WO1995011987A1 - Chimeric proteins including protease nexin-1 variants - Google Patents
Chimeric proteins including protease nexin-1 variants Download PDFInfo
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- WO1995011987A1 WO1995011987A1 PCT/US1994/011624 US9411624W WO9511987A1 WO 1995011987 A1 WO1995011987 A1 WO 1995011987A1 US 9411624 W US9411624 W US 9411624W WO 9511987 A1 WO9511987 A1 WO 9511987A1
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- 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
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/505—Erythropoietin [EPO]
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
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- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates generally to the field of proteins, protein analogs, and methods of specifically altering proteins in order to change their biological activity. More particularly, the invention relates to identifying the active site of a protein, for example, Protease Nexin-1, and altering the active site by
- the invention also relates to
- cysteine residues or amino acid residues which may be substituted by cystine without abolishing activity, and attaching polyethylene glycol to the thio group of cysteine, thereby increasing protein stability.
- proteases have specific functions in the body. For example, natural physiological functions such as tissue remodeling, inflammation, coagulation, and fibrinolysis require proteolytic enzymes. Of particular importance is a mechanistic class of proteases called serine proteases. It is also known that the active site of all functional members of the serine protease family contains a
- PN-1 Protease nexin-1
- Human foreskin cells Scott, R.W. et al., J Biol Chem (1983) 58:1043910444. It is a 42 kd glycoprotein which is released by fibroblasts, myotubes, heart muscle cells, and vascular smooth muscle cells. Its release, along with that of plasminogen activator, is stimulated by phorbol esters and by mitogens (Eaton, D.L. et al., J Cell Biol (1983) 123:128).
- Native PN-1 is an approximately 400 amino acid protein containing about 10% carbohydrate.
- PN-1 inhibits all the known activators of urokinase proenzyme, plasmin, trypsin, thrombin, and Factor Xa (Eaton, D.L. et al., J Biol Chem (1984) 259:6241). It also inhibits tissue plasminogen activator and urokinase. However, PN-1 does not inhibit elastase or cathepsin G.
- PN-1 is different from most serpins in that it is found in tissues, contains a high affinity heparin binding site which localizes it to tissues, and has a tissue clearance receptor that is responsible for endocytosis of protease-PN-1 complexes.
- PN-1 variants including variants which inactivate elastase and cathepsin G, and have moved well beyond our prior work to provide variants and methods for designing and producing such variants which have significantly altered protease specificity and second-order association rate constants with respect to a variety of serine proteases.
- variants and methods of producing such variants which have polyethylene glycol specifically attached to one or more cysteine residues, such cysteine residues being either present in the parent molecule or introduced on the surface of the protein by site-directed mutagenesis, and methods for determining appropriate sites for the introduction of cysteine residues.
- variants and methods of producing such variants which combine the specific localization ability of a receptor-binding protein with the protease-inhibiting activity of PN-1 or variant thereof, resulting in desired biological activities with particular
- a Type I variant of the invention is produced by site-directed mutagenesis wherein a single amino acid within the active site of PN-1 is substituted with an amino acid different from the naturally-occurring amino acid at that position.
- a Type II variant of the invention is similar to a Type I variant in that the active site of PN-1 is changed.
- Type II variant the active site of PN-1 is changed so as to match the active site of another serpin which change many require one or more amino acid substitutions, deletions or additions.
- a Type III variant of the invention is produced whereby the active site or a portion thereof of PN-1 is substituted with a sequence which corresponds to the substrate sequence for a particular protease.
- a Type IV variant is produced wherein a cysteine residue, which is either present in the native protein or introduced by site-specific
- Type V variant of the invention involves producing a fusion protein wherein PN-1 is fused to the receptor binding region of another protein in order to localize PN-1 to a different receptor.
- Variants of Type I, II or III are referred to herein as variants.
- the compounds of Type IV wherein polyethylene glycol is attached to a thio group are referred to as cysteine-PEGylation proteins, and the compounds of Type V are referred to as fusion proteins or chimeric proteins.
- Embodiments of the present invention provide pharmaceutical compositions which contain one or more variants of all or any of Type I, II, III, IV or V.
- An important object of the invention is to provide a wide range of different variants, and in particular PN-1-variants, having a particular and desired biological activity.
- Another object is to provide PN-1 variants using site-directed mutagenesis in order to change a single amino acid within the active site of PN-1.
- Another important object is to provide a PN-1variant wherein the active site of PN-1 is specifically modified so as to match the active site of another enzyme inhibitor, preferably another serpin.
- Yet another important object of the present invention is to provide variants such as protease nexin-1 variants which include, in PN-1, the substrate sequence for a different protease, making it possible to inhibit the activity of that protease.
- Still another important object is to provide proteins which are PEGylated by attachment to a thio group, i.e. the polyethylene glycol is attached to a cysteine amino acid within a protein, which cysteine amino acid of the protein is not involved in a disulfide bond.
- Another important object is to provide a method of attaching polyethylene glycol to a protein by first subjecting the protein to site-directed mutagenesis to add a cysteine residue at a position where the protein or a structurally related protein is normally glycosylated, and thereafter attaching the polyethylene glycol to the cysteine residue.
- Another important object is to provide a method of attaching PEG to a protein by first subjecting the protein to site-directed mutagenesis to add a cysteine residue at a position on the surface of the protein, and thereafter attaching the PEG to the cysteine residue.
- Another important object is to provide dimeric or multimeric proteins cross-linked by reaction with a reagent composed of PEG having two protein-reactive moieties.
- Yet another important object is to provide fusion proteins wherein the receptor binding region of another protein is connected to PN-1 in order to localize PN-1 to a different receptor.
- Another object of the present invention is to provide a pharmaceutical composition
- a pharmaceutical composition comprising excipient carrier materials having a compound of the invention dispersed therein.
- Another object of the present invention is to provide therapeutic methods of treatment which involve administering to a patient in need thereof a
- composition comprising excipients and a compound of the invention.
- a feature of the present invention is that the variants can be designed to have a specific receptor-binding domain while maintaining the natural biological activity of the protein to which the new binding domain is attached.
- An advantage of the present invention is that the variants have substantially different inhibitory effects on certain proteolytic enzymes as compared to the natural protein.
- Another object of the present invention is to provide variants useful in treating diseases associated with a specific biological activity.
- Yet another advantage of the present invention is to describe and disclose variants which are useful in treating elastase-related diseases.
- Another feature of the present invention is that certain variants have substantially altered protease specificity as compared with the natural protein.
- Another advantage of the present invention is that certain variants have substantially greater second order association rate constants with respect to particular serine proteases as compared with the second order association rate constant of natural proteins with respect to such serine proteases.
- Another advantage of the present invention is that certain variants have substantially slower second order association rate constants with respect to particular serine proteases as compared with the second order association rate constant of natural proteins with respect to such serine proteases.
- Yet another object is to provide methods of delivery such as by injection, internasal and interpulmonary delivery which methods are carried out using pharmaceutical compositions in the form of
- Another advantage is that biologically stabilized proteins can be produced by attaching the polyethylene glycol to a cysteine residue of the protein.
- Another advantage is to provide methodology for readily attaching polyethylene glycol molecules to proteins at a cysteine residue of the protein which are preferably located at native sites of glycosylation.
- amino acid residues for substitution with cysteine may be selected so that subsequent attachment of polyethylene glycol to the thio group of the substituted cysteine residue increases biological stability of the cysteine-PEGylated protein relative to wild type without abolishing biological activity.
- proteins which normally require glycosylation for biological stability may be produced commercially by expression in a prokaryotic host or other host which does not provide for glycosylated recombinant proteins. After expression of the protein by the prokaryotic host, biological stability of the protein can be increased by attachment of polyethylene glycol to a native or engineered cysteine residue in the protein.
- cysteine-PEGylated proteins can be produced without exposing the protein to highly toxic chemicals such as dioxane, cyanuric
- Figure 1 shows the nucleotide sequence of the coding region and the deduced amino acid sequence of PN-1 ⁇
- Figure 2 shows the nucleotide sequence of the coding region and the deduced amino acid sequence of PN-1 ⁇ .
- Figure 3 is a schematic drawing of a
- Figure 4 is a graph which shows the activity of samples of reaction mixtures containing cysteine-PEGylated PN-1 variants (N99C;N140C) produced by the method of the invention (open squares) and the activity of samples of reaction mixtures containing a PEGylated PN-1 variant (N99C;N140C) produced by a conventional method (closed diamonds).
- protease nexin-1 and “PN-1” are used interchangeably and refer to DNA codons and
- PN-1 is distinguishable from the two other protease nexin factors, PN-II and PN-III (Knauer, D.J. et al., J Biol Chem (1982) 257:15098-15104). which are also thrombin inhibitors, but are less strongly binding to this
- protease and are of different molecular weight, three-dimensional structure and mechanism of function.
