EP0638085A1 - Inhibition of cell adhesion by chemically-defined oligosaccharides, their derivatives, mimetics, and antibodies directed thereto - Google Patents

Inhibition of cell adhesion by chemically-defined oligosaccharides, their derivatives, mimetics, and antibodies directed thereto

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Publication number
EP0638085A1
EP0638085A1 EP93905988A EP93905988A EP0638085A1 EP 0638085 A1 EP0638085 A1 EP 0638085A1 EP 93905988 A EP93905988 A EP 93905988A EP 93905988 A EP93905988 A EP 93905988A EP 0638085 A1 EP0638085 A1 EP 0638085A1
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European Patent Office
Prior art keywords
carbohydrate
cells
elam
sle
antibody
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EP93905988A
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German (de)
French (fr)
Inventor
Naoya The Biomembrane Institute Kojima
Kazuko The Biomembrane Institute Handa
Sen-Itiroh The Biomembrane Institute Hakomori
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Biomembrane Institute
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Biomembrane Institute
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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
  • TACA leukocyte associated carbohydrate antigens
  • LACA leukocyte associated carbohydrate antigens
  • the instant invention is directed to and based on t inhibition of cell adhesion, for example, through TACA's
  • 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. For example:
  • high metastatic variants BL6 and F10
  • Adhesion of high metastatic variants to endothelial cells is greater than with low metastatic variants and the adhesion is inhibited by Me-jS-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/Ley'/Leb (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 tumor do not express or which weakly express those antigens
  • Those tumor-associated carbohydrate antigens are essentiall adhesion molecules which are recognized by targe cells, particularly platelets or endothelial cells Such is an example of a combination approach, that is interfering with (a) and (b) .
  • ELAM-1 or GMP-140 which are expressed on activate endothelial cells and activated platelets Sialosyl-Le x antigen has been known to be recognized b those LECCAM 1 s.
  • GMP-140 whose expression on platelet o endothelial cells is induced by thrombin, ADP or (AMP phorbol ester, may play an important role i 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 iso er 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 ⁇ , 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 ⁇ l ⁇ 3 or ⁇ l ⁇ 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 a important role in providing high affinity bindin sites to E-selectin. That role is particularl evident under high shear stress conditions.
  • Human endothelial cells are characterized by hig expression of H (Fuc ⁇ l ⁇ 2Gal) and many types of huma cancers are characterized by expression of Le y , H o 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 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.
  • 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.
  • Compounds, such as those set forth in Figure 20, which can be Le x /SLe x hybrids, or an appropriat mixture of the relevant components, such as Le x an SLe x provide high affinity adhesion binding sites, particularly under high shear stress conditions in dynamic flow system. Hence, such compounds bloc E-selectin-mediated adhesion of tumor cells o leukocytes to endothelial cells.
  • 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 (c) oligosaccharide components of those antigens, (d) conjugates of those antigens or oligosaccharides and (e) mimetics of the tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigens) , the agent inhibiting the metastasis potential of the preparation.
  • 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 variety of glycoconjugates useful for prolonging the in viv half-life of oligosaccharide components.
  • the conjugates compris an oligosaccharide coupled to polyethyleneglycol.
  • Additional oligosaccharide components for use within th methods and compositions of the instant invention included lactose, lacto-N-tetrose, methyl 0-D-lactoside and pheny ⁇ -D-thiolactoside. Oligosaccharide components may be use individually or in combination with one another.
  • the instant invention further provides a variety of method for inhibiting GMP-140-mediated or ELAM-1-mediated cel aggregation or adhesion causing metastasis at a tumor site an inflammatory responses at a site.
  • One suchmethod inhibits GMP-140-mediated or ELAM-1-mediate cell aggregation or adhesion within a biologic preparation an comprises incubating the biologic preparation with at least on agent selected from the group consisting of: (a) a hybrid suga molecule, such as one comprising Le x and SLe x (Structure 1 o Figure 20, a branched type II chain); (b) a mixture of th 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 specificall bind to a hybrid sugar, such as Le x /SLe x , or to the component thereof; (e) antibodies that specifically bind t monosialosyl-Le a I, Le a , Le
  • 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 provides a metho 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 epitope tumor associated carbohydrate antigen
  • Figure 1 graphically illustrates the effects of methyl jS-D-lactoside or methyl j9-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 ,3-D-lactoside ("Me- ⁇ -lactoside") or 0.1 M phenyl 3-D-thiolactoside ("phe-/S-S-lactoside) .
  • Twenty thousand cells were injected intravenously into C57B1 mice.
  • Lung colony numbers were counted at 21 days and colonies were classified on the basis of diameter (> 1 mm vs. ⁇ l 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 0-D-lactoside on the number and size of lung colony deposits of BL6 cells.
  • Methyl jS-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 represent control animals (not administered with methyl 3-D-lactoside)
  • groups B and C represent animals injected with 0.25 M and 0.5 methyl ,9-D-lactoside, respectively. For each group, column
  • Figure 3 graphically illustrates survival of cancer patient with or without expression of a defined tumor-associate
  • TACA carbohydrate antigen
  • Panel 3B represents sialosyl-Le x expression in colonic cance using antibody FH6.
  • Panel 3C represents sialosyl-Tn expressio
  • Panel 3D represent sialosyl-Tn level in sera of ovarian cancer patients.
  • Figure 4 graphically illustrates that melanoma cell adhesio on LacCer is based on GM3-LacCer interaction.
  • the order o metastatic potential is BL6>F10>Fl»Wa4.
  • Panel 4A shows th 20 order of melanoma cell adhesion on a LacCer-coated solid phase
  • Panel 4B shows the order of melanoma cell adhesion o LacCer/Fibronectin (FN) co-coated solid phase.
