WO1996009309A1 - The oligosaccharide structure of a ligand for e and p selectin - Google Patents

The oligosaccharide structure of a ligand for e and p selectin Download PDF

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Publication number
WO1996009309A1
WO1996009309A1 PCT/US1995/011401 US9511401W WO9609309A1 WO 1996009309 A1 WO1996009309 A1 WO 1996009309A1 US 9511401 W US9511401 W US 9511401W WO 9609309 A1 WO9609309 A1 WO 9609309A1
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ligand
essentially pure
types
selectin
oligosaccharides
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PCT/US1995/011401
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French (fr)
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Ake P. Elhammer
Jian-Guo Geng
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Pharmacia & Upjohn Company
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Priority to JP8510934A priority Critical patent/JPH10508580A/en
Priority to AU35486/95A priority patent/AU3548695A/en
Priority to EP95932441A priority patent/EP0783508A1/en
Publication of WO1996009309A1 publication Critical patent/WO1996009309A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00Drugs for disorders of the urinary system
    • A61P13/02Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00Drugs for genital or sexual disorders; Contraceptives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates to the field of cell adhesion biology, E and P selectin and the oligosaccharide structure of a ligand for E and P selectin.
  • GlyCAM-1 a mucin-like molecule
  • the Le x determinant (Gal ⁇ l,4( ⁇ l,3Fuc)GlcNAc) is an important element of the P-selectin ligand on myeloid cells.
  • the ligand for P-selectin may be the same or very similar to that for E-selectin.
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g. colon and mammary carcinomas.
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc) are known cancer antigens which appear in many carcinomas, e.g. colon and mammary carcinomas.
  • N-linked oligosaccharides Two common types of linkages for the attachment of oligosaccharide structures to glycoproteins are found in N-linked oligosaccharides, where the oligosaccharide structures are attached by an amide bond between the reducing N-acetylglucosamine on the oligosaccharide and an asparagine residue on the protein, and mucin type O-linked oligosaccharides which are conjugated via an ether bond between the reducing N-acetylgalactosamine on the oligosaccharide and a serine or threonine residue on the protein.
  • Other types of linkages between carbohydrate moieties and proteins do exist but these have little relevance for this discussion.
  • N-linked and mucin type O-linked oligosaccharides Several characteristics are shared by N-linked and mucin type O-linked oligosaccharides. Some similarities are: the majority of their component monosaccharides are the same; both types of structures, when synthesized by most untransformed mammalian cells, carries terminal sialic acids; both types of structures can carry blood group type substitutions such as Sialyl Lewis 1 (see below); and both types of oligosaccharides can contain poly-N-acetyllactosamine repeat structures. These observations, also suggested in the literature, indicate there may be a functional overlap between some N-linked and mucin type O-linked oligosaccharides. Fukuda, M. (1992) and Olden, K, Yeo, T.-K, and Yeo, -T.
  • Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. O-linked chains are of interest in view of the evidence that cell surface glycoproteins containing "clusters" of this type of substitution can form highly extended and rigid structures. Jentoft, N. (1990) Trends. Biochem. Sci. 15:291-294. Thus, these molecules are ideally positioned to perform recognition functions.
  • oligosaccharide structures can be created through the activities of a limited number of glycosyltransferases. Hence, these structures can be generated with relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell-cell interactions.
  • Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins, so called lectins, on interacting cells have been described Dodd, J., and Jessel, T.M., (1985) J. Neurosci., 5:3278; Regan, L.J., et al. (1986) Proc. Natl. Acad. Sci. USA, 83:2248; Constantine-Paton, M., et al. (1986) Nature, 324:159; and Tiemeyer, M., et al. (1989) J. Biol. Chem., 263:1671.
  • C-type lectins Animal lectins have been divided into three groups: C-type (Ca -dependent) lectins, S-type (soluble) lectins and P-type lectins. Drickamer, K. (1993) Annu. Rev. Cell Biol. 9:237-264.
  • An important group of C-type lectins are the selectins. The selectins have an important function in leucocyte extravasation at sites of inflammation.
  • Leukocytes are involved in the protection of the body against various microbial infections as well as diseases such as cancer.
  • white blood cells adhere to endothelial cells on the lumenal site of blood vessels, migrate to body tissues and accumulate there, they often cause damage, swelling, pain, and inflammation.
  • rheumatoid arthritis occurs when white blood cells enter the joints and attack the tissues.
  • cell adhesion receptors are called cell adhesion receptors.
  • the recruitment and extravasation of circulating leukocytes from the bloodstream across the endothelium into the target tissue, e.g. a site of injury or infection, are essential steps in the early response of inflammation. These processes are initiated by at least three families of cell surface adhesion molecules: the integrins, the immunoglobulin superfamily and the selectins. Springer, T.A. (1990) Nature 346:425; Hynes, R. O. and Lander, AD. (1992) Cell 68:303.
  • the selectins are a family of leukocyte-endothelial cell adhesion molecules, also referred to as LEC-CAM:s (Lectin EGF Complement regulatory-Cell Adhesion Molecule). Inflammation: Basic Principles and Clinical Correlates, Second Edition. J. I. Gallin et al, Eds, Raven Press, Ltd. NY 1992, pp 407-419. Selectin is the term favored by investigators working in the field. Bevilacqua M.P., et al. (1991) Cell 67:233. Selectins are generally believed to be primarily responsible for the initial interaction, called "rolling", of the leucocyte with the venular endothelium upon physiological and pathological stimuli, i.e.
  • leukocytes includes such cells as neutrophils, basophils, eosinophils, monocytes and T-cell subsets, de Bnujne-Admiraal, L.G. (1992) Blood 80:34.
  • L-selectin is a constitutively expressed lymphocyte homing receptor (a.k.a. peripheral lymph node homing receptor (pnHR), in a majority of leukocytes.
  • L- selectin is also known as: LECAM-1, LEC-CAM-1, LAM-1, gp90MEL, gplOOMEL, gpllOMEL, MEL-14, MEL- 14 antigen, Leu-8 (antigen), Ly-22 (antigen), TQ1 (antigen) and DREG.
  • E- selectin also known as ELAM-1, LECAM-2, and LEC-CAM-2
  • ELAM-1 a transient cytokine-inducible endothelial cell surface molecule which interacts with neutrophils, monocytes and memory T-cells.
  • P-selectin also known as LEC-CAM-3, LECAM-3, CD-62, GMP-140, and PADGEM, is rapidly expressed on the plasma membrane of endothelial cells and platelets during cellular activation and degranulation; it serves as a receptor for neutrophils and monocytes.
  • all selectins contain an NH 2 -terminal extracellular carbohydrate recognition domain (CRD) composed of 118 amino acid residues which is homologous to other C-type lectins, followed by a epidermal growth factor (EGF)- like domain, a variable number of complement regulatory protein-like repeats (different for different selectins), a transmembrane domain and a cytoplasmic tail.
  • CCD carbohydrate recognition domain
  • EGF epidermal growth factor
  • all selectins mediate Ca -dependent cell-cell contact by binding to oligosaccharide ligands on opposing cells.
  • selectins are known to interact with the oligosaccharide structures on other glycoproteins, called "selectin ligands," which are expressed on the surface of their opposing cells.
  • Immunoprecipitation experiments have shown that the construction of an L- ⁇ electin-IgG chimaera provides a compound that can be used to isolate selectin ligands.
  • a 50kD molecule and a 90kD molecule, both thought to be L-selectin ligands, has been specifically precipitated from mouse mesenteric lymph nodes using this type of compound. Imai, Y., et al. (1991) J. Cell Biol. 113:1213.
  • GlyCAM-1 mucin-like molecule
  • Recent work on the 90 kD ligand has revealed that the protein core of this molecule is identical to the vascular sialomucin CD34. Baumheuter, S., et al. (1993) Science 262:436.
  • a ligand has also been partially characterized for E-selectin.
  • a 150kD glycoprotein was isolated from a mouse neutrophil progenitor cell line using an E- selectin-IgG chimaera. Levinovitz, A., et al. (1993) J. Cell Biol. 121:449-459. See also Brandley et al., US 5,143,712, US 5,211,936, and US 5,211,937 patents.
  • a P- selectin ligand has similarly been identified.
  • a «120kD glycoprotein from human neutrophils has been identified in a 125 I-P-selectin blotting assay and by P-selectin affinity chromatography of H-glucosamine-labeled HL-60 cell extracts. Moore, K.L., et al. (1992) J. Cell. Biol. 118:445-456.
  • ligands for balh E- an ⁇ P-selectin have been described on mouse myeloid cells.
  • Two molecules, 150 kD and 160 kD which are monospecific ligands for E- and P-selectin, respectively, as well as two other molecules with molecular masses of 230 kD and 130 kD, respectively, that interact with both E- and P-selectin.
  • the present inventors report the structure of the O-linked oligosaccharides on a common ligand for E and P-selectin.
  • This molecule contains a heavy preponderance of O-linked oligosaccharides and fewer N-linked oligosaccharides.
  • the present inventors have found that the O-linked oligosaccharide structures on the ligand identified herein contains no Le x , Le a , sLe x or sLe a type substitutions.
  • the o-linked oligosaccharide structures on this ligand have little or no fucose.
  • the ligand described herein also contains little or no sulfated oligosaccharides.
  • the present inventors describe a truly unique ligand.
  • One object of the present invention is to provide a method for the purification of a selectin ligand. Another object of the invention is to provide a purified selectin ligand, specifically a common ligand for E and P-selectin. A further object of the present invention is to identify the 0-linked oligosaccharide structures on this ligand. Another aspect is to enable the preparation of glycosylation variants of selectin ligands, not otherwise found in nature. In another aspect, the invention provides a method of designing selectin inhibitors, mimicking carbohydrate based determinants of the selectin ligands.
  • the purified ligand, or fragments thereof, as well as carbohydrate and polypeptide components of the ligand, or antibodies to the ligand or to fragments thereof, can be used as inhibitors of binding of P- or E-selectin, or both, and in diagnostic assays and kits.
  • This invention encompasses those isolated molecules, fragments, combinations and variations thereof which adhere to E and or P selectin.
  • the isolated ligands can be generally characterized by the following embodiments, which may occur either separately or in various combinations and either in a free state or attached to a substrate.
  • 3GalNAc substituted with two sialic acids one sialic acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is linked to the 6 position of the reducing GalNAc.
  • Figure 2. Separation of the O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin on size -exclusion chromatography.
  • Figure 3. Size-exclusion separation of neutral and charged O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin.
  • Figure 4. Sequential exoglycosidase digestion of an O-linked 12.8 GU oligosaccharide isolated from the HL-60 common ligand for E- and P-selectin.
  • Figure 7 Purification of the approximately 120 kD Ligand for P- and E- selectins. Additional Description of the Invention and the Preferred Embodiments Definitions, Abbreviations and Sources of Materials Some standard abbreviations used in connection with the present invention include: BSA, bovine serum albumin; DEAE, diethylaminoethyl; DMSO, dimethylsulfoxide; ELAM-1, endotheUal leukocyte adhesion molecule- 1; NANA, N-acetylneuraminic acid; TFA, trifluoroacetic acid; Tris, tris (hydroxymethyl) aminomethane.
  • BSA bovine serum albumin
  • DEAE diethylaminoethyl
  • DMSO dimethylsulfoxide
  • ELAM-1 endotheUal leukocyte adhesion molecule- 1
  • NANA N-acetylneuraminic acid
  • TFA trifluoroacetic acid
  • Tris tris (hydroxymethyl) aminomethane
  • C-type calcium-type
  • Gal galactose
  • GlcN glucosamine
  • GalN galactosamine
  • GlcNAc N-acetylglucosamine
  • GalNAc N-acetylgalactosamine
  • Fuc fucose
  • Gal-6S galactose 6-sulfate
  • Glc ⁇ Ac-6S N-acetylglucosamine 6-sulfate
  • GlcU-S glucuronic acid monosulfate
  • HEN high endotheUal venule
  • HPAEC high- pH anion-exchange chromatography
  • N-acetyUactosamine high-acetyUactosamine.
  • ⁇ eu ⁇ Ac N-acetylneuraminic acid
  • SA siaUc acid
  • sialyl Lewis x or sLe x ⁇ eu5Ac ⁇ 2 ⁇ 3Gal ⁇ l ⁇ 4(Fuc ⁇ l ⁇ 3)Glc ⁇ Ac
  • Lewis x or Le x Gal ⁇ l ⁇ 4(Fuc ⁇ l ⁇ 3)GlcNAc
  • Lewis a or Le a Gal ⁇ l ⁇ 3(Fuc ⁇ l ⁇ 4(GlcNAc
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • Ugand is used to describe any molecular structure of any size or shape which can bind to an appropriate receptor.
  • a naturally occuring Ugand for a selectin usually has a polypeptide backbone with oUgosaccharide sugars which interact with the selectin.
  • Ugand may refer to any structure which can act in the desired fashion and have the desired effect.
  • the Ugand may have only an oUgosaccharide portion.
  • the Ugand may have an oUgosaccharide portion with a backbone composed of natural or synthetic polypeptides, natural or synthetic polymers, nonpeptide organic compounds, inorganic compounds, substrates, or other suitable backbone materials.
  • a size limitation is not inherent in the term Ugand unless specified. Materials.
  • D-[6- H]Glucosamine hydrochloride 32 Ci/mmol was purchased from Amersham Corp., and D-[2- H(N)]mannose (21.0 Ci/mmol) is from New England Nuclear (NEN). ConcanavaUn A-Separose and QAE-Sephadex is from Pharmacia. Biogel P-6 is from BioRad. Jack bean ⁇ -galactosidase and Jack bean ⁇ -N- acetylglucosaminidase were purchased from Sigma. Xanthomonas manihotis ⁇ - galactosidase is from New England Biolabs. Arthrobacter ureafaciens neuraminidase is from Boehringer.
  • DMEM Dulbecco's- modified Eagle's Medium
  • FBS heat inactivated fetal bovine serum
  • trypsin penicillin and streptomycin
  • Gibco Gibco.
  • SuppUes are from standard sources unless otherwise indicated.
  • This activity has therapeutic value in the prevention or treatment of various blood related diseases and disorders.
  • the compounds may be used to inhibit platelet leukocyte rosetting and leukocyte adhesion to endotheUum.
  • leukocyte includes such ceUs as neutrophils, basophils, eosinophils, monocytes and T-cell subsets. See, de Bnnjne-Admiraal, L.G., Blood, vol. 80. pp. 134-142 (Aug.
  • the compounds are thus important to and may be used in the preparation of, making and using diagnostic assays and kits. Many procedures known in the art may be used to demonstrate inhibition of platelet leukocyte rosetting and leukocyte adhesion, thus producing a diagnostic kit or assay.
  • cardiovascular disorders including restenosis foUowing percutaneous transluminal coronary angioplasty (PTCA), myocardial ischemia, stroke, thrombo-emboUsm, deep vein thrombosis, acceleration of thrombolysis, lung injury associated with cardiopulmonary bypass; rheumatoid arthritis, asthma, pulmonary hypersensitivity diseases, organ transplantation, particularly kidney, heart and Uver organs; shock (septic and hemorrhagic), multiple organ injury syndromes; thermal injury; autoimmune disease, multiple sclerosis; inflammatory bowel diseases; infectious diseases including; general inflammation, angiogenesis and cancer metastasis, particularly breast, colon and lung metastasis and neuroblastoma.
