WO1998008863A1 - Enzyme and dna sequence encoding same - Google Patents
Enzyme and dna sequence encoding same Download PDFInfo
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- WO1998008863A1 WO1998008863A1 PCT/US1997/015179 US9715179W WO9808863A1 WO 1998008863 A1 WO1998008863 A1 WO 1998008863A1 US 9715179 W US9715179 W US 9715179W WO 9808863 A1 WO9808863 A1 WO 9808863A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to purified nucleic acids encoding a k ⁇ ll-de ⁇ ved enzymes such as proteinases, which can be a multifunctional protein, and to pu ⁇ fied polypeptides
- a protein having "multifunctional activity,” is defined herein as including at least one of a chymotrypsm. trypsin. collagenase, elastase or exo peptidase activity, or asialo GM ⁇ ceramide binding activity
- Multifunctional proteins are useful for multiple purposes, including treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic mfections, including the p ⁇ marv and secondary infections of leprosy, colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, cystic fibrosis.
- viral infections such as herpes outbreaks, fungal, bacterial or parasitic mfections, including the p ⁇ marv and secondary infections of leprosy, colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, cystic fibrosis.
- sequences encoding k ⁇ ll-de ⁇ ved proteinases similar to those set forth in the Sequence Listmg have not been identified
- 6. 10, 20 22 or 24 or other isoforms thereof or chime ⁇ c polypeptides thereof are examples of such enzymes
- the invention is specified in terms of a reference protein sequence which is AA64-300 of SEQ ID NO 4 or a sequence differing therefrom bv at least one of the residue differences found m SEQ ID NOS 5. 20. 22. or 24
- the refenence reference sequence further has the differences relative to SEQ ID NO 4 that are found in SEQ ID NO 8 These differences are illustrated in Figures 6 and 7
- One preferred embodiment of the present invention is a substantially pure nucleic acid co p ⁇ sing a nucleic acid encoding a polypeptide having at least about 70% homology (such as identity or similanty) to a k ⁇ ll-de ⁇ ved multifunctional protein or a reference protein, such as the polypeptide of SEQ ID NOS 4, 5, 6. 8. 10. 20. 22 or 24 and in one embodiment especially SEQ ID NOS 4, 6. 10 or 22. and more preferably, at least about 80% homology. and most preferabK . at least about 90% homology Preferably. In another embodment embodiment, the homology is with reference to SEQ ID NOS 4. 5. 20.
- the nucleic acid comprises a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identitv with a reference protein or a k ⁇ ll-de ⁇ ved multifunctional protein and yet more preferably, at least about 80% identity, and still more preferably, at least about 90% identity. Yet still more preferably, the homology or identity is at least about 95% These levels of homology (such as identity) apply to all embodiments of the invention.
- the substantially pure nucleic acid comp ⁇ ses an engineered nucleic acid variant encoding a polypeptide differing from a reference protein or a krill- derived multifunctional protein or its isoforms by no more than about 33 ammo acid substitutions, and more preferably, no more than about 20 ammo acid substitutions.
- the engineered substitutions cause a conservative substitution in the amino acid sequence of a reference sequence or a multifunctional protein.
- Preferred substantially pure nucleic acids also include nucleic acid analogs.
- the nucleic acid comprises the open reading frames of SEQ ID NOS:l. 2. 3, 7, 9, 19, 21 or 23 and more preferably, SEQ ID NOS 1.
- the nucleic acid includes base pairs 1 0-900 of SEQ ID NO 1. base pairs 2-556 of SEQ ID NO 2. base pairs 190-900 of SEQ ID NO.3. base pairs 215-925 of SEQ ID NO 9, base pairs 1-633 of SEQ ID NO: 19, base pairs 196-906 of SEQ ID NO.21. or base pairs 215-535 of SEQ ID NO 23 (in one embodiment, 1 -900 of SEQ ID NO.1 , base pairs 2-556 of SEQ ID NO:2. base pairs 1-900 of SEQ ID NO:3, base pairs 2-925 of SEQ ID NO.9. base pairs 1 -633 of SEQ ID NO 19, base pairs 1-906 of SEQ ID NO:21.
- the invention additionally includes vectors capable of reproducing in a cell (such as a eukaryotic or prokaryotic cell) having a nucleic acid described above as well as transformed cells having such nucleic acid.
- the invention includes a substantially pure nucleic acid comprising a nucleic acid that hybridizes, under stringent conditions, to a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a k ⁇ ll-de ⁇ ved multifunctional protein, such as the polypeptide of SEQ ID NOS.4, 5, 6, 8, 10, 20, 22 or 24 and more preferably. SEQ ID NOS:4, 6, 10 or 22.
- the nucleic acid is a reference nucleic acid sequence including the open reading frame sequence of SEQ ID NO: 1 (or prfcrably preferably the portion from the NH 2 - IleValGlyGlvMet sequence through to the end) or a sequence diffenng therefrom by at least one of the bp differences found in SEQ ID NOS:2, 19, 21 or 23 (or prferably preferably the portion from the NHb-IleValGlyGly sequence through to the end).
- the nucleic acid includes a sequence with about 50%, about 60%. about 70%. about 80%, about 90% or about 95% identity to a reference sequence.
