WO1993017033A1 - Inhibition de l'adherence cellulaire par oligosaccharides definis chimiquement, leurs derives, des imitateurs et des anticorps - Google Patents

Inhibition de l'adherence cellulaire par oligosaccharides definis chimiquement, leurs derives, des imitateurs et des anticorps Download PDF

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Publication number
WO1993017033A1
WO1993017033A1 PCT/US1993/001375 US9301375W WO9317033A1 WO 1993017033 A1 WO1993017033 A1 WO 1993017033A1 US 9301375 W US9301375 W US 9301375W WO 9317033 A1 WO9317033 A1 WO 9317033A1
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carbohydrate
cells
elam
sle
antibody
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PCT/US1993/001375
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English (en)
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Naoya Kojima
Kazuko Handa
Sen-Itiroh Hakomori
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The Biomembrane Institute
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Priority to JP5514925A priority Critical patent/JPH07504222A/ja
Priority to EP93905988A priority patent/EP0638085A1/fr
Publication of WO1993017033A1 publication Critical patent/WO1993017033A1/fr

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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C07K16/2854—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed generally to the inhibition of tumor cell metastases and invasiveness and of inflammatory processes based on the inhibition of adhesion of tumor cells or inflammatory leukocytes to specific types of cells. More specifically, the invention is directed to such inhibition through the use of tumor-associated carbohydrate antigens, leukocyte-associated carbohydrate antigens, oligosaccharide derivatives thereof, mimetics of the tumor-associated carbohydrate antigens, leukocyte-associated carbohydrate antigens and antibodies directed to the tumor-associated carbohydrate antigens.
  • cancer Despite enormous investment of financial and human resources, cancer remains one of the major causes of death. Current cancer therapies cure only about half of all patients who develop a malignant tumor. In most human malignancies, metastasis is the major cause of death.
  • Metastasis is the formation of secondary tumor colonies at one or more distant sites. Metastasis is a multistep process of which tumor invasion is the first step. Tumor cells locally invade host tissue barriers, such as the epithelial basement membrane, to reach the interstitial stroma where the tumor cells gain access to blood vessels (or lymphatic channels) for further dissemination. After invading the endothelial layer of the vessel wall, the circulating tumor cells are dislodged into the circulation and arrest in the precapillary venules of the target organ by adhering to endothelial cell lumenal surfaces or exposed basement membranes. The tumor cells again invade the vascular wall to enter the organ parenchyma. Finally, the extravasated tumor cell grows in a tissue different from where the tumor originated.
  • host tissue barriers such as the epithelial basement membrane
  • both processes are triggered by adhesion of cells, leukocytes in the former case and tumor cells in the latter, to microvascular endothelial cells followed by transendothelial migration of the leukocytes or tumor cells into the tissue spaces. Both processes are enhanced by activating platelets.
  • TACA leukocyte associated carbohydrate antigens
  • LACA leukocyte associated carbohydrate antigens
  • the instant invention is directed to and based on the inhibition of cell adhesion, for example, through TACA's or
  • the instant invention provides compositions and methods of inhibiting metastatic potential and invasiveness of tumor cells based on blocking tumor cell adhesion by carbohydrate structures or antibodies directed thereto.
  • the instant invention also relates to compositions and methods of inhibiting inflammation potential of leukocytes based on blocking leukocyte adhesion by carbohydrate structures or antibodies directed thereto.
  • the rationale for the approach is to block (a) carbohydrate to carbohydrate interaction; (b) carbohydrate to selectin interaction; or (c) both.
  • i) In model experiments with mouse melanoma B16 variants with high and low metastatic potential, high metastatic variants, BL6 and F10, express more GM3 than low-metastatic or non-metastatic variants, F1 or
  • Adhesion of high metastatic variants to endothelial cells is greater than with low metastatic variants and the adhesion is inhibited by Me- ⁇ -lactoside, GM3 or LacCer (each within liposomes) or other lactoside derivations.
  • the sugars and derivatives also inhibit B16 melanoma metastatic potential.
  • tumor-associated carbohydrat antigens such as H/Le y /Le b (defined by monoclona antibody MIA-15-5), sialosyl Tn (defined by monoclona antibody TKH2) or sialosyl-Le x (defined by monoclona antibody FH6, SNH3 or SNH4), had a much shorte survival rate than those patients whose primary tumors do not express or which weakly express those antigens.
  • Those tumor-associated carbohydrate antigens are essentially adhesion molecules which are recognized by target cells, particularly platelets or endothelial cells Such is an example of a combination approach, that is, interfering with (a) and (b).
  • target cells particularly platelets or endothelial cells
  • LECCAM or selectin
  • ELAM-1 or GMP-140 which are expressed on activated endothelial cells and activated platelets.
  • Sialosyl-Le x antigen has been known to be recognized by those LECCAM's. Such is an example of (b), affecting a carbohydrate to selectin interaction.
  • GMP-140 whose expression on platelet or endothelial cells is induced by thrombin, ADP or (AMP) phorbol ester, may play an important role in platelet-tumor cell interaction and mediate tumor cell metastases. While the epitope recognized by that selectin was identified previously as sialosyl-Le x (Polley et al., Proc. Natl. Acad. Sci.
  • sialosyl-Le a also known as monosialosyl-Le a I
  • monosialosyl-Le a II a positional isomer of sialosyl-Le a
  • disialosyl-Le a also are recognized by GMP-140.
  • GMP-140 binds to sialosyl Le a better than to sialosyl-Le x . Such is another example of process (b).
  • ELAM-1 whose expression on endothelial cells is induced by interleukin-1, TGF- ⁇ , TNF- ⁇ or lipopolysaccharide, may play an important role in endothelial cell-leukocyte and endothelial cell-tumor cell interaction, mediate tumor cell metastasis, mediate endothelial cell-leukocyte interactions and mediate transendothelial migration of leukocytes and tumor cells. While the epitopes recognized by that selection previously were identified as sialosyl-Le x and sialosyl-Le a (Phillips et al.. Science 250:1130,
  • selectin epitopes also are internally sialylated, penultimate fucosylated type 1 or type 2 chains, such as monosialosyl-Le a II and disialosyl-Le a , particularly in a dynamic flow system. But the binding phenomenon is vibrant.
  • ELAM-1 also known as E-selectin recognizes primarily ⁇ 2 ⁇ 3 sialylated and ⁇ 1 ⁇ 3 or ⁇ 1 ⁇ 4 fucosylated carbohydrates, such as SLe x and SLe a .
  • molecules having formulae (I) or (II), see, for example, Figure 20, such as Le x /SLe x play an important role in providing high affinity binding sites to E-selectin. That role is particularly evident under high shear stress conditions.
  • H Human endothelial cells are characterized by high expression of H (Fuc ⁇ 1 ⁇ 2Gal) and many types of human cancers are characterized by expression of Le y , H or Le defined by monoclonal antibody MIA-15-5. Interaction of H with Le y or H with H has been established clearly, therefore, those human tumors expressing H/Le y /Le b may adhere to H-expressing endothelial cells which are mediated by Le y -H or H-H interaction. Such is an example of process (a), that is affecting a carbohydrate to carbohydrate interaction.
  • Monoclonal antibody MIA-15-5 directed to H/Le y /Le b inhibited lung metastasis of highly metastatic F10 and BL6 variant cells in the mouse. Furthermore, monoclonal antibody FH7 directed to disialosyl-Le a and monosialosyl-Le a II inhibited adhesion of human cancer cells expressing those antigens in a dynamic flow system.
  • the instant invention provides the following: a) Compositions and methods for inhibiting tumor cell metastasis based on tumor cell adhesion mediated by carbohydrate antigen by such oligosaccharides comprising GM3, H, Le y , Le b , monosialosyl-Le x (SLe x ),
  • Le a , Le x hybrid sugars, such as, Le x /SLe x hybrids (Structure 1 in Figure 20), monosialosyl-Le a I (SLe a ), monosialosyl-Le a II, sialosyl Tn, lactosyl and other structures as depicted in structures 1-14, in Example 3.
  • Such compounds block E-selectin-mediated adhesion of tumor cells or leukocytes to endothelial cells.
  • Utilization of antibodies recognizing those oligosaccharides comprising and representing tumor-associated carbohydrate antigens also may inhibit tumor cell adhesion to endothelial cells, platelets or target cells, and may inhibit metastasis.
  • a method for inhibiting tumor cell metastasis potential or inflammation within a biologic preparation comprises incubating the biologic preparation with at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigens) that exhibit differential prognostic significance,
  • oligosaccharide components of those antigens include cell cultures and biologic fluids.
  • Another aspect of the instant invention provides a method for inhibiting metastasis potential of tumor cells or inflammation in a warm-blooded animal.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigens) that exhibit differential prognostic significance, (b) antibodies that specifically bind to those antigens, (c) oligosaccharide components of those antigens,
  • the instant invention provides a variety of glycoconjugates useful for prolonging the in vivo half-life of oligosaccharide components.
  • the conjugates comprise an oligosaccharide coupled to polyethyleneglycol.
  • oligosaccharide components for use within the methods and compositions of the instant invention include lactose, lacto-N-tetrose, methyl ⁇ -D-lactoside and phenyl ⁇ -D-thiolactoside. Oligosaccharide components may be used individually or in combination with one another.
  • the instant invention further provides a variety of methods for inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion causing metastasis at a tumor site and inflammatory responses at a site.
  • One such method inhibits GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion within a biologic preparation and comprises incubating the biologic preparation with at least one agent selected from the group consisting of: (a) a hybrid sugar molecule, such as one comprising Le x and SLe x (Structure 1 of Figure 20, a branched type II chain); (b) a mixture of the components of the hybrid sugar of (a), such as, Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specifically bind to a hybrid sugar, such as Le x /SLe x , or to the components thereof; (e) antibodies that specifically bind to monosialosyl-Le a I, Le a , Le x , monosi
  • Another such method inhibits GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion at a tumor cell or inflammatory site in a warm-blooded animal thereby reducing metastatic potential or inflammation at the site and comprises administering to the warm-blooded animal an effective amount of at least one agent selected from the group consisting of: (a) a hybrid sugar, such as, SLe x /Le x ; (b) a mixture of the components of a hybrid sugar (a), such as, Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specifically bind to a hybrid sugar, such as Le x /SLe x , monosialosyl-Le a I, Le a , Le x , monosialos
  • the instant invention also provides a method of inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion at a site of inflammation in a warm-blooded animal thereby reducing inflammatory potential at the site and comprises administering to warm-blooded animal an effective amount of at least one agent selected from the group consisting of (a) a hybrid sugar, such as, Le x /SLe x ; (b) an appropriate mixture of sugars which are the components of a hybrid sugar (a), such as, Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specifically bind to a hybrid sugar, such as, Le x /SLe x , monosialosyl-Le 8 I, Le 8 , Le x , monosia
  • the instant invention provides a method for identifying a tumor associated carbohydrate antigen (TACA) epitope to which lectin activity of GMP-140 is directed, comprising: (A) constructing a fluorescent probe comprising fluorescent plastic beads coated with the TACA epitope suspected of being targeted by GMP-140; (B) incubating the fluorescent probe with a suspension of platelets; and (C) determining the degree of binding of the fluorescent probe to the platelets.
  • TACA tumor associated carbohydrate antigen
  • Figure 1 graphically illustrates the effects of methyl ⁇ -D-lactoside or methyl ⁇ -D-thiolactoside on the number and size of lung colony deposits of BL6 cells.
