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WO1992007572A1 - Agents et procedes de liaison a l'elam-1 - Google Patents

Agents et procedes de liaison a l'elam-1 Download PDF

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Publication number
WO1992007572A1
WO1992007572A1 PCT/US1991/007678 US9107678W WO9207572A1 WO 1992007572 A1 WO1992007572 A1 WO 1992007572A1 US 9107678 W US9107678 W US 9107678W WO 9207572 A1 WO9207572 A1 WO 9207572A1
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cells
cell
vol
elam
determinant
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PCT/US1991/007678
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English (en)
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John B. Lowe
Rory M. Marks
Lloyd M. Stoolman
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Regents Of The University Of Michigan
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Priority to AU90522/91A priority Critical patent/AU658383B2/en
Priority to JP4501038A priority patent/JPH06502857A/ja
Publication of WO1992007572A1 publication Critical patent/WO1992007572A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/16—Central respiratory analeptics
    • 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
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to compounds and methods for binding to ELAM-1.
  • the LEC-CAM/SELECTIN family of cell adhesion molecules mediates adhesive interactions between circulating leukocytes and the vascular endothelium (Stoolman, Cell, vol. 56, pp. 907-910 (1989); and Geng et al.. Nat re, vol. 343, pp. 757-760 (1990)). These molecules participate in the recruitment of neutrophils and monocytes to inflammatory lesions (Carlos and Harlan, Immunol. Rev.. vol. 114, pp. 1-24 (1990); and Jutila et al.. Transplantation. vol. 48, pp. 727-731 (1989)), in the adhesion of activated platelets to leukocytes (Larsen et al.. Cell, vol.
  • GMP-140/PADGEM Bosset et al. , Blood, vol. 73, pp. 1109-1112 (1989); and Johnston et al.. Cell, vol. 56, pp. 1033-1044 (1989)
  • LEC-CAM1 Consensus term referring to the antigen expressing the Mell4 epitope in the mouse (Bowen et al., J. Cell. Biol.. vol. 109, pp. 421-427 (1989); Lasky et al.. Cell, vol. 56, pp. 1045-1055 (1989)) and its human homologues (Leu8/TQ1; Camerini et al.. Nature, vol. 342, pp.
  • N-terminal domains of these molecules are homologous to one another and to a variety of calcium-dependent carbohydrate recognition domains (CRDs) containing a structural motif originally described by Drickamer and colleagues (Dricka er, J. Biol. Chem.. vol. 263, pp. 9552-9560 (1988)).
  • CCDs calcium-dependent carbohydrate recognition domains
  • LEC-CAM1 mediates adhesion to high endothelial venules (HEV) in vitro (Geoffroy and Rosen, J. Cell Biol.. vol. 109, pp. 2463-2470 (1989); and
  • ELAM-1 mediates the adhesion of blood leukocytes to inflammed endothelium. It is induced by a variety of soluble mediators of inflammation including but not limited to the immune cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) , and the bacterial endotoxins (LPS) .
  • the immune cytokines are elaborated by monocytes, keratinocytes and possibly fibroblasts in response to inflammatory stimuli such as infection, autoimmune response and tissue necrosis resulting from ischemic injury.
  • LPS refers to a heterogeneous group of lipopolysaccharides derived from the cell walls of gram negative bacteria.
  • ELAM-l on the microvasculature in such diseases as psoriasis, rheumatoid arthritis and delayed hypersensitivity reactions further indicates a role for this molecule in the development of pathologic inflammation.
  • ELAM-l dependent attachment of leukocytes to the endothelium may contribute to diverse illnesses in which the inflammatory response leads to tissue damage including (1) the acute respiratory distress syndrome (ARDS); (2) rheumatoid arthritis (Grober, J. et al., submitted; Koch, A.E. 1990, Am. J. Pathol.. in press); (3) Psoriasis and hypersensitivity disorders of the skin (Nickoloff, B. et al., submitted); (4) reperfusion injury resulting from myocardial infarction and (5) hypovolemic shock.
  • ARDS acute respiratory distress syndrome
  • rheumatoid arthritis Grober, J. et al., submitted; Koch, A.E. 1990, Am. J. Pathol.. in press
  • ELAM-l molecules which can bind to ELAM-l and block the attachment of leukocytes to human endothelial cells expressing ELAM-l.
  • These molecules may be used as therapeutic agents for the treatment of diseases mediated by the expression of ELAM-l on the surface of human endothelial cells or other cells.
  • cells which either naturally express or express as a result of recombinant DNA engineering a particular product, may be directed to the region of the vasculature in which the endothelial cells are expressing ELAM-l, so as to specifically deliver the particular product to the stressed region of the vasculature.
  • Such cells could also be used as hosts for the production of recombinant glycoproteins, or other glycoconjugate-containing molecules, that contain the ligand(s) for ELAM-l, by virtue of the fact that they maintain the necessary biochemical machinery necessary for the synthesis of such ligands.
  • ARDS Adult Respiratory Distress Syndrome
  • Figure la illustrates the structures and biosynthesis of type II-based sialyl Lewis X and Lewis X molecules.
  • Terminal galactose residues on neutral type II precursors may be substituted with ⁇ (2,3)-linked sialic acid via the action of an ⁇ (2,3)sialyltransferase ( ⁇ (2,3)sialyl-T, einstein et al. , J. Biol. Chem.. vol. 257, pp. 13845-13853 (1982)).
  • the subterminal GlcNAc residues on this sialylated precursor molecule, or on its neutral predecessor, may then each be substituted with ⁇ (l,3)-linked fucose residues to form the sialyl Lewis X (sLex) and Lewis X (Lex) determinants, respectively.
  • R glycoprotein or glycolipid moieties that may contain one or more additional lactosamine (Galj3l,4GlcNAqSl,3) repeat units.
  • Figure lb illustrates the structures of the type I-based sialyl Lewis a and Lewis a determinants.
