JPH03500002A - Novel thrombolytic protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] [Name of invention] Novel thrombolytic protein 〔Technical field〕 " The present invention relates to substances with tissue plasminogen activator-fm (tPA) activity. do. In particular, the invention is based on “recombinant” thrombolytic proteins, genetically engineered cells. A method for obtaining the protein (or proteins) from the protein, and the substance as a thrombolytic agent. Concerning the therapeutic use of.

[Disclosure of the invention]

+1 improvement compared to natural human tPA! Activated thrombus disintegration with aspects of vascular disintegration (These proteins are intended to be soluble → mulberry. This increases increased Fiber X affinity, decreased responsiveness to inhibitors of tPA, faster clot breakdown (rate of lysis → increased? fM fibril lytic activity, decreased or less (also accepted) as a level of fusion: IA hot bath decomposition and/or extended biological half-life. It will appear. The proteins of the invention are also more homogeneous than natural human tPA. It is intended to be easier to prepare in the form of These proteins if needed It is difficult to treat these proteins so that administration by large-scale injections of pharmaceutical compounds containing An improved comprehensive pharmacology profile is contemplated.

In an investigation involving lAIIM and studies of a series of tPA mutants in the buttocks, such The evaluation of the characteristics of a variant is characterized by favorable properties such as those described above. I discovered it. As explained in more detail below, the present invention provides the amino acids except within the mature N-terminus (a series of amino acids observed in natural peptide sequences). acids are deleted and/or substituted with new amino acids) Structurally characterized by an amino acid sequence substantially identical to that of PA provides a novel protein analogue of human tPA, resulting in a newly synthesized peptide region. It is similar to the cryoglue region of human plasminogen. one or more In this example at the above N-linked glycosylation site and/or plasmin cleavage site. The protein variant spans tPA sites 275-276.

The inventive protein contains both naturally occurring human tPA and other modifications of tPA. Improved fusion and fusion profile compared to the expected fusion profile. mutation An example of this is shown in Table 1 below.

The polypeptide backbone of native human tPA consists of four common A#% single-linked glycosylation molecules. It is known that it contains a binding site. Melanoma from white rice salivary mammalian cells These two sites are typically glycosylated in tPA. (j!IJchiΔ$1111? and Aesus). A# Town, 4 is sometimes Although glycosylated, Ja%21@ is typically not glycosylated.

tPA from melanoma Yumeharu mammalian cells (e.g. Bowes cells) is also referred to here as “heavenly”. It is also referred to as ``natural'' or 1 natural 2 human tPA.

As explained above (the present invention provides a new type of E-tPA with tPA-type thrombolytic activity). Contains protein analogs.

As illustrated in Table 1, the protein of the present invention has (1) natural S in its peptide sequence; As5 monochain glycol present in PA; up to three sylation sites; (υtPA) within the 91-amino acid mature N-terminus; (ill) Arg-275 and l Human tP in that it contains certain modifications at the proteolytic cleavage site spanning 1m-276. The structure is different from A. Amino acid numbering is shown in the single letter code sequence in Table 1. It is. For convenience, to retain the normal tPA amino acid numbering (e.g. glycodi the pentape between E-82 and E-85) Gaps within the petid sequence "CEEID" are also included in the numbering. However polypeptide between the structural regions that meet the bond between C-81 and C-95. It must be understood that the bonds will differ in amino acid composition and length. , the non-invention also includes compounds having such mutations.

[Best mode for carrying out the invention]

In one embodiment of the invention, the protein comprises all of the N-1g terminal region of tPA. or partially (spanning amino@1-91 of the native sequence) of plasminogen. A peptide region that is the same or substantially the same as the Kringle region (Kringle-1) of It is characterized by the fact that k is replaced by k. Examples of proteins of the invention In an illustrative example, amino acids 1!1-84 of fil PA are shown at the mature N-terminus of fil. In particular, 82 amino acid sequences have been essentially replaced. Therefore this These proteins have plasminogen clidiglue in the direction from the N-terminus to the C-terminus. 1 protein region! Fusion of the PA serine protease domain - in various embodiments II Other tP containing tPA kringle-1 and tPA kringle-2 regions in Some chimeric proteins characterized by being linked to the A region Ru.

As illustrated in Table 1, the protein variants of the invention also contain N-chained hydrocarbon moieties. It does not contain any glycosylation, or some of it is glycosylated compared to natural human*PA. It probably is. The phrase @partially glycosylated protein is used here. Contains fewer N-chain hydrocarbon moieties than natural human fog PA that is glycosylated to means protein. Loss of this glycosylation or only partial glycosylation is one of the common N-linked glycosylation sites present in 111 natural molecules or It is caused by substitution or deletion of more than one amino acid. one or more embodying such improvements at the N-linked glycosylation sites of The mutant tag of the present invention retains tPA-type thrombolytic activity with strong door fiber lytic activity. Proteins are more easily produced5 in a more homogeneous form than natural tPA, and in many cases was found to have a longer in vivo half-life than native t/A.

The N-linked glycosylation recognition site is currently believed to consist of a tripeptide sequence. It is specifically recognized by appropriate cellular glycosylation enzymes. These tips The lipeptidic sequence is either asparagine-X-threonine or asparagine-X-separate. phosphorus, where X is usually any amino acid. These within the peptide sequence The positions are shown as Ji1, R1 and R1 in Table 1, respectively. glyc; silation Mutation or deletion of one or more amino acids at three positions of the recognition site Glycosylation no longer occurs in the modified sequence. According to the example, one In an embodiment, A#%, 1. and 7 mouths 1.4 are both 61% independently. has been replaced by In the case of double G1m substitutions, the resulting glycoprotein (GE%ntGl town, 4) is a series of 2 or 3 N as in the case of natural tPA. Contains only one such moiety (in Ass1) rather than a chain glycosylation moiety It should be.

