JPH0811066B2 - Human uterine plasminogen activator produced by recombinant DNA - Google Patents

Description

The present invention relates to the application of recombinant DNA technology for the production of therapeutic proteins, in particular the production of modified human tissue plasminogen activator (mTPA). Recombinant DN for
A application of technology.

(Prior Art) Rijken et al .; Biochim. Biophys. Acta, 580,14
0 (1979) report partial purification of human tissue plasminogen activator (uTPA) from human uterine tissue. This activator is a single-chain zymogen enzyme that is activated by plasmin in the blood to a 2-chain form that dissolves the fibrin thrombus. Accordingly, TPA is useful as a therapeutic composition to dissolve thrombosis in patients suffering from various vascular diseases, especially myocardial infarction and deep vein thrombosis resulting from thrombosis in or around the heart.

MRNA from cancer cell line (Bowes melanoma) cells
Recombinant removal and use of this mRNA for the production of the cDNA-encoded Bowes TPA is described in Goedde
l) et al. in European Patent Application No. 0093619.

CONSTRUCTION OF THE INVENTION In general, the present invention encodes active human TPA, which comprises
-Encoding DNA molecule means Asn, Ser which encodes the N-linked carbohydrate recognition sequence Asn-X- (Ser or Thr) of a natural DNA molecule.
Alternatively, instead of one of the Thr codons, a codon encoding another amino acid was replaced to prevent the carbohydrate moiety from binding to TPA at a portion of the carbohydrate recognition sequence of TPA.

In a preferred embodiment, the carbohydrate recognition sequence is the carbohydrate recognition sequence closest to the carboxy terminus of TPA,
Or there are two such modified sequences, ie,
Sequences closest and intermediate to the amino terminus of TPA. Preferably, in the modified carbohydrate recognition sequence, the Asn codon is replaced with a non-Asn codon, such as a glutamine codon.

The recombinant DNA molecule of the present invention is carried by a replicable expression vector which, when infected into a host mammalian cell, expresses a DNA sequence encoding active human TPA which is expressed in the host cell. Active human TPA is a deletion of one or more of the TPA-linked N-linked carbohydrate moieties of native human TPA. Modified TP of the present invention
A may be mixed with a pharmaceutically acceptable carrier material to produce a thrombolytic therapeutic composition.

The mTPA of the present invention can be purified from a mammalian cell culture medium by a method applicable to any TPA by first performing hydrophobic affinity chromatography and then performing affinity chromatography for binding mTPA to an anti-TPA antibody. .

 The present invention will be described in detail below based on preferred embodiments.

Synthesis of human uterine TPA cDNA The DNA molecule of the present invention was constructed by site-directed mutagenesis (miutagenesis) of uTPA cDNA. That human uterus
The cDNA encoding TPA was produced and cloned by the following steps: 1) Isolation of total mRNA from human uterine tissue; 2) Enrichment of uTPA mRNA from this total mRNA; 3) cDNA from TPA mRNA Synthesize to obtain 3'cDNA and 5'cDNA sequences; 4) ligate the 5'and 3'cDNA sequences to obtain the complete cDNA sequence in the intermediate plasmid vector.

Isolation of uTPA mRNA mRNA from human uterine tissue was isolated as follows.
About 30 g of human uterine tissue was disrupted using a tissue homogenizer in 40 ml of a guanidine thiocyanate solution containing 4M guanidine thiocyanate, 1M2-mercaptoethanol, 50 mM sodium acetate (pH 5.0) and 1 mM EDTA. Then 1 g of CsCl per ml of homogenate was added and the homogenate was centrifuged at 3000 rpm for 10 minutes. The supernatant, 5
50 mM sodium acetate (pH 5.0), 1 mM EDT in 0 ml centrifuge tube
A, and CsCl cushion containing 1.592 g CsCl / ml solvent
Layered on 15 ml and centrifuged at 45,000 rpm at 20 ° C. for 24 hours. The white band containing RNA was collected, this CsCl solution was diluted 3 times with water, and the RNA was precipitated at −20 ° C.
Then, twice the amount of ethanol was added. The precipitate was collected by centrifugation. The dissolution and precipitation were repeated to sufficiently remove the inclusion of CsCl. About 11 mg of purified RNA was recovered.

Purified RNA was added to a high salt buffer (10 mM Tris (pH 8.0), 0.
Pass through an affinity column containing 1 ml oligo-dT cellulose in 5M NaCl and 0.2% sodium dodecyl sulfate), then concentrate by ethanol precipitation to 0.1M Tris HC.
l (pH 7.5) 300 μl, 1 mM EDTA, 1% sodium dodecyl sulfate and 50% formamide were obtained in SW41 tube. The tube was centrifuged at 15 ° C, 35,000 rpm for 16 hours. Separate the fractions and collect the RNs of each fraction.
The size of A was determined by ³ H labeled 4S, 18S and 28S standard markers.