  • the sialyl Lewis a (sLea) and Lewis a (Lea) molecules are thought to be constructed from sialylated and neutral type I precursors, respectively, by the action of a(1,4)fucosyltransferases, in a manner strictly analogous to the biosynthesis of the type II structures shown in Figure la (Hansson and Zopf, J. Biol. Chem.. vol. 260, pp. 9388-9392 (1985)).
  • R glycoprotein or glycolipid moieties that display the type I precursor oligosaccharide.
  • FIG. 2a illustrates the binding of HL-60 cell lines to human endothelial cell onolayers.
  • HL-60 lines A and B were tested for adherence to TNF ⁇ -activated HUVEC, using an adhesion assay that isolates the ELAM-l-dependent component of this interaction (Method II in Examples, vide infra) .
  • Cells were allowed to adhere to TNF ⁇ -treated HUVEC monolayers, at 7- 10°C, either in the presence of the anti-ELAM-l antibody BBll (solid bars) , or in the presence of the control antibody IgG2b (open bars) .
  • Non-adherent cells were removed after 45 minutes; the fraction of remaining adherent cells are shown (% HL-60 Bound) , and are mean determinations, +/- standard errors, representing a total of 14 (Hl-60 A) or 22 (HL-60 B) separate determinations from five independent experiments.
  • Figure 2b illustrates the results of the flow cytometry analysis of cell surface oligosaccharide determinants.
  • HL-60 cell lines A and B were subjected to indirect immunofluorescence using the monoclonal antibodies directed against carbohydrate determinants detailed in the inset. Analyses were performed as described in the Examples, using a Coulter Epics V instrument equipped with a 3-decade scale.
  • Figure 3a illustrates the results of the flow cytometry analysis of transfected COS-1 cells.
  • Cells were transfected with two fucosyltransferase expression vectors (pCDM7- (1,3/1,4)FT, or pCDNAl- ⁇ (l,3)FT; labeled, respectively, " ⁇ .(l,3/l,4)FT ,f , and " ⁇ (l,3)FT”) ) , or with the control vector pCDM7.
  • Transfected cells were then subjected to flow cytometry analysis with the monoclonal antibodies detailed in the inset, as described in the Examples.
  • Figure 3b illustrates the adhesion of transfected COS-1 cells to endothelial cell monolayers.
  • Untransfected COS-1 cells (NIL) or COS-1 cells transfected with glycosyltransferase expression vectors or their control vectors, were radiolabelled with 51 Cr, harvested, and tested for adhesion on HUVEC monolayers using Method I, as detailed in the Examples.
  • Transfected cells were applied to either TNFcc-treated (+) or to untreated (-) HUVEC monolayers and allowed to adhere for 20 minutes prior to washing.
  • Plasmids pCDM7- ⁇ (l,3/l,4)FT and pCDNAl- ⁇ (1,3)FT (labeled, respectively, " ⁇ (1,3/1,4)FT", and “ ⁇ (l,3)FT”) encode distinct (1,3)fucosyltransferases as described in the Detailed Description
  • Plasmid pCDM7- ⁇ GT encodes a murine ⁇ (l,3)galactosyltransferase (Larsen et al., Proc. Natl. Acad. Sci. USA, vol. 86, pp. 8227-8231 (1989)).
  • Plasmid pH3.4 encodes a human ⁇ (1,2)fucosyltransferase (Rajan et al., J. Biol. Chem.. vol. 264, pp. 11158-11167 (1989)).
  • COS-1 cells transfected with these latter two plasmids do not express Lex, sLex, Lea, or sLea determinants (data not shown) .
  • the adhesion of normal neutrophils (PMN) to HUVEC within the same experiment is also shown on a separately scaled ordinate. Error bars equal one standard deviation.
  • Figure 3c illustrates the specific anti-ELAM-l blocking of adhesion of pCDM7- ⁇ (l,3/l,4)FT transfected cells to TNF ⁇ - treated HUVEC monolayers.
  • 51 Cr-labeled COS-1 cells transfected with pCDM7- ⁇ (l,3/l,4)FT, or the pCDM7 control were assessed in the adhesion assay as outlined in Figure 3c, except that the HUVEC monolayers had been pretreated with antibody directed against ELAM-l (BBll) , with an isotope control (IgG2b) , or with antibodies against other endothelial- expressed polypeptides (anti-VCAMl 4B9, anti-ICAMl, and anti- HLA class 1) , as described in the Examples. Error bars equal one standard deviation.
  • Figure 4a illustrates the results of the flow cytometry analysis of transfected Chinese hamster ovary cells.
  • CHO cells stably transfected with the fucosyltransferase expression vector pCDM7- ⁇ (l,3/l,4)FT(CHO-FT) or with the control vector pCDM7 (CHO-V) were subjected to flow cytometry analysis with the monoclonal antibodies detailed in the figure and as described in the Examples.
  • the data presented here are the mean fluorescence intensities of the entire population of these transfected cells; virtually 100% of the CHO-FT cells stain with anti-sLex and anti-Lex antibodies, but not with the other three antibodies (data not shown) .
  • Figure 4b illustrates the time course of adhesion of CHO cells stably-transfected with pCDM7- ⁇ (1,3/1,4)FT(CHO-FT) or vector alone (CHO-V) .
  • Transfected cell lines were labeled with 51 Cr, harvested, and tested for adhesion on untreated (-) or TNF ⁇ -treated (+) HUVEC monolayers. Non-adherent cells were removed after the times shown, and adhesion was determined by Method I as described in the Examples. Error bars equal one standard deviation.
  • Figure 4c illustrates the specific anti-ELAM-l blocking of adhesion of CHO-FT cells to TNF ⁇ -treated HUVEC monolayers.
  • 51 Cr-labeled CHO-FT cells were subjected to adhesion for 10 minutes, as outlined in Figure 4b, except that the HUVEC monolayers had been pretreated with the anti-ELAM-l antibody BBll, the isotype control IgG2b, or with antibodies against VCAMl and HLA class 1 molecules, at the same concentrations used in Figure 3c.
