EP0668907A1 - Ligand clycoproteique pour la p-selectine et son procede d'utilisation - Google Patents

Ligand clycoproteique pour la p-selectine et son procede d'utilisation

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Publication number
EP0668907A1
EP0668907A1 EP94903270A EP94903270A EP0668907A1 EP 0668907 A1 EP0668907 A1 EP 0668907A1 EP 94903270 A EP94903270 A EP 94903270A EP 94903270 A EP94903270 A EP 94903270A EP 0668907 A1 EP0668907 A1 EP 0668907A1
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Prior art keywords
ligand
selectin
sds
linked oligosaccharides
molecular weight
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EP94903270A
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German (de)
English (en)
Inventor
Richard D. Cummings
Kevin L. Moore
Rodger P. Mcever
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University of Oklahoma
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University of Oklahoma
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the United States government has rights in this invention as a result of National Institutes of Health grants HL 34363 (R.P. McEver) and HL 45510 (R.P. McEver and K.L. Moore), CA 38701 (A. Varki) , IT4 RR 05351 (R.D. Cummings) , and GM 45914 (D.F. Smith) .
  • the selectins are three structurally related membrane glycoproteins that participate in leukocyte adhesion to vascular endotheliu and platelets, as reviewed by McEver in Thromb . Haemostas . , 66: 80-87 (1991) and in Curr. Opin . Cell Biol . , 4 ; 840-849 (1992).
  • P-selectin (CD62) , previously known as GMP-140 or PADGEM protein, is a receptor for neutrophils, monocytes and subsets of lymphocytes that is rapidly translocated from secretory granule membranes to the plasma membrane of activated platelets, as reported by Hamburger and McEver, Blood 75 : 550-554 (1990) ; Larsen et al., Cell 59 : 305-312 (1989) and endothelial cells, as reported by Geng et al., Nature, 343 : 757-760 (1990); Lorant et al., J. Cell Biol . , 115 : 223-234 (1991).
  • E-selectin is a cytokine-inducible endothelial cell receptor for neutrophils, as reported by Bevilacqua et al. , Proc. Natl . Acad. Sci . USA, 84 : 9238-9242 (1987), monocytes, as reported by Hession et al., Proc. Natl . Acad . Sci . USA, 87 : 1673-1677 (1990), and memory T cells, as reported by Picker et al.. Nature (London) , 349 : 796-799 (1991); Shi izu et al.. Nature (London) , 349 : 799-802 (1991).
  • L-selectin (LAM-1, LECAM-1) , a protein expressed on myeloid cells and most lymphocytes, participates in neutrophil extravasation into inflammatory sites and homing of lymphocytes to peripheral lymph nodes, as reported by Lasky et al., Cell, 56: 1045-1055 (1989); Siegelman et al., Science, 243 : 1165-1172 (1989); Kishimoto et al., Science, 245: 1238-1241 (1989); Watson et al., Nature (London) , 349 : 164-167 (1991) .
  • Each sele ⁇ tin functions as a Ca 2+ -dependent lectin by recognition of sialylated glycans. Both E- and P-selectin interact with sialylated, fucosylated lactosaminoglycans on opposing cells, including the sialyl Le x tetrasaccharide, as reported by Phillips et al., Science, 250 : 1130- 1132 (1990); Walz et al. , Science, 250 : 1132-1135 (1990); Lowe et al. , Cell, 63 : 475-484 (1990); Tiemeyer et al., Proc. Natl . Acad. Sci . USA, 88 :
  • P-selectin isolated from human platelets binds with apparent high affinity to a limited number of sites on neutrophils (Moore et al., J. Cell Biol . , 112 : 491-499 (1991); Skinner et al., J “ . Biol . Chem . , 266: 5371-5374 (1991) and HL-60 cells (Zhou et al., J. Cell Biol . , 115 : 557-564 (1991)). Binding is abolished by treatment of the cells with proteases (Moore et al., (1991)), suggesting that the glycans on myeloid cells recognized preferentially by P-selectin are on glycoprotein(s) rather than on glycolipids.
  • the number of binding sites for platelet P-selectin on neutrophils has been estimated at 10,000-20,000 per cell (Moore et al., 1991; Skinner et al., 1991), suggesting that these sites constitute a small component of the total cell surface protein.
  • the protein portion of this ligand(s) may be crucial for binding by presenting the glycan in an optimal configuration, clustering glycans to enhance avidity, favoring the formation of specific oligosaccharide structures by cellular gly ⁇ osyltransferases or modifying enzymes, and/or stabilizing the lectin-carbohydrate interaction through protein-protein interactions with P-selectin.
  • myeloid cells express one or more membrane glycoproteins not found on CHO cells that enhance the lectin- mediated interaction with P-selectin.
  • myeloid cells may express a glycosyltransferase or modifying enzyme not present in CHO cells.
  • P-selectin has been demonstrated to bind primarily to a single glycoprotein ligand on neutrophils and HL-60 cells, when assessed by blotting assays and by affinity chromatography of [ 3 H]glucosa ine-labeled HL-60 cell extracts on immobilized P-selectin. This molecule was characterized and distinguished from other well- characterized neutrophil membrane proteins with similar apparent molecular mass. The amino acid sequences of some tryptic peptides of the ligand were determined and found to be unrelated to other known amino acid sequences.
  • 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-selectin to cells and in diagnostic assay 1.
  • WO 92/01718 entitled "Peptides Selectively Interacting with Selectins" by Rodger P. McEver, described the ability of P-selectin (GMP-140) to mediate cell-cell contact by binding to carbohydrate ligands on target cells and specific binding to protease-sensitive sites on human neutrophils. Studies with antibodies and with neuraminidase indicated that P-selectin bound to carbohydrate structures related to sialylated, fucosylated lactosaminoglycans. As described in WO 92/01718 by Rodger P.
  • the glycoprotein was partially purified on a P- selectin affinity column. It appeared to be heavily glycosylated because it stained poorly with silver and Coomassie blue. It appeared to be heavily sialylated because it bound to a wheat germ agglutinin affinity column. Treatment of the glycoprotein ligand with low doses of sialidase slowed its mobility on SDS gels, a pattern consistent with partial desialylation of heavily O- glycosylated proteins. Binding of P-selectin to the glycoprotein ligand was Ca 2+ -dependent, blocked by monoclonal antibodies to P-selectin that also block P-selectin binding to leukocytes, and abolished by extensive treatment of the ligand with sialidase.
  • P-selectin The preferential binding of P-selectin to the 120,000 D glycoprotein ligand in myeloid cell extracts suggested that it contained special structural features that are recognized with high affinity by P-selectin. Such structures might not be present on every protein or lipid characterized by sialylated, fucosylated structures such as sLe x .
