EP0575557A1 - Verfahren zur hemmung von padgem-vermittelter interaktionen unter verwendung von einer 2,6-sialinsäure - Google Patents

Verfahren zur hemmung von padgem-vermittelter interaktionen unter verwendung von einer 2,6-sialinsäure

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Publication number
EP0575557A1
EP0575557A1 EP92910663A EP92910663A EP0575557A1 EP 0575557 A1 EP0575557 A1 EP 0575557A1 EP 92910663 A EP92910663 A EP 92910663A EP 92910663 A EP92910663 A EP 92910663A EP 0575557 A1 EP0575557 A1 EP 0575557A1
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EP
European Patent Office
Prior art keywords
padgem
cell
cells
inhibitor
sialic acid
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP92910663A
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English (en)
French (fr)
Inventor
Bruce Furie
Barbara C. Furie
Eric Larsen
Theresa Palabrica
Susan Sajer
Gary E. Gilbert
Denisa D. Wagner
Alessandro Celi
John Erban
Rosemary Gibson
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New England Medical Center Hospitals Inc
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New England Medical Center Hospitals Inc
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Application filed by New England Medical Center Hospitals Inc filed Critical New England Medical Center Hospitals Inc
Publication of EP0575557A1 publication Critical patent/EP0575557A1/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/78—Polymers containing oxygen of acrylic acid or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere

Definitions

  • LECCAMs or selectins including Mel-14 antigen, LAM-1 (LECAM1) , ELAM-1 (LECAM2), and PADGEM (LECAM3) , are a newly recognized class of cellular adhesion molecules that are characterized structurally by the presence of a lectin-like domain, an epidermal growth factor-like domain, a variable number of cysteine-rich repeats related to those found in a family of complement regulatory proteins, a transmembrane domain, and a short cytoplas ic tail (Osborn, L. , Cell 62; 306 (1990)). These cell adhesion molecules are thought to function in the adhesion of leukocytes to endothelial cells.
  • LAM-1 the Mel-14 antigen and its human analog, LAM-1, which are present on the surface of lymphocytes, are thought to be involved in the targeting of lymphocytes to endothelial cells within high endothelial venules (Siegelman, M.H. et al. , Science, 243: 1165-1172 (1989); Tedder, T.F. et al., J. Exp. Med., 170: 123-133,(1989)).
  • ELAM-1 endothelial-leukocyte adhesion molecule
  • the cell adhesion molecule PADGEM platelet activation dependent granule-external membrane protein
  • PADGEM platelet activation dependent granule-external membrane protein
  • the platelet activation dependent granule-external membrane protein, PADGEM has been cloned and has a typical LECCAM structure, with a lectin domain, an epidermal growth factor domain, nine complement binding repeat domains, a " transmembrane domain, and a cytoplasmic domain
  • PADGEM which is also referred to as GMP-140, CD62, or LECAM3 , is found on the endothelial cell surface as well as on the surface of platelets. PADGEM is ° also found in megakaryocytes, which are the precursors of platelets. (Beckstead et al. , Blood 67: 285-293 (1986)). In endothelial cells, PADGEM is stored as a component of the eibel-Palade bodies (Bonfanti, R. et al..
  • PADGEM and ELAM-1 are lectins that bind to lineage-specific carbohydrates on the surface of certain leukocytes (Larsen et al. , Cell 63: 467-474 (1990); Lowe et al.. Cell 63: 475-484 (1990)).
  • the data suggest both ligands have a common Le core.
  • surface carbohydrate structures could contribute to the specificity of the cell-cell interactions mediated by PADGEM and ELAM-1.
  • the present invention relates to a method of inhibiting (reducing or preventing) the interaction or adhesion of a PADGEM-bearing cell with a cell bearing a PADGEM ligand by contacting the
  • PADGEM-bearing cell with an inhibitor comprising a 2,6-linked sialic acid component under conditions whereby adhesion or interaction is inhibited.
  • a PADGEM-bearing cell such as a platelet or endothelial cell
  • a cell bearing a PADGEM ligand such as a white blood cell (leukocyte)
  • leukocytes e.g., neutrophils, monocytes
  • antibodies directed against the CD15 cell surface antigen inhibit the interaction of PADGEM-bearing cells (e.g. platelets and COS cells bearing PADGEM) with leukocytes (e.g. neutrophils, monocytes) .
  • PADGEM-bearing cells e.g. platelets and COS cells bearing PADGEM
  • leukocytes e.g. neutrophils, monocytes
  • LNF III Lacto-N-fucopentaose
  • PADGEM can interact with a Le core (e.g., GaL3l-4(Fuc ⁇ l-3)GlcNAc) and that inhibitors comprising a Le core can inhibit PADGEM-mediated
  • neuraminidase treatment of HL60 cells decreased PADGEM-mediated binding, suggesting that sialic acid may also be part of the PADGEM ligand.
  • ELAM-1 has also been shown to nr X X D recognize a sialyated Le (SLe ) core structure, sialyl 2,3 Le x and related structures, as shown herein, a distinct PADGEM ligand is implicated.