Compound AS R Tomi R1 Compound BIB* Bs1-ONSS NGS NR T1-41--Q--5--T1-I QSSNGSNRT 1-12 N5ANGSNRT1-2 NSS QGS NRT 1-i3 NSS NG A　NRTl-3N5SNGS(,lRT　1-14　N5SNGSVRA1- 4 QSS QGS NRT 1-15) isA) IGA NRTl-5Q SSNGS-RT 1-16 N5ANGSNRA1-6 N5S-GSQRT 1-17 N5VNGS) iRTl-7QSS-GS-RT 1-18 ES S) JGVNRVl-8----------1-19TSS NGS) iRT l-9N-QN-5N-T 1-20 TSSTGSNR Tl-10N--N- -N--1-21TSS TGS QRTJ = amino acid or peptide bond; λ lR and Rs independently represent a peptide bond, an amino acid, a dipeptide, or a tripeptide. Translated from the group consisting of: ll-111%-1", and 1--1"= Peptide bond. Furthermore, the mutant of the present invention has position 245 KM or r (shown above) and typically the 70 N-terminus is as shown (SEC ...starts at the N-terminus before: e.g. 5QEIBA-RFRRGAR5ECK, ,,,RGAR5ECJE...and/or most typically (,1R It starts with 5ECK-1.

Those skilled in the art will understand the same A#%44. Similar glycoproteins with monoglycosylation sites , deletion of amino acids at positions 117 and 184 or other 7 Substitution to Minoshun and/or each Gly;sylation recognition site (e.g. SmWH@ and deletion of one or more amino acids at other positions within Smr1ms). loss or substitution and/or one or more at the @x1 position of the tripeptide It will be appreciated that it will be produced by substitution or, more preferably, deletion.

In other embodiments, Ass at positions 117, 184 and 448 is 01%. has been replaced. The resulting mutants are 2 or 2 in the case of natural tPA. Rather than three N-chain hydrocarbon moieties, it should not contain any such moieties. It would be nice. In other embodiments, the potential glycosylation sites are separately modified. and in one presently preferred embodiment, the hl at position 117 is, for example, GL% in other embodiments at position 184, and in still other embodiments at position 448. It has been changed in the position. The present invention provides such non-glycosylated, monoglycosylated , diglycosylation, and triglycosylation variants are also included. of the present invention Three common N-linked glycosylation sequences found in various embodiments, Ht, B An exemplary modification in one or more of t and Bj is shown below in bag 2. .

In one aspect of the invention, the variants optionally include 17g-275 and 7fold g-27 6, the protein cleavage site can be found due to deletion of Artl-275 or other amino acids ( ArgK is preferably replaced with an amino acid other than Lya, Cym or E4m. It has been improved. Th de is for A de σ-275 in various embodiments of the present invention. It is currently a particularly preferred substituted amino acid. Natural tP A Lf) Arg Proteolytic cleavage at −275 produces a “2g” molecule as known in the art. Get what is called. It is characterized by being improved at this cleavage site. The protein of the present invention has a higher cleavage site than the corresponding protein without improved cleavage site. would be more easily produced in a uniform form and perhaps more importantly improved 5°R-27 has a fibrinolytic profile and pharmacodynamic properties. As an alternative to or supplement to the improvement at position 5, the mutants of the invention improve position 276 and/or or No. 277 or in published international application 1FO86101538 It will be improved as well.

Therefore, the present invention provides some novel thrombolytic proteins related to seven tPA. . This group consists of several types of proteins.

For purposes of clarity and convenience, the following nomenclature is used: R1, R1 and RB. ; and multiple part designations indicating improvements of the present invention (in that order) in position 275 (1) The variant is identified by 1Δ” indicates the absence of an amino acid, i.e. the part in question position is occupied by a peptide bond. Substituted amino acids are specifically indicated. Table 1 Compounds with a specially identified 1R'' group are called by our appropriate compound designation. It will be identified by the name that takes into consideration.

-1-one and---=peptide bond Conflict = any 7 mino knowledge j / = J a s, T A de or S- de (any 7 minutes F-A1 @%: Any amino acid or peptide bond excluded W = 5-de IN I or Peptide bond X+ = GJy # # or peptide bond Y = A de11 # I or peptide bond, Z = any 7 min except the or say Shun or peptide bond mt = wild strain, i.e. before mutagenesis Therefore, a variant containing all three common N-linked glycosylation sites is one compound 1 -0'' and is further improved by replacing N with Q in R1 and R3. The mutants included are 1 compound 1-42 or “Q-117, Q-184 mutants”. and is further improved by replacing R-275 with T. The variant is “compound eml-4,7-275” or @Q-117, Q-184, T- 275 variant”.

One genus of proteins is defined as peptides essentially identical to the peptide sequences shown in Table IK. Characterized by sequence.

Phrase 1 used here Characterized by a peptide sequence essentially identical to the peptide sequence in Table 1 1 refers to the specific peptide sequences in Table 1 or at least about 90% and preferably means a peptide sequence that is at least about 95% homologous thereto.

Encoded by a DNA sequence encoding a thrombolytic active derivative of the variant of the invention Also included are peptide sequences in which the DNA sequence is It can hybridize with the DNA sequences of the invention under hybridization conditions. here Phrase 1 “Stringent conditions” used are T, Ma*4*t4a, E, F, Seven reki error clonins by Ftitack and J, 5axkr@ok (Experiment Manual) (Cold Spring Harbor Laboratory 11982) 387-389. but the salt concentration in step 11 (page 388) is ~0.1 and ~5xss c or follow the precautions indicated in step 11 thereof.