  • Pretreatment with anti-ICAMl fails to inhibit binding to TNF ⁇ -treated HUVEC monolayers (data not shown) . Error bars equal one standard deviation.
  • one aspect of the present invention relates to molecules, which bind to ELAM-l as expressed on the surface of human endothelial cells and thus, are able to inhibit or prevent the adhesion of leukocytes to endothelial cells expressing ELAM-l on their surface.
  • ELAM-1-dependent adhesion was not manifested either by COS-1 cells transfected with pCDNAl- ⁇ l,3FT, or by the variant of the HL-60 cell line, when expression of the Lex determinant occurs in the absence of the sLex structure. It is thus concluded that expression of ⁇ (1,3)fucosyltransferases capable of modifying acceptors containing ⁇ (2,3)sialic acid-substituted lactosaminoglycans is a critical step in the synthesis of the ligand(s) for ELAM-l. Therefore, one or more members of the family of sialylated, fucosylated lactosaminoglycans constructed by such enzymes are identified as the ligands for the CRD of ELAM-l.
  • one aspect of the present invention relates to molecules which are able to bind to ELAM-l expressed on the surface of human endothelial cells and have the general formula (I) :
  • NeuAc represents a sialyl group (N-acetylneuraminyl group)
  • Gal represents a D-galactosyl group
  • Fuc represents a L-fucosyl group
  • GlcNAc represents an N-acetylglucosamine.
  • the greek letter ⁇ indicates that the sugar moiety on the left hand side of the linkage is linked to the sugar moiety on the right hand side of the linkage by an ⁇ -linkage
  • ⁇ indicates that the sugar moiety on the left hand side of the linkage is linked to the sugar moiety on the right hand side of the linkage by a jS-linkage.
  • the stereochemistry of the linkages is further defined by the number and arrow system, in which the first number indicates the position of left-hand sugar moiety involved in the linkage and the second number indicates the position of the right-hand sugar moiety involved in the linkage. It is to be understood that in formula (I) , R is bonded directly to carbon atom number 1 of the GlcNAc group.
  • R may be OH, a protected hydroxy group, a C J . J2 alkyl group, -0-(CH2) n -C0 2 " (wherein R" is C w alkyl and n is 2 to 12), -0-(CH 2 ) ]1 -CONHNH2 (n is 2 to 12), -0-(CH 2 ) n CON 3 (n is 2 to 12) , a lipid or glycolipid, a glycoconjugate consisting of a serine/threonine-linked oligosaccharide on a protein or peptide, a glycoconjugate consisting of a free oligosaccharide derived by enzymatic or chemical hydrolysis from a serine/threonine-linkage to a protein or peptide, a glycoconjugate consisting of an asparagine-linked oligosaccharide on a protein or peptide, a glycoconjugate consisting of a free oligo
  • Such glycoconjugates may include polylactosamine- type oligosaccharides whose internal GlcAc moieties may each be unsubstituted, or substituted with ⁇ (1,3)fucose linkages.
  • Such molecules include single (VIM-2, Macher, B.A. et al., __
  • Suitable protecting groups for the protected hydroxy group of R include those disclosed in European Patent Application 184,162, which is incorporated herein by reference.
  • R may represent a drug or a label linked to the molecule or a liposome encapsulating a drug or label.
  • suitable drugs include drugs encapsulated in or otherwise incorporated into liposomes (antiinflammatory agents such as corticosteroids, cytokine antagonists.
  • antiinflammatory polypeptides including some interleukins, indomethacin, gold salts, cyclosporine, and antiinflammatory eicosanoids or enzymes that would increase the local concentrations of antiinflammatory eicosanoids; cytotoxic/chemotherapeutic agents that include adriamycin, doxorubicin, cisplatin, vincristine, cytarabine, bleomycin, amikacin, penicillin derivatives, Amphotericin B, or methotrexate, for example (Ranade, V.V. , J. Clin. Pharmacol.. vol. 29, pp. 685-694 (1989)).
  • R may represent a drug or a label linked to the molecule
  • proteins, peptides, or pharmaceutical compounds including, by way of example, interleukins, antiinflammatory pharmaceuticals including corticosteroids, cytokine antagonists, indomethacin, and antiinflammatory eicosanoids or enzymes that would increase the local concentrations of antiinflammatory eicosanoids.
  • suitable labels include drugs encapsulated in or otherwise incorporated into liposomes (gadolinium diethylenetriaminepentaacetic acid or other paramagnetic chelates for magnetic resonance imaging; te ⁇ hnetium-99m and indium-lll-NTA for scintigraphy; radiopaque [iodinated or brominated compounds] for computed tomography (Seltzer, S.E., Radiology, vol. 171, pp. 19-21 (1989)).
  • the present compounds may be prepared by conventional processes as described by Palcic et al. Carbohydrate Res. , vol. 190, pp. 1-11 (1989); by Johnson et al, Biochem. Soc. Trans.. vol. 13, pp. 1119-1120 (1985); Lemieux et al, J. Am. Chem. Soc.. vol. 97, pp. 4056-4062, pp. 4063-4069, pp. 4069- 4075, p. 4076 (1975) ; Inman et al, Im unochemistrv. vol. 10, p. 165 (1973) ; Lemieux et al. Can. J. Biochem.. vol. 55 (1977) ; which are incorporated herein by reference.
  • R is an alkoxycarbonylalkyl group may be further derivatized as described by Lemieux et al, J. Am. Chem. Soc.. vol. 97, pp. 4076-4083 (1975); Bundle et al, J. Immunol.. vol.
  • the "NH" portion of R originates as an amino group on the protein.
