WO1987004722A1 - Novel thrombolytic proteins - Google Patents

Novel thrombolytic proteins Download PDF

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Publication number
WO1987004722A1
WO1987004722A1 PCT/US1987/000257 US8700257W WO8704722A1 WO 1987004722 A1 WO1987004722 A1 WO 1987004722A1 US 8700257 W US8700257 W US 8700257W WO 8704722 A1 WO8704722 A1 WO 8704722A1
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WIPO (PCT)
Prior art keywords
trat
gfagkcceid
garsyqvicr
nggtcqqaly
sgraqchsvp
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PCT/US1987/000257
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French (fr)
Inventor
Glenn R. Larsen
Tim J. Ahern
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Genetics Institute, Inc.
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=27505870&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO1987004722(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from US06/882,051 external-priority patent/US5002887A/en
Priority to AT8787902884T priority Critical patent/ATE104700T1/en
Priority to EP87902884A priority patent/EP0293394B2/en
Priority to DE3789664T priority patent/DE3789664T3/en
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to IE166087A priority patent/IE60017B1/en
Priority to ES8701920A priority patent/ES2004438A6/en
Priority to PT8522687A priority patent/PT85226B/en
Priority to GR871052A priority patent/GR871052B/en
Publication of WO1987004722A1 publication Critical patent/WO1987004722A1/en
Priority to DK198705118A priority patent/DK175784B1/en
Priority to NO874091A priority patent/NO175317C/en
Priority to US08/891,245 priority patent/US5837518A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • This invention relates to substances having tissue plasminogen activator-type (t-PA) activity. More specifically, this invention relates to "recoitibinant" thrombolytic proteins, a process for obtaining the proteins from genetically engineered cells, and the therapeutic use of the substances as thrombolytic agents.
  • t-PA tissue plasminogen activator-type
  • proteins are active thrombolytic agents which, it is contemplated, possess improved fibrinolyti ⁇ profiles relative to native human t-PA. This may be manifested as increased affinity to fibrin, decreased reactivity with inhibitors of t-PA, faster rate of thro bolysis, increased fibrinolytic activity and/or prolonged biological half-life. It is also contemplated that- proteins of this invention can be more conveniently prepared in more homogeneous form than can native human t-PA. An improved overall pharmacokinetic profile is contemplated for these proteins.
  • native human t-PA can be viewed as comprising an amino (N-) terminus of about 91 amino acid residues, two so- called “kringle” regions, and at the carboxy terminus a serine protease-type domain. * We have found that the N-terminus contains several sub-domains which play functional roles, inter alia, in fibrin binding and in the .in vivo clearance of the protein. Recently the recovery of another form of t-PA which lacks the native N-terminus and first kringle region has been reported, see European Published Patent Application No. 0 196 920 (published 08 October 1986) . According to that report the truncated form of t- PA, which begins with Ala-160 of native human t-PA, is fibrinolytically active.
  • this invention provides novel protein analogs of human t-PA which retain both kringle regions of native human t-PA, but contain modifications within the N-terminus. While in certain embodiments the modifications involve deletions in the N-terminus, the first kringle region is left intact, and the N-terminal deletion is never greater than 94 amino acids. Most embodiments involve significantlysmallerdeletion(s) and/oraminoacidsubstitution(s) .
  • the proteins of this invention selectively retain more of the desirable biological activities of native human t-PA and may be less immunogenic than more drastically modified analogs of t-PA. It is therefore contemplated that the proteins of this invention possess improved fibrinolytic and pharmacokinetic profiles relative to both native human t-PA and the truncated Ala-160 t-PA, as well as other modified forms of t- PA.
  • the polypeptide backbone of natural human t-PA also includes four consensus Asn-linked glycosylation sites. It has been shown that two of these sites are typically glycosylated in t-PA from melanoma-derived mammalian cells, i.e. at Asni ⁇ and Asn 448 . Asn 184 is glycosylated sometimes and Asn 2 ⁇ s is typically not glycosylated.
  • t-PA from melanoma-derived mammalian cells, e.g. Bowes cells is also referred to herein as "native" or "natural" human t-PA.
  • This invention involves novel protein analogs of human t-PA which possess t-PA-type thrombolytic activity.
  • the proteins of this invention differ in structure from human t-PA in that they contain modifications in peptide sequence (i) at up to three of the Asn-linked glycosylation sites present in native t-PA; (ii) within the N-terminus of the proteins corresponding to the 94 amino acid mature N-terminus of native t- PA; and/or (iii) at the proteolytic cleavage site spanning Arg- 275 and Ile-276.
  • the proteins are characterized by deletion of 1-94 amino acids within the peptide region spanning Gly-(-3) or Ser-1 through Thr-91, relative to native human t-PA.
  • Cys-51 through Asp-87 of native t-PA are deleted.
  • Cys-6 through Ser-50, and Cys-6 through Ile-86 are deleted, respectively.
  • more conservative modifications are present in the N-terminal region of the proteins.
  • certain proteins of this invention contain one or more amino acid deletions or substitutions within one or more of the following, more discrete subregions:
  • the protein variants of this invention may further contain no N-linked carbohydrate moieties or may be only partially glycosylated relative to natural human t-PA.
  • a "partially glycosylated" protein means a protein which contains fewer N-linked carbohydrate moieties than d oes fully-glycosylated native human t-PA. This absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at one or more of the concensus N-linked glycosylation recognition sites present in the native t-PA molecule.
  • variant proteins of this invention embodying such modification at one or more N-linked glycosylation sites retain t-PA-type thrombolytic activity with greater fibrinolytic activity in certain cases, may be more readily produced in more homogeneous form than native t-PA, and in many cases have longer in vivo half-lives than native t-PA.
  • N-linked glycosylation recognition sites are presently believed to comprise tripeptide sequences which are specifically recog ⁇ nized by the appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or aspar- agine-X-serine, where X is usually any amino acid. Their location within the t-PA peptide sequence is shown in Table 1. A variety of amino acid substitutions or deletions at one or more of the three positions of a glycosylation recognition site results in non-glycosylation at the modified sequence. By way of example, Asn 1 1 7 an(i As n-j_ 34 of t-PA have both been replaced with Thr in one embodiment and with Gin in another embodiment.
  • the resultant glycoprotein (Gln-j_ . 7 Gln-]_g 4 ) should contain only one N-linked carbohydrate moiety (at sn 44 g) rather than two or three such moieties as in the case of native t-PA.
  • analogous glycoproteins having the same Asn4 48 monogly ⁇ osy- lation may be prepared by deletion of amino acids or substitution of other amino acids at positions 117 and 184 and/or by deleting or substituting one or more amino acids at other positions within the respective glycosylation recognitions sites, e.g.
  • Asn at positions 117 , 184 and 448 are replaced with Gin.
  • the resultant variants should contain no N-linked carbohydrate moieties, rather than two or three such moieties as in the case of native t-PA.
  • potential glycosylation sites have been modified individually, for instance by replacing Asn, e.g. with Gin, at position 117 in one presently preferred embodiment, at position 184 in another embodiment and at position 448 in still another embodiment.
  • This invention encompasses such non-glycos lated, monoglycoslyated, diglycosylated and triglycosylated t-PA variants.
  • the variants are optionally modified at the proteolytic cleavage site spanning Arg-275 and Ile-276 by virtue of deletion of Arg-275 or substitution of another amino acid, preferably an amino acid other than Lys or His, for the Arg.
  • Thr is at present an especially prefered replacement amino acid for Arg-2 5 in the various embodiments of this invention.
  • Proteolytic cleavage at Arg-275 of native t-PA yields the so-called "two-chain" molecule, as is known in the art.
  • Proteins of this invention which are characterized by modification at this cleavage site may be more readily produced in more homogeneous form than the corresponding protein without the cleavage site modification, and perhaps more importantly may possess an improved fibrinolytic profile and pharmacokinetic characteristic.
  • This invention thus provides a family of novel thrombolytic proteins related to human t-PA.
  • This family comprises several genera of proteins.
  • the proteins are characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA, wherein Arg-275 is deleted or is replaced by a different amino acid, preferably other than lysine or histidine, and at least one of the consensus Asn-linked glycosylation sites is deleted or is modified to other than a consensus Asn-linked glycosylation sequence.
  • Exemplary proteins of this embodiment are depicted in Table 1 below.
  • proteins of this invention include analogs of t-PA characterized by the various modifications or combinations of modifications as disclosed herein, which may also contain other variations, e.g.
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of a peptide bond, amino acid, dipeptide or tripeptide, and at least one of R 1 , R 2 and R 3 are other than consensus N-linked glycosylation sequences; "-", "—” and " " - a peptide bond.
  • the proteins are characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA wherein one or more amino acids are deleted within the N-terminal region from Gly-(-3) through Thr-91 and wherein (a) one or more Asn-linked glycosylation sites are optionally deleted or otherwise modified to other than a consensus Asn-linked glycosylation site, and/or (b) Arg-275 is optionally deleted or replaced by a different amino acid, preferably other than lysine or histidine.
  • Exemplary proteins of this embodiment are shown below:
  • "-" indicates site of an amino acid deletion
  • This embodiment includes a subgenus of proteins wherein 1 to about 94 amino acids are deleted from the region Gly-(- 3) through Thr-91 and one or more of the Asn-linked glycosylation sites are deleted or otherwise modified to other than a consensus Asn-linked glycosylation sequence as previously described. Also included is a subgenus of compounds wherein 1 to about 94 amino acids are deleted from the region Gly-(-3) through Thr-9l, and Arg-275 is deleted or replaced with a different amino acid, preferably other than lysine or histidine.
  • a further subgenus of this embodiment is characterized by a deletion of 1 to about 94 amino acids from within the region Gly-(-3) through Thr-91, deletion or modification of one or more of the Asn-linked glycosylation sites (see e.g. the table on page 6) and deletion of Arg-275 or replacement thereof with a different amino acid.
  • Exemplary proteins of these subgenera are depicted in Tables 2 and 2.5, below.
  • This embodiment also includes a subgenus of proteins wherein the N-terminal deletion comprises a deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50. Also included is a subgenus of proteins wherein 1 to about 45 amino acids are deleted from within the region Ser-1 through Ser-50 and one or more glycosylation sites are modified as previously described. A further subgenus comprises proteins having a deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50, wherein Arg-275 is deleted or replaced with another amino acid. Additionally included is a subgenus having deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50 and wherein both of (a) one or more glycosylation sites, and (b) Arg-275, are optionally modified as previously described. Exemplary proteins of these subgenera are depicted in Table 3, below, as well as in Tables 2 and 2.5. 11
  • VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT 15 GARSYQVICR DEKTQMIYQQ H CWCN SGRAQCHSVP
  • Specific proteins of this invention may be referred to by a 3-part designation comprising a compound number from Table 2 followed by a designation of N-terminus and then identification of position 275.
  • compound No. 2-ll/N-6/Arg designates a protein wherein the 3 glycosylation sites are deleted ("2-11", See Table 2 ) , C-36 through C-43 are deleted (N-terminus #N-6) and Arg-275 is retained.
  • Table 3 Exemplary Proteins Having a Deletion of 1—45 Amino Acids in the Region Ser-1 Through Ser-50 and a Modification at either or both of (a) Arg-275 and (b) At Least One N-linked Glycosylation Site
  • Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild type (wt) sequence of Gly-(-3) through Thr-91:
  • N-termini With reference to the modified N-termini depicted in Table 3, it should be understood that more than one amino acid may be deleted. Where multiple amino acids are deleted, they may be adjacent to one another, or separated by one or more other amino acids. In designating compounds with such N-termini we indicate the size of the deletion by " ⁇ n" where "n” is the number of amino acids deleted. For example, N- terminus #24 wherein one amino acid is deleted as shown in Table 3 may be referred to as "N-24 ⁇ 1". Where two amino acids are deleted, e.g. S-l and Y-2, the N-terminus is referred to as "N-24 ⁇ 2", etc. Where combinations of deletions are made, e.g.
  • N- terminus may be referred to as "N-24 ⁇ 2, N-28 ⁇ 1".
  • specific compounds are designated by a 3-part code comprising a compound number from Table 2 followed by a designation of N-terminus #, e.g. from Table 2.5, and then identification of the status of position 275.
  • Compound 2-26/N-24 ⁇ 2, N-28 ⁇ 1/- thus designates the protein wherein all three glycosylation sites; R-275; S-l, Y-2 and 1-5 are deleted.
  • This embodiment further includes a subgenus of proteins wherein 1 to about 41 amino acids are deleted from the region Cys-51 through Thr-91. Proteins of this subgenus may optionally be modified such that Arg-275 is deleted or replaced with a different amino acid, preferably other than lysine or histidine. Proteins of this subgenus may, as an alternative to or in addition to the modification at Arg- 275, be modified such that (a) one or more N-linked glycosylation sites are abolished, as previously described and/or (b) one or more amino acids are deleted within the region Gly-(-3) through Ser-50. Exemplary proteins of this subgenus are similar to those depicted in Tables 2 and 3, but contain N-termini such as those depicted in Table 4, below.
  • Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild-type (wt) sequence of Gly-(-3) through Thr-91:
  • GARSYQVI CSEPRCF NGGTCQQALY —DFVCQCPE GFAGKCCEID TRAT
  • proteins containing a deletion of 1-37 amino acids (individually, consecutively, or in combination) from Cys- 51 through Asp-87 are depicted, as are proteins containing a deletion of 1-37 amino acids from Cys-51 through Arg-87 and a deletion of one or more amino acids within the region Gly-(-3) through Cys-51 (See N-76) .
  • proteins containing an N-terminus selected from N-termini N-76 through N-lll it should be understood that more than one amino acid may be deleted.
  • N-77 wherein one amino acid is deleted may be referred to as "N-77 ⁇ 1".
  • N-77 ⁇ 6 N-77 ⁇ 6
  • Specific proteins are designated as described following Tables 2 an 3. Examples wherein the three glycosylation sites and Arg-275 are deleted are shown below:
  • this embodiment further includes a subgenus of proteins wherein one or more deletions of less than about 20 amino acids are present within the region Gly-(-3) through Thr- 91. Proteins of this subgenus may also be modified at Arg- 275 and/or at one or more of the Asn-linked glycosylation sites. Exemplary proteins of this subgenus are similar to those depicted in Tables 2 through 4, but contain in place of the wild type N-terminus, an N-terminus such as those depicted in Table 5, below. Additional exemplary compounds of this subgenus are also listed above by their 3-part code designations.
  • the proteins are characterized by a peptide sequence substantially the same as. the peptide sequence of human t-PA wherein different amino acids are substituted for 1-94 of the amino acids in the region Gly- (-3) through Thr-91.
  • This embodiment includes a subgenus of compounds characterized by replacement of one or more amino acids within the above-mentioned N-terminus and by modification at Arg-275 as previously described. Also included is a subgenus of compounds characterized by the above-mentioned replacement of one or more amino acids within the N-terminus and modification, as previously described, at one or more of the consensus Asn-linked glycosylation sites.
  • a further subgenus of this embodiments is characterized by substitution of one or more amino acids within the N-terminus, and modifications as previously described, at both Arg-275 and at one or more of the N- Linked glycosylation sites.
  • the amino acid substitution(s) is/are within the region Gly-(-3) through Ser-50, with or without modification at Arg-275 and/or one or more of the N-linked glycosylation sites.
  • the amino acid substitution(s) is/are within the region Cys-51 through Thr-91, again, with or without modification at Arg-275 and/or at one or more of the N-linked glycosylation sites.
  • one to about eleven, preferably one to about 6 amino acids are replaced within one or more of the following regions, again with or without the other above- mentioned modification(s) : 26
  • the substitution(s) is/are present in one or more of the following regions: R-7 through S-20, W-21 through Y-33, N-37 through Q-42, and H- 44 through S-50.
  • the N-terminus is modified, again, by substitution for one to about eleven, preferably one to about six, amino acids in one or more of the above defined regions, and is further modified by deletion of one to 93, preferably 1 to about 45, and more preferably 1 to about 15, amino acids.
  • Table 5 Exemplary Proteins Having One or More Deletions of Less Than -20 Amino Acids Within the Region Gly-(-3) through Thr-91
  • Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild type (wt) sequence of Gly-(-3) through Thr-91:
  • R-7 S,T,Q,N,G,H,D or K S-52 G,A,Q,L,V,I or
  • T-91 N,S,L,G or A Table 6-B Exemplary Proteins Containing Substitution for one or more Amino Acids Within the Region Gly-(-3) through Thr- 91
  • Proteins of this invention embodying amino acid substitution(s) may be designated by a 3-part code as previously described.
  • n is the number of amino acids replaced, e.g. with the replacement amino acids such as (but not limited to) those depicted in Table 6-A.
  • N-terminus #N-122sl designates N-terminus 122 as depicted in Table 6-B
  • N- terminus #N-122s4 designates that N-terminus wherein S-l is replaced with G and the following three wt amino acids are replaced with other amino acids.
  • Proteins of this embodiment containing multiple amino acid substitutions may be designated by a string of N-terminus designations indicating specific replacements, as follows:
  • One subgenus of particular interest is characterized by replacement of one or more of Y-67 through S-69, with optional deletion of, and/or substitution for, one or more amino acids from Gly-(-3) through L-66, with or without modification as described above at one or more glycosylation sites and/or at Arg-275.
  • the proteins contain at least one so-called "complex carbohydrate” sugar moiety characteristic of mammalian glycoproteins.
  • Such "complex carbohydrate” glycoproteins may be produced by expression of a DNA molecule encoding the desired polypeptide sequence in mammalian host cells.
  • Suitable mammalian host cells and methods for transformation, culture, amplification, screening, andproductproductionandpurification are known in the art. See e.g. Gething and Sambrook, Nature 293:620-625 (1981), or alternatively, Kaufman et al.. Molecular and Cellular Biology 5 (7) :1750-1759 (1985) or Howley et al., U.S. Patent No. 4,419,446.
  • a further aspect of this invention involves t-PA variants as defined above in which each carbohydrate moiety is a processed form of the initial dolicol-linked oligosaccharide characteristic of insect cell-produced glycoproteins, as opposed to a "complex carbohydrate” substituent characteristic of mammalian glycoproteins, including mammalian derived t-PA.
  • Such insect cell-type glycosylation is referred to herein as "high mannose" carbohydrate for the sake of simplicity.
  • complex and high mannose carbohydrates are as defined in Kornfeld et al. , Ann. Rev. Biochem. 54: 631-64 (1985) .
  • High mannose variants in accordance with this invention are characterized by a variant polypeptide backbone as described above which contains at least one occupied N-linked glycosylation site. Such variants may be produced by expression of a DNA sequence encoding the variant in insect host cells. Suitable insect host cells as well as methods and materials for transfor- mation/transfection, insect cell culture, screening and product production and purification useful in practicing this aspect of the invention are known in the art.
  • Glycoproteins so produced also differ from natural t-PA and from t-PA produced heretofore by recombinant engineering techniques in mammalian cells in that the variants of this aspect of the invention do not contain terminal sialic acid or galactose substituents on the carbohydrate moieties or other protein modifications characteristic of mammalian derived glycoproteins.
  • the proteins of this invention which contain no N-linked carbohydrate moieties may also be produced by expressing a DNA molecule encoding the desired variant, e.g. compounds 1-6 through 1-11 of Table 1, in mammalian, insect, yeast or bacterial host cells, with eucaryotic host cells being presently preferred.