Therefore, the proteins of the present invention encode proteins under stringent conditions. The DNA present is a variant of the present invention; as far as possible, or if it is possible to use synonymous codons that reflect the degeneracy of genetic = -do. Vs. gene mutations such as those that retain thrombolytic activity (e.g. or further deletions, substitutions or insertions of amino acids) may also be included. tPAf characterized by various improvements or combinations of improvements as described above) It's an analogue. The invention therefore provides variants embodying the improvements described herein. It also includes a novel N-terminal linkage to the gPA Gringle-1 region. within the joining region and/or (&) before the new N-terminus and/or the joining region. and/or (1) by inclusion of a common N-linked glycosylation site within The inclusion of multiple copies of the N-terminal conjugate region may be improved.

In the second genus, proteins have peptide sequences essentially identical to those in Table 1. characterized by a column in which (1) one or more Jh% one-chain groups; The lycosylation site is optionally absent or otherwise common and/or (6) Arg-275 is optionally missing or a different amino acid (preferably lysine, cysteine or Substituted with K) other than histidine.

In one embodiment of the invention, the protein is at least one mammalian shield protein. contains sugar moieties commonly referred to as "complex hydrocarbons" characteristic of According to the following As exemplified in detail, such complex hydrocarbon glycoproteins are I) by expression of a NA molecule encoding the desired polypeptide sequence in the host cell. will be produced. Suitable mammalian host cells and transformation, culture, amplification, and Methods of cleaning and product production and purification are known in the art. example For example, Gathi%g and 1759 (1985) or Eo-Igy et al., USA See Patent No. 4419.446.

A further aspect of the invention includes a mammalian mammal in which each hydrocarbon moiety comprises mammalian-derived tPA. Contrary to the ``complex hydrocarbon'' substituents characteristic of glycoproteins, insect cell-produced glycoproteins It is a processed form of the primary dolichol single-chain oligosaccharide that is characteristic of protein KW. Contains the Fog PA variant previously defined. Such insect cell-induced glycosylation is For the purpose of simplicity, we will refer to these hydrocarbons as ``1-high-inose'' hydrocarbons for the purpose of this description. As such, there are complex and high mannose hydrocarbons. Main departure High mannose hydrocarbons according to It is characterized by a small number of (at least one occupied N-chain Gly; Contains sylation sites.

Such a variant is determined by I) the expression of the HA sequence encoding the variant in the insect host cell; will be produced by the present. Suitable insect host cells useful in this aspect of the invention include Transformation/transfection, insect cell culture, screening and products Production and purification methods and materials are well known in the art. in that way The tPA produced in mammalian cells is different from the tPA produced so far, and this The variants of this aspect of the invention are characterized by carbohydrate moieties or milk-based glycoproteins. other protein modifiers containing terminal sialic acid or galactose substituents. do not have.

Proteins of the invention that do not contain N-chained hydrocarbon moieties can also be used in combination with desired variants, e.g. For example, mammalian DNA molecules encoding compounds 1-7 to 1-11) in Table 1, in insect yeast or fungal host cells (eukaryotic host cells are currently preferred). ) can also be produced by expressing As previously indicated, this aspect of the invention Transformation/transfection, phagocytosis, screening and and methods and materials for product production and purification that are similarly suitable for mammalian and insect hosts. Cells and, in addition, suitable yeast and saline host cells are also known in the art.

As should be clear from the foregoing, all variants of the invention are naturally fewer or no potential glycosylation sites compared to PA and/or DNs encoding 5 analogs that may also contain deletions or substitutions of Arg-275 pi is produced by recombinant technology using the A sequence. Such a NA sequence is tPA The usual part of the DNA sequence encoding q! i Aberrant mutagenesis and synthesis will be produced by ligation to NA.

The DNA sequence encoding tPA has been cloned and characterized. example For example, D? -%%+fa et al., Nature (London): 214 (198 3) and R, Xts%/aaS et al.'s Molecular Cell Biology 5 ( 7) 1750 (1985). Thrombolytic activity 39891 is ranked 245th It is unique in that it contains a J(#! residue instead of Vag.Natural tPA The f*-coded DNA sequences are Gll, A island, Arg, Sur, 01%, Cys, starts with the song sequence, followed by the amino acid residues of the full-length protein, followed by the standard Typically contains a leader sequence that is processed (i.e. recognized and removed by the host cell) It's coded. Depending on the medium and host cell in which the DNA sequence is expressed, its The protein produced by K is G111 + Ala@Arg at the amino terminus. such that the starting or first three amino acid residues are proteolytically removed. will be further processed. In the latter case, the mature protein is SYQV,... 01. It has an amino terminus consisting of tPA mutant with both ano-termini is thrombolytic activity. In unexploited IM, mature proteins start earlier. It has an amino terminus. For example 5QEIEARFRRGAR5ECX ,,,,,,.

RGAR5ECK, , , 1, and/or (most typically) GAR5E C.K. et al. Variants according to the invention may also be similar to other variants (e.g. retain thrombolytic activity). Allelic variants or other amino acid substitutions or deletions) Mat! , , or Fajtai.

The present invention also encompasses compounds in which position 219 is modified as previously described. Ru. The compound of this embodiment has 7 molecules other than pro (preferably other than Cy&) at position 219. Characterized by the presence of nosan. Such compounds are therefore derived from me2normers. Aas-21 that is not glycosylated in tPA or its recombinant idle product 8 would be susceptible to N-linked glycosylation.

As previously described (1) the DNA sequences encoding the individual variants of the invention are human A normal sequence of DNA encoding gPA or an analog or variant thereof by site-directed mutagenesis and preferably encoding a novel N-Song terminal region. It will be produced by binding to a synthetic NA sequence. Such mutagenesis (1983); and DNA 3: 479-488 (1984), etc. method using single-stranded DNA, and Morixaga et al.