- Another preferred embodiment is a transformed cell (such as a prokaryotic or eukaryotic cell) comp ⁇ sing a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a k ⁇ ll-de ⁇ ved multifunctional protein
- the transformed cell expresses one of the enzymes desc ⁇ bed herem
- a vector capable of reproducing in a cell such as a eukaryotic or prokaryotic cell, the vector comp ⁇ sing a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a k ⁇ ll-de ⁇ ved multifunctional protein
- the inventive vector codes for expression, mtracellularly or extracellularlv, of one of the multifunctional proteins descnbed herem
- Another embodiment of the present invention is a polypeptide comprising a substantially pure lsoform of a reference sequence or a k ⁇ ll-de ⁇ ved multifunctional protein or engineered va ⁇ ant thereof
- the lsoform is a polypeptide comprising SEQ ID NOS 4, 5, 20. 22 or 24
- the invention further provides a pharmaceutical composition for treating an a mal comp ⁇ sing an effective amount of such a polypeptide together with a pharmaceutically acceptable earner
- Yet another prefened embodiment is a method of prepa ⁇ ng an enzyme such as a multifunctional protein, wherein the protem has at least about 70% homology to a reference sequence or a k ⁇ ll-de ⁇ ved multifunctional protein
- Such method comprises (a) transforming an appropriate eukaryotic or prokaryotic cell with an expression vector for expressing mtracellularly or extracellularlv a nucleic acid encoding the protein,
- Yet another preferred embodiment is a pharmaceutical composition for treating an animal comp ⁇ sing an effective amount of an expression vector compnsing a nucleic acid encoding a multifunctional protein, and a pharmaceutically acceptable earner
- the invention further provides a pharmaceutical composition for treating an animal comp ⁇ sing an effective amount of a nucleic acid encoding a polypeptide havmg at least about 70% homology to a reference sequence or a knll-de ⁇ ved multifunctional protein
- Figure 1 shows the DNA sequence of a first isoform (“p62") (SEQ ID NO 1) of a k ⁇ ll- denved multifunctional protein aligned with the DNA sequence of a second isoform (“p31”) (SEQ ID NO 2)
- Figure 2 shows the ammo acid sequence of the first isoform (“p62") (SEQ ID NO 4) of a k ⁇ ll-de ⁇ ved multifunctional protein aligned with the amino acid sequence of the second isoform (“p31”) (SEQ ID N0 5)
- Figure 3 shows the DNA sequence of a third isoform ("p5 la") (SEQ ID NO 7) of a k ⁇ ll- derived multifunctional protein aligned with the DNA sequence of the first isoform (“p62”) (SEQ ID NO 1)
- Figure 4 shows the amino acid sequence of the third isoform ("p5 la") (SEQ ID NO 8) aligned with the ammo acid sequence of the first isoform (“p62”) (SEQ ID NO 4) and the ammo acid sequence of the second isoform (“p31”) (SEQ ID NO 5)
- Figure 5 shows the ammo acid sequences of several proteins, namely, Factor VII, thrombin, kalhkrein. a Limulus pro-clotting enzyme, plasmin, hepsm and Factor XII. aligned with the amino acid sequence of the first isoform (“p62”) (SEQ ID NO 1)
- Figure 6 shows sequence comparisons between the nucleic acid sequences for p62 (SEQ ID NO l), pl3 (SEQ ID NO 19), p912 (SEQ ID NO 21), p5 lb (SEQ ID NO 23) and p31 (SEQ ID NO 2), as well as sequence compa ⁇ sions comparisons for the aligned peptide sequences (SEQ ID NOS 4. 20, 22, 24 and 5, respectively) Nuchc Nucleic acid sequence differences m the open reading frames relative to p62 are indicated by underlining, and differences in amino acid sequence are indicate with recitals of the diffenng residues
- Figure 7 shows sequence comparisons between the polypeptide sequences encoded by p62 (SEQ ID NO 4), pl3 (SEQ ID NO 20), p912 (SEQ ID NO 22), p5 lb (SEQ ID NO 24) and p31 (SEQ ID NO 5)
- Figure 8 illustrates a sequence alignment between the polypeptide sequences encoded by p62 (SEQ ID NO 4).
- p912 SEQ ID NO 22
- p5 la SEQ ID NO 8
- p31 SEQ ID NO 5
- a segment of a multifunctional protein having activity comprising at least one of a chymotrypsin. trypsin. collagenase, elastase or exo peptidase activity • hydrolase
- An enzyme that degrades bonds formed by dehydration reactions such as amide, ester, or ether bonds
- the term encompasses, but is not limited to, proteases such as trypsin and chymotrypsin • isoform
- the isoform shares at least about 80% identity, and more preferably, at least about 85% identity with SEQ ID NO 4 • krill-derived multifunctional protein
- a multifunctional protein having the same sequence as a protein isolated from k ⁇ ll having the properties of the protein desc ⁇ bed in the section entitled "Preferred Characteristics of the Multifunctional Protein" This protein is also referred to as the "k ⁇ ll-de ⁇ ved multifunctional hvdrolase" and includes all isoforms of the protein The ammo acid sequence included in SEQ ID NO 4.
- SEQ ID NO 8 or SEQ ID NO 10 or other isoforms thereof or chime ⁇ c polypeptides thereof are examples of k ⁇ ll-de ⁇ ved multifunctional prote s ⁇ multifunctional protein
- the nucleic acid sequence embodiments of the invention are preferably deoxy ⁇ bonucleic acid sequences, preferably double-stranded deoxy ⁇ bonucleic acid sequences
- they can also be ⁇ bonucleic acid sequences, or nucleic acid mimics, meaning compounds designed to preserve the hydrogen bonding and base-pai ⁇ ng properties of nucleic acid but which differ from natural nucleic acid m. for example, susceptibility to nucleases
- a reference protein sequence is AA64-300 of SEQ ID NO 4 or AA 1-300 of SEQ ID NO 4 or a sequence diffe ⁇ ng therefrom by at least one of the residue differences found in SEQ ID NOS 5.
- a reference protein is a protein having the reference protein sequence
- examples of reference proteins are (a) a protein with sequence of AA64-300 of SEQ ID NO 4 except that residue 128 is se ⁇ ne or (b) a protein with sequence of SEQ ID NO 4 except that before Leu 1 is senne
- the N-terminal additions from SEQ ID NOS 22 and 24 are NH Ala. NH HeAla, NH 2 -ArgIleAla. NH 2 -SerArgIleAla. NH : - ArgSerArglleAla, NH 2 -GlyArgSerArgIleAla or NH 2 -ProGlyArgSerArgIleAla
- Identity as known in the art. is a relaUonship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences, particularly, as determined b ⁇ the match between st ⁇ ngs of such sequences "Identity" is readily calculated by known methods ⁇ Computational Molecular Biology, Lesk, A M , ed Oxford University Press. New York, 1988. Bwcomputmg Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data. Part 1. Griffin, A M . and G ⁇ ffin, H G .
- the present invention provides DNA and corresponding amino acid sequences of a k ⁇ ll- de ⁇ ved enzymes such as proteinases, which can be multifunctional proteins Crustaceans including antarctic k ⁇ ll, are useful sources for the multifunctional protein of the invention
- a protein having "multifunctional activity.” is defined herein as including at least one of a chymotrypsin. trypsin, collagenase. elastase or exo peptidasc activity, or asialo GMj ceramide bmd g activity
- U S patent application Senal No 08/600.273 filed February 8. 1996) deFaire et al inventors entitled "Multifunctional Enzyme"
- the present invention provides nucleic acids (such as ⁇ bonucleic acids or deoxy ⁇ bonucleic acids) and polypeptides and analogs thereof including nucleic acids that bind to a multifunctional protein encoding nucleic acid, as well as pharmaceutical compositions, gene therapy and antibodies and antisera against the multifunctional protein
- nucleic acids and polypeptides are naturally occurring variants (isoforms) whereas others are non-natural occurring (engineered) variants 1.
- the nucleic acid embodiments of the invention are preferably deoxy ⁇ bonucleic acids (DNAs), both single- and double-stranded, and most preferably double-stranded deoxy ⁇ bonucleic acids
- DNAs deoxy ⁇ bonucleic acids
- RNAs ⁇ bonucleic acids
- Nucleic acids encoding a multifunctional protein include all multifunctional protc - encoding nucleic acids, whether native or synthetic, RNA DNA or cDNA, that encode a multifunctional protein, or the complementary strand thereof, including but not limited to nucleic acid found m a multifunctional protein-expressing organism For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic multifunctional protein-encoding nucleic
- the nucleic acid sequences of the invention can encode, for example one of several isoforms of a k ⁇ ll-de ⁇ ved protein SEQ ID NOS 4, 5. and 8 represent three isoforms that share about 88-89% identity with each other in overlapping amino acids See, for example.