  • a DNA molecule encoding the desired variant, e.g. compounds 1-6 through 1-11 of Table 1, in mammalian, insect, yeast or bacterial host cells, with eucaryotic host cells being presently preferred.
  • suitable mammalian and insect host cells, and in addition, suitable yeast and bacterial host cells, as well as methods and materials for transformation/transfection, cell culture, screening and product production and purification useful in practicing this aspect of the invention are also known in the art.
  • this invention also contemplates other t-PA variants which are characterized, instead of by amino acid deletion within the region Gly_ 3 or Ser ⁇ through Thrg ⁇ , by one or more amino acid substitutions within that region, especially in the region Arg ⁇ through Ser ⁇ Q , or by a combination of deletion and substitution.
  • cDNAs encoding these compounds may be readily prepared, e.g., by methods closely analogous to the muta- genesis procedures described herein using appropriate mutagenesis oligonucleotides.
  • the cDNAs may be optionally 41 mutagenized at one or more of the codons for R 1 , R 2 and R 3 , and/or Arg-275, and may be inserted into expression vectors and expressed in host cells by the methods disclosed herein. It is contemplated that these proteins will share the ad disadvantageous pharmacokinetic properties of the other compounds of this invention, and perhaps avoid undue anti- genicity upon administration in pharmacuetical preparations analogous to those disclosed herein.
  • variants of this invention are prepared by recombinant techniques using DNA sequences encoding the analogs which may also contain fewer or no potential glycosylation sites relative to natural human t-PA and/or deletion or replacement of Arg-275.
  • DNA sequences may be produced by conventional site-directed mutagenesis of DNA sequences encoding t-PA.
  • DNA sequences encoding t-PA have been cloned and characterized. See e.g., D. Pennica et al.. Nature (London) 101:214(1983) and R. Kaufman et al., Mol. Cell. Biol.5_(7) : 1750 (1985).
  • One clone, ATCC 39891 which encodes a thrombol- ytically active t-PA analog is unique in that it contains a Met residue at position 245 rather than Val.
  • the DNA sequence encodes a leader sequence which is processed, i.e., recognized and removed by the host cell, followed by the amino acid residues of the full length protein, beginning with Gly.Ala.Arg.Ser.Tyr.Gin... .
  • the protein so produced may begin with the Gly. la.Arg amino terminus or be further processed such that the first three amino acid residues are proteolytically removed.
  • the mature protein has an amino terminus comprising: Ser.Tyr.Gin.Leu... . t-PA variants having either amino terminus are thrombolytically active and are encompassed by this inven ⁇ tion.
  • Variants in accord with the present invention also 42 include proteins having either Met 245 or Val 245 ' as well as other variants, e.g. allelic variations or other amino acid substitutions or deletions, which still retain thrombolytic activity.
  • This invention also encompasses compounds as described above which contain a further modification in the polypeptide domain spanning Asn-218 through Thr-220.
  • compounds of this embodiment are further characterized by an amino acid other than Asn or a peptide bond at position 218 and/or an amino acid other than Pro or a peptide bond at position 219 and/or an amino acid other than Ser or Thr or a peptide bond at position 220.
  • Compounds of this embodiment thus lack the consensus N-linked glycosylation site which is typically not glycosylated in t-PA produced by melanoma-derived mammalian cells.
  • DNA sequences encoding individual variants of this invention may be produced by conventional site-directed mutagenesis of a DNA sequence encoding human t-PA or analogs or variants thereof.
  • Such methods of mutagenesis include the M13 system of Zoller and Smith, Nucleic Acids Res. 10.:6487-6500 (1982); Methods Enzymol. 100: 468-500 (1983) ; and DNA 1:479-488 (1984), using single stranded DNA and the method of Morinaga et al.. Bio/tech ⁇ nology, 636-639 (July 1984), using het ⁇ roduplexed DNA.
  • the mammalian cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art.
  • the components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol Biol.. 159:51-521 (1982); Kaufman, Proc Natl. Acad. Sci. 82.:689-693 (1985).
  • Established cell lines including transformed cell lines, are suitable as hosts.
  • Normal diploid cells cell strains derived from in. vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoetic stem cells) are also suitable.
  • Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
  • the host cells preferably will be established mammalian cell lines.
  • CHO Choinese hamster Ovary cells are presently preferred.
  • the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al.. Cell, 36:391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element.
  • mammalian cell lines include but are not limited to, HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cells lines and the like.
  • Stable transformants then are screened for expression of the product by standard immunological or enzymatic assays.
  • the 44 presence of the DNA encoding the variant proteins may be detected by standard procedures such as Southern blotting.
  • Transient expression of the DNA encoding the variants during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunologic assay of the proteins in the culture medium.
  • the DNA encoding the variant may be further modified to contain different codons for bacterial expression as is known in the art and- preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permitting bacterial expression, secretion and processing of the mature variant protein, also as is known in the art.
  • the compounds expressed in mammalian, insect, yeast or bacterial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.
  • These compounds have been found to bind to monoclonal antibodies directed to human t-PA, and may thus be recovered and/or purified by immunoaffinity chromatography using such antibodies. Furthermore, these compounds possess t-PA-type enzymatic activity, i.e., compounds of this invention effect ⁇ ively activate plasminogen in the presence of fibrin to evoke fibrinolysis, as measured in an indirect assay using the plasmin chromogenic substrate S-2251 as is known in the art.
  • compositions for thrombolytic therapy which comprise a therapeutically effective amount of a variant described above in admixture with a pharmaceutically acceptable parenteral carrier.
  • Such composition can be used in the same manner as that described for human t-PA and should other mammals known to be subject to thrombotic cardiovascular problems. It is contemplated that the compositions will be used both for treating and desirably for preventing thrombotic conditions.
  • the exact doseage and method of administration will be determined by the attending physician depending on the potency and pharmacokinetic profile of the particular compound as well as on various factors which modify the actions of drugs, for example, body weight, sex, diet, time of admini ⁇ stration, drug combination-, reaction sensitivities and severity of the particular case.
  • the nuclear polyhedrosis virus used was the L-l variant of the Auto ⁇ rapha Californica, and the insect cell line used was the spodoptera frugiperda IPLB-SF21 cell line (Vaughn, J.L. et al.. In Vitro (1977) 13, 213-217).
  • the cell and viral manipulations were as detailed in the literature (Pennock G.D., et al., supra; Miller, D.W., Safer, P., and Miller, L.K., Genetic Engineering. Vol. 8, pages 277-298, J.K. Setlow and A. Hollaender, eds. Plenum Press, 1986) .
  • the RF ml3 vectors, mpl8 and mp 11, are commercially available from New England Biolabs. However, those of ordinary skill in the art to which this invention pertains will appreciate that other viruses, strains, host cells, promoters and vectors containing the relevant cDNA, as discussed above, may also be used in the practice of each embodiment of this invention.
  • the DNA manipulations employed are, unless specifically set forth herein, in accordance with Maniatis et al.. Molecular Cloning: A Laborator Manual Cold S rin Harbor, NY 1982) . 46
  • oligonucleotides can be readily constructed for use in deleting one or more amino acids or inserting a different (i.e., replacement) amino acid at a desired site by deleting the codon(s) or substituting the codon for the desired replacement amino acid, respectively, in the oligonucleotide.
  • Other mutagenesis oligonucleotides can be designed based on an approximately 20-50 nucleotide sequence spanning the desired site, with replacement or deletion of the original codon(s) one wishes to change. Plas id Derivations
  • Mutagenesis of cDNAs at codons for the various amino acids was conducted using an appropriate restriction fragment of the cDNA in M13 plasmids by the method of Zoller and Smith. Deletions within the cDNA were effected by loopout mutagenesis using an appropriate restriction fragment, e.g. the SacI fragment, of the cDNA either in M13 vectors or by heteroduplex loop-out in plasmid pSVPA4.
  • an appropriate restriction fragment e.g. the SacI fragment
  • the plasmid pSVPA4 was constructed to allow the expression of t-PA glycoprotein in mammalian cells. This plasmid was made by first removing the DNA encoding the SV40 large T polypeptide from the plasmid pspLT5 (Zhu, Z. et al., 1984, J. Virology 5L:170-180) . This was accomplished by performing a total Xho 1 digest followed by partial Bam-Hl restriction endonuclease digestion.
  • the SV40 large T encoding region in pspLT5 was replaced with human t-PA-encoding sequence by ligating a cohesive Sail/ BamHl t-PA encoding restriction fragment, isolated by digesting plasmid J205 (ATCC No. 39568) with Sal I and BamHl, to the parent Xhol/BamHl cut vector pspLT5 prepared as described above. Consequently, t-PA will be transcribed in this vector under the control of the SV40 late promoter when introduced into mammalian cells. This final contruct is designated pSVPA4.
  • Plasmid pLDSG is an amplifiable vector for the expression of t-PA in mammalian cells such as CHO cells.
  • pLDSG contains a mouse DHFR cDNA transcription unit which utilizes the adenovirus type 2 major late promoter (MLP) , the simian virus 40 (SV40) enhancer and origin of replication, the SV40 late promoter (in the same orientation as the adenovirus MLP) , a gene encoding tetracy ⁇ lin resistance and a cDNA encoding human t-PA (Val-245) in the proper orientation with respect to the adenovirus type 2 MLP.
  • MLP major late promoter
  • SV40 simian virus 40
  • Plasmid pWGSM is identical to pLDSG except that the cDNA insert encodes Met-245 human t-PA.
  • pWSGM may be constructed using cDNA from plasmid J205 (ATCC No. 39568) or pIVPA/1 (ATCC No. 39891) .
  • pWGSM and pLDSG may be used interchangeably, although as indicated previously, the former vector will produce Val-245 proteins and the latter Met-245 proteins.
  • pIVPA/1 (ATCC No. 39891) is a baculoviral transplacement vector containing a t-PA-encoding cDNA. pIVPA/1 and mutagenized derivatives thereof are used to insert a desired cDNA into a baculoviral genome such that the cDNA will be under the transcriptional control of the baculoviral polyhedrin promoter.
  • the mutagenesis via heteroduplexed DNA of specfic areas in the t-PA expression plasmid, pSVPA4, involves the following steps: Preparation of a picillin sensitive pSVPA4 DNA
  • Plasmid pSVPA4 (15 ug) was linearized with Pvul to completion. This mixture was extracted with phenol/chloroform and the DNA was precipitated using two volumes of ethanol with 0.1 M NaCl present.
  • the DNA was resuspended in a 21 ul of water, 1 ul dNTB solution (containing 2mM dATP, dGTP, dTTP, dCTP) , 2.5 ul 10X nick translation buffer (0.5M Tris-Cl pH 7.5, 0.1 M MgS0 4 , 10 mM DTT, 500 ug/ml) and 0.5 ul (2 units) DNA polymerase 1-Large Fragment (New England Biolabs) . This mixture was incubated at room temperature for thirty minutes and then phenol/chlorform extracted followed by ethanol precipitation as described above. 3. The precipitated DNA was resuspended to 0.2 ug/ul by the addition of 75 ul water.
  • Plasmid pSVPA4 (15 ug) was digested with Sac I which cuts this plasmid twice within the t-PA encoding sequence to produce two restriction fragments, a 1.4 kbp t-PA encoding restriction fragment plus the parent vector. Following restriction digestion 1 ul (28 units) of calf intestine alkaline phospatase (Boehringer Mannheim) was added then incubated at 37 ⁇ C for five minutes. The two bands were separated by loading this mixture onto a 0.7% agarose gel.
  • the parent vector restriction fragment was excised from the gel and extracted by adsorption to silica dioxide at 4°C, which was folowed by elution in 50 mM Tris/lmM EDTA at 37"C for thirty minutes. The eluted DNA was adjusted to a final concentration of 0.2 ug/ul.
  • Each mutagenesis react.ion was adjusted to the following concentrations by the addition of 7 ul to the heteroduplex mixtures, 2mM MgCl/0.2 mM ATP/60uM dATP, dTTP,dGTP,dCTP/4 mM DTT/40 units/ml Klenow fragment of E. coli DNA polymerase I (B.R.L.), 2000 units/ml T4 DNA ligase (N.E.B.). This mixture was incubated at room temperature for 2 hours.
  • the filters were washed with 5X SSC, 0.1% SDS at a temperature 5" below the calculated melting temperature of the oligonucleotide probe.
  • DNA was prepared from positively hybridizing clones and analyzed initially by digestion with different restriction enzymes and agarose gel electrophoresis. DNA was transferred to nitrocellulose and filters were prepared and hybridized to the screening probes in order to ensure the mutagenic oligonucleotide was introduced in to the correct fragment.
  • DNA was then retransformed into E. coli and ampi ⁇ cillin resistant colonies were screened for hybridization to the screening oligonucleotide.
  • the following schematic restriction map illustrates a cDNA encoding human t-PA (above) with cleavage sites indicated for specific endonucleases (indicated below) :
  • mutagenesis at the N-terminus may be effected using the SacI fragment or the Bglll/Narl fragment, for example.
  • Mutagenesis at Arg-275 and/or at R 1 and/or R 2 may be effected using, e.g., the SacI fragment or Bglll/SacI fragment.
  • Mutagenesis at R 3 may be effected using, an EcoRI/Xmal or EcoRI/Apal fragment. The choice of restriction fragment may be determined based on the convenience of using particular vectors for mutagenesis and/or for expression vector construction.
  • the cDNA restriction fragment to be mutagenized may be excised from the full-length cDNA present, e.g., in pWGSM, pIVPA/1 or pSVPA4, using the indicated endonuclease enzyme(s) and then mutagenized, e.g. with the oligonucleo ⁇ tides shown in Table 7 or other oligonucleotides designed for the desired mutagenesis.
  • cDNA fragments I through IV are prepared by digesting pWGSM or pSVPA4 with SacI, inserting SacI fragment into M13 vector, utagenizing with desired oligonucleotide(s) , and digesting mutagenized M13/t-PA DNA with SacI; alternatively, I-IV may be excised from mutagenized M13/t-PA with Bglll and SacI and the Bglll/SacI fragment encoding the peptide domain spanning the N-terminus, R 1 , R 2 & Arg-275 may be inserted into Bglll/SacI-digested pIVPA; cDNA fragment V is prepared as described in Example 2, below.
  • the fragment may then be excised from the M13 vector and ligated back into an expression vector containing the full-length or partial cDNA previously cleaved with the same enzyme (s) as were used for excising the mutagenized fragment from the M13 vector.
  • the full-length cDNA, mutagenized as desired may be re-assembled using one or more mutagenized fragments as restriction fragment cassettes.
  • cDNAs encoding the following illustrative compounds may be prepared from the mutagenized fragments of Table 8 as follows:
  • -6/N-23/Arg (g) ligate mutagenized cDNA fragment IV -6/N-21/Arg (prepared using oligonucleotides #8,10 -6/N-22/Arg or 12 and oligonucleotides #3 and 5) into Sacl-digested pSVPA4 or excise fragment IV from mutagenized M13/t-PA as the Bglll/SacI fragment and ligate 55 same into Bglll/SacI-digested pIVPA/1.
  • Plasmids pIVPA or pSVPA4 in addition to utility as ex ⁇ pression vectors, may also be used as a "depot" in the construction of cDNAs having any desired permutation of mutagenized sites.
  • "pIVPA/ ⁇ ” or “pSVPA4/ ⁇ ", muta ⁇ genized (via M13 or heteroduplexing) plasmids containing a desired modification in the cDNA region encoding the N- terminal region may be digested with Narl (partial) and Xmal (Smal) (total) to remove the cDNA region encoding the protein domain spanning R 1 , R 2 and R 3 .
  • a second pIVPA or pSVPA4 plasmid mutagenized, if desired (via M13 or heteroduplexing), at any combination of Arg-275, R 1 , R 2 and R 3 -encoding regions may then be digested with Narl (total) and Xmal (Smal) (total) and the Narl/Xmal (Smal) fragment may then be identified, isolated and ligated into the Narl/Xmal (Smal) digested pIVPA/ ⁇ or pSVPA4/ ⁇ .
  • Such use of the Narl/Xmal (Smal) restriction fragment cassette allows the construction of desired mutagenized cDNAs in pIVPA or pSVPA4.
  • the mutagenized cDNA may then be transferred, e.g. as a Bglll/Xmal restriction fragment cassette into Bglll/Xmal-digested pWGSM for mammalian expression, if desired.
  • cDNA molecules encoding the polypeptide sequence of compounds 2-l/N-21/Arg, 2-l/N-22/Arg and 2-l/N-23/Arg were prepared using the oligonucleotide-directed mutagenesis method of Zoller and Smith.
  • the mutagenesis vector RF M13/t-PA containing the t-PA gene was constructed from the mammalian t-PA expression plasmid pSVPA4.
  • RF M13/t-PA was constructed by first digesting pSVPA4 to completion with the restriction endonuclease SacI.
  • the approximately 1, 436 base pair (bp) SacI fragment encodes a large portion of the polypeptide sequence of t-PA and includes the nucleotide sequences encoding the consensus N-l inked glycosylation sites encompassing asparagines 117 , 184 , and 218 .
  • This 1 , 436 bp (hereinafter 1.4 kbp) fragment was puri f ied by preparative agarose gel electrophoresis .
  • the ligation mixture was used to transform trans ⁇ formation competent bacterial JM101 cells.
  • M13 plaques containing t-PA-derived DNA produced from transformed cells were identified and isolated by analytical DNA restriction analysis and/or plaque hybridization.
  • Radiolabeled oligo ⁇ nucleotides ( ⁇ 17mers, of positive polarity) derived from within the SacI restriction sites of the t-PA-encoding nucleotide sequence depicted in Table 1 were used as probes when filter hybridization was employed to detect viral plaques containing tPA DNA. All oligonucleotides were 57 prepared by automated synthesis with an Applied Biosystems DNA synthesizer according to the manufacturer's instructions.
  • M13/t-PA bacteriophage obtained from the plaque purification procedure was used to infect JM101 cells. These infected cells produce cytoplasmic double-stranded "RF" M13/t-PA plasmid DNA. The infected cells also produce bacteriophage in the culturemediumwhich contain single-strandedDNAcomplimentary to the 1.4 kbp SacI fragment of t-PA and to M13 DNA. Single-stranded DNA was purified from the M13/t-PA-containing phage isolated from the culture medium.
  • This single— stranded M13/t-PA DNA was used as a template in a mutagenesis reaction according to the method of Zoller and Smith using oligonucleotide #3 of Table 7.
  • This mutagenesis event changes the Asn codon to a Gin codon at position 117 of the subsequently obtained coding strand of DNA by changing the DNA sequence from "AAC" to "CAG”.
  • the DNA was transformed into the bacterial strain JM 101.
  • the transformant plaques were screened by DNA hybridization using radiolabeled oligonucleotide #4 ofTable 7.
  • All exemplaryoligonucleotides in Table 7 are of positive polarity, i.e., represent portions of a coding rather than non-coding strand of DNA. All hybridization positive plaques were further purified by subsequent secondary infections of JM 101 cells with ' M13 phage containing mutagenized DNA.
  • RF M13/t-PA plasmid DNA was purified from JM 101 cells infected with purified M13 phage containing mutagenized t-PA cDNA.
  • the RF M13/t-PA plasmid thus obtained contains the Gln ⁇ mutagenized Sac I restriction fragment of t-PA 58
  • This mutagenized restriction fragment can then be further mutagenized, again by the method of Zoller and Smith, but using the oligonucleotides described below.