  • BL6 cells were preincubated with control medium, 0.1 M methyl ⁇ -D-lactoside ("Me- ⁇ -lactoside") or 0.1 M phenyl ⁇ -D-thiolactoside ("phe- ⁇ -S-lactoside). Twenty thousand cells were injected intravenously into C57Bl mice. Lung colony numbers were counted at 21 days and colonies were classified on the basis of diameter (> 1 mm vs. ⁇ 1 mm), as indicated for each bar. Colony numbers are expressed per single lung. Number of experiments ("n”) is indicated in parentheses.
  • Figure 2 graphically illustrates the effect of prior administration of methyl ⁇ -D-lactoside on the number and size of lung colony deposits of BL6 cells.
  • Methyl ⁇ -D-lactoside (1 ml dose) was injected intraperitoneally into C57B1 mice. After 10 minutes, BL6 melanoma cells were injected intravenously. Lung colonies were counted and sized at 19 days.
  • Group A represents control animals (not administered with methyl ⁇ -D-lactoside) and groups B and C represent animals injected with 0.25 M and 0.5 M methyl ⁇ -D-lactoside, respectively.
  • column 1 represents the total number of colonies
  • column 2 the number of colonies with diameter > 1 mm
  • column 3 the number of colonies with diameter ⁇ 1 mm. Number of experiments is expressed as "n".
  • Figure 3 graphically illustrates survival of cancer patients with or without expression of a defined tumor-associated carbohydrate antigen (TACA) in the tumors.
  • Panel 3A represents the expression of H/Le y /Le antigen in lung squamous cell carcinoma as determined by monoclonal antibody MIA-15-5.
  • Panel 3B represents sialosyl-Le x expression in colonic cancer using antibody FH6.
  • Panel 3C represents sialosyl-Tn expression in colonic cancer using antibody TKH2.
  • Panel 3D represents sialosyl-Tn level in sera of ovarian cancer patients.
  • Figure 4 graphically illustrates that melanoma cell adhesion on LacCer is based on GM3-LacCer interaction.
  • the order of metastatic potential is BL6>F10>Fl»Wa4.
  • Panel 4A shows the order of melanoma cell adhesion on a LacCer-coated solid phase.
  • Panel 4B shows the order of melanoma cell adhesion on LacCer/Fibronectin (FN) co-coated solid phase.
  • Panel 4C shows integrin-dependent adhesion.
  • Figure 5 graphically illustrates the melanoma cell (BL6) adhesion on LacCer (Panel 5A) and on endothelial cells (HuVEC) (Panel 5B) is inhibited by LacCer and GM3.
  • Figure 6 graphically illustrates the metastasis-inhibiting effect of methyl (Me)- ⁇ -lactoside.
  • Tumor cells were injected intravenously, followed by intraperitoneal injection of:
  • Figure 7 graphically illustrates H-Le y and H-H interaction.
  • Panel 7A shows H1-liposome binding to various glycolipids.
  • Panel 7B shows Le y -liposome binding to various glycolipids.
  • Figures 8A-8D are flow cytometric profiles of non-activated
  • Figure 9 graphically illustrates the binding indices of platelets with fluorescent beads coated with various GSL's.
  • the hatched bars represent non-activated platelets and the open bars represent activated platelets.
  • Figure 10 graphically illustrates the effects of various monoclonal antibodies on binding of activated platelets to sialosyl-Le a -coated beads.
  • the abscissa represents the percent inhibition.
  • Column 1 represents anti-GMP-140-mAb, IOP62;
  • column 2 represents anti-sialosyl-Le a monoclonal antibody, CA19-9;
  • column 3 represents anti-sialosyl-Le x monoclonal antibody, SNH4;
  • column 4 represents normal mouse IgG.
  • Figures 11A-11D illustrate experimental systems demonstrating dynamic adhesion of cells in a flow system.
  • Panel 11A shows the structure of the laminar flow chamber.
  • Panel 11B depicts a cross section of a laminar chamber in which the flow chamber body (16) is affixed tightly with the cover slip
  • Panel lie shows the entire assembly of the recording system.
  • Panel 11D is a schematic presentation of the flow of tumor cell in suspension passing over the cell layer or adhesion molecules.
  • Figure 12 is a graph showing the effect of various monoclonal antibodies on adhesion of human colon carcinoma Colo205 cells to interleukin-1-activated human umbilical vein endothelial cells in a dynamic flow system.
  • Open circles represent a mixture of irrelevant mouse IgG plus IgM (control)
  • the solid triangles represent monoclonal antibody CA19-9 directed to monosialosyl-Le a I
  • the open triangles represent monoclonal antibody SNH4 directed to sialosyl-Le x
  • the solid circles represent monoclonal antibody FH7 directed to monosialosyl-Le a II and disialosyl-Le a
  • the solid squares represent a mixture of irrelevant mouse IgG plus IgM and non-activated endothelial cells.
  • Figure 13 depicts binding of mAb's to HL60 cells and the effect of sialidase thereon. Binding activity was determined by flow cytometry. Abscissa: log fluorescence intensity. Ordinate: relative cell number.
  • Panel A Solid line, cells stained with mAb SNH4 as primary antibody. Dotted line, control cells stained with mouse IgG plus IgM [10 ⁇ g/ml] as primary antibody.
  • Panel B mAb SNH3 as primary antibody; control as in Pane A.
  • Panel C Solid line, cells treated with Newcastle Disease Virus (NDV) sialidase and then stained with mAb SNH4. Dotted line, control cells (as in Panel A, after sialidase treatment).
  • NDV Newcastle Disease Virus
  • Panel D NDV sialidase followed by mAb SNH3; control as in Panel C.
  • Panel E Vibrio cholerae (VC) sialidase followed by mAb SNH4.
  • Panel F VC sialidase followed by mAb SNH3. Note that expression of SLe x (defined by both SNH3 and SNH4) was abolished completely by both NDV and VC sialidases.
  • Figure 14 depicts adhesion of HL60 cells to E-selectin-coated plates in a static system. Abscissa, type of treatment. Ordinate, percent cell adhesion relative to untreated control cells. Panel A: effects of various sialidases. Panel B: effects of anti-Le x and anti-SLe x mAb's alone and in combination (incubated 90 min at 37oC). Panel C: effects of NDV sialidase plus mAb. Panel A: NDV sialidase (which cleaves ⁇ 2 ⁇ 3 sialosyl at a terminal Gal, eliminates the SLe x structure and abolishes reactivity with mAb's SNH3 and SNH4, see Figure 13, but did not abolish adhesion.
  • NDV sialidase which cleaves ⁇ 2 ⁇ 3 sialosyl at a terminal Gal, eliminates the SLe x structure and abolishes reactivity with mAb's SNH3 and SNH4, see Figure 13, but did not abolish adh
  • Panel B anti-SLe x mAb's were less effective than anti-Le x mAb's. Combinations of both types of mAb's were most effective.
  • Panel C Adhesion was inhibited most effectively by NDV sialidase plus anti-Le x mAb.
  • Figure 15 depicts adhesion of HL60 cells to E-selectin-coated plates in a dynamic flow system. Truncated E-selectin was coated onto marked areas (diameter of about 0.5 cm) on plastic plates and adhesion under defined wall shear stresses was assayed as described herein. Abscissa, shear stress (dynes/cm). Ordinate, number of cells adhered within 3 min. Panel A: hollow circle, control (untreated) cells; solid triangle, cells treated with NDV sialidase; solid circle, VC sialidase; and hollow triangle, AU sialidase.
  • Panel B hollow circle, control; solid triangle, cells cultured in medium containing anti-SLe x IgG 3 mAb SNH4; solid circle, anti-Le x IgM mAb FH2; and hollow triangle, anti-Le x IgG, mAb SH1.
  • Panel C hollow circle, control; solid triangle, NDV sialidase; solid circle, mAb SH1; and hollow triangle, NDV sialidase plus mAb SH1.
  • Panel D hollow circle, control; solid circle, mixture (1:1) of mAb's SNH4 and FH2; and hollow triangle, mixture (1:1) of mAb's SNH4 and SH1.
  • Figure 16 depicts reactivity of Colo201 cells with various mAb's, with or without sialidase treatment.
  • Colo201 cells were reactive strongly with anti-SLe a I mAb's CA19-9 and NKH1 (Panel A), anti-Le a mAb CA3F4 (Panel B) and anti-SLe a II mAb FH7 (Panel C).
  • Reactivity with CA19-9 was decreased by NDV sialidase (Panel D) and abolished by VC sialidase (Panel G).
  • Reactivity with CA3F4 was increased slightly by NDV and VC sialidases (Panels E and H).
  • Reactivity with FH7 was unchanged by NDV sialidase (Panel F) and decreased slightly by VC sialidase (Panel I).
  • Figure 17 depicts adhesion of Colo201 cells to E-selectin-coated plates in a static system. Abscissa and ordinate as in Figure 14.
  • Panel A effects of various sialidases (90 min. incubation, 37oC).
  • Panel B effects of sialidases (18 hr. incubation, 37°C), cells were first fixed with 0.5% paraformaldehyde for 10 minutes at room temperature.
  • Panel C effects of sialidases followed by mAb's.
  • NDV sialidase which cleaves ⁇ 2 ⁇ 3 sialosyl at terminal Gal, did not affect adhesion, whereas VC and AU sialidases, which cleave sialic acid residues regardless of location, abolished adhesion (Panel B).
  • Panel C most effective inhibition was observed with VC or AU sialidase plus mAb CA3F4.
  • Figure 18 depicts adhesion of Colo201 cells to E-selectin-coated plates in a dynamic flow system.
  • the adhesion assay is as described herein. Abscissa and ordinate as in Figure 15.
  • Panel A hollow circle, control; solid circle, NDV sialidase; hollow triangle, AU sialidase; and solid triangle, VC sialidase.
  • Panel B hollow circle, control; solid circle, anti-SLe a I mAb CA19-9; hollow triangle, anti-SLe a II mAb FH7; and solid triangle, anti-Le a mAb CA3F4.
  • Panel C hollow circle, control; solid circle, CA3F4; solid triangle, VC sialidase; hollow inverted triangle, VC sialidase plus CA19-9; and hollow triangle, VC sialidase plus CA3F4 (note that adhesion was most strongly inhibited by that combination).
  • Panel D hollow circle, control; solid triangle, NDV sialidase; solid inverted triangle, CA3F4; hollow inverted triangle, NDV sialidase plus CA19-9; solid circle, NDV sialidase plus FH7; and hollow triangle, NDV sialidase plus CA3F4 (note that adhesion was inhibited most strongly by that combination).
  • Figure 19 depicts the effect of Newcastle Disease Virus (NDV) sialidase, Vibrio cholerae (VC) sialidase or mAb's SNH4 or SH1 on HL60 binding to ELAM-coated plates in a dynamic flow system under various shear strength conditions.
  • the ordinate represents per cent cell binding relative to untreated control cells.
  • the antibodies were used at 15 ⁇ g/ml, NDV sialidase at 0.2 U/ml and VC sialidase at 0.1 U/ml. Each point represents the mean of three experiments. Number of untreated cells bound at shear stresses of 15.5, 7.75, 3.13, 1.56 and 0.78 dynes/cm 2 were 4.5, 27, 109.6 206.2 and 283.8 cells/mm 2 , respectively.
  • Figure 20 depicts various branched sugars.
  • the hybrid sugar, Le x /SLe x is depicted as structure 1.
  • the glycolipids containing such a structure were isolated from colon carcinoma or were prepared from G8 ganglioside presented in Structure 5 originally found in human erythrocytes (Watanabe et al., J. Biol. Chem., 254:8223, 1979) by enzyme catalyzed ⁇ 1 ⁇ 3 fucosylation.