  • the invention further relates to inhibitors of PADGEM-mediated cell-cell interaction.
  • Inhibitors of PADGEM-mediated cell-cell interaction useful in the present method comprise a 2,6-linked sialic acid (NeuAc) component.
  • useful inhibitors can comprise Neu5Acc.2,6Gal-, an ⁇ 2,6 sialylated Le core (e.g., NeuAc ⁇ 2,6Gal)Sl-4(Fuc ⁇ l-3)NAcGlc) or other ⁇ 2,6 sialylated ⁇ (l-3) fucosylated lactosamines or polylactosa ines.
  • An inhibitor comprising a 2,6-linked sialic acid component can further comprise a CD15 immunoreactive component, such as Le x or all or a portion of Le x or LNF-III.
  • Figure 1 illustrates the effects of a panel of anti-leukocyte antibodies on the interaction of neutrophils and activated platelets.
  • the percent adherence corresponds to the percentage of cells with two or more adherent platelets under the assay conditions.
  • Figure 2 illustrates the inhibitory effects of 80H5 monoclonal antibody on the interaction of thrombin-stimulated platelets with monocytes (Mono) , neutrophils (PMN) , U937 cells (U937) and HL60 cells (HL60) .
  • the height of the bar indicates the percent binding or percentage of cells with two or more adherent platelets in the absence of antibody (black bars) or in the presence of antibody (hatched bar) .
  • Figure 3 illustrates the effect of the concentration of anti-CD15 antibody 7C3 on the inhibition of the binding of activated platelets to neutrophils. The percent adherence corresponds to the percentage of cells with two or more adherent platelets under the assay conditions.
  • Figure 4 illustrates the inhibition of adherence
  • Figure 5 illustrates the results of a FACS analysis of the interaction of U937 cells with phospholipid vesicles containing purified PADGEM.
  • a histogram of log red fluorescence is given on the X axis and cell number is given on the Y axis.
  • U937 binding to phospholipid vesicles without PADGEM (dotted line)
  • to phospholipid vesicle containing PADGEM (dashed and dotted line)
  • to phospholipid vesicles containing PADGEM in the presence of anti-CD15 antibody solid line
  • Figure 6 illustrates the inhibitory effects of LNF isomers, LNF I (open squares) , LNF II (closed circles) , and LNF III (closed squares) , on the interaction of activated platelets and neutrophils.
  • Figure 7 illustrates the effects of LNF isomers LNF I (open squares) , LNF II (closed circles) , and LNF III (closed squares) , on the interaction of HL60 cells with COS cells expressing PADGEM. Standard errors of triplicate experiments are given by the error bars.
  • Figure 8 illustrates the results of an adhesion assay in which the adhesion of HL60 cells to CHO-PADGEM (hatched bar) or CHO-ELAM (black bar) cells treated with A. ureafaciens, V. cholerae or Newcastle disease virus neuraminidase was monitored. HL60 cell binding to neuraminidase-treated cells is recorded as a percent of binding observed with untreated control cells.
  • Figure 9 illustrates the inhibition of adhesion of HL60 cells to CHO-PADGEM (open circles) or
  • CHO-ELAM (filled circles) cells by purified PADGEM as a function of PADGEM concentration (xg/ml) .
  • Figure 10 illustrates the effect of Sambucus nigra lectin on the adhesion of HL60 cells to CHO-PADGEM (filled circles) or CHO-ELAM (open circles) as a function of the concentration ( ⁇ g/ml) of Sambucus nigra lectin. Binding of HL60 cells to lectin-treated cells is recorded as a percent of the HL60 cell binding to untreated control CHO-PADGEM or CHO-ELAM cells.
  • the present invention relates to a method of inhibiting (reducing or preventing) the interaction of a cell bearing PADGEM with its target ligand by contacting the cell with an inhibitor comprising a 2,6 linked sialic acid component.
  • the invention further relates to a method of inhibiting (reducing or preventing) the interaction or adhesion of endothelial cells or platelets with leukocytes (i.e.. white blood cells) , especially with nonlymphocytic leukocytes such as neutrophils and monocytes, by contacting the endothelial cells or platelets with an inhibitor comprising a 2,6-linked sialic acid component.
  • leukocytes i.e. white blood cells
  • nonlymphocytic leukocytes such as neutrophils and monocytes
  • a PADGEM-bearing cell such as a platelet or endothelial cell
  • a cell bearing a PADGEM ligand e.g., neutrophils and monocytes
  • a PADGEM ligand e.g., neutrophils and monocytes
  • CD15 is a carbohydrate antigen associated with glycolipids, glycoproteins, and proteoglycans (Kobata and Ginsburg, J. Biol. Chem., 244: 5496-5502 (1969) ; Yang and Hakomori, J. Biol. Chem., 246: 1192-1200 (1971); Huang et al.