- Figure 1 which compares the DNA sequence of the first isoform. SEQ ID NO 1, with the DNA sequence of the second isoform, SEQ ID NO 2. which share about 88% identical nucleotides See also, for example.
- Figure 3 which provides a comparison of the DNA sequence of the third isoform (SEQ ID NO 7) and the first isoform (SEQ ID NO 1).
- isoforms which share about 89% identical nucleotides
- isoforms all lack the initiation codon methionine
- two of these three isoforms contain a hydrophobic sequence which may function as a signal sequence, na eh .
- LLLALVAAASA which is amino acid residues 1-1 1 in the first isoform.
- SEQ ID NO 4. and PGRSRIALLLALVAATASA which is ammo acid residues 1-19 in the third isoform.
- pro-protein segment is the segment of the protein, other than the hydrophobic segment, that is present in the precursor protein but absent in the mature protein
- pro-protem segment may still be attached to the mature protein
- k ⁇ ll-de ⁇ ved multifunctional proteins may have two chains linked by a disulfide bond
- a cysteine in the pro-protem segment may participate in a disulfide bond in the mature protein
- the pro-protein segment has the following sequence, which corresponds to amino acid residues 12-63 in the first isoform, SEQ ID NO 4 AEWRWQFRHPTVTPNPRAKNPFRVTKSSPVQPPAVRGTKAVENCGPVAPRNK
- the third isoform has a pro-protein segment with the following sequence, which corresponds to amino acid residues 20-71 in SEQ ID NO 8 SEWRWQFRHPTVTPNPRANNPFRPSKVAPVQPPAV- RGTKAVENCGPVAPKNK
- the remaining ammo acid sequences of these polypeptides represent the mature protein See Figure 2. which provides a compa ⁇ son of the amino acid sequence of the first isoform and the second isoform. which share about 89% identical am o acids
- Figure 4 which provides a comparison of the amino acid sequences of all three isoforms
- nucleic acid sequences that encode polypeptides that are preferably present in the protein
- the following examples are derived from the pro-protcin segment of SEQ ID NO 4, and are polypepudes that are preferably present in the mature protein
- these polypeptides may form at least part of a first amino acid chain that is linked via a disulfide bond to a second amino acid chain which can be.
- the nucleic acid further encodes a polypeptide sequence such as AVENCGPVAPR (SEQ ID NO 11 ).
- AVENCGPVAPRNK SEQ ID NO 12
- GTKAVENCGPVAPR SEQ ID NO 13
- GTKAVENCGPVAPRNK SEQ ID NO 14
- SSPVQPPAVRGTKAVENCGPVAPR SEQ ID NO 15
- SSPVQPPAVRGTKAVENCGPVAPRNK SEQ ID NO 16
- AVENCGPVA SEQ ID NO 25
- the above-listed polypeptides may be linked to the remainder of the mature k ⁇ ll-de ⁇ ved multifunctional protein via a disulfide bond as follows
- the cysteme residue in one of these sequences may participate in a disulfide bond with, for example, a cysteine in the mature protein, such as a cysteine corresponding the cysteine at residue 171 of SEQ ID NO 4
- At least one of these sequences are therefore present in preferred embodiments of the invenUon See, for example.
- Figure 5 which shows the amino acid sequences of several proteins, namely, Factor VII. thrombin. kalhkrein, a Limulus pro-clotting enzyme from the Japanese horshoe horsehoe crab (Tachypleus tridentatus), plasmm. hepsin and Factor XII. aligned with the amino acid sequence of SEQ ID NO 4 All of the proteins aligned with the krill-de ⁇ ved multifunctional protein, except for the Limulus protein and Hepsin. are involved m the human blood coagulation pathway
- k ⁇ ll-denved multifunctional proteins include a larger N -terminus than that found in the first, second or the third isoform, SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 8. respectively NOS 4, 5 or 8. 20. 22 or 24
- the nucleic acids of the invention can encode engineered multifunctional proteins based on forming chime ⁇ c polypeptides from the above isoforms.
- the hydrophobic sequence or the pro-protein segment of one naturally occur ⁇ ng isoform can optionally be matched with the mature protein sequences of another naturally occurring isoform or isoforms
- the mature protein segment of SEQ ID NO 4 is ammo acids 64-300 SEQ ID NO 5, for mstance.
- certain embodiments of the mvention include a chime ⁇ c polypeptide in which the N-terminus of the polypeptide of SEQ ID NO 5 is linked to the remaining 25% of the length of the mature protein sequence found in SEQ ID NO 4, namely ammo acids 64-116
- a hypothetical chime ⁇ c sequence includes the first 63 amino acids of the protein of SEQ ID NO 4 together with the amino acid sequence of SEQ ID NO 5 See SEQ ID NO 6. which is a composite of the proteins of SEQ ID NO 4 and SEQ ID NO 5 See, for example.
- nucleic acids of the invention include nucleic acids that code for the mature protein, the protein including the pro-protem segment or the protein including the hydrophobic segment and the pro-protem segment, or portions thereof
- the nucleic acid of the first isoform SEQ ID NO 1. or the chime ⁇ c molecule.
- SEQ ID NO 3 are nucleic acids encoding the pro-protein, including the hydrophobic sequence and the pro-protem segment The chime ⁇ c molecule.
- SEQ ID NO 3 represents the first 344 nucleotides of SEQ ID NO 1. coding for the hydrophobic sequence and the pro-protein segment of the protein and the first 25% of the mature protein, together with the 599 nucleotides of SEQ ID NO 2, coding for the remaining 75% of the mature protein
- SEQ ID NO 8 can be attached to the mature protein sequences of SEQ ID NO 5, thereby forming a chime ⁇ c polypeptide, shown in SEQ ID NO 10
- the corresponding DNA sequence can be found SEQ ID NO 9
- ammo acid sequences of several isoforms can be used to create an engineered polypeptide
- the chime ⁇ c polypeptide of SEQ ID NO 6 can be further modified by adding to the N-terminus of the protein the amino acid sequence PGRSRIA, which is ammo acid residues 1-7 from the N-terminus of the third isoform.
- SEQ ID NO 10 is ammo acid residues 1-7 from the N-terminus of the third isoform.
- the native sequences of any of the isoforms can be used as a starting point and modified to suit particular needs.
- the nucleic acid sequence need not include the sequences encoding the 5' portion of the ammo acid sequence that is absent in the mature protein, including ammo acids 1-63 of SEQ ID NO 4
- the encoded polypeptide is homologous to or has the sequence of the mature protein only, and not the segments corresponding to the N-terminal portions that are removed during cellular processing, namely, the hydrophobic sequence and the pro-protein segment
- the sequences encoding the N-terminal portion of the ammo acid sequence that is absent in the mature protein including amino acids 1-63 of SEQ ID NO 4. are included m the nucleic acid sequences
- the amino acid sequence forming a synthetic multifunctional protein preferably includes an enzymatically active segment of a k ⁇ ll-de ⁇ ved multifunctional protein, such as amino acids 64- 300 of SEQ ID NO 4, particularly including the histidine at residue 104.