  • PTCA percutaneous transluminal coronary angioplasty
  • myocardial ischemia stroke
  • thrombo-emboUsm deep vein thrombosis
  • acceleration of thrombolysis lung injury associated with cardiopulmonary bypass
  • rheumatoid arthritis asthma, pulmonary hypersensitivity diseases, organ transplantation, particularly kidney,
  • oUgosaccharides should not be considered in isolation, but in conjunction with the protein to which they are attached. See Rademacher et al, Ann. Rev. Biochem. 57, 785-838 (1988).
  • the novel agents of the present invention thus have various potential therapeutic uses of which the foUowing are iUustrative: Treatment of toxic and cachectic syndromes where cytokines (e.g. TNF, tumor necrosis factor ) are impUcated. e.g. maUgnancy, pre-eclamptic toxaemia, septic shock, Uver disease, respiratory distress syndrome. Chemotherapy of immunosuppressed states, e.g.
  • maUgnancy-associated immune-suppression iatrogenic immune-suppression (i.e. drugs).
  • Use of glycoprotein (EP) or multivalent (EPC) oUgosaccharides e.g. treatment of patients with graft vs host reactions (transplants), treatment of patients with host vs graft reactions (transplants) and treatment of maUgnant disease via activation of TNF.
  • the compounds of this invention may be used to coat heart valves and joint implants, particularly hip, knee and temporal-mandibular, to prevent leukocyte attachment. Coating of these devices with oUgosaccharides would also involve a polymer preparation in which the oUgosaccharides were incorporated into the polymer and then bonded to the device.
  • compositions comprising the carbohydrates, oUgosaccharides, proteins, Ugands, fragments thereof or their formulations are provided by the present invention.
  • the compounds of the invention such as various Ugands having the carbohydrate structure described by this invention or described by the claims of this invention can be administered to a subject in need thereof to treat the subject by either prophylactically preventing inflammation and/or reUeving it after it has begun.
  • Various deUvery systems are known and can be used for therapeutic delivery of the compounds.
  • the Ugands are preferably administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a soUd carrier and/or IN. administration of a Uquid solution carrier.
  • Methods of administration include but are not Umited to intravenous routes.
  • the amounts of oUgosaccharides administered to adult humans will be in the range of about 0.1 to 20 mg/kg, i.v. for 4-14 days. A more preferred range would be 0.5 to 2.5 mg kg doseage level.
  • the formulation of choice can be accompUshed using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin ceUulose, magnesium carbonate, and the like.
  • Oral compositions may be taken in the form of solutions, suspensions, tablets, piUs, capsules, sustained release formulations, or powders.
  • the subject Ugand molecules may be administered directly in transdermal formulations with permeation enhancers such as DMSO.
  • treating inflammation shall mean preventing or ameUorating inflammation and/or symptoms associated with inflammation.
  • compositions of the instant invention wiU contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%.
  • the frequency of administration wiU be determined by the care given based on patient responsiveness.
  • Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials estabUshing dose response curves.
  • determining the dose of E or P selectin Ugands to be administered it must be kept in mind that one may not wish to completely block all of the E or P selectin receptors.
  • at least some of the white blood ceUs or neutrophils must be brought into the tissue in the areas where the wound, infection or disease state is occurring.
  • the amount of the E or P selectin Ugands administered as blocking agents must be adjusted carefully based on the particular needs of the patient while taking into consideration a variety of factors such as the type of disease that is being treated. It is beUeved that the Ugands or blocking agents of the present invention can be used to treat a wide range of diseases, including diseases such as rheumatoid arthritis and multiple sclerosis.
  • the compositions of the invention should be appUcable to treat any disease state wherein the immune system turns against the body causing the white cells to accumulate in the tissues to the extent that they cause tissue damage, swelling, inflammation and/or pain.
  • the inflammation of rheumatoid arthritis is created when large numbers of white blood ceUs quickly enter the joints in the area of disease and attack the surrounding tissues.
  • Formulations of the present invention might also be administered to prevent the undesirable after effects of tissue damage resulting from heart attacks.
  • a heart attack occurs and the patient has been revived, such as by the appUcation of anticoagulants or thrombolytic (e.g., tPA)
  • thrombolytic e.g., tPA
  • the endotheUal lining where a clot was formed has often suffered damage.
  • the antithrombotic has removed the clot, the damaged tissue beneath the clot and other damaged tissue in the endotheUal lining which has been deprived of oxygen become activated.
  • the activated endotheUal ceUs then express the E or P selectin within hours of the ceUs being damaged.
  • the selectin then extends into the blood vessels where they adhere to glycoprotein Ugand molecules on the surface of white blood ceUs.
  • Large numbers of white blood ceUs are quickly captured and brought into the tissue surrounding the area of activated endotheUal ceUs, resulting in inflammation, sweUing and necrosis which thereby decreases the likelihood of survival of the patient.
  • formulations of the invention In addition to treating patients suffering from the trauma resulting from heart attack, patients suffering from actual physical trauma could be treated with formulations of the invention in order to relieve the amount of inflammation and sweUing which no ⁇ naUy result after an area of the body is subjected to severe trauma.
  • Other disease states which might be treatable using formulations of the invention include various types of arthritis and adult respiratory distress syndrome.
  • the Ugand molecules of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions.
  • the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciUary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not Umited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • the Ugand molecules of the instant invention may also be administered as injectables.
  • injectable compositions are prepared as Uquid solutions or suspensions; soUd forms suitable for solution in, or suspension in, Uquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in Uposome vehicles.
  • the Ugands in the form of glycoUpids and carbohydrates, or more specifically compounds described in CHART, 1 can be mixed with compatible, pharmaceutically acceptable excipients.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiUary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiUary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack PubUshing Company, Easton, PA, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the Ugand molecules adequate to achieve the desired state in the subject being treated.
  • the various Ugand compounds of the present invention can be used by themselves or in combination with pharmaceutically acceptable excipient materials as described above. However, the Ugand compounds of the invention can be made as conjugates wherein the compounds of the invention are linked in some manner to a label.
  • the Ugand compounds of the invention act as biochemical delivery systems for the label so that a site of inflammation can be detected.
  • the Ugand molecules of the invention could also be used as laboratory probes to test for the presence of E or P selectin in a sample. Such probes are preferably labeled such as with a radioactive label.
  • Ligands in the form of glycoproteins having O-linked oUgosachharides which bind to E and P selectin are disclosed. More specificaUy, this invention relates to carbohydrate Ugands which bind to E and or P selectin and to compositions containing such Ugands which are useful in (a) determining the presence of E and or P selectin, (b) assaying for areas of inflammation, and (c) relieving inflammation by blocking the effects of E and or P selectin.
  • E and P selectin recognizes the oUgosaccharide structures on a mucin-like Ugand glycoprotein isolated from HL-60 ceUs in addition to a closely related protein, isolated from normal human leucocytes and developed the present invention.
  • Ligand molecules capable of binding to and interrupting the biological chain of events associated with E and or P selectin are disclosed.
  • the Ugand molecules act as biochemical blocking agents by binding to E and or P selectin and preventing circulating neutrophils from binding to stimulated endotheUal cells, thereby preventing a primary event of the inflammatory response.
  • the Ugands are in the form of O-Unked oUgosaccharides which can be labeled, bound to anti-inflammatory drugs and or formulated to provide: (1) compositions useful in assaying a sample for the presence of E and or P selectin, (2) compositions useful in detecting the site of inflammation in a patient, and (3) pharmaceutical composition useful in treating inflammation (or treating the inflammatory symptoms of certain diseases) or (4) blocking other effects involving the interaction of E and or P selectin and circulating neutrophils.
  • compositions which are useful in treating, preventing and/or alleviating any undesirable effects resulting from the interaction of E and or P selectin receptors and circulating neutrophils.
  • Such compositions are comprised of an inactive ingredient in the form of a pharmaceutically acceptable excipient material and a compound capable of binding to an E and or P selectin, the compounds having the general structural formula disclosed in CHART 1.
  • a primary object of the invention is to provide an E and or P selectin ligand in a useful formulation.
  • Another object is to provide a composition comprising an E and or P selectin Ugand which can be used to assay for the presence of E and or P selectin in a sample.
  • Another object is to provide a pharmaceutical formulation containing an E and or P selectin ligand which is useful in treating inflammation.
  • Ugands are in the form of non-toxic oUgosaccharides, and/or derivatives and mimetics thereof, which effectively bind the E and or P selectin and thereby block neutrophils from binding to the receptors in numbers which result in inflammation and/or other adverse effects.
  • a feature of the present invention is that the Ugand can be labeled and the labeled Ugand used in an assay to detect the presence of E and or P selectin in a sample.
  • Other features of the invention include the abiUty of pharmaceutical formulations of the invention to relieve the inflammatory symptoms of a wide range of diseases which are characterized by the binding of excessive amounts of leucocytes to a site.
  • a blood vessel waU is lined internally with endothelial cells.
  • the endotheUal ceUs can be activated, causing the ceUs to express E and or P selectin. Both red blood cells and white blood ceUs flow in the blood vessel.
  • the white blood ceUs display carbohydrate Ugands which have chemical and physical characteristics which aUow the Ugands to bind to the receptors, i.e. the selectins. Once the Ugand binds to the receptor, the white blood ceU is brought through the vessel wall into the surrounding tissue.
  • the white blood ceUs brought into the surrounding tissue can have positive effects, such as fighting infection, and negative effects, such as inflammation.
  • the present inventors have isolated and characterized Ugands apart from their presence on the surface of white blood ceUs. These isolated Ugands adhere to E and or P selectin by themselves and can be formulated into pharmaceutical compositions, which when administered will effectively block the E and or P selectin and prevent the adhesion of a receptor connected to a white blood ceU.
  • pharmaceuticaUy effective amounts of Ugands, some but not all, of the white blood ceUs wiU not reach the surrounding tissue.
  • inflammation can be prevented and or alleviated. It is known that for an acute inflammatory response to occur, circulating neutrophils must bind to and penetrate the vascular waU and access the site of injury.
  • the isolated Ugands can be generally characterized by the following embodiments, either separately or in various combinations:
  • CHART 1 The O-linked oUgosaccharide structures recovered from the common Ugand for E and P-selectin, isolated from HL-60 ceUs, are also shown and summarized in CHART 1.
  • CHART 1 summarizes five types of structures, named TYPES A-E, two additional types of structures not drawn in CHART 1 may also be present on the Ugand, they are referred to as TYPES F and G and are discussed in the section below labeled Obvious Variants and Preferred Compounds.
  • TYPES A-E two additional types of structures not drawn in CHART 1 may also be present on the Ugand, they are referred to as TYPES F and G and are discussed in the section below labeled Obvious Variants and Preferred Compounds.
  • CHART 1 contains two numbers separated by a slash, in a Unkage designation, it indicates that this Unkage has not been unequivocally determined; " ⁇ " at some terminal sialic acids indicates that this position may or may not be conjugated with
  • oUgosaccharides may be referred to by their corresponding size (without siaUc acids), in glucose units (GUs).
  • GU is a size designation.
  • a 12.8 GU oUgosaccharide does not necessarily contain any glucose, rather it would be the same size as a glucose oUgomer containing 12.8 units.
  • CHART 1 shows, from top to bottom, first, one type of oUgosaccharide, sized to 12.8 GU, that contains two sialic acid residues, as shown, this oUgosaccharide is named, TYPE A or 12.8 GU.
  • CHART 1 Second from the top in CHART 1 is an oUgosaccharide, sized to 9.8 GU, that may be in two forms, that is, it contains either one or two sialic acid residues at the locations shown, this oUgosaccharide is named, TYPE B or 9.8 GU. Third, is an oUgosaccharide, sized to 6.3 GU, that contains either zero, one (in either position) or two sialic acid residues, as shown, and is named, TYPE C or 6.3 GU. Fourth, is an oUgosaccharide, sized to 3.5 GU, that has either zero, one, or two sialic acid residue, named TYPE D or 3.5 GU.
  • the position of this substitution on the oUgosaccharide (on the terminal galactose or on the reducing N- acetylgalactosmine) has not been determined.
  • AU the oUgosaccharides except GalNAc by itself are core 1 based structures of comparatively simple design.
  • the 12.8, 9.8 and 6.3 GU oUgosaccharides are aU Unear, unbranched poly-N- acetyllactosamine repeat structures with the only variation between them being the number of N-acetyUactosamine repeat units and the sialylation pattern.
  • the o-linked oUgosaccharide structures on the HL-60 Ugand shares the N-acetyUactosamine feature previously reported for Ugands for E- and P-selectin but lacks Lewis type substitutions.
  • the m ⁇ ijority of the larger (12.8, 9.8 and 6.3 GU) structures are sialylated, again consistent with the reported requirement for sialic acid for selectin binding (Table 3).
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g colon and mammaiy carcinomas. See, Springer, G.F. (1984) Science 224:1198; Springer, G.F. (1989) Mol Immunol. 26:1; and Orntoft, T.F. et al. (1990) Int. J. Cancer 45:666.
  • CHART 1 may cooperatively generate a high affinity binding domain on the Ugand.
  • the Ugand described herein has features which may be described as mucin like: It is extensively O-glycosylated; there are three or fewer N-Unked oUgosaccharides on the Ugand.
  • the O-linked oUgosaccharides appear to form a tight cluster.
  • the molecule is largely resistant to degradation by pronase or trypsin.
  • neutrophils possess surface molecules which are referred to herein as Ugands and which Ugands have an affinity for and bind to receptor molecules on the surface of endotheUal ceUs which receptors are referred to herein as E or P selectin.
  • the larger molecules extracted which act as Ugands do so because they possess a particular molecular configuration at a particular point on the molecule required for adhesion to the E or P selectin.
  • the experiments herein were the first to isolate and characterize groups of molecules which do adhere to E or P selectin and thereafter specifically identify and characterize the oUgosaccharide structures of those molecules.
  • CHART 1 The general structural formula disclosed in CHART 1 encompasses a number of compounds which include characteristic features of isolated compounds which can adhere to E or P selectin receptors. Variations on the structures may also be possible provided those variations do not alter structural and or charge-related characteristics which would interfere with the adhesion of the Ugand to E or P selectin.
  • the present invention encompasses not only the molecules disclosed in CHART 1, and Types F and G discussed below, but various equivalents thereof which equivalents have an affinity for and an abiUty to adhere to E and or P selectin to the same or a greater degree than molecules of CHART 1 adhere to E and or P selectin.
  • the characteristics of such additional molecules can, of course, be confirmed using the adhesion assay described herein and/or related assays which test the adhesion of a putative Ugand to E and or P selectin under conditions of controlled detachment force.
  • each of the five or seven types of oUgosaccharides on the Ugand molecule are not intended to represent the total of the carbohydrates on the ligand.
  • the mucin like character of the Ugand suggests that it contains a large number of oUgosaccharide structures.
  • a similar Ugand molecule (which has been cloned) contains 53 predicted sites for o-glycosidic glycosylation. Sako et al, (1993) CeU 75:1179; Elhammer et al., (1993) J. Biol. Chem. 268:10029.
  • the selectins may be comprised of any combination of the different types of structures in any number.
