WO1994014472A1 - Procede pour inhiber l'adhesion cellulaire a des recepteurs renfermant des selectines - Google Patents

Procede pour inhiber l'adhesion cellulaire a des recepteurs renfermant des selectines Download PDF

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WO1994014472A1
WO1994014472A1 PCT/US1993/012464 US9312464W WO9414472A1 WO 1994014472 A1 WO1994014472 A1 WO 1994014472A1 US 9312464 W US9312464 W US 9312464W WO 9414472 A1 WO9414472 A1 WO 9414472A1
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enzyme
adhesion
leu
fucosidase
ser
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PCT/US1993/012464
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Dennis A. Carson
Ian Wicks
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01051—Alpha-L-fucosidase (3.2.1.51)

Definitions

  • the invention relates to a therapeutic method for the inhibtion of cell adhesion to cell receptors which contain selectins in mammals, particularly in humans.
  • it relates to a method of interfering with the adhesion of leukocytes to endothelial cell receptors via specific binding of a selectin in the receptor to its corresponding ligand, such as sialilated and fucosylated ligands.
  • the invention relates to the prevention of adhesion of any cell via interaction between carbohydrate residues in a cell surface ligand to receptors that contain selectins which will specifically bind such carbohydrate residues.
  • Inflammation resulting from the adhesion of neutrophils is known to play a role in acute conditions such as myocardial infarction, stroke, septic shock, anaphylactic reactions to drugs and allergens, and the "post-pump syndrome" which can develop following performance of a cardio-pulmonary bypass.
  • Inflammation is also a major cause of disability in chronic conditions such as rheumatoid arthritis, psoriasis, inflammatory bowel disease and chronic obstructive pulmonary disease.
  • Cell adhesion is also important to metastasis of tumors.
  • ELAM-1 A family of adhesion receptor glycoproteins critical to endothelium binding of leukocytes is known as the selectins.
  • One of the selectins in particular (ELAM-1) is thought to be synthesized by endothelial cells in response to the presence of inflammatory agents.
  • ELAM-1 is brought to the cell surface in its synthesis and serves as a receptor for corresponding carbohydrate adhesion ligands (hereafter, "selectin adhesion ligands”) on the cell surface of leukocytes.
  • the ligand for ELAM-1 contains carbohydrate residues which are part of a family of sialilated fucosylated polylactosamines, a critical determinant in which is sialyl - 2,3Gal ⁇ - ⁇ ,4 [fucose a 1 ,3] GlcNAc (hereafter, "sialyl Lewis X").
  • Sialyl Lewis X and Lewis X are ⁇ (1 ⁇ 3) fucosylated derivatives of polylactosamines found at the non-reducing termini of glycolipids.
  • Glycolipids and glycoproteins that contain ⁇ -L-fucose can be hydrolyzed by a lysosomal enzyme known as ⁇ -L-fucoside fucohydrolase (EC# 3.2.1.51 ), hereafter ⁇ -L-fucosidase.
  • Oligosaccharides, gangliosides and glycoproteins which contain sialic acid residues can also be hydrolyzed by a family of enzymes known as the sialidases (EC# 3.2.1.18; also known as neuraminidase).
  • cell adhesion is blocked by use of monoclonal antibodies specific to sialyl Lewis X or, as one set of researchers have attempted (Goelz, S.E., et al., Cell 63:1349-1356 (1990)) by use of a monoclonal antibody specific to an undefined carbohydrate structure in the selectin ligand.
  • Another approach inhibits cell adhesion by the use of soluble receptors (difucosylated sialyl Lewis X glycolipid or sialyl Lewis X-containing mucin) as competitors for binding of the natural ligand.
  • ARDS adult respiratory distress syndrome
  • the invention is a method for preventing or reducing adhesion of cells mediated by binding of a selectin adhesion ligand to an receptor containing selectin.
  • the method involves the administration of a therapeutically effective dosage of an anti- adhesion enzyme (in particular, a sialidase and/or a fucosidase) to a mammal
  • the invention will be principally described in the context of reducing inflammation through administration of the anti-adhesion enzymes of the invention.