  • Structure 2 was obtained by ⁇ 1 ⁇ 3 fucosylation of compound 6 originally obtained from human placenta. Structure 2 however did not exhibit high affinity binding to E-selectin.
  • Structures 3 and 4 depict analogs with high affinity binding sites having Le x and sialyl-Gal ⁇ 1 ⁇ 3GalNac within the same molecule (Structure 3), or the hybrid molecule Le a /SLe a , the positional isomer of structure 1.
  • Figure 21 depicts the relative adhesion of NS-1 cells expressing E-selectin on various "glyco-liposomes" coated on a plastic surface.
  • Panel 21A shows the result of such relative adhesion in a dynamic flow setting under middle shear stress conditions (7.75 dynes/cm 2 ).
  • the first seven bars indicate relative adhesion of NS-1 cells to SLe x on each glycoliposome as indicated.
  • Cpd I is structure 1 of Figure 20 and Cpd II is structure 2 of Figure 20.
  • Bars 8-10 show a mixture of Le x with different types of compounds as indicated.
  • the value of relative adhesion is expressed in comparison with the adhesion of SLe x -liposome as 100%. Values represent the mean of five determinations.
  • Panel 2IB indicates the same relative adhesion of NS-1 cells at high shear stress conditi .ons (11.8 dynes/cm 2 ). The value is expressed in terms of the adhesion on SLe x -coated plates. Values represent the mean of five determinations.
  • Figure 22 depicts the relative adhesion of NS-1 cells expressing E-selectin on various glycoliposomes coated on plastic plates at different shear stress conditions.
  • CPD I and CPD II are structures 1 and 2 of Figure 20. Enhancement of adhesion on CPD I-coated plates was noted only at middle to high shear stress conditions.
  • the ordinate indicates the relative adhesion as compared with that of the SLe x liposome.
  • the abscissa indicates the wall shear stress in dynamic flow in dynes/cm.
  • DSI represents disialosyl-I antigen.
  • Figure 23 depicts cell numbers bound per square millimeter on various glycoliposomes coated on a plastic surface with different glycolipid concentrations. Note that structure 1 of Figure 20 adheres E-selectin-expressing cells much more avidly than on SLe x -coated plates at high shear stress. The difference is not as stark at low shear stress. The ordinate indicates the number of cells bound per millimeter and the abscissa indicates glycolipid concentration in ⁇ m. Each point is the mean of five determinations.
  • Figure 24 depicts adhesion of NS-1 cells expressing E-selectin on glycoliposomes having a mixture of SLe x and various other glycolipids.
  • the ordinate shows the number of cells adhered per field.
  • the solid circle is SLe x + SPG.
  • the hollow circle is SLe x + H2.
  • the solid triangle is SLe x + Le x .
  • the hollow triangle is SLe x + Le y .
  • Each point is the mean of five determinations.
  • the instant invention in one aspect is directed to methods and compositions for the inhibition of tumor cell metastasis potential and invasiveness.
  • Numerous tumor cells possess the ability to metastasize, i.e., to form a secondary tumor colony at a distant site.
  • Sources of malignant tumor cells include melanoma, lung, breast, colorectal and urogenital cancers, such as bladder and prostate cancers.
  • TACA tumor-associated carbohydrate antigens
  • TACA tumor-associated carbohydrate antigens
  • LACA antibodies directed to those TACA's
  • oligosaccharide components of those TACA's oligosaccharide components of such TACA's
  • conjugates of such TACA's or of oligosaccharide components of such TACA's such as multivalent conjugates of lysyllysine or TACA-bearing glycosphingolipid (GSL) liposomes
  • GSL glycosphingolipid
  • mimetics of the TACA's Generally, unless indicated to the contrary, tumor cells and leukocytes are substantial equivalents inasmuch as both bind to endothelial cells by carbohydrate structures.
  • TACA epitopes play essential roles in tumor cell adhesion through interaction with endothelial cells, platelets and basement membranes, whereby tumor metastasis and invasion may occur.
  • the mechanism of adhesion may be based on carbohydrate (CHO) CHO-CHO interaction, CHO-lectin interaction or CHO-selectin family interaction.
  • Adhesion of various tumor cells to non-activated endothelial cells is mediated initially by carbohydrate to carbohydrate interactions, which in turn, trigger activation of endothelial cells to express selectins, such as ELAM-1 and GMP-140, Kojima & Hakomori, J. Biol. Chem., 266:17552, 1991; Kojima et al., J. Biol. Chem., 267:17264, 1992; Hakomori, Histochem. J., 24:771, 1992. Subsequently, adhesion of various tumor cells to activated endothelial cells and platelets is mediated primarily by the LECCAM or selectin superfamily (e.g., ELAM-1 and GMP-140).
  • selectins such as ELAM-1 and GMP-140
  • Tumor cell adhesion mediated by sialosyl-Le x is inhibited by anti-sialosyl-Le x monoclonal antibodies (FH6, CSLEX, SNH3 and SNH4) and tumor cell adhesion mediated by monosialosyl-Le a I is inhibited by monoclonal antibodies (CA19-9, CSLEA, NKH1 andNKH2) directed to that epitope.
  • Colo205 tumor cells which express predominantly type 1 chain sialosyl-Le a and to a lesser extent sialosyl-Le x , to endothelial cells is inhibited by anti-sialosyl-Le a monoclonal antibody and to a lesser extent by anti-sialosyl-Le x monoclonal antibody.
  • ELAM-1 and GMP-140 previously termed CD62 or PADGEM and also known as E-selectin and P-selectin.
  • GMP-140 is the major selectin (LECCAM) located on ⁇ -granules of platelets or Weibel-Pallade bodies of endothelial cells (EC's). On activation of those cells, GMP-140 is redistributed rapidly to the cell surface, where it plays an important role in adhesion of platelets or EC's to certain carbohydrate epitopes expressed on blood cells or tumor cells, resulting in aggregation of platelets or tumor cells, or adhesion thereof to capillary endothelia. GMP-140-mediated cell adhesion is believed by the instant inventors to be involved in initiation of metastatic deposition of tumor cells and initiation of inflammatory processes.
  • LECCAM major selectin
  • ELAM-1 is expressed on endothelial cells after activation with interleukin-1, TGF- ⁇ , TNF- ⁇ or lipopolysaccharide.
  • ELAM-1-mediated cell adhesion also is believed to be involved in initiation of metastatic deposition of tumor cells.
  • the instant invention in another aspect is directed to inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion, especially at tvimor cell sites.
  • GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion can be inhibited through the use of (a) a hybrid sugar, such as Le x /SLe x ; (b) a mixture of sugars which are the components of a hybrid sugar (a), such as Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specifically bind to a hybrid sugar (a), such as Le x /SLe x , monosialosyl-Le a I, Le a , Le x , monosialosyl
  • tumor metastasis and invasion is inhibited by blocking tumor cell adhesion thereby significantly reducing or eliminating the spread of metastatic cells.
  • tumor metastasis and invasion is minimized by inhibiting: (1) GMP-140-mediated tumor cell aggregation or adhesion at a tumor site due to: (a) adhesion of tumor cells to platelets, (b) adhesion of tumor cells to tumor cells via platelets, (c) adhesion of tumor cells to EC's via platelets and (d) adhesion of tumor cells to EC's directly via GMP-140; and (2) ELAM-1-mediated tumor cell aggregation or adhesion at a tumor site due to adhesion of cells to EC's directly via ELAM-1.
  • inflammation is minimized by inhibiting GMP-140-mediated leukocyte aggregation, adhesion or migration at a potential site of inflammation due to: (a) adhesion of leukocytes to platelets, (b) adhesion of leukocytes to endothelial cells (EC) via platelets, (c) adhesion of leukocytes to EC's directly via selectin and (d) transendothelial migration of leukocytes.
  • TACA's suitable for use within the instant invention are those showing differential prognostic significance (i.e., TACA's that may be correlated clearly with invasive or metastatic potential). Within the context of the instant invention, such TACA's may be distinguished through a comparison of invasiveness, metastasis and clinical prognosis of similar tumors showing expression vs. non-expression of such TACA's.
  • TACA's for use within the present invention include H/Ley/Le b , sialosyl-Le x (SA-Le x or SLe x ), Le a , Le x , monosialosyl-Le a I (SLe a or SA-Le a ) and sialosyl-Tn (SA-Tn or STn).
  • Derivatives of such TACA's include hybrid sugars, such as Le x /SLe x , dimeric Le x , sialosyl-dimeric Le x , trifuscosyl Le y , disialosyl-Le a and monosialosyl-Le a II.
  • TACA's for use within the instant invention exhibit a differential prognostic significance.
  • a differential prognostic significance may be illustrated by the fact that tumors expressing H/Le y /Le antigens (as defined by monoclonal antibody MIA-15-5) showed much worse patient prognosis than tumors not expressing those antigens.
  • FIG. 3A patients with squamous cell lung carcinoma expressing H/Le y /Le b had only an 11% survival over a 5-year period (i.e., 89% died) whereas comparable patients not expressing H/Le y /Le b had an approximately 62% survival over the same period.
  • antibodies or a mixture of antibodies to suitable TACA's may be employed within the context of the instant invention.
  • such antibodies include both monoclonal and poiyclonal antibodies and may be intact molecules, a fragment of such a molecule or a functional equivalent thereof.
  • the antibody may be engineered genetically. Examples of antibody fragments include F(ab') 2 , Fab', Fab and Fv.
  • poiyclonal antibodies may be produced by immunization of an animal and subsequent collection of sera therefrom. Immunization is accomplished, for example, by a systemic administration, such as by subcutaneous, intrasplenic or intramuscular injection, into a rabbit, rat or mouse. It is preferred generally to follow the initial immunization with one or more booster immunizations prior to sera collection. Succh methodology is well known and described in a number of references.
  • Monoclonal antibodies suitable for use within the instant invention include those of murine or human origin, or chimeric antibodies such as those which combine portions of both human and murine antibodies (i.e., antigen binding region of murine antibody plus constant regions of human antibody).
  • Human and chimeric antibodies may be produced using methods known by those skilled in the art. Human antibodies and chimeric human-mouse antibodies are advantageous because such antibodies are less likely than murine antibodies to cause the production of anti-antibodies when administered clinically.
  • Monoclonal antibodies may be produced generally by the method of Köhler and Milstein (Nature 256:495, 1975; Eur. J. Immunol. 6:511, 1976), as well as by various techniques which modify the initial method of K ⁇ hler and Milstein (see Harlow and Lane (eds.), "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988, which is herein incorporated by reference in its entirety).
  • the lymph nodes and/or spleen of an animal immunized with one of the TACA's or the oligosaccharide components thereof are fused with myeloma cells to form hybrid cell lines ("hybridomas" or "clones").
  • hybridoma secretes a single type of immunoglobulin and, like the myeloma cells, has the potential for indefinite cell division. It may be desirable to couple such molecules to a carrier to increase immunogenicity. Suitable carriers include keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof.
  • monoclonal antibodies suitable for use within the present invention include MIA-15-5 (Miyake & Hakomori, Biochem. 30:3328, 1991), as well as the monoclonal antibodies cited in Hakomori, Advances In Cancer Research 52:257-331, 1989.
  • oligosaccharide components of suitable TACA's also may be used in the instant invention.
  • oligosaccharide includes naturally derived oligosaccharides, synthetically prepared and mimetic derivatives of either, including portions of a TACA oligosaccharide component.
  • Additional oligosaccharide components useful in the instant invention include lactose and lactose derivatives, such as methyl ⁇ -D-lactoside, lact-N-tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc) and phenyl ⁇ -D-thiolactoside.