- variants proteins
- chimeric proteins are used interchangeably herein to refer to any amino acid sequence which corresponds to the amino acid sequence of a natural protein or a
- one or more amino acids within the natural sequence is replaced with one or more amino acids different from the amino acids present in the natural protein; and/or (2) one or more amino acids has been added to the natural sequence, and the addition of such amino acids changes the biological activity of the variant; and/or (3) one or more amino acids is deleted from the natural sequence; and/or (4) polyethylene glycol is bound to a thio group of a natural or artificially introduced cysteine residue of a sequence; and/or (5) two naturally occurring
- sequences are fused together, i.e. two sequences not normally connected are fused.
- protease nexin-1 variants and “analogs of protease nexin-1” are terms which are used synonymously herein to define a Type I variant and are thereby
- protease nexin-1 variants of the invention include substantially the same amino acid sequence as protease nexin-1 but for the substitution of different amino acids at or near the active site.
- substitutions of different amino acids can be made at any of P 1 , P 2 , P 3 , P 4 sites and/or made at the P 1 ', P 2 ', or P 3 ', P 4 ' sites.
- substitutions and deletions of amino acids in the sequence of protease nexin-1 are encompassed by this invention.
- protease nexin-1 variants of the invention are variants which have high activity relative to a substrate to which natural PN-1 has little or no activity such as variants which inhibit elastase and, more particularly, which inhibit elastase and have their ability to inhibit elastase enhanced in the presence of heparin and/or heparin-like compounds.
- Other preferred protease nexin-1 variants for example, have increased ability to inhibit urokinase and/or another serine protease as compared with protease nexin-1.
- Control sequence refers to a DNA sequence or sequences which are capable, when properly ligated to a desired coding sequence, of effecting its expression in hosts compatible with such sequences. Such control sequences include at least promoters in both procaryotic and eucaryotic hosts, and preferably, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be identified. As used herein, “control sequences” simply refers to whatever DNA sequence may be required to effect
- Cells or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context. These terms include the immediate subject cell, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or differences in environment. However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell. In the present case, for example, such a characteristic might be the ability to produce recombinant PN-1.
- pure refers to material which is free from substances which normally accompany it as found in its native state.
- pure PN-1-encoding DNA refers to DNA which is found in isolation from its native environment and free of association with DNAs encoding other proteins normally produced by cells natively producing PN-1.
- Pure PN-1 refers to PN-1 which does not contain materials normally associated with its in situ environment in human or other mammalian tissue.
- pure PN-1 may include materials in covalent association with it, such as glycoside residues or materials introduced for, for example, formulation as a therapeutic.
- pure also includes variants wherein compounds such as polyethylene glycol, Biotin or other moieties are bound to the variant in order to allow for the attachment of other compounds and/or provide for formulations useful in therapeutic treatment or
- additional sequence at the 5' and/or 3' end of the coding sequence which might result, for example, from reverse transcription of the noncoding portions of the message when the DNA is derived from a cDNA library or might include the reverse transcript for the signal sequence as well as the mature protein encoding sequence.
- “Degenerate with”, as referred to a DNA sequence, refers to nucleotide sequences encoding the same amino acid sequence as that referenced.
- operably linked refers to a juxtaposition wherein the components are configured so as to perform a desired function such as their natural biochemical function.
- control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
- Heparin heparan sulfate
- heparin-like compounds are terms which are used synonymously herein. Each of the terms singly or in combination with the others is intended to encompass a large group of compounds which are generally described as sulfated polysaccharides, which includes proteoglycans and
- glycosammoglycans which are alternating copolymers of a hexosamine and an aldouronic acid. These copolymers are found in sulfated forms and are synthesized as proteoglycans and are collectively referred to as
- mucopolysaccharides Other compounds such as dextran sulfate are considered “heparin-like" for purposes of the invention. Similar alternating copolymers, especially those which are highly sulfated and thus very negatively charged, are useful "heparin-like" compounds in this invention.
- glycosammoglycans are described in detail by Benito Casu, "Structure and Biological Activity of Heparin", published in Advances in Carbohydrate Chemistry and
- Imino Naturally occurring amino acids can be generally classified as being polar or non-polar as follows:
- Amino acid residues can be generally subclassified into four major subclasses as follows:
- Neutral/polar The residues are not charged at physiological pH, but the residue is attracted by aqueous solution so as to seek the outer positions in the
- Amino acid residues can be further subclassified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side chain substituent groups of the residues , and as small or large.
- the residue is considered small if it contains a total of 4 carbon atoms or less, inclusive of the
- Acidic Aspartic acid and Glutamic acid
- Basic/noncyclic Arginine, Lysine
- the gene-encoded amino acid proline although technically within the group neutral/non-polar/large/cyclic and nonaromatic, is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in this defined group, but is included as a group of its own.
- amino acid substitutions for those encoded in the gene can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme.
- Variants of the invention may include commonly encountered amino acids, which are not encoded by the genetic code, for example, ⁇ -alanine ( ⁇ -ala), or other omega-amino acids, such as 3-amino propionic, 4-amino butyric and so forth, ⁇ -aminoisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine (t-BuG),
- N-Melle N-methylisoleucine
- Phg phenylglycme
- Cha cyclohexylalanine
- Na norleucine
- Cya cysteic acid
- MSO methionine sulfoxide
- Sar and 0-ala are neutral/non-polar/small; t-BuA, t-BuG, N-Melle, Nle and Cha are neutral/ non-polar/large/nonaromatic;
- Orn is basic/noncyclic
- Phg is neutral/non-polar/large/aromatic.
- omega-amino acids are classified according to size as neutral/non-polar/small ( ⁇ -ala, i.e., 3-aminopropionic, 4-aminobutyric) or large (all others).
- Serine Proteases and their Inhibitors Although originally named for their mechanism of action, members of the serine protease family also show significant sequence and structural homology. Some serine proteases are very specific, cleaving only certain peptide bonds of a specific target protein while others are very nonspecific, degrading multiple target proteins into small peptides.
- Serine proteases are regulated at many levels. Some are synthesized as inactive proenzymes and are activated only during specific events and at specific locations. This allows the body to respond rapidly to a physiological perturbation by activating an already present reservoir of proteolytic activity. Coagulation, for example, is carried out when circulating proenzymes such as Factor X and prothrombin are sequentially
- proteolytic activity is often localized to specific sites, such as receptor binding sites which can cause high local concentrations of protease or proenzyme ready for activation.
- proteolytic activity be confined both spatially and temporally. This control is often achieved by the presence of specific inhibitors which block proteolytic activity.
- proteases serpins were first defined by their common mechanism of action but later turned out to be highly homologous both in terms of sequence and structure.
- Serpins all contain an inhibitor domain with a reactive peptide bond defined on either side by the variables P 1 and P 1 '.
- the amino acids are referred to as P 1 , P 2 , P 3 , etc.
- the P 1 residue is recognized by the substrate binding pocket of the target protease which attacks the reactive peptide bond as though a normal substrate.
- PN-1 Protease nexin-1
- PN-1 is a member of the serpin family.
- PN-1 is produced by many different cell types including fibroblasts, glial cells, platelets and
- PN-1 is secreted by cells into the
- PN-1-protease complexes then bind back to specific cell surface receptors where they are internalized and degraded.
- PN-1 is very similar, both structurally and functionally to antithrombin (AT-III).
- AT-III is the primary plasma inhibitor of blood coagulation.
- the inhibition of thrombin by AT-III in plasma is normally very weak but is accelerated significantly by the
- heparin as a blood "thinning" agent is due to its enhancement of AT-III activity.
- AT-III PN-1 has a high affinity heparin binding site and inhibits thrombin much more rapidly (50-100 fold) in the presence of heparin.
- PN-1 has therapeutic potential as an anticoagulant.
- PN-1 differs from AT-III in a number of ways. Unlike AT-III, PN-1 is also a good inhibitor of the fibrinolytic enzymes urokinase and plasmin, as well as trypsin. Furthermore, PN-1 is not found in significant quantities in plasma and may
- PN-1 The high affinity heparin binding site of PN-1 serves to localize it to connective tissues and cells which contain sulfated proteoglycans on their surface and surrounding
- PN-1 extracellular matrix.
- PN-1 s primary role seems to be in regulating proteolytic activity in tissues as opposed to blood. Further evidence for the role of PN-1 is found by the fact that it is present in brain tissue and may be involved in peripheral nerve regeneration and neurite extension.
- association constant has inverse-mole-seconds as its units, and the larger the k ass , the more rapid the
- a k ass value is always given as a value with respect to a particular enzyme and is zero if there is no inhibition of the enzyme.
- protease inhibitor reactions such as elastase- ⁇ -1 antitrypsin and plasmin- ⁇ -2-antiplasmin occur with rate constants as high as 1 ⁇ 10 7 M -1 S -1 or greater.