  • the oligonucleotides described below were designed to induce a deletion ("loop out") within the cDNA region encoding the N-terminal domain.
  • Oligonucleotide #12 of Table 7 can be used to generate a cDNA deletion encoding Cyssi through 59
  • Each of these mutagenized restriction fragments can then be ligated back into the mammalian expression vector pSVPA4 as a Sac I cassette by methods analogous to those described in Example #3B, or prepared for insertion into the insect cell expression vector pIVPA/1 (ATCC No.39891) as a Bgl II/Sac I cassette derived from modified RF M13/t-PA DNA.
  • the purified RF M13/t-PA containing the modified and truncated t-PA cDNA, prepared as described above, can be digested with the restriction endonucleases Bglll and Sac I.
  • the approximately 1.2 kbp Bglll/Sac I restriction fragment was purified by conventional preparative gel electrophoresis.
  • the Bglll/Sac I fragment so obtained constitutes a mutagenized cassette which lacks a 5' and 3' portion of the DNA which encodes the amino and carboxy termini of the translated protein.
  • Insect expression vector pIVPA/1 (ATCC No. 39891) contains a wild type tPA cDNA insert operatively linked to a polyhedrin promoter together with baculovirus flanking 60
  • pIVPA/1 was digested with Bglll and Sac I thereby excising a t-PA coding region spanning the N-terminus and R 1 and R 2 .
  • the Bglll/Sac I cassettes containing the mutagenized, N-terminally modified t-PA cDNA fragments may each then be ligated to pIVPA/1 expression vector DNA which had been previously purified following digestion with Bglll and SacI.
  • the resulting plasmids, pIVPA/ ⁇ FBR; Gln 117 , pIVPA/ ⁇ FBR/ ⁇ EGF; Gln 117 ; pIVPA/ ⁇ EGF, Gln 117 should contain the mutagenized cDNAs encoding compounds 2-1/N- 21/Arg, 2-l/N-22/Arg and 2-l/N-23/Arg, respectively, now operatively linked to the polyhedrinpromoter.
  • Thenucleotide sequence of each mutagenized cDNA insert may be confirmed by supercoil sequencing with plasmid as substrate. See e.g, E.Y. Chen et al., 1985, DNA 1(2) :165-170.
  • Each of the pIVPA plasmids containing the mutagenized cDNAs may be introduced into the insect virus by co-trans- fection with wild-type A ⁇ NPV in Spodoptera cells.
  • 1 ug of purified Autographa californica NPV DNA and lOug of the desired pIVPA DNA are introduced into Spodoptera cells growing on tissue culture dishes by a calcium phosphate transfection procedure (Potter, K.N. and Miller, L.K., J.Invertebr. Path. (1980), 3_6 431-432).
  • the progeny virus present in the media over the transfected cells are plaqued onto a fresh monolayer of cells at several different dilutions. Plaques are assayed, and the recombinants are picked based on the PIB-minus phenotype as follows: A virus which has lost its polyhedrin gene, as would a virus containing a mutagenized cDNA will not produce PIBs. Plaques that appear PIB deficient are selected, excised and amplified on fresh cells. The supernatant over these cells is then assayed for t-PA-type enzymatic activity. Positive assay results indicate that the glycoprotein is in fact being produced.
  • An alternative method of virus purification via the plaque lifting protocol differs slightly from the steps described above, and is described below. Plaque the progeny virus from transfeetion at suitable dilution onto cell culture dishes. Prepare a nitrocellulose replica of the cell monolayer and the virus plaques. Reserve the agarose overlay from the plate as the virus source after the results of the following steps are obtained.
  • Re-probe the filter with a radioactive DNA fragment which will identify viral plaques 62 regardless of the state of the polyhedrin gene.
  • a suitable fragment may be the EcoRI I fragment. Score these as progeny virus. Select those plaques which are positive for the foreign gene DNA probe, negative for the polyhedrin gene probe, and positive for the viral DNA probe. These are strong candidates for the desired genotype.
  • Antibodies have been used to demostrate the presence of the variant proteins in the extracellular media of infected cells.
  • Recombinant virus prepared as above, is used to infect cells grown in the usual TC-100 (Gibco) nutrient salts solution but instead of the standard media supplement of 10% fetal calf serum, this is replaced with a 50% egg yolk enrichment (to 1% total volume) ( Scott Biologicals) .
  • TC-100 Gibco
  • Egg yolk enrichment to 1% total volume
  • a fixed amount of activity units of this and control t-PA preparations are separated on an acrylamide gel. This gel is then stained with a silver-based reagent to display the protein pattern. This reveals that the virus, upon infection of insect cells, leads to the extracellular production of a protein having t-PA type activity.
  • Example 1 The partially glycosylated truncated proteins produced in Example 1 should have an increased gel mobility relative to the fully-glycosylated analog and to the non-glycosylated full-length analog.
  • Example 1 The mutagenesis methods of Example 1 can be used with other conventionally prepared synthetic oligonucleotides which modify the original t-PA DNA sequence to produce proteins modified at the N-terminal region and/or optionally modified at N-linked glycosylation sites and/or at Arg-275 with the appropriate codon change(s) described previously.
  • cDNA encoding Compounds D-6, D-l and D-3 may be prepared using the SacI restriction fragment in M13/t-PA and mutagenizing with oligonucleotides #S, 10 and 12 re ⁇ spectively, but not with oligonucleotide #3.
  • Arg-275 may be deleted or replaced, eg with Thr, using oligo's 14 or 15, respectively.
  • Vector construction, transfection and expression may be carried out as in Example 1 for insect cells or as described below in Example 3 for mammalian cells.
  • Single-stranded DNA generated from the M13 mutagenesis vector (RF M13/t-PA) , prepared as in Example 1, can also be used as a template to mutagenize, in a site specific manner, at Arg-275 and/or at glycosylation site(s) R 1 or R 2 or both.
  • the region encoding the consensus tripeptide which encompasses Asn 2 i 8 may be similarly mutagenized. To prepare multiple modifications of the protein at these sites an iterative process may be used.
  • cDNA encoding Compounds 2-2/N-23/Arg, 2- 2/N-21/Arg and 2-2/N-22/Arg may be prepared by the method of Example 1 but substituting mutagenesis oligonucleotide #5 for oligonucleotide #3 and screening oligonucleotide #6 for oligonucleotide #4.
  • cDNA encoding Compounds 2-6/N- 21/Arg, 2-6/N-22/Arg and 2-6/N-23/Arg may be prepared by twice mutagenizing the SacI fragment as described in Example 1 and addition mutagenizing and screening with oligonucleotides #5 and #6. Vector construction, transfection and expression are carried out as in Example 1 for insect cells or as described below for mammalian cells. See Routes (a)-(h), supra.
  • the RF M13/t-PA mutagenesis vector does not contain DNA sequence encoding R 3 , the N-linked glycosylation site of t-PA most proximal to the carboxy-terminus of the protein. Therefore in order to make DNA modifications at that site, a new M13/t-PA mutagenesis RF vector called M13/t-PA:Rl-Xma I was made. This vector was constructed by digesting the M13 vector M13mpll to completion with EcoRI and Xma I.
  • the Rl/Xmal digested M13 vector was ligated to a purified EcoRI/Xma I t-PA restriction fragment (approximately 439bp, hereinafter 0.4kbp) encoding a polypeptide region encom ⁇ passing glycosylation site R 3 .
  • This 0.4kbp restriction fragment was purified following digestion of the plasmid pWGSM with EcoRI and Xma I.
  • the mammalian expression plasmid pWGSM, encoding the t-PA gene is identical within the 439bp EcoRl/Xma I fragment to the plasmid pLDSG described by Kaufman et al., Mol. Cell Biol. 5.: 1750-1759 (1985) .
  • the ligation mixture was used to transform competent bacterial JM 101 cells. Several plaques were picked and analyzed for the presence of the 0.4kbp t-PA EcoRI/Xmal fragment by standard DNA restriction fragment analysis. Double-stranded RF M13 DNA was purified from cells containing the 0.4kbp t-PA fragment. This DNA was designated RF M13/t-PA:RI-Xma I mutagenesis vector. As previously indicated in Example 1A this vector, when transformed into competent JM101 cells, can be used to make M13/t-PA:RI-XmaI phage from which single-stranded M13/t-PA:RI-XmaI DNA can be purified. This single-stranded DNA can be used as template in the site-directed mutagenesis reaction to modify the t-PA DNA at the N-linked glycosylation site R 3 .
  • Modified R 3 coding sequences can be used to replace the wild-type R 3 sequences present in either modified pIVPA/1 as prepared in Example 1 (truncated and/or modified at R 1 and/or R 2 ) or wild-type pIVPA/1 plasmid DNA. This can be accomplished by first performing a total Sac I/Apa I digestion of the R 3 modified M13/t-PA:RI/XmaI mutagenesis plasmid vector, and isolating the R 3 modified 165 base pair t-PA restriction fragment so produced.
  • Example 2 can similarly be totally digested with Sac I and Apa I to excise the 165 bp wild-type t-PA restriction fragment encoding the unmodified R 3 site. Ligation of the purified insect expression vector lacking the 165 bp fragment to the modified R 3 165 bp fragment produces a new insect expression vector. Expression of the vector produces a truncated protein modified at the R 3 site, as well as at any or all of the other consensus N-linked glycosylation sites present in natural t-PA and/or at Arg-275.
  • the pIVPA plasmid containing the modified cDNA may also be used to generate the Bglll/Apal fragment of the modified t-PA cDNA which spans the deletion region in the N-terminal domain as well as the region encoding R 1 , R 2 and R 3 or the Narl/Xmal fragment which spans R 1 , R 2 and R 3 . Either of those fragments may be inserted into mammalian expression vectors such as pSVPA4 or pWGSM as described in Example 3.
  • cDNA molecules encoding the polypeptide sequences of compounds D-6, D-l and D-3 were prepared using mutagenesis oligonucleotides #8 , 10, and 12 , respectively, and the SacI fragment of the t-PA cDNA as template by the M13 method of Exampl e 1 or heterodupl ex mutagene s i s (Moranaga Heteroduplex Mutagenesis protocol ; both, supra) . Mutants s e l e c t e d by DNA hybrid i z ation us ing screening oligonucleotides 9, 11 and 13 respectively were confirmed by DNA sequence analysis to be correct in the modified DNA sequence.
  • Each modified cDNA prepared in Example 1A ( ⁇ , Gln ⁇ _i 7 ) or 3A ( ⁇ ) was first removed from the M13 mutagenesis vector RF M13/t-PA by total digestion of the vector with SacI.
  • the approximately 1.4kbp restriction fragment of each mutagenized cDNA was purified by gel ele ⁇ trophoresis and then ligated into pSVPA4 as follows. First, pSVPA4 was digested with SacI to remove the wild type t-PA 1.4kbp restriction fragment. The remaining portion of the SacI digested pSVPA4 was then ligated to the 1.4k p restriction fragment of the mutagenized cDNA. This ligation event can produce two orientations of the inserted fragment.
  • the appropriate orientation in each case may be identified using EcoRI and PvuII as the enzymes in conventional analytical restriction enzyme analysis.
  • This replacement allows the Sac I fragment to be used as a cassette ' fragment between the RF M13/t-PA mutagenesis vector and the pSVPA4 mammalian expression vector.
  • Modified M13 SacI fragments (truncated and optionally modified at R 1 and/or R 2 ) may be inserted into Sacl-digested pSVPA4 DNA which has been previously, or is subsequently, modified at R 3 if desired.
  • DNA previously modified at R 1 , R 2 and/or R 3 can be excised from vectors such as pIVPA or pSVPA4 as a Narl/Apal or Narl/Xmal fragment.
  • the fragment so obtained may then be inserted into vectors such as pSVPA4 or pWGSM previously digested with Narl (partial) and Apal or Xmal (total) .
  • vectors such as pSVPA4 or pWGSM previously digested with Narl (partial) and Apal or Xmal (total) .
  • COS-1 cells (ATCC CRL 1650) were transfectd by the method of Lopata, M.A. et al., Nucl. Acids Res. 12:5707-5717 (1984) with the vectors prepared in Example 3B, i.e., modified pSVPA4. Serum containing medium was replaced with 68 serum-free medium 24 hours after the transfection and conditioned medium was assayed for both the presence of plasminogen activating activity, using the chromogenic substrate S-2 ' 251, or the presence .of t-PA antigen by an ELISA assay, 48 and 72 hours post-transfection.
  • Serum containing medium was replaced with 68 serum-free medium 24 hours after the transfection and conditioned medium was assayed for both the presence of plasminogen activating activity, using the chromogenic substrate S-2 ' 251, or the presence .of t-PA antigen by an ELISA assay, 48 and 72 hours post-transfection.
  • Modified complex carbohydrate protein can be produced by infecting CVl cells (ATCC CCL 70) with SV40 viral stocks propagated as described by Gething and Sambrook (Nature 293:620-625, 1981). This has been carried out by first totally digesting modified pSVPA4 with the restriction endonuclease BamHl to remove the bacterial shuttle vector pXf3 from the SV40 viral DNA. Before transfecting this DNA into CVl cells, along with the helper virus SV40-rINS-pBR322 DNA (described below) , the Bam HI linearized SV40/t-PA DNA is circularized by ligation at dilute DNA concentrations (1 ug/ml) .
  • SV40-rINS is used to provide "late” SV40 proteins while pSVPA4 provides the "early” SV40 proteins necessary for virus DNA production while also encoding the proteins of this invention. Consequently when cells are transfected with both these DNA's as described by Gething and Sambrook, SV40 virus is produced which contains DNA from either viral vectors. Subsequent infection of CVl cells with amplified virus has produced proteinwith t-PA-type activity which can be assayed 72 hours post-infection as described in Example 3C.
  • cDNAs encoding various proteins of this invention have been prepared by the methods of Examples 1, 2 and 3.
  • the Bgl II/XmaI restriction fragment cassette may then be excised from either the pIVPA or pSVPA4 vector containing the cDNA encoding the truncated protein with or without modification at one or more glycosylation sites.
  • the excised Bglll/Xmal fragment may then be ligated into Bgl II/XmaI- ⁇ ut pSVPA4 or pWGSM for introduction into mammalian cells. Expression of such cDNAs in mammalian host cells, e.g.
  • cDNA encoding compound 2- l/N-23/Arg was prepared using mutagenesis oligonucleotide #12 and screening oligonucleotide #13 (Table 7) but by the heteroduplex method described above, with pSVPA4 previously mutagenized at position 117 (as above) as template.
  • cDNAs encoding Compounds D- 1 ( ⁇ FBR) and D-3 ( ⁇ EGF/FBR) were prepared by M13 mutagenesis, as described above, and inserted as the SacI fragment into Sacl-digested pSVPA4.
  • cDNA encoding Compound D-6 ( ⁇ EGF) was prepared by the heteroduplex method, described above, using pSVPA4 as template and mutagenesis oligonucleotide #12, and screening with oligonucleotide #13.
  • cDNA contained in pSVPA4 or pIVPA is excised as a Bglll/Xmal fragment and ligated into purified, Bglll/Xmal-digested pWGSM.
  • the resulting pWGSM vector is introduced into CHO cells and amplified by the method of Kaufman, supra.
  • the transformed and amplified CHO cells produce compounds D-6, D-l, D-3, 2-l/N-23/Arg, 2-l/N-21/Arg and 2-l/N-22/Arg respectively, which were detected in the culture medium by human t-PA specific antibodies.
  • the compounds may then be recovered and purified by immunoaffinity chromatography.
  • Example 4 may be repeated using cDNA encoding the proteins modified within the N-terminus and/or at R-275 with or without modification at R 1 , R 2 , and/or R 3 to produce the desired protein in CHO cells.
  • Mutagenized cDNAs may be prepared as described above.
  • cDNAs encoding Compounds 2-7/N-23/A.rg, 2-7/N-21/Arg and 2-7/N-22/Arg are prepared in pIVPA as described in Example 2.
  • the cDNAs may then be excised as the Bglll/Xmal fragment and ligated into purified, Bglll/Xmal-digested pWGSM, and the resultant vector transformed and amplified in CHO cells as in Example 4 to produce compounds 2-7/N-23/Arg, 2-7/N-21/Arg and 2- 7/N-22/Arg.

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Abstract

Thrombolytic proteins which have tissue plasminogen-type activity. The proteins are characterized by modification within the 94 amino acid N-terminus, and/or at Arg-275, and/or at one or more of the N-linked glycosylation sites. Methods for making these proteins are disclosed as are therapeutic compositions containing same.

Description

NOVEL THROMBOLYTIC PROTEINS
This invention relates to substances having tissue plasminogen activator-type (t-PA) activity. More specifically, this invention relates to "recoitibinant" thrombolytic proteins, a process for obtaining the proteins from genetically engineered cells, and the therapeutic use of the substances as thrombolytic agents.
These proteins are active thrombolytic agents which, it is contemplated, possess improved fibrinolytiσ profiles relative to native human t-PA. This may be manifested as increased affinity to fibrin, decreased reactivity with inhibitors of t-PA, faster rate of thro bolysis, increased fibrinolytic activity and/or prolonged biological half-life. It is also contemplated that- proteins of this invention can be more conveniently prepared in more homogeneous form than can native human t-PA. An improved overall pharmacokinetic profile is contemplated for these proteins.
The structure of native human t-PA can be viewed as comprising an amino (N-) terminus of about 91 amino acid residues, two so- called "kringle" regions, and at the carboxy terminus a serine protease-type domain. *We have found that the N-terminus contains several sub-domains which play functional roles, inter alia, in fibrin binding and in the .in vivo clearance of the protein. Recently the recovery of another form of t-PA which lacks the native N-terminus and first kringle region has been reported, see European Published Patent Application No. 0 196 920 (published 08 October 1986) . According to that report the truncated form of t- PA, which begins with Ala-160 of native human t-PA, is fibrinolytically active.
As described in greater detail hereinafter, this invention provides novel protein analogs of human t-PA which retain both kringle regions of native human t-PA, but contain modifications within the N-terminus. While in certain embodiments the modifications involve deletions in the N-terminus, the first kringle region is left intact, and the N-terminal deletion is never greater than 94 amino acids. Most embodiments involve significantlysmallerdeletion(s) and/oraminoacidsubstitution(s) . By retaining more of the structure of native human t-PA, it is contemplated that the proteins of this invention selectively retain more of the desirable biological activities of native human t-PA and may be less immunogenic than more drastically modified analogs of t-PA. It is therefore contemplated that the proteins of this invention possess improved fibrinolytic and pharmacokinetic profiles relative to both native human t-PA and the truncated Ala-160 t-PA, as well as other modified forms of t- PA.
The polypeptide backbone of natural human t-PA also includes four consensus Asn-linked glycosylation sites. It has been shown that two of these sites are typically glycosylated in t-PA from melanoma-derived mammalian cells, i.e. at Asni^ and Asn448. Asn184 is glycosylated sometimes and Asn2ιs is typically not glycosylated. t-PA from melanoma-derived mammalian cells, e.g. Bowes cells, is also referred to herein as "native" or "natural" human t-PA.