Biotechnology, 636-639 (July 1984) Includes methods used. @PAN-Achieve song end deletion or e.g. asparagus Used according to such methods to convert Gin residues into threonine or glutamine Some exemplary oligonucleotides are shown in Table 4. Of course, non-inventive DNA encoding each protein is injected into appropriately selected oligonucleotides. site-directed mutagenesis and/or one or more It is understood that analogs could be produced by one skilled in the art by combining synthetic NA sequences. Must be. D by conventional methods in clear mammals, mother, bacterial or insect host cell systems. The desired mutant is obtained by expression of NA. The mammalian expression system thus obtained and variants are currently preferred.

The mammalian cell expression vectors described herein are well known to those skilled in the art (and can be synthesized by known techniques). will be accomplished. Bacterial replicons, selection genes, engineered promoters, promoters, etc. The components of the vector may encode variants derived from nature or synthesized by known methods. DNA sequence that is coded (a protein that can control the invention of this DNA sequence in transformed cells) Such a vector containing a motor that is operably coupled to a motor is thus It could easily be made into VJ4. v4 production of such vectors, thereby making them suitable transformation of host cells under conditions that permit expression of the vector-carried DNA. Culture of recombinant cells is thus the usual method for the production of the mutants described here. It consists of

Defined cell lines (including transformed cell lines) are suitable as hosts. −Next explant (containing relatively undifferentiated cells such as hematopoietic stem cells) as well as primary tissues in vitro. Normal diploid cells, which are cell lines derived from culture, are also suitable. selective genetics (as long as the candidate cells are genotypically deleted in the selection gene) The host cell will preferably be an isolated mammalian cell line.

Stable integration and subsequent integration of vector DNA into chromosomal DNA by conventional methods. Cl0 (Chinese hamster) was used for amplification of the integrated vector DNA. -ovarian) cells are currently preferred. Or, vector DNA is bovine papilla. tumor virus gene (Lsaky et al., -ky, 36:391-401 (1984) ) may be contained in whole or in part, and C12 as a stable ebisome element. 7 in cell lines such as mouse cells. Other usable L-929m cells, Sui strain 313, B, 1b-C or NIH mouse %BHIr or Examples include, but are not limited to, Hag hamster cell lines.

Stable transformants are screened for product expression using standard immuno or enzymatic assays. -ning. The presence of DNA encoding the mutant protein is detected by Southern blot analysis. It will be detected by standard methods such as testing.

After introducing the expression vector DNA into suitable host cells such as CO5-1 monkey cells, Transient expression of the DNA encoding the variant during the day activity or immunoassay without selection.

In the case of bacterial expression, the DNA encoding the variant is derived from bacterial expression known in the art. further modified to include a different codon for the expression, and preferably again this Cellular expression, secretion and processing of mature mutant proteins is known in the field. operably readable into the nucleotide sequence that encodes the secretion leader polypeptide; will be combined within the frame. Expressed in mammalian, insect, yeast or bacterial host cells All compounds obtained are recovered, purified and/or purified by known methods. Characterized with respect to physicochemical, biochemical and/or clinical parameters.

These compounds are compatible with erythrina trypsin inhibitor series resins (existing in this field). known to bind to monoclonal antibodies against human tPA) and human tPA. have been observed and therefore affinity chromatography using such reagents will be recovered and/or purified by fees. Additionally, these compounds Hat? I was waiting for J-ffi moromi activity. Namely, 1 the compound of the present invention is present in the presence of fibrin. Effectively activates plasminogen and plasmin chromogen substrate S-2251 caused fibrinolysis as measured by a conventional indirect assay using .

Variants of the invention also include: Australian patent issue AU-,4-55514/86 , EP-i-0155388, EP-A-O152,736, J? PAs530 B533.01EPA8S308534.8 and EPJ0196920. can be derivatized to provide a conjugate such as may be formulated with excipients to provide other pharmaceutically useful compositions as described below. It will be provided.

Unexploited BA can also be prepared by mixing a therapeutically effective amount of said variant with a pharmaceutically suitable parenteral carrier. Also included are compositions for thrombolytic therapy comprising the compound. The prescription is GB86127 81°GB8513358, GB8521704. EPA211592 and G B2176703. Such a composition is It can be used in the same manner as described for human 1PA and does not cause thrombotic cardiovascular problems. humans or other mammals known to be susceptible, such as dogs, cats, and other mammals. It would be beneficial for other animals. This composition is effective against myocardial infarction, deep vein thrombosis and thrombolysis. Treatment of thrombotic conditions such as other indications where clinical therapy may be beneficial and desirable. It is intended that it could be used as a double-edged tool for prevention. exact dose and The method of administration is 1.For example, body weight, gender, food and drink, time of administration, combination of drugs, 1.response. There are various factors that modify the action of drugs, such as susceptibility and severity in particular cases. as determined by the physician depending on the potency and pharmacokinetic profile of the particular compound will be done.

The following examples are included to illustrate embodiments of the invention. These examples are For illustrative purposes only and the present invention is not to be considered limited thereto, the accompanying claims As shown in the range (limited).

The nuclear polyhedrosis virus is autoge2fa Karihoryuuka CAst@Ir”pλa It is an L-1 mutant of calfloras<ea), and is 213-217). Ta. Cell and virus handling are detailed in the literature (P-%segkG, D., et al., supra; Millar, D., W., 5afer, P., and and Miller, L.