The Furakushiyon containing TPA mRNA, Northern Bro Te queuing (Northern Blotting) a ³² p-labeled probe using connexion identified in the analysis, were pooled together these Furakushiyon. An equal volume of 0.4 MN was added to the gradient to recover the mRNA.
AOAc, pH 5 was added to dilute, and 2.5 times amount of ethanol was added to the diluted gradient solution for precipitation. The procedure of dissolution and precipitation was repeated to remove any formamide remaining in the recovered RNA.

Preparation of 3'sequence of uTPA cDNA The first strand of uTPA cDNA was prepared from the isolated mRNA as follows. RNA to 50 mM Tris HCl, pH 8.
3, 50 mM KCl, 8 mM MgCl ₂ , 1 mM each dATP, dCTP, dGTP
And dTTP, 25 μg / ml oligo-dT _12-18 , 50 μ / ml actinomycin D, 30 mM 2-mercaptoethanol, 0.5 mCi
/ ml [α ³² P] dCTP, 50 units RNasin and 40 units AMV
Incubated with reverse transcriptase in a total volume of 50 μl at 37 ° C. for 1 hour. The reaction was terminated by the addition of 20 mM EDTA. Then the reaction mixture was treated with 10 mM Tris-HCl (pH 8.0), 1 m
It was equilibrated with M EDTA and 0.1% sodium dodecyl sulfate and applied to a Sephadex G-100 column pre-chromatographed with 50 μg E. coli tRNA.

Fractions containing the m-RNA-cDNA hybrid were pooled and ethanol was added to precipitate. The recovered hybrid was treated with 0.5N NaOH at 37 ° C. for several hours, neutralized with glacial acetic acid, and then precipitated with ethanol.

Nucleotide triphosphates were removed by high salt-ethanol precipitation as follows. DNA first 20-5
It was dissolved in 0 μl of water and an equal volume of 4M ammonium acetate was added. Two times the amount of ethanol was added and left overnight at -70 ° C. The temperature of the solution was returned to room temperature, and a DNA pellet was obtained by centrifugation. This procedure was repeated twice more, and then the pellet was washed once with 70% ethanol to obtain a purified cDNA first strand corresponding to most of the 3'end of the TPA gene.

First strand of cDNA was added to 50 mM HEPES buffer (pH 6.
9), 10 mM MgCl ₂ , 2 mM DTT, 70 mM KCl, 0.5 mM dAT each
CDNA in a total volume of 100 μl containing P, dCTP, dGTP, dTTP
Used for second strand synthesis. 20 units of DNA polymerase-Klenow fragment were added to this mixture at 15 ° C. for 20 hours. DNA was extracted with a 1: 1 (v / v) mixture of phenol and chloroform and ethanol precipitated.

To treat the double-stranded cDNA with S1 nuclease to remove the hairpin structure, 30 mM NaOAc (pH
4.4), 0.3 M NaCl, 4.5 mM ZnCl 2, and 50 units of S1 nuclease at room temperature for 30 minutes. To stop the reaction, EDTA and 1 M Tris HCl (pH 8.0) were added to give final concentrations of 10 mM and 0.1 M, respectively. The DNA was purified by repeated phenol-chloroform extractions and ethanol precipitation at high salt concentrations as described above.

The purified double-stranded cDNA sequence was cloned as follows. First, in a 100 μl reaction solution containing 60 mM cacodylic acid-0.14 M Tris (pH 7.6), 1 mM dithiothreitol, 0.1 mM dCTP, and 0.2 μCi / μl [ ³ H] dCTP in a double-stranded cDNA, oligo- It has a dC tail. 10 mM CoCl ₂ was added to a final concentration of 1 mM and 50 units of terminal deoxytrasferase was added at 15 ° C. for 10 minutes. To stop the reaction, EDTA (10 mM) and sodium dodecyl sulfate (0.1%) were added, and the mixture was diluted 2-fold with water. The reaction mixture was then subjected to agarose gel electrophoresis and c-sized to 500 bp or longer.
DNA was recovered from the gel.

This oligo-dC tailed cDNA was preliminarily linearized with PstI and annealed to oligo-dG tailed pBR322 DNA, so in a total volume of 0.5 ml 15 mM Tris HCl (pH 7.5), 1
It was treated at 65 ° C. for 10 minutes in a solution containing 50 mM NaCl and 1 mM EDTA, and then at 42 ° C. for 2 hours. Annealed DNA
Were stored frozen and treated with pre-CaCl ₂ E. coli (M
C1061) used to transform cells. The mixture of cells and DNA was left on ice for 20 minutes, heat-shocked at 37 ° C for 7 minutes, then incubated with 20 volumes of L-broth for 45 minutes at 37 ° C and containing 15 µg / ml tetracycline. Plated on L-plate.