This invention, as mentioned above, involves novel protein analogs of human t-PA which possess t-PA-type thrombolytic activity. The proteins of this invention differ in structure from human t-PA in that they contain modifications in peptide sequence (i) at up to three of the Asn-linked glycosylation sites present in native t-PA; (ii) within the N-terminus of the proteins corresponding to the 94 amino acid mature N-terminus of native t- PA; and/or (iii) at the proteolytic cleavage site spanning Arg- 275 and Ile-276. These features of the proteins of this invention are described in greater detail below. Notwithstanding the various modifications, the numbering of amino acids as shown in the one- letter code sequence of Table 1 is retained.
A. Modifications at the N-terminus
In one aspect of this invention the proteins are characterized by deletion of 1-94 amino acids within the peptide region spanning Gly-(-3) or Ser-1 through Thr-91, relative to native human t-PA. In one embodiment, for example, Cys-51 through Asp-87 of native t-PA are deleted. In two other specific embodiments Cys-6 through Ser-50, and Cys-6 through Ile-86 are deleted, respectively. In other embodiments, more conservative modifications are present in the N-terminal region of the proteins. For instance, certain proteins of this invention contain one or more amino acid deletions or substitutions within one or more of the following, more discrete subregions:
re ion from to reαion from to
1 Gly-(-3) Gln-3 7 Cys-34 Cys-43
2 Val-4 Lys-10 8 His-44 Ser-50
3 Thr-11 His-18 9 Cys-51 Cys-62
4 Gln-19 Leu-22 10 Gln-63 Val-72
5 Arg-23 Arg-27 11 Cys-73 Cys-84
6 Ser-28 Tyr-33 12 Glu-85 Thr-91
These and other modifications within the N-terminus spanning Gly- (-3) through Thr-91 are described in greater detail hereinafter.
B. Modifications at N-linked Glycosylation Sites
The protein variants of this invention may further contain no N-linked carbohydrate moieties or may be only partially glycosylated relative to natural human t-PA. A "partially glycosylated" protein, as the phrase is used herein, means a protein which contains fewer N-linked carbohydrate moieties than does fully-glycosylated native human t-PA. This absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at one or more of the concensus N-linked glycosylation recognition sites present in the native t-PA molecule. We have found that variant proteins of this invention embodying such modification at one or more N-linked glycosylation sites retain t-PA-type thrombolytic activity with greater fibrinolytic activity in certain cases, may be more readily produced in more homogeneous form than native t-PA, and in many cases have longer in vivo half-lives than native t-PA.
N-linked glycosylation recognition sites are presently believed to comprise tripeptide sequences which are specifically recog¬ nized by the appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or aspar- agine-X-serine, where X is usually any amino acid. Their location within the t-PA peptide sequence is shown in Table 1. A variety of amino acid substitutions or deletions at one or more of the three positions of a glycosylation recognition site results in non-glycosylation at the modified sequence. By way of example, Asn 117 an(i Asn-j_34 of t-PA have both been replaced with Thr in one embodiment and with Gin in another embodiment. At least in the case of the double Gin replacement, the resultant glycoprotein (Gln-j_ .7Gln-]_g4) should contain only one N-linked carbohydrate moiety (at sn44g) rather than two or three such moieties as in the case of native t-PA. Those skilled in the art will appreciate that analogous glycoproteins having the same Asn448 monoglyσosy- lation may be prepared by deletion of amino acids or substitution of other amino acids at positions 117 and 184 and/or by deleting or substituting one or more amino acids at other positions within the respective glycosylation recognitions sites, e.g. at Ser*j_19 and Serisg, as mentioned above and/or by substitution, or more preferably by deletion, at one or more of the "X" positions of the tripeptide sites. In another embodiment Asn at positions 117, 184 and 448 are replaced with Gin. The resultant variants should contain no N-linked carbohydrate moieties, rather than two or three such moieties as in the case of native t-PA. In other embodiments, potential glycosylation sites have been modified individually, for instance by replacing Asn, e.g. with Gin, at position 117 in one presently preferred embodiment, at position 184 in another embodiment and at position 448 in still another embodiment. This invention encompasses such non-glycos lated, monoglycoslyated, diglycosylated and triglycosylated t-PA variants.
Exemplary modifications at one or more of the three consensus N- linked glycosylation sequences. R1, R2 and R3 , as found in various embodiments of this invention are depicted below:
Exemplary ' Modifications at N-linked Glycosylation Sites
Rl 2 R3
(wt) (Asn Ser Ser) (Asn Gly Ser) (Asn Arg Thr)
I U Ser Ser V Gly Ser V Arg Thr
II Asn W Ser Asn X Ser Asn Y Thr
III Asn Ser Z Asn Gly Z Asn Arg U
IV Asn W Z Asn X Z Asn Y U
V - U * - * - - * -
VI - Asn * - * * - * *
VII u * * - - * - - *
VIII Asn * * * - * * -
IX _ - - - - - _ _ _.
-,— and = a peptide bond
* = any amino acid
U - any amino acid except Asn, Thr or Ser
Asn, or a peptide bond Ser, or a peptide bond Gly, or a peptide bond Arg, or a peptide bond Thr or Ser, or a peptide bond
=* wild type, i.e., prior to mutagenesis C. Modification at the Arg-275/Ile-276 Cleavage Site
In one aspect of this invention the variants are optionally modified at the proteolytic cleavage site spanning Arg-275 and Ile-276 by virtue of deletion of Arg-275 or substitution of another amino acid, preferably an amino acid other than Lys or His, for the Arg. Thr is at present an especially prefered replacement amino acid for Arg-2 5 in the various embodiments of this invention. Proteolytic cleavage at Arg-275 of native t-PA yields the so-called "two-chain" molecule, as is known in the art. Proteins of this invention which are characterized by modification at this cleavage site may be more readily produced in more homogeneous form than the corresponding protein without the cleavage site modification, and perhaps more importantly may possess an improved fibrinolytic profile and pharmacokinetic characteristic.
This invention thus provides a family of novel thrombolytic proteins related to human t-PA. This family comprises several genera of proteins.
In one embodiment the proteins are characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA, wherein Arg-275 is deleted or is replaced by a different amino acid, preferably other than lysine or histidine, and at least one of the consensus Asn-linked glycosylation sites is deleted or is modified to other than a consensus Asn-linked glycosylation sequence. Exemplary proteins of this embodiment are depicted in Table 1 below. By "characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA," as the phrase is used herein, we mean the peptide sequence of human t-PA, or a peptide sequence encoded by a DNA sequence encoding human t-PA or a DNA sequence capable of hybridizing thereto under stringent hybridization conditions. Thus the proteins of this invention include analogs of t-PA characterized by the various modifications or combinations of modifications as disclosed herein, which may also contain other variations, e.g. allelic variations or additional deletion(s) , substitution(s) or insertion(s) of amino acids which still retain thrombolytic activity, so long as the DNA encoding those proteins (prior to the modification of the invention) is still capable of hybridizing to a DNA sequence encoding human t-PA under stringent conditions.
Table 1: Illustrative Proteins Containing Modification at Arg-275 and at Least One N-linked Glycosylation Site
-3 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP
48 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRATCYEDQG
98 IS RGTWSTA ESGAECTNW- R^-ALAQKPYS GRRPDAIRLG LGNHNYCRNP
148 DRDSKPWCYV FKAGKYSSEF CSTPACSEGN SDCYFG-R2A YRGTHSLTES
198 GASCLPWNSM ILIGKVYTAQ NPSAQALGLG KHNYCRNPDG DAKPWCHVLK
248 NRTLTWEYCD VPSCSTCGLR QYSQPQFJIK GGLFADIASH PWQAAIFAKH
298 RRSPGERFLC GGILISSCWI LSAAHCFQER FPPHHLTVIL GRTYRWPGE
348 EEQKFEVEKY IVHKEFDDDT YDNDIALLQL KSDSSRCAQE SSWRTVCLP
398 PADLQLPDWT ECELSGYGKH EALSPFYSER LKEAHVRLYP SSRCTSQHLL
448 ~R3VTDNMLC AGDTRSGGPQ ANLHDACQGD SGGPLVCLND GRMTLVGIIS
498 WGLGCGQKDV PGVYTKVTNY LDWIRDNMRP
compound R2 R3 compound R-*- R^ R3 wt NSS NGS NRT 1-12 NSA NGS NRT
1-1 QSS NGS NRT 1-13 NSS NGA NRT
1-2 NSS QGS NRT 1-14 NSS NGS NRA
1-3 NSS NGS QRT 1-15 NSA NGA NRT
1-4 QSS QGS NRT 1-16 NSA NGS NRA
1-5 QSS NGS -RT 1-17 NSV NGS NRT
1-6 NSS -GS QRT 1-18 NSS NGV NRV
1-7 QSS -GS -RT 1-19 SS NGS NRT
1-8 1-20 TSS TGS NRT
1-9 N-Q N-S N-T 1-21 SS TGS QRT
1-10 N— N— N—
1-11 —Q —S —
J = other than Arg, preferably other than Arg, His or Lys R1, R2, and R3 are independently selected from the group consisting of a peptide bond, amino acid, dipeptide or tripeptide, and at least one of R1, R2 and R3 are other than consensus N-linked glycosylation sequences; "-", "—" and " " - a peptide bond. In a second embodiment the proteins are characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA wherein one or more amino acids are deleted within the N-terminal region from Gly-(-3) through Thr-91 and wherein (a) one or more Asn-linked glycosylation sites are optionally deleted or otherwise modified to other than a consensus Asn-linked glycosylation site, and/or (b) Arg-275 is optionally deleted or replaced by a different amino acid, preferably other than lysine or histidine. Exemplary proteins of this embodiment are shown below:
Illustrative Proteins Having N-terminal Deletions
The following proteins have the peptide sequence shown in Table 1, wherein R1, R2 and R3 represent the wt tripeptide sequences, but wherein the N-termini (Gly-(-3) through Thr-91) are replaced with: compound N-terminal sequence
D-l
GARSYQVI CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
D-2
GARSYQ SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
D-3
GARSYQVI
Figure imgf000011_0001
D-4 D TRA
D-5
D-6
GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS TRAT
"-" indicates site of an amino acid deletion This embodiment includes a subgenus of proteins wherein 1 to about 94 amino acids are deleted from the region Gly-(- 3) through Thr-91 and one or more of the Asn-linked glycosylation sites are deleted or otherwise modified to other than a consensus Asn-linked glycosylation sequence as previously described. Also included is a subgenus of compounds wherein 1 to about 94 amino acids are deleted from the region Gly-(-3) through Thr-9l, and Arg-275 is deleted or replaced with a different amino acid, preferably other than lysine or histidine. A further subgenus of this embodiment is characterized by a deletion of 1 to about 94 amino acids from within the region Gly-(-3) through Thr-91, deletion or modification of one or more of the Asn-linked glycosylation sites (see e.g. the table on page 6) and deletion of Arg-275 or replacement thereof with a different amino acid. Exemplary proteins of these subgenera are depicted in Tables 2 and 2.5, below.
This embodiment also includes a subgenus of proteins wherein the N-terminal deletion comprises a deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50. Also included is a subgenus of proteins wherein 1 to about 45 amino acids are deleted from within the region Ser-1 through Ser-50 and one or more glycosylation sites are modified as previously described. A further subgenus comprises proteins having a deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50, wherein Arg-275 is deleted or replaced with another amino acid. Additionally included is a subgenus having deletion of 1 to about 45 amino acids from within the region Ser-1 through Ser-50 and wherein both of (a) one or more glycosylation sites, and (b) Arg-275, are optionally modified as previously described. Exemplary proteins of these subgenera are depicted in Table 3, below, as well as in Tables 2 and 2.5. 11
Table 2:Illustrative Proteins Containing a Deletion of 1 to 94 Amino Acids at the N-Terminus and Optional Modification at Either or Both of Arg-275 and at Least One N-Linked Glycosylation Site
(for general sequence , see Table 1) amino compound J E1 R2 S3 terminus
( t) R NSS NGS NRT G-(-3) thru T-91
2-0 R NSS NGS NRT *
2-1 R QSS NGS NRT *
2-2 R NSS QGS NRT *
2-3 R NSS NGS QRT *
2-4 R NSS QGS QRT *
2-5 R QSS NGS QRT *
2-6 R QSS QGS NRT *
2-7 R QSS QGS QRT *
2-8 R QSS NGS -RT *
2-9 R NSS -GS QRT *
2-10 R QSS -GS -RT *
2-11 R — — - *
2-12 — wt Wt wt *
2-13 G wt wt wt *
2-14 A wt wt wt *
2-15 T wt wt wt *
2-16 # QSS NGS NRT *
2-17 # NSS QGS NRT *
2-18 # NSS NGS QRT *
2-19 # NSS QGS QRT *
2-20 # QSS NGS QRT *
2-21 # QSS QGS NRT *
2-22 # QSS QGS QRT *
2-23 # QSS NGS -RT *
2-24 # NSS -GS QRT *
2-25 # QSS -GS -RT *
2-26 # * Table 2 (Cont'd)
compound J s1 s2 s3 N-terminus
2-27 R U Ser Ser wt wt *
2-28 # U Ser Ser wt wt *
2-29 R Asn W Ser wt wt *
2-30 # Asn W Ser wt wt *
2-31 R Asn Ser Z wt wt *
2-32 # Asn Ser z wt wt *
2-33 R Asn W z wt wt *
2-34 # Asn W z wt wt *
2-35 R U A wt wt *
2-36 # U A wt wt *
2-37 R - Asn A wt wt *
2-38 # - Asn A t wt *
2-39 R U A A wt wt *
2-40 # U A A wt wt *
2-41 R Asn - wt wt *
2-42 # Asn A - wt wt *
2-43 R - wt wt *
2-44 # — wt wt *
* indicates a N-terminus which is different at at least one amino acid relative to the wt N-terminus, illustrative examples of which are depicted below in Table 2.5 and in Tables 3-5 & 6-B; "#" represents a peptide bond or an amino acid other than Arginine (R) ; "-" represents a peptide bond; U is any amino acid except Asn, Thr or Ser; W is a peptide bond or any amino acid except Ser; Z is a peptide bond or any amino acid except Thr or Ser; A is any amino acid; and wt indicates wild type. Table 2.5:Illustrative N-Termini Containing a Deletion of 1-94 Amino Acids
N-terminus Designation
_J
GARSYQVICR DEKTQM WLRPVLR SNRVEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
2 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SG P
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
3 GAR NRVEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
4 GAR VEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
5 GAR EYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
6 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCW HSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
7 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWC HSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
8 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWC GRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
9 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN —RAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
10 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS CQQALY FSDFVCQCPE GFAGKCCEID TRAT
11 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVC EID TRAT
12 GARSYQVIC N SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
13 GARSYQVI— PRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
14 GARSYQVICR DEK VEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT 15 GARSYQVICR DEKTQMIYQQ H CWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
16 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQ CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
17 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRC- QCPE GFAGKCCEID TRAT
18 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSC PE GFAGKCCEID TRAT
19 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRC VCQCPE GFAGKCCEID TRAT
20 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS QCPE GFAGKCCEID*TRAT
21 GARSYQVI CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
22 GARSYQVI— D TRAT
23 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP
VKS TRAT
424 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
425 GARSYQVICR DEKTQMIYQQ HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
426 GARSYQVICR DEKTQMIYQQ HQSWLRP—R SNR—YCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
427 GARSYQVICR DEKTQMIYQ- HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
428 GARSYQVICR DEKTQMI—Q HQSWLRP--R SNR—YCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
Specific proteins of this invention may be referred to by a 3-part designation comprising a compound number from Table 2 followed by a designation of N-terminus and then identification of position 275. For example, compound No. 2-ll/N-6/Arg designates a protein wherein the 3 glycosylation sites are deleted ("2-11", See Table 2 ) , C-36 through C-43 are deleted (N-terminus #N-6) and Arg-275 is retained. Table 3: Exemplary Proteins Having a Deletion of 1—45 Amino Acids in the Region Ser-1 Through Ser-50 and a Modification at either or both of (a) Arg-275 and (b) At Least One N-linked Glycosylation Site
(for general sequence, see Table 1)
Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild type (wt) sequence of Gly-(-3) through Thr-91:
N-terminus Designation
_ L_
24 GAR-YQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
25 GARS-QVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
26 GARSY-VICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
27 GARSYQ-ICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
28 GARSYQV-CR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
29 GARSYQVI-R DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
30 GARSYQVIC- DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
31 GARSYQVICR -EKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
32 GARSYQVICR D-KTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
33 GARSYQVICR DE-TQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
34 GARSYQVICR DEK-QMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
35 GARSYQVICR DEKT-MIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 3 (Cont'd)
36 GARSYQVICR DEKTQ-IYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
37 GARSYQVICR DEKTQM-YQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
38 GARSYQVICR DEKTQMI-QQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
39 GARSYQVICR DEKTQMIY-Q HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
40 GARSYQVICR DEKTQMIYQ- HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
41 GARSYQVICR DEKTQMIYQQ -QSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
42 GARSYQVICR DEKTQMIYQQ H-SWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
43 GARSYQVICR DEKTQMIYQQ HQ-WLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
44 GARSYQVICR DEKTQMIYQQ HQS-LRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
45 GARSYQVICR DEKTQMIYQQ HQSW-RPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
46 GARSYQVICR DEKTQMIYQQ HQSWL-PVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
47 GARSYQVICR DEKTQMIYQQ HQSWLR-VLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
48 GARSYQVICR DEKTQMIYQQ HQSWLRP-LR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
49 GARSYQVICR DEKTQMIYQQ HQSWLRPV-R SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
50 GARSYQVICR DEKTQMIYQQ HQSWLRPVL- SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
51 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR -NRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 3 (cont'd)
52 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR S-RVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
53 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SN-VEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
54 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNR-EYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
55 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRV-YCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
56 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVE-CWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
57 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEY-WCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
58 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYC-CN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
59 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCW-N SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
60 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWC- SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
61 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN -GRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
62 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN S-RAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
63 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SG-AQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
64 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGR-QCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
65 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRA-CHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
66 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQ-HSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
67 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQC-SVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 3 (cont'd)
68 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCH-VP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
69 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHS-P VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
70 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV- VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
71 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP -KSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
72 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP V-SCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
73 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VK-CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
74 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS-SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
With reference to the modified N-termini depicted in Table 3, it should be understood that more than one amino acid may be deleted. Where multiple amino acids are deleted, they may be adjacent to one another, or separated by one or more other amino acids. In designating compounds with such N-termini we indicate the size of the deletion by "Δn" where "n" is the number of amino acids deleted. For example, N- terminus #24 wherein one amino acid is deleted as shown in Table 3 may be referred to as "N-24Δ1". Where two amino acids are deleted, e.g. S-l and Y-2, the N-terminus is referred to as "N-24Δ2", etc. Where combinations of deletions are made, e.g. wherein S-l, Y-2 and 1-5 are deleted, the N- terminus may be referred to as "N-24Δ2, N-28Δ1". Asindicated in the text following Table 2.5, specific compounds are designated by a 3-part code comprising a compound number from Table 2 followed by a designation of N-terminus #, e.g. from Table 2.5, and then identification of the status of position 275. Compound 2-26/N-24Δ2, N-28Δ1/- thus designates the protein wherein all three glycosylation sites; R-275; S-l, Y-2 and 1-5 are deleted.