-Page 298, J, Smtltrv and A, Eol 1tsasd are edition L 7” Renum Press, 1986). RFwh13 vector, tph p18 and 5yl are commercially available from New England Biolabs . However, one of ordinary skill in the art to which this invention pertains will be aware of other viruses, strains, host cells, etc. , promoter and associated #I)A'A vectors were also discussed above. It will be appreciated that the invention can be used in any of the various implementations of the invention as discussed. cormorant. Unless otherwise indicated, the DNA used herein was handled by Maniatia. L-Molekyu? -/Roning: Guide to the Phantoms (Coldsfring Harbor-#r1 982).

used to screen for the mutations indicated in parentheses (screening o Ligonucleotides not shown, identical for mutagenesis and screening nucleotides are used). Codons for substituted amino acids are underlined. and Δ indicates the deletion site. Those skilled in the art will understand the codons in an oligonucleotide. Deleting or deleting the desired position by substituting a codon for the desired replacement amino acid deletion of one or more amino acids or insertion of a different amino acid (i.e. It is recognized that oligonucleotides can be easily constructed for use in Probably. Other mutagenic oligonucleotides span the desired position approximately 20- You can plan based on the 50 nucleotide sequence and replace the original codon you want to change. or delete it.

Plasmid induction Mutagenesis of a　D　N　A at various 7 amino acid codons and -1) appropriate regulation of NA in M13 vector by the method of Sv*itk. It was carried out using limited fragments.

, I) Deletions within the NA are made using appropriate restriction fragments of the cDNA in the M13 vector (e.g. Out-of-loop mutagenesis using Saml fragment or in plasmid psVPA4 This is accomplished by heteroduplex out-of-loop mutagenesis.

Plasmid psV to enable expression of tPA glycoprotein in mammalian cells. PA4 was constructed. This plasmid was initially extracted from plasmid papLT5 by SV 40: By removing the DNA encoding the y-diT polypeptide, the complete Xh6I Digestion followed by partial B.ya-E.5A restriction endonuclease digestion. achieved. Plasmid J205 (ATCC number 39568) was added to 5all and Adherent Sa order/Ba□■tPA coat isolated by digestion with Ba1llEl. The parent f) SV405'-Sif in papLT5 from IC by linking to papLT5 w-)” region is replaced by the tPA-coding sequence. As a result, the mammalian When introduced into similar cells, tPA under 11jiI of 5V4Q late pro7-- is transcribed in this vector. This final construct is designated 5VPA4.

Plasmid, LX) SG is used for expression of tPA in mammalian cells such as CHO cells. It is an amplifiable vector for

pLDsG is a mouse gene that utilizes the adenovirus 2m late promoter (NLP). DEFRgDNA transcription unit, simian virus 40 (SV40) enhancer and origin of replication, 5V4Q late promoter (same orientation as adenovirus MLP) k), the gene encoding nato2cyclin hyporesistance and the adenocylus :, human tP A CMm t-24 in the appropriate orientation with respect to type MLPIIC It contains a cDNA encoding 5').

, CVSVL2CATCC number 39813) and a encoding tPA j) A'　LDSGf) production from A is the co-production of LDSG in the CEOIIB cell. With transformation and amplification (1985).

The plasmid, IP'GsM, contains an aDNA insert containing ral-245 human tPA. It is identical to pLDsG except that it is coded. II'GSM is a plasmid 1205 (ATCC sauce number 3956g) or IVPA/1 (ATCC number 39 891) (with the desired mutagenesis at position 245). will be accomplished. Book F! pM’G SM and LDSG can be exchanged through AIIa book. However, as shown earlier, the former vector is Val- 245 protein and the latter would produce Net-245 protein.

plVPA/1 (ATCC number 39891) is a buffer containing gPA-coded eDNA. It is a culovirus transplacement vector. pl　V　P　A　 /l and its mutagenized derivatives are derived from the baculovirus genome of the desired cDNA. The cDNA is used for insertion into the baculovirus polyhetrium protein. It will be under the transcriptional control of the lomotor.

pMT2p is SF'- containing the adenovirus-VA gene and enhancer. Adenovirus containing 40 origins of replication, a trisegmental leader and a 5r donor splice site S major late promoter, 3' splice acceptor site, DHFR cDNA insert, S A mammalian expression vector containing the 1'40u polyazonylation signal and the pBR322 sequence. He is a master. pMT22G contains a unique PJ1 cloning site.

This vector is included in the American Type Culture Collection with ATcc number 4034. It has been deposited as No. 8.

pMT2pc-tPA and its derivatives themselves.

Destroy the BgtH site present on 2M72pc and digest 9M72p with p. Then, the linearized DNA was ligated to a blunt-end linker, and the linker-attached DNA%ptp Ba1l of WGSM containing A-encoding cDNA or mutagenic derivative thereof It is a derivative of p,%IT2pt: produced by binding to a restriction fragment.

The vectors so produced are given a restriction map of tPA (e.g. (shown below) by conventional restriction analysis.

Preparation of expression vector The mammalian expression vector p-pMT2pe-FE787' was initially created using 10μ of plasmid p. By digesting M72 pg DNA with restriction endonuclease BglN. It is made by

Following this, the cut ends were treated with DNA polymerase I (Klenow cleavage) in the presence of nucleotides. (piece). The cut and plugged plasmids are diluted and ligated. Ru. The plasmid was used to transform E. coli HB101, and the EQ was Select colonies containing plasmids. The purified plasmid was cut with Pxtl. Ru. This is followed by phenol/chloroform extraction and ethanol precipitation.

The blunt-ended adapter described below was then mixed 50:1 (adapter) with the Pstl linearized vector. Combine at a molar ratio of vector: vector): (5’) HO-CTAGAGGCCTCTGCA-OH (3’) (3’) The HOGATCTCCGG-P(5') vector was then cut with Ba1l and mixed. The compound was run on a 0.7 thiagarose gel and the ~4.8 Kbp expression vector DN Cut out and purify A. This vector now accepts t, P, 46 DNA inserts be able to.