  • NeoLewis CHO cells a cell line expressing sialylated, fucosylated lactosaminoglycans, described in WO 92/017178
  • the NeoLewis cells express higher levels of sLe x antigen, as reported by Zhou et al., J. Cell Biol . , 115 : 557-564 (1991).
  • fluid- phase [ 125 I]P-selectin binds with high affinity to a limited number of sites on myeloid cells, whereas it binds with lower affinity to a higher number of sites on NeoLewis CHO cells.
  • the 120,000 D glycoprotein ligand for P-selectin in neutrophil extracts is likely to correspond to the limited number of protease-sensitive, high affinity binding sites for P-selectin on intact neutrophils. Interaction of P-selectin with these sites may be required for efficient adhesion of leukocytes in flowing blood to P-selectin expressed by activated platelets or endothelial cells.
  • a method for purifying the glycoprotein ligand for P-selectin and structural features including the amino acid sequence of tryptic peptides of the ligand are described below.
  • the purified ligand, or fragments thereof, including both the carbohydrate and protein components, or antibodies to the ligand, or fragments thereof, can be used as inhibitors of binding of P-selectin to cells.
  • WGA wheat germ agglutinin
  • pepstatin aprotinin
  • N-acetylglucosamine N-acetylglucosamine
  • leupeptin antipain
  • benzamidine MOPS
  • Pipes BSA, EDTA, EGTA, and Ponceau S were purchased from Sigma Chemical Co. (St. Louis, MO) .
  • Endo- ⁇ - galactosidase 150 U/mg, EC 3.2.1.103
  • Bacteroides fragills 4-methyl-umbelliferyl ⁇ -N- acetylneuraminic acid
  • 2,3-dehydro-2,3-dideoxy- N-acetylneuraminic acid Ne2en5Ac
  • Peptide :N glycosidase F from FlavoJba ⁇ terium eningosepticum (EC 3.2.2.18, N-glycanase) and endo- ⁇ -W- acetylgalactosaminidase from Diplococcus pneumoniae (EC 3.2.1.97, O-glycanaseTM) were purchased from Genzy e (Cambridge, MA) .
  • HBSS was obtained from Gibco Laboratories (Grand Island, NY) .
  • Vecta-Stain ABC kits were purchased from Vector Laboratories Inc. (Burlingame, CA) .
  • Phycoerythrin-streptavidin was obtained from Becton Dickinson & Co.
  • the anti-P-selectin murine MAbs S12 and Gl, and goat anti-human P-selectin IgG were prepared and characterized as described by McEver and Martin, J. Biol . Chem . , 259 : 9799-9804 (1984); Geng et al. (1990); Lorant et al. (1991).
  • Rabbit polyclonal antisera and murine MAbs to human lamp-1 (CD3) described by Carlsson et al., J. Biol . Chem . , 263 : 18911-18919 (1988) , and lamp-2 (BB6) , Carlsson and Fukuda, J. Biol . Chem.
  • Erythrocyte membranes were isolated from leukocyte-depleted human erythrocytes as described by Rollins and Sims, J. Immunol . , 144 : 3478-3483 (1990) and extracted with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 1% LubrolTM PX. Detergent-insoluble material was removed by centrifugation at 16,000 x g for 10 min.
  • the cells were centrifuged at 500 x g for 5 min and resuspended in ice-cold HBSS containing 5 mM EDTA and 10 mM MOPS, pH 7.5. Diisopropylfluorophosphate was then added to a final concentration of 2 mM and the cell suspension incubated for 10 min on ice.
  • the cells were centrifuged at 500 g for 5 min at 4°C and resuspended in ice-cold 100 mM KCl, 3 mM NaCl, 1 mM NazATP, 3.5 mM MgCl 2 , 10 mM Pipes, pH 7.3 (relaxation buffer) .
  • protease inhibitors were added at the indicated final concentrations: 2 mM diisopropylfluorophosphate, 20 ⁇ M leupeptin, 30 ⁇ M antipain, and l mM benzamidine.
  • the cell suspension was pressurized with N 2 at 350 psi in a cell disruption bomb (model 4635; Parr Instrument Company, Moline, IL.) for 40 min at 4°C with constant stirring as described by Borregaard et al., J. Cell Biol . , 97: 52-61 (1983).
  • the cavitate was collected into EGTA (2 mM final concentration) and nuclei and undisrupted cells were pelleted at 500 g for 10 min at 4°C.
  • the cavitate was fractionated as described by Eklund and Gabig, J. Biol . Chem . , 265 : 8426-8430 (1990). Briefly, it was layered over 40% sucrose in relaxation buffer containing 2 mM EGTA, 20 ⁇ M leupeptin, 30 ⁇ M antipain, and 1 mM benzamidine, and centrifuged at 104,000 x g (at r av ) for 45 min at 4°C in a rotor (model SW28; Beckman Instruments, Inc., Palo Alto, CA) .
  • the top layer (FX,) , the 40% sucrose layer (FX 2 ) , and the granule pellet (FX 3 ) were collected and assayed for lactate dehydrogenase as a cytoplasmic marker, alkaline phosphatase as a plasma membrane marker, and myeloperoxidase as a marker for azurophilic granules as described by Borregaard et al., (1983); Geng et al., (1990).
  • Table I shows the distribution of marker enzymes in the various fractions.
  • FX 2 enriched for alkaline phosphatase, was diluted with four volumes of 0.1 M NaCl, 10 mM MOPS, pH 7.5, and centrifuged at 111,000 x g (at r, v ) for 60 min at 4°C in a rotor (model 50.2 Ti; Beckman Instruments, Inc.). The supernatant was collected and the membrane pellet was extracted with 1% LubrolTM PX, 0.1 M NaCl, 10 mM MOPS, pH 7.5, 0.02% sodium azide, 20 ⁇ M leupeptin, 30 ⁇ M antipain, 1 mM benzamidine, and stored at 4°C.
  • HL-60 cells maintained in suspension culture in RPMI-1640 supplemented with 10% FCS, 100 IU/ml penicillin, and 100 ⁇ g/ml streptomycin, were washed in HBSS, 10 mM MOPS, pH 7.5, and membranes were isolated exactly as described for neutrophils. Partial Purification of P-selectin Ligand
  • Neutrophil or HL-60 cell membrane extracts were applied to a wheat germ agglutinin (WGA) affinity column (0.9 x 20 cm. 7.6 mg lectin/ml resin) equilibrated at room temperature with 0.5 M NaCl, 10 mM MOPS, pH 7.5, 0.02% sodium azide, 0.1%
  • WGA wheat germ agglutinin
  • LubrolTM PX The column was washed with five column volumes of equilibration buffer, followed by two column volumes of 0.1 M NaCl, 10 mM MOPS, pH 7.5, 5 mM EDTA, 0.02% sodium azide, 0.01% LubrolTM PX. The column was then eluted with the above buffer containing 100 mM N-acetylglucosamine. Protein- containing fractions were pooled and extensively dialyzed against 0.1 M NaCl, 10 mM MOPS, pH 7.5, 0.02% sodium azide, 0.01% LubrolTM PX at 4°C.