  • the invention will be understood to not be so limited; i.e., the inventive method for administration of anti-adhesion enzymes may be utilized to inhibit adhesion of any cells wherein the adhesion occurs via formation of a bond between a carbohydrate residue in a cell surface ligand on the cell and a corresponding receptor which contains selectin.
  • a therapeutically effective dosage of the anti-adhesion enzyme will be administered by intravenous, intramuscular or other parenteral routes. Enzymatic activity in the plasma is verified and monitored by monitoring means such as spectrophometry as well as changes in clinical signs and symptoms. Because when administered to a human the enzyme will preferably be a recombinant one of human origin, no immune response should be induced by its administration. Further, because a single enzyme molecule can cleave many different adhesion ligand residues, the potency of the composition is expected to be relatively high, and required dosage relatively low, as compared to prior art proposals for methods of inhibiting cell adhesion by leukocytes.
  • FIGURE 1 depicts the pH activity curve of human ⁇ -L-fucosidase.
  • FIGURE 2 depicts the in vitro effect of substantially pure recombinant ⁇ -L-fucosidase in varying concentrations on adherence of neutrophils to human umbilical vein endothelial cells.
  • FIGURE 3 depicts the effect of sialidase on neutrophil content in lung tissue in rats injected with cobra venom factor (ARDS Model).
  • the anti-adhesion enzymes of the invention can be extracted from many mammalian tissues, including liver and placental tissues. For purposes of this application, however, the need for a consistent, high volume production source dictates that the recombinant form of the enzymes modified and produced as taught herein will be preferred.
  • amino acid sequence shown in SEQ.ID.NO.1 is derived from sequence information obtained through isolation of several human ⁇ -L-fucosidase cDNA clones reported by Occhiodoro et al. at Biochem. Biophys. Res. Comm. 164:439-445 (1989), the disclosure of which is incorporated herein by this reference. Potential glycosylation sites are at nucleotides 754-756, 805-807 and 1147-1149.
  • the full-length nucleotide sequence for ⁇ -L-fucosidase is publicly available from the GENBANK molecular sequence database under Accession No. M28099.
  • the fucosidase whose amino acid sequence is depicted in SEQ.ID.NO.1 is preferred for use in the method of the invention for therapy in humans because it is of human origin. It will be appreciated by those skilled in the art, however, that other fucosidases are known and could be of potential therapeutic value in the inventive method, particularly for use in short-term treatment of acute inflammatory conditions where the immunogenicity of the enzyme may not be of as great concern as it would be if used in a treatment regime of longer duration.
  • Fucosidase activity in an enzyme to be used in the method of the invention may be determined according to conventional means for determining enzyme activity which are well-known in the art; for example, see Methods of Enzymology (New York Acad. Press, 1981). However, a particularly preferred and well-known method for determining fucosidase activity in an enzyme is disclosed in Zielke, et al., J. Lab. Clin.
  • Sialidases have isolated from several vertebrate and microbial sources and cloned and the amino acid/nucleotide sequences reported in publicly accessible molecular sequence databanks such as GENBANK.
  • sialidases cloned from microbial sources include the gene from Actinomyces viscosus (GENBANK Accession NOS. S73643, S854402, S69682, X64360, S55807, S55811 , S55812, S55814 and S55817); Salmonella typhimurim LT2 (GENBANK Accession No.M55342); Clostridium septicum (expressed in E.coli; GENBANK Accession Nos.
  • sialidases for use in the inventive method can be readily synthesized or cloned by one of ordinary skill in the art through use of the methods described below without undue experimentation.
  • each of the above- referenced references, including the related sialidase sequence data from GENBANK and equivalent sequence databases, are incorporated herein as examples of sialidases useful in this invention.
  • Purified non-human sialidases are also commercially available (from, for example, Sigma Chemical). However, as noted above with respect to the use of fucosidase, use of the human enzyme will be preferred, most preferably in recombinant form.
  • Sialidase activity in an enzyme to be used in the method of the invention may be determined according to conventional means for determining enzyme activity which are well-known in the art; for example, see Methods of Enzymology (New York Acad. Press, 1981 ).
  • the method of the invention encompasses the use of any catalytically active anti-adhesion enzyme which will cleave bonds between selectins and carbohydrate residues in selectin adhesion ligands, in particular sialidases and fucosidases. It is not intended, therefore, that the invention be considered as limited to the use of the specific enzymes whose amino acid sequences are referred to herein. Rather, functional derivatives of these enzymes and enzymes which are substantially similar in function are encompassed herein.
