AAC TAC TTC TTG GAC GTG GAG CTG GGC CGA ACC ACG TGT ACC AAG ACC CAG CCC AAC TIG Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr Thr Pys Thr List Thr Gin Pro Asn .Leu 80 455 GAC AAC TGC CCC TTC CAT GAC CAG CCA CAT CTG AAA AGG AAA GCA TTC TGC TCT TTC CAG 35 Asp Asn Cys Pro Phe His Asp Gin Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gin 100 515 ATC TAC GCT GTC (XT TGG CAG GGC ACA ATG ACC TTG TCG AAA TCC ACC TGT CAG GAC GCC Ile Tyr Ala Val Pro Trp Gin Gly Thr With Thr Leu Ser Lys Ser Thr Cys Gin Asp Ala 120 593 A polypeptide according to claim 1, characterized in that it has the same structure as 3-des-hydroxy-cystatin C with the change that one or more amino acids at positions 5-17 and / or 55-59 are replaced by another amino acid. DNA sequence according to claim 4, characterized in that it has a structure as claimed in claim 2, but with the change that it comprises codons encoding a modified 3-des-OH-cystatin C as claimed in claim third Process for producing 3-des-hydroxy-cysteine C or a modification thereof, characterized in that a host organism containing a DNA sequence as claimed in claims 2 or 4 is grown in a substrate and that it formed 3-dec. -hydroxy-cystatin C or the modification thereof is then recovered from the culture. 5 TAG GGGTnnCTACCGGGOGGCCltJIXJCCTATCAttnxnTATCCAC ^ CCTCXX ^ ^ CXXXXrrCTATrCCX CXXXnGGAC 672 TOTIGGCCOTrGCCTTGGGGAAGGTCIXXXX ^ ^ TGTGCCnXACCAGGAGACAGACAGAGAAGGCAGCAGGCC CCTrrG 751 TTGCTCAGCAAGGGGCTCIXXXXritXXnXX7rTCCTTCTTGCITCTCATAGCCXX3SGlX7nXG5GTGCATACACCCCCACC 10,777 TCCTGCAATAAAATAGTAGCATCOCC Plasmid for introduction into a host organism used for biosynthetic production of 3-des-hydroxy-cystatin C or a modification thereof, characterized in that it contains the DNA structure of claim 2 or 4 as well as a promoter and signal sequence, etc. DK 164283 B Microorganism, characterized in that it contains a plasmid as claimed in claim 6. Use of 3-des-hydroxy-cystatin C or a modification thereof as defined in claims 1 or 3 for the preparation of a therapeutic composition. 10 15 20 25 30 35