This embodiment further includes a subgenus of proteins wherein 1 to about 41 amino acids are deleted from the region Cys-51 through Thr-91. Proteins of this subgenus may optionally be modified such that Arg-275 is deleted or replaced with a different amino acid, preferably other than lysine or histidine. Proteins of this subgenus may, as an alternative to or in addition to the modification at Arg- 275, be modified such that (a) one or more N-linked glycosylation sites are abolished, as previously described and/or (b) one or more amino acids are deleted within the region Gly-(-3) through Ser-50. Exemplary proteins of this subgenus are similar to those depicted in Tables 2 and 3, but contain N-termini such as those depicted in Table 4, below.
Table 4: Exemplary Proteins Having a Deletion of 1—41 Amino Acids From the Region Cys-51 through Thr-91
(for general sequence, see Table 1)
Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild-type (wt) sequence of Gly-(-3) through Thr-91:
N-terminus Designation
#
75 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP
VKS TRAT
10 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS CQQALY FSDFVCQCPE GFAGKCCEID TRAT
11 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVC EID TRAT
17 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRC QCPE GFAGKCCEID TRAT Table 4 (cont'd)
18 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSC PE GFAGKCCEID TRAT
19 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRC- VCQCPE GFAGKCCEID TRAT
20 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS QCPE GFAGKCCEID TRAT
21 GARSYQVIC- PRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
22 GARSYQVI— D TRAT
23 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS TRAT
76 GARSYQVI SWLRPVLR SN CHSVP
VK QQALY FSDFVCQCPE GFAGKCCEID TRAT
74 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS-SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
77 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSC-EPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
78 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCS-PRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
79 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSE-RCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
80 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEP-CF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
81 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPR-F NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
82 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRC- NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
83 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF -GGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
84 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF N-GTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
85 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NG-TCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 4 (cont'd)
86 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGG-CQQALY FSDFVCQCPE GFAGKCCEID TRAT
87 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGT-QQALY FSDFVCQCPE GFAGKCCEID TRAT
88 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTC-QALY FSDFVCQCPE GFAGKCCEID TRAT
89 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQ-ALY FSDFVCQCPE GFAGKCCEID TRAT
90 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQ-LY FSDFVCQCPE GFAGKCCEID TRAT
91 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQA-Y FSDFVCQCPE GFAGKCCEID TRAT
92 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQAL- FSDFVCQCPE GFAGKCCEID TRAT
93 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY -SDFVCQCPE GFAGKCCEID TRAT
94 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY F-DFVCQCPE GFAGKCCEID TRAT
95 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FS-FVCQCPE GFAGKCCEID TRAT
96 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSD-VCQCPE GFAGKCCEID TRAT
97 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDF-CQCPE GFAGKCCEID TRAT
98 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFV-QCPE GFAGKCCEID TRAT
99 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVC-CPE GFAGKCCEID TRAT
100 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQ-PE GFAGKCCEID TRAT
101 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCP- GFAGKCCEID TRAT Table 4 (cont'd)
102 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE -FAGKCCEID TRAT
103 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE G-AGKCCEID TRAT
104 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GF-GKCCEID TRAT
105 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFA-KCCEID TRAT
106 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAG-CCEID TRAT
107 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGK-CEID TRAT
108 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKC-EID TRAT
109 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCC-ID TRAT
110 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCE-D TRAT
111 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEI- TRAT
312 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQAL DFVCQCPE GFAGKCCEID TRAT
313 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY —DFVCQCPE GFAGKCCEID TRAT
314 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQAL- -SDFVCQCPE GFAGKCCEID TRAT
315 GARSYQVI CSEPRCF NGGTCQQAL- FSDFVCQCPE GFAGKCCEID TRAT
316 GARSYQVI CSEPRCF NGGTCQQALY -SDFVCQCPE GFAGKCCEID TRAT
317 GARSYQVI CSEPRCF NGGTCQQALY F-DFVCQCPE GFAGKCCEID TRAT Table 4 (cont'd)
318 GARSYQVI— CSEPRCF NGGTCQQALY —DFVCQCPE GFAGKCCEID TRAT
319 GARSYQVI — CSEPRCF NGGTCQQAL DFVCQCPE GFAGKCCEID TRAT
With reference to Table 4, above, it should be noted that several subclasses of proteins are disclosed. For example, proteins containing a deletion of 1-37 amino acids (individually, consecutively, or in combination) from Cys- 51 through Asp-87 are depicted, as are proteins containing a deletion of 1-37 amino acids from Cys-51 through Arg-87 and a deletion of one or more amino acids within the region Gly-(-3) through Cys-51 (See N-76) . With reference to compounds containing an N-terminus selected from N-termini N-76 through N-lll it should be understood that more than one amino acid may be deleted. For example, N-77 wherein one amino acid is deleted, as shown in Table 4, may be referred to as "N-77Δ1". Where six amino acids are deleted, e.g. S-52 through F-57, the N-terminus is referred to as "N-77Δ6", etc. Specific proteins are designated as described following Tables 2 an 3. Examples wherein the three glycosylation sites and Arg-275 are deleted are shown below:
Compound Designation Deletions
2-26/N-75/- C-51 through D-87
2-26/N-74Δ6/- C-51 through C-56
2-27/N-74Δ1, N-76Δ6/- C-51,E-53 through N-58
Illustrative compounds wherein no modifications are present at glycosylation sites, but which contain various deletions are shown below: Compound Designation # Modification: deletion of :
2-0/N-42 /Arg P-47 through S-50
2-0/N-425/Arg L-26 & V-31
2-0/N-426/Arg V-25 , L-26 , V-31 & E-32
2-0/N-427/Arg Q-17 , L-26 & V-31
2-0/N-428/Arg Y-15 , Q-16 , V-25 , L-26 , V-31&E-32
2-0/N-63Δ2 ,N-92Δ3/Arg R-40 ,A-41 , Y-67 , F-68 & S-69
2-0/N-63Δl, N-92Δ2/Arg R-40 , Y-67 & F-68
2-0/N-63Δl ,N-92Δl/Arg R-40 & Y-67
Illustrative compounds with the same deletions as above, but whi ch are al s o modi f ied at the f irst N- l inked glycosylation site, here by replacing Asn-117 with Gin, are listed below:
2-l/N-424/Arg 2-l/N-425/Arg 2-l/N-426/Arg 2-l/N-427/Arg 2-l/N-428/Arg 2-1/N-63Δ2,N-92Δ3/Arg 2-l/N-63Δl,N-92Δ2/Arg 2-1/N-63Δ1,N-92Δ1/Arg
Illustrative compounds with the same deletions as above, but which are also modified at all three glycosylation sites, here by replacement of Asn's with Gin's, are listed below:
2-7/N-424/Arg 2-7/N-425/Arg 2-7/N-426/Arg 2-7/N-427/Arg 2-7/N-428/Arg 2-7/N-63Δ2,N-92Δ3/Arg 2-7/N-63Δl,N-92Δ2/Arg 2-7/N-63Δ1,N-92Δ1/Arg
Thus, this embodiment further includes a subgenus of proteins wherein one or more deletions of less than about 20 amino acids are present within the region Gly-(-3) through Thr- 91. Proteins of this subgenus may also be modified at Arg- 275 and/or at one or more of the Asn-linked glycosylation sites. Exemplary proteins of this subgenus are similar to those depicted in Tables 2 through 4, but contain in place of the wild type N-terminus, an N-terminus such as those depicted in Table 5, below. Additional exemplary compounds of this subgenus are also listed above by their 3-part code designations.
In a third embodiment the proteins are characterized by a peptide sequence substantially the same as. the peptide sequence of human t-PA wherein different amino acids are substituted for 1-94 of the amino acids in the region Gly- (-3) through Thr-91. This embodiment includes a subgenus of compounds characterized by replacement of one or more amino acids within the above-mentioned N-terminus and by modification at Arg-275 as previously described. Also included is a subgenus of compounds characterized by the above-mentioned replacement of one or more amino acids within the N-terminus and modification, as previously described, at one or more of the consensus Asn-linked glycosylation sites. A further subgenus of this embodiments is characterized by substitution of one or more amino acids within the N-terminus, and modifications as previously described, at both Arg-275 and at one or more of the N- Linked glycosylation sites. In one aspect of this embodiment the amino acid substitution(s) is/are within the region Gly-(-3) through Ser-50, with or without modification at Arg-275 and/or one or more of the N-linked glycosylation sites. In another aspect, the amino acid substitution(s) is/are within the region Cys-51 through Thr-91, again, with or without modification at Arg-275 and/or at one or more of the N-linked glycosylation sites. In a further aspect, one to about eleven, preferably one to about 6 amino acids are replaced within one or more of the following regions, again with or without the other above- mentioned modification(s) : 26
reαion - from to reαion from to
1 Gly-(-3) Gln-3 7 Cys-34 Cys-43
2 Val-4 Lys-10 8 His-44 Ser-50
3 Thr-11 His-18 9 Cys-51 Cys-62
4 Gln-19 Leu-22 10 Gln-63 Val-72
5 Arg-23 Arg-27 11 Cys-73 Cys-84
6 Ser-28 Tyr-33 12 GlU-85 Thr-91
In a further aspect of this embodiment, the substitution(s) is/are present in one or more of the following regions: R-7 through S-20, W-21 through Y-33, N-37 through Q-42, and H- 44 through S-50. In an additional aspect of this embodiment, the N-terminus is modified, again, by substitution for one to about eleven, preferably one to about six, amino acids in one or more of the above defined regions, and is further modified by deletion of one to 93, preferably 1 to about 45, and more preferably 1 to about 15, amino acids.
Illustrative amino acid substitutions are shown in Table 6- A, below, and exemplary proteins are depicted in Table 6-B. Of the replacement amino acids for R-40, A-41 and Q-42, presented in Table 6-A, S is a preferred replacement for R-40, and V and L are preferred replacements for A-41 and Q-42, respectively. It should be noted that proteins of this invention embodying the substitutions identified for R-40, A-41 and Q-42 in Table 6A are presently preferred, alone, or, as in other aspects and subgenera of this embodiment, in combination with other substitution(s) and/or deletions within the N-terminus, and/or modifications at R-275 and/or at at least one glycosylation site. It is contemplated that to the extent that our proteins are modified by substitution rather than deletion, our proteins retain more of the native t-PA conformation and selectively retain more of the desirable biological activities of native t-PA.
Table 5: Exemplary Proteins Having One or More Deletions of Less Than -20 Amino Acids Within the Region Gly-(-3) through Thr-91
(for general sequence, see Table 1)
Illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild type (wt) sequence of Gly-(-3) through Thr-91:
N-terminus Designation
#
112 GARSYQVICR QSWLRPVLR SNRVEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
113 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR GRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
114 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS CQQALY FSDFVCQCPE GFAGKCCEID TRAT
115 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDF CCEID TRAT
116 GARSYQVICR HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP
VKS GGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
117 GARSYQVICR HQSWLRPVLR SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
118 GARSYQVICR HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSDFV CCEID TRAT
119 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SGRAQCHSVP
VKSCSEPRCF NGGTCQQALY FSD GKCCEID TRAT
120 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SGRAQCHSVP
VKSCS CQQALY FSDFVCQCPE GFAGKCCEID TRAT
121 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCS GGTCQQALY FSDFV CCEID TRAT Table 5 (cont'd) And, with reference to Tables 2.5, 3 and 4:
N-l N-2
N-6 through N-ll N-15 through N-17 N-2 Δ1 through Δ19 N-27Δ1 through Δ19 N-44Δ1 through Δ19 N-73Δ1 through Δ19 N-95Δ1 through Δ19 NlllΔl through Δ5
Table 6-A: Illustrative Amino Acid Substitutions
wt —> replacement wt —>replacement
C-6 S,T,G or A
R-7 S,T,Q,N,G,H,D or K S-52 G,A,Q,L,V,I or
D-8 It II E-53 S,T,Q,N,G,H,D 0
E-9 II II P-54 A,G,Y,D or S
K-10 II II C-56 S,T,G or A
T-ll N,S,L,G or A R-55 S,T,Q,N,G,H,D or K
T-ll N,S,L,G or A F-57 Y,I,W,H,D or R
Q-12 N,S,L,G,A or T N-58 G,A,Q,L,V,I or T
M-13 II II G-59 A,S,T,D,V or P
1-14 II II G-60 II If
Q-16 11 II T-61 N,S,L,G or A
Q-17 II II C-62 S,T,G or A Q-63 N,S,L,G,A or T
H-18 II II Q-64 II II
R-23 S,T,Q,N,G,H,D or K A-65 G,S,T,H,N or Q
R-27 II II L-66 N,S,L,G,A or T
R-30 II II L-67 Y,I,W,H,D or R
V-31 II II F-68 II II
E-32 S,T,Q,N,G,H,D or K S-69 G,A,Q,L,V,I or T
Y-33 F,S,H or L D-70 S,T,Q,N,G,H,D or K
C-34 S,T,G or A
W-35 T,V,Ior Q
C-36 S,T,G or A F-71 Y,I,W,H,D or R
N-37 G,A,Q,L,V,I or T V-72 N,S,L,G,A or T C-73 S,T G or A
S-38 II II Q-74 N,S,L,G or A C-75 S, T, G or A
G-39 A,S,T,D,V or P P-76 A,G,Y,D or S
R-40 S,T,N,G,K or D E-77 S,T,Q,N,G,H,D or K
A-41 G,S,T,H,N or Q G-78 A,S,T,D,V or P
Q-42 N,S,L,G,A or T F-79 Y,I,W,H,D or R
H-44 II II A-80 G,S,T,H,N or Q
S-45 G,A,Q,L,V,I or T G-81 A,S,T,D,V or P
V-46 N,S,L,G,A orT K-82 S,T,Q,N,G,H,D or K
P-47 A,G,Y,D or S C-83 S, T, G or A
V-48 N,J,L,G,A or T C-84 S, T, G or A
K-49 S,T,Q,N,G,H,D orK E-85 S,T,Q,N,G,H,D or K
S-50 G,A,Q,L,V,I orT 1-86 N,S,L,G,A or T
C-51 S, T, G or A D-87 S,T,Q,N,G,H,D or K T-88 N,S,L,G or A R-89 S,T,N,G or D A-90 G,S,T,H,N or Q
T-91 N,S,L,G or A Table 6-B: Exemplary Proteins Containing Substitution for one or more Amino Acids Within the Region Gly-(-3) through Thr- 91
(for general sequence, see Table 1) illustrative proteins are as defined in Table 2, but with the following N-termini replacing the wild type (wt) sequence of Gly-(-3) through Thr-91
N-terminus Designation
_L_
122 GARGYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY _FSDFVCQCPE GFAGKCCEID TRAT
123 GARSFQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
124 GARSYNVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
125 GARSYQJICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
126 GARSYQVSCR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
127 GARSYQVISR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
128 GARSYQVICT DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
129 GARSYQVICR NEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
130 GARSYQVICR DQKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
131 GARSYQVICR DETTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
132 GARSYQVICR DEKAQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
133 GARSYQVICR DEKTLMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 6-B (cont ' d)
134 GARSYQVICR DEKTQGIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
135 GARSYQVICR DEKTQMAYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
136 GARSYQVICR DEKTQMISQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
137 GARSYQVICR DEKTQMIYLQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
138 GARSYQVICR DEKTQMIYQL HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
139 GARSYQVICR DEKTQMIYQQ NQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
140 GARSYQVICR DEKTQMIYQQ HDSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
141 GARSYQVICR DEKTQMIYQQ HQNWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
142 GARSYQVICR DEKTQMIYQQ HQSYLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
143 GARSYQVICR DEKTQMIYQQ HQSWERPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
144 GARSYQVICR DEKTQMIYQQ HQSWLGPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
145 GARSYQVICR DEKTQMIYQQ HQSWLRTVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
146 GARSYQVICR DEKTQMIYQQ HQSWLRPYLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
147 GARSYQVICR DEKTQMIYQQ HQSWLRPVDR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
148 GARSYQVICR DEKTQMIYQQ HQSWLRPVLS SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
149 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR PNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
150 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SDRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 6-B (cont'd)
151 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNSVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
152 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRLEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
153 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVSYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
154 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVESCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
155 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYSWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
156 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCTCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
157 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWTN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
158 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCD SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
159 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN PGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
160 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SARAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
161 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGSAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
162 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRVQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
163 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRALCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
164 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQTHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
165 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCSSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
166 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHIVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT Table 6-B (cont'd)
167 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSNP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
168 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVD VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
169 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP NKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
170 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VDSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
171 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKVCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
172 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VTSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
173 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVQYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
174 GARSYQVICR NEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
175 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQAKY FSDFVCQCPE GFAGKCCEID TRAT
176 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALA FSDFVCQCPE GFAGKCCEID TRAT
177 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALG FSDFVCQCPE GFAGKCCEID TRAT
178 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY GSDFVCQCPE GFAGKCCEID TRAT
179 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY ASDFVCQCPE GFAGKCCEID TRAT
180 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSFFVCQCPE GFAGKCCEID TRAT
181 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEGRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
182 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGHCCEID TRAT
183 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGQCCEID TRAT 34
184 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGNCCEID TRAT
185 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPT GFAGKCCEID TRAT
186 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEIH TRAT
187 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPTCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
188 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSSFVCQCPE GFAGKCCEID TRAT
189 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPHCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
190 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF VGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
191 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSTPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
192 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSQPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
193 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VHSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
194 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY HSDFVCQCPE GFAGKCCEID TRAT
195 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY ISDFVCQCPE GFAGKCCEID TRAT
196 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY PSDFVCQCPE GFAGKCCEID TRAT
197 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY RSDFVCQCPE GFAGKCCEID TRAT
198 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FIDFVCQCPE GFAGKCCEID TRAT
199 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRALCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
200 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQELY FSDFVCQCPE GFAGKCCEID TRAT 201 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQANY FSDFVCQCPE GFAGKCCEID TRAT
201 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKSCSEPSCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT
Proteins of this invention embodying amino acid substitution(s) may be designated by a 3-part code as previously described.