  • this carbohydrate is a marker for adenocarcinoma of the lung, colon and stomach, and for certain forms of lymphoma (Hall and Ardenne, J. Clin. Pathol., 40: 1298-1304 (1987); Sanders et al. , J. Pathol., 154: 255-266 (1988)).
  • the CD15 antigen is a component of glycolipids (Fukuda et al. , J_-_ Biol. Chem., 260: 1067-1082 (1985)), glycoprotein
  • O-linked oligosaccharides (Carlsson et al. , J. Biol. Chem. , 261: 1287-12951986), and glycoprotein N-linked oligosaccharides (Fukuda et al. , J. Biol. Chem. , 260: 12957-12967 (1985)) on human granulocytes.
  • glycoproteins present on the leukocyte surface have been shown to carry CD15 antigens and include LFA-1, Mac-1, gpl50,95 (CD11/CD18) , and CR1 (the C3b receptor) as well as proteins with molecular weights of 105,000 and 145,000 and a phosphotyrosine- containing protein of about 180,000 (Albrechtsen and Kerr, Br. J. Haematol., 72: 312-320 (1989); Skubitz et al., J. Immunol., 141: 4318-4323 (1988)). Although these proteins may be distributed among many vascular cell types, only on specific leukocytes, such as neutrophils and monocytes, do their structures include the complex carbohydrate LNF III.
  • LNF III As shown in Example 4, purified forms of LNF III inhibit the interaction of activated platelets with neutrophils and monocytes. COS cells expressing PADGEM were shown to bind to HL60 and U937 cells, whereas COS cells not expressing PADGEM did not; this interaction was inhibited by LNF III or anti-CD15 antibodies (Example 4) . Thus, inhibition by LNF III involves a process that is mediated by PADGEM on activated platelets.
  • LNF III or a portion thereof is a component of the PADGEM ligand.
  • the LNF isomers are structurally closely related. They are composed of the same monosaccharides, but differ in the covalent linkages of these monosaccharides to form the pentassaccharide chain. LNF III binds more tightly to PADGEM, whereas LNF I demonstrates little or no interaction with PADGEM. LNF II, however, demonstrated slight inhibitory activity, particularly when the LNF to PADGEM ratio was high. Possibly minor contamination of the LNF II preparation with LNF III could account for this observation.
  • LNF-III has features preferentially recognized by PADGEM.
  • a Le core comprising GalSl-4(Fuc ⁇ l-3)GlcNAc, is unique to LNF III.
  • ⁇ (l-3) fucosylated structures such as ⁇ (l-3) fucosylated lactose or lactosamine are recognized by PADGEM.
  • PADGEM ligand comprises a CD15 immunoreactive carbohyd ate, such as Le x or all or portion of LNF-III.
  • An inhibitor comprising this structure or one which mimics the CD15 positive antigen on the surface of leukocytes can interfere with PADGEM-mediated interactions.
  • PADGEM-bearing cell e.g., a platelet, an endothelial cell
  • a PADGEM ligand such as a neutrophil or a monocyte
  • an inhibitor comprising a Le core component.
  • LNF-III a complex carbohydrate which comprises a Lex core component and i.s recogni•zed by CD15 antibodies, inhibits the binding of stimulated platelets to neutrophils ( Figure 6) .
  • LNF III also inhibits the interaction of HL60 cells (monocyte-like cells) with COS cells that were transfected with PADGEM ( Figure 7) .
  • COS cells are fibroblast-like SV40-transformed African Green Monkey kidney cells. Therefore, LNF III inhibits the adhesion involving cells which naturally express PADGEM (e.g. neutrophils and monocytes) , as well as adhesion involving cells artificially induced to express PADGEM (e.g. PADGEM-transfected cells).
  • useful inhibitors can comprise a CD15 immunoreactive carbohydrate, such as LNF III.
  • ELAM also recognizes a Le core structure on the surface of leukocytes.
  • ⁇ 2,3 sialyl Le x (SLe x ) and related structures have been suggested as the ELAM ligand (Lowe et al.. Cell 63: 475-484 (1990); Phillips et al. , Science 250: 1130-1132 (1990); alz et al. , Science 250: 1132-1135 (1990)).
  • neuraminidase treatment of leukocytes greatly decreases PADGEM-mediated interaction has suggested that sialic acid may also be a part of the PADGEM ligand (Corral et al. , Biochem. Biophys. Res. Comm.
  • SLe x could be the ligand for both PADGEM and ELAM. Since PADGEM and ELAM both appear to interact with monocyte and neutrophil surface structures, the question arises of whether there are structural differences between the PADGEM and ELAM ligands, and what those differences are.
  • FIG. 8 shows the results of experiments showing that neuraminidase treatment of HL60 cells, which cleaves sialic acid residues from cell surface molecules markedly diminished the interaction of HL60 cells with CHO cells transfected with PADGEM or ELAM, consistent with the presence of a sialic acid residue in both ligands.
  • results of a competition assay indicate that the dominant PADGEM and ELAM ligands, while sharing some features are distinguishable.
  • purified PADGEM almost completely interfered with the ability of HL60 cells to bind to CHO-PADGEM transfectants.