- the thrombin-PN-1 reaction occurs at a similar high rate in the presence of heparin.
- Figures 1 and 2 respectively, show the amino acid sequence of PN-1 ⁇ and PN-1 ⁇ .
- the ⁇ and ⁇ forms differ by the substitution of thr 310 -gly 311 in PN-1 ⁇ for arg 310 in PN-1 ⁇ .
- the "P 1 " site (arginine at position 345 for PN-1 ⁇ and 346 for PN-1 ⁇ ) has been confirmed by sequencing of the peptide fragment released from PN-1 upon dissociation of complexes with thrombin.
- PN-1 normally inhibits only enzymes which cleave at arginine (the P 1 residue), such as thrombin, plasmin, trypsin, plasminogen activators, and plasma kallikrein.
- proteolytic activity is often localized to specific sites such as receptor binding sites. Once a proteolytic enzyme is activated, it is extremely important that the enzyme activity be confined both spatially and temporally. Such confinement is in part brought about by the inhibitory effect of serpins.
- All serpins contain an inhibitor domain with a reactive peptide bond defined on either side by P 1 and P 1 ' residues.
- the P 1 residue (such as arginine at position 345 for PN-1 ⁇ and 346 for PN-1 ⁇ ) is recognized by the substrate binding pocket of the target protease.
- the protease Upon recognition of the "reactive" site (of the inhibitor by the protease) the protease attacks the reactive peptide bond of the inhibitor as if it were a normal substrate.
- serpin hydrolysis of the peptide bond and release of the protease does not proceed to completion.
- the normal deacylation step is so slow that the reaction becomes essentially irreversible and the protease becomes trapped with the inhibitors in a stable, covalent, equal molar complex. Since the P 1 residue is the predominant determinant residue recognized by the substrate binding pocket of the target protease,
- a first protein the receptor binding region of a second protein.
- biochemical function and protein binding specificity of different proteins are known, the methodology of the present
- a first protein might be known to have virtually no binding affinity with respect to a given receptor and a second protein might have very high binding affinity with
- a receptor can be any protein or a portion thereof, ligand, cell surface area or
- a specific situation to which variants of the invention might be applied is as follows.
- the movement of monocytes and neutrophils from the bloodstream into inflamed tissue requires activation events at the site of interaction. These events include expression of adhesion molecules and recruitment of transmigration machinery.
- uPA-plasminogen activator (uPA)-plasmin system plays a major role in regulating extracellular proteolysis i.e. uPA-plasmin is important in breaking intracellular connections so that neutrophils can migrate through the endothelium blood vessel lining into surrounding tissues. Although the migration of some neutrophils cells through the
- a variant which would (1) bind to activated endothelium cells and (2) inhibit uPA-plasmin would be useful in preventing and/or reducing inflammation at a particular site.
- Tumor cell invasiveness is also dependent upon the uPA-plasmin system as shown in tumor cell metastasis model systems (Ossowski and Reich, 1983; Hearing, et al., 1988) and extracellular matrix degradation and basement membrane invasion (Gergman, et al., 1986; Mignatti, et al., 1986; Reich, et al., 1988; Cajot, et al, 1990).
- the levels of uPA are significantly higher in human breast cancer tissues than in normal tissues.
- Increased amounts of uPAR correlate with increased invasiveness of
- proteolytic activity of uPA or plasmin may protect against extracellular matrix degradation and basement membrane invasion and aid in preventing inflammation.
- urokinase with the urokinase receptor might block metastasis.
- a variant protein of the present invention could be designed to block the urokinase receptor and inhibit urokinase.
- a variant could be produced which combined the ability to block the urokinase receptor with the ability to inhibit urokinase and plasmin, and thereby have an effect on alleviating or preventing inflammation.
- Such a variant would be more effective in reducing or preventing inflammation than would either protein by itself.
- the variant will localize to the desired specific site of extracellular matrix degradation, specifically preventing further degradation by inhibiting enzymes and preventing the binding of enzymes which cause degradation, thereby having a dual effect on alleviating or preventing
- a chimeric protein of the present invention To produce a chimeric protein of the present invention the central role of uPA and uPAR in cancer invasion and inflammation were recognized. With this information in mind it was understood that the present invention should provide a chimeric protein which would interfere with the binding of uPA to uPAR and inhibit both uPA and plasmin generated at sites of cellular invasion.
- the receptor binding region of uPA has been localized to the 135 residue amino-terminal fragment (ATF).
- ATF 135 residue amino-terminal fragment
- This region binds to the uPAR with high affinity (K d 0.1 to 1 nM) and can competitively inhibit the binding of uPA to the uPAR.
- PN-1 blocks tumor cell-mediated extracellular matrix degradation and tumor cell migration in vitro, in that PN-1 inhibits the uPA-plasmin system.
- the present invention provides a chimeric protein consisting of the amino- terminal fragment of uPA and PN-1 (ATF-PN1). Due to the presence of the ATF portion, this chimeric protein has a high affinity to sites of cellular invasion; and due to the PN-1 protein it inhibits uPA and plasmin.
- This aspect of the invention involves the manipulation of the amino acid sequence of the PN-1, so that the reactive site is in some way altered, to change the protease specificity or the degree of inhibitory effect of PN-1 on serine proteases. More specially, the present invention involves substituting one or more amino acids within protease nexin-1 and/or deleting or adding amino acids to the sequence of protease nexin-1 in order to obtain an effect on the reactive site of protease nexin-1 so that the protease specificity of protease nexin-1 and/or the degree of inhibitory effect of
- protease nexin-1 on a serine protease is changed.
- the change in protease specificity or degree of inhibitory effect is obtained by substituting an amino acid at the P 1 , P 2 , P 3 , P 4 or, alternatively, P 1 ', P 2 ', P 3 ', P 4 ' sites.
- the invention involves substituting one or both of the "P 1 " site arginine residue or "P 1 '" site serine residue with a different residue resulting in PN-1 variants with radically different protease specificities and/or inhibitory effects on particular serine proteases.
- the PN-1 variants of the invention can also be described in terms of their functionality. Importantly, some of the PN-1 variants of the invention are capable of inhibiting elastase. Within this general group are PN-1 variants wherein the ability to inhibit elastase is greatly enhanced in the presence of heparin and/or heparin-like compounds. Another group of PN-1 variants of the invention include PN-1 variants which have an enhanced ability to inhibit serine proteases as compared with PN-1. The PN-1 variants of the invention are designed to inhibit serine proteases such as urokinase. Factor Xa plasmin, kallikrein. Factor XIIa, Factor XIa, Factor Va, tPA, elastase, cathepsin and contrapsin.
- serine proteases such as urokinase.
- Functional objectives of the invention such as the production of a compounds which inhibit serine proteases such as elastase and whose ability to inhibit such is enhanced in the presence of other compounds such as heparin, are obtained, in general, by manipulating DNA.
- the DNA encoding an enzyme is manipulated by including within the DNA a sequence of DNA which encodes a substrate for a particular serine protease.
- the recombinant DNA is then expressed to produce a variant of the invention which will include a substrate for the particular serine protease.
- protease can be specifically inhibited by the variant while the variant maintains its natural biological activity.
- the arginine residue is a polar, basic amino acid. Substitution of the polar arginine with a non-polar residue has a dramatic effect on the degree of serine protease inhibition obtainable.
- the R345I variant i.e., isoleucine has been substituted for arginine a position 345 of PN-1 ⁇
- the NCY2010 variant is a good inhibitor of
- NCY2010 The activity of NCY2010 should be viewed with the understanding that the relative efficiency at which protease inhibitors (such as the PN-1 variants of the invention) inhibit serine proteases are measured by a known standard. That standard is the second order
- k ass equal to or greater than 1 ⁇ 10 5 M -1 S -1 for a particular protease-inhibitor reaction is considered to be physiologically significant (Travis and Salveson Ann. Rev. Biochem. (1983)
- rate constants Many physiologically important protease-inhibitor reactions such as elastase- ⁇ -1-antitrypsin and plasmin- ⁇ -2-antiplasmin occur with rate constants as high as 1 ⁇ 10 7 M -1 S -1 or greater.
- the thrombin-PN-1 reaction occurs at a similar high rate, or faster, in the presence of heparin.
- Native PN-1 has essentially no effect with respect to inhibiting the activity of elastase.
- the R345I variants of the present invention clearly provide not just a new biological activity for the serpin, i.e., its ability to inhibit elastase, but clearly provide an extremely potent elastase inhibitor.
- the ability of the R345I variants to inhibit elastase to such a degree was in itself a surprising finding. However, it was clearly unexpected to find that, in addition to providing such a potent elastase inhibitor, these variants had still further increased potency with respect to inhibiting elastase while in the presence of heparin.