  • the dialyzed WGA column eluate was made 1 mM in CaCl 2 and MgCl 2 and applied to a human serum albumin AffigelTM 15 precolumn (0.9 x 11 cm, 25 mg protein/ml resin) hooked in series to a P selectin- Affigel 15TM column (0.6 x 13 cm, 2 mg protein/ml resin).
  • the columns were equilibrated with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.02% sodium azide, 0.01% LubrolTM PX.
  • the membranes were blocked overnight at 4°C in 0.1 M NaCl, 10 mM MOPS, pH 7.5, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.02% sodium azide, 10% (wt/vol) CarnationTM nonfat dry milk, and then washed with the same buffer containing 0.1% Tween- 20 without milk.
  • HL-60 cells (1-2 x 10 6 cells/ml) in 100-mm tissue culture dishes were labeled for 48 h with 50 ⁇ Ci/ml [6- 3 H]glucosamine at 37°C in RPMI-1640 containing 10% FCS, 2 mM glutamine, 100 IU/ l penicillin, and 100 ⁇ g/ml streptomycin.
  • the cells were washed three times by centrifugation and resuspension in ice-cold PBS.
  • the cell pellet was solubilized with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 4 mM CaCl 2 , 4 mM MgCl 2 , 1% Triton X-100TM, 20 ⁇ g/ml aprotinin, 20 ⁇ g/ml leupeptin, 8 ⁇ g/ml pepstatin, 2 mM PMSF, 10 mM benzamidine, and 0.5 mM dichloroisocoumarin.
  • the solubilized cells were allowed to sit on ice for 1-2 h and then sonicated for 20 min at 4°C in a water bath sonicator.
  • the cell extract was centrifuged for 5 min at 16,000 x g and the supernatant was applied to a P-selectin-Affigel 15TM column (0.25 x 13 cm, 2 mg protein/ml resin) equilibrated with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.1% Triton X-100.
  • the column was washed with 10-20 column volumes of equilibration buffer and bound material was eluted with equilibration buffer containing 10 mM EDTA. Fractions (1 ml) were collected and monitored for radioactivity by liquid scintillation counting.
  • Metabolically labeled proteins eluted from the P-selectin column (above) were precipitated in the presence of 0.1 mg/ml BSA by addition of cold TCA (10% final concentration) .
  • the resulting pellets were washed with 1 ml acidified acetone (0.2%), solubilized in 0.1 M NaOH and electrophoresed under reducing and nonreducing conditions on 10% SDS- polyacrylamide gels.
  • the gels were stained with
  • samples analyzed by P- selectin blotting were pretreated with exo- or endo-glycosidases before SDS-PAGE.
  • samples analyzed by P- selectin blotting were dialyzed against 0.15 M NaCl, 50 mM acetate, pH 6.0, 9 mM CaCl 2 , 0.02% azide, 0.01% LubrolTM PX, and incubated for various times at 37°C in the presence or absence of 200 mU/ml of enzyme.
  • samples were first reduced and denatured by bailing in 0.5% SDS, 0.5% ⁇ -mercaptoethanol for 5 min, and then a 7.5- fold molar excess of NP-40 was added.
  • the samples were incubated for 16 h at 37°C with either PNGaseF (20 U/ml at pH 8.6) or endo- ⁇ -N acetylgalactosaminidase (70 U/ml at pH 6.5) in the presence of 5 mM PMSF and 5 mM 1,10 phenanthroline.
  • HBSS/FCS/Az Human neutrophils, isolated as described by Hamburger and McEver, (1990) , were suspended (10 6 /ml) in HBSS containing 1% FCS and 0.1% sodium azide (HBSS/FCS/Az) . 1 ml of neutrophil suspension was underlaid with 100 ⁇ l FCS and centrifuged at 500 g for 5 min.
  • the neutrophil pellet was resuspended in 50 ⁇ l of purified P selectin (10 ⁇ l/ml, in HBSS/FCS/Az) , and then incubated sequentially with 50 ⁇ l of biotin-conjugated S12 (10 ⁇ g/ml, in HBSS/FCS/Az) and 20 ⁇ l of phycoerythrin-streptavidin (neat) .
  • the neutrophils were preincubated for 10-15 min with antisera or antibodies before the addition of P-selectin. Between each step the cells were diluted with one ml of HBSS/FCS/Az, underlaid with 100 ⁇ l FCS, and centrifuged at 500 g for 5 min.
  • WGA eluate was incubated with 10 ⁇ g of anti- leukosialin (Leu22) or an isotype matched control monoclonal antibody for 1 h at 37°C.
  • the mixture was then incubated with protein A-SepharoseTM CL4B beads saturated with rabbit anti-mouse IgG for 1 h at 37°C.
  • the beads were pelleted, washed four times with 1 ml of 0.1 M NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100TM, and bound material eluted by boiling 5 min in 2% SDS, 60 mM Tris, pH 6.8, and 5% ⁇ -mercaptoethanol.
  • Immunoprecipitates and immunosupernatants were then analyzed by P-selectin blotting and by Western blotting using Leu22 as a probe.
  • the column was then eluted with the above buffer containing 500 mM N- acetylglucosamine. Protein-containing fractions were pooled and subjected to an additional affinity chromatographic step using a P-selectin-EmphazeTM column. The pooled fractions were made 8 mM in
  • the 125 I-labeled ligand was subjected to gel filtration on a SuperoseTM 6 column.
  • the availability of highly purified [ 125 I]P-selectin ligand allowed various functional and structural analyses to be carried out on the ligand.
  • 75 to 90 percent of the [ 125 I]P-selectin ligand re-bound to a P-selectin-immobilized affinity column (see above) and was eluted with EDTA. This material specifically bound to recombinant soluble P-selectin immobilized on microtiter plates in both a time- and dose- dependent fashion.
  • Binding was abolished with EDTA and anti-P-selectin monoclonal antibodies which inhibit P-selectin function, but not anti-P- selectin monoclonal antibodies which do not inhibit function.
  • P-selectin Ligand Amino Acid Sequencing Ligand-containing fractions from the Mono Q PC 1.6/5 column were pooled, then diluted with 2 parts HPLC grade H 2 0 and centrifuged for 10 min at 16,000 x g. The sample was applied to a ProspinTM Sample Preparation Cartridge (Applied Biosystems) after wetting the PVDF membrane with HPLC grade methanol. The cartridge was centrifuged at 4500 x g for one hour in a Fisher Model 59A Microfuge equipped with a swing-out rotor. After the sample was applied, the PVDF membrane was washed twice with 400 ⁇ l of HPLC grade H 2 0.