  • this reaction may proceed with multiple additions when the protein, P, has more than one free amino group to yield compounds with a structure (NeuAc ⁇ 2 ⁇ 3Gal/3l-*4 (Fuc ⁇ l-»3)GlcNAc ⁇ l ⁇ O(CH 2 ) n CONH) ⁇ -P in which x is limited by the number of free amino groups on the protein.
  • R includes a protein
  • the present compounds in which R includes a protein may be prepared by treating a glycoprotein which contains a suitable substrate with the appropriate enzyme, e.g., the fucosyl transferase encoded by the plasmid pCDM7- ⁇ (l,3/l,4FT) .
  • R is -0(CH 2 ) n CONHNH 2 or -0(CH 2 ) n CON 3
  • drugs or labels may be attached directly to the R group by conventional techniques (see: Science, vol. 144, p. 1344 (1964), Im unochem.. vol. 6, p. 53 (1969); and Peptides and Amino Acids. Benjamin, N.Y. (1966) , which are incorporated herein by reference) or via any suitable bifunctional spacer. Suitable bifunctional spacers are disclosed in Haughland, Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Inc., Eugene, Oregon (1989) and Pierce Immunotechnology Catalog and Handbook. Pierce, Rockford, II (1990) , which are incorporated herein by reference. -
  • chemiluminescent labels examples include those which may be used in chemiluminescent or fluorescent assays.
  • Suitable drugs include any which contain a reactive group which can couple directly with either -0-(CH 2 ) n -CONHNH 2 ,
  • the drug may be one which has been derivatized to contain a reactive group for the purpose of linking, such as those described in U.S. Patent 4,331,808, incorporated herein by reference.
  • the present invention relates to liposomes which contain on their surface a liquid for ELAM-l, such as the sialyl Lewis X determinant. The production of liposomes is discussed in U.S. Patent Nos. 4,743,560 and 4,429,008; Batzri et al, Biochimica Biophvsica Acta.. vol. 298, pp.
  • the present liposomes may be prepared by reacting liposomes which contain thiol reactive groups (see U.S. Patent 4,429,008) with a compound to introduce free amino groups on the surface of the liposome, and then reacting the liposome having free amino groups on the surface with a compound of formula (I) in which R is
  • a liposome having maleimide groups on its surface may be reacted with a compound such as H 2 NCH 2 CH 2 SH, to obtain a liposome having free -NH 2 groups.
  • the present liposomes may be prepared by incorporating a molecule such as a ceramide which is bonded, either directly or through a suitable bifunctional spacer, to a ligand of ELAM-l, such as the sialyl Lewis X determinant (see: Eggens et al, J. Biological Chem.. vol. 264, pp. 9476- 9484 (1989) ; Batzri et al Biochim. Biophys.
  • the present liposomes may also contain drugs or other molecules as described in U.S. Patent Nos. 3,993,754 and 4,263,428, which are incorporated herein by reference. Additionally, the present liposomes may also encapsulate labels for NMR imaging or radionucleotide scanning. Suitable labels for use as radioactive diagnostics are disclosed in U.S. Patent 4,094,965 and Canadian Patent Application 946,741, which are incorporated herein by reference. Suitable labels for NMR imaging are disclosed in French Patent Application 2,612,400, Eur. J. Med.. vol. 24, pp. 241-247 (1989), UK Patent Application 2,214,507, and Radiology, vol. 175, pp. 483-488 (1990) , which are incorporated herein by reference.
  • transfected cells which do not normally exhibit ELAM-1-dependent adhesion to endothelial cells, but which as a result of their transfection exhibit ELAM-1-dependent adherence to endothelial cells and a method for the production of such cells.
  • such cells are those which either naturally or as a result of transfection produce a suitable substrate and have been transfected with either cDNA or genomic DNA encoding an enzyme capable of converting said suitable substrate into the sLex determinant.
  • the process for preparing the present cells involves:
  • the starting cells may be any which do not express the sLex determinant but do produce a substrate which may be converted to a molecule containing the sLex determinant, by the enzyme encoded by the heterologous DNA which is introduced into the cells.
  • suitable starting cells include COS-1 and Chinese hamster ovary (CHO) line Ade" C.
  • pCDM7- ⁇ (l,3/l,4)FT transfection induces expression of ligands for ELAM-l on non-hematopoietic cells indicates generalized distribution of suitable "acceptor" molecules for the ⁇ (1,3/1/4)FT.
  • ⁇ (1,3/1,4)FT cDNA into (and expression in) a variety of normal cells and cell lines will result in ELAM-l binding activity.
  • Suitable examples of the enzyme include the fucosyltransferase encoded by pCDM7- ⁇ (l,3/l,4FT) .
  • the plasmid pCDM7- ⁇ (l,3/l,4FT) has been deposited in the E. coli strain MC 1069/P3 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and has been given the accession number 68448.
  • ⁇ (1,3)fucosyltransferase capable of constructing Fuc ⁇ (l,3)GlcNAc linkages on ⁇ (2,3)sialylated lactosamine molecules, of the type that is expressed in human myeloid cells (Potvin et al., J. Biol. Chem.. vol. 265, 1615- 1622 (1990)) or in other species (Howard et al., J. Biol. Chem.. vol. 262, pp. 16830-16837 (1987).
  • the fucosyltransferase encoded by pCDM7- ⁇ (l,3/l,4FT) is preferred and represents the product of the human Lewis blood group locus (Kukowska-Latallo et al.. Genes Dev.. vol. 4, pp. 1288-1303 (1990)). It exhibits a uniquely broad spectrum of acceptor substrate requirements that encompasses the requirements exhibited by members of two other general classes of ⁇ (1,3)fucosyltransferases (Prieels et al., J. Biol. Chem.. vol. 256, pp. 10456-10463 (1981); and Palcic et al., Carbohvd. Res.. vol. 190, pp. 1-11 (1989)).
  • suitable starting cells are those that produce substrates which contain carbohydrate moieties corresponding to the sialylated type II precursor shown in Figure la:
  • the starting cells are immune effector cells whose cell surface oligosaccharide structure does not allow them to be targeted to endothelium expressing ELAM-l.