It should be understood of course that more than one wt amino acid may be replaced. In designating compounds with such N- termini we indicate the number of substitutions by "sn" where
"n" is the number of amino acids replaced, e.g. with the replacement amino acids such as (but not limited to) those depicted in Table 6-A. For example, N-terminus #N-122sl designates N-terminus 122 as depicted in Table 6-B, while N- terminus #N-122s4 designates that N-terminus wherein S-l is replaced with G and the following three wt amino acids are replaced with other amino acids. Proteins of this embodiment containing multiple amino acid substitutions may be designated by a string of N-terminus designations indicating specific replacements, as follows:
Illustrative compounds with multiple N-terminal substitutions, with Asn-117 replaced with Gin and Arg-275 replaced with Thr: substitutions: compound # wt replacement
2-16/N-128,N-130/Thr R-7 T
E-9 Q
2-16/N-129,N-130/Thr D-8 N
E-9 Q
2-16/N-130,N-131/Thr E-9 Q K-10 T
2-16/N-131,N-153/Thr K-10 T
E-32 s
2-16/N-161,N-165/Thr " R-40 s H-44 s 36
(cont'd) -16/N-159,N-161/Thr S-38 P
R-40 s -16/N-161,N-163/Thr R-40 s
Q-42 L -16/N-161,N-172/Thr R-40 s
K-49 T -16/N-138,N-142/Thr Q-17 L
W-21 Y -16/N-133,N-138/Thr Q-12 L
Q-17 L -16/N-128,N-133/Thr R-7 T
Q-12 L -16/N-131,N-162/Thr K-10 T
A-41 V -16/N-165,N-170/Thr H- 4 s
K-49 D -16/N-143,N-146/Thr L-22 E
V-25 Y -16/N-173,N-165/Thr E-32 Q
H-44 s -16/N-165,N-172/Thr H-44 s
K-49 T
2-16/N-148,N-151/Thr R-27 s
R-30 s
2-16/N-143,N-153/Thr L-22 E
E-32 s
2-16/N-161,N-170/Thr R-40 s
K-49 D
2-16/N-153,N-161/Thr E-32 S
R-40 S
2-16/N-129,N-131/Thr D-8 N
K-10 T (cont 'd) 2-16/N-131 , N-143/Thr K-10 T
L-22 E
2-16/N-128,N-131/Thr R-7 T
K-10 T
2-16/N-128,N-161/Thr 5-7 T
R-40 s
2-16/N-175,N-176/Thr L-66 K
Y-67 A -16/N-176,N-178/Thr Y-67 A
F-68 G -16/N-177 ,N-170/Thr Y-67 G
D-70 F -16/N-177,N-179/Thr Y-67 G
F-68 A -16/N-181,N-182/Thr P-54 G
K-82 H -16/N-202,N-183/Thr R-55 S
K-82 Q -16/N-185,N-184/Thr E-77 T
K-82 N -16/N-184,N-186/Thr K-82 N
D-87 H -16/N-187,N-186/Thr R-55 T
D-87 H -16/N-188 , N-201/Thr L-66 N
D-70 S -16/N-189 ,N-190/Thr R-55 H
N-58 V -16/N-191,N-189/Thr E-53 T
R-55 H -16/N-193 , N-192/Thr K-49. H
E-53 Q 38
-16/N-161,N-175/Thr R-40 S
L-66 K -16/N-148,N-194/Thr R-27 S
F-68 H -16/N-131,N-184/Thr K-10 T
F-68 H -16/N-174,N-195/Thr D-8 N
F-68 I -16/N-144,N-195/Thr R-23 G
F-68 I -16/N-151,N-196/Thr R-30 s
F-68 P -16/N-161,N-198/Thr R-40 s
S-69 I -16/N-199,N-200/Thr Q-42 L
A-65 E -16/N-161,N-197/Thr R-40 s
F-68 R
One subgenus of particular interest is characterized by replacement of one or more of Y-67 through S-69, with optional deletion of, and/or substitution for, one or more amino acids from Gly-(-3) through L-66, with or without modification as described above at one or more glycosylation sites and/or at Arg-275.
In one aspect of the invention the proteins contain at least one so-called "complex carbohydrate" sugar moiety characteristic of mammalian glycoproteins. As exemplified in greater detail below, such "complex carbohydrate" glycoproteins may be produced by expression of a DNA molecule encoding the desired polypeptide sequence in mammalian host cells. Suitable mammalian host cells and methods for transformation, culture, amplification, screening, andproductproductionandpurification are known in the art. See e.g. Gething and Sambrook, Nature 293:620-625 (1981), or alternatively, Kaufman et al.. Molecular and Cellular Biology 5 (7) :1750-1759 (1985) or Howley et al., U.S. Patent No. 4,419,446.
A further aspect of this invention involves t-PA variants as defined above in which each carbohydrate moiety is a processed form of the initial dolicol-linked oligosaccharide characteristic of insect cell-produced glycoproteins, as opposed to a "complex carbohydrate" substituent characteristic of mammalian glycoproteins, including mammalian derived t-PA. Such insect cell-type glycosylation is referred to herein as "high mannose" carbohydrate for the sake of simplicity. For the purpose of this disclosure, complex and high mannose carbohydrates are as defined in Kornfeld et al. , Ann. Rev. Biochem. 54: 631-64 (1985) . "High mannose" variants in accordance with this invention are characterized by a variant polypeptide backbone as described above which contains at least one occupied N-linked glycosylation site. Such variants may be produced by expression of a DNA sequence encoding the variant in insect host cells. Suitable insect host cells as well as methods and materials for transfor- mation/transfection, insect cell culture, screening and product production and purification useful in practicing this aspect of the invention are known in the art. Glycoproteins so produced also differ from natural t-PA and from t-PA produced heretofore by recombinant engineering techniques in mammalian cells in that the variants of this aspect of the invention do not contain terminal sialic acid or galactose substituents on the carbohydrate moieties or other protein modifications characteristic of mammalian derived glycoproteins.
The proteins of this invention which contain no N-linked carbohydrate moieties may also be produced by expressing a DNA molecule encoding the desired variant, e.g. compounds 1-6 through 1-11 of Table 1, in mammalian, insect, yeast or bacterial host cells, with eucaryotic host cells being presently preferred. As indicated above suitable mammalian and insect host cells, and in addition, suitable yeast and bacterial host cells, as well as methods and materials for transformation/transfection, cell culture, screening and product production and purification useful in practicing this aspect of the invention are also known in the art.
Additionally, as should be clear to those of ordinary skill in this art, this invention also contemplates other t-PA variants which are characterized, instead of by amino acid deletion within the region Gly_3 or Ser^ through Thrg^, by one or more amino acid substitutions within that region, especially in the region Argγ through Ser^Q , or by a combination of deletion and substitution. cDNAs encoding these compounds may be readily prepared, e.g., by methods closely analogous to the muta- genesis procedures described herein using appropriate mutagenesis oligonucleotides. The cDNAs may be optionally 41 mutagenized at one or more of the codons for R1, R2 and R3, and/or Arg-275, and may be inserted into expression vectors and expressed in host cells by the methods disclosed herein. It is contemplated that these proteins will share the adavantageous pharmacokinetic properties of the other compounds of this invention, and perhaps avoid undue anti- genicity upon administration in pharmacuetical preparations analogous to those disclosed herein.
As should be evident from the preceding, all variants of this invention are prepared by recombinant techniques using DNA sequences encoding the analogs which may also contain fewer or no potential glycosylation sites relative to natural human t-PA and/or deletion or replacement of Arg-275. Such DNA sequences may be produced by conventional site-directed mutagenesis of DNA sequences encoding t-PA.
DNA sequences encoding t-PA have been cloned and characterized. See e.g., D. Pennica et al.. Nature (London) 101:214(1983) and R. Kaufman et al., Mol. Cell. Biol.5_(7) : 1750 (1985). One clone, ATCC 39891, which encodes a thrombol- ytically active t-PA analog is unique in that it contains a Met residue at position 245 rather than Val. Typically, the DNA sequence encodes a leader sequence which is processed, i.e., recognized and removed by the host cell, followed by the amino acid residues of the full length protein, beginning with Gly.Ala.Arg.Ser.Tyr.Gin.. . Depending on the media and host cell in which the DNA sequence is expresed, the protein so produced may begin with the Gly. la.Arg amino terminus or be further processed such that the first three amino acid residues are proteolytically removed. In the latter case, the mature protein has an amino terminus comprising: Ser.Tyr.Gin.Leu... . t-PA variants having either amino terminus are thrombolytically active and are encompassed by this inven¬ tion. Variants in accord with the present invention also 42 include proteins having either Met245 or Val 245' as well as other variants, e.g. allelic variations or other amino acid substitutions or deletions, which still retain thrombolytic activity.
This invention also encompasses compounds as described above which contain a further modification in the polypeptide domain spanning Asn-218 through Thr-220. Specifically, compounds of this embodiment are further characterized by an amino acid other than Asn or a peptide bond at position 218 and/or an amino acid other than Pro or a peptide bond at position 219 and/or an amino acid other than Ser or Thr or a peptide bond at position 220. Compounds of this embodiment thus lack the consensus N-linked glycosylation site which is typically not glycosylated in t-PA produced by melanoma-derived mammalian cells.
As mentioned above, DNA sequences encoding individual variants of this invention may be produced by conventional site-directed mutagenesis of a DNA sequence encoding human t-PA or analogs or variants thereof. Such methods of mutagenesis include the M13 system of Zoller and Smith, Nucleic Acids Res. 10.:6487-6500 (1982); Methods Enzymol. 100: 468-500 (1983) ; and DNA 1:479-488 (1984), using single stranded DNA and the method of Morinaga et al.. Bio/tech¬ nology, 636-639 (July 1984), using hetεroduplexed DNA. Several exemplary oligonucleotides used in accordance with such methods to effect deletions in the N-terminus or to convert an asparagine residue to threonine or glutamine, for example, are shown in Table 7. It should be understood, of course, that DNA encoding each of the glycoproteins of this invention may be analogously produced by one skilled in the art through site-directed mutagenesis using(an) appropriately chosen oligonucleotide(s) . Expression of the DNA by conven¬ tional means in a mammalian, yeast, bacterial, or insect host 43 cell system yields the desired variant. Mammalian expression systems and the variants obtained thereby are presently preferred.
The mammalian cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol Biol.. 159:51-521 (1982); Kaufman, Proc Natl. Acad. Sci. 82.:689-693 (1985).
Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in. vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoetic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.
The host cells preferably will be established mammalian cell lines. For stable integration of the vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO (Chinese hamster Ovary) cells are presently preferred. Alternatively, the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al.. Cell, 36:391-401 (1984) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other usable mammalian cell lines include but are not limited to, HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cells lines and the like.
Stable transformants then are screened for expression of the product by standard immunological or enzymatic assays. The 44 presence of the DNA encoding the variant proteins may be detected by standard procedures such as Southern blotting. Transient expression of the DNA encoding the variants during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunologic assay of the proteins in the culture medium.
In the case of bacterial expression, the DNA encoding the variant may be further modified to contain different codons for bacterial expression as is known in the art and- preferably is operatively linked in-frame to a nucleotide sequence encoding a secretory leader polypeptide permitting bacterial expression, secretion and processing of the mature variant protein, also as is known in the art. The compounds expressed in mammalian, insect, yeast or bacterial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.
These compounds have been found to bind to monoclonal antibodies directed to human t-PA, and may thus be recovered and/or purified by immunoaffinity chromatography using such antibodies. Furthermore, these compounds possess t-PA-type enzymatic activity, i.e., compounds of this invention effect¬ ively activate plasminogen in the presence of fibrin to evoke fibrinolysis, as measured in an indirect assay using the plasmin chromogenic substrate S-2251 as is known in the art.
This invention also encompasses compositions for thrombolytic therapy which comprise a therapeutically effective amount of a variant described above in admixture with a pharmaceutically acceptable parenteral carrier. Such composition can be used in the same manner as that described for human t-PA and should other mammals known to be subject to thrombotic cardiovascular problems. It is contemplated that the compositions will be used both for treating and desirably for preventing thrombotic conditions. The exact doseage and method of administration will be determined by the attending physician depending on the potency and pharmacokinetic profile of the particular compound as well as on various factors which modify the actions of drugs, for example, body weight, sex, diet, time of admini¬ stration, drug combination-, reaction sensitivities and severity of the particular case.
The following examples are given to illustrate embodiments of the invention. It will be understood that these examples are illustrative, and the invention is not to be considered as restricted thereto except as indicated in the appended claims.
In each of the examples involving insect cell expression, the nuclear polyhedrosis virus used was the L-l variant of the Autoαrapha Californica, and the insect cell line used was the spodoptera frugiperda IPLB-SF21 cell line (Vaughn, J.L. et al.. In Vitro (1977) 13, 213-217). The cell and viral manipulations were as detailed in the literature (Pennock G.D., et al., supra; Miller, D.W., Safer, P., and Miller, L.K., Genetic Engineering. Vol. 8, pages 277-298, J.K. Setlow and A. Hollaender, eds. Plenum Press, 1986) . The RF ml3 vectors, mpl8 and mp 11, are commercially available from New England Biolabs. However, those of ordinary skill in the art to which this invention pertains will appreciate that other viruses, strains, host cells, promoters and vectors containing the relevant cDNA, as discussed above, may also be used in the practice of each embodiment of this invention. The DNA manipulations employed are, unless specifically set forth herein, in accordance with Maniatis et al.. Molecular Cloning: A Laborator Manual Cold S rin Harbor, NY 1982) . 46
Table 7: Exemplary Oligonucleotides for Mutagenesis
No. Sequence Mutation
1. ACC AAC TGG ACC AGC AGC GCG Asn117 : ►Thr
2. CTAC TTT GGG ACS GGG TCA GC Asn184 * ► hr
3. GTGCACCAACTGGCAGAGCAGCGCGTTGGC
Figure imgf000048_0001
4. CAACTGGCAGAGCAGCG (#3)*
5. ACTGCTACTTTGGGCAGGGGTCAGCCTACC Asn184 i ►Gin
6. CTTTGGGCAGGGGTCAG (#5)*
7. CATTTACTTCAGAGAACAGTC A ASsnn44488— > ->GQin
8. GGA GCC AGA TCT TAC CAA GTG ATC TGCAAGC (Δ FBR)
GAG CCA AGG TGT TTC AAC GGG GGC
9. TGATC TGC AGC GAG CC (#8)*
10. A AGA GGA GCC AGA TCT TAC CAA GTG ATCAGAT ACC AGG GCC ACG TGC TAC GAG (ΔFBR/EGF)
11. CAA GTG ATCAGAT ACC AG (#10) *
12. TCA GTG CCT GTC AAA AGT ACC AGG GCC
ACG TGC TAC (Δ EGF)
13. GTC AAA AGT±ACC AGG G (#12)*
14. GC CAG CCT CAG TTTAATC AAA GGA GGG C (R-275 del)
15. CT CAG TTT1 λC.r. ATC AAA G (—» T-275)
*Used for screening the mutation indicated in parenthesis (where a screening oligonucleotide is not indicated, the same oligonucleotide is used for mutagenesis and screening) . Codons for replacment amino acids are underlined, A indicates site of deletion. As those skilled in this art will appre¬ ciate, oligonucleotides can be readily constructed for use in deleting one or more amino acids or inserting a different (i.e., replacement) amino acid at a desired site by deleting the codon(s) or substituting the codon for the desired replacement amino acid, respectively, in the oligonucleotide. Other mutagenesis oligonucleotides can be designed based on an approximately 20-50 nucleotide sequence spanning the desired site, with replacement or deletion of the original codon(s) one wishes to change. Plas id Derivations
Mutagenesis of cDNAs at codons for the various amino acids was conducted using an appropriate restriction fragment of the cDNA in M13 plasmids by the method of Zoller and Smith. Deletions within the cDNA were effected by loopout mutagenesis using an appropriate restriction fragment, e.g. the SacI fragment, of the cDNA either in M13 vectors or by heteroduplex loop-out in plasmid pSVPA4.
The plasmid pSVPA4 was constructed to allow the expression of t-PA glycoprotein in mammalian cells. This plasmid was made by first removing the DNA encoding the SV40 large T polypeptide from the plasmid pspLT5 (Zhu, Z. et al., 1984, J. Virology 5L:170-180) . This was accomplished by performing a total Xho 1 digest followed by partial Bam-Hl restriction endonuclease digestion. The SV40 large T encoding region in pspLT5 was replaced with human t-PA-encoding sequence by ligating a cohesive Sail/ BamHl t-PA encoding restriction fragment, isolated by digesting plasmid J205 (ATCC No. 39568) with Sal I and BamHl, to the parent Xhol/BamHl cut vector pspLT5 prepared as described above. Consequently, t-PA will be transcribed in this vector under the control of the SV40 late promoter when introduced into mammalian cells. This final contruct is designated pSVPA4.
Plasmid pLDSG is an amplifiable vector for the expression of t-PA in mammalian cells such as CHO cells. pLDSG contains a mouse DHFR cDNA transcription unit which utilizes the adenovirus type 2 major late promoter (MLP) , the simian virus 40 (SV40) enhancer and origin of replication, the SV40 late promoter (in the same orientation as the adenovirus MLP) , a gene encoding tetracyσlin resistance and a cDNA encoding human t-PA (Val-245) in the proper orientation with respect to the adenovirus type 2 MLP. The preparation of pLDSG from pCVSVL2 in detail as has cotransformation with, and amplification of, pLDSG in CHO cells.. Kaufman et al., Mol. and Cell. Bio. 5(7) : 1750-1759 (1985).
Plasmid pWGSM is identical to pLDSG except that the cDNA insert encodes Met-245 human t-PA. pWSGM may be constructed using cDNA from plasmid J205 (ATCC No. 39568) or pIVPA/1 (ATCC No. 39891) . Throughout this disclosure pWGSM and pLDSG may be used interchangeably, although as indicated previously, the former vector will produce Val-245 proteins and the latter Met-245 proteins.
pIVPA/1 (ATCC No. 39891) is a baculoviral transplacement vector containing a t-PA-encoding cDNA. pIVPA/1 and mutagenized derivatives thereof are used to insert a desired cDNA into a baculoviral genome such that the cDNA will be under the transcriptional control of the baculoviral polyhedrin promoter.
Heteroduplex Mutagenesis
The mutagenesis via heteroduplexed DNA of specfic areas in the t-PA expression plasmid, pSVPA4, involves the following steps: Preparation of a picillin sensitive pSVPA4 DNA
1. Plasmid pSVPA4 (15 ug) was linearized with Pvul to completion. This mixture was extracted with phenol/chloroform and the DNA was precipitated using two volumes of ethanol with 0.1 M NaCl present.
2. The DNA was resuspended in a 21 ul of water, 1 ul dNTB solution (containing 2mM dATP, dGTP, dTTP, dCTP) , 2.5 ul 10X nick translation buffer (0.5M Tris-Cl pH 7.5, 0.1 M MgS04, 10 mM DTT, 500 ug/ml) and 0.5 ul (2 units) DNA polymerase 1-Large Fragment (New England Biolabs) . This mixture was incubated at room temperature for thirty minutes and then phenol/chlorform extracted followed by ethanol precipitation as described above. 3. The precipitated DNA was resuspended to 0.2 ug/ul by the addition of 75 ul water.
Preparation of ampicillin resistant pSVPA4 DNA
1. Plasmid pSVPA4 (15 ug) was digested with Sac I which cuts this plasmid twice within the t-PA encoding sequence to produce two restriction fragments, a 1.4 kbp t-PA encoding restriction fragment plus the parent vector. Following restriction digestion 1 ul (28 units) of calf intestine alkaline phospatase (Boehringer Mannheim) was added then incubated at 37βC for five minutes. The two bands were separated by loading this mixture onto a 0.7% agarose gel. The parent vector restriction fragment was excised from the gel and extracted by adsorption to silica dioxide at 4°C, which was folowed by elution in 50 mM Tris/lmM EDTA at 37"C for thirty minutes. The eluted DNA was adjusted to a final concentration of 0.2 ug/ul.
Heteroduplex Annealing
1. Mix 6 ul (1.2 ug) of ampicillin sensitive pSVPA4 DNA with 6 ul (1.2 ug) ampicillin resistant pSVPA4 DNA.
2. Add an equal volume (12 ul) of 0.4 M NaOH. Incubate at room temperature for ten minutes.
3. Slowly add 4.5 volumes (108 ul) of 0.1 M Tris-Cl pH 7.5/20 mM HC1.
4. 50 piσomoles (5 ul) of phosphorylated mutagenic oligonucleotide was added to 45 ul of heteroduplex mixture.