  • a “fragment” of a molecule such as any of the anti- adhesion enzyme encoding DNA sequences of the present invention, includes any nucleotide subset of the molecule.
  • a “variant” of such molecule refers to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule refers to a non-natural molecule which is substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the. molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical. As used herein, a molecule is also said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule.
  • Such moieties may improve the molecule's solubility, absorption, biological half life, etc.
  • the moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc.
  • Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).
  • Minor modifications of the primary amino acid sequences for the anti-adhesion enzymes of the invention may result in proteins which have substantially equivalent activity as compared to the specific anti-adhesion enzymes described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of an anti-adhesion enzyme (i.e., the ability to specifically cleave bonds between carbohydrate residues and selectins) still exists. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which may not be required for the enzyme to exert the desired catalytic activity.
  • a "functional derivative" of a polynucleotide sequence which encodes an anti-adhesion enzyme of the present invention includes “fragments”, “variants”, or “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • anti- adhesion enzyme includes any functional derivative, fragments, variants, analogues, chemical derivatives which may be substantially similar to the anti-adhesion enzymes described herein and which possess similar activity.
  • DNA sequences for use in producing anti-adhesion enzymes other than those identified herein can be obtained by several methods.
  • the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to:.1 ) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features and 3) synthesis by the polymerase chain reaction (PCR).
  • DNA sequences encoding the anti-adhesion enzymes of the invention can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • DNA sequences encoding the anti-adhesion enzymes can be expressed in vitro by
  • Hos cells are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, in other words when the foreign DNA is continuously maintained in the host, are known in the art.
  • the genes encoding the anti-adhesion enzymes of the invention may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the appropriate genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • Polynucleotide sequences encoding the anti-adhesion enzymes of the invention can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences encoding the anti-adhesion enzymes of the invention.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCI 2 method by procedures well known in the art.
  • CaCI 2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be cotransformed with DNA sequenc ⁇ es encoding the anti-adhesion enzymes of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • the polynucleotides encoding the anti-adhesion enzymes of the invention will preferably contain additional restriction sites to facilitate cloning which are appropriate to the recombinant expression vector to be used, which is Not-I for the preferred vectors.
  • suitable eukaryotic recombinant expression vectors in addition to those used by Occhiodoro et al. are selectable eukaryotic expression vectors available from InVitrogen of San Diego, California (1992 product catalog) such as pRc/CMV, pc DNAI/Neo or pRc/RSV, all of which contain a Not-I cloning site.
  • the oligonucleotide for the Not-I enzyme has the sequence 5'-GCGGCCGC-3' and 3'- CGCCGGCG-5".
  • These latter recombinant expression vectors are preferred for two reasons. First, they contain a neomycin selection marker for stable expression in mammalian cells.
  • a eukaryotic vector is generally preferred over prokaryotic vectors because use of a eukaryotic vector will allow for glycosylation of the recombinant enzyme.
  • a prokaryotic recombinant expression vector for transfection into bacteria preferably E. coli
  • suitable prokaryotic expression vectors are available from InVitrogen of San Diego, California and include its pSE280 and pSE380 vector products.
  • Anti-adhesion enzymes of the invention whose amino acid sequences are known can also be synthesized by the well known solid phase peptide synthesis methods described Merrifield, J. Am. Chem. Soc, 85:2149, (1962), and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969, pp.27-62), using a copoly(styrene-divinylbenzene) containing 0.1 -1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0°C.
  • the peptides are extracted from the polymer with 1 % acetic acid solution which is then lyophilized to yield the crude material.
  • This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be charac ⁇ terized by such standard techniques as amino acid analysis, thin layer chromato- graphy, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
  • An anti-adhesion enzyme clone having a desirable mutation as described further below may be amplified using a conventional polymerase chain reaction (PCR) and a primer pair corresponding to the 3' and 5' regions of the cDNA.
  • PCR polymerase chain reaction
  • the preferred method of amplification is the overlap extension PCR technique described by Ho, et al., Gene 77:51 -59 (1989), the disclosure of which is incorporated herein by this reference.