  • the E or P selectin When the Ugand binds with the receptor, the E or P selectin, it binds using a selected combination of carbohydrates. Some combination is certainly needed, the range can be from one to seven types of oUgosaccharide(s), with five types, A-E, being preferred.
  • Varki concluded that this type of interaction between the selectins and their Ugands appear to be the most likely; he also described how the mucin like features of some of the Ugand molecules described to date easfly could accomodate this type binding "patch.”
  • Varki A(1994) Proc. Natl. Acad. Sci. USA 91:7390.
  • the ligand described herein has features which may be described as mucin like: It is extensively O-glycosylated; significant amounts of N-linked oUgosaccharides are also present on the molecule. The O-Unked oUgosaccharides appear to form a tight cluster and the molecule is largely resistant to degradation by pronase or trypsin.
  • Examples of specific oUgosaccharide Ugand structures are hereby provided.
  • Also described and claimed are the polyvalent forms where the oUgosaccharide combinations repeat themselves any number of times
  • possibiUties A, AA, AAA, B, BB, BBB, C, etc.
  • the order of the oUgosaccharides may be important.
  • One embodiment would aUow 3 of 7 possible oUgosaccharides in 3 unique possible combinations, repeated any number of times. This would aUow 7 x (7x7) or
  • possibilites 343 possibilites. Some of those possibiUties are shown here. AAA, AAB, AAC, AAD, AAE, AAF, AAG,
  • HL-60 ceUs were labeled with H-glucosamine (50 ⁇ Ci/ml) in normal RPMI medium for 48 hours. Preparation of the P-selectin ligand.
  • a P-selectin affinity column (10 mg of purified human platelet P-selectin coupled onto 10 ml of Affi-Gel 15 resins) and a bovine serum albumin guard column were prepared as described by Ma et al, (1994) J. Biol. Chem. 269, 27739-27746.
  • the labeled ceUs were lysed with detergent as described by Moore et al. ((1992) J. CeU Biol. 118, 445-456) and the ceU lysates were appUed to the P-selectin affinity column through a BSA guard column in the presence of CaCl 2 .
  • Glycopeptides were prepared from the intact purified radiolabeled ligand molecule by digestion with Pronase (2 mg ml) in 100 mM Tris-HCl, pH 8.0, 20 mM CaCl 2 at 55°C for 4 hours.
  • FIGURE 7 Purification of -120 kD Ligand for P- and E-selectins HL-60 ceUs labeled with [3H]GlcNAc were lysed with Triton X-100 and separatedly loaded onto P-selectin affinity column or E-selectin receptor-globuUn affinity column in the presence of Ca . After extensive washing, the bound materials were eluated with EDTA and were subject to 7% SDS-PAGE (under reducing conditions) foUowed by autoradiography. Lane 1, the eluate from P-selectin affinity column; lane 2, eluates from E-selectin receptor-globuUn affinity column. Enzyme Treatments.
  • Digestion with Arthrobacter ureafaciens neuraminidase (2 U/ml) was done in 100 mM sodium acetate buffer, pH 5.0 for 18 hours; jack bean ⁇ -galactosidase (400mU/ml) was in 50 mM citrate-phosphate, pH 4.6, for 48 hours; jack bean ⁇ -N- acetylglucosaminidase (4.15 mU/ml) was in 50 mM sodium citrate, pH 5.6, for 18 hours; Xanthomonas manihotis ⁇ -galactosidase (167 U/ml) was in 50 mM sodium citrate, pH 4.5, for 18 hours; almond ⁇ -fucosidase (0.2 mU/ml) was in 50 mM sodium citrate, pH 5.0, for 18 hours. AU enzyme digestions were carried out at 37° C under toluene atmosphere.
  • Glycopeptides prepared by digestion of the intact [ 3 H]-glucosamine labeled Ugand molecule with pronase, were separated on ConcanavaUn A-Sepharose. This resulted in essentially all of the appUed radioactivity eluting in the break-through fractions from the column, insignificant amounts of radioactivity was eluted by the hapten sugars, ⁇ -methylmannoside and ⁇ -methylglucoside, suggesting that the Ugand molecule does not contain any high-mannose, hybrid or bi-antennary complex type oUgosaccharides.
  • the total O-Unked oUgosaccharides were cleaved from the purified [ H]- glucosamine labeled Ugand molecule by mild alkaline sodium borohydride treatment.
  • the released oUgosaccharides were then fractionated on QAE-Sephadex and charged structures were eluted with step-wise increases NaCl concentration ( Figure 1 and Table 1).
  • FIGURE 1 Separation of labeled O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin, on QAE-Sephadex. Charge separation of the O-linked oligosaccharide structures.
  • Re-chromatography of the charged fractions after digestion with Arthrobacter ureafaciens neuraminindase, resulted in the majority of the radioactivity in both charged fractions eluting in the QAE-Sephadex column break-through fractions, and the remaining portion (24% and 25% for the 70 mM and 20 mM eluates, respectively) eluted in the fractions containing material with one charge.
  • the radioactive material in these neuraminidase resistant, charged fractions was identified by paper chromatography.
  • FIGURE 2 shows separation of the complete mixture of O-Unked oUgosaccharides on the molecule.
  • the mixture of sugars separated into five peaks eluting in the positions of oligomers composed of 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units (GU), respectively.
  • TABLE 2 shows the distribution of radioactivity in the five peaks on the chromatogram.
  • FIGURE 3 shows a size separation of the portions of the structures containing none, one and two charges, respectively. See, TABLE 3, FIGURE 2, and FIGURE 3.
  • FIGURE 2 Separation of the O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin on size -exclusion chromatography.
  • Total O-Unked oUgosaccharides were isolated from the purified in vivo [ H]- glucosamine labeled E/P-selectin Ugand, siaUc acids were removed by neuraminidase digestion and the resulting neutral oUgosaccharides were separated on a GlycoMap column.
  • the five radioactive peaks (from right to left) on the chromatogram have elution volumes corresponding to glucose oUgomers composed of 12.8, 9.8, 6.3. 3.5 and 2.5 units respectively.
  • FIGURE 3 Size-exclusion separation of neutral and charged O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin.
  • the total [ H]-glucosamine labeled O-Unked oUgosaccharides from the H -60 Ugand were fractionated on QAE-Sephadex as described in FIGURE 1.
  • FoUowing removal of sialic acid from the charged oUgosaccharides by neuraminidase digestion, neutral oUgosaccharides (Panel A) and oUgosaccharides containing one (Panel B) and two siaUc acids (Panel C) were fractionated separately on the GlycoMap column. Only the portions of the chromatograms containing measurable amounts of radioactivity are shown in the figure.
  • FIGURE 4 shows the sequential degradation of the largest (12.8 GU) oUgosaccharide. Initially, this structure was digested sequentially with jack bean ⁇ - galactosidase and jack bean ⁇ -N-acetylglucosaminidase; this resulted in a one and two GU shift in elution volume, respectively.
  • a 2 GU radioactive digestion product was released by the ⁇ -N-acetylglucosaminidase digestion. These are the predicted results for the removal of one terminal N-acetyUactosamine unit (FIGURE 4, top panel). The released radioactivity was identified as N-acetylglucosamine by paper chromatography. The product from these two digestions eluted at 9.8 GU (FIGURE 4, top panel). As is apparent from FIGURE 4, the first ⁇ -N-actylglucosaminidase digestion of the 12.8 GU oUgosaccharide did not result in degradation of all the radiactive material in this peak; some radioactivity also eluted at 11.8 GU after digestion.
  • FIGURE 4 Sequential exoglycosidase digestion of an O-Unked 12.8 GU oUgosaccharide isolated from the HL-60 common Ugand for E- and P-selectin.
  • the 12.8 GU oUgosaccharide isolated from fractionation of the total O-Unked oUgosaccharides on the HL-60 Ugand was subjected to sequential exoglycosidase digestion and re-chromatography on the GlycoMap column as described in the Experimental Procedures.
  • the elution positions of the glucose oUgomer standards are indicated by the arrows at the top of the chromatograms; the numbers identifies the size (in GU) of the individual standard molecules.
  • the key identifies elution profiles obtained for the undigested molecule and for products from digestions with jack bean ⁇ -N-acetylglucosaminidase and jack bean ⁇ -galactosidase, respectively.
  • E elution volume (ml); R, radioactivity.
  • the two GU fragments released by the ⁇ -N-acetylglucosaminidase digestions and the final 3.5 GU degradation product were identified as N-acetylglucosamine and Gal ⁇ l,3GalNAc-ol, respectively, on paper chromatography (FIGURE 5, panels A and B; the chromatography standards are: 1, Gal ⁇ l,3GalNAc-ol and 2, GalNAc-ol).
  • FIGURE 5 Separation of radioactive fragments obtained from exoglycosidase digestion of the 12.8 GU O-linked oUgosaccharide on the HL-60 common Ugand for E- and P-selectin.
  • the 2 and 3.5 GU fragments generated by exoglycosidase digestion of the 12.8 GU oUgosaccharide were separated on descending paper chromatography in pyridine-ethyl acetate-glacial acetic acid-water (5:5:1:3).
  • Panels A and B shows separations of the 2 and 3.5 GU exoglycosidase digestion fragments, respectively.
  • the standards were 1, Gal ⁇ l,3GalNAc-ol; 2, GalNAc-ol.
  • Digestion of the 6.3 GU oUgosaccharide with jack bean ⁇ -galactosidase resulted in a one GU shift in elution volume on the column, indicating the removal of one terminal galactose.
  • Digestion of the 6.3 GU oUgosaccharide with Xanthomonas manihotis ⁇ -galactosidase did not result in a shift in elution volume indicating that the terminal galactose on this structure is linked 1,4 to N- acetylglucosamine.
  • the two smaUer O-Unked structures on the Ugand molecule were identified on paper chromatography.
  • the 3.5 GU oUgosaccharide co-migrated with the Gal ⁇ l, 3GalNAc-ol standard and digestion of this structure with jack bean and bovine testis ⁇ -galactosidase followed by re-chromatography on paper, revealed its identity with this standard.
  • FIGURE 6 Identification of the 3.5 and 2.5 GU O-Unked oUgosaccharides on the HL-60 common Ugand for E- and P-selectin.
  • a Numbers in parenthesis represents percent of total identified structures.

Abstract

A specific ligand for E- and P-selectin was isolated from in vivo[3H]-glucosamine labeled HL-60 cells by a combination of weat germ agglutinin agarose and E- or P-selectin rIg-agarose chromatography. The total O-linked oligosaccharides on the purified molecule were released by alkaline hydrolysis in the presence of NaBH¿4? and analyzed by ion-exchange, size-exclusion and paper chromatography in combination with specific exoglycosidase digestions. Various derived ligands and carbohydrates are claimed.

Description

THE OLIGOSACCHARIDE STRUCTURE OF A LIGAND FOR E AND P SELECTTN
Field of the Invention This invention relates to the field of cell adhesion biology, E and P selectin and the oligosaccharide structure of a ligand for E and P selectin.
Information Disclosure Hemmerich, Stefan, et. al., "Identificaiton of the Sulfated Monosaccharides of GlyCAM-1, an Endothelial-Derived Ligand for L-Selectin," (1994) Biochemistry, Vol. 33, pp. 4820-4829.
Hemmerich, Stefan, and Rosen, Steven, "61- Sulfated Sialyl Lewis x Is a Major Capping Group of GlyCAM-1." (1994) Biochemistry, Vol. 33, pp. 4830-4835. Lasky, Laurence A., et al., US 5,304,640, issued 19 April 1994. "DNA Sequence Encoding a Selectin Ligand." This patent claims DNA sequences of a possible selectin ligand.
Lasky, Laurence A., et al., WO 94/11498-A1, issued to University of Oklahoma State, "New glycoprotein for P-selectin." This application attempts to characterize some of the amino acids and glycoproteins for a possible P-selectin ligand. Brandley, et al., US 5,143,712, issued 1 Sept. 1992; US 5,211,936, issued 18
May 1993; and US 5,211,937 issued 18 May 1993. "Method of Determining a Cite of Inflammation Utilizing ELAM-1 Ligands."
Varki, Ajit, (1994) Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 7390-7397. "Selectin Ligands" Reviews selectin ligand literature and discusses hypothesis that specificity may involve recognition of clustered saccharide patches.
Imai, Y., et al. (1991) J. Cell Biol. 113:1213. Precipitation from mouse mesenteric lymph nodes of a 50kD molecule and a 90kD molecule, both thought to be L-selectin ligands.
Lasky L.A., et al. (1992) Cell 69:927. Molecular cloning shows that the 50kD protein called "GlyCAM-1" is a mucin-like molecule.
Baumhueter, S., et al. (1993) Science 262:436. The protein core of a 90 kD ligand for L-selectin is identical to the vascular sialomucin CD34.
Levinovitz, A., et al. (1993) J. Cell Biol. 121:449-459. A 150kD glycoprotein ligand for E-selectin was isolated from a mouse neutrophil progenitor cell line using an E-selectin-IgG chimaera.
Moore, KL., et al. (1992) J. Cell. Biol. 118:445-456. Partial characterization of a P-selectin ligand.
Lenter, M., et al. (1994) J. Cell Biol. 125:471. Partial description of two molecules, 150 kD and 160 kD, which are monospecific ligands for E- and P-selectin, respectively, as well as two other molecules with molecular masses of 230 kD and 130 kD, respectively, that interact with both E- and P-selectin.
Tiemeyer et al. ,(1991) Proc. Natl. Acad. Sci. USA 88:1138, purification of several myeloid-derived glycolipids have ligand activity for E-selectin transfected cells in a solid-phase assay.
Larsen et al. (1990) Cell 63:467. The Lex determinant (Galβl,4(αl,3Fuc)GlcNAc) is an important element of the P-selectin ligand on myeloid cells.
Polley et al. (1991) Proc. Natl. Acad. Sci. USA 88:6224. The ligand for P-selectin may be the same or very similar to that for E-selectin.
Sako, et al., (1993) Cell 75:1179.
Varki, A. (1994) Proc. Natl. Acad. Sci. USA, 91:7390. The oligosaccharide structures on the HL-60 ligand shares the N-acetyllactosamine feature previously reported for ligands for E- and P-selectin but lacks Lewis type substitutions. Springer, G.F. (1984) Science 224:1198; Springer, G.F. (1989) Mol Immunol.
26:1; and Orntoft, T.F. et al. (1990) Int. J. Cancer 45:666. Two structures, frequently referred to as Tn (GalNAc) and T (Galβl,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g. colon and mammary carcinomas. El-hammer et al., (1993) J. Biol. Chem. 268:10029. van Halbeek, H. et al. (1982) Eur. J. Biochem. 127:7.
A ano, J. et al. (1991) J. Biol. Chem. 266:11461.