  • the PVDF membrane was removed using a ProspinTM Membrane Removal Punch (Applied Biosystems) and washed ten times with 1 ml of HPLC grade H 2 0. After the last wash was removed, the PVDF membrane was frozen on dry ice.
  • the sample (designated gpl20) was shipped on dry ice to Harvard Microchem (16 Divinity Avenue, Cambridge, MA 02138) for N-terminal sequencing and in situ trypsin digestion and HPLC separation of peptides.
  • P-selectin Ligand To identify proteins from myeloid cells which bind P-selectin, neutrophil and HL-60 cell membrane extracts were electrophoresed on 7.5% SDS- polyacrylamide gels, transferred to ImmobilonTM membranes, and probed with [ 125 I]P-selectin. When samples were analyzed without reduction, P-selectin bound preferentially to a glycoprotein species with an approximately 250,000 M, from both neutrophil and HL-60 cell membranes as determined by SDS-PAGE.
  • Cell membrane extracts (80 ⁇ g protein/lane) were electrophoresed on 7.5% SDS-polyacrylamide gels under nonreducing or reducing conditions, transferred to ImmobilonTM membranes, and probed with [ 125 I]P-selectin. Under nonreducing conditions P-selectin also bound to proteins at the stacking gel interface and to a minor species with an approximately 160,000 M t . When samples were analyzed after reduction, P-selectin preferentially bound to a glycoprotein with an approximately 120,000 M.. Minor bands were observed at approximately 250,000 and approximately 90,000 r . Under both reducing and nonreducing conditions P- selectin also bound to the blots at the dye front.
  • P-selectin binding proteins were not detected when an equivalent amount of erythrocyte membrane protein was analyzed in parallel.
  • the total proteins in the neutrophil cavitate were also solubilized with SDS and analyzed for their ability to interact with P-selectin with the blotting assay.
  • P-selectin bound only to proteins with apparent molecular weights of 120,000 and 90,000 under reducing conditions. Although the sensitivity of this analysis was limited by the amount of protein that could be run on the gel, the results indicate that major ligands that were either not enriched in the membrane fraction (FX 2 ) or not effectively solubilized by nonionic detergent were not excluded.
  • neutrophil membrane extracts electrophoresed under reducing conditions were probed with [ 125 I]P-selectin in the presence or absence of EDTA or anti-P-selectin MAbs.
  • Neutrophil membrane extracts 200 ⁇ g protein/lane were electrophoresed on 7.5% SDS-polyacrylamide gels under reducing conditions, transferred to
  • [ 125 I]P-selectin binding to the major 120-kD and the minor 250-kD species was Ca 2+ -dependent, a characteristic of all selectin-dependent cellular interactions. Binding to both species was also blocked by Gl, a MAb to P-selectin that inhibits adhesion of myeloid cells to P-selectin, but not by S12, a MAb to P-selectin that does not block adhesion.
  • the membrane fraction constituted approximately 5% to 7% (n>10) of the protein in the cavitate. This fractionation depleted both cytosolic proteins and azurophilic granules as shown by Table I. Proteins binding P-selectin were not detected in the cytosolic fraction (FXj) with the blotting assay. The final membrane pellet was solubilized with nonionic detergent and applied to a WGA column which bound 4-5% of the protein in the membrane extract. P-selectin blotting assays of reduced proteins demonstrated that both the major 120,000 D and the minor 250,000 D ligands bound quantitatively to WGA. However, the 90,000 D band and the band at the dye front observed in the membrane extract were not bound by WGA.
  • the WGA eluate was applied to an Affigel 15TM precolumn in series with a P- selectin affinity column. Approximately 2% of the protein in the WGA eluate bound to the P-selectin column and could be eluted with EDTA. Both the 250,000 D and the 120,000 D ligands bound quantitatively to the P-selectin column. Quantitative analysis of the protein recovered from the P-selectin eluate indicated that the ligand(s) formed less than 0.01% of the total protein in the neutrophil cavitate.
  • the amounts of protein loaded onto the lanes were as follows: membrane extract and WGA flow through, 200 ⁇ g; WGA eluate and P-selectin flow through, 50 ⁇ g; P- selectin eluate, 2 ⁇ g.
  • the same samples (10 ⁇ g protein/lane) were also analyzed by SDS-PAGE under the reducing conditions followed by silver staining.
  • the major silver-stained band in the P- selectin eluate had an approximately 150,000 M. which is similar to that of P-selectin itself.
  • the P-selectin eluate was analyzed by SDS- PAGE under both reducing and nonreducing conditions, followed by silver staining, Western blotting with goat anti-P-selectin IgG, and P- selectin blotting.
  • the major silver-stained protein in the P-selectin eluate was indeed P- selectin.
  • Purified P-selectin migrates with an approximately 120,000 ⁇ f r under nonreducing conditions; a minor component migrates with an approximately 250,000 M t . After reduction the protein migrates more slowly with an approximately 150,000 M t .
  • the two nonreduced bands and the one reduced band detected by silver staining of the P- selectin eluate co-migrated with purified P- selectin and were recognized by anti-P-selectin IgG.
  • the P-selectin ligand identified in the blotting assay was not detected by silver staining and migrated differently than P-selectin under both reducing and nonreducing conditions.
  • P- selectin eluate was electrophoresed without reduction, P-selectin did not bind to proteins at the stacking gel interface. Therefore, the P- selectin binding proteins at the stacking gel interface, observed in extracts of neutrophil membranes, were probably an artifact due to the relatively high amount of protein loaded on the gel.
  • the ligand on intact target cells requires sialic acids to interact with P-selectin.
  • Neutrophil WGA eluate (50 ⁇ g) was either sham-treated or digested with 200 mU/ml of sialidase or with 20 U/ml of PNGaseF for 16 h, then electrophoresed on 7.5% SDS polyacrylamide gels under reducing conditions, transferred to Immobilon membranes, and probed with [ 125 I]P-selectin.
  • Sialidase digestion for 30 min increased the apparent molecular weight of the major 120,000 D ligand, a shift characteristic of heavily sialylated glycoproteins. Longer sialidase digestion did not further alter the electrophoretic mobility of the ligand but did abolish its ability to bind [ 125 l]P-selectin.
  • Sialidase treatment had a similar effect on the minor 250 kD ligand.
  • neutrophil membrane glycoproteins which bound to WGA were digested with PNGaseF. This treatment did not affect [ 125 I]P-selectin binding but did decrease the apparent molecular weight of the ligand by approximately 3000 D, consistent with the enzymatic removal of one or two N-linked glycan chains. This demonstrates that the ligand contains at least one N-linked oligosaccharide chain that is not required for P-selectin binding. Although one could not directly assess whether N-linked glycans were quantitatively removed from the ligand, conditions that normally cleave such glycans from most proteins were used.
  • P-selectin blotting of denatured membrane proteins from myeloid cells may not detect molecules whose ability to bind P-selectin is dependent on secondary and/or tertiary structure.