  • the present process results in such cells expressing the sLex determinant and being targeted to the areas of inflammation at sites of pathology, where their immune effector functions are therapeutically beneficial.
  • suitable immune effector cells include lymphokine- activated killer cells (LAK cells) and tumor infiltrating lymphocytes (TIL cells) in the treatment of advanced cancer, which produce substrates which may be converted to molecules containing the sLex determinant by the enzymes:
  • the starting cells may also be transfected with another heterologous DNA sequence which encodes for a desired polypeptide.
  • heterologous DNA sequences include, e.g., EPO, insulin, and interleukin 2.
  • the present invention provides a method for targeting a glycoprotein to cells expressing ELAM- 1.
  • a cell which produces a first glycoprotein which can serve as a substrate for an enzyme which will convert the first glycoprotein to a second glycoprotein having a ligand for ELAM-l will produce the second glycoprotein after transfection with a suitable enzyme, e.g., the fucosyl transferase encoded by the plasmid pCDM7- ⁇ (l,3/l,4FT) .
  • a suitable enzyme e.g., the fucosyl transferase encoded by the plasmid pCDM7- ⁇ (l,3/l,4FT) .
  • the cell may produce the first glycoprotein either naturally or as a result of genetic engineering.
  • the cell may be transfected to produce a second enzyme or a series of enzymes which will convert a first protein or glycoprotein, which is not directly convertable to a glycoprotein having the sialyl Lewis X determinant, to a glycoprotein which can serve as substrate for an enzyme which will convert it to a glycoprotein having the sialyl Lewis X determinant.
  • the present invention provides cells which produce glycoproteins which are targeted to ELAM-l and glycoproteins which are targeted to ELAM-l.
  • transfection of the starting cells with the DNA sequence encoding for the enzyme responsible for converting the substrate into the ligands for ELAM-l and any other desired heterologous DNA sequences may be accomplished by any conventional transfection technique. Suitable methods are discussed in Davis et al, Basic Methods in Molecular Biology. Elsevier Publishing Co., NY, NY (1986).
  • the carbohydrate ligands for ELAM- 1 are linked to recombinant polypeptide antiinflammatory molecules vector by expressing such recombinant proteins, in a form suitable to be expressed with N- and/or 0-linked oligosaccharides, in an eukaryotic host that also expresses the enzymes that construct the oligosaccharide ligand(s) for ELAM-l (i.e., CHO-FT cells, for example).
  • the present cells are those which express a substrate on their surface which may be converted to the ligands for ELAM-l through direct enzymatic action of soluble or immobilized recombinant ⁇ (1,3/1,4)FT or its _ analogous myelod specific enzyme.
  • the present cells may be produced by a process involving treating a cell with an enzyme which is capable of converting a determinant found on the surface of the cell into a ligand for ELAM-l. It is also possible to treat an oligosaccharide precursor by itself or on another molecule with a recombinant enzyme to form an ELAM-l ligand.
  • the starting cells need not be transfected.
  • the present invention also relates to targeting of therapeutics to endothelium expressing ELAM-l through the construction and linkage of type II structures to liposomes, macromolecular carriers and single molecules.
  • the identification of alpha 1,3 fucosyl transferases as the rate limiting step in the synthesis of ELAM-l specific ligands, and the demonstration that a cloned alpha 1,3/1,4 fucosyl transferase can generate ligands from type II precursors in a stable transfection system provides both the knowledge and the means to synthesize targeted therapeutics.
  • antiinflammatory agents with systemic toxicity such as glucocorticoids, ethotrexate, cyclosporin and gold salts, could be delivered in high concentration directly to sites of active inflammation.
  • the present cells and liposomes may contain a label suitable for use in an imaging technique in addition to or in replacement of a drug or therapeutic agent.
  • an imaging technique such as NMR or radionucleotide scanning, may be used to specifically locate the affected area.
  • Such molecules may be shed from malignant tumors in part as a consequence of the "aberrant" expression of the sialyl Lex determinant frequently seen in association with malignant transformation (Fukushi et al., J. Biol. Chem.. vol. 259, pp. 4681-4685 (1984); Fukushima et al.. Cancer Res.. vol. 44, pp. 5279-5285 (1984); and Kannagi et al.. Cancer Res.. vol. 46, pp. 2619- 2626 (1986) ) .
  • Such molecules may participate in specific interactions with ELAM-l acting to inhibit normal interactions between leukocytes and the vascular wall, and thereby act to blunt inflammation-induced leukocyte recruitment in these patients.
  • Adenocarcinoma cells have also been shown to exhibit ELAM-1-dependent HUVEC adhesion (Rice and Bevilacqua, Science, vol. 246, pp. 1303-1306 (1989)).
  • the present invention relates to a method of treating a disease mediated by the expression of ELAM-l by endothelium.
  • diseases include, e.g., adult respiratory distress syndrome, vasculitis, and myocardial infarction.
  • the treatment of the disease involves administering an effective amount of the compound of the formula (I) .
  • R is not a moiety derived from a drug or therapeutic agent
  • the compound of formula (I) binds to the ELAM-1 on the surface of the affected endothelial cells and acts to block the adhesion of the neutrophils.
  • R is a moiety derived from a drug or represents a drug attached to the sLex determinant via a linking molecule
  • the compound also acts to specifically deliver the drug to the affected area, in addition retarding the adhesion of the neutrophils to the endothelium.
  • the present treatment involves administering cells or liposomes which have been treated according to the present invention so that they carry the ligand(s) for ELAM-l.
  • the present cells and liposomes which carry the carbohydrate ligands on their surfaces as blocking agents.
  • the cells and liposomes used in the present treatment contain a drug effective for the treatment of the affected area, in addition to carrying the carbohydrate ligand(s) on their surfaces.