Hirano, T. et al. (1993) Eur J. Biochem. 214:763. References cited herein are incorporated by reference. Background of the Invention
Protein-protein interactions in cell recognition have been recognized for some time, but only recently has the role of carbohydrates in cell recognition been seriously investigated. See Brandley, B.K., and Schnaar, R.L. (1986) J. Leuk. Biol. 40:97; Sharon, N., and Lis, H. (1989) Science 246:227. In recent years, a major focus for researchers in the area of carbohydrate chemistry has been the glycoproteins. Glycoproteins constitute a distinct population of molecules synthesized by eucaryotic cells. A common feature of these proteins is that they all contain a covalently linked carbohydrate moiety, the size of which can range from one to more than twentyfive monosaccharides. Two common types of linkages for the attachment of oligosaccharide structures to glycoproteins are found in N-linked oligosaccharides, where the oligosaccharide structures are attached by an amide bond between the reducing N-acetylglucosamine on the oligosaccharide and an asparagine residue on the protein, and mucin type O-linked oligosaccharides which are conjugated via an ether bond between the reducing N-acetylgalactosamine on the oligosaccharide and a serine or threonine residue on the protein. Other types of linkages between carbohydrate moieties and proteins do exist but these have little relevance for this discussion.
Several characteristics are shared by N-linked and mucin type O-linked oligosaccharides. Some similarities are: the majority of their component monosaccharides are the same; both types of structures, when synthesized by most untransformed mammalian cells, carries terminal sialic acids; both types of structures can carry blood group type substitutions such as Sialyl Lewis1 (see below); and both types of oligosaccharides can contain poly-N-acetyllactosamine repeat structures. These observations, also suggested in the literature, indicate there may be a functional overlap between some N-linked and mucin type O-linked oligosaccharides. Fukuda, M. (1992) and Olden, K, Yeo, T.-K, and Yeo, -T. (1992) in Glycocoiyugates, Allen, H.J., and Kisailu, E.C., Eds., pp. 379-401 and 403- 420, Marcel Dekker Inc., New York. On the other hand, several functions and characteristics of O-linked oligosaccharides are distinct from those of N-linked structures, Jentoft, N. (1990) Trends. Biochem. Sci. 15:291, and the biosynthetic pathways for the two types of structures are quite different. Kornfeld, R. and Kornfeld, S. (1985) Ann. Rev. Biochem. 54:631; Schachter, H., and Brockhausen, 1.(1992) in Glycoconjugates, Allen, H.J., and Kisailu, E.C., Eds., pp. 263-332, Marcel Dekker Inc., New York. Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. O-linked chains are of interest in view of the evidence that cell surface glycoproteins containing "clusters" of this type of substitution can form highly extended and rigid structures. Jentoft, N. (1990) Trends. Biochem. Sci. 15:291-294. Thus, these molecules are ideally positioned to perform recognition functions. A quite diverse number of oligosaccharide structures can be created through the activities of a limited number of glycosyltransferases. Hence, these structures can be generated with relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins, so called lectins, on interacting cells have been described Dodd, J., and Jessel, T.M., (1985) J. Neurosci., 5:3278; Regan, L.J., et al. (1986) Proc. Natl. Acad. Sci. USA, 83:2248; Constantine-Paton, M., et al. (1986) Nature, 324:159; and Tiemeyer, M., et al. (1989) J. Biol. Chem., 263:1671.
Animal lectins have been divided into three groups: C-type (Ca -dependent) lectins, S-type (soluble) lectins and P-type lectins. Drickamer, K. (1993) Annu. Rev. Cell Biol. 9:237-264. An important group of C-type lectins are the selectins. The selectins have an important function in leucocyte extravasation at sites of inflammation.
Leukocytes (white blood cells) are involved in the protection of the body against various microbial infections as well as diseases such as cancer. However, when white blood cells adhere to endothelial cells on the lumenal site of blood vessels, migrate to body tissues and accumulate there, they often cause damage, swelling, pain, and inflammation. For example, rheumatoid arthritis occurs when white blood cells enter the joints and attack the tissues. It is believed that several molecules on white blood cells recognize their receptors on the surface of the endothelial cell, allowing them to migrate through the blood vessel wall to the site of inflammation. These cell surface molecules on leukocytes and endothelial cells are called cell adhesion receptors. It is believed that if the recognition between these cellular receptors can be blocked, the inflammatory response can be reduced or eliminated. Different adhesion receptors are presented on the endothelial cell surface at different times and may be recognized by different types of white blood cells. It is possible that two or more receptors may work together to perform this task. Several molecules are already known to act in this fashion.
The recruitment and extravasation of circulating leukocytes from the bloodstream across the endothelium into the target tissue, e.g. a site of injury or infection, are essential steps in the early response of inflammation. These processes are initiated by at least three families of cell surface adhesion molecules: the integrins, the immunoglobulin superfamily and the selectins. Springer, T.A. (1990) Nature 346:425; Hynes, R. O. and Lander, AD. (1992) Cell 68:303.
The selectins are a family of leukocyte-endothelial cell adhesion molecules, also referred to as LEC-CAM:s (Lectin EGF Complement regulatory-Cell Adhesion Molecule). Inflammation: Basic Principles and Clinical Correlates, Second Edition. J. I. Gallin et al, Eds, Raven Press, Ltd. NY 1992, pp 407-419. Selectin is the term favored by investigators working in the field. Bevilacqua M.P., et al. (1991) Cell 67:233. Selectins are generally believed to be primarily responsible for the initial interaction, called "rolling", of the leucocyte with the venular endothelium upon physiological and pathological stimuli, i.e. the earliest interaction of circulating leukocytes with endothelium of small vessel walls. Lawrence, M.B. and Springer T.A. (1990), Cell 65:859-873. The word leukocytes includes such cells as neutrophils, basophils, eosinophils, monocytes and T-cell subsets, de Bnujne-Admiraal, L.G. (1992) Blood 80:34.
The selectin family, so far, consists of three cell surface membrane proteins. Lasky, L.A. (1992), Science 258:964; McEver, R.P., (1991) Thromb. Heamostasis 66:80. L-selectin is a constitutively expressed lymphocyte homing receptor (a.k.a. peripheral lymph node homing receptor (pnHR), in a majority of leukocytes. L- selectin is also known as: LECAM-1, LEC-CAM-1, LAM-1, gp90MEL, gplOOMEL, gpllOMEL, MEL-14, MEL- 14 antigen, Leu-8 (antigen), Ly-22 (antigen), TQ1 (antigen) and DREG. 56 (antigen), but the term "L-selectin" is preferred. E- selectin, also known as ELAM-1, LECAM-2, and LEC-CAM-2, is a transient cytokine-inducible endothelial cell surface molecule which interacts with neutrophils, monocytes and memory T-cells. P-selectin, also known as LEC-CAM-3, LECAM-3, CD-62, GMP-140, and PADGEM, is rapidly expressed on the plasma membrane of endothelial cells and platelets during cellular activation and degranulation; it serves as a receptor for neutrophils and monocytes.
Structurally, all selectins contain an NH2-terminal extracellular carbohydrate recognition domain (CRD) composed of 118 amino acid residues which is homologous to other C-type lectins, followed by a epidermal growth factor (EGF)- like domain, a variable number of complement regulatory protein-like repeats (different for different selectins), a transmembrane domain and a cytoplasmic tail. Functionally, all selectins mediate Ca -dependent cell-cell contact by binding to oligosaccharide ligands on opposing cells.
The selectins are known to interact with the oligosaccharide structures on other glycoproteins, called "selectin ligands," which are expressed on the surface of their opposing cells. Immunoprecipitation experiments have shown that the construction of an L-βelectin-IgG chimaera provides a compound that can be used to isolate selectin ligands. A 50kD molecule and a 90kD molecule, both thought to be L-selectin ligands, has been specifically precipitated from mouse mesenteric lymph nodes using this type of compound. Imai, Y., et al. (1991) J. Cell Biol. 113:1213. Molecular cloning has demonstrated that the 50kD protein is a mucin-like molecule, and it is referred to as "GlyCAM-1." Lasky L.A., et al. (1992) Cell 69:927. Recent work on the 90 kD ligand has revealed that the protein core of this molecule is identical to the vascular sialomucin CD34. Baumheuter, S., et al. (1993) Science 262:436.
A ligand has also been partially characterized for E-selectin. A 150kD glycoprotein was isolated from a mouse neutrophil progenitor cell line using an E- selectin-IgG chimaera. Levinovitz, A., et al. (1993) J. Cell Biol. 121:449-459. See also Brandley et al., US 5,143,712, US 5,211,936, and US 5,211,937 patents. A P- selectin ligand has similarly been identified. A «120kD glycoprotein from human neutrophils has been identified in a 125I-P-selectin blotting assay and by P-selectin affinity chromatography of H-glucosamine-labeled HL-60 cell extracts. Moore, K.L., et al. (1992) J. Cell. Biol. 118:445-456.
In addition, ligands for balh E- anύ P-selectin have been described on mouse myeloid cells. Two molecules, 150 kD and 160 kD, which are monospecific ligands for E- and P-selectin, respectively, as well as two other molecules with molecular masses of 230 kD and 130 kD, respectively, that interact with both E- and P-selectin. Lenter, M., et αl. (1994) J. Cell Biol. 125:471.
The finding that simple monomeric sugars, such as mannose-6-phosphate (M6P) and fructose- 1-phosphate, can block the interactions of murine and human lymphocytes with HEN of peripheral lymph nodes (pin) (Stoohnan et αl. (1993) J. Cell Biol. 96:722 ; Stoohnan et αl. (1994) J. CeU Biol. 99:1535; Stoohnan et αl. (1987) Blood 70:1842) suggest that the endothelial ligand recognized by L-selectin is carbohydrate-based. Furthermore, in a series of experiments, Rosen and colleagues demonstrated that the homing receptor-dependent binding of lymphocytes to pin HEN was abolished by either in vitro or in vivo treatment with broad spectrum sialidases. -Rosen et al. (1985) Science 228:1005; Rosen et al. (1989) J. Immunol. 142:1895. This strongly suggests that terminal sialic acids are a critical elements for recognition. These investigators also presented data suggesting that the oligosaccharide structures on the L-selectin ligand were sulfated and that sulfation was important for selectin recognition (compare below).
Several laboratories, using a wide range of approaches, have concluded that a blood group oligosaccharide structure known as sialyl Lewis* (sLex; NeuAcα2,3Galβl,4(Fuc cd,3)GlcNAc) and some related structures, can function as ligands for E-selectin. Lowe et al. , (1990) Cell 63:475, transfected non-myeloid cells with an αl,3/4-fucosyltransferase and generated ligand activity for E-selectin, which correlated with the expression of the sLex determinant. Goeltz et al., (1990) Cell 63:1349, identified and cloned an αl,3- fucosyltransferase that appeared to be involved in the synthesis of an ELAM-1 ligand in myeloid cells. Using more direct approaches, Phillips et al, (1990) Science 250:1130, and Walz et al. , (1990) Science 250:1132, were able to show inhibition of E-selectin dependent adhesion with either sLex-containing glycocoηjugates or antibodies to sLex.
Tiemeyer et al ,(1991) Proc. Natl. Acad. Sci. USA 88:1138, purified several myeloid-derived glycolipids that had ligand activity for E-selectin transfected cells in a solid-phase assay. Mass spectroscopic analysis of the purified, E-selectin binding glycolipid revealed that the minimal structure necessary for activity was a sialylated lactosamine with a second internal N-acetyllactosamine unit containing an αl,3-linked fucose on the N-acetylglucosamine (CD65). Tiemeyer concluded, like other studies involving sLez type substitutions, both the sialic acid and fucose were essential for binding activity of the putative ligand. Other researchers have concluded that fucose, sulfate and sialic acid are present in the 0-linked oligosaccharides of E-selectin ligands and that fucose, like sialic acid, is required for full ligand activity. See, Lasky, et al. US patent 5,304,640. Finally, Hemmerich and co-workers recently demonstrated that 6-sulfated sLex is a major capping group on the L-selectin ligand GlyCAM-1. Hemmerich, S. et al. (1994) Biochemistry 33:4820; Hemmerich, S. and Rosen, S.D. (1994) Biochemistry 33:4830.
Progress has also been made in the identification of ligands for P-selectin. There is a possibility that the ligand for P-selectin is the same or very similar to that for E-selectin, especially since both selectins bind to a very similar spectrum of cell types. Polley et al. (1991) Proc. Natl. Acad. Sci. USA 88:6224. The remarkable 96/09309 PCI7US95/11401
homology in selectin structures as well as the already demonstrated similarities in the ligands suggest that these molecules will contain related and yet subtly different structures. All of the references and articles cited herein are incorporated by reference. The present inventors, in one aspect of their invention, have now isolated a common ligand for E and or P-selectin and they have identified the oligosaccharide portions of that ligand.
SUMMARY OF INVENTION The present inventors report the structure of the O-linked oligosaccharides on a common ligand for E and P-selectin. This molecule contains a heavy preponderance of O-linked oligosaccharides and fewer N-linked oligosaccharides. Contrary to the reported findings for other ligands for E and/or P-selectin, the present inventors have found that the O-linked oligosaccharide structures on the ligand identified herein contains no Lex, Lea, sLex or sLea type substitutions. Surprisingly, the o-linked oligosaccharide structures on this ligand have little or no fucose. The ligand described herein also contains little or no sulfated oligosaccharides. Thus, the present inventors describe a truly unique ligand.
One object of the present invention is to provide a method for the purification of a selectin ligand. Another object of the invention is to provide a purified selectin ligand, specifically a common ligand for E and P-selectin. A further object of the present invention is to identify the 0-linked oligosaccharide structures on this ligand. Another aspect is to enable the preparation of glycosylation variants of selectin ligands, not otherwise found in nature. In another aspect, the invention provides a method of designing selectin inhibitors, mimicking carbohydrate based determinants of the selectin ligands. These and further objects of the present invention, discussed herein, will be apparent to one skilled in the art after studying this document.
The purified ligand, or fragments thereof, as well as carbohydrate and polypeptide components of the ligand, or antibodies to the ligand or to fragments thereof, can be used as inhibitors of binding of P- or E-selectin, or both, and in diagnostic assays and kits.
This invention encompasses those isolated molecules, fragments, combinations and variations thereof which adhere to E and or P selectin. In addition to their ability to adhere to E and or P selectin, in addition to their mucin type O-linked oligosaccharides, in addition to their lack of fucosylated o-linked oligosaccharides, in addition to their lack of Lex or a or sLex or a , in addition to their lack of sulfated oligosaccharides, the isolated ligands can be generally characterized by the following embodiments, which may occur either separately or in various combinations and either in a free state or attached to a substrate.
(1) The monosaccharide GalNAc.
(2) The disaccharide Galβl, 3GalNAc.
(3) The disaccharide Galβl, 3GalNAc substituted with, zero or one sialic acid, at the 3 on the Gal and, zero or one sialic acid linked to GalNAc. (4) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc.
(5) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with one sialic acid, the substitution being at either the 3 position of the terminal (non-reducing) Gal Q∑. linked to the reducing GalNAc.
(6) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with two sialic acids, one sialic acid is at the 3 position of the terminal (non- reducing) Gal and one sialic acid is linked to the 6 position of the reducing GalNAc.
(7) The hexasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with one sialic acid at either the 3 position of the terminal (non-reducing) Gal fir at the reducing GalNAc. (8) The hexasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 4GlcNAcβl, 3/6Galβl,
3GalNAc substituted with two sialic acids, one sialic acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is linked to the 6 position of the reducing GalNAc.
(9) The octasaccharide Galβl, 4GlcNAcβl, 3/6 Galβl, 4GlcNAcβl, 3/6Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with two sialic acids, one sialic acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is linked to the reducing GalNAc.