  • HL-60 cells were metabolically labeled with [ 3 H]glucosamine, solubilized with nonionic detergent, and applied to a P-selectin affinity column. After extensive washing, bound material was eluted with EDTA and analyzed by SDS-PAGE followed by fluorography.
  • Samples were electrophoresed on 10% SDS polyacrylamide gels under both nonreducing and reducing conditions and analyzed by fluorography. Other samples were either sham treated or digested with 1 U/ml of sialidase for 24 h or with 3.3 U/ml of PNGaseF for 24 h, and then electrophoresed on 10% SDS polyacrylamide gels under reducing conditions and analyzed by fluorography.
  • the properties of the major 120,000 D P-selectin ligand were compared with those of three well- characterized neutrophil membrane proteins with similar apparent molecular weight.
  • the first two molecules, lamp-1 and lamp-2 are abundant neutrophil proteins that are predominantly localized in lysosomal membranes but are also expressed in small amounts on the cell surface. These proteins have a large number of complex N- linked glycan chains, many of which carry the sialyl Le x tetrasaccharide.
  • Membrane extracts (200 ⁇ g protein/lane) were electrophoresed on 7.5% SDS-polyacrylamide gels under nonreducing or reducing conditions, transferred to ImmobilonTM membranes, and probed with [ 125 1 P-selectin or murine monoclonal antibodies directed against human lamp-1 (CR3 ) , human lamp-2 (BB6) , human L-selectin (DREG-200) , or human leukosialin (Leu22) .
  • Western blot analysis of neutrophil membranes with MAbs to lamp-1 and lamp-2 showed that the electrophoretic mobilities of these proteins under nonreducing conditions were distinct from that of the P-selectin ligand.
  • lamp-1 and lamp-2 are not ligands for P-selectin even though they carry many sialyl Le x structures.
  • the third molecule whose apparent molecular weight is similar to the 120,000 D P-selectin ligand is CD43 (leukosialin, sialophorin) , a heavily sialylated membrane protein present on platelets and all leukocytes. It carries numerous O-linked sugar chains and is differentially glycosylated by cells of various hematopoietic lineages. Like the P-selectin ligand, treatment of leukosialin with sialidase increases its apparent molecular weight. However, in contrast to the P- selectin ligand, the electrophoretic mobility of leukosialin was unaffected by reduction.
  • Monospecific polyclonal anti-human leukosialin antisera (1:5 dilution) did not inhibit P-selectin binding to neutrophils as assessed by flow cytometry. Furthermore, im unodeple ion of leukosialin from neutrophil membrane extracts did not deplete P-selectin ligand as assessed by the blotting assay. Finally, leukosialin purified from HL-60 cells did not bind P-selectin.
  • Neutrophil WGA eluate (50 ⁇ g) and leukosialin purified from HL-60 cells (0.5 ⁇ g) were electrophoresed under reducing conditions on 7.5% SDS-polyacrylamide gels, transferred to ImmobilonTM, and probed with [ 125 I]P-selectin. The same membrane was then probed with the monoclonal anti-human leukosialin antibody Leu22.
  • L-selectin is an important glycoprotein ligand on myeloid cells for P-selectin by Picker et al., Ceil, 66: 921-933 (1991). Although L-selectin is present in membrane extracts and WGA eluates of neutrophil membranes, as detected by Western blotting, [ 125 13P-selectin did not bind to L-selectin in the blotting assay.
  • the anti-L-selectin MAb DREG-56 (100 ⁇ g/ml) had no effect on the binding of purified P- selectin to quiescent neutrophils as assessed by flow cytometry. Neutrophils were preincubated for 15 min with buffer alone, 100 ⁇ g/ml of the anti-L- selectin monoclonal antibody DREG-56, or 100 ⁇ g/ml of the anti-P-selectin MAb Gl before addition of buffer or P-selectin.
  • P- selectin ligand contains a limited number of N- linked glycan chains and that enzymatic removal of these chains with PNGaseF did not affect the ability of the ligand to bind [ 125 13P-selectin using the P-selectin blotting assay.
  • [ 125 I]P-selectin ligand was digested with PNGaseF either with or without prior denaturation with SDS. As before, the PNGaseF digestion decreased the apparent molecular weight of the ligand as assessed by SDS-PAGE and autoradiography.
  • HL-60 cells were cultured in media containing radioactive [6- 3 H3glucosamine, as described above.
  • This precursor is efficiently converted by cells to radioactive GlcNAc, GalNAc, and sialic acid.
  • the ligand was then purified by affinity chromatography on a column of P-selectin-Affigel 15TM and the radiolabeled material was digested with the commercial protease preparation called Pronase.
  • the Pronase-derived glycopeptides were treated with A. ureafaciens neuraminidase which released approximately 25% of the radioactivity as N- acetylneuraminic acid.
  • glycopeptides were then hydrolyzed in strong acid (2 N HC1 for 4 h at 100'C and the hydrolyzed material (minus the sialic acid which is destroyed by this treatment) was analyzed by both high performance anion exchange chromatography on a PA-1 Dionex column and by descending paper chromatography of the material after reacetylation by treatment with acetic anhydride according to standard procedures.
  • the remaining radioactivity in the glycopeptides was composed of N-acetylgalactosamine (GalNAc) and N- acetylglucosamine (GlcNAc) in the approximate ratio of 1:2, respectively.
  • each mole of ligand contains approximately 23 moles of fucose, 7 moles of GalNAc and 20 moles of GlcNAc.
  • the P-selectin ligand from HL-60 cells was purified from cells grown in media containing either [2- 3 H]mannose or [ 14 C]fucose. These precursors allow specific radiolabeling of mannose and fucose residues, respectively. Both radioactive mannose and fucose were recovered in the purified P-selectin ligand, confirming that it, like the neutrophil ligand, contains both mannose and fucose.
  • 3 H-fucose-labeled ligand is treated with mild base and sodium borohydride to effect beta-elimination, 3 H-fucose- labeled oligosaccharides are released that are both high molecular weight and moderate molecular weight, as estimated by chromatography on a column of BioGelTM P-10.
  • the unreleased N-linked oligosaccharides (now contained on a base-hydrolyzed peptide) elute in a peak near the void volume.
  • the / ⁇ -elimination reaction was also performed on the 120 kD glycoprotein ligand derived from human neutrophils. In that case the ligand was post-radiolabeled on its sialic acid by periodate oxidation followed by reduction with NaB 3 H 4 .
  • the 3-eliminated material from the neutrophil ligand eluted in a similar position on the BioGelTM P-10 column.
  • O-sialoglycoprotease a novel protease from Pasteurella hemolytica termed O-sialoglycoprotease. This enzyme cleaves the peptide backbone of proteins containing relatively "clustered" sialylated Ser/Thr-linked oligosaccharides (Norgard, et al., J. Biol . Chem . , 268 : 12764-12774 (1993); Sutherland, D.R. , et al., J. Immunol . , 148, 1458-1464 (1992).