- Variants of the invention are clearly capable not only of providing improved potency with respect to acting as elastase inhibitors, but of providing such activity site-specifically in that their activity is greatly enhanced in the presence of heparin, heparin-like
- sulfated proteoglycans such as other heparin-like compounds normally found on the surface and surrounding extracellular matrix would provide not only increased potency with respect to the ability of the variants of the invention to inhibit elastase but provide site-specific activity due to the affinity of these variants to heparin and heparin-like compounds.
- the inhibitory effect of the R345I variant on elastase is increased approximately two orders of
- Non-polar residues which can be used include G, A, V, L, I, M, F and W, and more preferably (due to "R" group structures similar to valine) G, A, V and L, and most preferably I.
- PN-1 variants which are capable of acting as potent elastase inhibitors. More specifically, the invention encompasses such PN-1 variants which act as elastase inhibitors and further wherein the ability to inhibit elastase is greatly increased in the presence of heparin and heparin-like materials. Still more
- the invention encompasses PN-1 variants which act as elastase inhibitors and which variants have their ability to inhibit elastase increased 10 fold or more in the presence of heparin, preferably 50 fold or more in the presence of heparin and more preferably 100 fold or more in the presence of heparin.
- PN-1 variants which act as elastase inhibitors and which variants have their ability to inhibit elastase increased 10 fold or more in the presence of heparin, preferably 50 fold or more in the presence of heparin and more preferably 100 fold or more in the presence of heparin.
- formulations of the invention include PN-1 variants formulated in pharmaceutical compositions along with heparin and heparin-like compounds such as various
- heparin-like compounds are highly sulfated, thus providing high negative charges.
- P 1 variants of the invention which include non-polar residues such as valine substituted for the polar arginine can be used to treat individuals due to the ability of these P 1 variants to inhibit the activity of elastase.
- other proteases such as urokinase and plasmin which are inhibited by PN-1 are not heparin activatable.
- the P 1 variants of the present invention are effective in inhibiting elastase due to the cationicity of elastase which promotes its binding to heparin which is anionic.
- the active site should be substituted with other residues which are non-polar and have similar "R" groups in order to have a reasonable expectation of similar activity.
- variants of the invention such as elastase inhibitors of any PN-1 were not known. Further, it was not known whether any such variants would provide any activity, let alone the type of activity obtained. In fact, elastase cleaves native PN-1. This cleavage inactivates PN-1 toward thrombin and other proteases. It was quite possible that, in changing Arg 345 to Ile(R345I), PN-1 could be changed into an even better substrate for elastase, leading to even quicker inactivation of PN-1.
- the present invention not only provides variants wherein active sites have been replaced, but shows that such variants have activity and that the activity is
- variants can be produced wherein substitution is made at both the P 1 and P 1 ' sites. Such double substitutions could be put forth in a variety of different ways.
- One approach to producing such variants is to substitute one of the sites with a residue which is substantially different from the residue present such as including a non-polar residue in place of a polar residue while substituting the other site with a residue which is substantially similar to the residue present there both in terms of being polar or non-polar and in terms of having a similar "R" group.
- Another approach is to substitute both sites with residues which are
- variants which continue to have activity with respect to a given protease or gain substantial activity with respect to another protease.
- the PN-1 variants which have activity are variants which have (1) substantially increased potency with respect to inhibiting tPA, urokinase, and/or other related enzymes; (2) substantially increased potency with respect to inhibiting elastase; or most preferably (3) substantially increased potency with respect to inhibiting elastase and which potency is still further increased dramatically in the presence of heparin.
- the present invention has demonstrated that it is possible to produce PN-1 variants which inhibit elastase and has further
- protease nexin-1 variants of the invention are listed in Table 2 along with "indication" of the variant.
- the examples could be continued to include all 20 amino acid substitutions at each position within the active site, that is, all 20 naturally-occurring amino acids could be substituted, using site-directed mutagenesis, at positions P 1 , P 2 , P 3 , P 4 , P 1 ', P 2 ', P 3 ', and P 4 '.
- variants of the present invention can be tested by comparing the variants with a panel of proteases and determining their second-order rate
- Type II variants of the invention are produced in a manner similar to Type I variants. However, the active site of PN-1 is modified in a manner so that it matches the sequence of the active site of another protease, and preferably another serpin. Examples of Type II variants of the present invention include the following:
- ⁇ indicates the sequence is preferably followed by
- Type II variants can be used in the production of Type II variants. Further, the methodology disclosed within the above-cited patents (incorporated herein by reference) can be used to produce Type II variants. The methodology is modified only by first determining the active site of another serpin. After determining the amino acid sequence of the active site of a different serpin and studying the activity of that different serpin it is possible to produce a Type II variant having a particular and desired changed activity as compared with the naturally occurring PN-1. Testing Type II Variants
- NCY 2202 (particularly in the presence of heparin) is analogous in some ways to that of NCY 2322 in that both inhibit catheprin G but not
- Type III Variants Incorporation of a Substrate Sequence
- Type III variants of the invention are produced by first determining the substrate sequence of a protease.
- the substrate sequences of some proteases which are known and which would be useful in connection with the present invention are put forth below in Table 3A. TABLE 3A
- PROTEASE substrate sequence Thrombin D-Phe-Pip-Arg-pNA
- substrate sequences can be determined by determining the best artificial small molecule peptide substrates (i.e. Ala-Ala-Pro-Phe-pNA) as determined by k cat and k ⁇ , or by examining the sequence of natural protein substrates (e.g. fibrinogen for thrombin).
- Type III variants are as follows: TABLE 3B
- Type IV variants of the invention are quite different from all of the other variants in that the amino acid sequence may be the same as the naturally-occurring protein or different therefrom.
- Type IV is quite different from all of the other variants in that the amino acid sequence may be the same as the naturally-occurring protein or different therefrom.
- variants may be generated by covalently binding (i.e., coupling) PEG to any protein, protein fragment, or
- peptide in general when increased biological stability is desired.
- proteins, protein fragments, and peptides which are useful in therapeutic applications, such as serine protease
- the proteins PN-1, human growth hormone (hGH), erythropoietin (EPO), and antithrombin-III (ATIII) are of particular interest.
- Specific exemplary proteins of interest, as well as exemplary classes of proteins, which can be modified to create a Type IV variant using the methods of the invention are provided in Table 4A.
- Type IV variants are created by attaching polyethylene glycol to a thio group on a cysteine residue of the protein.
- the PEG moiety attached to the protein may range in molecular weight from about 200 to
- the PEG moiety will be from about 1,000 to 8,000 MW, more preferably from about 3,250 to 5,000, most preferably 5,000 MW.
- the protein is modified by
- a cysteine residue is added at a position which is normally glycosylated (e.g.
- the polyethylene glycol is attached to the thio group of the added cysteine residue.
- the protein of interest is one member of a family of structurally related proteins, glycosylation sites for any other member can be matched to an amino acid on the protein of interest, and that amino acid changed to cysteine for attachment of the polyethylene glycol.
- a crystal structure has been determined for the protein of interest or a related protein, surface residues away from the active site or binding site can be changed to cysteine for the
- the protein may be biotinylated by attaching biotin to a thio group of a cysteine residue.
- Type IV variants are as follows:
- records numbers 1 and 2 are of a general type known in the art in that the polyethylene glycol or biotin is attached to lysine position of the peptide.
- record numbers 3 and 4 are,
- polyethylene glycol or biotin are connected at a cysteine group.
- PEG polyethylene glycol
- lymphoblastic leukemia interferon alpha 2b (IFN- ⁇ 2b) as an anticancer and antiviral drug
- IFN- ⁇ 2b interferon alpha 2b
- superoxide dismutase as an anticancer drug
- streptokinase tPA
- urokinase uricase
- hemoglobin interleukins
- interferons TGF- ⁇
- EGF EGF
- this modification of a protein by attachment of a PEG moiety involves activating PEG with a functional group which will react with lysine residues on the surface of the protein. If the modification of the protein goes to completion, the activity of the protein is usually lost. Current modification procedures allow only partial PEGylation of the protein. Usually this results in only 50% loss of activity and greatly
- An unavoidable result of the partial modification is that there will be a statistical distribution of the number of PEG groups per protein, each PEG attached to a lysine residue. Also, there will be a random usage of lysine residues on the surface. For instance, when adenosine deaminase is optimally modified, there is a loss of 50% activity when the protein has about 14 PEG per protein, with a broad distribution of actual PEG per individual protein and a broad distribution of actual lysine residues used.
- Zalipsky, et al. (Zalipsky, S., Seltezer, R. & Nho, K. (1991) Polymeric Drugs and Drug Delivery Systems. ACS.) describe the use of succinimydyl carbonates of PEG to produce stable carbamate linkages with proteins via free lysine residues.
- succinimydyl carbonates of PEG to produce stable carbamate linkages with proteins via free lysine residues.