- the protein need not include the I drophobic sequence or pro-protem segment that are present in a k ⁇ ll-de ⁇ ved protein before cellular processing occurs, although the hydrophobic sequence and the pro-protein segment are preferably present
- the nucleic acids will encode polypeptides having at least about 70% homology, more preferably, at least about 80% homolog ⁇ . even more preferably, at least about 85% homology, yet more preferably at least about 90% homology. and most preferably at least about 95% homology to a reference protein or a krill-de ⁇ ved multifunctional protem, such as the polypeptides of SEQ ID NO 4, SEQ ID NO 5.
- nucleic acids will encode polypeptides sharing at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 85% identity, still more preferably at least about 90% identity, and most preferably at least about 95% identity with a k ⁇ ll-de ⁇ ved multifunctional protein
- the invention includes a substantial ly pure nucleic acid comprising a nucleic acid that binds to a nucleic acid encoding a polypeptide having at least about 70% homology to a reference protem or a krill-de ⁇ ved multifunctional protein Even more preferably, the nucleic acid binds to a nucleic acid encoding a polypeptide having at least about 80% homology.
- nucleic acid binds to a nucleic acid encoding a polypeptide sha ⁇ ng at least about 70% amino acid identity, and more preferably, at least about 80% ammo acid identity, and yet more preferably, at least about 90% amino acid identity with a krill-de ⁇ ved multifunctional protem, such as the polypeptide of SEQ ID NO 4, SEQ ID NO 5.
- nucleic acid that binds to a nucleic acid that encodes a polypeptide homologous to a krill-de ⁇ ved multifunction protein can be used as a probe, for example, to identify additional multifunctional proteins or to determine multifunctional protein expression
- the mature protein of the polypeptide of SEQ ID NO 4 is about 61% identical to the chymotrypsin-Iike serine protemase in the shrimp Penaeus vannamei according to the sequence provided by Genbank (Mountain View, CA), database acquisition no X6641 . and about 60% identical to the collagenolytic se ⁇ ne protemase in the fiddler crab Uca pugilator.
- the amino acid sequence of the pro-protein of SEQ ID NO 4 is about 53% identical to the precursor of the chymotrypsin-hke se ⁇ ne protemase in the sh ⁇ mp Penaeus vannamei, and about 51% identical to the precursor of the collagenolytic se ⁇ ne protemase in the fiddler crab Uca pugilator
- the nucleic acids encoding polypeptides having multifunctional activity are less than about 70% lden cal to the above-identified proteinases of Penaeus vannamei or Uca pugilator
- the present invention includes nucleic acids encoding polypeptides that are homologous to a reference protein or a knll- de ⁇ ved multifunctional protem or that share a percentage identity with a reference protem or a k ⁇ ll-de ⁇ ved multifunctional protein Further, the present invention includes nucleic acids that encode a portion of a multifunctional protem or a variant thereof, such as the enzymaticallv acUve portion of the protein or the portion of the protein that provides asialo GM j ceramide binding activity
- the invention also is directed to a nucleic acid encoding a k ⁇ ll-de ⁇ ved multifunctional protem that has at least one of the following activities chymotrypsin, trypsin. collagenase. elastase and exopeptidase activity or asialo GM j ceramide binding activity
- die encoded polypeptide will be effective to remove or inactivate a cell-surface adhesion molecule, and most preferably, the encoded polypeptide will be pharmaceutically effective
- deletion mutants are constructed by subcloning restriction fragments of a multifunctional protein cDNA The deletional constructs are cloned into expression vectors and tested for their multifunctional activity
- the nucleic acid sequences can be further mutated for example, to incorporate useful restriction sites See Maniatis ct al Molecular Cloning a Laboratory Manual (Cold Spring Harbor Press, 1989) Such restriction sites can be used to create "cassettes", or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and hgation reactions
- the cassettes can be used to substitute synthetic sequences encoding mutated multifunctional protein ammo acid sequences
- the multifunctional protein-encoding sequence can be. for instance, substantially or fully synthetic See. for example. Goeddel et al , Proc Nail Acad Sci USA, 76, 106-1 10. 1979
- codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic multifunctional protein-encoding nucleic acid
- numerous nucleic acid sequences can be used to create the same amino acid sequence
- numerous methods are known to delete sequence from or mutate nucleic acid sequences that encode a polypeptide and to confirm the function of the polypeptides encoded by these deleted or mutated sequences
- the invention also relates to a mutated or deleted version of a multifunctional prote nucleic acid that encodes a polypeptide that retains multifunctional protein activity
- Conservative mutations of the naturally occurring isoforms are preferred for engineered va ⁇ ants
- Such conservative mutations include mutations that switch one ammo acid for another within one of the following groups
- substitutions selected may be based on the analysis of the frequencies of ammo acid substitutions between homologous proteins of different species developed by Schulz et al . Principles of Protein Structure, Sp ⁇ nger-Verlag. 1978, pp 14-16, on the analyses of structure- forming potentials developed by Chou and Fasman. Biochemistry 13, 21 1, 1974 or other such methods reviewed by Schulz et al. Principles in Protein Structure, Springer- Verlag. 1978, pp 108-130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doohttle, J Mol Biol 157 105-132, 1982 2.
- Polypeptides of the invention include all polypeptides having multifunctional activin whether native or synthetic, including but not limited to poKpeptidcs purified from a multifuncUonal protein-expressing organism
- a preferred embodiment of the invention provides a polypeptide comprising a substantially pure isoform of a reference protein or a k ⁇ ll-de ⁇ ved multifunctional protein or engineered variant thereof, and more preferably, a polypeptide comprising SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 SEQ ID NO 8 or SEQ ID NO 10
- polypeptides of the invention preferably comprise at least one of the ammo acid sequences of SEQ ID NOS 1 1-16 and 25
- the present invention includes poly peptides that are homologous to a reference protein or a krill-de ⁇ ved multifunctional protein or that share a percentage ldentif. with a reference protein or a krill-de ⁇ ved multifunctional protein Further, the present invention includes portions of the a reference protein or a multifunctional protein or a variant thereof, such as the enzymatically active portion of the protein or the portion of the protein that provides asialo GM j ceramide binding activity
- the present invention includes engineered va ⁇ ants of multifunctional proteins that retain multifunctional activity
- these engineered variants lack, for example, no more than about 63 amino acid residues at the N-termmal end of SEQ ID NO 4
- the va ⁇ ants will have at least about 70% homology, more preferably, at least about 80% homology. even more preferably, at least about 85% homology, still more preferably at least about 90% homology. and most preferably at least about 95% homology to a k ⁇ ll-denved multifunctional protein, such as the polypeptides of SEQ ID NO 4. SEQ ID NO 5. SEQ ID NO 6.