  • purified PADGEM only partially inhibited the adhesion of HL60 cells to CHO-ELAM transfeetants.
  • the PADGEM and ELAM ligands both characterized by a terminal sialic acid and a branched trisaccharide structure consisting of Gal/3l-4[Fuc ⁇ l-3]GlcNAc (Le x ) , differ in the linkage of the sialic acid.
  • Figure 10 shows the results of an adhesion assay in which a highly specific lectin, which requires a disaccharide of the structure Neu5Ac ⁇ 2-6Gal or Neu5Ac ⁇ 2-6GalNAc, is shown to inhibit PADGEM-leukocyte interaction. Under the same conditions, the specific lectin did not significantly decrease ELAM-leukocyte adhesion.
  • PADGEM-mediated Interactions are components of a protein, characterized by a Le x core component, and a terminal sialic acid linked ⁇ 2,6 to a galactose.
  • the 2,6-linked sialic acid residue could be linked to the galactose of the Le x core. This structure is distinct from sialyl 2,3 Le x previously identified as the ELAM ligand. Inhibition of PADGEM-mediated Interactions
  • An inhibitor comprising all or a portion of a natural PADGEM ligand or one which mimics features of the deduced structure of the PADGEM ligand can inhibit the interaction of a PADGEM-bearing cell with a second cell bearing a PADGEM ligand.
  • a PADGEM-bearing cell For example, the interaction of a platelet or endothelial cell with a cell, such as a monocyte or neutrophil can be inhibited by contacting the platelet or endothelial cell with an inhibitor comprising a 2,6 sialic acid component.
  • PADGEM need not be associated with a cell (e.g., present at the cell surface as a transmembrane protein) for inhibition of the interaction with its target ligand to occur.
  • the interaction may be inhibited by contacting the molecule with an inhibitor.
  • an inhibitor for example, a cDNA encoding a form of PADGEM which lacks the transmembrane region has been isolated from a human umbilical vein endothelial cell cDNA library (Johnston, et al. , Cell 56: 1033-1044) and soluble forms of PADGEM can be constructed using recombinant techniques.
  • the interaction of such truncated versions of PADGEM with a PADGEM-ligand can also be inhibited, reduced or prevented using an inhibitor comprising a 2,6 linked sialic acid component. This method can be useful in counteracting the effect of soluble forms of PADGEM.
  • Inhibitors useful in the present method can be identified by their ability to inhibit (reduce or prevent) the interaction of PADGEM with its target ligand.
  • PADGEM can be in several forms, including, but not limited to, a soluble form, incorporated into a vesicle, such as a liposome or phospholipid vesicle, or associated with a cell (e.g., as a transmembrane protein) .
  • the ligand can also be in several forms, including, but not limited to, a soluble form or associated with a cell (e.g., attached to a cell surface structure such as a glycoprotein or glycolipid) .
  • inhibitors of the interaction of PADGEM with its target.ligand can also be in several forms.
  • inhibitors useful in the present method can comprise a terminally located sialic acid, in which the sialic acid is at the non-reducing end of a saccharide having two or more monosaccharide units.
  • an internally located 2,6-linked sialic acid residue e.g., X-NeuAc2,6-X, where X is at least a monosaccharide
  • inhibitors comprising an internal 2,6 linked sialic acid residue can be used also.
  • Inhibitors useful in the present method comprise a 2,6-linked sialic acid component or residue (i.e., N-acetyl neuraminic acid, NeuAc, or NANA) , especially an or 2-6 linked sialic acid residue.
  • inhibitors useful in the present method can further comprise a galactosyl residue (e.g., galactose, N-acetylgalactose) linked to sialic acid
  • the inhibitor can comprise a sialyl ⁇ 2,6 galactosyl component, in which a sialic acid residue linked to a galactose residue at the C-6 position of the galactose.
  • the inhibitor comprises a sialyl ⁇ 2,6 galactosyl component in addition to a Le x core component.
  • a sialyl ⁇ 2,6 galactosyl component and a Le core component can be part of a single oligosaccharide chain or on separate chains.
  • inhibitors of the present invention can comprise a 2,6-linked sialic acid component and a Le core.
  • an inhibitor can comprise a 2,6-linked sialic acid component and a
  • Lex core in a contiguous sequence such as ⁇ 2,6 sialyl Le (e.g., a branched tetrasaccharide NeuAc ⁇ 2 ,6Galff1-4(Fuc ⁇ l-3)GlcNAc) , or in a non-contiguous sequence within a single saccharide chain or on separate chains (e.g., as in a glycoprotein) .
  • ⁇ 2,6 sialyl Le e.g., a branched tetrasaccharide NeuAc ⁇ 2 ,6Galff1-4(Fuc ⁇ l-3)GlcNAc
  • a Le core component refers to a structure comprising a Le antigen (e.g., a trisaccharide Gal/31-4(Fuc ⁇ l-3)NAcGlc, an ⁇ (1-3)fucosylated lactosamine) or other ⁇ (1-3)fucosylated lactosamines exhibiting similar biological function.