  • the enzyme cleaves the peptide backbone of proteins containing relatively "clustered" sialylated Ser/Thr-linked oligosaccharides (Norgard, et al., J. Biol . Chem . , 268 : 12764-12774 (1993); Sutherland, D.R. , et al., J. Immunol . , 148, 1458-1464
  • [ 3 H3glucosamine-labeled P-selectin ligand was purified as described above from HL-60 cells and from human neutrophils. The latter were radiolabeled as above with periodate/NaB 3 H 4 treatment. The radiolabeled glycoproteins were analyzed by SDS-PAGE and fluorography before and after treatment with the O-sialoglycoprotease. Treatment with the O-sialoglycoprotease caused extensive degradation of the ligand. Interestingly, treatment of intact HL-60 cells with the 0- sialoglycoprotease abolished their interaction with purified membrane P-selectin, as evidenced by flow cytometric analysis and cell adhesion to immobilized P-selectin (Norgard, et al.
  • the 120 kP glycoprotein ligand from human neutrophils was purified by affinity chromatography on a column of immobilized soluble truncated P-selectin (tPS) (Ushiyama et. al. J. Biol . Chem . , 268 : 15229-15237 (1993)).
  • tPS immobilized soluble truncated P-selectin
  • the material was analyzed by SDS-PAGE in reducing conditions, transferred to ImmobilonTM membrane, and probed for its reactivity with radioiodinated P-selectin and the monoclonal antibody CSLEX-1, which reacts with SLe x .
  • the 120 kD glycoprotein eluted from the P-selectin affinity column reacts with 125 I-P-selectin.
  • the radiolabeled ligand bound to both immobilized antibodies, as well as to immobilized P-selectin, as expected.
  • Reactivity with immobilized CSLEX-1 was destroyed by A. ureafaciens neuraminidase treatment of the ligand.
  • Reactivity to the LeuM-1 was destroyed by treatment of the radioiodinated ligand with the Streptomyces sp. ⁇ l,3/4 fucosidase. Fucosidase treatment did not affect binding to P- selectin. This is not surprising, since other data indicated that the Streptomyces sp. 0.1,3/4 fucosidase cannot release fucose from sialylated oligosaccharides containing SLe x epitopes.
  • P-selectin Ligand Contains Poly-N-acetyllactosamine Seguences
  • This enzyme is an endoglycosidase that cleaves certain oligosaccharides containing the repeating unit [ ⁇ 3Galj8l ⁇ 4GlcNAcj8l ⁇ 3 n , where n > 2, at internal ⁇ -galactosyl residues. These chains constitute the so-called poly-N-acetyllactosamine sequence (or polylactosaminoglycan) .
  • these polyfucosylated and sialylated polylactosaminoglycans are not highly sensitive to endo-/3-galactosidase because of the terminal sialic acids.
  • HL-60 cells were metabolically-radiolabeled with Na 35 S0 4 to examine whether the 120 kP ligand for P- selectin is sulfated. Approximately 2 x 10 6 cells/ml were grown in media containing 0.15 mCi/ml of Na 35 S0 4 for 48 h in RPMI supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin. The P- selectin glycoprotein ligand was then purified as described above. Fractions (1 ml) from the P-selectin-immobilized affinity column were collected and radioactivity monitored by liquid scintillation counting.
  • the metabolically- radiolabeled glycoprotein eluted from the P- selectin column was precipitated by addition of ice-cold trichloroacetic acid (10% final) .
  • the pellets were washed with 1 ml of cold acetone, 0.2% HC1, resuspended in Laemmli sample buffer, and analyzed by SPS-PAGE in 7.5% acryla ide.
  • the gel was processed for fluorography with EN 3 HANCETM according to the manufacturer's instructions.
  • the dried gel was exposed to Fuji RX film at -80'C for 7 days. The results indicated that the 120 kP glycoprotein bound by P-selectin is radiolabeled by
  • the differential mobility of the major ligand during SDS-PAGE in the presence and absence of reducing agents indicates that the native ligand is a disulfide-linked homodimer.
  • a homodimeric ligand with two equivalent binding sites might enhance the avidity of the interaction with P-selectin.
  • the ability of [ 125 I]P-selectin to bind to the ligand after reduction and denaturation with SDS suggests that higher order structural features of the protein are not critical for recognition.
  • the blotting assay also detected two minor ligands.
  • the first has an approximately 250,000 M r under reducing conditions. Because its mobility is identical to that of the major ligand under nonreducing conditions, it may represent a subpopulation of the major ligand that is resistant to reduction.
  • the second has an approximately 160,000 M t under nonreducing conditions. Binding of P-selectin to both minor ligands was Ca 2+ -dependent and blocked by the MAb Gl.
  • L-selectin which is expressed on leukocytes and binds to sialylated structures on endothelial cells, interacts preferentially with 50,000 D and 90,000 D sulfated, fucosylated glycoproteins from murine peripheral lymph nodes (Imai, et al., J. Cell Biol . , 113 : 1213-1222 (1991)).
  • P-selectin and L-selectin appear to interact with a small subset of glycoprotein ligands.
  • L-selectin on neutrophils carries the sialyl Le x epitope and that a MAb to L-selectin partially blocks neutrophil adhesion to cells transfected with P-selectin cDNA (Picker, et al., Cell , 66: 921-933 (1991)). Based on these observations, it was proposed that L- selectin on neutrophils is a predominant ligand for P-selectin. However, no direct interaction of L- selectin with P-selectin was demonstrated. Binding of P-selectin to L-selectin in neutrophil membrane extracts was not detectable.
  • a recombinant P-selectin IgG chimera was shown to bind to myeloid cells and to a sulfatide, Gal(3- S0 4 ) Bl-Ceramide by Aruffo et al., Cell , 67: 35-44 (1991) .
  • Sulfatide also inhibited interaction of the chimera with monocytoid U937 cells, as reported by Aruffo et al., (1991). It was not demonstrated whether binding of the P-selectin chimera to the cells or to sulfatide was Ca 2+ dependent, a fundamental characteristic of selectin-dependent cellular interactions.
  • Protease digestion of intact cells should increase the accessibility of P-selectin to potential glycolipid ligands such as sulfatides.
  • protease treatment abolishes binding of P-selectin to neutrophils and HL-60 cells as well as adhesion of neutrophils to immobilized P-selectin.
  • erythrocytes and platelets express sulfatides, they do not specifically interact with P-selectin. Thus, it seems unlikely that sulfatides are the principal mediators of adhesion of myeloid cells to P-selectin. It remains to be determined whether sulfatides inhibit binding of P-selectin to myeloid cells by specific competition with a glycoprotein ligand or by indirect effects. Because the P- selectin ligand described herein is sulfated, it may contain structural features that are mimicked by sulfatides.