Brief Description of the Drawings Figure 1. Separation of labeled O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin, on QAE-Sephadex.
Figure 2. Separation of the O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin on size -exclusion chromatography. Figure 3. Size-exclusion separation of neutral and charged O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin. Figure 4. Sequential exoglycosidase digestion of an O-linked 12.8 GU oligosaccharide isolated from the HL-60 common ligand for E- and P-selectin.
Figure 5. Separation of radioactive fragments obtained from exoglycosidase digestion of the 12.8 GU O-linked oligosaccharide on the HL-60 common ligand for E- and P-selectin.
Figure 6. Identification of the 3.5 and 2.5 GU O-linked oligosaccharides on the HL-60 common Ugand for E- and P-selectin.
Figure 7. Purification of the approximately 120 kD Ligand for P- and E- selectins. Additional Description of the Invention and the Preferred Embodiments Definitions, Abbreviations and Sources of Materials Some standard abbreviations used in connection with the present invention include: BSA, bovine serum albumin; DEAE, diethylaminoethyl; DMSO, dimethylsulfoxide; ELAM-1, endotheUal leukocyte adhesion molecule- 1; NANA, N-acetylneuraminic acid; TFA, trifluoroacetic acid; Tris, tris (hydroxymethyl) aminomethane.
Other abbreviations: C-type, calcium-type; Gal, galactose; GlcN, glucosamine; GalN, galactosamine; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Gal-6S, galactose 6-sulfate; GlcΝAc-6S, N-acetylglucosamine 6-sulfate; GlcU-S, glucuronic acid monosulfate; HEN, high endotheUal venule; HPAEC, high- pH anion-exchange chromatography; N-acetyUactosamine.
Other abbreviations: ΝeuδAc, N-acetylneuraminic acid; SA, siaUc acid; sialyl Lewis x or sLex, Νeu5Acα2→3Galβl→4(Fucαl→3)GlcΝAc; Lewis x or Lex, Galβl→4(Fucαl→3)GlcNAc; Lewis a or Lea, Galβl→3(Fucαl→4(GlcNAc; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
With the exception of fucose, which is in the L-configuration, all sugars are in the D-configuration. The term "Ugand" is used to describe any molecular structure of any size or shape which can bind to an appropriate receptor. A naturally occuring Ugand for a selectin usually has a polypeptide backbone with oUgosaccharide sugars which interact with the selectin. Here the term Ugand may refer to any structure which can act in the desired fashion and have the desired effect. The Ugand may have only an oUgosaccharide portion. More preferably, the Ugand may have an oUgosaccharide portion with a backbone composed of natural or synthetic polypeptides, natural or synthetic polymers, nonpeptide organic compounds, inorganic compounds, substrates, or other suitable backbone materials. A size limitation is not inherent in the term Ugand unless specified. Materials.
D-[6- H]Glucosamine hydrochloride (32 Ci/mmol) was purchased from Amersham Corp., and D-[2- H(N)]mannose (21.0 Ci/mmol) is from New England Nuclear (NEN). ConcanavaUn A-Separose and QAE-Sephadex is from Pharmacia. Biogel P-6 is from BioRad. Jack bean β-galactosidase and Jack bean β-N- acetylglucosaminidase were purchased from Sigma. Xanthomonas manihotis β- galactosidase is from New England Biolabs. Arthrobacter ureafaciens neuraminidase is from Boehringer. Almond α-fucosidase is from Oxford Glycosystems. Dulbecco's- modified Eagle's Medium (DMEM), heat inactivated fetal bovine serum, (FBS), trypsin, penicillin and streptomycin is from Gibco. SuppUes are from standard sources unless otherwise indicated.
Utility, Compositions and Administrations The carbohydrates, oUgosaccharides, proteins, ligands, and fragments thereof, that is the compounds described in this disclosure and in the claims of this appUcation, exhibit binding and blocking activity to E and P-selectin. This activity has therapeutic value in the prevention or treatment of various blood related diseases and disorders. Specifically, the compounds may be used to inhibit platelet leukocyte rosetting and leukocyte adhesion to endotheUum. The word leukocyte includes such ceUs as neutrophils, basophils, eosinophils, monocytes and T-cell subsets. See, de Bnnjne-Admiraal, L.G., Blood, vol. 80. pp. 134-142 (Aug. 1992). The compounds are thus important to and may be used in the preparation of, making and using diagnostic assays and kits. Many procedures known in the art may be used to demonstrate inhibition of platelet leukocyte rosetting and leukocyte adhesion, thus producing a diagnostic kit or assay.
Various diseases and disorders may also be treated or controUed with the compounds of this invention. In particular, embodiments of the invention may be administered to a patient for treatment of blood related diseases such as: cardiovascular disorders, including restenosis foUowing percutaneous transluminal coronary angioplasty (PTCA), myocardial ischemia, stroke, thrombo-emboUsm, deep vein thrombosis, acceleration of thrombolysis, lung injury associated with cardiopulmonary bypass; rheumatoid arthritis, asthma, pulmonary hypersensitivity diseases, organ transplantation, particularly kidney, heart and Uver organs; shock (septic and hemorrhagic), multiple organ injury syndromes; thermal injury; autoimmune disease, multiple sclerosis; inflammatory bowel diseases; infectious diseases including; general inflammation, angiogenesis and cancer metastasis, particularly breast, colon and lung metastasis and neuroblastoma. The function of oUgosaccharides should not be considered in isolation, but in conjunction with the protein to which they are attached. See Rademacher et al, Ann. Rev. Biochem. 57, 785-838 (1988). The novel agents of the present invention thus have various potential therapeutic uses of which the foUowing are iUustrative: Treatment of toxic and cachectic syndromes where cytokines (e.g. TNF, tumor necrosis factor ) are impUcated. e.g. maUgnancy, pre-eclamptic toxaemia, septic shock, Uver disease, respiratory distress syndrome. Chemotherapy of immunosuppressed states, e.g. maUgnancy-associated immune-suppression, iatrogenic immune-suppression (i.e. drugs). Use of glycoprotein (EP) or multivalent (EPC) oUgosaccharides. e.g. treatment of patients with graft vs host reactions (transplants), treatment of patients with host vs graft reactions (transplants) and treatment of maUgnant disease via activation of TNF.
Furthermore, the compounds of this invention may be used to coat heart valves and joint implants, particularly hip, knee and temporal-mandibular, to prevent leukocyte attachment. Coating of these devices with oUgosaccharides would also involve a polymer preparation in which the oUgosaccharides were incorporated into the polymer and then bonded to the device.
Pharmaceutical compositions comprising the carbohydrates, oUgosaccharides, proteins, Ugands, fragments thereof or their formulations are provided by the present invention. The compounds of the invention such as various Ugands having the carbohydrate structure described by this invention or described by the claims of this invention can be administered to a subject in need thereof to treat the subject by either prophylactically preventing inflammation and/or reUeving it after it has begun. Various deUvery systems are known and can be used for therapeutic delivery of the compounds. The Ugands are preferably administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a soUd carrier and/or IN. administration of a Uquid solution carrier.
Methods of administration include but are not Umited to intravenous routes. In general, the amounts of oUgosaccharides administered to adult humans will be in the range of about 0.1 to 20 mg/kg, i.v. for 4-14 days. A more preferred range would be 0.5 to 2.5 mg kg doseage level. Those skilled in the art will be able to readily identify disorders to be treated and optimal dosages can be routinely determined. The formulation of choice can be accompUshed using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin ceUulose, magnesium carbonate, and the like. Oral compositions may be taken in the form of solutions, suspensions, tablets, piUs, capsules, sustained release formulations, or powders. The subject Ugand molecules may be administered directly in transdermal formulations with permeation enhancers such as DMSO.
Other topical formulations can be administered to treat dermal inflammation. A sufficient amount of Ugand molecules should be administered to bind to a substantial portion of the E or P selectin expected to cause or actually causing inflammation so that inflammation can either be prevented or ameliorated. Thus, "treating inflammation" as used herein shall mean preventing or ameUorating inflammation and/or symptoms associated with inflammation.
TypicaUy, the compositions of the instant invention wiU contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%. The frequency of administration wiU be determined by the care given based on patient responsiveness. Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials estabUshing dose response curves. In determining the dose of E or P selectin Ugands to be administered, it must be kept in mind that one may not wish to completely block all of the E or P selectin receptors. In order for a normal healing process to proceed, at least some of the white blood ceUs or neutrophils must be brought into the tissue in the areas where the wound, infection or disease state is occurring. The amount of the E or P selectin Ugands administered as blocking agents must be adjusted carefully based on the particular needs of the patient while taking into consideration a variety of factors such as the type of disease that is being treated. It is beUeved that the Ugands or blocking agents of the present invention can be used to treat a wide range of diseases, including diseases such as rheumatoid arthritis and multiple sclerosis. The compositions of the invention should be appUcable to treat any disease state wherein the immune system turns against the body causing the white cells to accumulate in the tissues to the extent that they cause tissue damage, swelling, inflammation and/or pain.
The inflammation of rheumatoid arthritis, for example, is created when large numbers of white blood ceUs quickly enter the joints in the area of disease and attack the surrounding tissues. Formulations of the present invention might also be administered to prevent the undesirable after effects of tissue damage resulting from heart attacks. When a heart attack occurs and the patient has been revived, such as by the appUcation of anticoagulants or thrombolytic (e.g., tPA), the endotheUal lining where a clot was formed has often suffered damage. When the antithrombotic has removed the clot, the damaged tissue beneath the clot and other damaged tissue in the endotheUal lining which has been deprived of oxygen become activated. The activated endotheUal ceUs then express the E or P selectin within hours of the ceUs being damaged. The selectin then extends into the blood vessels where they adhere to glycoprotein Ugand molecules on the surface of white blood ceUs. Large numbers of white blood ceUs are quickly captured and brought into the tissue surrounding the area of activated endotheUal ceUs, resulting in inflammation, sweUing and necrosis which thereby decreases the likelihood of survival of the patient.
In addition to treating patients suffering from the trauma resulting from heart attack, patients suffering from actual physical trauma could be treated with formulations of the invention in order to relieve the amount of inflammation and sweUing which noπnaUy result after an area of the body is subjected to severe trauma. Other disease states which might be treatable using formulations of the invention include various types of arthritis and adult respiratory distress syndrome. After reading the present disclosure, those skiUed in the art wiU recognize other disease states and/or symptoms which might be treated and/or mitigated by the administration of formulations of the present invention.
Other modes of administration will also find use with the subject invention. For instance, the Ugand molecules of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciUary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not Umited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
The Ugand molecules of the instant invention may also be administered as injectables. TypicaUy, injectable compositions are prepared as Uquid solutions or suspensions; soUd forms suitable for solution in, or suspension in, Uquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in Uposome vehicles. The Ugands in the form of glycoUpids and carbohydrates, or more specifically compounds described in CHART, 1 can be mixed with compatible, pharmaceutically acceptable excipients. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiUary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack PubUshing Company, Easton, PA, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the Ugand molecules adequate to achieve the desired state in the subject being treated. The various Ugand compounds of the present invention can be used by themselves or in combination with pharmaceutically acceptable excipient materials as described above. However, the Ugand compounds of the invention can be made as conjugates wherein the compounds of the invention are linked in some manner to a label. By forming such conjugates, the Ugand compounds of the invention act as biochemical delivery systems for the label so that a site of inflammation can be detected. The Ugand molecules of the invention could also be used as laboratory probes to test for the presence of E or P selectin in a sample. Such probes are preferably labeled such as with a radioactive label. The instant invention is shown and described herein in what is considered to be the most practical and preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.
Compounds of the Invention Ligands in the form of glycoproteins having O-linked oUgosachharides which bind to E and P selectin are disclosed. More specificaUy, this invention relates to carbohydrate Ugands which bind to E and or P selectin and to compositions containing such Ugands which are useful in (a) determining the presence of E and or P selectin, (b) assaying for areas of inflammation, and (c) relieving inflammation by blocking the effects of E and or P selectin. The present inventors have found that E and P selectin recognizes the oUgosaccharide structures on a mucin-like Ugand glycoprotein isolated from HL-60 ceUs in addition to a closely related protein, isolated from normal human leucocytes and developed the present invention.
Ligand molecules capable of binding to and interrupting the biological chain of events associated with E and or P selectin are disclosed. The Ugand molecules act as biochemical blocking agents by binding to E and or P selectin and preventing circulating neutrophils from binding to stimulated endotheUal cells, thereby preventing a primary event of the inflammatory response. The Ugands are in the form of O-Unked oUgosaccharides which can be labeled, bound to anti-inflammatory drugs and or formulated to provide: (1) compositions useful in assaying a sample for the presence of E and or P selectin, (2) compositions useful in detecting the site of inflammation in a patient, and (3) pharmaceutical composition useful in treating inflammation (or treating the inflammatory symptoms of certain diseases) or (4) blocking other effects involving the interaction of E and or P selectin and circulating neutrophils.
An important aspect of the invention is pharmaceutical compositions which are useful in treating, preventing and/or alleviating any undesirable effects resulting from the interaction of E and or P selectin receptors and circulating neutrophils. Such compositions are comprised of an inactive ingredient in the form of a pharmaceutically acceptable excipient material and a compound capable of binding to an E and or P selectin, the compounds having the general structural formula disclosed in CHART 1. A primary object of the invention is to provide an E and or P selectin ligand in a useful formulation.
Another object is to provide a composition comprising an E and or P selectin Ugand which can be used to assay for the presence of E and or P selectin in a sample. Another object is to provide a pharmaceutical formulation containing an E and or P selectin ligand which is useful in treating inflammation.
Other objects include providing methods to treat inflammation and to determine the site of inflammation. An advantage of the invention is that the Ugands are in the form of non-toxic oUgosaccharides, and/or derivatives and mimetics thereof, which effectively bind the E and or P selectin and thereby block neutrophils from binding to the receptors in numbers which result in inflammation and/or other adverse effects. A feature of the present invention is that the Ugand can be labeled and the labeled Ugand used in an assay to detect the presence of E and or P selectin in a sample. Other features of the invention include the abiUty of pharmaceutical formulations of the invention to relieve the inflammatory symptoms of a wide range of diseases which are characterized by the binding of excessive amounts of leucocytes to a site. These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the isolation, structure, formulation and usage as more fully set forth below, references being made to the accompanying figures and general structural formulae forming a part hereof wherein like symbols refer to like molecular moieties throughout.
Before the present E and or P selectin Ugands, the oUgosaccharies, proteins, DNA, fragments thereof and composition containing such Ugands and processes for isolating and using such are described, it is to be understood that this invention is not Umited to the particular compositions, methods or processes described as such. Compositions and methods may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
It must be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a Ugand" includes mixtures of ligands, reference to "E and or P selectin Ugand" includes reference to mixtures of such molecules, reference to "the formulation" or "the method" includes one or more formulations, methods and/or steps of the type described herein and/or which wiU become apparent to those persons skiUed in the art upon reading this disclosure, references cited herein, other relevant references and so forth.
Overview. A blood vessel waU is lined internally with endothelial cells. The endotheUal ceUs can be activated, causing the ceUs to express E and or P selectin. Both red blood cells and white blood ceUs flow in the blood vessel. The white blood ceUs display carbohydrate Ugands which have chemical and physical characteristics which aUow the Ugands to bind to the receptors, i.e. the selectins. Once the Ugand binds to the receptor, the white blood ceU is brought through the vessel wall into the surrounding tissue. The white blood ceUs brought into the surrounding tissue can have positive effects, such as fighting infection, and negative effects, such as inflammation.