  • a Le x core refers to a structure comprising a structural analog of a Le x antigen, which, alone or as a component of an inhibitor, can inhibit PADGEM-mediated interactions.
  • the inhi•bi•tors can comprise a LeX core or a larger carbohydrate comprising a Lex core component.
  • useful inhibitors can comprise a CD15 immunoreactive carbohydrate comprising a Le core, although the inhibitor itself need not be CD15 immunoreactive.
  • CD15 immunoreactive carbohydrates comprising a Le core are LNF III, or a portion of LNF III comprising a Le x core, and a Le antigen (e.g., Gal / 51-4(Fuc ⁇ l-3)GlcNAc)
  • CD15 antigen is a component of glycolipids, glycoprotein O-linked oligosaccharides, and glycoprotein N-linked oligosaccharides on human granulocytes.
  • a PADGEM ligand is protease sensitive. Possibly, additional carbohydrate, protein or lipid structures of the actual ligand or ligands can contribute to the interaction with surface molecule such as PADGEM, and enhance the specificity of the interaction.
  • useful inhibitors can comprise, for example, a protein or peptide, with a carbohydrate moiety comprising a 2,6-linked sialic acid component or other embodiment described above (e.g., a glycoprotein with N-linked and/or O-linked oligosaccharide(s) ) .
  • a carbohydrate moiety comprising a 2,6-linked sialic acid component or other embodiment described above (e.g., a glycoprotein with N-linked and/or O-linked oligosaccharide(s) ) .
  • NeuAc ⁇ 2,6Gal3l-4(Fuc ⁇ l-3)GlcNAc- can be linked to a protein via the GlcNAc (N-acetylglucosamine) moiety or incorporated into a larger saccharide chain on a protein.
  • inhibitors can comprise a lipid portion (e.g., a phospholipid, ceramide, or sphingolipid) , such as NeuAc ⁇ 2,6Galj8l-ceramide.
  • Inhibitors comprising more than one Le x core or 2,6-linked sialic acid component may have enhanced activity due to multivalency.
  • Inhibitors useful in the method e.g., glycoproteins, glycolipids, carbohydrates
  • Inhibitors can be purified from natural sources.
  • sialylated fucosyl lactosaminoglycans can be isolated from granulocytes (Fukuda, et al. , J. Biol. Chem. 259: 10,925-10,935 (1984)).
  • they can be synthesized chemically or enzymatically using techniques known in the art (Toone, E. et al. , Tetrahedron Rep., 45: 5365-5422
  • the activity of an inhibitor may be monitored using an appropriate assay.
  • the adhesion assays described in Example 2 can be used to assay the inhibitory activity of candidates upon
  • a candidate inhibitor may be identified by its ability to interfere with the interaction between an identified inhibitor (e.g., LNF III) and PADGEM (e.g., purified PADGEM, PADGEM on a cell, PADGEM in a liposome) in a competitive binding assay.
  • an identified inhibitor e.g., LNF III
  • PADGEM e.g., purified PADGEM, PADGEM on a cell, PADGEM in a liposome
  • PADGEM on the surface of platelets is thought to be have an important role in the clotting process. Moreover, PADGEM, which is also present in endothelial cells is thought to be involved in the recruitment of neutrophils and monocytes to sites of inflammation.
  • white blood cells i.e., leukocytes, such as monocytes and neutrophils
  • the inhibition of the interaction of activated platelets with neutrophils and monocytes is inhibited by contacting the platelets with an inhibitor comprising a 2,6 linked sialic acid component.
  • Activated platelets can bind to injured endothelial and subendothelial surfaces through mechanisms involving glycoprotein lb and von Willebrand factor.
  • the expression of PADGEM on these platelets at the site of vascular injury could lead to the binding of monocytes and neutrophils.
  • monocytes and neutrophils are capable of initiating the tissue factor-mediated extrinsic pathway of blood coagulation.
  • the inhibitors of this invention can interfere with platelet-neutrophil or platelet-monocyte interactions to block adhesion and thereby interrupt the coagulation process. Thus, it is possible to inhibit pathological thrombosis using the present method.
  • activated platelets or endothelial cells at the site of tissue injury or inflammation could recruit leukocytes from the blood stream, resulting in the release of inflammatory mediators and causing further tissue damage.
  • an inhibitor comprising a 2,6 linked sialic acid component can inhibit the adhesion of monocytes and neutrophils to platelets or endothelial cells, to prevent or minimize inflam ⁇ mation.
  • autoimmune and inflammatory diseases or conditions can be treated by the present method.
  • Tissue injury such as neutrophil-mediated ischemia-reperfusion damage due to blood vessel occlusion and reperfusion could be inhibited by interfering with adhesion of neutrophils.
  • Contacting platelets bearing PADGEM with an inhibitor comprising a 2,6 linked sialic acid component and/or a Le core can inhibit neutrophil adhesion, minimizing damage in the region of the thrombus.