Recombinant clones were transferred to nitrocellulose filters and one set of filters was placed on tetracycline plates to preserve viable clones. The second set of filters containing the replica recombinant clones was placed on chloramphenicol plates and subjected to plasmid amplification overnight at 37 ° C. The filters were processed to expose the DNA. The pre-hybridization of the filter is 50
% Formamide, 3XSSC, 5X Denhardt's solution, 0.1% sodium dodecyl sulfate, and 100 μg / ml E. coli tRNA were applied at 42 ° C. for 2 hours or more. To this prehybridization solution was added ³² P-labeled probe having TPA cDNA sequence, and D
Hybridized with NA (42 ° C, 18 hours or more). The filter was then washed with 3XSSC for 4 hours, 0.1% SDS at 65 ° C for 30 minutes, and again with 3XSSC for 30 minutes at room temperature. The filter was air dried and exposed to a Kodak XAR-5 X-ray film using an intensifying screen for 16 hours at -70 ° C.

Ten candidate positive clones were then isolated, subcloned and rescreened as described above. Only clones showing a positive signal repeatedly were analyzed by restriction endonuclease cleavage. The clone designated pUPA327 contained the largest cDNA insert (2.15 kb).

Preparation of 5'sequence of uTPA cDNA Since the full-length cDNA clone did not contain the entire coding sequence of uTPA, the following method was used to isolate the missing 5'sequence. MRNA was isolated as above except size selection was not performed. Subsequently, the first cDNA strand was synthesized by the primer extension method shown below. TPA cD in a reaction mixture containing 80% formamide, 10 mM PIPES buffer pH 6.4, 0.4 M NaCl, 0.25 μg 72 bp fragment, and 70 μg mRNA in 100 μl.
A 72 bp PstI fragment of NA (nucleotides 552-624) was annealed to the mRNA. Hybridization was performed by heating the mixed solution at 85 ° C. for 5 minutes and incubating at 50 ° C. for 20 hours. The recovery of DNA-RNA hybrid is
The buffer was diluted 3 times with water, and ethanol precipitation was carried out at -20 ° C overnight. The mRNA dissolution and precipitation operations were repeated again to remove contaminants. The conditions for first strand cDNA synthesis are the same as those used for the 3'sequence, except that oligo-dT was omitted.

Second-strand cDNA is synthesized by DNA polymerase I-
Klenow fragment primer (nucleotide 1-1
5) Synthetic pentadecamer (CTG TGA AGC AAT CAT)
It was done using. 50 mM HEPES buffer (pH = 100 μl)
6.9), 10 mM MgCl ₂ , and 70 mM KCl in 500 ng
At 65 ° C for 5 minutes, 65 ° C to 43 ° C for 15 minutes, and 43 ° C for annealing the pentadecamer of
Treated for 15 minutes and stored the mixture on ice until use. Double-stranded cDNA was synthesized and cloned in substantially the same manner as described above for the 3'sequence.

After plating and screening and subcloning, a clone designated pUPA432 was identified. This clone contained the missing 5'coding sequence and had a 60 bp overlap with clone pUPA372.

Production of full length uTPA cDNA Prior to producing the cDNA clone encoding the full length human uterine TPA gene, the 5'and 3'portions of the gene were sequenced. To this end, clones pUPA432 (5 'coding sequence) and pUPA372 (3' coding sequence) were analyzed in combination with the Maxam-Gilbert chemical degradation method and the Sanger dideoxynucleotide thie termination method. The nucleotide sequence obtained is shown in FIG.

After determining the sequence of the gene, a complete cDNA gene was produced as described in FIG. Plasmid pUPA432
The (5 'sequence) was first digested with a combination of BglI and BglII. 400 bp BglI-BglII from polyacrylamide gel
The fragment was isolated and further partially digested with HaeIII. The resulting BglII-HaeIII fragment (nucleotide
117-387) was purified by electrophoresis on a polyacrylamide gel. Fragment PstI-NarI (nucleotides 321-44
8) was isolated by a combination of PstI and NarI digestions and subsequent gel electrophoresis. Add this fragment to Ha
Digested with eIII to generate a 61 bp HaeIII-NarI fragment. BglII-HaeIII fragment and HaeIII-NarI
Both fragments were cloned into pBR322 linearized with a combination of BamHI and NarI digests. From the obtained clone (named pUPA-BglII / NarI), BglII-NarI
The fragment (nucleotides 117-448), and the larger BglII-SalI fragment were isolated.

Nucleotide 2 of plasmid pUPA372 was generated by digesting plasmid pUPA372 with BglII, forming blunt ends with DNA polymerase I, adding a SalI linker, and then cloning into E. coli to generate pUPA372-SalI.
The BglII site of 091 was converted into a SalI site. Then pUPA372-
Cleavage of SalI with a double digestion of NarI and SalI yields NarI-S
The alI fragment (nucleotides 448-2091) was obtained.