  • this technique accomplishes site-specific mutagenesis of the clone by utilizing a 3' primer to add the mismatched mutating bases (primer B in the Ho article, which is used with the 5' primer A in the first PCR cycle described).
  • Amplification using the A and B primers yields an AB fragment.
  • a second PCR cycle uses a primer (D) from the 3' end of the gene and a 5' mutated primer (C) complementary to primer B.
  • the resulting amplification product (fragment CD) will overlap the AB fragment.
  • the resulting fusion product (AD) will contain the full-length cDNA sequence and the desired mutation.
  • any site of N-linked glycosylation in the anti-adhesion enzyme polypeptides of the invention may be necessary to mutate any site of N-linked glycosylation in the anti-adhesion enzyme polypeptides of the invention to prevent mannosylation of the protein. This will prevent uptake of the recombinant enzyme by monocytes and macrophages that have receptors for mannose-phosphate, thus extending the half-life of the enzyme in a patient's circulation.
  • the overlap extension PCR technique described above may be used to eliminate the sites for N-linked glycosylation in the anti-adhesion enzyme produced according to the method described herein.
  • the polymerase chain reaction technique may be modified to change 1 to 3 of the asparagines in the sites of N-linked glycosylation to lysines or arginine (e.g., to change the AAT or AAC codons for asparagine to AGA or AGG, the codons for arginine).
  • Another suitable approach to modification of the lysines or arginines is described by Shaw in U.S. Patent No. 4,904,584 ("Site-Specific Homogenenous Modification of Polypeptides"), the disclosure of which is incorporated herein by this reference for purposes of illustration.
  • glycosylation of the enzyme enhances its desired functional activity
  • the interest in extending the half-life of the enzyme may be outweighed. In that instance, therefore, the sites for N-linked glycosylation will not be modified and a eukaryotic vector will be used to express the anti-adhesion enzyme.
  • Another useful mutation of the recombinant enzyme is one which will raise the pH optimum for the enzyme's activity from pH 5-5.5 to pH 7-7.4, the normal pH for human plasma (see, e.g., FIGURE 4 for the normal pH activity curve for ⁇ -L-fucosidase in human serum [as determined at varying pH values in citrate-phosphate buffers for 30 minutes at 37°C]).
  • This change to enhance the enzyme's activity in more alkaline environments is achieved by separately mutating single charged amino acids selected from the group consisting of lysine, arginine, histidine, glutamic and aspartic acid.
  • the anti-adhesion enzyme will be purified by means well-known in the art to a preferred specific activity.
  • purification is best achieved by affinity chromatography using agarose to which aminocaproyl fucosamine has been coupled (suitable columns for use in this procedure are available from Sigma Chemical of St. Louis, Missouri or from Miles
  • the tissue extract, transformed cell line supernatants, transformed bacterial lysates, or other source of the synthetic or recombinant fucosidase may be applied to an affinity column and washed with 10 mM phosphate buffer (pH 5.5) until all protein has been eluted. The column is then washed again with the phosphate buffer supplemented with 50 mM fucose available from Sigma) to elute the native or recombinant fucosidase, which is then concentrated using an ultrafiltration device (such as the ultrafiltration apparatus and microconcentrators available from Amicon of Danvers, Massachusetts) to a preferred concentration of 1.5 to 2.0 mg of enzyme per milliliter of buffer.
  • an ultrafiltration device such as the ultrafiltration apparatus and microconcentrators available from Amicon of Danvers, Massachusetts
  • the purified anti-adhesion enzyme will preferably have a specific activity of 25,000 - 30,000 units/mg of protein, wherein one unit (U) of enzyme activity is equivalent to the amount of enzyme which will hydrolyze 1 nmole of P-nitrophenyl- ⁇ -L-fucopyranoside or sialic acid substrate (available from Sigma) per minute at 37° C.
  • U one unit of enzyme activity is equivalent to the amount of enzyme which will hydrolyze 1 nmole of P-nitrophenyl- ⁇ -L-fucopyranoside or sialic acid substrate (available from Sigma) per minute at 37° C.
  • the enzyme is suspended in a buffer suitable for administration to humans, preferably isotonic phosphate buffered saline (pH 7.4) at a concentration of 1 to 10 mg enzyme/ml buffer, not to exceed 100 ml of buffer.