  • Treatment with clot-dissolving drug, such as tissue plasminogen activator or streptokinase can be accompanied by treatment with an inhibitor comprising a Le core to inhibit reperfusion injury.
  • the inhibitor can also act together with clot-dissolving drugs to inhibit clotting.
  • Atherosclerosis In a model of atherosclerosis, injured endothelial cells in a vessel wall express PADGEM on their surface. Monocytes bearing a PADGEM ligand are recruited to the site by virtue of PADGEM-PADGEM ligand interaction, and adhere to the endothelial cells. The monocytes become pathological foam cells by ingestion of lipids, platelet fragments, and other molecules.
  • the atherosclerotic process can be inhibited by contacting the PADGEM-bearing endothelial cells with an inhibitor of the present invention, which inhibits PADGEM-mediated adhesion.
  • CD15 antigen comprising a Le core
  • CD15 antigen is a marker for adenocarcinoma of the lung, colon .and stomach, and for certain forms of lymphoma
  • ELAM-1 supports the adhesion of a human colon carcinoma cell line to endothelial cells (Rice and Bevilacqua, Science, 246: 1303-1306 (1989) ) .
  • Adhesion to vessel walls and estravasation by certain tumor types may be facilitated by their expression of a ligand for PADGEM. It is possible to disrupt the metastatic process by inhibiting the interaction of PADGEM with complementary tumor cell antigens by the method of the present invention.
  • inhibitors of the present invention are administered by an appropriate route (e.g., intravenously, parenterally or topically) .
  • Treatment is under appropriate conditions and in amounts sufficient to reduce or prevent adhesion and thereby, reduce or prevent the disease process.
  • an inhibitor can be combined with a suitable carrier, incorporated into a liposome, or polymer release system for administration. The invention is further and more specifically described in the following examples.
  • Antibody 80H5 was purchased from AMAC, Inc. Other antibodies were the generous gifts of Drs. Dennis Hickstein and John Harlan (7C3) , Dr. Paul Guyre (PM81, 168, AML-2-23), and Dr. Douglas Faller
  • Platelets were isolated by gel filtration from fresh anticoagulated blood obtained from normal human donors (Hsu-Lin et al. , J. Biol. Chem. , 259: 9121-9126 (1984)). Activated platelets were prepared by incubating cells without stirring for 20 minutes at 22° C with thrombin at a final concentration of 0.25 U/ml. Fresh platelets were used in cell adhesion assays within 30 minutes of preparation. Neutrophils were prepared by the method of English and Anderson (J. Immuno1. Method, 5_: 249-252 (1974)) . The neutrophil preparations were greater than 95% pure by light microscopy.
  • Monocytes were prepared by washing the mono-nuclear leukocyte fraction twice with human serum-5mM EDTA and incubating the cells in RPMI 1640-10% fetal calf serum in sterile plasmid dishes for 2 hours at 37° C. The dishes were washed three times with PBS at 37° C to remove nonadherent cells. PBS at 0° C was added, and the cells were incubated at 4° C for 1 hour. Adherent cells were gently detached with a rubber policeman, washed in PBS, and resuspended in RPMI 1640-1% fetal calf serum.
  • Lymphocytes were obtained by washing the nonadherent cells with PBS and resuspending these cells in RPMI 1640-1% fetal calf serum. The purity of these preparations was established to be greater than 90% by light microscopy using Wright esterase and nonspecific esterase stains.
  • the PADGEM cDNA was cloned from a human umbilical vein cDNA library in ⁇ gtll using oligonucleotides based upon the published DNA sequence (Johnston et al. , Cell 56: 1033-1044 (1989)). Approximately 3 x 10 plaques from an oligo(dT)-primed human umbilical vein endothelial cell cDNA library were transferred to nitrocellulose filters for screening. Duplicate filters were hybridized with either a 32P-labeled 24 nudeotide probe derived from the 5' end of the translated sequence or one from the 3' end of the translated sequence (Johnston et al. , Cell 56: 1033-1044 (1989)).
  • the sequence of the full-length PADGEM cDNA was established in its entirety.
  • the nudeotide sequence obtained was identical to that of Johnston et al. (Cell 56: 1033-1044 (1989)), with the exception of five nudeotides within the coding sequence: T at 1088, C at 1832, C at 1850, C at 99, and C at 859.
  • the latter two sequence differences result is amino acid sequence differences, such that a proline is encoded at residue -21 instead of serine, and a threonine is encoded at residue 233 instead of isoleucine.
  • the other three base changes do not alter the predicted amino acid sequence.
  • the full-length PADGEM cDNA was inserted into a modified form of the expression vector CDM8 (Tedder and Isaacs, J. Immunol., 143: 712-717 (1989)).
  • COS cells (1 X 10 ) were transfected with 40 ⁇ g of the resulting PADGEM expression vector by calcium phosphate precipitation. Coverslips (12 x 12 mm) were added to each culture. After 48 hours of growth in DMEM-10% fetal calf serum, the COS cells were confluent.