5. This mixture was incubated at 68βC for two hours then slowly cooled to room temperature. Mutagenesis
1. Each mutagenesis react.ion was adjusted to the following concentrations by the addition of 7 ul to the heteroduplex mixtures, 2mM MgCl/0.2 mM ATP/60uM dATP, dTTP,dGTP,dCTP/4 mM DTT/40 units/ml Klenow fragment of E. coli DNA polymerase I (B.R.L.), 2000 units/ml T4 DNA ligase (N.E.B.). This mixture was incubated at room temperature for 2 hours.
2. The reaction was then phenol/chloroform extracted which was followed by ethanol precipitation. The precipitated DNA was resuspended in 12 ul 50mM Tris-Cl/lmM EDTA. 4ul of this was used to transform competent HB101 bacteria.
3. Ampicillin resistant colonies were screened with 1x10f*5 σpm/ml of a 32P-labeled screening oligonucleotide in 5X SSC, 0.1% SDS, 5Xdenhardt's reagent, and 100 ug/ml denatured salmon sperm DNA.
4. The filters were washed with 5X SSC, 0.1% SDS at a temperature 5" below the calculated melting temperature of the oligonucleotide probe.
5. DNA was prepared from positively hybridizing clones and analyzed initially by digestion with different restriction enzymes and agarose gel electrophoresis. DNA was transferred to nitrocellulose and filters were prepared and hybridized to the screening probes in order to ensure the mutagenic oligonucleotide was introduced in to the correct fragment.
6. DNA was then retransformed into E. coli and ampi¬ cillin resistant colonies were screened for hybridization to the screening oligonucleotide.
7. Final mutations were confirmed by DNA sequencing (Sanger) . Preparation of Mutagenized cDNAs: M13 method
The following schematic restriction map illustrates a cDNA encoding human t-PA (above) with cleavage sites indicated for specific endonucleases (indicated below) :
ATG R1
I 1 Sac Bgl Nar -*2 Ec-o Erco- Sa-c R3— I Apa X
I II I Rl Rl I I (S
The initiation codon, ATG, and the cDNA regions encoding (a) , R1, R2 and R3 are indicated. Thus, mutagenesis at the N-terminus may be effected using the SacI fragment or the Bglll/Narl fragment, for example. Mutagenesis at Arg-275 and/or at R1 and/or R2 may be effected using, e.g., the SacI fragment or Bglll/SacI fragment. Mutagenesis at R3 may be effected using, an EcoRI/Xmal or EcoRI/Apal fragment. The choice of restriction fragment may be determined based on the convenience of using particular vectors for mutagenesis and/or for expression vector construction.
Generally, the cDNA restriction fragment to be mutagenized may be excised from the full-length cDNA present, e.g., in pWGSM, pIVPA/1 or pSVPA4, using the indicated endonuclease enzyme(s) and then mutagenized, e.g. with the oligonucleo¬ tides shown in Table 7 or other oligonucleotides designed for the desired mutagenesis.
Exemplary mutagenized cDNA fragments which may thus be prepared are shown in Table 8, below. Table 8: Exemplary Mutagenized cDNA Fragments
Figure imgf000054_0001
(II)
Figure imgf000054_0002
(HI)
(
Figure imgf000054_0003
(V)
*
3 i i B
Eco Sac " Apa r Xma r(SmaΙ) Rl I I I
* indicates site of mutagenesis; cDNA fragments I through IV are prepared by digesting pWGSM or pSVPA4 with SacI, inserting SacI fragment into M13 vector, utagenizing with desired oligonucleotide(s) , and digesting mutagenized M13/t-PA DNA with SacI; alternatively, I-IV may be excised from mutagenized M13/t-PA with Bglll and SacI and the Bglll/SacI fragment encoding the peptide domain spanning the N-terminus, R1, R2 & Arg-275 may be inserted into Bglll/SacI-digested pIVPA; cDNA fragment V is prepared as described in Example 2, below. Following mutagenesis the fragment, with or without further mutagenesis, may then be excised from the M13 vector and ligated back into an expression vector containing the full-length or partial cDNA previously cleaved with the same enzyme (s) as were used for excising the mutagenized fragment from the M13 vector. By this method the full-length cDNA, mutagenized as desired, may be re-assembled using one or more mutagenized fragments as restriction fragment cassettes.
cDNAs encoding the following illustrative compounds (see Table, page 9 and Tables 2.0, 2.5 & 3) may be prepared from the mutagenized fragments of Table 8 as follows:
Compound Route
D-6, (a) ligate mutagenized cDNA fragment I
D-l (prepared using oligonucleotides #8,10
D-3 or 12) into Sacl-digested pSVPA4, or excise fragment I from mutagenized M13/t-PA as the Bglll/SacI fragment and insert same into Bglll/SacI-digested pIVPA/1.
2-l/N-23/Arg (b) ligate mutagenized cDNA fragment II 2-l/N-21/Arg (prepared using oligonucleotides #8,10
2-l/N-22/Arg or 12) and then oligonucleotide #3) into Sacl-digested pSVPA4 , or excise fragment II from mutagenized M13/t-PA as the Bglll/SacI fragment and insert same into Bglll/SacI-digested pIVPA/1.
2-2/N-23/Arg (c) ligate mutagenized cDNA fragment III 2-2/N-21/Arg (prepared using oligonucleotides #8,10
2-2/N-22/Arg or 12 and oligonucleotide #5) into Sacl- digested pSVPA4 or excise fragment III from mutagenized M13/t-PA as the Bglll/ 54
SacI fragment and insert same into Bglll/SacI-digested pIVPA/1.
-3/N-23/Arg (d) digest mutagenized pIVPA/1 or pSVPA4 -3/N-21/Arg produced by Route (a) with EcoRI -3/N-22/Arg (partial digest) and Xmal (Smal) or Apal (total digest) to remove wild type R3 coding region, and ligate thereto mutagenized cDNA fragment V (prepared using oligonucleotide #7) as the EσoRI/Apal or EσoRI/Xmal (Smal) fragment .
-4/N-23/Arg (e) digest mutagenized pIVPA or pSVPA -4/N-21/Arg prepared as in Route (c) with EcoRI -4/N-22/Arg (partial digest) and Xmal (Smal) or Apal (total digest) to remove wild type R3 -coding region, and ligate thereto cDNA fragment V (prepared using oligonucleotide #7) as the EcoRI/Apal or EcoRI/Xmal (Smal) fragment.
-5/N-23/Arg (f) digest mutagenized pIVPA or pSVPA4 -5/N-21/Arg prepared by Route (b) with EcoRI -5/N-22/Arg (partial digest) and Xmal (Smal) or Apal (total digest) and ligate thereto mutagenized cDNA fragment V (prepared using oligonucleotide #7) as the EcoRI/ Apal or EcoRI/Xmal (Smal) fragment.
-6/N-23/Arg (g) ligate mutagenized cDNA fragment IV -6/N-21/Arg (prepared using oligonucleotides #8,10 -6/N-22/Arg or 12 and oligonucleotides #3 and 5) into Sacl-digested pSVPA4 or excise fragment IV from mutagenized M13/t-PA as the Bglll/SacI fragment and ligate 55 same into Bglll/SacI-digested pIVPA/1. 2-7/N-23/Arg (h) ligate mutagenized cDNA fragment IV 2-7/N-21/Arg (prepared using oligonucleotides #8,10 2-7/N-22/Arg or 12 and oligonucleotides #3 and 5) into Sacl-digested pSVPA4 prepared by Routes (d) , (e) or (f) or ligate fragment IV so produced as the Bglll/SacI fragment into Bglll/SacI-digested pIVPA produced by Route(s) (d) , (e) , of (f) .
Plasmids pIVPA or pSVPA4, in addition to utility as ex¬ pression vectors, may also be used as a "depot" in the construction of cDNAs having any desired permutation of mutagenized sites. Thus, "pIVPA/Δ" or "pSVPA4/Δ ", muta¬ genized (via M13 or heteroduplexing) plasmids containing a desired modification in the cDNA region encoding the N- terminal region may be digested with Narl (partial) and Xmal (Smal) (total) to remove the cDNA region encoding the protein domain spanning R1, R2 and R3. A second pIVPA or pSVPA4 plasmid mutagenized, if desired (via M13 or heteroduplexing), at any combination of Arg-275, R1, R2 and R3-encoding regions may then be digested with Narl (total) and Xmal (Smal) (total) and the Narl/Xmal (Smal) fragment may then be identified, isolated and ligated into the Narl/Xmal (Smal) digested pIVPA/Δ or pSVPA4/Δ. Such use of the Narl/Xmal (Smal) restriction fragment cassette, for example, allows the construction of desired mutagenized cDNAs in pIVPA or pSVPA4. The mutagenized cDNA may then be transferred, e.g. as a Bglll/Xmal restriction fragment cassette into Bglll/Xmal-digested pWGSM for mammalian expression, if desired. EXAMPLES Example 1
Preparation of Gln*j_ 7 Deletions Variants A. Preparation of Gln-117 truncated cDNA
cDNA molecules encoding the polypeptide sequence of compounds 2-l/N-21/Arg, 2-l/N-22/Arg and 2-l/N-23/Arg were prepared using the oligonucleotide-directed mutagenesis method of Zoller and Smith. Specifically, the mutagenesis vector RF M13/t-PA containing the t-PA gene was constructed from the mammalian t-PA expression plasmid pSVPA4. RF M13/t-PA was constructed by first digesting pSVPA4 to completion with the restriction endonuclease SacI. The approximately 1, 436 base pair (bp) SacI fragment encodes a large portion of the polypeptide sequence of t-PA and includes the nucleotide sequences encoding the consensus N-l inked glycosylation sites encompassing asparagines 117 , 184 , and 218 . This 1 , 436 bp (hereinafter 1.4 kbp) fragment was puri f ied by preparative agarose gel electrophoresis .
The Sac I fragment of the t-PA cDNA, obtained as a SacI fragment, above, was ligated to a linearized double-stranded RF M13mpl8 DNA vector which had been previously digested with Sac I. The ligation mixture was used to transform trans¬ formation competent bacterial JM101 cells. M13 plaques containing t-PA-derived DNA produced from transformed cells were identified and isolated by analytical DNA restriction analysis and/or plaque hybridization. Radiolabeled oligo¬ nucleotides (~17mers, of positive polarity) derived from within the SacI restriction sites of the t-PA-encoding nucleotide sequence depicted in Table 1 were used as probes when filter hybridization was employed to detect viral plaques containing tPA DNA. All oligonucleotides were 57 prepared by automated synthesis with an Applied Biosystems DNA synthesizer according to the manufacturer's instructions.
Several of the positive plaques detected by restriction or hybridization analysis were then further cloned by conventional plaque purification. Purified M13/t-PA bacteriophage obtained from the plaque purification procedure was used to infect JM101 cells. These infected cells produce cytoplasmic double-stranded "RF" M13/t-PA plasmid DNA. The infected cells also produce bacteriophage in the culturemediumwhich contain single-strandedDNAcomplimentary to the 1.4 kbp SacI fragment of t-PA and to M13 DNA. Single-stranded DNA was purified from the M13/t-PA-containing phage isolated from the culture medium. This single— stranded M13/t-PA DNA was used as a template in a mutagenesis reaction according to the method of Zoller and Smith using oligonucleotide #3 of Table 7. This mutagenesis event changes the Asn codon to a Gin codon at position 117 of the subsequently obtained coding strand of DNA by changing the DNA sequence from "AAC" to "CAG". Following the mutagenesis reaction, the DNA was transformed into the bacterial strain JM 101. To identify mutagenized cDNA's , the transformant plaques were screened by DNA hybridization using radiolabeled oligonucleotide #4 ofTable 7. All exemplaryoligonucleotides in Table 7 are of positive polarity, i.e., represent portions of a coding rather than non-coding strand of DNA. All hybridization positive plaques were further purified by subsequent secondary infections of JM 101 cells with'M13 phage containing mutagenized DNA.
RF M13/t-PA plasmid DNA was purified from JM 101 cells infected with purified M13 phage containing mutagenized t-PA cDNA. The RF M13/t-PA plasmid thus obtained contains the Gln^ mutagenized Sac I restriction fragment of t-PA 58
DNA. This mutagenized restriction fragment can then be further mutagenized, again by the method of Zoller and Smith, but using the oligonucleotides described below. The oligonucleotides described below were designed to induce a deletion ("loop out") within the cDNA region encoding the N-terminal domain.
Deletion Mutagenesis 1: Oligonucleotide #8 of Table 7 induced a cDNA deletion encoding Cys-6 through Ser-50 inclu¬ sive. Following this second mutagenesis reaction the DNA is transformed into JM 101 cells. To identify mutagenized cDNAs, the transformant plaques were screened as above, but using radiolabeled oligonucleotide #9 of Table 7. Hybrid¬ ization positive plaques can be further purified by subsequent secondary infections of JM 101 cells with M13 phage containing the twice mutagenized t-PA cDNA. The cDNA prepared as described below which contains this mutagenized restriction fragment encodes compound 2-l/N-21/Arg in which Ile-5 is covalently bonded to Cys-51 by a peptide bond.
Deletion Mutagenesis 2: Oligonucleotide #10 of Table 7 induced a cDNA deletion encoding Cysg through Ile86, inclu¬ sive. Following this second mutagenesis reaction the DNA is transformed into JM 101 cells. To identify mutagenized cDNAs, the transformant plaques were screened as above; but using radiolabeled oligonucleotide #11 of Table 7. Hybrid¬ ization positive plaques can be further purified by subsequent secondary infections of JM 101 cells with M13 phage containing the twice mutagenized t-PA cDNA. The cDNA prepared as described below which contains this mutagenized fragment encodes compound 2-l/N-22/Arg in which Ile5 is covalently bonded to Asp8 by a peptide bond.
Deletion Mutagenesis 3: Oligonucleotide #12 of Table 7 can be used to generate a cDNA deletion encoding Cyssi through 59
Asp87, inclusive. Following this second mutagenesis reaction the DNA is transformed into JM 101 cells. To identify mutagenized cDNAs, the transformant plaques were screened as above, but using radiolabeled oligonucleotide #13 of Table 7. Hybridization positive plaques can be further purified by subsequent secondary infections of JM 101 cells with M13 phage containing the twice mutagenized t-PA cDNA. The cDNA prepared as described below which contains this mutagenized restriction fragment encodes compound 2-l/N-23/Arg in which Ser50 is covalently bonded to Thrgg by a peptide bond.
Each of these mutagenized restriction fragments can then be ligated back into the mammalian expression vector pSVPA4 as a Sac I cassette by methods analogous to those described in Example #3B, or prepared for insertion into the insect cell expression vector pIVPA/1 (ATCC No.39891) as a Bgl II/Sac I cassette derived from modified RF M13/t-PA DNA.
B. Preparation of Vectors Used for Expression of High Mannose Glnι17 Deletion Variants
The purified RF M13/t-PA containing the modified and truncated t-PA cDNA, prepared as described above, can be digested with the restriction endonucleases Bglll and Sac I. The approximately 1.2 kbp Bglll/Sac I restriction fragment was purified by conventional preparative gel electrophoresis. The Bglll/Sac I fragment so obtained constitutes a mutagenized cassette which lacks a 5' and 3' portion of the DNA which encodes the amino and carboxy termini of the translated protein.
Insect expression vector pIVPA/1 (ATCC No. 39891) contains a wild type tPA cDNA insert operatively linked to a polyhedrin promoter together with baculovirus flanking 60
DNA sequences. pIVPA/1 was digested with Bglll and Sac I thereby excising a t-PA coding region spanning the N-terminus and R1 and R2. The Bglll/Sac I cassettes containing the mutagenized, N-terminally modified t-PA cDNA fragments may each then be ligated to pIVPA/1 expression vector DNA which had been previously purified following digestion with Bglll and SacI. The resulting plasmids, pIVPA/Δ FBR; Gln117, pIVPA/Δ FBR/Δ EGF; Gln117; pIVPA/Δ EGF, Gln117 should contain the mutagenized cDNAs encoding compounds 2-1/N- 21/Arg, 2-l/N-22/Arg and 2-l/N-23/Arg, respectively, now operatively linked to the polyhedrinpromoter. Thenucleotide sequence of each mutagenized cDNA insert may be confirmed by supercoil sequencing with plasmid as substrate. See e.g, E.Y. Chen et al., 1985, DNA 1(2) :165-170.
B. Introduction of the Mutagenized cDNA into the Insect Virus
Each of the pIVPA plasmids containing the mutagenized cDNAs may be introduced into the insect virus by co-trans- fection with wild-type AσNPV in Spodoptera cells. 1 ug of purified Autographa californica NPV DNA and lOug of the desired pIVPA DNA are introduced into Spodoptera cells growing on tissue culture dishes by a calcium phosphate transfection procedure (Potter, K.N. and Miller, L.K., J.Invertebr. Path. (1980), 3_6 431-432). The joint intro¬ duction of these DNAs into the cells results in a double recombination event between the pIVPA plasmid (containing the mutagenized cDNAs) and the viral DNA at the regions of ho ology between the two; that is, the polyhedrin gene region of the progeny virus from the recombination event contains the mutagenized cDNA insert from the pIVPA plasmid. Isolation of Virus Containing the Nucleotide Sequence Encoding the Proteins of this Invention
The progeny virus present in the media over the transfected cells are plaqued onto a fresh monolayer of cells at several different dilutions. Plaques are assayed, and the recombinants are picked based on the PIB-minus phenotype as follows: A virus which has lost its polyhedrin gene, as would a virus containing a mutagenized cDNA will not produce PIBs. Plaques that appear PIB deficient are selected, excised and amplified on fresh cells. The supernatant over these cells is then assayed for t-PA-type enzymatic activity. Positive assay results indicate that the glycoprotein is in fact being produced.
An alternative method of virus purification via the plaque lifting protocol differs slightly from the steps described above, and is described below. Plaque the progeny virus from transfeetion at suitable dilution onto cell culture dishes. Prepare a nitrocellulose replica of the cell monolayer and the virus plaques. Reserve the agarose overlay from the plate as the virus source after the results of the following steps are obtained.
Probe the nitrocellulose filter with radioactive DNA fragments representative of the gene being placed into the viral chromosome. Score positives as those containing the foreign gene. Remove the hybridized probe. Re-probe the filter with radioactive DNA representative of a portion of the viral chromosome removed by substitution with the foreign DNA. One would score positives as those which still have a polyhedrin gene.
Remove the hybridized probe. Re-probe the filter with a radioactive DNA fragment which will identify viral plaques 62 regardless of the state of the polyhedrin gene. A suitable fragment may be the EcoRI I fragment. Score these as progeny virus. Select those plaques which are positive for the foreign gene DNA probe, negative for the polyhedrin gene probe, and positive for the viral DNA probe. These are strong candidates for the desired genotype.
C. Production and Characterization of High Mannose Glyco- protein
Antibodies have been used to demostrate the presence of the variant proteins in the extracellular media of infected cells. Recombinant virus, prepared as above, is used to infect cells grown in the usual TC-100 (Gibco) nutrient salts solution but instead of the standard media supplement of 10% fetal calf serum, this is replaced with a 50% egg yolk enrichment (to 1% total volume) ( Scott Biologicals) . Previous experiments had demonstrated a more intact protein under these conditions. The supernatant from the infected cells is fractionated on an affinity column bearing an attached monoclonal antibody to natural human t-PA. Protein specifically retained by the column is eluted and assayed for t-PA enzymatic activity. A fixed amount of activity units of this and control t-PA preparations are separated on an acrylamide gel. This gel is then stained with a silver-based reagent to display the protein pattern. This reveals that the virus, upon infection of insect cells, leads to the extracellular production of a protein having t-PA type activity.