The present inventors have isolated and characterized Ugands apart from their presence on the surface of white blood ceUs. These isolated Ugands adhere to E and or P selectin by themselves and can be formulated into pharmaceutical compositions, which when administered will effectively block the E and or P selectin and prevent the adhesion of a receptor connected to a white blood ceU. By administering pharmaceuticaUy effective amounts of Ugands, some but not all, of the white blood ceUs wiU not reach the surrounding tissue. By slowing the rate at which the white blood ceUs reach the surrounding tissue, inflammation can be prevented and or alleviated. It is known that for an acute inflammatory response to occur, circulating neutrophils must bind to and penetrate the vascular waU and access the site of injury. Several molecules have been impUcated in this interaction, including a family of putative carbohydrate ligands and their receptors, i.e. selectins. The present invention involves the isolation and characterization of such Ugands. This invention encompasses those isolated molecules, fragments, combinations and variations thereof which adhere to E and or P selectin. In addition to their ability to adhere to E and or P selectin, in addition to their mucin type O-linked oUgosaccharides, in addition to their lack of fucosylated o-linked oUgosaccharides, in addition to their lack of Lex or a or SLex or a , in addition to their lack of sulfated oUgosaccharides, the isolated Ugands can be generally characterized by the following embodiments, either separately or in various combinations:
(1) The monosaccharide GalNAc.
(2) The disaccharide Galβl,3GalNAc.
(3) The disaccharide Galβl, 3GalNAc substituted with, zero or one sialic acid, at the 3 position on the Gal and, zero or one siaUc acid linked to GalNAc.
(4) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc.
(5) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with one siaUc acid, the substitution being at eifchεi the 3 position of the terminal (non-reducing) Gal β∑ Unked to the reducing GalNAc. (6) The tetrasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with two sialic acids, one siaUc acid is at the 3 position of the terminal (non- reducing) Gal and one siaUc acid is linked to the reducing GalNAc.
(7) The hexasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with one siaUc acid at either the 3 position of the terminal
(non-reducing) Gal Q∑ linked to the reducing GalNAc.
(8) The hexasaccharide Galβl, 4GlcNAcβl, 3/6Galβl, 4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with two siaUc acids, one siaUc acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is linked to the reducing GalNAc. (9) The octasaccharide Galβl,4GlcNAcβl, 3/6 Galβl, 4GlcNAcβl, 3/6Galβl,
4GlcNAcβl, 3/6Galβl, 3GalNAc substituted with two sialic acids, one siaUc acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is Unked to the reducing GalNAc.
The O-linked oUgosaccharide structures recovered from the common Ugand for E and P-selectin, isolated from HL-60 ceUs, are also shown and summarized in CHART 1. CHART 1 summarizes five types of structures, named TYPES A-E, two additional types of structures not drawn in CHART 1 may also be present on the Ugand, they are referred to as TYPES F and G and are discussed in the section below labeled Obvious Variants and Preferred Compounds. Where CHART 1 contains two numbers separated by a slash, in a Unkage designation, it indicates that this Unkage has not been unequivocally determined; "±" at some terminal sialic acids indicates that this position may or may not be conjugated with siaUc acid.
These five types of oUgosaccharides may be referred to by their corresponding size (without siaUc acids), in glucose units (GUs). GU is a size designation. A 12.8 GU oUgosaccharide does not necessarily contain any glucose, rather it would be the same size as a glucose oUgomer containing 12.8 units. Thus, CHART 1 shows, from top to bottom, first, one type of oUgosaccharide, sized to 12.8 GU, that contains two sialic acid residues, as shown, this oUgosaccharide is named, TYPE A or 12.8 GU. Second from the top in CHART 1 is an oUgosaccharide, sized to 9.8 GU, that may be in two forms, that is, it contains either one or two sialic acid residues at the locations shown, this oUgosaccharide is named, TYPE B or 9.8 GU. Third, is an oUgosaccharide, sized to 6.3 GU, that contains either zero, one (in either position) or two sialic acid residues, as shown, and is named, TYPE C or 6.3 GU. Fourth, is an oUgosaccharide, sized to 3.5 GU, that has either zero, one, or two sialic acid residue, named TYPE D or 3.5 GU. And fifth, is an oUgosaccharide, sized to 2.5 GU, and which has no siaUc acid residues, named TYPE E or 2.5 GU. The size given, in glucose units, GUs, does not include siaUc acids.
The relative distribution of siaUc acid residues for each carbohydrate shown in CHART 1 is provided in TABLE 3.
For the structures carrying one siaUc acid, the position of this substitution on the oUgosaccharide (on the terminal galactose or on the reducing N- acetylgalactosmine) has not been determined. AU the oUgosaccharides except GalNAc by itself are core 1 based structures of comparatively simple design. The 12.8, 9.8 and 6.3 GU oUgosaccharides are aU Unear, unbranched poly-N- acetyllactosamine repeat structures with the only variation between them being the number of N-acetyUactosamine repeat units and the sialylation pattern.
Given the abundance of data in the Uterature suggesting Lewisx or Lewisa type fucose substitutions on the oUgosaccharide Ugands for E- and P-selectin, it is noteworthy that these type of substitutions appear to be absent from the structures recovered from this molecule. Several experiments, all of which failed, were carried out aimed at demonstrating the presence of fucose on the oUgosaccharides from the HL-60 Ugand. Those experiments are summarized here.
3H-mannose could not be incorporated into identifiable oUgosaccharide structures on the molecule and none of the [ H]-glucosamine labeled oUgosaccharides isolated from the molecule was susceptible to α-fucosidase digestion. Furthermore, aU of the structures can be completely degraded by alternate digestions with β-galactosidase and β-N-acetylglucosaminidase, this suggests there are no branching fucoses. Finally, none of the o-Unked oUgosaccharides isolated from the ligand molecule bound to, or in any other way interacted with Aurelia auarantia lectin. This lectin specifically recognizes oUgosaccharides containing terminal fucose.
Thus, the o-linked oUgosaccharide structures on the HL-60 Ugand shares the N-acetyUactosamine feature previously reported for Ugands for E- and P-selectin but lacks Lewis type substitutions. Varki, A. (1994) Proc. Natl. Acad. Sci. USA, 91:7390. The mεijority of the larger (12.8, 9.8 and 6.3 GU) structures are sialylated, again consistent with the reported requirement for sialic acid for selectin binding (Table 3).
It should be noted however, that none of the GalNAc (2.5 GU) and almost 75% of the Galβl,3GalNAc (3.5 GU) does not contain any siaUc acid. These two structures, frequently referred to as Tn (GalNAc) and T (Galβl,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g colon and mammaiy carcinomas. See, Springer, G.F. (1984) Science 224:1198; Springer, G.F. (1989) Mol Immunol. 26:1; and Orntoft, T.F. et al. (1990) Int. J. Cancer 45:666.
Refer to TABLE 3, for a summary of the distribution of siaUc acids on the O- linked oUgosaccharides on the HL-60 common Ugand for E- and P-selectin.
An additional feature that is shared by all of the structures on the Ugand is that they are unbranched. As mentioned above, aU structures (except GalNAc) are based on the same core, core 1, and there are no branches except that the reducing N-acetylgalactosamine probably is subsituted with sialic acid on the structures containing two siaUc acids; it is not known if this position is substituted with sialic acid on any of the structures containing one siaUc acid. Unbranched, lactosamine containing, O-Unked oUgosaccharides seem to have a relatively limited distribution in mammals. A search for matching structures in the Complex Carbohydrate Database revealed that the 6.3 GU oUgosaccharide has been described previously on various preparations of human respiratory tract mucins, van Halbeek, H. et al. (1982) Eur. J. Biochem. 127:7; LambUn, G. et al. (1984) Eur. J. Biochem. 143:227; LambUn, G. et al. (1984) J. Biol. Chem. 259:9051; Berg, J. et al. (1987) Eur. J. Biochem. 168:57; Klein, A. et al. (1988) Eur. J. Biochem. 171:631; Lehrmitte, M. et al. (1991) Glycobiology 1:277; human saUvary mucins, Klein, A. et al. (1992) Biochemistry 31:6152; human seminal mucins, Hanish, F.G. et al. (1986) Eur. J. Biochem. 155:239; human meconium glycoproteins, Lawson, A.M. et al. (1991) Carbohydr. Res. 221:191; human milk βl,4 galactosyltransferase, Amano, J. et al. (1991) J. Biol. Chem. 266:11461; and porcine zona pellucida, Hirano, T. et al. (1993) Eur J. Biochem. 214:763.
The 9.8 GU oUgosaccharide has been reported on human milk βl,4 galactosyltransferase, Amano, J. et al. (1991) J. Biol. Chem. 266:11461; porcine zona peUucida, Hirano, T. et al. (1993) Eur J. Biochem. 214:763; Collocalia mucin,
Hanisch, F.G. and Uhlenbruck, G. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365:119; and on the human leucocyte common antigen, Childs, R.A. et al. (1983) Biochim. Biophys. Res. Commun. 110:424.
Only one previous description of the 12.8 GU oUgosaccharide was found, on porcine zona peUucida, Hirano, T. et al (1993) Eur J. Biochem. 214:763.
The lack of unusual features and the presence of these oUgosaccharide structures on several other mammalian glycoproteins (see above) indicates that binding specificity and affinity may be associated with more than a single oUgosaccharide on the Ugand. Two or more of the carbohydrates identified in
CHART 1 may cooperatively generate a high affinity binding domain on the Ugand.
The Ugand described herein has features which may be described as mucin like: It is extensively O-glycosylated; there are three or fewer N-Unked oUgosaccharides on the Ugand. The O-linked oUgosaccharides appear to form a tight cluster. The molecule is largely resistant to degradation by pronase or trypsin.
Additional evidence for a high affinity binding domain comprised of a type of binding "patch" is provided by the foUowing experiments showing it is not possible to identify a single oUgosaccharide structure(s) or a glycopeptide fragments) capable of binding to immobiUzed E- or P-selectin. Re-application of the purified, [ H]-glucosamine labeled Ugand molecule to either an E- or a P-selectin column resulted in considerable binding to the affinity matrix. Trypsin generated fragments that retained some binding activity. Similarly, appUcation of the total purified O-linked Ugand oUgosaccharides, i.e. aU five structures together, to an E-selectin column, resulted in recovery of essentiaUy aU the appUed radioactivity in the break-through fractions from the column. Taken together, these results are consistent with the view that the more preferable Ugand has more than a single oUgosaccharide structure, perhaps presented in a specific arrangement, which may be needed for high-affinity interaction with the selectin. Documents cited herein are incorporated by reference. Obvious Variants and Preferred Compounds
The discussion above demonstrates that neutrophils possess surface molecules which are referred to herein as Ugands and which Ugands have an affinity for and bind to receptor molecules on the surface of endotheUal ceUs which receptors are referred to herein as E or P selectin. The larger molecules extracted which act as Ugands do so because they possess a particular molecular configuration at a particular point on the molecule required for adhesion to the E or P selectin. The experiments herein were the first to isolate and characterize groups of molecules which do adhere to E or P selectin and thereafter specifically identify and characterize the oUgosaccharide structures of those molecules. The general structural formula disclosed in CHART 1 encompasses a number of compounds which include characteristic features of isolated compounds which can adhere to E or P selectin receptors. Variations on the structures may also be possible provided those variations do not alter structural and or charge-related characteristics which would interfere with the adhesion of the Ugand to E or P selectin. Thus, in its broadest sense, the present invention encompasses not only the molecules disclosed in CHART 1, and Types F and G discussed below, but various equivalents thereof which equivalents have an affinity for and an abiUty to adhere to E and or P selectin to the same or a greater degree than molecules of CHART 1 adhere to E and or P selectin. The characteristics of such additional molecules can, of course, be confirmed using the adhesion assay described herein and/or related assays which test the adhesion of a putative Ugand to E and or P selectin under conditions of controlled detachment force.
Other such molecules which can be extracted and/or synthesized may be used directly and/or formulated and deUvered to a patient so that the E and or P selectin receptors of the patient are blocked, at least to a certain degree, thus preventing the adhesion of the selectin Ugands on the surface of the E or P selectin and thereby avoiding the adverse responses (e.g., inflammation) which such neutrophils eventuaUy create. There are five types of oUgosaccharides discussed above and shown in
CHART 1, types A-E.
In addition to these five types there may be smaller amounts of at least two other sUghtly different types of oUgosaccharides, types F and G.
Referring now to CHART 1, and FIGURE 4, upon β-N-acetylglucosaminidase digestion of the 11.8 and 8.8 GU degradation products from the 12.8 and 9.8 GU oUgosaccharides, respectively, aU of the radioactivity in the 11.8 GU and 8.8 GU peaks did not display a shift in elution volume, hence it is possible that two peaks (9.8 and 12.8 GU) contain additional structures. These two peaks represent types F and G oUgosaccharides. Types F and G oUgosaccharides have the same distribution of siaUc acid as types B or A, they are the same size, and they contain no fucose and no sulphate. (Type A - 12.8 GU correspondes to type G and type B - 9.8 GU is the same size as type F.)
One copy each of the five or seven types of oUgosaccharides on the Ugand molecule are not intended to represent the total of the carbohydrates on the ligand. The mucin like character of the Ugand suggests that it contains a large number of oUgosaccharide structures. A similar Ugand molecule (which has been cloned) contains 53 predicted sites for o-glycosidic glycosylation. Sako et al, (1993) CeU 75:1179; Elhammer et al., (1993) J. Biol. Chem. 268:10029. Thus, it is likely that the five or seven oUgosaccharides are present in more than one copy on the Ugand molecule and the feature recognized by the selectins may be comprised of any combination of the different types of structures in any number.
When the Ugand binds with the receptor, the E or P selectin, it binds using a selected combination of carbohydrates. Some combination is certainly needed, the range can be from one to seven types of oUgosaccharide(s), with five types, A-E, being preferred.
The lack of unusual features and the presence of these oUgosaccharide structures on several other mammaUan glycoproteins suggests that the characteristics conferring binding specificity and affinity may not be associated with a single oUgosaccharide on the Ugand but rather that more than one structure may cooperatively generate a high affinity binding domain.
In a recent review on selectin Ugands Varki concluded that this type of interaction between the selectins and their Ugands appear to be the most likely; he also described how the mucin like features of some of the Ugand molecules described to date easfly could accomodate this type binding "patch." Varki, A(1994) Proc. Natl. Acad. Sci. USA 91:7390. The ligand described herein has features which may be described as mucin like: It is extensively O-glycosylated; significant amounts of N-linked oUgosaccharides are also present on the molecule. The O-Unked oUgosaccharides appear to form a tight cluster and the molecule is largely resistant to degradation by pronase or trypsin.