Next, the GC tail was removed from pUPA432 and a SalI site was introduced to genetically engineer the leader sequence of the uterine TPA cDNA. This SalI site was introduced by isolating the cDNA from the PstI digest, cleaving it with SfaN1 in the 5'untranslated region, forming a blunt end with DNA polymerase I, and
Add linkers and digest with SalI and BglII combination to give B
This was done by cloning into the pBR322 vector linearized by double digestion with amHI and SalI.

This genetically engineered uterine TPA leader, XhoII
The vector was excised with a combination of and SalI digestion.
This SalI-BglII fragment (nucleotides 7-117)
SalI-BglII formed by ligating each fragment of BglII-NarI, NarI-SalI and BglII-SalI as described above.
It was spliced into the fragment (nucleotides 117-2091). The full-length uterine TPA gene with SalI sites at both ends was then cloned into the SalI site of pUC9 vector. See FIG. This pUC9-uTPA plasmid was subsequently used for the production of recombinant DNA encoding mTPA.

Preparation of mTPA DNA In Figure 5, native human uTPA is N-glycosylated at three asparagine residues, 120,187 and 451 of the molecule (these positions are amino terminal next to the carboxy terminus of the leader sequence). It is counted from glycine residues and corresponds to positions 114, 181 and 445 in Fig. 1, respectively. In Fig. 1, the position immediately following the third leader cleavage site is designated as position 1.). The recognition sequence for N-linked glycosylation is Asn-X- (Ser or Thr). To prevent glycosylation at one or more of these positions, the inventors have added the AAC or AAT codon encoding asparagine in the glycosylation recognition sequence of the uTPA cDNA to the CAA codon (encoding glutamine). I adopted the method of replacing. As described in more detail below, these mutant sites (A, B and C in FIG. 5) were created by in vitro site-specific mutagenesis using the following Mismatch primers: (a) at mutant position A In contrast, for TCGGTGACTGTTCTTTGAAGTAAATGTTGT (b) mutant position B, for CAACGCGCTGCTTTGCCAGTTGGTGCA (c) mutant position C, GTAGGCTGACCCTTGCCCAAAGTAGCA mutant A single-stranded DNA was used as a template,
Miutant AB, AC, and ABC were also manufactured respectively. Finally, reconnecting the mutants B and C to
I got BC. Thus all seven possible non- or partially glycosylated human TPA mutants were prepared. The symbols A, B, and C of the miutant indicate that the miutant is the symbol part A.
Meaning to have Gln codon instead of sn codon, otherwise identical to natural DNA: hence mutant A
B has Gln codons at sites A and B. Gln was used for convenience only and other non-Asn codons can be used as well. Further, instead of replacing the Asn codon, the Ser or Thr codon two downstream from this Asn codon may be replaced with another codon, for example, Gln, and similar results can be obtained.

Site-specific mutagenesis In vitro site-specific mutagenesis is performed by modifying the method originally reported by Smith et al., Gene, 8, 81-87, 99-106 (1979) as follows. It was

The mismatched oligonucleotide primer described above was applied to the Applied Biosystems (Ap
plied Biosystems) was used for the synthesis.

As described in Fig. 3, D encodes human uterine TPA.
Use NA (above) for Phage M13mp18 (Pharmacia
ia) or New England Biolabs (New En
gland Biolabs)]. Plasmid pUC9 uTPA was digested with SalI and the fragment containing the uTPA gene was isolated and digested with SalI.
It was inserted into the two-strand form of the 3-mp18 vector. next
Single-stranded phage DNA containing the sense strand of uTPA was isolated and used as a template for oligonucleotide-mediated miteresis. Each mismatched oligonucleotide primer was incubated at 65 ° C for 10 minutes, then at room temperature for 10 minutes at 20 mM.
Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM dithiothreitol, 0.2 pM single-stranded M13-mp1
It was annealed to the template by treatment in a reaction containing 8-TPA DNA and 20 pM 5'phosphate mismatched oligonucleotide. Next, 30 mM Tris-HCl, pH
7.5, 10 mM MgCl ₂ , 2 mM mercaptoethanol, 1 mM each d
An equal volume of buffer PE containing ATP, dCTP, dGTP, dTTP and 1 mM ATP was added to the annealing reaction mixture. 5-10 units
DNA polymerase-Klenow fragment and 3-5 units of T4 ligase were added and the primer was extended by incubating overnight at 16 ° C.

The reaction mixture was diluted and used to transform competent JM105 cells. For the plaques of the phage, ³² P- by the in-situ in situ miutation-oligonucleotide plaque hybridization method.
The desired misutant was screened using labeled mismatched primers. This method ensures that the desired mutant is perfectly complementary to the primer,
Is based on containing one or more mismatched moieties. That is, under appropriate hybridizing conditions, the extent to which a mismatched primer forms a hybrid with a mutant is greater than that of a native form. This difference in hybrid forming ability enables selection of a desired mutant.

Candidate miutants are isolated and identified by restriction endonuclease digestion and DNA sequencing. The desired mutant uTPA gene was isolated by SalI digestion of the replicative form of the appropriate M13mp18-TPA mutant.