- SEQ ID NO 8 SEQ ID NO 10, or other isoforms, or amino acid sequences 64-300 of SEQ ID NO 4
- the analogs will share at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 85% identity, still more preferably at least about 90% identity, and most preferably at least about 95% identity with a krill-de ⁇ ved multifunctional protein
- the polypeptide has the sequence of a contiguous stretch of at least about 237 ammo acids of the following mature proteins in SEQ ID NO 4, amino acid residues 64-300. in SEQ ID NO 6, amino acid residues 64-300, and in SEQ ID NO 10. ammo acid residues 72-308
- the present invention provides a pharmaceutical composition for treating an animal compnsing an effective amount of a polypeptide comp ⁇ sing a substantially pure isoform of a krill-de ⁇ ved multifunctional protem or engineered variant thereof and a pharmaceutically acceptable carrier
- the polypeptide comprises SEQ ID NO 4, SEQ ID NO 5 SEQ ID NO 6.
- SEQ ID NO 8 or SEQ ID NO 10 and even more preferably, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 10, and the polypeptide preferably comp ⁇ ses at least one of the amino acid sequences of SEQ ID NO 1 1-16 3.
- the polypeptides of the invention are made as follows, using a gene fusion
- fusion to maltose-binding protein MBP
- MBP maltose-binding protein
- the hvb ⁇ d protem can be purified, for example, using affinity chromatography using the binding protein's substrate See, for example, Gent? 67 21-30 (1988)
- a cross-linked amylose affinity chromatography column can be used to punfy the protein
- the cDNA specific for a given multifunctional protein or analog thereof can also be linked using standard means to a cDNA for glutathione s-transferase ("GST"), found on a commercial vector, for example
- GST glutathione s-transferase
- the fusion protein expressed by such a vector construct includes the multifunctional protem or analog and GST. and can be treated for pu ⁇ fication Should the MBP or GST portion of the fusion protein interfere with function, it is removed by partial proteolytic digestion approaches that preferentially attack unstructured regions such as the linkers between MBP or GST and the multifunctional protem
- the linkers are designed to lack structure, for instance using the rules for secondary structure-forming potential developed by Chou and Fasman. Biochemistry 13. 21 1.
- the linker is also designed to lncorporatc protease target amino acids, such as trypsm. arginme and lysine residues
- standard synthetic approaches for making oligonucleotides are employed together with standard subcloning methodologies
- Other fusion partners other than GST or MBP can also be used
- the multifunctional proteins can be directly synthesized from nucleic acid (by the cellular machinery) without use of fusion partners
- nucleic acids having the sequence of SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 9 are subcloned into an approp ⁇ ate expression vector having an approp ⁇ ate promoter and expressed m an appropriate organism
- Antibodies against the k ⁇ ll multifunctional protein can be employed to facilitate purification Additional purifications techniques are applied as needed, including without limitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala.
- HPLC e g . using gel filtration, reverse-phase or mildly hydrophobic columns
- gel filtration differential precipitation (for instance, “salting out” precipitations)
- ion-exchange chromatography and affinity chromatography (including affinity chromatography using the RE1 duplex nucleotide sequence as the affinity gand).
- a polypeptide or nucleic acid is "isolated” in accordance with the invention in that the molecular cloning of the nucleic acid of interest, for example, involves taking a multifunctional protem nucleic acid from a cell, and isolating it from other nucleic acids This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example
- a polypeptide or nucleic acid is "substantially pure” in accordance with the invention if it is predominantly free of other polypeptides or nucleic acids, respectively A macromolecule. such as a nucleic acid or a polypeptide.
- the polypeptide or nucleic acid of the present invention constitutes at least about 60% by weight of the total polypeptides or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%. still more preferably about 90%, yet more preferably about 95%. and most preferably about 100%
- Such compositions are referred to herein as being polypeptides or nucleic acids that are 60% pure. 80% pure. 90% pure. 95% pure, or 100% pure. any of which are substantially pure 4.
- Knll. including without limitation k ⁇ ll of the genuses Euphasia (such as superba. crystallorphias.frigida. triacantha. vellantmi. lougirostns. lucens. similis. spimfera. recurva and the like). Meganyctiphanes (such as norvegica and the like) and Tysanoessa (such as macurura. vicma, gregana and the like), are a preferred source of k ⁇ ll-de ⁇ ved multifunctional proteins
- the protein has a molecular weight between about 20 kd and about 40 kd. and more preferably from about 26 kd to about 32 kd, and most preferably about 29 kd, as determined by sodium dodecyl sulfate ("SDS") polyacrylamide gel electrophoresis (“PAGE”)
- SDS sodium dodecyl sulfate
- PAGE polyacrylamide gel electrophoresis
- the protem preferably has substantial homology to a k ⁇ ll-de ⁇ ved multifunctional protein
- Preferred proteins are hydrolases. and preferably, proteases
- the protein is selectively reactive with cell-surface receptors such as polypeptides or glycolipids
- Protease activity can be determined by incubating a protein preparation with casein (concentration 1 % w/v) at 30 C for 20 hours and measuring the release of amino acids or peptides (which can be measured by the increase in coloromet ⁇ cally determinablc amino groups)
- Isolated multifunctional protein of 95% pu ⁇ ty will typically have a specific activity of at least about 25 Casein Units per mg Casein Units are defined in Biochem J , 173 291-298, 1978 (using azocasein as the substrate)
- tryptic protease activity can be measured against tyrosine-arginine-methyl- ester ("TAME")
- TAME tyrosine-arginine-methyl- ester
- the multifunctional protein (of at least about 95% purity) will preferably have specific activity of at least about 60 TAME Units per mg
- tryptic activity can be measured using Benzoyl-Val-Gly-Arg-p-N ⁇ 2-an ⁇ l ⁇ de as the substrate Using this substrate and
- the multifunctional protein will preferably have specific activity at least about 260 Units per mg Elastase activity can be measured using Boc-Ala-Ala- Pro-Ala-p-N ⁇ 2-an ⁇ hde as the substrate Using this substrate and the method of J Biol Chem . 269 19565-19572, 1994, the multifunctional protein will preferably have specific activity of at least about 270 Units per mg
- the multifunctional protein will be sufficiently stable so that at least about 50% of the proteolytic activity is retained after incubation at 50° C for 24 hours at pH 7 0 at a concentration of 5 mg/ml Preferably at least about 50% of the proteolytic activity is retained after incubation at 60° C for 5 hours at pH 7 0 at a concentration of 5 mg/ml
- the pH optimum of the multifunctional protein is substrate dependent For the substrate azocasein.
- the pH optimum is preferably from about 3 5 to about 6 5. more preferably from about 4 0 to about 6 0
- the pH optimum is preferably in excess of about 8 0, more preferably in excess of about 9 0
- the pH optimum is preferably between about 6 0 and about 7 0, more preferably about 7 0
- the K m at about pH 9 5 in the presence of 2 mM Ca ⁇ + is preferably between about 200 and about 240 ⁇ M
- the K m at pH 9 5 in the presence of 2 mM Ca ⁇ + is preferably between about 250 and about 290 ⁇ M
- the multifunctional protein has a temperature optimum for activity against casein of between about 45° C and about 60° C Generally the protein retains at least about 50% of its activity when incubated at 5 mg/ml for 18 hours at a pH ranging from about 5 0 to about 9 5 at 25 C
- HL60 cells When HL60 cells are pretreated with k ⁇ ll-de ⁇ ved multifunctional hydrolase. their binding to TNF stimulated endothehal cells is inhibited by more than about 60%
- treatment of HL60 or endothehal cells with the multifunctional protein of the invention will inhibit HL60 cell binding to TNF stimulated endothehal cells by at least about 20%. more preferably at least about 40%. still more preferably at least about 60%.