  • a buffer suitable for administration to humans preferably isotonic phosphate buffered saline (pH 7.4) at a concentration of 1 to 10 mg enzyme/ml buffer, not to exceed 100 ml of buffer.
  • the buffer may also be supplemented with stabilizers and/or anti-bacterial agents well known to those skilled in the art to maintain purity and extend shelf life of the anti-adhesion enzyme suspension.
  • the enzyme will be modified to delay its clearance from the patient's bloodstream via the kidneys, thus extending the patient's exposure to the enzyme and reducing the number of times that the composition will have to be administered for effective treatment.
  • the enzyme may be covalently attached to activated polyethylene glycol (PEG), which couples to the lysines and arginines in the protein.
  • PEG polyethylene glycol
  • Activated PEG is available from, for example, Sigma Chemical, in five different forms; an exemplary form for use ' in this embodiment is methoxypolyethylene glycol- succimidyl succinate. Whichever form of PEG is chosen, its selection should be made with a view toward chemically coupling at least 50% of the lysines and arginines in the protein.
  • the actual percentage of these amino acids which will be coupled in the final enzyme product will be that percentage which will help to maintain availability of the enzyme to the patient without destroying the catalytic activity of the enzyme.
  • the point at which enzyme activity is significantly reduced can be determined by the assay procedures for enzyme activity described above (see, e.g., Aldaheff, et al., J. Biol. Chem. 250:7106-7113 (1975)).
  • Coupling of the lysines and arginines in the enzyme product to PEG may be achieved by use of several methods known to those skilled in the art (see, e.g., Savoca, et al., Biochem. Biophys. Acta. 578:47-53 (1979) and methods used in U.S. Patent 4,766,106).
  • a suitable adaptation of these methods to conjugation of the anti- adhesion enzymes of the invention would be performed as follows. Approximately 25 mg of purified recombinant enzyme is dissolved in 5 ml of 50 mM phosphate buffered saline (pH 7.2).
  • the dissolved enzyme is then incubated with approximately 500 mg of activated polyethyleneglycol (such as methoxypolyethylene glycol-succinimidye succinate, available from Sigma), then isolated by means known in the art (see, e.g., Abuchowski et al., "Enzymes as Drugs", John Wiley and Sons (1981)).
  • activated polyethyleneglycol such as methoxypolyethylene glycol-succinimidye succinate, available from Sigma
  • the anti-adhesion enzyme composition will be administered to the mammal in a therapeutically effective dosages, preferably by intra-arterial infusion.
  • a "therapeutically effective dosage” refers to that amount of anti-adhesion enzyme which will produce a detectable reduction in inflammation without substantial toxicity to the patient. Generally, this dosage will comprise about 0.6 to 234 U/kg of the patient's body weight.
  • the infusion will be performed for an extended period of up to about 4 hours, although those of ordinary skill in the clinical art will know or be able to readily determine how to adjust the course of treatment to suit the therapy needs of particular patients.
  • Administration of this dosage will preferably be repeated at intervals of two times per week to- once a month, depending on the patient's presenting condition and responsiveness to the therapy.
  • Enzymatic activity in the plasma is verified and monitored by spectrophotometry according to means well- known in the art; changes in clinical signs and symptoms will provide a further basis for assessment of functional enzyme activity.
  • the anti-adhesion enzyme suspension will be effective in therapy for acute inflammatory conditions associated with septic and endotoxin shock, post-pump syndrome following cardiopulmonary bypass, the acute rejection of transplanted organs, reprofusion injury following myocardial infarction or stroke and peripheral vascular injuries. It is also expected to be of therapeutic value in treatment of chronic inflammation associated with rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and inflammatory ulceris.
  • tumor cells having metastasic capability rat mammary adenocarcinoma cell line R13762
  • bovine fucosidase see data reported in Wright, et al. J. Cell. Biochem. 37:49-59, (1988)
  • the human fucosidase composition and, possibly, the sialidase composition disclosed herein will be of value in limiting metastasis of tumor cells including, but not limited to, those associated with cancers of the breast, colon, prostate, lung, stomach and pancreas.