  • sialyl Le x inhibits interactions of myeloid cells with P-selectin.
  • CHO cells transfected with a fucosyltransferase express sialyl Le x yet bind P- selectin with significantly lower affinity than do myeloid cells (Zhou et al., (1991)).
  • HT-29 cells which also express sialyl Le x , do not interact at all with P-selectin (Zhou et al., 1991) .
  • neutrophil membrane proteins known to carry the sialyl Le x structure are distinct from the major glycoprotein ligand identified herein and do not bind P-selectin in the assays described here. These observations suggest that the ligand contains structural features in addition to the sialyl Le x tetrasaccharide that enhance the affinity and/or specificity of its interaction with P-selectin.
  • a blotting assay of neutrophil and HL-60 cell membrane extracts was used to search for ligands for P-selectin. As described previously in WO 92/01718, [ 125 1 P-selectin bound preferentially to a glycoprotein of Mr 120,000 as assessed by SDS-PAGE under reducing conditions.
  • the ligand for P-selectin had an apparent Mr of 250,000, suggesting that it is a disulfide-linked homodimer.
  • the ligand was partially purified by serial affinity chromatography on wheat germ agglutinin (WGA) and P-selectin affinity columns. Proteins bound to the P-selectin column were eluted with EDTA. The glycoprotein ligand was greatly enriched in the EDTA eluate from the P-selectin column, as assessed by the intensity of the band identified by [ 125 13P-selectin blotting.
  • the ligand stained poorly with silver, consistent with its being an unusually heavily glycosylated protein.
  • the only contaminating protein present noted by silver staining of the gel was a small amount of P-selectin itself which had been leached from the affinity column.
  • the ligand has now been isolated free from contaminants. This conclusion is based on observation that there are no silver staining bands present but the ligand is clearly identified by its ability to interact with [ 125 13P-selectin in the blotting assay.
  • a form of the ligand in which the carbohydrate components are radiolabeled has also been purified by P-selectin affinity chromatography, as described above. SDS-PAGE analysis of the P-selectin column eluate, followed by fluorography, indicates that the only labeled protein has an Mr of 250,000 under nonreducing conditions and 120,000 under reducing conditions.
  • the radiolabeled ligand has the same shifts in electrophoretic mobility following treatment with sialidase or PNGase F. Thus, all the features of the radiolabeled ligand correspond to those of the ligand identified by the P-selectin blotting assay.
  • the glycoprotein ligand for P- selectin from myeloid cells has the characteristics of a disulfide-linked homodimer with each subunit having an apparent Mr of 120,000 as assessed by SDS-PAGE.
  • the protein has some N-linked carbohydrate but its most striking feature is the presence of a large number of clustered sialylated O-linked glycans, most of which appear to be larger than the usual simple O-linked chains cleaved by O- glycanase.
  • the ligand contains the sLe x structure, the data indicate that additional structural features in the ligand are required to confer high affinity binding to P-selectin.
  • Antibodies to the ligand, fragments thereof, or its carbohydrate or polypeptide components can be prepared by methods known in the art (e.g., Harlow, E. and Lane, D., in Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) . Such antibodies can be used for the detection of human disorders in which P- selectin ligands might be defective. Such disorders would most likely be seen in patients with increased susceptibility to infections in which leukocytes might not be able to bind to activated platelets or endothelium. Cells to be tested, usually leukocytes, are collected by standard medically approved techniques and screened. Detection systems include ELISA procedures, binding of radiolabeled antibody to immobilized activated cells, flow cytometry, immunoperoxidase or immunogold analysis, or other methods known to those skilled in the arts.
  • Antibodies directed specifically to protein or carbohydrate components of the ligand can be used to distinguish defects in expression of the core protein or in glycosyltransferases and/or modifying enzymes that construct the proper oligosaccharide chains on the protein.
  • the antibodies can also be used to screen cells and tissues other than leukocytes for expression of the protein or carbohydrate components of the ligand for P- selectin.
  • nucleic acid probes for use in cloning and detecting nucleic acid sequences, e.g., in genomic or cDNA libraries, encoding the polypeptide component of the P- selectin ligand.
  • nucleic acid sequences e.g., in genomic or cDNA libraries
  • degenerate nucleic acid probes can be synthesized which possess all possible codons for each amino acid sequence, using standard oligonucleotide synthetic methods (see, e.g., Sambrook et al., In Molecular Cloning: A Laboratory Manual, second ed..
  • Such synthetic probes can be labeled by any of a variety of methods and used to screen human genomic and cDNA libraries for nucleic acid molecules encoding the structural coding sequence for the P-selectin ligand (see, e.g., Sambrook et al., 1989).
  • antibodies directed against the ligand, or its carbohydrate or polypeptide components can also be used to screen cDNA libraries for expression of ligand protein to identify clones containing nucleic acid molecules encoding the ligand.
  • Complementary DNA clones encoding the polypeptide component of the ligand can be isolated and sequenced.
  • RNA transcripts for the ligand can be used as diagnostic reagents to examine expression of RNA transcripts for the ligand in leukocytes and other tissues by standard procedures such as Northern blotting of RNA isolated from cells and in situ hybridization of tissue sections.
  • a similar approach can be used to determine qualitative or quantitative disorders of P-selectin itself.
  • the glycoprotein ligand, carbohydrates, or appropriate derivatives thereof, is labeled and tested for its ability to bind to P-selectin on activated platelets from patients with disorders in which P-selectin might be defective.
  • the ligand, or components thereof, can also be used in assays of P-selectin binding to screen for compounds that block interactions of P-selectin with the ligand.
  • P-selectin has several functions related to leukocyte adherence, inflammation, tumor metastases, and coagulation
  • compounds which interfere with binding of P-selectin and/or the other selectins including E-selectin and L- selectin, such as the carbohydrates, can be used to modulate these responses.
  • These compounds include the P-selectin ligand, antibodies to the ligand, and fragments thereof.
  • the glycoprotein ligand, or components thereof, particularly the carbohydrate moieties can be used to inhibit leukocyte adhesion by competitively binding to P-selectin expressed on the surface of activated platelets or endothelial cells.
  • antibodies to the ligand can be used to block cell adhesion mediated by P-selectin by competitively binding to the P-selectin ligand on leukocytes or other cells.
  • These therapies are useful in acute situations where effective, but transient, inhibition of leukocyte-mediated inflammation is desirable.
  • treatment of chronic disorders may be attained by sustained administration of agents, for example, by subcutaneous or oral administration.
  • An inflammatory response may cause damage to the host if unchecked, because leukocytes release many toxic molecules that can damage normal tissues. These molecules include proteolytic enzymes and free radicals.