- the multifunctional protein will preferably have at least about 30% of the adhesion- lnhibiting activity of the krill-de ⁇ ved multifunctional hydrolase More preferably, the multifunctional protein shall have at least about 60% of the adhesion inhibiting activity of the k ⁇ ll- de ⁇ ved multifunctional hydrolase, still more preferably at least about 80%, yet more preferably at least about 100%
- the multifuncUonal protein of the invention effectively removes or inactivates certain cell- surface adhesion molecules, such as ICAM-1 (t e , CD 54).
- ICAM-1 t e , CD 54.
- ICAM-2 t e , CD 54.
- VCAM-1 VCAM-1, CD4.
- This adhesion site removal or inactivation phenomenon is believed to provide at least a partial explanation for the protein's effectiveness against many, though probably not all.
- the present invention provides methods for identifying polypeptides that are homologous to the multifunctional protein Such polypeptides may be found, for example, in fish and crustaceans
- the method by which multifunctional protein cDNA was isolated illustrates how readily multifunctional proteins are identified For instance, see Example 1
- the same methodology can be used to identify other sequences from other sources that have multifunctional activity
- probes for multifunctional protein expression can be used, for example, to detect the presence of a multifunctional protem
- probes include antibodies directed against multifunctional protein or fragments thereof, nucleic acid probes that hyb ⁇ dize to multifunctional protein mRNA under st ⁇ ngent conditions, and oligonuclcotides that specifically prime a PCR amplification of multifunctional protein mRNA Nucleic acid molecules that bind to a multifunctional protein-encoding nucleic acid under high stringency conditions are identified functionally, or by using the hyb ⁇ dization rules reviewed in Sambrook et al . Molecular Cloning A Laboratory Manual, 2nd ed . Cold Sp ⁇ ng Harbor Press. 1989
- nucleic acid sequences for a multifunctional protein are effective hybridization probes for multifunctional protein-encoding nucleic acid
- the present invention relates to nucleic acids that hybridize with such multifunctional prote -encodmg nucleic acids under stringent conditions
- the nucleic acid of the present invention hybridizes with at least a segment of the nucleic acid described as SEQ ID NO 1.
- SEQ ID NO 2 SEQ ID NO 3.
- “St ⁇ ngent conditions” refers to conditions that allow for the hybridization of substantially related nucleic acids, where relatedness is a function of the sequence of nucleotides in the respective nucleic acids For mstance. for a nucleic acid of 100 nucleotides. such conditions will generally allow hybridization thereto of a second nucleic acid havmg at least about 85% homology. and more preferably having at least about 90% homology Such hybridization conditions are desc ⁇ bed by Sambrook et al . Molecular Cloning A -Laboratory Manual. 2nd ed .
- PCR polymerase chain reaction
- PCR methods of amplifying nucleic acids utilize at least two p ⁇ mers one p ⁇ mer and often at least two One of these p ⁇ mers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of p ⁇ ming protem-d ⁇ ven nucleic acid synthesis in a first direction
- the other is capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is smgle stranded, this sequence is initially hypothetical, but is synthesized in the first amplification cycle) and of p ⁇ ming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first p ⁇ mer
- Conditions for conducting such amplifications are well known See, for example, PCR Protocols
- Antibodies against multifunctional proteins can also be used to identify polypeptides that are homologous to multifunctional protein
- Antigens for eliciting the production of antibodies against the a reference protein or a multifunctional protem can be produced recombmantly by expressing all of or a part of the nucleic acid of a reference prote or a multifunctional protein in a prokaryote such as bacteria or a eukaryote such as yeast
- the recombinant protein is expressed as a fusion protein, with the non-multifunctional functional protein portion of the protein serving either to facilitate purification or to enhance the immunogenicity of the fusion protein
- the non-multifunctional functional protein portion comprises a protein for which there is a readily-available binding partner that is utilized for affinity pu ⁇ fication of the fusion protem
- the antigen includes an "antigenic determinant," I e . a minimum portion of ammo acids sufficient to bind specifically with an anti-multifunctional protein antibody
- Antisera to a reference protein or a reference protein or a multifunctional protein can be made, for example, by creating a multifunctional protein antigen by linking a portion of the cDNA for human multifunctional protein to a cDNA for glutathione s-transfcrase ("GST") found on a commercial vector
- GST glutathione s-transfcrase
- the resulting vector expresses a fusion protein containing an antigenic segment of multifunctional protein and GST that is readily purified from the expressing bacteria using a glutathione affinity column
- the purified antigenic fusion protein is used to immunize rabbits
- the same approach is used to make antigens based on other segments of the multifunctional protem Procedures for making antibodies and for identifying antigenic segments of proteins are well known See. for instance, Harlow, Antibodies. Cold Spring Harbor Press. 1989 6. Gene Therapy
- the invention also encompasses the use of gene therapy approaches to insert a gene expressing a multifunctional protem or a polypeptide with multifunctional protein activity
- medical workers prefer to incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a polypeptide missing from the cell or useful to the cell or organism when expressed in greater amounts
- transfection methods See, generally, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd cd , Cold Spring Harbor Press, 1989
- dosages are generally from about 1 g to about 1 mg of nucleic acid per kg of body mass
- dosages are generally from about 1 g to about 100 mg of nucleic acid per kg of body mass 7.
- the multifunctional protein polypeptides and nucleic acid compositions of the invention can be administered orally, topically, rectally, vaginally. by instillation (for instance into the urinary tract or into fistulas), by pulmonary route by use of an aerosol, by application of drops to the eye. or systemically. such as parenterally, including, for example, intramuscularly, subcutaneously, intraperitoneally. lntraarte ⁇ ally or intravenously
- the multifunctional protein composition can be administered alone, or it can be combined with a pharmaceutically-acceptable earner or excipient according to standard pharmaceutical practice
- the multifunctional protein composition can be used in the form of tablets. capsules, lozenges, chewing gum.
- troches powders, syrups, elixirs, aqueous solutions and suspensions, and the like
- carriers that is used include lactose, sodium citrate and salts of phosphoric acid
- Various dismtegrants such as starch, and lubricating agents such as magnesium stearatc and talc, are commonly used in tablets
- useful diluents are lactose and high molecular weight polyethylene glycols
- certain sweetening and/or flavoring agents are added
- sterile solutions of the multifunctional protein are usually prepared, and the pHs of the solutions arc suitably adjusted and buffered For intravenous use.
- ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers
- Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid EDTA or benzylchromum chlonde, and the usual quantities of diluents and/or carriers
- diluents and or carriers will be selected to be appropriate to allow the formation of an aerosol
- the multifunctional protein is typically administered m aqueous form or m a hydrogel
- a preferred hydrogel comprises an aqueous suspension of from about 1% (w/v) to about 10% of low molecular weight hydrolyzed starch
- Suppository forms of the multifunctional protein are useful for vaginal, urethral and rectal administrations
- Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature
- the substances commonly used to create such vehicles include theobroma oil, glyce ⁇ nated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weighty and fatty acid esters of polyethylene glycol. See. Remington's Pharmaceutical Sciences, 16th Ed.. Mack Publishing. Easton. PA. 1980, pp. 1530-1 33 for further discussion of suppository dosage forms
- Analogous gels or cremes can be used for vaginal, urethral and rectal administrations
- a suitable dose of multifunctional protein per application ranges from about 0.1 ⁇ g/cm ⁇ to about 1 mg/cm preferably from about 1 ⁇ g/cm ⁇ (for example, using about 10 ⁇ g/ml) to about 1 mg/cm ⁇ (for example, using about 10 mg/ml). more preferably from about 5 ⁇ g cm ⁇ (for example, using about 50 ⁇ g/ml) to about 100 ⁇ g/cm ⁇ (for example, using about 1 mg/ml), yet more preferably from about 1 ⁇ g/cm ⁇ to about 250 ⁇ g/cm ⁇ .
- dosages ill generally be selected to maintain a serum level of multifunctional protein between about 0 1 ⁇ g/lOOcc and about 5 ⁇ g/lOOcc. preferably between about 0.5 ⁇ g lOOcc and about 2 0 ⁇ g/l OOcc In an alternative measure of preferred systemic administration amounts, preferably from about 0 1 mg/kg to about 10 mg/kg, more preferably about 1 mg/kg.
- a suitable dose of multifunctional protein per application ranges from about 0 01 mg per eye to about 5 mg per eye, preferably from about 0.1 mg per eye to about 2 0 mg per eye.
- suitable flushing/ instillation solutions of the multifunctional protein will generally have concentrations from about 1 ⁇ g/ml to about 15 mg/ml. preferably from about 100 ⁇ g/ml to about 3 mg/ml.
- suitable mouthwash solutions will generally have concentration of multifunctional protein from about 1 mg/ml to about 15 mg/ml preferably from about 2 mg ml to about 10 mg/ml.
- Lozenges will typically contain from about 100 ⁇ g to about 1 mg of multifunctional protein. Aerosols will generally be made from solutions having protein concentrations from about 0.1 mg/ml to about 15 mg/ml. preferably from about 1 mg ml to about 10 mg/ml.
- the protein composition will generally be applied from about 1 to about 10 times per day, preferably from about 2 to about 5 times per day.
- the multifunctional protein can be administered systemically or in a manner adapted to target the affected tissue
- the composition is preferably applied to the lungs or airways
- the composition may be applied systemically or in a manner adapted to target the affected tissue
- the primary administration route will generally be the topical route
- the composition will be injected into the scar or keloid.
- the composition will generally be administered ocularly without injection
- the composition will generally be administered systemically by a route or m a manner adopted to target the affected tissue
- the composition will generally be administered systemically.
- the site of administration mav be chosen to administer the highest dosages to the portion of the circulatory system most at ⁇ sk For asthma, the general route of administration will be pulmonary For treating pseudomonas infections, the infection will typically be a lung infection and the administration route pulmonary For reperfusion injury, the composition will generally be administered systemically. although the site of administration may be designed to administer the highest dosages to the portion of the body that suffered an ischemic event For treating the painful symptoms of malaria, the administration mode will generally by systemic
- the multifunctional protein is preferably be applied more often than simply the time at which the wound is first dressed Preferably, the multifunctional protein is applied at least about every time the wound dressing is changed The multifunctional protein can also be applied at least about every other day, more preferably, every day
- the multifunctional protem is administered to a wound substantially free of necrotic tissue
- the phrase "substantially free of necrotic tissue" shall mean sufficiently lacking in necrotic tissue so that an ordinarily-skilled pathologist would consider any residue of necrotic tissue to be irrelevant to determining a wound-healing prognosis
- the organ to be transplanted will preferably be bathed in a solution of the multifunctional protem for between about 10 minutes and about 5 hours
- the protein solution will preferably contain between about 0 01 mg/ml and about 25 mg ml of the multifunctional protein, more preferably, between about 0 5 mg/ml and about 5 mg/ml
- the multifunctional protem will preferably be administered systemically us
- the multifunctional protein can be used to treat (a) the build up of viscous fluids in the lungs and (b) associated pulmonary infections
- treatments of CF and COPD patients include pulmonary treatments with an aerosol of the multifunctional protein, but can include other routes of administration including systemic administrations
- sexually-transmitted diseases such as Candida, gonorrhea, chlamydia. syphilis, t ⁇ chomonas. chancroid, HIV, herpes or hepatitis mfections
- these viral diseases are particularly preferred targets for transmission prevention.
- HIV is a still more preferred target For this use.
- the body cavity involved in sexual activity is generally rinsed or flushed with a composition containing the multifunctional protem. or a creme, gel or suppository designed to localize the composition to the body cavity is used
- the composition can be used soon before, in conjunction with, or soon after, sexual activity, although prior or concurrent use is preferred
- the viral targets include HSV-1. which p ⁇ manly manifests as oral herpes, HSV-2, which p ⁇ manly manifests as genital herpes, and herpes zoster
- treatment targets include without limitation rheumatoid arthritis, multiple sclerosis, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, rheuma c arth ⁇ tis. scleroderma. systemic lupus erythematosus, Hashimoto's thyroiditis, p ⁇ mary myxedema. thyroroxicosis, pernicious anemia Addison's disease, premature onset of menopause, autoimmune male infertility, insulin-dependent diabetes, type B insulin resistance of acanthosis nig ⁇ cans. alopic allergy, myasthenia gravis. Lambert-Eaton syndrome.
- the cells or viruses involved can include, without limitation. endothehal cells, lymphocytes, including T-cells, tumor cells, microbial cells, viruses, including HIV and herpes Adhesion processes are believed to be involved in tissue invasion, for instance. by immune cells, microbes, and tumor cells
- an effective amount is an amount effective to either ( 1 ) reduce the symptoms of the disease sought to be treated, (2) induce a pharmacological change relevant to treating the disease sought to be treated, (3) inhibit or prevent infection or re-infection by an infective agent, or (4) prevent the occurrence of a non-mfectious disease (for instance a disease treatable by blocking a cell adhesion phenomenon)
- an effective amount further includes an amount effective to prevent or limit metastasis, for instance, to reduce the level of metastasis, reduce the size of a tumor, slow the growth of a tumor, and increase the life expectancy of the affected animal
- an effective amount includes an amount which, if regularly applied, prevents the occurrence of infection
- an effective amount includes an amount effective to reduce the average time it takes for a wound to heal
- the multifunctional protein can be used in many vete ⁇ nary contexts to treat animals, preferably to treat mammals, as will be recognized by those of ordinary skill in light of the present disclosure
- the PHIM polypeptide was pu ⁇ fied and the polypeptide was partially sequenced, as desc ⁇ bed in U S patent application Se ⁇ al No 08/600.273 (filed February 8, 1996).