Radiolabeled protein is produced for further character¬ ization by first incubating spodoptera frugjperda cells infected with the virus (m.o.i=l) for 48 hours. The culture plates are then rinsed with methionine-deficient media. Methionine-deficient media supplemented with 35S- methione is then added to the culture plates. The cell cultures are incubated for 4 hours. The supernatant con¬ taining the radiolabeled glycoprotein may be analyzed by SDS-PAGE (7.5%) against wild type (i.e. full-length fully glycosylated) t-PA analogously produced in insect cells and against mammalian t-PA produced e.g. by the method of R. Kaufman et al., Mol. Cell. Biol. 5(7) :1750(1985) . , but in the presence of tunicamycin (non-glycosylated) . The partially glycosylated truncated proteins produced in Example 1 should have an increased gel mobility relative to the fully-glycosylated analog and to the non-glycosylated full-length analog.
EXAMPLE 2
PREPARATION OF OTHER PROTEINS OF THIS INVENTION.
A. Preparation of other cDNA's
The mutagenesis methods of Example 1 can be used with other conventionally prepared synthetic oligonucleotides which modify the original t-PA DNA sequence to produce proteins modified at the N-terminal region and/or optionally modified at N-linked glycosylation sites and/or at Arg-275 with the appropriate codon change(s) described previously.
See, e.g. "Preparation of Mutagenized cDNAs: M13 Method" and Routes (a)-(h), supra.
For example, cDNA encoding Compounds D-6, D-l and D-3 may be prepared using the SacI restriction fragment in M13/t-PA and mutagenizing with oligonucleotides #S, 10 and 12 re¬ spectively, but not with oligonucleotide #3. Arg-275 may be deleted or replaced, eg with Thr, using oligo's 14 or 15, respectively. Vector construction, transfection and expression may be carried out as in Example 1 for insect cells or as described below in Example 3 for mammalian cells.
Single-stranded DNA generated from the M13 mutagenesis vector (RF M13/t-PA) , prepared as in Example 1, can also be used as a template to mutagenize, in a site specific manner, at Arg-275 and/or at glycosylation site(s) R1 or R2 or both. The region encoding the consensus tripeptide which encompasses Asn2i8 may be similarly mutagenized. To prepare multiple modifications of the protein at these sites an iterative process may be used. For example, following the identification and the purification of M13 phage containing a modified R1 site, single-stranded DNA containing this modified site can be purified from phage and used as template to initiate a second round of mutagenesis within the R2 site and/or at Arg-275. This process can be repeated until all desired modifications are obtained. Thus, cDNA encoding Compounds 2-2/N-23/Arg, 2- 2/N-21/Arg and 2-2/N-22/Arg may be prepared by the method of Example 1 but substituting mutagenesis oligonucleotide #5 for oligonucleotide #3 and screening oligonucleotide #6 for oligonucleotide #4. cDNA encoding Compounds 2-6/N- 21/Arg, 2-6/N-22/Arg and 2-6/N-23/Arg may be prepared by twice mutagenizing the SacI fragment as described in Example 1 and addition mutagenizing and screening with oligonucleotides #5 and #6. Vector construction, transfection and expression are carried out as in Example 1 for insect cells or as described below for mammalian cells. See Routes (a)-(h), supra.
The RF M13/t-PA mutagenesis vector does not contain DNA sequence encoding R3, the N-linked glycosylation site of t-PA most proximal to the carboxy-terminus of the protein. Therefore in order to make DNA modifications at that site, a new M13/t-PA mutagenesis RF vector called M13/t-PA:Rl-Xma I was made. This vector was constructed by digesting the M13 vector M13mpll to completion with EcoRI and Xma I. The Rl/Xmal digested M13 vector was ligated to a purified EcoRI/Xma I t-PA restriction fragment (approximately 439bp, hereinafter 0.4kbp) encoding a polypeptide region encom¬ passing glycosylation site R3. This 0.4kbp restriction fragment was purified following digestion of the plasmid pWGSM with EcoRI and Xma I. The mammalian expression plasmid pWGSM, encoding the t-PA gene, is identical within the 439bp EcoRl/Xma I fragment to the plasmid pLDSG described by Kaufman et al., Mol. Cell Biol. 5.: 1750-1759 (1985) .
The ligation mixture was used to transform competent bacterial JM 101 cells. Several plaques were picked and analyzed for the presence of the 0.4kbp t-PA EcoRI/Xmal fragment by standard DNA restriction fragment analysis. Double-stranded RF M13 DNA was purified from cells containing the 0.4kbp t-PA fragment. This DNA was designated RF M13/t-PA:RI-Xma I mutagenesis vector. As previously indicated in Example 1A this vector, when transformed into competent JM101 cells, can be used to make M13/t-PA:RI-XmaI phage from which single-stranded M13/t-PA:RI-XmaI DNA can be purified. This single-stranded DNA can be used as template in the site-directed mutagenesis reaction to modify the t-PA DNA at the N-linked glycosylation site R3.
Modified R3 coding sequences can be used to replace the wild-type R3 sequences present in either modified pIVPA/1 as prepared in Example 1 (truncated and/or modified at R1 and/or R2) or wild-type pIVPA/1 plasmid DNA. This can be accomplished by first performing a total Sac I/Apa I digestion of the R3 modified M13/t-PA:RI/XmaI mutagenesis plasmid vector, and isolating the R3 modified 165 base pair t-PA restriction fragment so produced. The insect expression vector pIVPA/1 or pIVPA/1 plasmid DNA modified, e.g. as in Example 1, can similarly be totally digested with Sac I and Apa I to excise the 165 bp wild-type t-PA restriction fragment encoding the unmodified R3 site. Ligation of the purified insect expression vector lacking the 165 bp fragment to the modified R3 165 bp fragment produces a new insect expression vector. Expression of the vector produces a truncated protein modified at the R3 site, as well as at any or all of the other consensus N-linked glycosylation sites present in natural t-PA and/or at Arg-275.
The pIVPA plasmid containing the modified cDNA may also be used to generate the Bglll/Apal fragment of the modified t-PA cDNA which spans the deletion region in the N-terminal domain as well as the region encoding R1, R2 and R3 or the Narl/Xmal fragment which spans R1, R2 and R3. Either of those fragments may be inserted into mammalian expression vectors such as pSVPA4 or pWGSM as described in Example 3.
EXAMPLE 3
PREPARATION OF COMPOUNDS D-6, D-l and D-3 IN MAMMALIAN CELLS
A. Preparation of cDNA.
cDNA molecules encoding the polypeptide sequences of compounds D-6, D-l and D-3 were prepared using mutagenesis oligonucleotides #8 , 10, and 12 , respectively, and the SacI fragment of the t-PA cDNA as template by the M13 method of Exampl e 1 or heterodupl ex mutagene s i s (Moranaga Heteroduplex Mutagenesis protocol ; both, supra) . Mutants s e l e c t e d by DNA hybrid i z ation us ing screening oligonucleotides 9, 11 and 13 respectively were confirmed by DNA sequence analysis to be correct in the modified DNA sequence.
B. Modified t-PA Vector Preparation
Each modified cDNA prepared in Example 1A (Δ, Glnι_i7) or 3A (Δ) was first removed from the M13 mutagenesis vector RF M13/t-PA by total digestion of the vector with SacI. The approximately 1.4kbp restriction fragment of each mutagenized cDNA was purified by gel eleσtrophoresis and then ligated into pSVPA4 as follows. First, pSVPA4 was digested with SacI to remove the wild type t-PA 1.4kbp restriction fragment. The remaining portion of the SacI digested pSVPA4 was then ligated to the 1.4k p restriction fragment of the mutagenized cDNA. This ligation event can produce two orientations of the inserted fragment. The appropriate orientation in each case may be identified using EcoRI and PvuII as the enzymes in conventional analytical restriction enzyme analysis. This replacement allows the Sac I fragment to be used as a cassette' fragment between the RF M13/t-PA mutagenesis vector and the pSVPA4 mammalian expression vector. Modified M13 SacI fragments (truncated and optionally modified at R1 and/or R2) may be inserted into Sacl-digested pSVPA4 DNA which has been previously, or is subsequently, modified at R3 if desired. - Alternatively, DNA previously modified at R1, R2 and/or R3 can be excised from vectors such as pIVPA or pSVPA4 as a Narl/Apal or Narl/Xmal fragment. The fragment so obtained may then be inserted into vectors such as pSVPA4 or pWGSM previously digested with Narl (partial) and Apal or Xmal (total) . By this method any combination of N-terminal deletion and/or substitution and/or glycosylation site mutagenesis and/or Arg-275 mutagenesis may be achieved.
C. Transfection of COS (SV40 transformed African Green Monkey Kidney) Cells
COS-1 cells (ATCC CRL 1650) were transfectd by the method of Lopata, M.A. et al., Nucl. Acids Res. 12:5707-5717 (1984) with the vectors prepared in Example 3B, i.e., modified pSVPA4. Serum containing medium was replaced with 68 serum-free medium 24 hours after the transfection and conditioned medium was assayed for both the presence of plasminogen activating activity, using the chromogenic substrate S-2'251, or the presence .of t-PA antigen by an ELISA assay, 48 and 72 hours post-transfection.
D. Viral propagation in CV1 (African Green Monkey Kidney) cells.
Modified complex carbohydrate protein can be produced by infecting CVl cells (ATCC CCL 70) with SV40 viral stocks propagated as described by Gething and Sambrook (Nature 293:620-625, 1981). This has been carried out by first totally digesting modified pSVPA4 with the restriction endonuclease BamHl to remove the bacterial shuttle vector pXf3 from the SV40 viral DNA. Before transfecting this DNA into CVl cells, along with the helper virus SV40-rINS-pBR322 DNA (described below) , the Bam HI linearized SV40/t-PA DNA is circularized by ligation at dilute DNA concentrations (1 ug/ml) . This process was repeated with the insulincontainingSV40vectorSV40-rINS-pBR322 (Horowitz, M. et al., 1982, Eukaryotic Viral Vectors, pp. 47-53, Cold Spring Harbor Laboratory) . The bacterial shuttle vector pBR322 in SV40-rINS-pBR322 was removed by a total EcoRI digestion. The linearized insulin/SV40 viral DNA was then circularized by ligation at a DNA concentration of 1 ug/ml. It is necessary to transfect CV-1 cells with circular ligated pSVPA4 and SV40-rINS DNAs, at equimolar amounts in order to generate viral stocks. SV40-rINS is used to provide "late" SV40 proteins while pSVPA4 provides the "early" SV40 proteins necessary for virus DNA production while also encoding the proteins of this invention. Consequently when cells are transfected with both these DNA's as described by Gething and Sambrook, SV40 virus is produced which contains DNA from either viral vectors. Subsequent infection of CVl cells with amplified virus has produced proteinwith t-PA-type activity which can be assayed 72 hours post-infection as described in Example 3C.
Example 4
Preparation of Other Proteins
cDNAs encoding various proteins of this invention have been prepared by the methods of Examples 1, 2 and 3. The Bgl II/XmaI restriction fragment cassette may then be excised from either the pIVPA or pSVPA4 vector containing the cDNA encoding the truncated protein with or without modification at one or more glycosylation sites. The excised Bglll/Xmal fragment may then be ligated into Bgl II/XmaI-σut pSVPA4 or pWGSM for introduction into mammalian cells. Expression of such cDNAs in mammalian host cells, e.g. by the method of Example 3 or by the method of Kaufman et al., supra, (CHO host cells) or by the method of Howley et al., U.S. Patent No. 4,419,446 (1983) (BPV expression systems) yields the corresponding mammalian-derived truncated proteins. Thus, cDNAs encoding compounds 2-l/N-21/Arg (Δ FBR, Gln117) and 2-l/N-22/Arg (Δ FBR/EGF, Gln117) were prepared and inserted into pSVPA4 as described above. cDNA encoding compound 2- l/N-23/Arg (Δ EGF, Gln*n7) was prepared using mutagenesis oligonucleotide #12 and screening oligonucleotide #13 (Table 7) but by the heteroduplex method described above, with pSVPA4 previously mutagenized at position 117 (as above) as template. Similarly, cDNAs encoding Compounds D- 1 (Δ FBR) and D-3 (Δ EGF/FBR) were prepared by M13 mutagenesis, as described above, and inserted as the SacI fragment into Sacl-digested pSVPA4. cDNA encoding Compound D-6 (Δ EGF) was prepared by the heteroduplex method, described above, using pSVPA4 as template and mutagenesis oligonucleotide #12, and screening with oligonucleotide #13.
To prepare the cDNAs encoding the proteins for amplif¬ ication and expression in mammalian cells, cDNA contained in pSVPA4 or pIVPA is excised as a Bglll/Xmal fragment and ligated into purified, Bglll/Xmal-digested pWGSM. In each case the resulting pWGSM vector is introduced into CHO cells and amplified by the method of Kaufman, supra. The transformed and amplified CHO cells produce compounds D-6, D-l, D-3, 2-l/N-23/Arg, 2-l/N-21/Arg and 2-l/N-22/Arg respectively, which were detected in the culture medium by human t-PA specific antibodies. The compounds may then be recovered and purified by immunoaffinity chromatography.
71
Example 5
Example 4 may be repeated using cDNA encoding the proteins modified within the N-terminus and/or at R-275 with or without modification at R1, R2, and/or R3 to produce the desired protein in CHO cells. Mutagenized cDNAs may be prepared as described above. Thus, cDNAs encoding Compounds 2-7/N-23/A.rg, 2-7/N-21/Arg and 2-7/N-22/Arg are prepared in pIVPA as described in Example 2. The cDNAs may then be excised as the Bglll/Xmal fragment and ligated into purified, Bglll/Xmal-digested pWGSM, and the resultant vector transformed and amplified in CHO cells as in Example 4 to produce compounds 2-7/N-23/Arg, 2-7/N-21/Arg and 2- 7/N-22/Arg.

Claims

What is claimed- is:
1. A thrombolytic protein having tissue plasminogen activator-type activity characterized by a peptide sequence substantially the same as the peptide sequence of human t- PA, wherein
(a) Arg-275 is deleted or is replaced by a different amino acid, and
(b) at least one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site.
2. A thrombolytic protein of claim 1, wherein one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site.
3. A thrombolytic protein of claim 1, wherein two of the consensus N-linked glycosylation sites are modified to other than consensus N-linked glycosylation sites.
4. A thrombolytic protein of claim 1, wherein three of the consensus N-linked glycosylation sites are modified to other than consensus N-linked glycosylation sites.
-5. A thrombolytic protein having tissue plasminogen activator-type activity characterized by a peptide sequence substantially the same as the peptide sequence of human t- PA, wherein one or more amino acids are deleted within the region Gly-(-3) through Thr-91.
6. A thrombolytic protein of claim 5 which is further characterized in that
(a) Arg-275 is deleted or is replaced by a different amino acid, and/or (b) at least one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site.
7. A thrombolytic protein of claim 5, wherein 1 to about 45 amino acids are deleted from the region Cys-6 through Cys-51..
8. A thrombolytic protein of claim 5, wherein 1 to about 37 amino acids are deleted from the region Cys-51 through Asp-87.
9. A thrombolytic protein of claim 5, wherein one or more deletions of less than about 20 amino acids are present within the region Gly-(-3) through Thr-91.
10. A thrombolytic protein having tissue plasminogen activator-type activity characterized by a peptide sequence substantially the same as the peptide sequence of human t- PA, wherein one or more amino acids within the region Gly- (-3) through Thr-91 are replaced with different amino acids.
11. A thrombolytic protein of claim 10, wherein the amino acid replacement(s) are within the region Gly-(-3) through Cys-51.
12. A thrombolytic protein of claim 10, wherein the amino acid replacement(s) are within the region Cys-51 through Thr-91.
13. A thrombolytic protein of claim 10, wherein the number of amino acids replaced is from 1 to about fifteen.
14. A thrombolytic protein of claim 11, wherein the number of amino 'acids replaced is from 1 to about five.
15. A thrombolytic protein of claim 13, wherein the region Gly-(-3) through Thr-91 is further modified by deletion of 1-93 amino acids.
16. A thrombolytic protein of claims 10 - 15 wherein the protein is further modified in that:
(a) Arg-275 is deleted or is replaced by a different amino acid, and/or
(b) at least one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site.
17. A DNA molecule encoding a protein of claim 1-16.
18. A thrombolytic protein having tissue plasminogen-type activity produced by expression of a DNA molecule of claim 17 in a host cell selected from the group consisting of mammalian, yeast, insect, fungal or bacterial cells.
19. A therapeutic compostition for the treatment of thrombotic conditions which comprises an effective amount of a protein of claims 1-16 in admixture with a pharmaceutically acceptable carrier.
PCT/US1987/000257 1986-01-31 1987-01-30 Novel thrombolytic proteins WO1987004722A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
AT8787902884T ATE104700T1 (en) 1986-01-31 1987-01-30 NEW THROMBOLYTIC PROTEINS.
EP87902884A EP0293394B2 (en) 1986-01-31 1987-01-30 Novel thrombolytic proteins
DE3789664T DE3789664T3 (en) 1986-01-31 1987-01-30 NEW THROMBOLYTIC PROTEINS.
IE166087A IE60017B1 (en) 1986-07-03 1987-06-22 Processes for the preparation of a thrombolytic protein having tissue plasminogen activator-type activity
PT8522687A PT85226B (en) 1986-07-03 1987-07-01 PROCESS FOR THE PREPARATION OF NEW THROMBOLITIC PROTEINS
ES8701920A ES2004438A6 (en) 1986-07-03 1987-07-01 A method to produce a thrombolic protein (Machine-translation by Google Translate, not legally binding)
GR871052A GR871052B (en) 1986-07-03 1987-07-03 Novel thrombolytic proteins
DK198705118A DK175784B1 (en) 1986-01-31 1987-09-29 Hitherto unknown thrombolytic proteins
NO874091A NO175317C (en) 1986-01-31 1987-09-29 DNA molecule encoding a thrombolytic protein
US08/891,245 US5837518A (en) 1986-01-31 1997-07-10 Thrombolytic proteins

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US82510486A 1986-01-31 1986-01-31
US825,104 1986-01-31
US85378186A 1986-04-18 1986-04-18
US853,781 1986-04-18
US86169986A 1986-05-09 1986-05-09
US861,699 1986-05-09
US06/882,051 US5002887A (en) 1986-01-31 1986-07-03 Truncated thrombolytic proteins
US882,051 1992-05-12

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EP0293394B2 (en) 2003-10-29
DK511887D0 (en) 1987-09-29
JPH0746989A (en) 1995-02-21
DK175784B1 (en) 2005-02-21
EP0293394A4 (en) 1989-11-14
JPS63501335A (en) 1988-05-26
JP2527454B2 (en) 1996-08-21
DE3789664T3 (en) 2004-09-16
JP2679915B2 (en) 1997-11-19
DE3789664T2 (en) 1994-09-15
AU7239587A (en) 1987-08-25
JPH104960A (en) 1998-01-13
DK511887A (en) 1987-09-29
JPH05268959A (en) 1993-10-19
AU612974B2 (en) 1991-07-25
EP0293394B1 (en) 1994-04-20
EP0293394A1 (en) 1988-12-07
JP2568382B2 (en) 1997-01-08
DE3789664D1 (en) 1994-05-26

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