  • lactose and lactose derivatives such as methyl ⁇ -D-lactoside, lact-N-tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc) and phenyl ⁇ -D-thiolactoside.
  • lactose derivatives such as methyl ⁇ -D-lactoside, lact-N-tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc) and phenyl ⁇ -D-thiolactoside.
  • lactose derivatives block binding of melanoma cells
  • oligosaccharide components suitable for inhibiting metastasis potential of cells of a particular tumor may be identified based on determining the structure of specific carbohydrate chain(s) which are involved in the ability of the tumor to metastasize.
  • the identification of carbohydrate-containing molecules involved in the ability of a tumor to metastasize may be accomplished in a variety of ways, including through the use of glycosidases and inhibitors of glycosy1transferases.
  • the structure of carbohydrates bound to either lipids or proteins may be determined based on degradation, mass spectrometry, including electron-impact direct-probe (El) and fast atom bombardment (FAB), and methylation analysis (techniques described, for example, in Nudelman et al., J. Biol. Chem. 261:5487, 1986).
  • Degradation analysis may be accomplished chemically and/or enzymatically, e.g., by glycosidases.
  • the carbohydrate sequence suggested by degradation analysis may be determined by methylation analysis (Hakomori, J. Biochem. 55:205, 1964) followed by chemical ionization mass spectrometry of permethylated sugars (Stellner et al.. Arch. Biochem. Biophys. 155:464, 1974; Levery et al., Meth. Enzymol. 138:13, 1987).
  • EI mass spectrometry may be performed on permethylated glycans or after the appropriate degradation of intact glycans (Kannagi et al., J. Biol. Chem. 259:8444, 1984; Nudelman et al., J. Biol. Chem.263:13942, 1988). Homogeneity of the carbohydrate sequence may be demonstrated based on various chemical and physical criteria, including proton NMR spectroscopy of intact or methylated glycans and FAB mass spectrometry. Once the carbohydrate sequence has been determined, it will be evident to those of ordinary skill in the art to select an appropriate oligosaccharide for inhibiting the metastasis potential of a tumor cell.
  • conjugates of suitable TACA's or oligosaccharide components thereof such as multivalent conjugates with lysyllysine or TACA-bearing glycosphingolipid (GSL) liposomes (or glyco-liposomes), also may be used in the instant invention.
  • GSL glycosphingolipid
  • the components of the conjugate may be coupled covalently to one another either directly or via a linker group.
  • a direct reaction between components is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one component may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acyl halide, or with an alkyl group containing a leaving group, e.g., a halide, on the other.
  • a linker group can serve to increase the chemical reactivity of a substituent and thus increase the coupling efficiency.
  • An increase in chemical reactivity also may facilitate the use of functional groups on components which would not otherwise be possible.
  • a carboxyl group may be activated. Activation of a carboxyl group includes formation of an "active ester", such as a succinimidyl ester.
  • active ester is known to refer to esters which are highly reactive in nucleophilic substitution reactions.
  • TACA's may be incorporated into the outer surface of glycosphingolipid (GSL) liposomes.
  • oligosaccharides may be coupled to (i.e., covalently bonded to) a straight-chain amphophilic polymer, such as polyethyleneglycol.
  • a straight-chain amphophilic polymer such as polyethyleneglycol.
  • a representative example of a method for producing an oligosaccharide-polyethyleneglycol conjugate is the reaction of an oligosaccharide, which has been derivatized to contain a succinimidyl group, with a polyethyleneglycol having a terminal amino group.
  • the latter compound has a general formula of NH 2 -(CH 2 CH 2 -O) n -CH 3 , where n typically averages 44.7 (i.e., molecular weight of about 2,000) to 112.9 (i.e., molecular weight of about 5,000).
  • any structure which may show more effective blocking activity of the lectin domain than naturally occurring epitopes are useful in the present invention.
  • Such unnatural synthetic compounds termed "mimetics", of, for example, sialosyl-Le x or sialosyl-Le a I or II, which mimic the surface structure of naturally occurring epitopes but show better blocking activity of carbohydrate-dependent adhesion, can be considered.
  • Examples of useful mimetics include, but are not limited to, sialosyl-Le x or monosialosyl-Le a I or II having trifluoro-L-fucose, N-trifluoro-acetyl-glucosamine or a heterocyclic or aromatic ring structure having a sialic acid analog and fucose analog at the same distance and spacial configuration as those found in naturally occurring sialosyl-Le x , monosialosyl-Le a I and II, or the H/Le y /Le b structure having trifluoro-L-fucose, N-trifluoro-acetyl-glucosamine or sialosyl-Tn analogs containing N-trifluoro-acetyl-neuraminic acid.
  • a modified carbohydrate epitope, or any other "mimetic" mimicking the surface structure of a carbohydrate epitope, which blocks cell adhesion through tumor-associated carbohydrates more efficiently than a naturally occurring epitope is within the scope of the instant invention.
  • the inhibition of metastasis potential of tumor cells and GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion have a variety of in vitro and in vivo uses, e.g., treatment of isolated tumor cells or tumor-bearing hosts and treatment of disease processes involving GMP-140 or ELAM-1.
  • the instant invention provides a method for inhibiting tumor cell metastasis potential within a biologic preparation.
  • the method comprises incubating a biologic preparation with at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to those antigens, (c) oligosaccharide components of those antigens, (d) conjugates of those antigens or oligosaccharide components and (e) mimetics of the tumor-associated carbohydrate antigens, the agent inhibiting the metastasis potential of the preparation.
  • at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to those antigens, (c) oligosaccharide components of those antigens, (d) conjugates of those antigens or oligosaccharide components and (e) mimetics of the tumor-associated carbohydrate antigens,
  • the instant invention also provides a method for inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion in a biologic preparation.
  • the method comprises incubating the biologic preparation with at least one agent selected from the group consisting of (a) a hybrid sugar, such as Le x /SLe x ; (b) sugar components of a hybrid sugar (a), such as Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specifically bind to a hybrid sugar, such as Le x /SLe x or to the component sugars thereof, monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or
  • Suitable biologic preparations include cell cultures and cell suspensions in biologic fluids, such as blood, urine, lymph, synovial and cerebrospinal fluid.
  • TACA's, oligosaccharides or conjugates thereof generally will be incubated at a final concentration of about 0.1 to IM, and typically at about 0.2 to 0.5 M. Incubation is performed typically for 5 to 15 minutes at 37°C.
  • the preparation may be injected or implanted in an animal, e.g., to confirm effectiveness of the inhibition of metastasis potential.
  • the instant invention also provides a method for inhibiting tumor cell metastasis potential in a warm-blooded animal, such as a human.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance,
  • oligosaccharide components of those antigens (c) oligosaccharide components of those antigens, (d) conjugates of those antigens or the oligosaccharide components and (e) mimetics of monosialosyl-Le a I, Le a , Le x , monosialosyl-Le 8 II, disialosyl-Le a or sialosyl Le x , the agent inhibiting the metastasis potential of the preparation.
  • the instant invention also provides a method for inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion at a tumor cell site in a warm-blooded animal.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent selected from the group consisting of (a) a hybrid sugar, such as Le x /SLe x ; (b) component sugars of a hybrid sugar (a), such as Le x and SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specificall bind to a hybrid sugar, such as Le x /SLe x , monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disi
  • the instant invention also provides a method for inhibitin GMP-140-mediated cell aggregation or adhesion at an inflammatio site in a warm-blooded animal.
  • the method comprises administering to warm-blooded anima an effective amount of at least one agent selected from the grou consisting of: (a) a hybrid sugar, such as Le x /SLe x ; (b) component sugars of a hybrid sugar (a), such as Le x an SLe; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x ; (d) antibodies that specificall bind to a hybrid sugar, such as Le x /SLe x , monosialosyl-Le a I, Le a , Le x , monosialosyl-Le a II, disialosyl-Le a or sialosyl Le x (e) oligosaccharide components of a hybrid sugar, such a Le x /SLe x , monosi
  • TACA's, oligosaccharides or conjugates thereof generally will be administered at a concentration of about 0.1 to 1 M and typically at about 0.2 to 0.5 M. It will be evident to those skilled in the art how to determine the optimal effective dose for a particular substance, e.g., based on in vitro and in vivo studies in non-human animals. A variety of routes of administration may be used. Typically, administration will be intravenous or intracavitary, e.g., in the pleural or peritoneal cavities, in the bed of a resected tumor or at a site of inflammation.
  • a TACA, antibody, oligosaccharide or derivative as discussed above may be administered in combination with a pharmaceutically acceptable carrier or diluent, such as physiologic saline.
  • a pharmaceutically acceptable carrier or diluent such as physiologic saline.
  • the agents that inhibit or reduce metastatic potential may be administered in combination with an immunotherapeutic or chemotherapeutic substance, and the agents that reduce inflammatory potential may be administered in combination with an anti-inflammatory substance.
  • each compound may be administered sequentially, simultaneously or combined and administered as a single composition. Diagnostic techniques, such as CAT scans, may be performed prior to and subsequent to administration to confirm the effectiveness of the inhibition of metastatic potential or inflammatory potential.
  • One in vitro system for measuring adhesion or aggregation of tumor cells to other cells (e.g. EC's), or for determining successful inhibition of adhesion or aggregation is a dynamic flow system similar to that described by M. B. Lawrence et al. (Blood 70:1284, 1987) and which is shown in Figures 11A, 11B, 11C and 11D.
  • a parallel-plate laminar flow chamber (1) (shown upside down for convenience) connected to a pressure pump (2) via tubing (18) is used to simulate the flow shear stresses present in physiological microvascular environments.
  • the flow chamber consists of a plastic or glass cover slip (3) resting on chamber body (16) on which a parallel, transparent plastic surface (4) is attached with a rubber or silicone gasket (5); there is a 114 ⁇ m gap between the two surfaces, and this gap is connected to an inlet slot (6) connected to an inlet manifold (8) and outlet slot (7) connected to an outlet manifold (19) ( Figure 11A).
  • a laminar flow with defined rate and wall shear stress is achieved by manipulation of the pressure pump (2), which is connected to the inlet manifold (8) of the flow chamber via tubing (18).
  • Figure 11B depicts the configuration of an assembled flow chamber (1).
  • Cells e.g., endothelial cells
  • a tumor cell suspension in medium flows from inlet manifold (16) to outlet manifold (19).
  • the structure of the flow chamber (1) in Figure 11B is shown upside down for convenience.
  • the chamber is placed under an inverted microscope stage, right side up ( Figure 11C), and the flow of tumor cells over the cell layer (e.g., endothelial cell layer) is observed under the microscope.
  • the observed pattern of rolling and stopping (i.e., pattern of adhesion) of tumor cells can be recorded on videotape.
  • the cells (9) are grown as a monolayer, or adhesion molecules are affixed, on the cover slip (3) and a laminar flow of tumor cell suspension (14), maintained in a vessel in a water bath (17), is passed through the chamber via tubing (18).
  • Cell movements are observed under an inverted phase-contrast microscope (10) and recorded by time-lapse videocassette recorder (11) using a video camera (12) and a digital image processor (13).
  • Adhesion is observed as rolling followed by stopping of cells. Number of cells bound during a set time, e.g. 3 minutes, at different shear stresses, e.g., from 0.4 to 4.8 dynes/cm, are counted from several fields recorded on videotape ( Figure 11B).
  • Figure 11D schematically shows laminar flow of tumor cell suspension (14) through a chamber in which one surface is coated with endothelial cells (9). Rolling or stopped cells (15) are observed under an inverted microscope and recorded on videotape, as described above. The arrows indicate the direction of flow of the tumor cell suspension (14).