The mutant tPA sequence for insertion into the vector prepared as described above. It's coded. The DNA insert was a restriction vector of the plasmid, WGSM-FE787'. Produced by digestion with endonuclease Ba1l~2. I Kbp limit Purification of the fragment by agarose gel produces lpl.

pWGsljl-FE787' lacks amino acids 6-86 and has an N substitute at position 117. encodes a cPA variant containing M instead of Q and V at position 245. Ru. It is a derivative of WGSH. This mutant (compound 2-17N-227R-27 5) is published international patent application number roB7704'122KCeDNA and vector preparation and its expression) are described. , WGSM-FE78'l The BαII fragment of ' is described in published international patent application F087104722. The DNA sequence required for full-length translation of the <Q-117*PJ mutant sequence, but the “flip” observed at the 91-amino acid N-end of the natural protein The ``Finger'' and ``Epidermal Growth Factor 1'' regions are missing.

Vector pMT2pa-F was created by inserting the BcLIL fragment into pMT2pc. E787' is produced. (pMT2pc- in IJ ByE* and Apal By digesting FE'187', the mature N-terminus has a third compound starting with Encoding the majority of gPA variants extending to the N-linked glycosylation site Remove the DNA sequence that is present. and (2J to that location, WGSM or other The corresponding Ertl*/ApaI restriction fragment from the 5PA-encoding vector of By inserting 1M72pc-J'E787' into pMT2pc-tPA or will be converted to its derivative. Alternatively, achieve the desired mutagenesis Therefore, heteroduplex mutagenesis on PMT2pc-FE7B7' (e.g. one or more glycosylation sites and/or or plasmin cleavage site).

Preparation of tPA cDNA derivatives: M13 method The diagrammatic restriction map below shows human tPA (above) and describes the cDNA encoding the specific endonuclease. The cleavage sites for the enzyme (shown below) are also shown: the start codon (ATG) and The cDNA regions encoding R1, R1 and Bs are also shown. Therefore , N-terminus mutagenesis e.g. Sacl fragment or BQEH/A'sr This would be accomplished using l fragments. Arg-275 and/or RI and Mutagenesis at R2 and/or R2 can be carried out using e.g. Sagl fragment or Eggl/ Mutagenesis in Ra, which would be achieved using the Sac 1 fragment, is analogous to This can be achieved using Sag summer/X%Gl or 5acl/Apa l fragments. Probably. Selection of restriction fragments for mutagenesis and/or expression vector construction The decision will be based on the convenience of the particular vector used.

Generally the 6 DNA restriction fragments to be mutagenized are - e.g. pWGsM, p MT2pc-*PA, plVPA/1 or pSFFPAJ The cDNA is excised from the full-length cDNA using the indicated endonuclease enzyme, and the appropriate The oligonucleotides shown in Table 4, for example, otide or other oligonucleotides designed for the desired mutagenesis. more mutagenized.

An exemplary mutagenized cDNA fragment thus prepared is shown in the following table. 5 is shown. Such a fragment is pMT2p6-@PA or as described above. In particular, it is used to replace nucleotide sequences in its derivatives that have undergone mutagenesis. It will be done.

(!) (IT) (V) (vI) Middle shows the site of mutagenesis; cDNA fragment I fragment ■ is pWGsM or or pSVP A4 was digested with 5acI.

Insert the Sac I fragment into the M13 vector and overprint with the desired oligonucleotide. After natural mutagenesis, the mutated M13/lPA DNA was digested with Sac I. or ■-■ are BglN and BglN from mutated M13/lPA. and 5aCI, across N-Song end, R1, R8 and Ayg-275. Bgt@l5a6 which encodes the peptide region! Fragment is Byl II/ 5aGI digested pI V P A will be inserted into: cDNA fragment V and and ■ are prepared as described in Examples 2 and 1, respectively. New N-terminus The sequence has the C1a i site first.

Following mutagenesis, the fragment (with or without further mutagenesis) becomes M 13 and used to excise the mutagenic fragment from the 1M13 vector Full-length or partial eDNA that has been previously cut with the same enzyme that was ligated back into the containing expression vector. This method allows full-length cDNA (desired and (mutagenized) limit one or more mutagenized fragments. It is used as a fragment cassette and reconstituted.

cDNA encoding exemplified compound 0 (see Table 1) was prepared as follows. .

The overlapping oligos shown in Table 4A were used to construct the DNA encoding compound 1-1. A set of nucleotides is prepared using a commercially available automatic DNA synthesizer according to the instructions in a conventional manner. first synthesized. Odd numbered oligonucleotides are sense strands , even numbered oligonucleotides are one anti-sense strand. oligo Nucleotides 2.3.4 and 5 were separately phosphorylated using conventional methods. origone The cleotide pairs 1 and 2.3 and 4.5 and 6 respectively are similar to each other under normal conditions. Anneal. For example, 85 x M) squirrel, pH 7, 5, 50 m N aC1゜&　5　aMMgCI, and 4.2 pmol (each nucleotide of )/λ solution, heated to 80°C and then gradually cooled to 37°C over 2 hours. Form a set of overlapping composite binaries: The individual mixtures of the binaries are combined and concentrated, and the binaries are combined with each other under standard conditions. For example, 50 taM Tris.

pH7,4,10vshM MgC1, 10mM DTT and 1mnATP and 5 Weiss units of T4 ligase (New England BioLabs) at 4°C - overnight. (viewed until 16:00). The mixture was heated through a 2% low gelling temperature agarose gel FJR: Run and cut out the ~250 hp band from the gel. produced in that way The extracted DNA molecule encodes the synthetic kringle region on the cassette, with the 5′ and EavnBl and C1ai overhang at the and 3′ ends, respectively. .

data such as E. coli GM161 - 1M72pc produced v4 in a miniature host - FE781' Plasmid DNA-digested with kBgEl and C1al (partial) and starts with the Arrt-(-) codon present in pMT2pa-FE787'. 11m15) - AlpC87) Cut out the coding region that continues to the fusion site. .