  • coverslips were washed with RPMI 1640, and duplicate coverslips were assayed for 111In acti.vi.ty.
  • samples were evaluated for HL60 cell adherence by fluorescence and phase-contrast microscopy using a Zeiss Axioscope microscope in a blind assay.
  • PADGEM was incorporated into phospholipid vesicles as previously described (Larsen et al. , Cell 59: 305-312 (1989)) with some modifications. Briefly, 5 mg of egg phosphatidylcholine (Avanti Polar Lipids) and 0.025 mg of Di IC 16 (3) (1,l'-dihexadec l-3,3,3',3'-tetramethyl-lindocarbocy- anine perchlorate) (Molecular Probes) in chloroform were mixed, and the chloroform was removed by evaporation at 37° C under nitrogen. The dried lipids were resuspended in methylene chloride, and the solvent was removed by evaporation.
  • Vesicles were separated from free protein by gel filtration on a Sepharose 4B column. Phospholipid vesicles (50 ⁇ l) with or without PADGEM were incubated with 2 x 10 5 U937 cells in RPMI 1640, 1% fetal calf serum, 2% bovine serum albumin for 30 minutes at 23° C. For experiments with 80H5 antibody, U937 cells were incubated with the antibody (5 ⁇ g/ml) for 1 hour; phospholipid vesicles were added, and the incubation was continued for an additional 30 minutes. Prior to analysis on a
  • Anti-CD15 Antibodies Inhibit the Platelet-Leukocyte
  • Thrombin-activated platelets bind to human neutrophils, monocytes, HL60 cells and U937 cells in an interaction that is mediated by PADGEM on the surface of the platelet (Larsen et al. , Cell 59: 305-312 (1989) . This interaction is inhibited by anti-PADGEM antibodies and purified PADGEM. Unstimulated platelets, which do not express PADGEM on the platelet surface, do not interact with these leukocytes.
  • the antibodies which were tested and their corresponding antigens are as follows: TS1/18, LFA-1 ( ⁇ ) ; OKM15, CR3; TS2/9, LFA-3; W6/32, HLA class I; LB3.1, HLA class II; GAP8.3, T200; 4F2, 4F2; 63D3, 63D3; 168, 168; AML-2-23, 2-23; PM81, CD15; 7C3, CD15; 80H5, CD15.
  • These immunochemical reagents included antibodies of the IgG and IgM isotype.
  • COS/PADGEM cells were constructed as described in Example 1.
  • the COS cell-PADGEM adhesion assay is described in Example 2.
  • anti-CD15 antibody 80H5 inhibited COS/PADGEM binding to U937 cells, indicating that the anti-CD15 antibodies specifically interfere with PADGEM-mediated interactions.
  • anti-CDl5 antibodies are directed against the PADGEM ligand, and not a ligand of other proteins that have been implicated in platelet-leukocyte interaction (Silverstein and Nachman, J. Clin. Invest., 79: 867-874 (1987)).
  • PADGEM anti-CD15 antibody inhibition of leukocyte-platelet interaction
  • Purified PADGEM was incorporated into fluorescently labelled phospholipid vesicles and adhesion of vesicles to U937 cells was monitored on a fluorescence-activated flow cytometer as described in Example 2.
  • anti-CDl5 antibodies inhibited the interaction of U937 cells with phospholipid vesicles containing PADGEM.
  • Phospholipid vesicles lacking PADGEM did not interact with U937 cells, confirming previous results (Larsen et al. f Cell 59: 305-312 (1989)).
  • CD15 antigen has been identified as a complex carbohydrate; CD15 antibodies react with lacto-N-fucopentaose III (LNF III) .
  • This carbohydrate has the structure Galj8l-4(Fuc ⁇ l-3)GlcNAqSl-3Galj3l-4Glc.
  • purified CD15 antigen e.g., LNF III
  • LNF III was an effective inhibitor of the adherence of activated platelets to neutrophils, as determined using the direct cell adhesion assay (closed squares) . Half-maximal inhibition was observed at about 50 ⁇ g/ml.
  • Two LNF III isomers, known as LNF I (Fuc ⁇ l-2Gal/3l-3NAcGlcj3l-4Glc) and LNF II (GaL3l-3(Fuc ⁇ l-4)NAcGlc/?l-3Galj3l-4Glc) were also tested for inhibitory activity. The three LNF isomers are structurally closely related.
  • LNF III inhibited the interaction of activated platets and neutrophils, LNF III did not alter cell viability, as determined using the trypan blue exclusion method. In addition, a similar inhibitory effect of LNF III on platelet-HL60 cell and platelet-U937 cell interactions was demonstrated (data not shown) .
  • COS/PADGEM cells to HL60 cells was also studied.
  • LNF III significantly inhibited the binding of radiolabeled HL60 cells to COS/PADGEM cells (closed square) .
  • LNF I open squares
  • LNF II closed circles
  • the inhibitory effect of LNF II was more apparent when the LNF to PADGEM ratio was high.