  • substantially pure ⁇ -L-fucosidase composition was prepared as discussed above without coupling to PEG or mutation to enhance optimal pH activity or to prevent mannosylation.
  • substantially pure refers to a fucosidase composition which consists essentially of catalytically active fucosidase, but may also contain catalytically active sialidase.
  • the pRc/CMV or pcDNAI/Neo eukaryotic expression vectors available from InVitrogen of San Diego, California were used to express the enzyme.
  • the fucosidase composition was injected in a dosage of 0.01-0.1 mg/kg of body weight into New Zealand white rabbits of 2-3 kg body weight.
  • carrageenan available from Sigma in a preparation of 2.5 mg carrageenan/0.25 ml water
  • the plural exudate was carefully removed by pipetting and assayed for total volume of exudate (in milliliters) and for total cellular influx.
  • HUVEC Human umbilical vein endothelial cells
  • EGMUV medium also available from Clonetics
  • IL-1 interleukin-1
  • Neutrophils were isolated from human peripheral blood by Ficoll-Hypaque sedimenta ⁇ tion according to means well known in the art.
  • the cell pellet was treated for 10 seconds with water to lyse erythrocytes, and then was washed two times in Hank's balanced salt solution (HBSS) supplemented with 1 % human plasma.
  • HBSS Hank's balanced salt solution
  • the isolated neutrophils were incubated with bovine kidney fucosidase at the concentrations indicated in FIGURE 2 for one hour in HBSS with 1% autologous plasma.
  • the cells were then washed and then added at a density of 200,000 cells/ml to the HUVEC monolayers.
  • neutrophil myeloperoxidase was measured spectrophotometrically by the oxidation of diaminobenzidine with H 2 O 2 in the presence of the enzyme, using a commercial assay (the kit for which is available from Sigma, St. Louis). Since myeloperoxidase is present in neutrophils and not endothelial cells, the total enzyme level is directly proportional to the number of adherent neutrophils in each culture. As indicated in FIGURE 2, pretreatment of the neutrophils with the fucosidase composition blocked their ability to adhere to HUVEC. EXAMPLE III
  • a sialidase composition of microbial origin (Clostridium) was prepared as described elsewhere above.
  • ARDS adult respiratory distress syndrome
  • CVF COBRA VENOM FACTOR
  • a tradenamed product of Calbiochem of San Diego was administered to rats as follows.
  • CVF activates the complement system.
  • the resulting formation of the complement factor C5A induces neutrophil activation.
  • the activated neutrophils accumulate in the lung vasculature, causing tissue damage.
  • CVF was diluted in phosphate buffered saline (PBS) to 32 ⁇ /ml and dialyzed overnight at 4°C in PBS. The final concentration of the CVF was 22.8 ⁇ /ml.
  • the sialidase was resuspended in PBS to a volume of 50 ⁇ /ml and injected into the rat's tails in volume of 200 ⁇ l of PBS. Control rats received injections of 0.5 ml PBS.
  • Lung tissue was dissected out with a specimen fixed in formalin for histological study and a second specimen place in PBS for myeloperoxidase activity assay. Rat lungs in PBS were frozen at -20 °C for 2 days. After 2 days, rat lung specimens were thawed in a 37 °C circulating water bath. Approximately 500 mg of lung tissue was minced to fine pieces approximately 1 mm 2 using sterile scalpel and forceps. Lung pieces were then sonicated at a setting of 4.5 on a microtip sonicator for 45, 30 and
  • SEQUENCE ID NO 1 is the polynucleotide sequence which will encode human ⁇ -L- fucosidase, including the signal peptide (bases 19 to 84) and the mature protein (bases 85 to 1401).
  • SEQUENCE ID NO 2 is the deduced amino acid sequence for human ⁇ -L-fucosidase, including the signal peptide and mature protein.