- There are several other methods for modification of proteins with PEG through free lysine residues but each suffers from the problems associated with partial, random modification of proteins and the potential for losing activity if lysine residues are important.
- PEG-modify other residues such as His, Trp, Cys, Asp, Glu, etc. in such a manner that activity is not lost. It is anticipated or likely that many of these residues will be at or near the active site or that these residues will not be at the surface or in sufficient number to significantly affect serum half-life, or that the modification chemistry is not specific enough for the target residue, or the chemistry is too harsh for the protein to withstand without loss of activity.
- glycosylation residues are glycosylated when part of a well known N-glycosylation signal (NP!T/SP!). Replacement of these Asn residues by cysteine, followed by cysteine-specific PEGylation is expected to lead to proteins with significantly increased serum half-life with a minimum loss in activity. To date, this is the closest thing to in vitro glycosylation, and in some respects may be better since glycosylation with
- glycosylation not found in the protein of interest If these "new" potential sites are in a region which is reasonably conserved (i.e. not part of an insertion or with a sequence which is so different that it is likely to have a different structure) it is expected that replacement of the residue equivalent to the Asn with cysteine followed by PEGylation will result in a more highly PEG modified protein without significant loss in activity.
- amino acid sequences in the native protein which are recognized by antibodies are usually on the surface of the protein.
- Modification of proteins with PEG can also be used to generate dimers and multimeric complexes of proteins, fragments, and/or peptides which have increased
- multimeric proteins of the invention may be naturally occurring dimeric or multimeric proteins.
- the dimer or multimer may be composed of cross-linked subunits of a protein (e.g., hemoglobin).
- the dimeric and multimeric proteins may be composed of two proteins which are not normally cross-linked (e.g., a dimer of cross-linked EPO protein.
- Dimeric proteins of the invention may be produced by reacting the protein with (Maleimido) 2 -PEG, a reagent composed of PEG having two protein-reactive moieties.
- R 1 -S-PEG-S-R 2 where R 1 and R 2 may represent the same or different proteins and S represents the thio group of a cysteine either present in the native R 1 or R 2 protein, or
- the proteins R 1 and R 2 may each vary in size from about 6 to 1,000 amino acids, preferably about 20 to 400 amino acids, more preferably 40 to 200 amino acids. In dimeric molecules, R 1 and R 2 are preferably from about 100 to 200 amino acids. Dimers and multimers of particular interest include those composed of proteins, protein fragments and/or peptides which are less than about 40,000
- multimeric proteins where the proteins (represented by R 1 , R 2 , acrossR n ) may be the same or different can be generated.
- the proteins represented by R 1 , R 2 , acrossR n may vary in size from about
- Such multimeric proteins may be of the following general formula:
- R 1 represents the protein having multiple free cysteines, for example from 2 to 20 free cysteines, usually from 5 to 7 free cysteines.
- R 2 may represent a protein the same as or different from R 1 .
- each of the R 2 proteins attached to R 1 may be the same proteins, or represent several different proteins.
- the degree of multimeric cross-linking can be controlled by the number of cysteines either present and/or engineering into the protein and by the
- the Maleimido-PEG+ (Maleimido) 2 -PEG reagents may be used in the same reaction with proteins for formation of couplings of proteins having simple PEG moieties as well as PEG cross-links between proteins within the complex.
- the dimeric or multimeric protein generated will have an increased half-life relative to the native, protein, due at least in part to its
- Dimeric and multimeric proteins may be generated by reaction with Maleimido-PEG or (Maleimido) 2 -PEG.
- Exemplary proteins for dimeric and/or multimeric complex formation using the method of the invention include PN-1, PN-1 variants, hemoglobin, and erythropoietin (EPO), as well as any of the proteins or members of the protein classes exemplified in Table 4A.
- the protein generated by the method of the invention is a PEGylated cross-linked complex of the "a" and "b" chains of
- hemoglobin multimeric complexes of hemoglobin having intermolecular and/or intramolecular cross-links may also be generated by the subject method.
- the method of identifying cysteine residues for PEG modification, and/or identifying amino acid residues to be replaced with cysteine which are subsequently modified by attachment of PEG provides for generation of a PEGylated protein which can be reasonably expected to retain most or all of the activity of the native protein.
- the sites selected for modification and/or substitution with cysteine are selected on the basis of the structure of the protein, i.e. the selected sites are solvent accessible residues which are not involved in the active site.
- mutations located outside of the active site are generally predictable in that they generally do not change the primary activity of the protein.
- structural mutations described herein are within solvent-accessible regions of the protein (i.e. on the protein "surface") which have limited or no interaction with other residues in the protein molecule. Thus, mutations at these positions are unlikely to affect the conformation of any other amino acid in a protein.
- Type V variants of the invention are produced by fusing all or a fragment of another protein to PN-1.
- the amino terminal fragment of a protein such as urokinase is fused to PN-1 in order to localize PN-1 to a different receptor, i.e. to the urokinase receptor.
- a protein such as urokinase
- it is possible to fuse the amino terminal fragment of proteins such as tPA, Factor IX, Factor X, and Protein C. Examples of Type V variants are as follows:
- the type V variants of the invention can be produced in manner similar to variants of type I, II and III. However, it is more preferable to produce such variants by chemically fusing an N-terminal fragment of a different protein to the PN-1.
- Type V variants can also be produced by chemical linkage of purified preparations of both protein components. Such linkage is conveniently accomplished by using bi-functional cross-linking reagents. Methods for chemically establishing such linkages are well known to those skilled in the art. Specific variants which might be produced in this manner include variants produced by fusing PN-1 to any one of: EGF; Factor IX; Factor X; and APC.
- the method of PEGylation of the invention is intended to be a general procedure and as such is
- Type V variants of the invention are chimeric proteins wherein PN-1 is covalently bound to another protein.
- the same panel of proteases indicated above were used to test one of the Type V variants of the invention, and the results are put forth below.
- chimeric proteins, PN-1 variants, and cysteine-PEGylated proteins of the invention can provide different effects.
- P 1 variants with non-polar residues such as valine substituted for the polar arginine residue could be used as heparin activatable inhibitors.
- inhibitors could be used to treat individuals suffering from elastase-related diseases. Although not limited to such diseases, such variants could be used to treat emphysema, congenital ⁇ -1-antitrypsin deficiency, inflammation, arthritis and septic shock.
- chimeric proteins, PN-1 variants, and/or cysteine-PEGylated versions of these proteins of the invention is the inclusion of such within various topical formulations such as creams or gels, or a combination of such formulations, with various bandages for application to wounds to aid in wound healing and decrease
- chimeric proteins, PN-1 variants, and cysteine-PEGylated versions of these proteins of the invention are believed to be effective in decreasing inflammation, injectable formulations
- containing the chimeric proteins, PN-1 variants, and/or cysteine-PEGylated versions of these proteins of the invention may be injected directly into inflamed joints or other inflamed areas of the body in order to decrease the inflammation. Further the formulations of the invention may be used prophylactically by providing the chimeric proteins, PN-1 variants, and/or
- cysteine-PEGylated versions of these proteins to a particular site which may be subjected to trauma, (such as in surgery), and thus inflammation, to prevent the inflammation from occurring.
- the pharmaceutical compositions containing the chimeric proteins, PN-1 variants, and/or cysteine-PEGylated proteins of the invention will be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium, preferably at a pH of about 5 to 8, more preferably 6 to 8, although the preferred pH of the pharmaceutical composition may vary according to the protein employed and condition to be treated.
- cysteine-PEGylated proteins and pharmaceutical compositions containing these protein.
- PEGylation of proteins generates proteins which are ready for immediate therapeutic use (i.e. do not require reconstitution), have increased solubility and have an increased half-life and are reduced in immunogenicity and antigenicity relative to the unmodified protein (Nucci et al. 1991 Adv. Drug Delivery res . 6:133-151).
- the increased half-life of PEGylated proteins decreases the amount of protein needed for an effective dosage, reduces the number and frequency of administrations required, and decreases the patient's exposure to the protein, thus decreasing the potential for allergic reactions, toxic effects, or other side effect.
- PEGylated proteins allow for long-term use of the protein with less potential for undesirable side effects related to protein immunogenicity and/or toxicity.
- Exemplary proteins for which an increase half-life has effected by PEGylation of the protein include: hGH, insulin,
- interferon-alpha2A interferon-alpha-2A
- interferon-alpha2B interferon-alpha-2B
- tPA interferon-alpha2A
- EPO interferon-alpha2B
- G-CSF antigen E
- arginase arginase
- asparaginase adenosine deaminase
- batroxobin bovine serum albumin
- catalase catalase
- elastase factor VIII
- galactosidase alpha-galactosidase, beta-glucuronidase, IgG, honeybee venom, hemoglobin, interleukin-2, lipase, phenylalanine ammonia lyase, alphaj-proteinase inhibitor, pro-urokinase, purine nucleoside phosphorylase, ragweed allergen, streptokinase, superoxide dismutase, tPA, D-alpha-tocopherol, trypsin, tryptophanase, uricase, and urokinase (see in general Nucci et al. ibid. ; see also Davis et al. 1981 Clin . Exp.