  • the present compound, cells or liposomes are being used for the treatment of a disease or the imaging of the affected area, all and any of these may be administered by any conventional means and in any conventional form suitable for placing the compound, cell or liposome in the vasculature.
  • the compounds, cells, and liposomes are administered by injection in a form suitable therefore, such as a solution or suspension.
  • a form suitable such as a solution or suspension.
  • the exact dosage of the compound, cells, and liposomes will depend on whether the compound, cells, or liposomes are being used as a blocking agent or to target drug or label delivery, the identity of the drug or label being delivered, if any, and the size and health of the patient. For any of the above-described purposes or cells, compounds or liposomes, the exact dosage to be administered may be easily determined by one of ordinary skill.
  • HUVEC HUVEC were passaged with trypsin/EDTA (Gibco) , and plated in 96 well plates
  • Plasmid pCDNAl- ⁇ (l,3)FT contains a 3.6 kb Pstl human genomic DNA restriction fragment that encodes an ⁇ (1,3)fucosyltransferase (Lowe et al., unpublished). This fragment was isolated from a human lambda phage rescued from a genomic DNA library probed at low stringency with the insert in pCDM7- ⁇ (l,3/l,4)FT. The gene segment is cloned in the appropriate transcriptional orientation into the mammalian expression vector pCDNAl (InVitrogen) .
  • the anti-Le x antibody anti-SSEA-1 (mouse monoclonal IgM as ascites; Solter and Knowles, Proc. Natl. Acad. Sci. USA, vol. 75, pp. 5565-5569 (1978)) was provided by Dr. D. Solter (Philadelphia) .
  • Anti-sialyl Lex antibody CSLEX1 (mouse monoclonal IgM, HPLC purified; Fukushima et al.. Cancer Res.. vol. 44, pp. 5279-5285 (1984)
  • anti-sialyl Lea antibody CSLEA1 (mouse monoclonal IgG3, ammonium sulfate precipitate; Galton et al.
  • Antibodies Protective. Destructive, and Regulatory Role. Ninth Int. Convoc. Immuno. , Amherst, NY, pp. 117-125, Karger, Basel (1985); and Chia et al.. Cancer Res.. vol. 45, pp. 435-437 (1985)) were provided by Dr. P. Terasaki (Los Angeles) .
  • Anti-H and anti-Lea a antibodies both mouse monoclonal IgM, antigen affinity purified
  • Anti-ELAM-l antibody BBll (Benjamin et al., Biochem. Biophys. Res. Commun.. vol. 171, pp. 348-353 (1990)) was the gift of Dr. Roy Lobb (Biogen, Inc., Cambridge, MA).
  • IgG2b, anti-CDllb, anti-CD19, and anti-CD33 antibodies were purchased from Coulter Corp
  • Anti-VCAM antibody 4B9 (Carlos and Harlan, Immunol. Rev. , vol. 114, pp. 1-24 (1990)) was provided by Dr. John Harlan (University of Washington, Seattle) .
  • Anti-ICAMl antibody 84H10 (Makgoba et al. , Nature. vol. 331, pp. 86-88 (1988)) was purchased from AMAC, Inc.
  • Anti-HLA class I antibody W6/32 (Parham et al. , J. Immunol., vol. 123, pp. 342- 349 (1979)) was purchased from SeraLab, Inc. (U.K) .
  • COS-1 cells were transfected with various plasmids using the DEAE-dextran procedure (Davis et al., Basic Methods in
  • Labeled or unlabeled, transfected COS-1 cells were harvested for HUVEC binding assays, or for flow cytometry analyses, by washing the cell monolayers with calcium, magnesium free PBS (CMF-PBS) containing 2 mM EDTA, and then 5. incubating the washed monolayers with CMF-PBS/2 mM EDTA. Detached cells were washed once by centrifugation through CMF-PBS/2 mM EDTA, and then resuspended in buffers compatible with flow cytometry or binding analyses.
  • CMF-PBS calcium, magnesium free PBS
  • CHO Ade-C cells (Oates and Patterson, Somatic Cell Genet. , vol. 3, pp. 561-577 (1977); and Van Keuren et al.. Am. J. Hum. Genet.. vol. 38, pp. 793-784 (1986)) were transfected (Chen and Okayama, Mol. Cel. Biol. , vol. 7, pp. 2745-2752 5 (1987)) with Xhol-linearized pCDM7- ⁇ (l,3/l,4)FT
  • Cell extracts prepared from CHO-FT contained substantial amounts of 0 ⁇ (1,3)fucosyltransferase activity when assayed with the acceptor N-acetyllactosamine (Kukowska-Latallo et al.. Genes Dev.. vol. 4, pp. 1288-1303 (1990)), whereas extracts prepared from the parental cell line and from the CHO-V cells contained no detectable fucosyltransferase activity.
  • CHO-FT or CHO-V (6 x 10 6 ) were labeled with 51 Cr by incubating them for 5-6 hours in 25 ⁇ Ci/ml of Na 51 Cr. Labelled cells were then harvested using the procedure described above for COS-1 cells.
  • Method I was used for analyses with transfected cells, and is a conventional HUVEC adhesion assay utilizing 51 Cr labelled cells incubated at 37°C.
  • HUVEC in 96-well plates were placed in growth media without growth factors, with or without 20 ng/ml TNF ⁇ (Genentech) , for 4 to 6 hours prior to binding assays.
  • TNF ⁇ human ng/ml TNF ⁇
  • antibodies were added in 50 ⁇ l PBS containing 100 ⁇ g per ml each of Ca and Mg (PBS/Ca/Mg) , 0.5% human serum albumin (endotoxin-free. Cutter) , and incubated with HUVEC at 4°C for 1 hour.
  • Antibodies were added to a final concentration of 1 ⁇ g/ml, except the anti-HLA class I antibody, which ascites used at a 1:1000 dilution. These represent saturating concentrations, as determined by radioimmunoassay. After treating with antibodies, HUVEC monolayers were washed three times with PBS/Ca/Mg. Cells to be tested for adhesion were harvested as detailed above, resuspended in PBS/Ca/Mg, and held at 4°C for the shortest ti e possible until used in the assay.