  • LNF I, II, and III The inhibitory effects of LNF I, II, and III on the interaction of HL60 cells and COS/PADGEM was also studied morphologically in a blind assay. In these experiments, the binding of fluorescently labeled HL60 cells to COS/PADGEM cells was scored (Example 2) . LNF III demonstrated significant inhibitory activity. In contrast, LNF I and LNF II demonstrated no inhibitory activity, and cell adhesion was comparable to assays in which LNF was absent. Mock-transfected COS cells did not bind HL60 cells. The data demonstrate that LNF III specifically interferes with PADGEM-mediated cell-cell interactions.
  • CD15 antigen e.g., LNF III
  • HL60 cells were treated with trypsin or proteinase K and then tested for their ability to bind activated platelets. HL60 cells were incubated with trypsin or proteinase K for 5-120 minutes at 24°C. Protease digestion of HL60 cells destroyed the ability of activated platelets to bind to HL60 cells (data not shown) . These results suggest that the complete PADGEM ligand is located on a glycoprotein and not on glycolipid associated with the cell surface.
  • the adhesion assay used was similar to the COS-PADGEM adhesion assay using 111In-labelled HL60 cells.
  • CHO-PADGEM and CHO-ELAM cells were constructed by transfecting CHO-DUKX cells with a cDNA encoding either PADGEM or ELAM-1.
  • Vibrio cholerae and Arthrobacter ureafaciens neuraminidases are of broad specificity and Newcastle disease virus neuraminidase is thought to be specific for ⁇ 2,3- or ⁇ 2,8-linked sialic acid, the possibility of non-specific cleavage or contaminating activities makes these results difficult to interpret. However, these results are consistent with the results of Corral et al. , in indicating that the removal of sialic acid from HL60 cells markedly diminished PADGEM-mediated cell interaction (Biochem. Biophys. Res. Commun. , 172: 1349-1356 (1990)). The data confirm the contribution of sialic acid to the PADGEM-PADGEM ligand interaction.
  • a competition assay was employed to determine whether purified PADGEM can inhibit the interaction of CHO-ELAM with HL60 cells, as it does with activated platelets.
  • the results of this assay are shown in Figure 9.
  • the data indicate that purified PADGEM inhibits the interaction of HL60 cells with CHO-PADGEM (open circles) .
  • Only partial inhibition of CHO-ELAM binding to HL60 cells was observed in the presence of purified PADGEM (closed circles) .
  • half-maximal inhibition of binding of CHO-PADGEM cells to HL60 cells was observed at 2 ⁇ g/ml of PADGEM, approximately 50-fold greater concentrations of PADGEM were necessary to effect comparable inhibition of CHO-ELAM binding to HL60 cells.
  • CHO-DUKX Chinese hamster ovary cells-DUKX
  • CHO-ELAM Chinese hamster ovary cells transfected with cDNA for ELAM
  • CHO-PADGEM Chinese hamster ovary cells transfected with cDNA for PADGEM
  • Tritiated thymidine (5 ⁇ Ci per milliliter) was added to the HL60 cell suspension and the cells were grown overnight. The following day the HL60 cells were washed trice in serum free RPMI and resuspended in a volume of 5 mis of RPMI. After assuring that the free tritium in the cell suspension is no more than
  • the cell count of the cell suspension was established and the cell density was adjusted to 1 x 10 cells per milliliter.
  • HL60 cells 100,000 to 300,000 cells
  • HL60 cells 100,000 to 300,000 cells
  • HL60 cells were removed by aspiration and the wells were washed trice with serum free RPMI. The adherent
  • CHO cells and any bound HL60 cells were detached from the surface of the wells with 250 ⁇ l of phosphate buffered saline containing 1 mM EDTA. An aliquot of the detached cells (200 ⁇ l) was analyzed for tritium content in a j3-scintillation counter. The level of nonspecific binding of tritiated HL60 cells was taken as the level of binding seen in the wells containing the CHO-DUKX cells (parent cell line of CHO-PADGEM and CHO-ELAM transfectants) . This value was subtracted from the level of tritiated HL60 cells bound in wells containing CHO-ELAM or CHO-PADGEM cells. The decrease in binding induced by the presence of the lectin was determined by comparing the level of binding of HL60 cells in the presence of the lectin to that observed in its absence.
  • Sambucus nigra lectin which requires a disaccharide Neu5Ac ⁇ 2-6Gal or Neu5Acc ⁇ 2-6GalNAc for binding, inhibited the interaction of CHO-PADGEM with HL60 cells; half-maximal inhibition under the conditions employed was observed at 1-2 ⁇ g/ml ( Figure 10) .
  • This lectin exhibited minimal inhibitory effect on CHO-ELAM binding to HL60 cells.
  • CHO cells expressing neither PADGEM nor ELAM failed to bind HL60 cells in the presence or absence of Sambucus nigra lectin (not shown) .
  • 3'-sialyllactose i.e., NeuAc ⁇ 2-3Gal01-4Glc
  • 6'-sialyllactose i.e., NeuAc ⁇ 2-6Gal/3l-4Glc

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