Examples of specific oUgosaccharide Ugand structures are hereby provided. In some embodiments of the invention there are only 1, or only 2, or only 3, in any combination, of the seven types (A-G), more preferably of the five types (A- E), of oUgosaccharides (only types A-E are shown in CHART 1), combined in any number and combination from 1 to 3, more preferably 2, on the putative Ugand. Also described and claimed are the polyvalent forms where the oUgosaccharide combinations repeat themselves any number of times
For example, consider the embodiment where there is only 1 of the 7 of the seven types (A-G) of oUgosaccharides, in any number and combination from 1 to 3, on the putative Ugand. Only 1 of the 7 types of oUgosaccharides in 3 possible combinations, repeated any number of times, would be present. This aUows only 3 x
7 or 21 possibiUties (A, AA, AAA, B, BB, BBB, C, etc.). More preferably 1 of only 5 types in 2 possible combinations would be embodied. This would aUow only 5 possibiUties (AA, BB, CC, DD, EE).
Even more preferable is the embodiment where only 2 of the 5 types of oUgosaccharides are present in only 2 possible combinations, possibly repeated any number of times. This embodiment would aUow only 15 unique possibiUties (AA,
AB, AC, AD, AE, BB, BC, BD, BE, CC, CD, CE, DD, DE, EE) for the putative Ugands.
The order of the oUgosaccharides may be important.
One embodiment would aUow 3 of 7 possible oUgosaccharides in 3 unique possible combinations, repeated any number of times. This would aUow 7 x (7x7) or
343 possibilites. Some of those possibiUties are shown here. AAA, AAB, AAC, AAD, AAE, AAF, AAG,
ABA,ABB,ABC,ABD,ABE,ABF,ABG,
ACA,ACB,ACC,ACD,ACE,ACF,ACG
ADA,ADB,ADC,ADD,ADE,ADF,ADG
AEA,AEB,AEC,AED,AEE,AEF,AEG AFA,AFB,AFC,AFD,AFE,AFF,AFG
AGA,AGB,AGC,AGD,AGE,AGF,AGG
BAA,BAB,BAC,BAD, BAE, BAF,BAG
BBA,BBB,BBC,BBD, BBE,BBF,BBG
BCA,BCB,BCC,BCD,BCE,BCF,BCG BDA,BDB, BDC, BDD,BDE,BDF, BDG
BEA,BEB,BEC,BED,BEE,BEF,BEG
BFA,BFB,BFC,BFD, BFE,BFF,BFG
BGA, BGB, BGC,BGD, BGE, BGF,BGG
This series continues for aU the possible types, that is, CAA...CCC...CGG etc.; DAA...DDD...DGG etc.; EAA...EEE...EGG etc.; FAA...FFF...FGG etc.;
GAA...GGG...GGG etc. The three combination series for types A-E being preferred over the types A-F.
Preparation of the Compounds and Analytical Procedures
Analytical Procedures Metabolic labeling, isolation of the ligand molecule and preparation of radiolabeled oligosaccharides and glycopeptides.
HL-60 ceUs were labeled with H-glucosamine (50 μCi/ml) in normal RPMI medium for 48 hours. Preparation of the P-selectin ligand.
A P-selectin affinity column (10 mg of purified human platelet P-selectin coupled onto 10 ml of Affi-Gel 15 resins) and a bovine serum albumin guard column were prepared as described by Ma et al, (1994) J. Biol. Chem. 269, 27739-27746. The labeled ceUs were lysed with detergent as described by Moore et al. ((1992) J. CeU Biol. 118, 445-456) and the ceU lysates were appUed to the P-selectin affinity column through a BSA guard column in the presence of CaCl2. After washing with 50 ml of PBS, pH 7.4, 1 mM CaCl2, 0.01 % Brij-35 and 0.02 % NaN3, the bound materials were eluted with the same buffer containing 10 mM EDTA as described by Ma et al, ((1994) J. Biol. Chem. In Press). The purity of the Ugand molecule was routinely determined on SDS-PAGE; the purified preparations contained only one radioactive protein band (Figure 7). The purified radiolabeled molecule was then precipitated, using the procedure described by Wessel and Flugge, (1984) Anal. Biochem. 138:141, to remove salts and detergent and the O-linked oUgosaccharides were released by mild alkaline sodium borohydride treatment, essentially as described by Carlson. (1968) J. Biol. Chem. 24:616. The recovery of the Ugand molecule in the precipitation step (as determined by SDS-PAGE) was invariable >90%. The released oUgosaccharide structures were purified on SepPak Clg cartridges prior to analysis.
Glycopeptides were prepared from the intact purified radiolabeled ligand molecule by digestion with Pronase (2 mg ml) in 100 mM Tris-HCl, pH 8.0, 20 mM CaCl2 at 55°C for 4 hours.
FIGURE 7. Purification of -120 kD Ligand for P- and E-selectins HL-60 ceUs labeled with [3H]GlcNAc were lysed with Triton X-100 and separatedly loaded onto P-selectin affinity column or E-selectin receptor-globuUn affinity column in the presence of Ca . After extensive washing, the bound materials were eluated with EDTA and were subject to 7% SDS-PAGE (under reducing conditions) foUowed by autoradiography. Lane 1, the eluate from P-selectin affinity column; lane 2, eluates from E-selectin receptor-globuUn affinity column. Enzyme Treatments. Digestion with Arthrobacter ureafaciens neuraminidase (2 U/ml) was done in 100 mM sodium acetate buffer, pH 5.0 for 18 hours; jack bean β-galactosidase (400mU/ml) was in 50 mM citrate-phosphate, pH 4.6, for 48 hours; jack bean β-N- acetylglucosaminidase (4.15 mU/ml) was in 50 mM sodium citrate, pH 5.6, for 18 hours; Xanthomonas manihotis β-galactosidase (167 U/ml) was in 50 mM sodium citrate, pH 4.5, for 18 hours; almond α-fucosidase (0.2 mU/ml) was in 50 mM sodium citrate, pH 5.0, for 18 hours. AU enzyme digestions were carried out at 37° C under toluene atmosphere.
Column chromatography. Ion-exchange chromatography on QAE-Sephadex was carried out essentially as decribed by Varki, A. and Kornfeld, S. (1983) J. Biol. Chem. 258, 2808-2818. Charged oUgosaccharides were eluted step-wise with increasing concentrations of NaCl in 2 mM Tris base.; 1.5 ml fractions were collected; the column bed volume was 1 ml. SiaUc acid was removed by digestion with Arthrobacter ureafaciens neuraminidase.
Size exclusion chromatography and sequential exoglycosidase digestion of desialylated oUgosaccharides was carried out on a GlycoMap Chromatograph using a Biogel P-4 type column (Glycan Sizing Column, Oxford Glycosystems) and the High- Resolution Program (30 μl min for 366 min foUowed by a linear increase to 200 ul min over 234 minutes); 2 drop fractions (approximately 90 μl) were collected. Paper chromatography.
For determining total radioactive amino sugar composition, samples were hydrolyzed in 6 N HCL at 100 °C for 4 hours (strong acid hydrolysis), re-acetylated and separated on borate impregnated papers as described by Cummings, R.D., and Kornfeld, S. (1982) J. Biol. Chem. 257, 11230-11234. Neutral sugar (mannose and fucose) composition was determined by separating H-mannose labeled oUgosaccharide hyrdolysates (2 M trifluoroacetic acid, 100°C for 4 hours) on descending paper chromatography in ethyl acetate-pyridine-water (8:2:1) for 16 hours. Small radioactive oUgosaccharides were separated on descending paper chromatography in pyridine-ethyl acetate-glacial acetic acid-water (5:5:1:3) for 19-20 hours. SiaUc acid was separated on descending paper chromatography in isoamyl acetate-galacial acetic acid-water (3:3:1) for 20 hours.
Preparation of Preferred Embodiments Characterization of the oligosaccharide structures on the HL-60 ligand for E- and P-selectin.
Separation of radioactive monosaccharides (amino sugars) obtained from strong acid hydrolysis of the total oUgosaccharide structures on the HL-60 Ugand molecule, by borate paper chromatography, resulted in a large portion of the radioactivity co-migrating with the GalNAc standard; the GalNAc:GlcNAc ratio was approximately 1.8:1.
Glycopeptides, prepared by digestion of the intact [3H]-glucosamine labeled Ugand molecule with pronase, were separated on ConcanavaUn A-Sepharose. This resulted in essentially all of the appUed radioactivity eluting in the break-through fractions from the column, insignificant amounts of radioactivity was eluted by the hapten sugars, α-methylmannoside and α-methylglucoside, suggesting that the Ugand molecule does not contain any high-mannose, hybrid or bi-antennary complex type oUgosaccharides.
Results from a partial characterization of the N-linked glycans on the Ugand molecule suggest that these structures are predominantly of multiantennary complex type with extended poly-N-acetyUactosamine containing outer chains; a large portion of these structures are core-fucosylated.
The total O-Unked oUgosaccharides were cleaved from the purified [ H]- glucosamine labeled Ugand molecule by mild alkaline sodium borohydride treatment. The released oUgosaccharides were then fractionated on QAE-Sephadex and charged structures were eluted with step-wise increases NaCl concentration (Figure 1 and Table 1).
FIGURE 1. Separation of labeled O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin, on QAE-Sephadex. Charge separation of the O-linked oligosaccharide structures.
Total O-Unked oUgosaccharides were isolated from the purified in vivo [ H]- glucosamine labeled common Ugand or E and P-selectin and fractionated on QAE- Sephadex. Neutral and oUgosaccharides containing one and two oUgosaccharides were eluted in the column break-through fractions and with 20 and 70 mM NaCl, respectively.
Approximately 12% of the radioactivity loaded on the column did not interact with the ion-exchange matrix and was recovered in the break-through fractions. The remaining radioactivity eluted in fractions containing structures with one and two charges, respectively. See Table 1 and Figure 2. Some variations in the distribution of radioactivity in the charged and neutral fractions from QAE chromatography was observed in different Ugand preparations.
Control experiments showed that the radioactivity in the smaU peak eluted with 140 mM NaCl partly represents a portion of the later eluting fractions in the 70 mM NaCl eluate and partly (glyco)peptide fragments.
Re-chromatography of the charged fractions, after digestion with Arthrobacter ureafaciens neuraminindase, resulted in the majority of the radioactivity in both charged fractions eluting in the QAE-Sephadex column break-through fractions, and the remaining portion (24% and 25% for the 70 mM and 20 mM eluates, respectively) eluted in the fractions containing material with one charge. The radioactive material in these neuraminidase resistant, charged fractions, was identified by paper chromatography.
Neuraminidase resistant radioactivity from the fractions containing structures with either one or two charges contained over 95% of the radioactivity chromatographed, as free sialic acid. This means the neuraminidase digestion completely removed the sialic acids from the oUgosaccharides in the samples and that the radioactivity which retained its charge after neuraminidase treatment represented the released sialic acid only. See, TABLE 1. Charge separation of the O-Unked oUgosaccharide structures isolated from the HL-60 common Ugand for E- and P-selectin.
Size exclusion chromatography of the O-linked oligosaccharide structures.
The neuraminidase digested oUgosaccharides were subsequently separated by size-exclusion chromatography. FIGURE 2 shows separation of the complete mixture of O-Unked oUgosaccharides on the molecule. The mixture of sugars separated into five peaks eluting in the positions of oligomers composed of 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units (GU), respectively. TABLE 2 shows the distribution of radioactivity in the five peaks on the chromatogram. Also included in this table, based on the final characterization of the oUgosaccharide structures on the molecule, is the number of amino sugars in each structure and the distribution (in per cent of the total oUgosaccharides) of the individual oUgosaccharides on the molecule. FIGURE 3 shows a size separation of the portions of the structures containing none, one and two charges, respectively. See, TABLE 3, FIGURE 2, and FIGURE 3.
FIGURE 2. Separation of the O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin on size -exclusion chromatography. Total O-Unked oUgosaccharides were isolated from the purified in vivo [ H]- glucosamine labeled E/P-selectin Ugand, siaUc acids were removed by neuraminidase digestion and the resulting neutral oUgosaccharides were separated on a GlycoMap column. The five radioactive peaks (from right to left) on the chromatogram have elution volumes corresponding to glucose oUgomers composed of 12.8, 9.8, 6.3. 3.5 and 2.5 units respectively.
FIGURE 3. Size-exclusion separation of neutral and charged O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin. The total [ H]-glucosamine labeled O-Unked oUgosaccharides from the H -60 Ugand were fractionated on QAE-Sephadex as described in FIGURE 1. FoUowing removal of sialic acid from the charged oUgosaccharides by neuraminidase digestion, neutral oUgosaccharides (Panel A) and oUgosaccharides containing one (Panel B) and two siaUc acids (Panel C) were fractionated separately on the GlycoMap column. Only the portions of the chromatograms containing measurable amounts of radioactivity are shown in the figure.
Structure determination of the isolated oligosaccharides. In order to determine the structure of the oUgosaccharides isolated from the HL-60 ligand, the three larger peaks (12.8, 9.8 and 6.3 GU) were subjected to sequential exoglycosidase digestion and re-chromatography on the GlycMap. FIGURE 4 shows the sequential degradation of the largest (12.8 GU) oUgosaccharide. Initially, this structure was digested sequentially with jack bean β- galactosidase and jack bean β-N-acetylglucosaminidase; this resulted in a one and two GU shift in elution volume, respectively. A 2 GU radioactive digestion product was released by the β-N-acetylglucosaminidase digestion. These are the predicted results for the removal of one terminal N-acetyUactosamine unit (FIGURE 4, top panel). The released radioactivity was identified as N-acetylglucosamine by paper chromatography. The product from these two digestions eluted at 9.8 GU (FIGURE 4, top panel). As is apparent from FIGURE 4, the first β-N-actylglucosaminidase digestion of the 12.8 GU oUgosaccharide did not result in degradation of all the radiactive material in this peak; some radioactivity also eluted at 11.8 GU after digestion. (FIGURE 4, top panel) Thus it is likely that the 12.8 GU peak contains additional oUgosaccharide structures. FIGURE 4. Sequential exoglycosidase digestion of an O-Unked 12.8 GU oUgosaccharide isolated from the HL-60 common Ugand for E- and P-selectin. The 12.8 GU oUgosaccharide isolated from fractionation of the total O-Unked oUgosaccharides on the HL-60 Ugand (see Figure 2), was subjected to sequential exoglycosidase digestion and re-chromatography on the GlycoMap column as described in the Experimental Procedures. The elution positions of the glucose oUgomer standards are indicated by the arrows at the top of the chromatograms; the numbers identifies the size (in GU) of the individual standard molecules. The key identifies elution profiles obtained for the undigested molecule and for products from digestions with jack bean β-N-acetylglucosaminidase and jack bean β-galactosidase, respectively. E, elution volume (ml); R, radioactivity.
Degradation of the radioactivity in the 9.8 GU peak was initiated by digestion with jack bean or S. pneumoniae β-galactosidase. This resulted in a one GU shift in elution volume for 50-75% (the numbers varied in different preparations) of the radioactivity in the peak. The remaining 25-50% of the radioactivity was resistant to β-galactosidase digestion, again suggesting that the 9.8 GU peak contains an additional oUgosaccharide(s).