- PEGylated proteins may be administered for the treatment of a wide variety of diseases. Exemplary disease conditions and the proteins useful in treatment of these diseases are provided in Table 6A.
- chimeric proteins As discussed above, chimeric proteins, PN-1 variants, and cysteine-PEGylated proteins may be used.
- the protein as well a various factors which may modify the effectiveness of the protein as a drug, such as disease state (e.g. severity) of the patient, age, weight, gender, diet, time of administration, drug combination, reaction sensitivities, tolerance to
- the clearance rate i.e. the half-life of the protein
- the clearance rate can be varied to give ultimate flexibility to fit the particular need of the patient by changing, for example, the number of PEG moieties on the cysteine-protein, the size of the PEG moiety
- cysteine-PEGylated proteins are employed, the daily regimen should generally be in the range of the dosage for the natural, recombinant, or PEGylated
- Normal dosage amounts may vary from 0.1 to 100 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
- EPO, GM-CSF, G-CSF, and multi-CSF for treatment of pancytopenia may be found in USPN 5,198,417; guidance for administration of EPO for treating iron overload may be found in USPN 5,013,718; guidance for administration of EPO in the treatment of
- hemoglobinopathies may be found in USPN 4,965,251;
- pancreatin, rennin, papain, trypsin, pancrelipase, chymopapain, bromelain, chymotrypsin, streptokinase, urokinase, tissue plasminogen activator, fibrinolysin, deoxyribonuclease, sutilains, collagenase, asparaginase, or heparin in a cryogel bandage for treatment of sites of trauma may be found in USPN 5,260,066; guidance for the administration of superoxide dismutase,
- glucocerebrosidase asparaginase, adenosine deaminase, interferons (alpha, beta, and gamma), interleukin
- tissue necrosis factor TNF-alpha or TNF-beta
- colony stimulating factors CSF, G-CSF, GM-CSF
- USPN 5,225,212 guidance for administration of asparaginase or insulin in the treatment of neoplastic lesions may be found in USPN 4,978,332; guidance for administration of asparaginase in the reduction of tumor growth may be found in USPN
- interleukin-1 administration of interleukin-1 as a therapy for
- immunomodulatory conditions including T cell mutagenesis, induction of cytotoxic T cells, augmentation of natural killer cell activity, induction of interferon-gamma, restoration or enhancement of cellular immunity, and augmentation of cell-mediated anti-tumor activity may be found in USPN 5,206,344; guidance for the administration of interleukin-2 in the treatment of tumors may be found in USPN 4,690,915; and guidance for administration of interleukin-3 in the stimulation of hematopoiesis, as a cancer chemotherapy, and in the treatment of immune disorders may be found in USPN 5,166,322.
- PEGylated proteins have already been approved for use by the U.S. Food and Drug Administration (FDA). These PEGylated proteins include: hGH, insulin, interferon-alpha2A, interferon-alpha2B, tPA, EPO, G-CSF, and a hepatitis B vaccine which contains PEGylated proteins (Nucci et al. ibid) .
- PN-1 is not found in significant quantities in plasma and may function
- connective tissues and cells which contain sulfated proteoglycans on their surface and surrounding
- PN-1 extracellular matrix
- PN-1 variants or chimeric proteins in order to facilitate peripheral or central nerve regeneration.
- Formulations, routes of administration and dosages for use of PN-1 in the treatment of inflammation and wounds are described in USPNs 5,206,017; 5,196,196; and 5,112,608; each of which are incorporated herein by reference to the extent that such methods of treatment using PN-1 are described.
- chimeric proteins or protease nexin-1 and its variants by oral delivery systems.
- Such proteins are generally digested in the GI tract (unless formulated with special carriers) and do not enter the cardiovascular system in their original forms due to such digestion.
- Chimeric proteins, PN-1 variants, and/or cysteine-PEGylated versions of these proteins can be administered by any type of injection, such as intramuscular or intravenous, thus avoiding the GI tract.
- Transmucosal administration include transdermal and transmucosal administrations provided by patches and/or topical cream compositions.
- Transmucosal administrations can include nasal spray formulations which include the chimeric proteins, protease nexin-1 variant, and/or cysteine-PEGylated proteins within a nasal formulation which contacts the nasal membranes and diffuses through those membranes directly into the cardiovascular system. PEGylated proteins may have an increased ability to cross membranes and thus may enter the body more easily.
- Formulations which include the chimeric proteins, PN-1 variants within aerosols for intrapulmonary delivery are also contemplated by this invention, as are intraocular delivery systems wherein the chimeric proteins or PN-1 variants are included within ophthalmic formulations for delivery in the form of eye drops.
- the formulations can be designed to provide the chimeric proteins or PN-1 variants systemically or to a particular site. Further, the formulations can be designed so as to provide the chimeric proteins or PN-1 variants as quickly as possible or in a sustained release or timed released manner.
- topical formulations could be created whereby the chimeric proteins or PN-1 variants of the invention were incorporated or disbursed throughout topical polymer formulations capable of slowly releasing the chimeric proteins or PN-1 variants to a wound site in order to continually aid in wound healing and in
- chimeric proteins, PN-1 variants, and/or cysteine-PEGylated protein can be administered in a variety of different manners in order to introduce the chimeric proteins, PN-1 variants, and/or cysteine-PEGylated protein into the cardio vascular system.
- the chimeric proteins, PN-1 variants, and/or cysteine-PEGylated proteins are
- proteins PN-1 variants, and/or cysteine-PEGylated versions of these proteins are useful for their
- PN-1 was purified to homogeneity from serum-free medium conditioned by human foreskin fibroblasts in microcarrier cultures by affinity chromatography on heparin-agarose, followed by gel exclusion
- the purified protein shows an M T of 42-43 kd, based on sedimentation equilibrium analysis, or of 47 kd, estimated from gel-exclusion chromatography.
- purified material shows the properties exhibited by PN-1 when contained in conditioned medium, including formation of sodium dodecylsulfate-stable complexes with thrombin, urokinase, and plasmin; inhibition of protease activity; heparin-enhanced inhibition of thrombin; and cellular binding of protease-PN complexes in a heparin-sensitive reaction.
- N-terminal amino acid sequence of the isolated, purified protease nexin was determined for the first 34 amino acids to be: Ser-His-Phe-Asn-Pro-Leu-Ser-Leu-Glu-Glu-Leu-Gly-Ser-Asn-Thr-Gly-Ile-Gln-Val-Phe-Asn-Gln-Ile-Val-Lys-Ser-Arg-Pro-His-Asp-Asn-Ile-Val-Ile.
- the PN-1 variants of the present invention can be synthesized by utilizing the pure PN-1 which has been isolated and purified in the manner indicated above.
- the variants can be obtained by cleaving the purified PN-1 protein at the P 1 or P 1 ' site and replacing the arginine, serine or both residues at that site with the desired non-polar substitute residue. After replacement of the desired residue with the desired non-polar residue, the segments can be fused utilizing protocols known to those skilled in the art.
- protocols known to those skilled in the art.
- cDNA encoding the complete human PN-1 protein was obtained from a foreskin fibroblast DNA library. The retrieval of this clone took advantage of probes based on the amino acid sequence determined in the native protein.
- the cloned cDNA is amenable to expression in recombinant cells of both procaryotic and eucaryotic organisms by excising the coding sequence from the carrier vector and ligating it into suitable expression systems.
- the PN-1 can be directly produced as a mature protein preceded by a Met N-terminal amino acid (which may or may not be processed, depending on the choice of expression systems) may be produced as a fusion protein to any desirable additional N-terminal or C-terminal sequence, or may be secreted as a mature protein when preceded by a signal sequence, either its own, or a heterologous sequence provided by, for example, the known signal sequence associated with the bacterial-lactamase gene or with secreted human genes such as insulin or growth hormones.
- a signal sequence either its own, or a heterologous sequence provided by, for example, the known signal sequence associated with the bacterial-lactamase gene or with secreted human genes such as insulin or growth hormones.
- restriction sites at appropriate locations with respect to the desired coding sequence by site-directed
- mutagenesis are well understood, and the coding sequence can thus be provided with suitable sites for attachment to signal sequence or fusion sequence, or into expression vectors.
- the protein produced may include the N-terminal Met. Modification of the protein produced either intracellularly or as
- polysaccharide substances by refolding using techniques to sever and reform disulfide bonds, or other post-translational ex vivo processing techniques. If the protein is produced in mammalian or other
- the cellular environment is such that post-translational processing can occur in vivo, and a glycosylated form of the protein is most likely produced.