- deFaire et al . inventors, entitled “Multifunctional Enzyme” Degenerate ohgonucleotide p ⁇ mers were constructed based on the partial ammo acid sequence The p ⁇ mers had the following sequences CACGCCTACCCITGGCA (SEQ ID NO 17) and GTGTTGGACTCGATCCAGATC (SEQ ID NO- 18) The p ⁇ mers were used to screen a k ⁇ ll cDNA library that was constructed in lambda zap.
- a recombinant multifunctional protein was expressed in an E cob as follows, using the BamHI and Xho I sites of a pET23c vector provided by Novagen (Abingdon. Oxford. U K )
- the pET23c vector includes a gene 10 tag for facilitating pu ⁇ fication of the expressed recombinant protein
- the pET vector places the recombinant multifunctional protein under the control of bactc ⁇ ophage T7 transcription and translation signals Once established in a non-expression host. E coli MC 1061. the plasmid was then transferred to an expression host.
- E coli BL21 (DE3 ) pLYS S having a chromosomal copy of the T7 polymerase gene under lacUV5 control Expression was induced by the addition of 1 mM IPTG at an optical density of 0 5 at wavelength 600
- the cells were harvested after 2 hours at an optical density of 1 0
- the recombinant protein was insoluble in the lysate and after harvesting, it was washed and dissolved in 6 M urea
- Refolding of the recombinant protem was carried out by 200-fold dilution using a buffer containing 100 mM t ⁇ s HCl pH 9 5, 100 mM CaCl2 0 3 mM oxidized glutathione and 3 mM reduced glutathione. followed by stir ⁇ ng overnight at 4 C Example 3.
- the recombinant protein was shown to cleave the model substrate succinyl-ala-ala-pro- phe- ?-n ⁇ troanihde, thereby demonstrating its proteolytic act ⁇ v ⁇ t ⁇
- the proteolytic activity was inhibited by the protease inhibitor Eghn
- SEQ ID NOs The nucleic acid or amino acid sequences referred to herein by SEQ ID NOs are as follows
- ADDRESSEE Dechert Price & Rhoads
- TTC TTC ACC CAC GAG AAC TGG AAC TCC TTC ACC CTC ACC AAT GAT CTT 481 Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Thr Asn Asp Leu 145 150 155 160
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Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10511922A JP2001500373A (en) | 1996-08-28 | 1997-08-28 | Enzyme and DNA sequence encoding the same |
GB9828022A GB2329896A (en) | 1996-08-28 | 1997-08-28 | Enzyme and DNA sequence encoding same |
US09/242,999 US6524814B1 (en) | 1997-08-28 | 1997-08-28 | Enzyme and DNA sequence encoding krill-derived multifunctional protein |
AU41679/97A AU4167997A (en) | 1996-08-28 | 1997-08-28 | Enzyme and dna sequence encoding same |
EP97939637A EP0925307A4 (en) | 1996-08-28 | 1997-08-28 | Enzyme and dna sequence encoding same |
DK199900148A DK199900148A (en) | 1996-08-28 | 1999-02-04 | Enzyme as well as DNA sequence encoding this |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/705,875 | 1996-08-28 | ||
US08/705,875 US6040155A (en) | 1996-08-28 | 1996-08-28 | Multifunctional protein and DNA sequence encoding same |
US76831896A | 1996-12-17 | 1996-12-17 | |
US08/768,318 | 1996-12-17 |
Publications (1)
Publication Number | Publication Date |
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WO1998008863A1 true WO1998008863A1 (en) | 1998-03-05 |
Family
ID=27107590
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/015179 WO1998008863A1 (en) | 1996-08-28 | 1997-08-28 | Enzyme and dna sequence encoding same |
Country Status (7)
Country | Link |
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EP (1) | EP0925307A4 (en) |
JP (1) | JP2001500373A (en) |
AU (1) | AU4167997A (en) |
CA (1) | CA2259151A1 (en) |
DK (1) | DK199900148A (en) |
GB (1) | GB2329896A (en) |
WO (1) | WO1998008863A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6232088B1 (en) | 1995-02-08 | 2001-05-15 | Phairson Medical, Inc. | Treatment and prevention of immune rejection reactions |
WO2006022947A1 (en) * | 2004-07-22 | 2006-03-02 | University Of Chile | Protein and nucleic acid sequence encoding a krill-derived cold adapted trypsin-like activity enzyme |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995033470A1 (en) * | 1994-06-07 | 1995-12-14 | Kristian Hellgren | Composition for dental use comprising krill enzyme |
WO1996024371A1 (en) * | 1995-02-08 | 1996-08-15 | Phairson Medical, Inc. | Multifunctional enzyme |
-
1997
- 1997-08-28 EP EP97939637A patent/EP0925307A4/en not_active Withdrawn
- 1997-08-28 CA CA002259151A patent/CA2259151A1/en not_active Abandoned
- 1997-08-28 WO PCT/US1997/015179 patent/WO1998008863A1/en not_active Application Discontinuation
- 1997-08-28 JP JP10511922A patent/JP2001500373A/en active Pending
- 1997-08-28 GB GB9828022A patent/GB2329896A/en not_active Withdrawn
- 1997-08-28 AU AU41679/97A patent/AU4167997A/en not_active Abandoned
-
1999
- 1999-02-04 DK DK199900148A patent/DK199900148A/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995033470A1 (en) * | 1994-06-07 | 1995-12-14 | Kristian Hellgren | Composition for dental use comprising krill enzyme |
WO1996024371A1 (en) * | 1995-02-08 | 1996-08-15 | Phairson Medical, Inc. | Multifunctional enzyme |
Non-Patent Citations (2)
Title |
---|
DERMATOLOGICAL RESEARCH, 1989, Vol. 281, ANHELLER et al., "Biochemical and Biological Profile of a New Enzyme Preparation from Antarctic Krill (E. Superba) Suitable for Debridement of Ulcerative Lesions", pages 105-110. * |
See also references of EP0925307A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6232088B1 (en) | 1995-02-08 | 2001-05-15 | Phairson Medical, Inc. | Treatment and prevention of immune rejection reactions |
WO2006022947A1 (en) * | 2004-07-22 | 2006-03-02 | University Of Chile | Protein and nucleic acid sequence encoding a krill-derived cold adapted trypsin-like activity enzyme |
Also Published As
Publication number | Publication date |
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CA2259151A1 (en) | 1998-03-05 |
EP0925307A4 (en) | 2002-10-16 |
AU4167997A (en) | 1998-03-19 |
GB9828022D0 (en) | 1999-02-10 |
GB2329896A (en) | 1999-04-07 |
EP0925307A1 (en) | 1999-06-30 |
JP2001500373A (en) | 2001-01-16 |
DK199900148A (en) | 1999-02-04 |
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