The thus digested pMT 2pc-FET 87' was subjected to Ba Connect to Hl-CL a 1 resultant force set.

The above construction resulted in a new 2M72pe vector, pMT2p6-PKl-FE7. 87' is created, where Arg-(1) and /! ＃　-5()1m- 5-Asp-87 fusion site) has been re-created, while the codon for force-7 A cutout U is inserted. Therefore, the resulting vector now carries compound 1-1;- Contains an improved cDNA insert that is coded.

Below shows the restriction map of the coding sequence of 1M72pe-FE'187': Here, 1*” is the position of the sequence encoding the Nol-(5)-Agp(87) fusion. and R1 are as described (modified).

The restriction map of the synthetic cassette is as below (: Below is Bathll / (: '　j　　9M72pc-PKl-FE7879 that imports the G I cassette A restriction map of the t-encoding sequence is shown: Here °＊”’I”! The position of the synthesis cassette is shown. cassette into vector The EamB1/BQEI fusion at the site of 5′ Song end binding results in Bα-81 and Care must be taken that the BQIl and BQIl restriction sites are destroyed. Further in the vector By inserting the cassette into the 3′ Song end of the cassette, the CEa 1 site is recreated, A new N (,,, 1 site was introduced.

If further mutations are desired (e.g. one or more common N-series linkages) at the cocculation site and/mataha plasmin cleavage site), 9M72pc-PHI- Usually using FE78T' and appropriate oligonucleotides as shown in Table 4. may conveniently be achieved by heteroduplex mutagenesis of

Plasmids plVPA, psVPA4t and @pMT2pc-tPA are expression vectors. In addition to its use as a vector, it can also be used to construct cDNAs with desired mutagenic permutations. It will also be used as a storage tank 1. Therefore, the desired modified cDNA (e.g. Arg-275, R1, R1 and/or R3 coding region) mutagenesis (via M2S or heteroduplex) plasmids (in any combination) is digested with one or more appropriate restriction enzymes, the desired fragment is identified, and a single separated and digested to correspond as 9M72pc-PKl-FE787'. ligated into an expression vector.

cDNA encoding the polypeptide sequence of Δ6-86, Q-117tPA Oligonucleotide-directed mutagenesis of ZOllmr and Sio1 molecules (Essentially as described in FORT104722) Ku]. In particular, the mutagenesis vector R containing 1 Mat-2<5tPA gene F M137tPA is constructed from the septate, P1 expression plasmid psVPA4 .

RF M13/gPA first uses psVPA4 as restriction endonuclease H4nd Constructed by complete digestion with III and Xms l. Approximately 1,860 The Hi%d■/Xmtz■ fragment of 4-group Cbp) is a polypeptide of M-245tPA. encodes the majority of the tide sequences, including asparagines 117, 184 and 218 %: Contains nucleotide sequences encoding common N-linked glycosylation sites. I'm reading. This ~1,860hp (hereinafter 1.9A6p) fragment was placed on a preparative agarose plate. Purified by gel electrophoresis.

84% of the tPA cDNA obtained as above [I/XtnaI fragment was Linear double @RF M digested with Bi%dm and Xtml 13 vs*p 18 DIV Connect with A vector. The ligation mixture was transformed Competent Mi) is used to transform AflO1 cells. from transformed cells The produced fog PA-derived DNA-containing M13 plaques were analyzed by analytical DNA restriction analysis and identified and isolated by plaque hybridization. tPA DNA included Filter hybridization was used to detect viral plaques with If 8i%d of the gPA-encoding nucleotide sequence [17Xma■ within the restriction site Radiolabeled oligonucleotide (~l 7 marl, positive one) tropism) was used as a probe. All oligonucleotides are as specified by the manufacturer. ℃Applied Biosystems DNA 47f? , automatic synthesis by machine Manufactured by.

Some positive plaques detected by restriction or hybridization safe 3F analysis One plaque was further cloned by conventional plaque purification. Is it a plaque purification process? The purified M13/lPA bacteriophage obtained was used to infect JM 101 cells. used. These infected cells contain cytoplasmic duplex ``RF'M13/!FARF'M 13/! Cells that received the FA plasmid also contained 1.9 Kbp of gPA cDNA. A medium containing the Bind m/Xml fragment and single-stranded DNA complementary to M13 DNA. Bacteriophages were also produced in the culture medium. - The key DNA is isolated from the culture medium. The M13/gPA-containing phages were purified from isolated M13/gPA-containing phages. This -1iL head M13/ 1PiDNA is #1 in Table 7 described in international patent application WO37104722. and Zollar and 5tntth using oligonucleotide #10. used as a casting base in two consecutive rounds of mutagenesis according to the method. This mutation For induction, the 117th position is changed to A3B:yton, and then the resulting DNA code is The nucleotides encoding amino acids 6 to 86 of the binding chain are deleted. sudden change In the allogenic reaction, the DNA is transformed into bacterial strain JM 101.

H13 sJ from transformant pools to identify mutagenic cDNAs. Array types.

RF M13/lrA plasmid DNA contains mutagenized PA cDNA JMlol infected with purified M13 phage is then purified. That's how you get it The resulting RFM137@FA plasmid was mutagenized as described above. gPA　DHAf)H4nd　Tri/Xtm　I restriction fragment. this Mutagenic restriction fragments can be prepared, if necessary, using suitable oligonucleotides such as those listed in Table 4. published, again by the ZO1la de and 5vhitldQ method, using For example, one or more is the further N-linked glycosylation site or the plus of ArfJ-275 The min cleavage site can undergo further mutagenesis. particularly interesting The first (R”), the first and second (J?l and R1), or all three (81% R1 and R3) common N-linked glycosylation sites This is a variant of the present invention in which a site other than the glycosylation site has been modified.