  • the instant invention also provides a method for identifying a TACA epitope to which lectin activity of a selectin, such as GMP-140, is directed.
  • the TACA epitopes were studied based on the inhibitory effect of various glycosphingolipids (GSL's), GSL oligosaccharides or GSL-containing liposomes on adhesion of blood cells or tumor cells to a solid phase (e.g., a plastic surface) coated with activated platelets.
  • GSL's glycosphingolipids
  • GSL oligosaccharides or GSL-containing liposomes bind readily to a gelatin-coated solid phase via GpIIb/IIIa, the major platelet integrin receptor.
  • sialosyl-Le x is the carbohydrate epitope defined by GMP-140 (Polley et al., Proc. Natl. Acad. Sci. USA 88: 6224, 1991).
  • Fluorescent plastic e.g. polystyrene
  • GSL's are known to be adsorbed strongly on such beads, which allows construction of fluorescent probes containing specific GSL's.
  • Platelets activated or non-activated are incubated with such GSL-coated beads, followed by determination of platelet fluorescence intensity by flow cytometry.
  • activated platelets were found to show much stronger binding to fluorescent beads coated with monosialosyl-Le a I (see Table 3) than to beads coated with any related GSL.
  • the binding of platelets to sialosyl-Le a -coated beads was inhibited by anti-GMP-140 monoclonal antibody or anti-sialosyl-Le a monoclonal antibody, but not by anti-sialosyl-Le x monoclonal antibody.
  • binding of activated platelets to sialosyl-Le x -coated beads was observable, the level of binding was much lower than binding to sialosyl-Le a -coated beads.
  • Those results indicate that the primary epitope structure defined by GMP-140 is sialosyl-Le a , rather than sialosyl-Le x .
  • ELAM-1 (E-selectin) is expressed on the surface of activated endothelial cells.
  • ELAM-1 has a carbohydrate-binding domain at the amino terminal region and indeed ELAM-1 is known to bind SLe x and SLe a .
  • the instant invention is a result of systematic studies on selectin-dependent adhesion under static and dynamic circumstances.
  • the methods employed include, (i) adhesion of tumor cells to IL-1-activated human umbilical cord endothelial cells (HUVEC); (ii) adhesion of tumor cells to E-selectin-coated solid supports, for example, by using recombinant ELAM-1; (iii) adhesion of fluorescent particulate solid supports coated with glycoliposomes with activated platelets or HUVEC's expressing P-selectin or E-selectin; and (iv) adhesion of NS-1 myeloma cells, transfected with E-selectin coding sequences and permanently expressing E-selectin onto plates coated with glycoliposomes.
  • HUVEC human umbilical cord endothelial cells
  • the systems (i), (ii) and (iii) were employed to assess the effect on adhesion of various mAb's directed to SLe x , SLe a I, SLe a II, Le x , Le a and related structures; combinations of such mAb's; sialidases with various substrate specificities; or combinations of various sialidases and mAb's.
  • the method of (iv) was used to compare the intensity of adhesion under dynamic conditions.
  • the instant invention relates to carbohydrates defined by formulae (I), (II) and (III) below which are characterized by internal sialosyl residues or a branched structure.
  • Formula (I) relates to a type 1 or extended type 1 chain with internal ⁇ 2 ⁇ 6 sialosyl substitutions and an ⁇ 1 ⁇ 4 fucosyl substitution.
  • R 1 is H or a sialic acid residue in ⁇ 2 ⁇ 3 linkage
  • R 2 is H or a sialic acid residue in ⁇ 2 ⁇ 6 linkage
  • n is equal to or greater than 0
  • R 3 is H or a fucosyl residue in ⁇ 1 ⁇ 4 linkage.
  • Formula (II) relates to a type 2 chain structure with internal sialosyl and fucosyl substitutions.
  • R 2 is as defined for formula I, R 4 is H or a fucosyl residue in ⁇ l ⁇ 3 linkage and R 5 is H, a sialic acid residue in ⁇ 2 ⁇ 3 linkage, NeuAc ⁇ 2 ⁇ 8NeuAc in ⁇ 2 ⁇ 3 linkage or
  • R 6 ⁇ NeuAc in ⁇ 2 ⁇ 3 linkage, wherein R 6 is one or more sugars other than a sialic acid residue and n is equal to or greater than 0.
  • Formula (III) relates to a type 2 chain structure which is a hybrid molecule comprising a branch wherein each branch comprises an epitope of a single carbohydrate antigen as disclosed herein.
  • a hybrid molecule does not necessarily comprise the entirety of the two component sugars that comprise the hybrid. Instead, the hybrid comprises the epitopes of the component sugars.
  • structure 1 comprises the epitopes of Le x and SLe x , however it will be noted that with reference to the diagrammatic structures of the various sugars set forth hereinbelow, not all of the Le x or SLe x molecules are found in the hybrid.
  • epitope is that portion of the sugar which interacts in the adhesion phenomenon.
  • each of R 10 and R 11 comprises galactose, Gal ⁇ 1 ⁇ 4GlcNAc or Gal ⁇ 1 ⁇ 3GlcNAc;
  • R 8 comprises Gal or GalNAc;
  • R comprises lactosyl ceramide or an O-linked sugar.
  • R 10 and R 11 may comprise fucosyl and sialic acid residues.
  • the hybrid structures are identified by the respective epitopes contained therein. Hence, structure 1 of Figure 20 is denoted SLe x /Le x , or Le x /SLe x .
  • Formula I is based on inhibition by various mAb's and sialidases and combinations thereof of E-selectin-dependent adhesion of tumor cells (e.g., Colo201 cells) which express exclusively type 1 chain, i.e., Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal, repeats thereof and substitutions thereof.
  • E-selectin-dependent adhesion of Colo201 cells was inhibited only minimally by mAb CA19-9 (directed to SLe a ) and moderately inhibited by mAb FH7 (directed to disialosyl Le a and monosialosyl Le a II).
  • Colo201 adhesion was inhibited most strongly by mAb CA3F4 (directed to monosialosyl Le a II and Le a or by a combination of CA19-9 plus CA3F4.
  • mAb CA3F4 directed to monosialosyl Le a II and Le a or by a combination of CA19-9 plus CA3F4.
  • Specific reactivities of FH7 with disialosyl Le a and monosialosyl Le a II, and of CA3F4 with monosialosyl Le a II, were described previously (Nudelman et al., J. Biol. Chem., 261: 5487, 1986).
  • NDV sialidase in combination with mAb's CA19-9 or CA3F4 strongly inhibited the adhesion.
  • the binding dynamics of selectins is vibrant, as revealed in dynamic flow systems which simulate more closely physiologic conditions, that is, for example, leukocytes or tumor cells can be moving at considerable speed in large and unoccluded small vessels and at a slower speed in occluded vessels and in tissue spaces.
  • cell interactions may be mediated by interaction with a first set of molecules that share a common characteristic
  • cell interactions may be mediated by interaction with a second set of molecules that share a common characteristic, different from that shared by the first set of molecules.
  • the binding requirements may vary depending on the speed at which the cells are moving.
  • E-selectin (ELAM)-mediated adhesion of HL60 cells is dependent on different carbohydrate structures when the cells are reacted in a stationary or slow moving setting or are reacted while the cells are in rapidly moving setting.
  • ELAM binds preferentially to ⁇ 2 ⁇ 3 sialylated and ⁇ 1 ⁇ 3 fucosylated structures, such as SLe x
  • ELAM preferentially binds to other structures, such as Le x , Le y , H and to various hybrid structures, such as Le x /SLe x .
  • Formula II is based on inhibition by various mAB's and sialidases and combinations thereof of E-selectin-dependent adhesion of HL60 tumor cells, which express only type 2 chain, i.e. Gal ⁇ 1 ⁇ 4GlcNAc ⁇ 1 ⁇ 3Gal and repeats thereof, and substitutions thereof.
  • Treatment of HL60 cells with NDV sialidase, which cleaves NeuAc ⁇ 2 ⁇ 3Gal (R., in Formula II) completely abolished reactivity of the cells with anti-SLe x mAb's, although the cells remained strongly adherent to E-selectin-coated plates and to activated EC's.
  • NDV sialidase treatment of HL-60 cells which removes NeuAc ⁇ 2 ⁇ 6Gal, completely abolished reactivity of cells with anti-SLe x mAb although the cells remained adherent to E-selectin plates and activated endothelial cells.
  • Adhesion was inhibited effectively with a combination of mAb's directed to Le x and SLe x .
  • type 2 chain structures with internally sialic acid residues were hitherto unknown. Data presented in the instant application indicate the natural occurrence of such epitopes.
  • the structures bindable to ELAM-1 can be synthesized using known techniques.
  • the carbohydrates can be synthesized chemically using known and commercially available reagents or can be synthesized using known and available enzymes to effect the appropriate linkage.
  • known sialosyl transferases and fucosyl transferases can be used to derivatize the basic carbohydrate backbone.
  • the carbohydrates bindable to ELAM-1 can be isolated using ELAM-1 as an absorbent.
  • ELAM-1 purified ELAM-1, cells expressing ELAM-1 or membrane preparations of cells expressing ELAM-1 can be used.
  • the ELAM-1 can be immobilized to a solid phase, such as an inert bead matrix or the inside wall of a vessel, to enhance separation.
  • suitable carbohydrates bindable to ELAM-1 such as extracts of HL60 or Colo201 cells obtained by known techniques, are exposed to the ELAM-1 affinity matrix. Following a washing procedure to remove unwanted and non-specifically bound components, the ELAM-1 together with carbohydrates bindable thereto are collected.
  • the carbohydrates bound to the ELAM-1 are separated from the ELAM-1, for example, by altering the salt concentration of the holding buffer, and collected.
  • the various carbohydrate species can be discriminated using known procedures, such as chromatography.
  • cells known to express predominantly type 1 chain structures or type 2 chain structures are grown and membrane preparations are obtained therefrom using known techniques.
  • the glycolipid and glycoprotein fraction of the membrane prep is obtained using known techniques and exposed to an affinity column wherein antibodies directed to carbohydrate epitopes, such as those described herein, are affixed to a matrix, such as agarose beads, to form an affinity matrix.
  • affinity chromatography procedure the bound materials are eluted and separated further by known techniques, such as HLPC and TLC.
  • the separated molecules in the separation medium can be exposed to ELAM-1-expressing cells that are labelled to serve as a tag, for example, the cells can be labelled metabolically with a radioisotope.
  • the ELAM-1-expressing cells will bind to the respective sites of the separation medium where separated ELAM-1 epitopes are found.
  • the TLC matrix can be autoradiographed to locate such sites of cell binding to identify ELAM-1 epitope-bearing molecules.
  • the respective sites of the TLC matrix can be excised and the molecules extracted.
  • the carbohydrates of formulae I and II can be derivatized to provide oligosaccharides with more desirable therapeutic properties.
  • portions of the structures comprising formula I or II can be substituted, for example, with sulfur-containing sugars or fluorine-containing sugars.
  • the oligosaccharide derivatives can be prepared using the methods disclosed hereinabove but substituting for the naturally occurring components the appropriate reagent comprising an altered substituent, such as 6-trifluoro-fucosyl which is incorporated into either of formula I or II as the fucosyl residues.
  • the carbohydrates bindable to ELAM-l can be used as immunogens to obtain antibodies bindable to the carbohydrates bindable to ELAM-1.
  • Either poiyclonal or monoclonal antibodies can be generated, using methods such as those described hereinabove, and in the references cited herein, which are incorporated by reference. Monoclonal antibodies are preferred.