As shown in Figure 4B, each isolated mutant uTPA
The gene was inserted into the XhoI cloning site of plasmid pCLH3ax (plasmid pCLH3ax contains pBR322 sequence, 2000 base pairs containing the 5'regulatory sequence of the mouse metallothionein gene and 237 base pairs containing the 3'regulatory sequence of SV40.
with hoI cloning site). Plasmid pCLH3ax
Was prepared from plasmid pCL28 as shown in FIG. 4A: the starting plasmid pCL28 was cut with HindIII and religated by changing the orientation of the metallothionein gene; this new plasmid was cut with BglII and BamHI. We inserted a 237 base pair polyA region of SV40; finally BglI
The I site was converted to an XhoI site by adding a XhoI linker. Each of the isolated plasmids was digested with XhoI.
The mTPA gene was inserted. Each construct containing each mutant DNA is named pCT [Miutant]. Here, [myutant] indicates a site where each mTPA is modified to prevent glycosylation. For example, a construct containing a modification at site A is named pCTA.

The final step in construction is the addition of sequences encoding bovine papillomavirus (BPV), which causes infection of mammalian host cells. Plasmid pB2-2 was digested with the enzymes BamHI and SalI to generate a 7.9 kb fragment containing the BPV genome. This fragment was ligated into pCT [Minutant] which had been digested with the same two enzymes. This final construct (pCAT [Miutant]) contains the 5'metallothionein regulatory signal portion, the modified uTPA gene, the 3'signal portion of SV40 (along with the poly A addition site), and the complete BPV genome. These pCAT [miutant] expression vectors were then used to infect and express mammalian host cells and isolate the modified uTPA protein.

Infection of Mammalian Cells (Transfection) Using a modified method of the infection technique of Wigler et al., Cell, 11, 223 (1977), pCAT [miutant] plasmid DNA was introduced into mouse C127 cells as follows. did.

5-20 μg of pCAT [myutant] DNA was added into 0.5 ml of 240 mM CaCl ₂ containing 10 μg of carrier salmon sperm DNA. Add this solution to an equal volume of 2X HBS (280 mM NaCl, 20 mM Hepe
s and 1.5 mM sodium phosphate; pH 7.1). Calcium phosphate precipitates were allowed to form for 30 minutes at room temperature and 5 × 10 ⁵ C127 cells were plated 24 hours before infection. While calcium phosphate precipitation is occurring,
The cell growth medium was changed. Calcium phosphate precipitate was added to the cells and incubated at 37 ° C for 6-8 hours. DNA is removed and cells are in phosphate buffered saline (PBS), pH 7.
Exposed to 20% glycerol for 1-2 minutes at room temperature. Cells in PBS
And washed with 10% fetal calf serum (MA Biologicals), penicillin / streptomycin, and 10 ml of Dulbecco's modified medium containing 10 mM glutamine (GIBCO). The medium was changed after 24 hours and every 3-4 thereafter. Focuses were detected after 14-21 days and isolated 21 days later by the cloning ring method. This focus was propagated for analysis.

Infected cells were cultivated by conventional means and mTPA was continuously harvested from the culture medium by conventional means. The transformed C127 cells containing pCAT [myutant] produced mTPA at a high rate. BPV
The strong metallothionein promoter regulated production of mTPA in the vector, which effects increased DNA copy number, and the continuous growth characteristics of transformed cells with BPV are:
Aimatsu provides the optimum system for mass production of mTPA.

Extraction and purification of mTPA from culture medium Activity modified TPA produced by transformed mammalian cells
Were recovered from the host cell culture medium. Purification of active mTPA from cell extracts or culture medium generally involves the steps of 1) hydrophobic affinity chromatography and 2) antibody affinity chromatography. This method can be applied to wild-type TPA and other modified TPA. In detail, these steps are performed as follows.

Hydrophobic Affinity Chromatography This step allows binding of mTPA in host cell culture medium to mTPA, such as Trisacryl Blue (LKB), Macrosolve Blue, or CM Affigel Blue (BioRad), in the culture medium. It has the step of complexing with a dye that does not bind to some other protein. (Afigel blue was isolated from human neuroblastoma line in a separate purification of plasminogen activator by (NH ₄ ) ₂ SO ₄ precipitation and p-aminobenzamidine-sepharose chromatography. Used in combination with; Biochim.Biophys.Acta, 704, 450-460,
1980. 3.) The medium was clarified over time and then used without dilution or concentration. 50 ml / l of trisacryl blue matrix (LKB) equilibrated with phosphate buffered saline, 0.01% Tween, pH 7.1 was added. The supernatant was added to Tris acrylic blue matrix (20 ml / mg TPA) (batch binding) and the solution was gently stirred at 4 ° C. for 2 hours. The matrix is then 20 mM phosphate buffer, pH 7.4, 0.15 M NaC.
and washed with 0.01% Tween 80. This TPA-containing substance was then injected into the gel permeation column at a load factor of 50%, and gel permeation was performed at a linear velocity of 85 cm / hour. 0.5M Arginine from Tris Acrylic Blue Matrices, 0.
02M phosphate buffer, 0.15M NaCl, 0.01% Tween, pH7.4
It was eluted with. The recovery of TPA was generally 90-95%.