Construction of B0 expression vector pM72pc-FE787' In this case, Δ1-86, Q Is the mutagenic cDNA encoding -117tPA an M13 vector? It is excised as a Bindl/Xvpbal fragment. This fragment is Binds to Hi sd [[/X%Gl-digested pWGsM -(pWGsM-FE 787'. pMT2pc-FE787' was then combined with the above pM Preparation of T2pe'A and pWGsM-FE7B7'C6 synthesis cassette For the production of a vector encoding compound 1-1.

The overlapping oligonucleotides shown in Table 4A were first produced in a commercially available automated DNA synthesizer. Synthesized by conventional methods using according to the original description. Odd numbered ori The oligonucleotides are the “sense” strand, and even numbered oligonucleotides Tido is “anti-sense tin.

Oligonucleotide pairs 1 and 2; 3 and 4; and 5 and 6 are each After annealing and appropriate phosphorylation, the binary synthesized under normal conditions as previously described. form a set of Merging mixtures of individual dyads and forming the dyads as described previously combine with each other. The mixture was electrophoresed through two low gelling temperature agarose gels. The ˜250 hp band is excised from the gel. D thus produced The NA molecule encodes a synthetic Ringer region on the cassette, with 5' and 3F:i There are %H1 and C1al overhangs at each end.

D, Preparation of pMT2pc-PI-1-FE787' grown in E. coli (, H161). pMT2pc-FE787' was digested with BQIn and ctalc (partially). and starts within the Ary-(-) codon present in 2M72pc-FE787'. the law of nature.

11a(5)-AspC8'l) fusion site into the Jim-5 codon. Extract the code area. The thus digested 2M72pc-FE787' It is then ligated to the B, Hl-ClcLl synthesis cassette using a standard method. to this construction A newer pM72pc vector, pMT2pe-PKl-FB787', created; there Ary-(-1) and lig-5()1m-5-A lp-R7 fusion site] has been recreated, and there is a cassette between them. A cut array has been inserted. The vector from which it is released contains the modified cDNA insert. , which now encodes compound 1-1.

As mentioned earlier, pM72 pc-PK 1-FE 787' is Teloduplex mutagenesis and other conveniently generated synthetic oligonucleotides are conveniently mutagenized using a DNA sequence that modifies the DNA sequence as desired. . For example, restoration of a common N-linked glycosylation site in R1 and/or R2 and conversion of one or both of R3 to other than N-contiguous glycosylation sites, and /or modification of the plasmin cleavage site spanning position 275.

pM72pc derivation containing a cDNA molecule encoding the desired polypeptide sequence Expression vectors such as vectors are constructed as previously described.

Transformation of mammalian host cells, selection of transformants, and genetic cell culture are performed using Ka%f%an (cited above) (CBO host cells) or now Nirira's method, US patent No. 4.419.446 <1983) (using the EPV expression system). Corresponding mammalian-derived mutant proteins can be obtained. For CHO cell expression, vector vector DNA was introduced into CEO cells by normal protoplast fusion and %Ka%/% G% method (cited above). Transformed and amplified CH OIF5 cells produced the desired mutants in good yields, which were shown to contain human tPA in the culture medium. % heterologous antibodies (probably directed against epitopes other than the N-terminus) . The mutants are then recovered and analyzed by immunoaffinity chromatography or FTI. Purified by other conventional methods, such as those involving resins. Similarly, one of the present invention Other mutants are also expressed in sentinel cells and recovered and purified in such a manner. ・ C transformed with 2M72pc-PKl-FE787' as described above. The mutants expressed by HOa follicles were as determined in rat and rabbit models. Due to its in vivo half-life! # Requisitioned, less than that of wild type customer FA also approximately 10 to 20 times longer, by indirect Kuromogae 7 substrate assay using 5-2251. The specific activity measured was significantly higher than that of the WHOεPA standard, e.g. Zoo is expensive.

Procedural amendment (c) ■ October 1990/8B

Claims (9)

[Claims] 1. Among thrombolytic proteins with tissue plasminogen activator-type activity, L Amino acids 1-82 of PA contain a sequence that is identical or essentially equivalent to the following sequence: Peptide of the human LPA sequence except for the mature N-terminal region, which has been replaced with a amino acid sequence. A protein characterized by having a peptide sequence that is essentially the same as a peptide sequence. [There is an array]. 2. One or more common N-linked glycosylation sites are common N-linked glycosylation sites The thrombolytic protein according to claim 1, which is modified at a site other than the sylation site. 3. The common N-linked glycosylation site on Asn-117 The thrombolytic protein according to claim 2, which has been modified at a site other than the cosylation site. 4. Three common conditions for Asn-117, Asn-184 and Asn-448 Each N-linked glycosylation site is modified to a common N-linked glycosylation site. The thrombolytic protein according to claim 2. 5. Arg-275 is deleted or replaced with a different amino acid, The thrombolytic protein according to claim 1, characterized in that: 6. It is further shown that Arg-275 is deleted or replaced with a different amino acid. The thrombolytic protein according to claim 2, characterized in: 7. A DNA molecule encoding a protein according to claims 1 to 6. 8. produced by expression of the DNA molecule according to claim 7 in a mammalian host cell. A thrombolytic protein with tissue plasminogen-type activity. 9. An effective amount of the protein described in claims 1 to 6 or 8 and a pharmaceutically suitable carrier. A therapeutic composition for the treatment of thrombotic conditions characterized in that it is mixed with