  • ELAM-1 may serve to mediate intercellular interactions
  • carbohydrates bindable to ELAM-l, ELAM-l, antibody to ELAM-1 or antibody to carbohydrates bindable to ELAM-l for example, can be used to interrupt binding between ELAM-l and carbohydrates bindable thereto.
  • the carbohydrates bindable to ELAM-1, ELAM-1, antibody to ELAM-1 or antibody to carbohydrates bindable to ELAM-l are administered in therapeutically effective amounts and via routes that are determinable readily and routinely practicing settled methods of the pharmaceutic arts.
  • the terminal sialic acid is not essential in a carbohydrate bindable to ELAM-1.
  • Key elements held in common are the terminal galactose, glucosamine, ⁇ 2 ⁇ 6sialic acid and fucose residues.
  • antibodies capable of binding to such a structure are effective in inhibiting ELAM-1-mediated interactions. Suitable antibodies are CA3FA and FH7.
  • antibodies bindable to Le x such as, SH-1 and FH-2, and to SLe x , such as FH-6, SNH-4 and SNH-3, are effective cooperatively in inhibiting ELAM-1-mediated adhesion at high shear stress conditions.
  • ELAM and GMP-140 are carried by 0-linked sugar chains and selectin-dependent cell adhesion can be blocked by inhibitors of O-glycosylation (Kojima et al., Biochem. Biophys. Res. Commun. 182:1288, 1992). Hence, it often is preferable to have compounds of formulae (I), (II) and (III) carried on 0-linked carbohydrate chains.
  • a further means of interrupting ELAM-1 mediated interactions is using a combination of carbohydrates or antibodies to interfere with ELAM-1 binding to relevant carbohydrates.
  • the carbohydrates or antibodies are related to ELAM-1 or carbohydrates bindable thereto or in certain circumstances may be carbohydrates or antibodies that are not specifically those carbohydrates believed to bind ELAM-1.
  • SLe x and Le x are effective in inhibiting ELAM-1 interaction.
  • Suitable SLe x antibodies are SNH3 and SNH4; and suitable Le x antibodies are SH1 and FH2.
  • the skilled artisan can determine other suitable combinations practicing the methods taught herein using reagents disclosed herein, with particular attention drawn to the working examples set forth hereinbelow.
  • Heptaacetyllactosylimidate (Zimmermann et al., J. Carbohydr. Chem. 7:435, 1988) was reacted with methanol in dry dichloromethane containing trimethylsilyl trifluoromethanesulfonate according to a standard procedure (Grundler & Schmit, Liebigs. Ann. Chem. 1984:1826, 1984). Purification by silica gel column chromatography (toluene/EtOAc, 1:1 by vol.), followed by de-O-acetylation with 0.01 M sodium methoxide, gave methyl ⁇ -D-lactoside in 68% yield from the imidate: m.p. 211-212°C (lit.
  • Lactose octaacetate (Hudson & Kunz, J. Am. Chem Soc. 47:2052, 1926) was treated with thiophenol and SnCl 4 (Nicolaou et al., J. Am. Chem Soc. 110:7910, 1988) in dichloromethane at 0oC to give phenyl heptaacetyl ⁇ -D-thiolactoside in 80% yield.
  • the product was deacetylated with NaOMe in MeOH and neutralized with Amberlyst ® 15.
  • the polyethyleneglycol derivative of ⁇ -D-lactoside was arepared from readily available 3-succinimidooxycarbonylpropyl O-(2, 3, 4, 6-tetra-O-acetyl-O- ⁇ -D-galactopyranosyl)-(1 ⁇ 4)-2,3,6-tri-O-acetyl- ⁇ -D-glucopyranoside 1 and polyethyleneglycol methyl ether (average M.W. 2000; Aldrich Chemical, Milwaukee, WI) having a terminal amino group 2. (Zalipsky et al., Eur. Polym. J. 19:1177, 1983). Treatment of 1 (100 mg, 0.12 mmol) and 2.
  • the highly metastatic BL6 clone of the B16 melanoma cell line was obtained originally from Dr. Jean Starkey (Montana State
  • a suspension of BL6 cells (1-3 ⁇ 10 6 cells/ml RPMI 1640 medium) was prepared and aliquots were incubated in the presence or absence of various oligosaccharides at various concentrations, at 37oC for 5-10 minutes. Following incubation, typically, 3 ⁇ 10 4 or 2 ⁇ 10 4 cells (with or without oligosaccharide pretreatment) per 200 ⁇ l were injected via a tail vein into 8-week-old female mice. After 18-21 days, the mice were killed, the lungs were fixed in 10% formaldehyde in PBS (pH 7.4) and tumor cell colonies were counted under a dissecting microscope, thereby providing background values of metastatic melanoma colony number in lung under those conditions. Data on the number and the size of colonies were treated statistically by an analysis of variance (ANOVA) procedure. Colonies with a diameter of 1 mm or greater were considered large-size and those with a diameter less than 1 mm were considered small-size.
  • ANOVA analysis of variance
  • BL6 cells were incubated with various concentrations of lactose, lacto-N-tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4GLc), methyl ⁇ -D-lactoside or phenyl ⁇ -D-thiolactoside for various durations.
  • lactose lacto-N-tetrose
  • methyl ⁇ -D-lactoside methyl ⁇ -D-lactoside
  • phenyl ⁇ -D-thiolactoside methyl ⁇ -D-lactoside
  • phenyl ⁇ -D-thiolactoside phenyl ⁇ -D-thiolactoside
  • Lactose and lacto-N-tetrose showed 26% and 36% reductions, respectively, of metastatic colonies in lung when BL6 cells were preincubated with those sugars followed by intravenous injection of cells under identical conditions.
  • Treatment of BL6 cells with 0.1 M, 0.01 M or 0.005 M methyl ⁇ -D-lactoside under the same conditions as above resulted in (respectively) a 43%, 16% and 8% reduction of metastatic lung colony number compared to control.
  • the significant reduction caused by 0.1 M methyl ⁇ -D-lactoside was reproduced in three separate experiments and the reduction was found to be consistently between 35% and 45%.
  • the effect of methyl ⁇ -D-lactoside on melanoma cell metastasis was determined after administration of the oligosaccharide, followed by inoculation with tumor cells. Specifically, a one ml dose of methyl ⁇ -D-lactoside (at a concentration of 0.25 M or 0.5 M) was injected intraperitoneally in mice.
  • mouse melanoma B16 variants showing different degrees of metastatic potential showed the same order of expression of GM3 ganglioside, which was previously identified as a melanoma-associated antigen (Hirabayashi et al., J. Biol. Chem. 260:13328, 1985; Nores et al., J. Immunol. 139:3171, 1987).
  • Capillary endothelial cells are strongly reactive with antibodies directed to H/Le y /Le b , such as antibody MIA-15-5.
  • Liposomes comprising H-1 or Le y were made and exposed to plates to which various glycolipids had been affixed at a range of concentrations.
  • H-bearing liposomes bound to H or Le y coated onto plates were found to bind only to H-coated plates.
  • H and paragloboside are related, the only difference being the presence of a terminal fucose residue in H.
  • cells expressing H, Le y or Le can adhere to endothelial cells expressing H and possibly to Le y as well.
  • KUM-LK-2 is a human non-adenocarcinoma cell line characterized by producing spontaneous lung metastasis in nude mice. After screening 35 human carcinoma cell lines grown in nude mice, only that cell line produced metastatic deposits in nude mouse lung. KUM-LK-2 was used as the parent cell line to obtain, by limiting dilution technique, sub-cell lines producing lung metastasis on IV injection.
  • KUM-LK-2 was cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) at 37'C in a 5% CO 2 /95% air atmosphere. Cells were treated briefly with 2 mM EDTA solution and washed twice with RPMI 1640 to make a single cell suspension in RPMI with 10% FCS. Cell viability was > 98% as determined by trypan blue exclusion staining. A cell suspension containing 1 cell per 100 ⁇ l was transferred to each well of a 96-well microtiter plate (Corning Glass Works, Corning, NY) and cultured continuously for 24 hours. Each well then was examined by phase contrast microscopy.
  • HAL-8, HAL-24 and HAL-33 Three cell lines (HAL-8, HAL-24 and HAL-33) with different metastatic potential ("MP") were selected out of 25 clones obtained by limiting dilution technique on the basis of stable cell morphology.
  • the 25 clones were selected originally from 63 clones showing stable morphology as well as consistent in vitro cell growth.
  • mice 46 5.8 (3-8) a Nude mice were injected (2 ⁇ 10 5 cells) via the tail vein at various generation times as indicated.
  • mice Fifty-six days after injection, mice were killed and metastatic nodules on lung surface were counted under dissecting microscope. b Mean of 6 animals (range in parentheses)
  • the cell surface expression of various carbohydrate epitopes was analyzed by cytofluorometry using various monoclonal antibodies (mAb's) directed to Le x (mAb SH1), sialosyl-Le x (mAb SNH4), sialosyl-dimeric Le x (mAb FH6), T (mAb HH8), Tn (mAb 1E3) and sialosyl-Tn (mAb TKH2). All antibodies used were culture supernatants from the respective hybridomas, adjusted as 10 ⁇ g/ml of immunoglobulin.
  • the structures of sialosyl-Le x structure 1), sialosyl-dimeric-Le x (structure 2), dimeric-Le x (structure 3), trifucosyl-Le y (structure 4),
  • R represents a carrier molecule.
  • Structure 11 NeuAc ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 4Glc ⁇ 1 ⁇ Cer Structure 12: NeuAc ⁇ 2 ⁇ 3Gal ⁇ 1 ⁇ 4GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ R Structure 13 :
  • Cells were detached from culture flasks with 0.25% trypsin, 2mM EDTA solution and 1 ⁇ 10 cells were prepared for each mAb treatment. Cells were incubated with a mAb for 1 hour at 4oC and washed 2 times with RPMI 1640. Goat anti-mouse IgG or IgM-FITC (Boehringer-Mannheim, Indianapolis, IN), diluted 50 times with PBS, then was added and incubated 30 minutes at 4oC. Finally, cells were washed 3 times, resuspended with PBS and analyzed in an EPICS PROFILE flow cytometer (Epics, Hialeah, FL). The experiments were repeated with three different cell generations.
  • Patterns of expression of six carbohydrate epitopes (defined by the respective mAb's) on sub-cell lines HAL-8, HAL-24 and HAL-33 showed nearly identical profiles (as did the protein profiles for the three sub-cell lines) except in the case of sialosyl-dimeric-Le x .
  • HAL-8, HAL-24 and HAL-33 were found to express highly and equally sialosyl-Le x and sialosyl-Tn structures.
  • Each of the three lines expressed low quantities of Le x and Tn, and did not express T.
  • expression of sialosyl-dimeric Le x was high on HAL-8, moderate on HAL-33 and low on HAL-24.
  • sialosyl residues were assessed in the following manner. Cells were detached using 2 mM EDTA in PBS, washed and resuspended in 9 volumes of PBS. One ml of cell suspension was incubated 5 minutes at 37oC with 0.2 U/ml of Clostridium perfringens sialidase (type X, Sigma Chemical Co., St. Louis, MO) . After incubation, cells were washed three times, resuspended with RPMI 1640 and investigated for MP and expression of sialosyl-dimeric-Le x . MP of HAL-8 and HAL-33 was inhibited completely by sialidase treatment of cells (see Table 2 below). Expression of sialosyl-dimeric-Le x appears to play an important role in blood-borne metastasis.