As mentioned above, matrices other than Tris acrylic blue may be used in this step. One example is CM Afigel Blue (Bio Rad), which binds mTPA equally well and elutes mTPA in a pattern similar to trisacryl blue.

Affinity chromatography The next step in achieving a high degree of purification is affinity chromatography using an anti-TPA antibody immobilized on a solid support column. Either polyclonal or monoclonal anti-TPA antibodies made by conventional means can be used.

The preferred affinity chromatography step is performed as follows. The material eluted from Tris-acryl blue matrix was used for all mT contained in the eluate.
Pass through the monoclonal anti-TPA antibody Sepharose matrix capable of binding PA activity very gently at 4 ° C. Suitable antibodies are commercially available from Bioscott, Ltd of Scottland or American Diagnostica. This antibody is bound to CNBr-activated Sepharose in an amount of 5 mg antibody / ml resin. Antibody matrix is 0.1M Tris, pH 8.
Pre-equilibrated with 0 and 0.01% Tween 80, 0.5M NaCl,
Wash with 0.01% Tween 80, 0.1M Tris, pH 8.0 and elute with 3.0M KSCN, 0.01% Tween 80, 0.1M Tris, pH 8.0. The elution is performed at a linear velocity of 60 cm / hour and a recovery rate higher than 70-80% is obtained. Then add this concentrated mTPA to 0.
Dialyze against 3M Tris HCl, pH 8.0, 0.25M NaCl, and 0.01% Tween 80.

The purified mTPA recovered from the antibody / Sepharose column is
According to SDS PAGE electrophoresis, it has less than 5% of contaminant components. Under reducing conditions, some 2-chain TPA (active form; single chain is the preferred form) was observed. The 2-chain form can be prevented by adding aprotinin to the buffer used in the purification step.

Characterization of Uterine TPA cDNA and Modified uTPA As described above, FIG. 1 shows the nucleotide sequence of the uterine TPA cDNA along with the amino acid sequence of the uterine TPA that was calculated back by the cDNA. The tentative preprosequence is from amino acid -38
Equivalent to 1. In Figure 1, amino acids +1, -3 and-
Each vertical line before 6 indicates a cleavage site. Possible glycosylation sites are shown in the CHO of Asn residues; the 215 site is not actually glycosylated. The plasmin cleavage site (the site where single-chain uTPA is converted to 2-chain uTPA) is indicated by an arrow. The conserved sequence of the active centers of serine proteases is underlined. Figure 1 also shows the uterine TPA cDNA
And the difference in the nucleotide sequence between the recombinant DNA molecule in which various asparagine codons are replaced by glutamine codons according to the present invention. Removal of these specific asparagine codons can reduce the degree of N-linked glycosylation of the encoding TPA molecule.

The effect of partial or non-glycosylation on the half-life of mTPA was investigated by measuring half-lives and comparing their half-lives with the known half-lives of fully glycosylated uTPA. The half-life was measured by using rabbits, collecting blood samples at various times after TPA injection, and examining the activity and amount of TPA present by a fibrin plate and an ELISA assay, respectively. As an example of this comparison, the analysis results of Myutant A and BC are shown below.

Analysis of Miutant A TPA and Miutant BC TPA To investigate whether glycosylation affects the half-life of uTPA in vivo, rabbits were tested as an animal model [Collen et al., J. Phar
m.Exp.Ther., 231: 146-152, 1984].

New Zealand White Rabbits (4-5 lbs), 50-200 μg of modified TPA or native type T via vein around ear
An amount of 50-200 μg of PA was injected. From this rabbit aorta
1 ml blood sample directly into 3.8 ml sodium citrate at time 0 and 30 seconds after injection, 1 minute, 2 minutes, 3 minutes, 4
Samples were collected after a set time of 5 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes and 20 minutes. Samples were centrifuged to obtain cell-free plasma, which was stored frozen until assayed. At least 3 rabbits were injected for each wild type TPA and modified TPA. When plotting the data, each TPA tested showed a similar half-life of 2-3 minutes. However, for a long time (20
Curve, the concentration of modified TPA in plasma was higher than that of wild-type TPA. Therefore, the initial rapid elimination rate was similar for all TPAs tested, but the elimination rate by 20 minutes was lower with the modified TPA. An example thereof is shown in FIG. Table 1 shows Miutant A and BC
This is a summary of the results suggesting that P. has a significantly slower elimination rate than wild-type TPA.

The following experiment was performed to confirm that the modified TPA in blood detected by ELISA assay was biologically active. TPA activity was determined in a fibrin clot lysis assay with ¹²⁵ I-fibrin and an indirect amide substrate lysis assay (amidolyticassay). The results are shown in Table 1 and FIG. 6, showing that Miutant A and BC showed a longer elimination time compared to wild type uterine TPA regardless of the assay method.