- the recombinant cells are cultured under
- the protein is recovered from the cellular lysate or from the medium, as determined by mode of expression.
- DNA segments coding for the production of PN-1 have been inserted into bacterial hosts, multiple copies of the segments can, of course, be cloned by growing the bacteria.
- the segments can be extracted from the bacteria by the use of conventional methodology whereby the DNA is extracted by subjecting disrupted cells to centrifugation and then subjecting the extracted DNA to enzyme digestion, which will result in obtaining the desired segments by subjecting the digested DNA to separation processes such as gel electrophoresis and blotting.
- the segments coding for the production of PN-1 can then be subjected to conventional recombinant
- PN-1 variants might be made by using recombinantly produced PN-1 and then substituting only the desired "R” group (e.g., -OH of serine 346) with a non-polar "R” group (e.g.,
- the cDNA sequence In order to produce PN-1 and/or PN-1 variants in insect cells, the cDNA sequence must first be inserted into a suitable plasmid expression vector, such as pAC373. Appropriate restriction sites for this insertion can be created by standard site-directed mutagenesis procedures.
- a suitable expression vector include a transcriptional promoter such as the polyhedron gene promoter of pAC373, and flanking homologous sequences to direct recombination into the baculovirus genome.
- a polyadenylation signal such as the one from the polyhedron gene present in this plasmid vector, may or may not be necessary for expression of the recombinant gene.
- a marker gene such as the ⁇ -galactosidase gene of E.
- coli juxtaposed to regulatory sequences including a transcriptional promoter and possibly a polyadenylation signal, may be included in the vector but is not essential for expression of a convected gene.
- a chimeric baculovirus is created by homologous recombination between the expression plasmid containing the PN-1 target gene and wild type baculovirus DNA.
- Plasmid and wild type baculovirus DNA are co-precipitated by the calcium phosphate technique and added to
- cells will exhibit a cytopathic morphology and contain the nuclear occlusion bodies typically produced by viral infection.
- the cell-free culture media containing both wild type and recombinant virus is harvested.
- Clonal isolates of virus can be obtained from this co-transfection stock by plaque purification on Sf9 cell monolayers overlaid with agarose.
- Candidate plaques for analysis will be identified by a plaque morphology negative for occlusion bodies. If the expression plasmid contains a marker gene such as ⁇ -galactosidase,
- recombinant plaques will be indicated by the blue color produced from a chromogenic substrate such as 5-bromo- 4-chloryl-3-indolyl-b-D-galactopyranoside (X-gal) in the agarose plating medium.
- a chromogenic substrate such as 5-bromo- 4-chloryl-3-indolyl-b-D-galactopyranoside (X-gal) in the agarose plating medium.
- Picked plaques will be used for inoculation of cells in multiwell dishes.
- the resulting cell lysates and infected cell supernatants can be evaluated for expression of recombinant PN-1, using standard activity or immunological assays. Positive wells may require additional rounds of plaque
- Sf9 cells are adapted to growth in serum-free, low protein medium such as ExCell (J.R. Scientific). Cells are collected from suspension culture by gentle
- PN-1 is purified from the cell-free supernatant by standard means.
- Variants of PN-1 may be created and produced in the same manner as described above.
- PN-1 produced in insect cells using a baculovirus expression system is a glycosylated protein of
- N-terminal amino acid sequence is identical to that of mature mammalian cell PN-1, indicating correct processing of the signal sequence.
- the specific activity vs is identical to that of mature mammalian cell PN-1, indicating correct processing of the signal sequence.
- PN-1 The gene for PN-1 was generated by PCR from the CHO
- the pGEMEX E. coli expression vector contains three RNA polymerase promoters.
- the T7 promoter is positioned upstream from the gene 10 leader fragment.
- pT7-NK This plasmid is referred to as pT7-NK.
- pT7-NK was cut with Ndel and BamHI to remove the gene 10 fusion protein region.
- the linear vector was isolated and ligated with the PCR-generated PN-1 linear fragment, cut with Ndel and Bell, described above.
- This plasmid is referred to as pT7PN-1.
- the correct sequence was confirmed by sequencing the entire coding region for PN-1.
- the native signal sequence was removed using PCR and the following oligos:
- the expression of the resulting protein is expected to be intracellular, either in inclusion bodies or as soluble protein.
- the plasmid pT7PN1 has an f1 ori for the
- substitutions in the region of P4 to P4' are generated by site directed mutagenesis. In general, substitutions at the P1 site will have the most dramatic effects.
- sequences found at the active site region of other serpins were grafted onto PN-1.
- a number of combinations must be created to determine how much of the sequence at the active site must be changed to change the specificity and kinetics.
- the P 4 to P 4 ' region is generally found to be most important, but amino acids residues outside this region can have pronounced affects on protease inhibition.
- sequences which have been found to be particularly good substrates are added to or used to replace a sequence of PN-1. Prior to making these mutants, it was not clear if these changes would ruin the inhibitory effects of PN-1 and turn PN-1 from an inhibitor of proteolysis into a substrate. In fact, incorporation of Ala-Ala-Pro-Phe, a good substrate sequence for subtilisin, into PN-1 results in a molecule which is cleaved particularly well by subtilisin.
- PN-1 variants have now been obtained which are good inhibitors of mammalian serine proteases based upon this approach.
- optimum inhibitor sequences can be generated by using a phage display system. Since PN-1 forms covalent interactions with the target
- protease it is important that one is not selecting for mutants which bind more tightly than the parent PN-1 molecule. Rather, one selects for PN-1 variants which bind more rapidly to the target protease by allowing phage-displayed variant PN-1 library to interact with the immobilized target protease for only short times. Thus, only rapid-binding variants will be selected. This is a novel application of the phage display system.
- I343F;A344P FPR, thrombin
- JM109 (DE3) contains a chromosomal copy of the gene which codes for T7RNA polymerase under the control of the inducible lac promoter.
- JM109 (DE3) containing pT7PN-1 (or a variant of PN-1) was grown overnight in 2xYT + 0.2% glucose + 100 mg/ml carbenicillin at 28-32°C. Low temperature and high nutrient containing solution is helpful in generating productive innoculants.
- the inoculum was diluted 1:250 to 1:500 and grown to OD 600 ⁇ 1 in a shake flask or -50 in a fermentor at 26-37°C, and induced with IPTG at 0.1-1.0 mM for 4-16 hours.
- the bacteria were collected by centrifugation, resuspended in 10 mM TRIS, pH 8, 1 mM EDTA, and disrupted by high pressure homogenization. Inclusion bodies were collected by centrifugation, washed with 1 M NaCl, 0.05%
- PN-1 was purified by capture on Fasts sepharose and eluted with 0.6 M NaCl, diluted to 0.25 M NaCl and passed over FastQ sepharose to remove endotoxin and recaptured on Fasts sepharose and eluted with 0.6 M NaCl or a gradient of 0.25 to 1 M NaCl.
- PN-1 can be generated in a soluble form within E. coli by adjusting the fermentation
- PN-1 The production of PN-1 was about 50 mg per gram of cell paste. This corresponds to about 50 mg per liter of production at a cell density of 1 OD 600 or up to 2.5 grams of soluble PN-1 per liter of fermentation.
- Refolded or soluble protein was tested for capacity to inhibit thrombin in a standard assay.
- PN-1 variants were tested for their ability to inhibit urokinase using the substrate S-2444, plasmin using the substrate S-2390, tPA using the substrate S2288, Factor Xa using the substrate S-2222 or S-2765, kallikrein using the
- the t 1/2 was estimated from resulting curves.
- the k assoc was estimated according to the equation ln2/ [PN-1] ⁇ t 1/2 .
- the apparent first order rate constant was determined from the slope of a plot of log (normalized activity) vs time.
- the second order rate constant was calculated by dividing the apparent first order rate constant by the PN-1 (or variant) concentration used.
- pT7ATF-PN1 The correct sequence was verified by
- ATF 48 -PN1 was made by introducing a Mlul site (underlined) by site directed mutagenesis at codon 48 using the
- the resulting plasmid was cut with Mlul to remove a
- the plasmid pT7ATF-PN1 has an f1 ori for the production of single-stranded DNA.
- pT7ATF-PN1 was transformed into the E. coli strain CJ236 for the
- PN-1 variants of interest can be subcloned into pT7ATF-PN-1 by using standard molecular biology
- the resultant plasmid was transformed into the E. coli strain JM109 (DE3), grown to OD ⁇ -1 in a shake flask or ⁇ 50 in a fermentor, and induced with IPTG at 0.1-1.0 mM for 4-16 hours at 26-37°C.
- the bacteria were collected by centrifugation, resuspended in 10 mM TRIS, pH 8, 1 mM EDTA, and disrupted by high pressure
- ATP-PN1 was purified by capture on Fasts sepharose and eluted with 0.6 M NaCl, diluted to 0.25 M NaCl and passed over FastQ sepharose to remove endotoxin and recaptured on Fasts sepharose and eluted with 0.6 M NaCl or a gradient of 0.25 to 1 M NaCl.