  • Cells (3 x 10 5 neutrophils, 1 x 10 5 COS-1 cells, 3 x 10 5 CHO cells) were added to HUVEC monolayers in 100 ⁇ l of PBS/Ca/Mg, and incubated at 37°C. Microscopic titration was used to determine the numbers of cells added of each type (sufficient to just allow cells to form an essentially.confluent monolayer overlying the HUVEC monolayer, providing the majority of added cells an opportunity to interact with the underlying endothelium without multilayering) . Cells were allowed to adhere for various times (10 to 25 minutes, see Figure legends) . Unbound cells were removed by exchanging the wells three times with 150 ⁇ l of PBS/Ca/Mg. Each well was then counted in a gamma counter, and the number of bound cells was calculated based upon a previous determination of the number of cpms incorporated per radiolabeled cell.
  • Method II maximizes carbohydrate-dependent adhesion by adopting principles used to measure the lectin-dependent adhesion of lymphocytes to frozen sections of lymph nodes (Stoolman et al. , Blood . vol. 70, pp. 1842-1850 (1987)). This method was used to determine ELAM-1-dependent adhesion of the HL-60 myeloid cell lines. HUVEC were plated in 96-well tissue culture plates; 24 hours later, the confluent HUVEC monolayers were incubated with 20 ng/ml of TNF ⁇ for 4-6 hours prior to initiating binding assays.
  • HL-60 cells were washed in MEM+ (minimal essential medium buffered to pH 7.3 with Tricine, 40 mM, and supplemented with 1 mg/ml bovine serum albumin) immediately prior to fixation.
  • MEM+ minimal essential medium buffered to pH 7.3 with Tricine, 40 mM, and supplemented with 1 mg/ml bovine serum albumin
  • cells in suspension were fixed in freshly prepared paraformaldehyde (0.5% paraformaldehyde in 0.15 M cacodylate buffer, pH 7.4; 20 minutes, 4°C) , washed extensively, and resuspended at 10 6 cells/ml in MEM+.
  • the TNF ⁇ -treated HUVEC monolayers were preincubated at 7-10°C with 50 ⁇ l of either IgG2b or BBll monoclonal antibodies at a concentration of 20 ⁇ /ml.
  • Fixed cells (10 5 cells in 50 ⁇ l) were generally added directly to the plates after 15-30 minutes of preincubation (washing the HUVEC monolayers free of unbound antibody prior to instituting the binding assays had no effect on the level of inhibition, data not shown) .
  • Added cells were allowed to settle and interact with the HUVEC for 45 minutes at 7-10°C prior to quantitation of adhesion.
  • Unattached cells were removed by aspirating the cell suspensions and gently washing the monolayers three times with 200 ⁇ l of MEM+ at room temperature.
  • the wash solution was applied with an 8-channel multipipettor to the upper wall of the wells while maintaining the plates at a 30° angle.
  • the solutions were then drawn off the lower wall without contacting the monolayer.
  • the number of cells recovered from each well was determined by counting the pooled washes on a Coulter ZBI cell counter. The number of bound cells represents the difference between the number of cells applied to each well and the number recovered from the well.
  • Transfected COS-1 cells, or CHO transfectants were subjected to flow cytometry analysis with mouse monoclonal IgM antibodies directed against carbohydrate epitopes, using procedures described previously (Ernst et al., J. Biol. Chem.. vol. 264, pp. 3436-3447 (1989); and Kukowska-Latallo et al.. Genes Dev.. vol. 4, pp. 1288-1303 (1990)).
  • HL-60 cells Flow cytometry-based indirect immunofluorescence analyses of HL-60 cells, were conducted in microtiter plates. Both primary and secondary antibodies were used at saturating concentrations. Antigen specific mouse anti-human monoclonal antibodies (50 ⁇ L) were combined with cells (2.5 x 10 5 in 50 ⁇ L) and incubated at 4°C for 30 minutes in PBS+ (phosphate buffered saline supplemented with 1% fetal bovine serum and 0.05% Na azide) .
  • PBS+ phosphate buffered saline supplemented with 1% fetal bovine serum and 0.05% Na azide
  • Fluorescence quantitation was performed on a Coulter Epics V Flow Cytometer equipped with a three-decade scale.
  • the ELAM-l receptor mediates the adhesion of neutrophils, monocytes, and the related cell lines HL-60 and U937 to cytokine-stimulated human umbilical vein endothelial cells (HUVEC) (Bevilacqua et al., Proc. Natl. Acad. Sci. USA, vol. 84, pp. 9238-9242 (1987); and Bevilacqua et al.. Science, vol. 243, pp. 1160-1165 (1989)).
  • VEC cytokine-stimulated human umbilical vein endothelial cells
  • the surfaces of these leukocytic cells are unusually rich in fucosylated derivatives of neutral and ⁇ (2,3)sialylated polylactosamine ([Gal/3l,4GlcNAc) n ]) moieties (Fukuda et al. J. Biol. Chem.. vol. 259, pp. 10925- 10935 (1984); Fukuda et al., J. Biol. Chem.. vol. 260, pp. 1067-1082 (1985); and Spooncer et al., J. Biol. Chem.. vol. 259, pp. 4792-4801 (1984)).
  • the structurally and biosynthetically related members of this group include the sialyl Lewis X tetrasaccharide (NeuAc ⁇ 2 ⁇ 3Gal/3l ⁇ 4(Fuc ⁇ l ⁇ 3)GlcNAc, sLex; Fukushima et al., Cancer Res.. vol. 44, pp. 5279-5285 (1984); Figure la), its non-sialylated trisaccharide analogue Lewis X (Galj ⁇ l ⁇ 4(Fuc ⁇ l ⁇ 3)GlcNAc, Lex or SSEA-1; Gooi et al.. Nature, vol. 292, pp.