  • MOLECULE TYPE DNA (genomic)
  • ATC ATT TCG GAA CTG GTT CAG ACA GTA AGT TTG GGA GGC AAC TAT CTT 1011 lie He Ser Glu Leu Val Gin Thr Val Ser Leu Gly Gly Asn Tyr Leu 320 325 330
  • GAA AGG CTT CTT GCT GTT GGG AAA TGG CTG AGC ATC AAT GGG GAG GCT 1107 Glu Arg Leu Leu Ala Val Gly Lys Trp Leu Ser He Asn Gly Glu Ala 350 355 360
  • TCT GTA TGG TAT ACC TCA AAG GGA TCG GCT GTT TAT GCC ATT TTT CTG 1203 Ser Val Trp Tyr Thr Ser Lys Gly Ser Ala Val Tyr Ala He Phe Leu 30 380 385 390 395
  • CAGTTGTCAC CAACAGTCTT TCTGCAAAGG GCAGGAGAGC TTTGGGGGAA AGGAAAAGGC 1871

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Abstract

Dans un procédé pour inhiber l'adhésion des cellules à des récepteurs renfermant des sélectines chez des mammifères, une enzyme anti-adhésion leur est administrée en un dosage thérapeutiquement efficace. Dans un mode préféré de réalisation, l'enzyme anti-adhésion sera administrée pour traiter une pathologie inflammatoire chronique ou aiguë (de préférence cette dernière). Dans ce contexte, le dosage thérapeutiquement efficace sera un dosage suffisant pour obtenir une réduction détectable de l'inflammation sans toxicité notable. L'enzyme administrée va cliver spécifiquement les résidus glucidiques qui sont impliqués dans la formation des liaisons entre les résidus glucidiques dans les ligands (généralement sur les leucocytes) qui sont spécifiques contre les récepteurs contenant des sélectines (généralement sur les cellules endothéliales), notamment les liaisons formées par les résidus fucosylés et/ou sialilés. Cette enzyme sera de préférence une fucosidase ou une sialidase et s'utilise de préférence sous forme recombinée. Sont également décrits des moyens permettant de stimuler l'activité de l'enzyme pour qu'elle opère de manière optimale à pH 7-7,4 (c'est-à-dire le pH du plasma). L'enzyme peut également être modifiée pour prolonger sa demi-vie in vivo et sa durée de conservation. La même enzyme devrait cliver de nombreux résidus d'adhésion.
PCT/US1993/012464 1992-12-22 1993-12-21 Procede pour inhiber l'adhesion cellulaire a des recepteurs renfermant des selectines WO1994014472A1 (fr)

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WO2000063351A2 (fr) * 1999-04-21 2000-10-26 Incyte Genomics, Inc. Enzymes modifiant les hydrates de carbone
AU754480B2 (en) * 1998-10-22 2002-11-14 Glaxo Group Limited Fluticasone lotion having improved vasoconstrictor activity
US6774210B1 (en) * 1998-01-22 2004-08-10 Good Humor-Breyers Ice Cream Division Of Conopco., Inc. Frozen food product
US7332179B2 (en) 2003-12-12 2008-02-19 Kimberly-Clark Worldwide, Inc. Tissue products comprising a cleansing composition
US7642395B2 (en) 2004-12-28 2010-01-05 Kimberly-Clark Worldwide, Inc. Composition and wipe for reducing viscosity of viscoelastic bodily fluids
EP2141237A1 (fr) * 2008-07-03 2010-01-06 Mogam Biotechnology Research Institute Procédé pour la réduction de la teneur en fructose de protéines recombinantes

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6774210B1 (en) * 1998-01-22 2004-08-10 Good Humor-Breyers Ice Cream Division Of Conopco., Inc. Frozen food product
AU754480B2 (en) * 1998-10-22 2002-11-14 Glaxo Group Limited Fluticasone lotion having improved vasoconstrictor activity
WO2000063351A2 (fr) * 1999-04-21 2000-10-26 Incyte Genomics, Inc. Enzymes modifiant les hydrates de carbone
WO2000063351A3 (fr) * 1999-04-21 2001-08-09 Incyte Genomics Inc Enzymes modifiant les hydrates de carbone
US7332179B2 (en) 2003-12-12 2008-02-19 Kimberly-Clark Worldwide, Inc. Tissue products comprising a cleansing composition
US7642395B2 (en) 2004-12-28 2010-01-05 Kimberly-Clark Worldwide, Inc. Composition and wipe for reducing viscosity of viscoelastic bodily fluids
EP2141237A1 (fr) * 2008-07-03 2010-01-06 Mogam Biotechnology Research Institute Procédé pour la réduction de la teneur en fructose de protéines recombinantes

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