The above experiments show that the modified TPA retains biological activity in plasma. Further tests of fibrinolytic activity of miutant were performed by dissolution of the crot in vitro and in vivo. The modified TPA was first tested in vitro on ¹²⁵ I-fibrin blots. Fibrin blots were made from a mixture of 1.5 μCi of ¹²⁵ I fibrin (IBRIN, Amersciam) and 10 μl of thromboplastin in 0.5 ml of normal human pool plasma containing 10 mM CaCl ₂ . Crots were kept in 3 ml plasma for the duration of the experiment. After pre-incubating at 37 ° C for 30 minutes to stabilize the baseline of the radioactivity released, the same amount of wild-type or mutant TPA was added to 0.1-
2 μg protein was added to the ¹²⁵ I-fibrin blot and the incubation was continued at 37 ° C. At various times after the addition of TPA, 50 μl samples were removed and counted for radioactivity in solution. The percent clot dissolution at time t was calculated by adding the counts in solution at time t or adding the total released radioactivity counts (100
% Soluble). Fig. 7 shows wild type TP
The results of in vitro clot lysis comparing the TPA of A, Myutant A, and the TPA of Myutant BC are shown. As can be seen in the graph in Figure 7, for each specific amount, the modification
The fibrinolytic activity of TPA was similar to that of wild-type TPA.

The modified TPA was then tested for in vivo fibrinolytic activity on fibrin clots. The same 0.5 or 1 ml ¹²⁵ I-fibrin blots were made for in vivo clot lysis experiments, as a sensitivity enhancement of the in vivo clot lysis test was observed when using rabbit plasma. The clot was applied to the jugular vein of a 4lb New Zealand white rabbit using a 12 gauge needle. TPA was administered via a vein around the ear and plasma samples were withdrawn each time between 30 seconds and 3 hours through a catheter in the carotid artery. After testing various parameters of TPA infusion, a large dose of 45 μg of TPA followed by a continuous infusion of 110 min at a rate of 0.25 mg / hr was shown to increase fibrinolytic activity of wild type and miutant A in rabbits. It turned out to be the best for comparison. As shown in FIG. 8, it was observed that the fibrinolytic activity of three rabbits injected with TPA of myutant A was higher than that of rabbits injected with the same amount of wild-type TPA. Furthermore, during the infusion, not only the amidolytic activity in plasma samples containing myutant A protein was greater than that of wild-type TPA, but also the amidolytic activity present in the blood 40-60 minutes after the end of the infusion. A TPA was larger than that in the plasma sample of rabbits treated with wild-type TPA (Fig. 9).

The results of these in vivo clot dissolution tests clearly show the long biological half-life of Myutant A,
This leads to an increase in the accumulation of TPA in the plasma during the continuous injection. Similar results were observed for Myutant BC. From both in vitro and in vivo biological assays, two TPA mutants A and BC
It is theorized that it always has a longer excretion time regardless of the assay and retains its biological activity. As mentioned above, hepatic excretion of fully glycosylated TPA from circulating blood is very fast with a biological half-life of approximately 2 minutes (Thromb. Haemostas. 46,658,1981). Therefore, TPA
Is usually not given by injection, but by infusion of a large amount over 3 hours. The long half-life of mTPA allows treatment by injection rather than by infusion, as it requires less to achieve clot lysis.

Use The mTPA of the present invention is used for the therapeutic purpose of dissolving thrombus in a human patient in need of thrombosis treatment, for example, a postoperative patient, a patient who has recently suffered myocardial infarction due to thrombosis, and a patient who is apt to have deep vein thrombosis. mTPA is a pharmaceutically acceptable carrier substance,
For example, by mixing with a salt solution and administering orally, intravenously, etc.,
It is administered by injection into the damaged artery or heart. An exemplary embodiment is shown below.

Example 1 For emergency treatment of thrombosis by bolus administration, 5-10 mg of lyophilized mTPA was mixed with saline and placed in the barrel of a syringe for intravenous administration of large doses of mTPA to patients.

Example 2 For injection administration aiming at immediate dissolution of coronary artery thrombosis,
About 100 mg / hour of lyophilized mTPA was infused intravenously over about 1 hour, and then about 50 mg / hour of intravenous infusion over about 3 hours.

Example 3 For infusion administration aiming at immediate dissolution of coronary thrombosis,
According to the formulation of Example 2, except that about 10 mg of bulk mTPA in saline was intravenously injected prior to infusion.

Example 4 For infusion treatment aiming at gentle dissolution of deep vein thrombosis, freeze-dried mTPA dissolved in physiological saline was intravenously infused at a rate of about 15 mg / hour for about 12 to 24 hours.

 Other embodiments are within the scope of the claims.