  • FIG. 2 depicts a series of experiments with two spontaneously arising variants of the HL-60 line. Both cell lines expressed the myeloid differentiation antigens CDllb, CD19 and CD33 (data not shown) .
  • HL-60 line B cells bound avidly to TNF ⁇ -treated HUVEC, with 55% of the added cells attached in the presence of the control antibody IgG2b. This adhesion was largely ELAM-1-dependent since only 11% of the cells adhered in the presence of the anti-ELAM-l antibody BBll (Benjamin et al. , Bioche .
  • HL-60 A exhibited a lower absolute level of adhesion (mean of 16%) which was not significantly reduced by treatment of the HUVEC with BBll (14%) .
  • BBll BBll
  • the LECH mutant exhibits de novo expression of a specific ⁇ (1,3)fucosyltransferase, termed Fuc-TI, that determines the synthesis of surface-localized neutral and ⁇ (2,3)sialylated polylactosaminoglycans substituted with ⁇ (1,3)-linked fucose residues ( Figure la).
  • Fuc-TI a specific ⁇ (1,3)fucosyltransferase
  • sialyl Lex moiety The penultimate step in the biosynthesis of these structures, which include the sialyl Lex moiety, is thought to be catalyzed by a widely distributed sialyltransferase that attaches sialic acid in an ⁇ (2,3) linkage to terminal gala ⁇ tose residues in the polylactosamine substrate (Weinstein et al., J. Biol. Chem.. vol. 257, pp. 13845-13853 (1982)). These sialylated molecules can then serve as acceptors for some (Holmes et al., J. Biol. Chem.. vol. 261, pp. 3737-3743 (1986); and Howard et al., J. Biol. Chem.. vol.
  • ⁇ (1,3)fucosyltransferases can generate the Lex moiety and its polyfucosylated analogues from neutral polylactosamine precursors. These neutral fucosylated molecules are not, however, substrates for any known ⁇ (2,3)sialyltransferases. These considerations suggest a critical, regulatory role for expression of specific ⁇ (1,3)fucosyltransferases in the biosynthesis of the family of sialylated, fucosylated lactosaminoglycans that represent putative ELAM-l ligands.
  • Non-myeloid hosts were chosen for these experiments in order to isolate the role of the oligosaccharide molecules in ELAM-l-dependent adhesion from other myeloid-specific molecules known to participate in leukocyte-endothelial cell interactions (Springer, Nature. vol. 346, pp. 425-434 (1990)).
  • COS-1 cells were used for the first of these experiments since these cells do not express detectable ⁇ (1,3)fucosyltransferase activity nor cell surface oligosaccharides that contain the cognate ⁇ (1,3)fucose linkages (Kukowska-Latallo et al.. Genes Dev.. vol. 4, pp. 1288-1303 (1990)).
  • COS-1 cells do not exhibit ELAM-l-dependent adhesive properties. They do, however, express the oligosaccharide substrates necessary for ⁇ (1,3)fucosyltransferase-dependent biosynthesis of the family of oligosaccharides represented by the Lex and sialyl Lex molecules (Fukuda et al., J. Biol. Chem.. vol. 263, pp. 5314- 5318 (1988); and Kukowska-Latallo et al.. Genes Dev.. vol. 4, pp. 1288-1303 (1990)).
  • Stably-transfected CHO Cells Determines ELAM-l Dependent Cell Adhesion and sLex Expression
  • CHO-FT transfected mammalian cell line
  • Ade The parental CHO cell line Ade " C (Oates et al.. Somatic Cell Genet.. vol. 3, pp. 561-577 (1977); and Van Keuren el al.. Am. J. Hum. Genet.. vol. 38, pp.
  • Each CHO transfectant line was assessed for adhesion to untreated and TNF ⁇ -treated HUVEC ( Figures 4b and 4c) .
  • Cells were allowed to adhere for between 5 and 25 minutes, before washing off unbound cells.
  • CHO-FT cells exhibited marked adhesion to TNF ⁇ -treated HUVEC, but not to untreated endothelium ( Figure 4b) .
  • Adhesion of CHO-FT cells was well- established within 5 minutes, peaked at 10 minutes, and was resistant to repeated and vigorous washing (vide supra) .
  • control transfectants that do not express significant amounts of the Lex or sialyl Lex molecules ( Figure 4a)
  • Figure 4b did not exhibit meaningful binding to either TNF ⁇ -treated or untreated HUVEC ( Figure 4b) at early time points, and minimal, non-specific adhesion at later ones.
  • This adhesion was ELAM-1-dependent since it was inhibited by pretreatment of the HUVEC with the BBll anti-ELAM-l antibody, but not by the control antibody IgG2b, nor with antibodies directed against other adhesion receptors (VCAMl and ICAMl) , or to HLA class I determinants ( Figure 4c and data not shown) .

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Abstract

L'identification du déterminant sialyle Lewis X comme déterminant responsable de l'adhésion, dépendant de l'ELAM-1, de neutrophiles à l'endothélium, a eu pour résultat le développement d'un nombre d'agents de liaison à l'ELAM-1 et à des procédés de traitement de maladies à médiation par l'ELAM-1.
PCT/US1991/007678 1990-10-25 1991-10-25 Agents et procedes de liaison a l'elam-1 WO1992007572A1 (fr)

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NZ250685A (en) 1996-12-20
IE913722A1 (en) 1992-05-22
ZA918478B (en) 1992-07-29
JPH06502857A (ja) 1994-03-31
IL99847A0 (en) 1992-08-18
EP0555385A1 (fr) 1993-08-18
EP0555385A4 (en) 1996-03-27
IL99847A (en) 1999-03-12
AU9052291A (en) 1992-05-26
NZ240316A (en) 1996-12-20
AU658383B2 (en) 1995-04-13
CA2094911A1 (fr) 1992-04-26

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