  • HAL-33 0 a Nude mice were injected (2 x 10 cells) via the tai l vein. Fifty-six days after injection, were killed and metastatic nodules on lung surface were counted under dissecting microscope. b Mean of 6 animals (range in parentheses). EXAMPLE 4
  • Platelets were isolated from "platelet-rich plasma” obtained from the Oregon Red Cross (Portland, OR). Contaminating red blood cells were removed by centrifugation at 80 ⁇ g for 10 min. Platelets were centrifuged at 300 ⁇ g for 10 min and suspended in Tyrode's buffer (pH 6.5) containing 22 mM citrate buffer with 0.35% bovine serum albumin (BSA). The platelet suspension (1 ⁇ 10 /ml) was incubated (pH 7.2, 37oC, 5 min) after addition of thrombin (final concentration 1 U/ml). The mixture then was incubated at 37oC for 10 min without stirring.
  • Tyrode's buffer pH 6.5
  • BSA bovine serum albumin
  • the thrombin-activated platelets were fixed with an equal volume of 2% formaldehyde in phosphate-buffered saline (PBS), pH 7.2, and washed 2 ⁇ with PBS containing 1% BSA.
  • Activated platelets (but not non-activated platelets) showed strong reactivity with 2.5 ⁇ g/ml anti-GMP-140 mAb AC1.2 (isotype IgG 1 ; Beckton-Dickinson, San Jose, CA) when incubated at 37oC for 30 min., followed by reaction with 50 ⁇ l of fluorescence-labeled goat anti-mouse Ig (Tago, Burlingame, CA).
  • Flow cytometric profiles of activated vs. non-activated platelets with mab AC1.2 are shown in Figures 8A-8D.
  • Activated and non-activated platelets were fixed with paraformaldehyde in Ca 2+ -free PBS, pH 7.2, washed 2 ⁇ with Ca 2+ -containing PBS with 1% BSA, resuspended in CA 2+ -PBS with 1% BSA and 0.1% azide and the number of platelets adjusted to ⁇ 1 ⁇ 10 9 /ml.
  • the cell suspension was stored at 4oC and the binding assay performed within 24 hr.
  • the dried residue was suspended in 2 ml Ca 2+ -PBS with 3% BSA and 0.1% azide, sonicated for 10 min and allowed to stand at 37°C for 60 min to block the bead surface with BSA.
  • the suspension was centrifuged at 3000 ⁇ g for 10 min, the bead pellet was washed 2 ⁇ with Ca 2+ -PBS containing 1% BSA and azide and finally suspended in 500 ⁇ l of the same medium and stored at 4oC.
  • the binding index (BI) was calculated as mean fluorescence intensity (MFI) of platelets incubated with fluorescent GSL-coated beads divided by MFI of platelets incubated with fluorescent non-GSL-coated (control) beads.
  • MFI mean fluorescence intensity
  • BI values for various GSL's are shown in Table 3 and in Figure 9. In Figure 9, the hatched bars represent non-activated platelets and the open bars represent activated platelets.
  • SA-Le x SA-Le a , SPG, GM3 and Le x also is shown in the "Ratio A/NA" column.
  • Non-activated) GM3 1.0 ⁇ 0.1 0.7 ⁇ 0.5
  • mAb's affected platelet binding to fluorescent GSL-coated beads. Platelets were incubated with anti-GMP-140 mAb IOP62 (Immunotech, Marseille, France) at 37°C for 30 min and a binding assay was performed using GSL-coated beads, as described hereinabove. Non-specific mouse IgG (10 ⁇ g/ml) was used in a control binding assay. Also, 10 ⁇ l of SA-Le a -coated beads (2 ⁇ 10 7 ) were incubated with 20 ⁇ l of anti-SA-Le a mAb CA19-9 (20 ⁇ g/ml) (mouse IgG 1 ;
  • Activated platelets showed high expression of GMP-140 as evidenced by high reactivity with anti-CD62 mAb ( Figures 8A-8D). Activated platelets expressing GMP-140 showed strong binding with fluorescent beads coated with SA-Le x ( Figure 9).
  • Adhesion was measured using the dynamic flow experimental system shown in Figures 11A to 11D.
  • the coefficient of viscosity was 1.0 P, the half channel height was 5.7 ⁇ 10 -3 cm and the channel width was 1.3 cm.
  • HUVEC's (Cell Systems, Kirkland, WA) were cultured to confluency in 48-well plates (Costar, Cambridge, MA) and stimulated with 1 U/ml IL-1 for 4 hr. Non-simulated HUVEC's were used as a control. Expression of E-selectin (ELAM-1) on IL-1-stimulated HUVEC's was confirmed by reactivity with anti-E-selectin mAb 3B7 (IgG 2a ) (Graber et al. J. Imm. 145:819, 1990).
  • HL60 and Colo201 cells were labeled metabolically by culture in the presence of [ 3 H]-thymidine after pretreatment with glycosylation modifier and added to HUVEC-coated plates. After 15 min incubation, plates were washed with PBS and adherent cell number estimated by conversion from radioactivity count.
  • 96-well plates (Falcon, Lincoln, NJ) were coated with 0.1-1 ⁇ g/ml of a truncated, recombinant E-selectin lacking transmembrane and cytoplasmic domains (Shimizu et al. Nature 349:799, 1991) for 18 hr.
  • HL60 cells were pretreated with 2mM benzyl- ⁇ -GalNAc for 72 hr and labeled with [ 3 H]thymidine. After washing with PBS, 1 ⁇ 10 6 cells were added to each well and plates were incubated for 30 min at room temp. After washing to remove unbound cells, bound cells were detached with trypsin and counted by liquid scintillation counter.
  • a parallel-plate laminar flow chamber connected to an infusion pump (Model 935, Harvard Apparatus, Cambridge, MA) was used to simulate the flow shear stresses present in physiological microvascular environments.
  • the flow chamber consists of a glass plate on which a parallel, transparent plastic surface is attached with a Silastic rubber gasket; there is a 114 ⁇ m gap between the two surfaces and the gap is connected to an inlet and outlet.
  • a laminar flow with defined rate and wall shear stress is achieved by manipulation of the infusion pump, which is connected to the inlet of the flow chamber.
  • EC's are grown as a monolayer, or adhesion molecules are coated, on the glass plate, and a laminar flow of a cell suspension is passed through the chamber.
  • T 3 ⁇ Q/2ba 2
  • ⁇ coefficient of viscosity (1.0 cP)
  • Q volumetric flow rate (cm /sec)
  • a half channel height (for the experiments reported herein, 5.7 x 10 3 cm)
  • b channel width (1.3 cm).
  • Promyelocytic leukemia cell line HL60 has been shown to express only type 2 chain and sialosylated/fucosylated derivatives as probed by specific mAb's (Symington et al., J. Immunol. 134:2498, 1985) and has been extensively used as a model of leukocyte adhesion mediated by E-selectin and P-selectin
  • NDV sialidase eliminates only the ⁇ 2 ⁇ 3 sialosyl residue linked to the terminal Gal whereas both Vibrio and Arthroacter sialidase completely eliminate terminal and internal sialic acid residues, notably, ⁇ 2 ⁇ 6 linked sialic acid residues.
  • the findings indicate that SLe x and SLe a are not the sole epitopes of E-selectin and P-selectin.
  • anti-SLe x mAb's e.g., SNH3 and SNH4
  • SNH3 and SNH4 should inhibit completely selectin-dependent adhesion.
  • SNH4 also should inhibit E-selectin-dependent cell adhesion.
  • fibronectin (FN), laminin (LN), truncated E-selectin and GSL's used, 10-50 ⁇ l of a solution having a concentration of 20-200 ⁇ g/ml was placed on a marked area (0.5 cm diameter) on a glass plate (38 ⁇ 75 mm; Corning Glassworks, Corning, NY) and dried in a refrigerator at 4oC. Dried plates were immersed in PBS at 37oC for 1 hr and washed extensively with several changes of PBS.
  • GSL-liposomes were prepared from 200 ⁇ g GSL, 200 ⁇ g cholesterol and 400 ⁇ g phosphatidylcholine in 1 ml PBS. Ten ⁇ l of GSL-liposome solution was placed on a glass plate, dried at 4oC and the plates were washed with PBS, as described above.
  • the quantity of adsorbed molecules was determined using 125 I labeling for lectins, FN or LN, or [ 3 H]cholesterol labeling for GSL-liposomes. Under those conditions, almost the entire quantity of protein, regardless of whether FN, LN or lectin, was adsorbed on the glass plate. For example, when 100 ⁇ g/ml FN was applied, 12.5 + 1.8 ng/mm was adsorbed. Likewise, almost all GLS-liposome dried on the glass plate was adsorbed; e.g., when 200 ⁇ g/ml GLS-liposome was applied, 31.3 + 5.2 ng GSL/mm was adsorbed. EC's were coated by placing 100 ⁇ l of a suspension containing 2 ⁇ 10 5 mouse or human EC's on glass plates and culturing in a CO 2 incubator at 37° C until confluency was achieved.
  • B16 melanoma cells were harvested from culture using 0.02% EDTA in PBS, and suspended in PBS at a concentration of 1 ⁇ 10 /ml.
  • sialic acid in the carbohydrate epitope is important in providing binding specificity to E-selectin.
  • the sialic residue is not required to be ⁇ 2 ⁇ 3 linked at the terminal Gal; the sialic acid residue alternatively could be present at an internal location, e.g., linked to internal Gal or GlcNAc.
  • ⁇ 1 ⁇ 3 fucosylation at GlcNAc is essential.
  • NDV sialidase had an inhibitory effect only at low shear stress whereas VC or AU sialidase significantly reduced adhesion even at high shear stress.
  • Anti-Le x IgG mAb SH1 strongly inhibited adhesion even at high shear stress, whereas the effect of anti-SLe x IgG 3 mAb SNH4 was minimal. Strongest inhibition was produced by a combination of NDV sialidase plus anti-Le x mAb SH1.
  • a mixture of anti-Le x plus anti-SLe x mAb's produced stronger inhibitory effect than either mAb alone.
  • Colo201 adhesion to E-selectin-coated plates under static conditions In contrast to HL60 cells (which express predominantly type 2 chain structure), Colo201 cells express mainly type 1 chain, and E-selectin-dependent Colo201 adhesion is through type 1 chain epitopes.
  • Colo201 cells were treated with various mAb's following exposure to various sialidases and were assessed for residual binding.
  • Colo201 reactivity with mAb CA19-9 (directed to SLe a I) was inhibited almost completely by Vibrio sialidase, and to a lesser extent by Arthrobacter and NDV sialidases.
  • Colo201 reactivity with mAb FH7 was reduced by Arthrobacter sialidase but minimally affected by Vibrio or NDV sialidases.
  • Colo201 reactivity with mAb CA3F4 was enhanced by sialidase treatment.
  • mAb CA19-9 inhibited Colo201 adhesion slightly and was influenced only minimally by Vibrio sialidase ( Figure 16).
  • the SLE a epitope present at the surface of Colo201 cells is organized in such as way that it is (i) not susceptible to CA19-9 for E-selectin-dependent adhesion and (ii) not sensitive to sialidase treatment.
  • the inhibitory effects of mAb FH7 (Nudelman et al. supra) and, more strikingly, mAb CA3F4 on Colo201 adhesion to E-selectin-coated plates were enhanced by pretreatment of cells with Vibrio sialidase. Arthrobacter sialidase reduced but did not abolish Colo201 adhesion. See Figure 17.
  • NDV sialidase had no effect on Colo201 adhesion, particularly at high shear stresses.
  • mAb CA3F4 At both low and high shear stresses, the strongest inhibition of adhesion was observed for mAb CA3F4, which is directed to Le a with an ⁇ 2 ⁇ 6 sialosyl substitution at the penultimate GlcNAc.