  • Examples of pathological situations in which leukocytes can cause tissue damage include injury from ischemia and reperfusion, bacterial sepsis and disseminated intravascular coagulation, adult respiratory distress syndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.
  • Reperfusion injury is a major problem in clinical cardiology.
  • Therapeutic agents that reduce leukocyte adherence in ischemic myocardium can significantly enhance the therapeutic efficacy of thrombolytic agents.
  • Thrombolytic therapy with agents such as tissue plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death.
  • tissue plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death.
  • many such patients still suffer myocardial neurosis despite restoration of blood flow.
  • This "reperfusion injury” is known to be associated with adherence of leukocytes to vascular endothelium in the ischemic zone, presumably in part because of activation of platelets and endothelium by thrombin and cytokines that makes them adhesive for leukocytes (Romson et al.. Circulation, 67 : 1016-1023 (1983)). These adherent leukocytes can migrate through the endothelium and destroy ischemic myocardium just as it is being rescued by restoration of blood flow.
  • Leukocyte-dependent organ damage is an important feature of these conditions.
  • LAK cells interleukin-2 treated LAK cells (lymphokine-activated lymphocytes) .
  • LAK cells are known to adhere to vascular walls and release products that are presumably toxic to endothelium. Although the mechanism by which LAK cells adhere to endothelium is not known, such cells could potentially release molecules that activate endothelium and then bind to endothelium by mechanisms similar to those operative in neutrophils. Tumor cells from many malignancies, including carcinomas, lymphomas, and sarcomas, can metastasize to distant sites through the vasculature.
  • Platelet-leukocyte interactions are believed to be important in atherosclerosis. Platelets might have a role in recruitment of monocytes into atherosclerotic plaques; the accumulation of monocytes is known to be one of the earliest detectable events during atherogenesis. Rupture of a fully developed plaque may not only lead to platelet deposition and activation and the promotion of thrombus formation, but also the early recruitment of neutrophils to an area of ischemia. Another area of potential application is in the treatment of rheumatoid arthritis.
  • the glycoprotein ligand of P-selectin comprising peptides having the sequences HMYPVR and PGLTPEP, or fragments of the ligand that retain P-selectin binding ability, can be administered to block selectin-dependent interactions by binding competitively to P-selectin expressed on activated cells.
  • carbohydrate components of the ligand which play a key role in recognition by P-selectin, can be administered alone, as well as attached to all or a fragment of the polypeptide component of the ligand.
  • natural or synthetic analogs of the ligand or its fragments which bind to P- selectin can also be administered to a patient to block P-selectin dependent interactions.
  • antibodies to the polypeptide and/or carbohydrate components of the ligand, or fragments thereof can be administered.
  • the antibodies are preferrably of human origin or modified to delete those portions most likely to cause an immunogenic reaction.
  • the ligand, or fragments thereof, carbohydrate components of the ligand, and antibodies to the ligand molecule or its carbohydrate or polypeptide components, in an appropriate pharmaceutical carrier are preferably administered intravenously where immediate relief is required. Other modes of administration include intramuscularly, intraperitoneally, subcutaneously, and orally.
  • the carbohydrate component of the ligand may also be conjugated to a carrier molecule, or incorporated into a drug delivery device for more effective and prolonged delivery to a patient.
  • the carbohydrate can also be modified chemically to increase its in vivo half-life.
  • the carbohydrate can be isolated from cells expressing the carbohydrate, either naturally or as a result of genetic engineering as described in the transfected COS cell examples, or, preferably, by synthetic means. These methods are known to those skilled in the art.
  • a large number of glycosyltransferases have been cloned (J.C. Paulson and K.J. Colley, J. Biol . Chem . , 264 : 17615-17618 (1989)). Accordingly, workers skilled in the art can use a combination of synthetic chemistry and enzymatic synthesis to make pharmaceuticals or diagnostic reagents.
  • the P-selectin glycoprotein ligand and protein fragments of the ligand can also be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • Carbohydrate and polypeptide components and fragments of the glycoprotein ligand of P-selectin that are biologically active are those which, like the full-length P-selectin glycoprotein ligand, inhibit binding of leukocytes to P-selectin.
  • Suitable pharmaceutical vehicles for administration to a patient are known to those skilled in the art.
  • a biologically active carbohydrate or protein fragment of the P- selectin glycoprotein ligand, or the entire P-selectin ligand will usually be dissolved or suspended in sterile water or saline.
  • a carbohydrate component of the P- selectin glycoprotein ligand, the P-selectin glycoprotein ligand, and fragments thereof will be incorporated alone, or in combination into an inert carrier in tablet, liquid, or capsular form.
  • Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
  • the carbohydrate, ligand, or fragments thereof can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
  • a carbohydrate component of the ligand, the ligand, or fragments thereof may also be administered in, on or as part of, liposomes or microspheres (or microparticles) .
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream.
  • the carbohydrate components of the P-selectin ligand, the ligand, or fragments thereof can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patent No. 4,906,474, 4,925,673, and 3,625,214.
  • the carbohydrates should be active when administered parenterally in amounts above about 1 ⁇ g/kg of body weight.
  • the dosage range will be between 0.1 to 30 mg/kg of body weight.
  • a dosage of 70 mg/kg may be required for some of the carbohydrates characterized in the examples.
  • the criteria for assessing response to therapeutic modalities employing the P-selectin glycoprotein ligand, fragments thereof, carbohydrate components of the P-selectin glycoprotein ligand, or antibodies to the ligand or its carbohydrate or polypeptide components is dictated by the specific physiological and pathological condition of the patient and will generally follow standard medical practices.
  • the criteria for the effective dosage to prevent extension of myocardial infarction would be determined by one skilled in the art by looking at marker enzymes of myocardial necrosis in the plasma, by monitoring the electrocardiogram, vital signs, and clinical response.
  • NAME Pabst, Patrea L.

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Abstract

On a découvert par des techniques de transfert et par chromatographie par affinité d'extraits de cellules HL-60 marquée par la [3H]glucosamine sur de la P-sélectine immobilisée que la P-sélectine se lie principalement à un seul ligand glycoprotéique sur des neutrophiles et des cellules HL-60. Cette molécule a été caractérisé et distinguée d'autres protéines membranaires de neutrophiles bien caractérisées, de masse moléculaire apparente similaire. On peut utiliser le ligand purifié, ou des fragments de celui-ci, y compris les composants glucidique et protéiques, ou encore des anticorps dirigés contre le ligand, ou des fragments ou des composants de ceux-ci, en tant qu'inhibiteurs de fixation de la P-sélective à des cellules.
EP94903270A 1992-11-16 1993-11-16 Ligand clycoproteique pour la p-selectine et son procede d'utilisation Ceased EP0668907A1 (fr)

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AU681369B2 (en) 1997-08-28
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WO1994011498A1 (fr) 1994-05-26
CA2151142A1 (fr) 1994-05-26

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