- the cell supernatant from the disruption step was clarified by centrifugation and filtration, and the soluble protein was purified as above.
- Refolded or soluble protein was tested for capacity to inhibit thrombin in a standard assay. Briefly, serial 2-fold dilutions of ATF-PN1 were added to microtiter plate wells (50 ⁇ l/well), followed by 50 ⁇ l of a 30 ⁇ g/ml heparin solution, followed by 1 NIH unit of thrombin in 50 ⁇ l. These are allowed to incubate at 25°C for 15 minutes. Residual thrombin activity is measured by the addition of 50 ⁇ l S-2238 (kabi Pharmaceuticals) at 0.625 mg/ml. ATF-PN1 was tested for its ability to inhibit urokinase using the substrate S-2444, or plasmin using the substrate S-2390, in a similar manner, without the addition of heparin.
- Refolded or soluble protein was tested for the ability to bind to a soluble form of the urokinase receptor as measured by ELIZA, or the ability to inhibit the binding of urokinase to the soluble urokinase
- ATF-PNl was also tested for its ability to inhibit uPA or DFP/PMFS treated uPA binding to cells such as HT1080, U937, or THP-1 expressing uPA receptor.
- the amount of the components above (particularly 1) and 2)) and the volume indicated may be varied.
- the difference in the ratio of methoxypolyethylene amine to GMBS can vary by up to ten to 100-fold. Normally, about a two-fold excess of 1) above to GMBS is preferred.
- various buffers may be substituted for the Caps buffer, it is important that Tris buffers are not used in this mixture, as Tris buffers will quench the reaction.
- the pH of the buffer used may vary considerably, although buffers having a pH of 10.0 are preferable over buffers having a pH of 8.0.
- the mixture above may contain up to 50% DMSO as a cosolvent. It is particularly important that the reaction mixture does not contain a reducing agent such as dithiothreitol (DTT) or ⁇ -mercaptoethanol ( ⁇ ME) .
- DTT dithiothreitol
- ⁇ ME ⁇ -mercaptoethanol
- the mixture was incubated at 37°C for 30 minutes, although the reaction temperature may be as low as 4°C and the reaction time may be extended for up to one hour or more. After incubation, 12 mg of Tris free base or ethanolamine was added to the mixture to quench the NH 3 moiety. This quenching step may be omitted.
- the reacted mixture is purified by elution through a PD-10 column (G-25) (BioRad) equilibrated with 20 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% Tween. The eluant was collected in 0.5 ml fractions and assayed for
- the purified protein Prior to reaction of the protein with the Maleimido PEG reagent, the purified protein was diluted to a concentration of about 200 ⁇ g/ml to 1 mg/ml in any suitable buffer which does not contain DTT or ⁇ ME.
- the buffer was composed of 20 mM PIPES pH 6.75, 0.6 M NaCl, and 1% glycerol. Approximately 10 ⁇ l to 40 ⁇ l of the diluted protein was used for the PEGylation reaction.
- the Maleimido-PEG reagent described in section F.l was diluted in a series of 2-fold dilutions using 10 ⁇ l transfers of solution containing approximately 1 ⁇ l Maleimido-PEG in a 10 ⁇ l volume of buffer composed of 20 mM Tris pH 7.4, 0.1 M NaCl, and 0.01% Tween.
- the ratio of the maleimido-PEG to protein may be varied according to the preferred level of PEGylation of the protein desired. Up to 20-fold excess of maleimido-PEG to protein still provided for specific reaction of the reagent with cysteine residues of the protein.
- the protein and maleimido-PEG were incubated for one hour at room temperature, although this reaction may be performed at 4°C for longer periods of time.
- a sample of the reacted mixture may be analyzed by SDS-PAGE to determine the minimal amount of maleimido-PEG reagent needed for complete coupling.
- the reaction described above may be used to determine the proper ratio of Maleimido-PEG to protein, and then scaled up to produce commercially acceptable amounts of PEGylated protein.
- (Maleimido) 2 -PEG is prepared by mixing the following:
- the amount of the components above (particularly 1) and 2)) and the volume indicated may be varied.
- the difference in the ratio of 10 and 2) can vary by up to 10 to 100 fold, although an excess of GMBS to 1) above is preferred, normally about a 2-fold excess.
- various buffers may be substituted for the Caps buffer, it is important that Tris buffers are not used in this mixture, as Tris buffers will quench the reaction.
- the pH of the buffer used may vary
- the mixture above may contain up to 50% DMSO as a
- reaction mixture does not contain a reducing agent such as dithiothreitol (DTT) or ⁇ -mercaptoethanol (/3ME) .
- a reducing agent such as dithiothreitol (DTT) or ⁇ -mercaptoethanol (/3ME) .
- the mixture is incubated at 37°C for 30 minutes, although the reaction temperature may be as low as 4°C and the reaction time may be extended for up to one hour or more. After incubation, 12 mg of Tris free base or ethanolamine is added to the mixture to quench the NH 3 moiety. This quenching step may be omitted.
- the reacted mixture is purified by elution through a PD-10 column (G-25) (BioRad) equilibrated with 20 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% Tween. The eluant is collected in 0.5 ml fractions and production of
- (Maleimido) 2 -PEG is assayed by precipitation with 50% TCA.
- the resulting (Maleimido) 2 -PEG (Mal-PEG) reagent is then used in to modify a selected protein by attachment of PEG to a cysteine residue(s).
- the purified protein e.g. a PN-1 mutant containing a cysteine residue at position 99
- the purified protein is diluted to a concentration of about 200 ⁇ g/ml to
- the buffer is composed of 20 mM PIPES pH 6.75, 0.6 M NaCl, and 1% glycerol.
- section F.l is diluted in a series of 2-fold dilutions using 10 ⁇ l transfers of solution containing approximately 1 ⁇ l (Maleimido) 2 -PEG in a 10 ⁇ l volume of buffer composed of 20 mM Tris pH 7.4, 0.1 M NaCl, and 0.01% Tween.
- the ratio of the maleimido-PEG to protein may be varied according to the preferred level of
- the protein and (Maleimido) 2 -PEG are incubated for one hour at room temperature, although this reaction may be performed at 4°c for longer periods of time.
- a sample of the reacted mixture may be analyzed by SDS-PAGE to determine the minimal amount of (Maleimido) 2 -PEG reagent needed for complete coupling.
- reaction described above may be used to determine the proper ratio of (Maleimido) 2 -PEG to protein, and then scaled up to produce commercially acceptable amounts of PEGylated dimeric or multimeric proteins.
- PN-1 ⁇ and PN-1 ⁇ contain N-glycosylation sites at amino acid residue positions 99 and 140. Therefore, these sites were selected for site-directed mutagenesis to replace the asparagine at one or both of these
- Amino acid residue D192 was selected for replacement with cysteine since the proteins angiotensin and Rab ORF1, each which are homologous to PN-1, are N-glycosylated at the amino acids corresponding to this residue in PN-1.
- Amino acid residue E230 was selected for replacement with cysteine since baboon ⁇ 1 -antitrypsin ( ⁇ 1 -AT), which is homologous to PN-1, is glycosylated at the amino acid residue corresponding to this position in PN-1.
- Amino acid residue H252 was selected for replacement with cysteine since ⁇ 2 - antiplasmin ( ⁇ 2 -AP), another protein homologous to PN-1, is glycosylated at the corresponding residue.
- amino acid residues were selected for replacement with cysteine on the basis of the position of the amino acid within the three-dimensional structure of PN-1 as determined by X-ray crystallography ( Figure 3).
- the approximate position of the amino acid residues selected for cysteine substitution are indicated by their corresponding amino acid residue number.
- the particular amino acid residues identified for mutagenesis in the present example were selected on the basis of the
Abstract
Description
Claims
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EP94931834A EP0730660A4 (en) | 1993-10-29 | 1994-10-28 | Chimeric proteins including protease nexin-1 variants |
AU80769/94A AU685187B2 (en) | 1993-10-29 | 1994-10-28 | Chimeric proteins including protease nexin-1 variants |
JP7512667A JPH09504174A (en) | 1993-10-29 | 1994-10-28 | Chimeric protein containing protease nexin 1 mutant |
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US14475893A | 1993-10-29 | 1993-10-29 | |
US08/144,758 | 1993-10-29 |
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EP (1) | EP0730660A4 (en) |
JP (1) | JPH09504174A (en) |
AU (1) | AU685187B2 (en) |
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AU8076994A (en) | 1995-05-22 |
EP0730660A4 (en) | 1998-02-25 |
AU685187B2 (en) | 1998-01-15 |
JPH09504174A (en) | 1997-04-28 |
EP0730660A1 (en) | 1996-09-11 |
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