[Brief description of drawings]

1A to 1C are a series of nucleotide sequence diagrams showing a nucleotide sequence of a natural human uterine TPA gene including a flanking region, together with an example of a codon replacement portion in a recombinant DNA molecule of the present invention, 2A and 2B are a series of process diagrams showing a method for producing a complete uTPA cDNA sequence, FIG. 3 is a process diagram showing a method for producing a recombinant DNA molecule encoding mTPA, and FIG. And B are a series of process charts showing the method for producing the mammalian expression vector of the present invention, and FIG. 5 shows the human uterus in which the N-glycosylation site and the disulfide binding site and the glycosylation mutant site of the present invention are entered. FIG. 6 is a schematic diagram of TPA, FIG. 6 is a graph for determining the in vivo half-life of uTPA and mTPA myutant A, and FIG. 7 is an in vitro thrombolytic test of uTPA and mTPA myutant A and BC. FIG. 8 is a graph showing data, FIG. 8 is a graph showing thrombolysis test data for investigating the in vivo elimination rate of uTPA and mTPA myutant A and BC, and FIG. 9 is uTPA and mTPA myutant A and BC. 3 is a graph showing indirect amide substrate dissolution test data for investigating the in vivo disappearance rate of

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 38/00 38/46 C07H 21/04 B C12N 5/10 9/64 Z // A61K 35 / 50 7431-4C (C12N 9/64 C12R 1:91) A61K 37/00 37/54 (72) Inventor Vermuri B. Reddy Two John McQueen Circle, 01701, Framingham, Massachusetts, USA (no address) (72) Inventor Jeffrey F. Lemont 02165, West Newton, Fairway Drive, Massachusetts, USA 165 (72) Inventor William Duckowski, Massachusetts, USA 01721, Ashland, Oregon Street 73 (72) Inventor Chard Douglas United States Massachusetts 01772, Edgeborough Road, Southborough, 46 (72) Inventor Edward S. Coul United States Massachusetts 01756, Mendon, Military Street 11 (72) Inventor Richard Dee Purcell Jr. Massachusetts United States State 02158, Newton, Maple Avenue 17 (72) Inventor David Thay Yui Row, USA Massachusetts State 01752, Marlborough, Westerview Drive 12 (56) References Biochemistry, 23 (1984) p. 6191 ~ 6195

Claims (6)

[Claims] 1. A modified active human TPA different from native human TPA, wherein at least one of the plurality of N-glycosylation sites of native human TPA is not glycosylated and at least one is Glycosylated and at least one amino acid residue of Asn-X- (where X represents Ser or Thr) at the non-glycosylated glycosylation site is replaced by another amino acid residue A modified active human TPA that differs from native human TPA in that respect. 2. Native human TPA in that the glycosylation site near the carboxy terminus of native human TPA is glycosylated, but at least one of the glycosylation sites near the amino terminus and near the center is unglycosylated. Modified active human TPA according to claim 1, which is different from. 3. The glycosylation sites near the amino terminus and near the center of natural human TPA are glycosylated,
The modified active human TPA of claim 1, wherein the glycosylation site near the carboxy terminus differs from native human TPA in that it is not glycosylated. 4. A modified active human TPA different from native human TPA, wherein at least one of the plurality of N-glycosylation sites of native human TPA is non-glycosylated and at least one Is glycosylated and at least one amino acid residue of Asn-X- (where X represents Ser or Thr) at the non-glycosylated glycosylation site is replaced by another amino acid residue A method for producing a modified active human TPA which differs from natural human TPA in that: a) an N-linked carbohydrate recognition sequence of a DNA molecule of natural human TPA
The above carbohydrate of TPA, wherein at least one but not all of the Asn, Ser or Thr codons encoding Asn-X- (where X represents Ser or Thr) is replaced with a codon encoding another amino acid. Transforming mammalian cells with an expression vector containing a recombinant DNA molecule that encodes an active human TPA, which differs from the DNA molecule that encodes the native TPA in that it prevents the carbohydrate moiety from binding at the appropriate position in the recognition sequence. B) expressing the TPA by expressing the DNA sequence in the mammalian cell
Culturing in a medium under conditions that give rise to, and c) isolating TPA from said cells or medium. 5. The step of isolating TPA from the medium comprises subjecting the medium to hydrophobic affinity chromatography to obtain TPA.
5. The method according to claim 4, comprising obtaining a composition having an increased specific activity of, and then subjecting the composition to affinity chromatography to bind the TPA to an anti-TPA antibody. 6. A modified active human TPA different from natural human TPA, wherein the modified TPA is not glycosylated at at least one of the plurality of N-glycosylation sites of natural human TPA, At least one is glycosylated and the non-glycosylated glycosylation site
A modified active human different from natural human TPA in that at least one amino acid residue of Asn-X- (where X represents Ser or Thr) is replaced with another amino acid residue. A thrombolytic therapeutic agent comprising TPA together with a pharmaceutically acceptable carrier substance.