Due to difficulties in obtaining amounts of the [ H]-glucosamine labeled 12.8 and 9.8 GU oUgosaccharides sufficient for a complete exoglycosidase digestion of these structures individually, the 9.8 GU product from the partial degradation of the 12.8 GU oUgosaccharide (see above) was pooled with the 8.8 GU β-galactosidase digestion product from the 9.8 GU oUgosaccharide (see above) prior to continued exoglycosidase degradation. The sequential exoglycosidase digestion of the pooled structures is shown in FIGURE 4, middle and bottom panel. Two sequential cycles of digestion with jack bean β-galactosidase followed by jack bean β-N-acetylglucosaminidase resulted, for both digestion cycles, in shifts in elution volumes of one and two glucose units, respectively (FIGURE 4).
The two GU fragments released by the β-N-acetylglucosaminidase digestions and the final 3.5 GU degradation product were identified as N-acetylglucosamine and Galβl,3GalNAc-ol, respectively, on paper chromatography (FIGURE 5, panels A and B; the chromatography standards are: 1, Galβl,3GalNAc-ol and 2, GalNAc-ol).
FIGURE 5. Separation of radioactive fragments obtained from exoglycosidase digestion of the 12.8 GU O-linked oUgosaccharide on the HL-60 common Ugand for E- and P-selectin. The 2 and 3.5 GU fragments generated by exoglycosidase digestion of the 12.8 GU oUgosaccharide were separated on descending paper chromatography in pyridine-ethyl acetate-glacial acetic acid-water (5:5:1:3). Panels A and B shows separations of the 2 and 3.5 GU exoglycosidase digestion fragments, respectively. The standards were 1, Galβl,3GalNAc-ol; 2, GalNAc-ol.
Digestion of the 6.3 GU oUgosaccharide with jack bean β-galactosidase resulted in a one GU shift in elution volume on the column, indicating the removal of one terminal galactose. Digestion of the 6.3 GU oUgosaccharide with Xanthomonas manihotis β-galactosidase did not result in a shift in elution volume indicating that the terminal galactose on this structure is linked 1,4 to N- acetylglucosamine.
Subsequent digestion (of the jack bean β-galactosidase digestion product) with jack bean β-N-acetylglucosaminidase produced an additional two GU shift, indicating the removal of one N-actylglucosamine; the radioactivity released from this digest eluted at two GU and was identified as N-acetylglucosamine on paper chromatography.
Again, the remaining 3.5 GU fragment (of the 6.3 GU oUgosaccharide) was identified as Galβl,3GalNAc-ol by separation on paper chromatography.
The two smaUer O-Unked structures on the Ugand molecule were identified on paper chromatography. The 3.5 GU oUgosaccharide co-migrated with the Galβl, 3GalNAc-ol standard and digestion of this structure with jack bean and bovine testis β-galactosidase followed by re-chromatography on paper, revealed its identity with this standard.
FinaUy, the 2.5 GU structure co-migrated with the GalNAc-ol standard on paper chromatography indicating the same structure (FIGURE 6, panels A and B, respectively; the chromatography standards are: 1, Galβl, 3GalNAc-ol and 2, GalNAc-ol). It is noteworthy that none of the GalNAc and a significant portion of the Galβl,3GalNAc on the Ugand does not contain any sialic acid. Hence the HL-60 Ugand molecule carries both the T and Tn antigen. FIGURE 6. Identification of the 3.5 and 2.5 GU O-Unked oUgosaccharides on the HL-60 common Ugand for E- and P-selectin. The 3.5 and 2.5 GU structures isolated from fractionation of the total O-Unked oUgosaccharides on the HL-60 Ugand (see Figure 2) were subjected to descending paper chromatography in pyridine-ethyl acetate-glacial acetic acid-water (5:5:1:3). Panels A and B shows separations of the 2.5 and 3.5 GU oUgosaccharides, respectively. The standards were 1, Galβl,3GalNAc-ol; 2, GalNAc-ol. [F]
Analysis of possible fucose branching.
Since considerable amounts of data in the Uterature suggest that the interaction between the Ugands for E- and P-selectin, including Ugand molecules isolated from HL-60 ceUs, with the selectins, is dependent on the presence of an outer (Lewis type) fucose branch on the ligand oUgosaccharides, we carried out experiments specificaUy aimed at detecting this type of substitution.
The intact 12.8, 9.8 and 6.3 GU oUgosaccharides isolated from the HL-60 Ugand were individually digested with Almond α-fucosidase; this failed to produce a shift in elution volume for any of the three oUgosaccharides. In addition we were unable to incorporate any mannose or fucose linked radioactivity into the O-linked Ugand oUgosaccharides by in vivo labeUng of the HL-60 cells with [3H]-mannose prior to isolation of the ligand.
Tables 1-3.
TABLE 1. Charge separation of the O-Unked oUgosaccharide structures isolated from the HL-60 common Ugand for E- and P-selectin.
Fraction % of total radioactivity
QAE O 12
QAE 20 20
QAE 70 52
QAE 140 16
TABLE 2. Distribution of O-linked oUgosaccharides on the HL-60 common Ugand for E- and P-selectin.
Elution volume # of aminosugars % of total % of structures
(GU) radioactivity
12.8 4 11.7 3.4
9.8 3 15.2 6.7
6.3 2 50.5 47.7
3.5 1 20.2 38.1
2.5 1 2.2 4.1
TABLE 3. Distribution of siaUc acids on the O-linked oUgosaccharides on the HL- 60 common Ugand for E- and P-selectin.
Structure Non-sialylated Mono-sialylated Di-sialylated
12.8 GU (5.2)a - - 100.0 (5.2)
9.8 GU (9.2) - 28.8 (2.6) 71.2 (6.6)
6.3 GU (45.4) 12.0 (5.5) 23.6 (10.7) 64.4 (29.2)
3.5 GU (36.3) 72.6 (26.4) 27.4 (9.9) -
2.5 GU (3.9) 100.0 (3.9) - -
a Numbers in parenthesis represents percent of total identified structures.
CHART 1
SA α2-3 (Gal β 1-4GlcNAc βl -3/6 )3Gal β 1
Figure imgf000038_0001
+ SAα 2-3 (Gal β1 -4GlcNAc β1 -3/6) Gal β 1 -3GalNAc
Contains one or two SA.
Figure imgf000038_0003
(in either position) ±SA
Figure imgf000038_0002
+ SA α 2 -3 Gal β 1 - 4GlcNAc β 1 -3/6 Gal β 1 -3 Gal NAc
May contain zero, one, or two SA. / (in either position) + SA α 2
Figure imgf000038_0004
+ SA α 2 -3 Galβ 1 -3GalNAc-
Figure imgf000038_0006
May contain zero, one or two SA. ± SA α 2
Figure imgf000038_0005
(in either position)
Figure imgf000038_0007

Claims

Claims
1. An essentially pure ligand that binds with both E and P selectin and is comprised of N and O-linked oligosaccharides where the majority of oligosaccharides are O- linked.
2. An essentially pure ligand of claim 1 where the ligand contains less than 2 percent Lex, Lea, sLe or sLea type substitutions.
3. An essentially pure ligand of claim 2 where the ligand contains essentially no Lex, Lea, sLex or sLea type substitutions.
4. An essentially pure ligand of claim 1 where the o-linked oligosaccharides on the ligand contains less than 2 percent fucose.
5. An essentially pure ligand of claim 4 where the o-linked oligosaccharides on die ligand contains essentially no fucose.
6. An essentially pure ligand of claim 1 where the ligand contains less than 2 percent sulfated oligosaccharides.
7. An essentially pure ligand of claim 6 where the ligand contains essentially no sulfated oligosaccharides.
8. An essentially pure ligand of claim 1 where, on the in vivo H-glucosamine labeled molecule, the ratio of GalNAc.-GlcNAc associated radioactivity is about 1.8:1.
9. An essentially pure ligand of claim 1 where the o-linked oligosaccharide structures of the ligand exhibits no branching, said branching does not include sialic acid substitutions.
10. An essentially pure ligand of claim 1 where the ligand contains, a) less than 2 percent Lex, Lea, sLex or sLea type substitutions, and b) less than 2 percent sulfated oligosaccharides and c) where the o-linked oligosaccharides on the ligand contains less than 2 percent fucose.
11. An essentially pure ligand of claim 10 where, on the in vivo H-glucosamine labeled molecule, the ratio of GalNAc: GlcN Ac associated radioactivity is about 1.8:1.
12. An essentially pure ligand of claim 11 where the oligosaccharides structures of die ligand exhibits no branching, said branching does not include sialic acid substitutions.
13. An essentially pure ligand of claim 12 where the ligand contains, a) essentially no Lex, Lea, sLex or sLea type substitutions, and b) essentially no fucosylated o-linked oligosaccharides and c) essentially no sulfated oligosaccharides.
14. An essentially pure ligand of claim 13, which comprises, any number of any seven types, of types A-G, as shown in CHART 1 , of oligosaccharides, including seven of the same type of oligosaccharide.
15. An essentially pure ligand of claim 14, which comprises, any number of any six types, of types A-G, as shown in CHART 1, of oligosaccharides, including seven of the same type of oligosaccharide.
16. An essentially pure ligand of claim 15, which comprises, any number of any five types, of types A-E, as shown in CHART 1 , of oligosaccharides, including five of the same type of oligosaccharide.
17. An essentially pure ligand of claim 16, which comprises, any number of any four types, of types A-E, as shown in CHART 1 , of oligosaccharides, including four of the same type of oligosaccharide.
18. An essentially pure ligand of claim 17, which comprises, any number of any three types, of types A-E, as shown in CHART 1 , of oligosaccharides, including three of the same type of oligosaccharide.
19. An essentially pure ligand of claim 18, which comprises any number of any two types, of types A-E, as shown in CHART 1, of oligosaccharides, including two of the same type of oligosaccharide.
20. An essentially pure ligand of claim 19, which comprises any number of any one type, of types A-E, of oligosaccharides.
21. An essentially pure ligand of claim 12 which comprises die following oligosaccharide,
SA α2-3 (Gal β 1-4GlcNAc βl -3/6 ), Gal β 1
Figure imgf000041_0001
22. An essentially pure ligand of claim 12 which comprises the following oligosaccharide,
+ SA α 2 - 3 (Gal β1 -4GlcNAc β 1 -3/6 ) Gal β 1 -3Gal NAc -
Contains one or two SA.
Figure imgf000041_0003
(in either position) ± SA 2
Figure imgf000041_0002
23. An essentially pure ligand of claim 12 which comprises die following oligosaccharide,
+ SA α 2 -3 Gal β 1 - 4GlcNAc β 1 -3/6 Gal β 1 -3 Gal NAc-
May co vntain zero, one, or two SA. / /
(in either position) *" ± SA α 2
Figure imgf000042_0001
24. An essentially pure ligand of claim 12 which comprises the following oligosaccharide,
May
(i
Figure imgf000042_0002
25. An essentially pure ligand of claim 12 which comprises the following oligosaccharide,
Figure imgf000042_0003
26. An essentially pure ligand of claim 12 comprising any number of any five of the five types of oligosaccharides represented by the five structures shown in CHART 1 , in any combination.
27. An essentially pure ligand of claim 26 comprising any number of any four of the five types of oligosaccharides represented by die five structures shown in CHART 1, in any combination.
28. An essentially pure ligand of claim 27 comprising any number of any three of the five types of oligosaccharides represented by the five structures shown in CHART 1 , in any combination.
29. An essentially pure ligand of claim 28 comprising any number of any two of the five types of oligosaccharides represented by the five structures shown in CHART 1 , in any combination.
30. An essentially pure ligand of claim 29 comprising any number of any one of the five types of oligosaccharides represented by the five structures shown in CHART 1.
31. An essentially pure ligand of claim 29 comprising any number of any of the following oligosaccharide types AA, AB, AC, AD, AE, BB, BC, BD, BE, CC, CD, CE, DD, DE, or EE.
32. An essentially pure ligand of claim 31 comprising any number of any of the following oligosaccharide types AA, AB, AC, AD, or AE.
33. An essentially pure ligand of claim 31 comprising any number of any of the following oligosaccharide types BA, BB, CB, DB, or EB.
34. An essentially pure ligand of claim 31 comprising any number of any of the following oligosaccharide types CA, CB, CC, DC, or EC.
35. An essentially pure ligand of claim 31 comprising any number of any of the following oligosaccharide types DA, DB, DC, DD, or ED.
36. An. essentially pure ligand of claim 31 comprising any number of any of the following oligosaccharide types EA, EB, EC, ED, or EE.
37. A pharmaceutical composition comprising the ligand of claim 1.
38. A pharmaceutical composition comprising any of the carbohydrates shown in CHART 1 , in any number of any combinations of five carbohydrates or less.
39. A mediod of treating disease comprising the administration of a ligand of claim 1 to a patient in need thereof.
PCT/US1995/011401 1994-09-20 1995-09-15 The oligosaccharide structure of a ligand for e and p selectin WO1996009309A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0847766A2 (en) * 1996-12-10 1998-06-17 SORIN BIOMEDICA S.p.A. An implantation device and a kit including the device
WO1999043834A2 (en) * 1998-02-27 1999-09-02 Genetics Institute, Inc. P-selectin ligand protein, including tetrameric fusion proteins
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
WO2002089819A1 (en) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugates and uses thereof
WO2004033473A1 (en) 2002-10-11 2004-04-22 Yamanouchi Europe B.V. Glucose-based compounds with affinity to p-selectin

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019501A1 (en) * 1990-06-15 1991-12-26 Cytel Corporation Intercellular adhesion mediators
WO1992022563A1 (en) * 1991-06-10 1992-12-23 Alberta Research Council Modified sialyl lewisa compounds
WO1994026760A1 (en) * 1993-05-14 1994-11-24 Cytel Corporation SIALYL Lex ANALOGUES AS INHIBITORS OF CELLULAR ADHESION
WO1994026759A1 (en) * 1993-05-14 1994-11-24 The Regents Of The University Of California Selectin receptor modulating compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019501A1 (en) * 1990-06-15 1991-12-26 Cytel Corporation Intercellular adhesion mediators
WO1992022563A1 (en) * 1991-06-10 1992-12-23 Alberta Research Council Modified sialyl lewisa compounds
WO1994026760A1 (en) * 1993-05-14 1994-11-24 Cytel Corporation SIALYL Lex ANALOGUES AS INHIBITORS OF CELLULAR ADHESION
WO1994026759A1 (en) * 1993-05-14 1994-11-24 The Regents Of The University Of California Selectin receptor modulating compositions

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
US6303584B1 (en) 1996-11-20 2001-10-16 The University Of Montana Water soluble lipidated arabinogalactan
EP0847766A2 (en) * 1996-12-10 1998-06-17 SORIN BIOMEDICA S.p.A. An implantation device and a kit including the device
EP0847766A3 (en) * 1996-12-10 2000-08-30 SORIN BIOMEDICA S.p.A. An implantation device and a kit including the device
US6251142B1 (en) 1996-12-10 2001-06-26 Sorin Biomedica Cardio S.P.A. Implantation device and a kit including the device
WO1999043834A2 (en) * 1998-02-27 1999-09-02 Genetics Institute, Inc. P-selectin ligand protein, including tetrameric fusion proteins
WO1999043834A3 (en) * 1998-02-27 1999-11-18 Genetics Inst P-selectin ligand protein, including tetrameric fusion proteins
WO2002089819A1 (en) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugates and uses thereof
WO2004033473A1 (en) 2002-10-11 2004-04-22 Yamanouchi Europe B.V. Glucose-based compounds with affinity to p-selectin

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