  • Vibrio sialidase which efficiently cleaves terminal ⁇ 2 ⁇ 3 sialosyl linkages but is less effective at removing internal sialic acid residues, reduced adhesion to some extent at high shear stress, but less so at low shear stress.
  • mAb CA3F4 inhibited adhesion strongly at high shear stress but much less at low shear stress.
  • a combination of Vibrio sialidase plus mAb CA3F4 produced strong inhibition at both high and low shear stress (Figure 18).
  • Truncated, recombinant ELAM-l lacking the transmembrane and cytoplasmic domains is used to coat beads, for example, capable of packing into a standard chromatography columns.
  • the ELAM-1 at a concentration of 0.1-1 ⁇ g/ml is mixed with the beads and the mixture is incubated to allow binding of ELAM-1 to the bead matrix.
  • a suitable incubation period is 12-24 hours at 4oC - room temperature.
  • the beads are washed to remove unbound ELAM-1, optionally can be blocked with an inert carrier, such as BSA, and washed again.
  • the ELAM-1 coated beads can be used in a batch process or packed into a suitably-sized column.
  • Cells known to carry carbohydrates bindable to ELAM-1, such as HL60 and Colo201, are obtained.
  • the cells are lysed to obtain a membrane fraction using known methods, such as repeated freeze-thaw cycles.
  • the membrane fraction is obtained, for example, by centrifugation.
  • the membrane prep may be a suitable source without further purification.
  • the membrane prep is treated using known methods to obtain a membrane component preparation, and in particular, a fraction that contains cell surface carbohydrate.
  • the carbohydrate-rich fraction is mixed with or passed over the ELAM-1 affinity matrix, depending on the format, the exposed matrix is washed and the carbohydrates bound to the matrix are eluted, for example, by exposing the matrix to a high salt buffer.
  • the resultant preparation comprises carbohydrate bindable to ELAM-1 and the various species are separable using known techniques, such as TLC or HPLC.
  • Carbohydrates bindable to ELAM-1 either prepared chemically using known reagents and methods, see, for example, Example 1 hereinabove, prepared enzymatically or obtained from suitable cells, see, for example.
  • Example 8 hereinabove, or whole cells known to express carbohydrate bindable to ELAM-l serve as immunogen in suitable hosts to generate antibody thereto. Either poiyclonal or monoclonal antibody can be obtained and the selection of a suitable host is premised on known methods and preferences.
  • the carbohydrates, cells, cell lysates or membrane preps are administered to the host, either with or without adjuvant, in a schedule that will generate an immune response.
  • the blood is collected, serum separated and tested.
  • the spleens of the host animals are removed and cells therefrom are fused with a suitable myeloma cell using known techniques.
  • Specificity of the antibodies can be tracked using an ELISA comprising, for example, purified recombinant ELAM-1 and mAb 3B7 with the appropriate labeled reagents and reporter molecules.
  • Antibody directed to carbohydrates of formulae (I), (II) and (III) can be obtained by using specific carbohydrate species as antigen and in the screening ELISA.
  • the antisera can be made "monospecific" by absorption with cells carrying only SLe x and/or SLe a or with a solid matrix to which SLe x and/or SLe 8 is bound.
  • the resultant residual activity directed to carbohydrates bindable to ELAM-1 can be attributed in part to antibodies directed to carbohydrates of formula (I), (II) or (III).
  • NS-1 cells were obtained from the ATCC (Rockville, MD) and maintained in RPMI 1640:Dulbecco's MEM (1:1) supplemented with 10% HI FCS. Fifty ⁇ g of a plasmid comprising cDNA of E-selectin in vector pCDM8 (R & D Systems, Minneapolis, MN) and 5 ⁇ g of pSV2-neo (ATCC) were co-transfected into NS-1 cells (1 ⁇ 10 7 ) by electroporation. After 48 hours in culture, the cells were transferred to medium containing 650 ⁇ g/ml G418 (Gibco, Grand Island, NY).
  • E-selectin-dependent adhesion using transfected NS-1 cells onto SLe a , SLe x , Le y , Le x , H-2, sialylparagloboside (SPG), disialosyl I (Structure 6 of Figure 20) and dimeric SLe x were compared at various shear stress conditions.
  • the number of cells adhered per mm is expressed relative to adhesion onto SLe x -coated plates which is regarded as 100%.

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Abstract

De nombreux antigènes d'hydrate de carbone associés aux leucocytes et aux tumeurs agissent comme des molécules d'adhérence cellulaire, reconnues par les lectines (interaction protéine-hydrate de carbone) ou les hydrates de carbone complémentaires (interaction hydrate de carbone-hydrate de carbone). Les antigènes associés aux tumeurs et ceux associés aux leucocytes ont des structures communes. Le potentiel métastasique des cellules tumorales ainsi que la migration transendothéliale des leucocytes ont été supprimés par des agents ou une combinaison d'agents appartenant aux groupes suivants: (a) antigènes d'hydrate de carbone; (b) anticorps dirigés contre ces antigènes; (c) composants oligosaccharides de ces antigènes; et (e) imitateurs des antigènes ou des oligosaccharides. Les oligosaccharides et leurs dérivés qui inhibent l'adhérence et l'aggrégation cellulaire transmises par la sélectine-P (GMP 140) et la sélectine-E (ELAM-1) sont également décrits. Les agents efficaces pour ces applications se composent de sucres hybrides contenant de multiples épitopes comme Lex/SLex qui est une combinaison de sucres individuels contenant un sucre hybride qui peut se présenter sur des liposomes, ainsi que d'anticorps ou d'une combinaison d'anticorps.
PCT/US1993/001375 1992-02-19 1993-02-19 Inhibition de l'adherence cellulaire par oligosaccharides definis chimiquement, leurs derives, des imitateurs et des anticorps WO1993017033A1 (fr)

Priority Applications (2)

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JP5514925A JPH07504222A (ja) 1992-02-19 1993-02-19 化学的に規定されるオリゴ糖，該オリゴ糖の誘導体，擬態物及び該オリゴ糖に対する抗体による細胞接着の抑制
EP93905988A EP0638085A1 (fr) 1992-02-19 1993-02-19 Inhibition de l'adherence cellulaire par oligosaccharides definis chimiquement, leurs derives, des imitateurs et des anticorps

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US83697892A 1992-02-19 1992-02-19
US07/836,978 1992-02-19
US95072092A 1992-09-25 1992-09-25
US07/950,720 1992-09-25
US99690392A 1992-12-29 1992-12-29
US07/996,903 1992-12-29

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6008203A (en) * 1995-07-14 1999-12-28 Glycotech Corp. Methods for treatment of EGF receptor associated cancers
WO2000055149A1 (fr) * 1999-03-12 2000-09-21 Biota Scientific Management Pty. Ltd. Composes dimeres en tant qu'inhibiteurs de la neuraminidase
WO2002038168A2 (fr) * 2000-11-07 2002-05-16 Nemod Immuntherapie Ag Utilisation de proteines produites pendant la grossesse, fixant la selectine, de liposomes, de fragments de mucine natifs et de composes mimetiques pour traiter et prevenir des maladies infectieuses, pour empecher la metastatisation et pour prevenir des maladies tumorales
WO2003016464A2 (fr) * 2001-08-17 2003-02-27 Biotie Therapies Corp. Sequences d'oligosaccharides specifiques pour le cancer et leur utilisation
US7294615B1 (en) 1999-06-30 2007-11-13 Nikolai Vladimirovich Bovin Compounds that associate on the intermolecular level and aggregate bodies that contain them
US7326563B2 (en) 2000-11-08 2008-02-05 Surface Logix, Inc. Device and method for monitoring leukocyte migration
US7374906B2 (en) 2000-11-08 2008-05-20 Surface Logix, Inc. Biological assays using gradients formed in microfluidic systems

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5331293B2 (ja) * 2006-04-28 2013-10-30 公益財団法人野口研究所 多様性を表現するオリゴ糖またはその誘導体
JP5894732B2 (ja) * 2010-11-11 2016-03-30 学校法人東京女子医科大学 細胞培養基材の評価方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Cancer Research, Volume 48, issued 15 January 1988, T.S. KIM et al., "Le(x) and Le(y) Antigen Expression in Human Pancreatic Cancer", pages 475-483, entire document. *
Clin. Exp. Metastasis, Volume 9, No. 3, issued May-June 1991, H. INUFUSA et al., "Human Lung Adenocarcinoma Cell Lines with Different Lung Colonization Potential and a Correlation Between Expression of Sialosyl Dimeric Le(x) and LCP", Abstract, entire document. *
J. Biol. Chem., Volume 264, No. 31, issued 05 November 1989, E.D. NUDELMAN et al., "A Series of Disialogangliosides with Binary 2-3 Sialosyllactosamine Structure, Defined by Monoclonal Antibody NUH2, are Oncodevelopmentally Regulated Antigens", pages 18719-18725, entire document. *
J. Biol. Chem., Volume 266, No. 13, issued 05 May 1991, M.R. STROUD et al., "Extended Type 1 Chain Glycosphingolipids: Dimeric Le(a) as Human Tumor Associated Antigen", pages 8439-8446. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6008203A (en) * 1995-07-14 1999-12-28 Glycotech Corp. Methods for treatment of EGF receptor associated cancers
US6281202B1 (en) 1995-07-14 2001-08-28 Glycotech Corp. Pharmaceutical compositions for treatment of EGF receptor associated cancers
WO2000055149A1 (fr) * 1999-03-12 2000-09-21 Biota Scientific Management Pty. Ltd. Composes dimeres en tant qu'inhibiteurs de la neuraminidase
US6548476B1 (en) 1999-03-12 2003-04-15 Biota Scientific Management Pty. Ltd. Dimeric inhibitors of influenza neuraminidase
US7294615B1 (en) 1999-06-30 2007-11-13 Nikolai Vladimirovich Bovin Compounds that associate on the intermolecular level and aggregate bodies that contain them
WO2002038168A2 (fr) * 2000-11-07 2002-05-16 Nemod Immuntherapie Ag Utilisation de proteines produites pendant la grossesse, fixant la selectine, de liposomes, de fragments de mucine natifs et de composes mimetiques pour traiter et prevenir des maladies infectieuses, pour empecher la metastatisation et pour prevenir des maladies tumorales
WO2002038168A3 (fr) * 2000-11-07 2004-06-10 Nemod Immuntherapie Ag Utilisation de proteines produites pendant la grossesse, fixant la selectine, de liposomes, de fragments de mucine natifs et de composes mimetiques pour traiter et prevenir des maladies infectieuses, pour empecher la metastatisation et pour prevenir des maladies tumorales
US7326563B2 (en) 2000-11-08 2008-02-05 Surface Logix, Inc. Device and method for monitoring leukocyte migration
US7374906B2 (en) 2000-11-08 2008-05-20 Surface Logix, Inc. Biological assays using gradients formed in microfluidic systems
WO2003016464A2 (fr) * 2001-08-17 2003-02-27 Biotie Therapies Corp. Sequences d'oligosaccharides specifiques pour le cancer et leur utilisation
WO2003016464A3 (fr) * 2001-08-17 2003-05-22 Carbion Oy Sequences d'oligosaccharides specifiques pour le cancer et leur utilisation
US8313912B2 (en) 2001-08-17 2012-11-20 Glykos Finland Oy Cancer specific oligosaccharide sequences and use thereof

Also Published As

Publication number Publication date
CA2129987A1 (fr) 1993-09-02
EP0638085A1 (fr) 1995-02-15
JPH07504222A (ja) 1995-05-11

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