  • Panel 4C sho integrin-dependent adhesion.
  • Figure 5 graphically illustrates the melanoma cell (BL6 25 adhesion on LacCer (Panel 5A) and on endothelial cells (HuVEC * (Panel 5B) is inhibited by LacCer and GM3.
  • FIG. 6 graphically illustrates the metastasis-inhibiti effect of methyl(Me)- / 8-lactoside.
  • Tumor cells were inject intravenously, followed by intraperitoneal injection of:
  • Panel 7A shows Hl-liposome binding to various glycolipids.
  • Panel 7B shows Le y -liposome binding to various glycolipids.
  • Figures 8A-8D are flow cyto etric 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 8 monoclonal antibody, CA19-9;
  • column 3 represents anti-sialosyl-Le x monoclonal antibody, SNH4; and
  • 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 variou monoclonal antibodies on adhesion of human colon carcinom Colo205 cells to interleukin-1-activated human umbilical vei endothelial cells in a dynamic flow system.
  • Open circle represent a mixture of irrelevant mouse IgG plus IgM (control) the solid triangles represent monoclonal antibody CA19-9 directe to monosialosyl-Le 8 I, the open triangles represent monoclona antibody SNH4 directed to sialosyl-Le , the solid circle represent monoclonal antibody FH7 directed to monosialosyl-Le 8 I and disialosyl-Le 8 and the solid squares represent a mixture o irrelevant mouse IgG plus IgM and non-activated endothelia cells.
  • Figure 13 depicts binding of mAb's to HL60 cells and th effect of sialidase thereon. Binding activity was determined b flow cyto etry.
  • Abscissa log fluorescence intensity Ordinate: relative cell number.
  • Panel A Solid line, cell stained with mAb SNH4 as primary antibody. Dotted line, contro cells stained with mouse IgG plus IgM [10 ⁇ g/ml] as primar antibody.
  • Panel B mAb SNH3 as primary antibody; control as i Pane A.
  • Panel C Solid line, cells treated with Newcastl Disease Virus (NDV) sialidase and then stained with mAb SNH4 Dotted line, control cells (as in Panel A, after sialidas treatment).
  • Panel D NDV sialidase followed by mAb SNH3 control as in Panel C.
  • Panel E Vibrio cholerae (VC) sialidas followed by mAb SNH4.
  • Panel F VC sialidase followed by mA 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 37 ⁇ C) . 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
  • 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 SHI.
  • Panel C hollow circle, control; solid triangle, NDV sialidase solid circle, mAb SHI; and hollow triangle, NDV sialidase plu mAb SHI.
  • Panel D hollow circle, control; solid circle, mixtur (1:1) of mAb's SNH4 and FH2; and hollow triangle, mixture (1:1 of mAb's SNH4 and SHI.
  • Colo201 cells were reactive strongly with anti-SLe a I mAb's CA19-9 and NKH (Panel A) , anti-Le 8 mAb CA3F4 (Panel B) and anti-SLe 8 II mAb FH (Panel C) .
  • Reactivity with CA19-9 was decreased by NDV sialidas (Panel D) and abolished by VC sialidase (Panel G) .
  • Reactivit with CA3F4 was increased slightly by NDV and VC sialidase (Panels E and H) .
  • Reactivity with FH7 was unchanged by ND sialidase (Panel F) and decreased slightly by VC sialidas (Panel I) .
  • Figure 17 depicts adhesion of Colo201 cells t E-selectin-coated plates in a static system. Abscissa an ordinate as in Figure 14.
  • Panel A effects of variou sialidases (90 min. incubation, 37 ⁇ C).
  • Panel B effects o 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 SHI on HL60 binding to ELAM-coated plates in a dynamic flow system under various shear strength conditions.
  • the ordinat represents per cent cell binding relative to untreated contro cells.
  • the antibodies were used at 15 ⁇ g/ml, NDV sialidase a 0.2 U/ml and VC sialidase at 0.1 U/ml. Each point represents th mean of three experiments. Number of untreated cells bound a 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 hybri sugar, Le x /SLe x is depicted as structure 1.
  • the glycolipid containing such a structure were isolated from colon carcinom or were prepared from G8 ganglioside presented in Structure originally found in human erythrocytes (Watanabe et al., J. Biol Chem., 254:8223, 1979) by enzyme catalyzed ⁇ l ⁇ 3 fucosylation Structure 2 was obtained by ⁇ l ⁇ 3 fucosylation of compound originally obtained from human placenta. Structure 2 however di not exhibit high affinity binding to E-selectin.
  • Structures and 4 depict analogs with high affinity binding sites having L and sialyl-Gal31 ⁇ 3GalNac within the same molecule (Structure 3) or the hybrid molecule Le a /SLe a , the positional isomer o structure 1.
  • Figure 21 depicts the relative adhesion of NS-1 cell expressing E-selectin on various "glyco-liposomes" coated on plastic surface.
  • Panel 21A shows the result of such relativ adhesion in a dynamic flow setting under middle shear stres conditions (7.75 dynes/cm ) .
  • the first seven bars indicate relative adhesion of NS-1 cells to SLe x on each glycoliposome a indicated.
  • Cpd I is structure 1 of Figure 20 and Cpd II i structure 2 of Figure 20.
  • Bars 8-10 show a mixture of Le x wit different types of compounds as indicated.
  • the value of relative adhesion is expressed in comparison with the adhesion of SLe x -liposome as 100%.
  • Panel 2IB indicates the same relative adhesion of NS-1 cells at high shear stress conditi .ons (11.8 dynes/cm2) . 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 hollo circle is SLe x + H2.
  • the solid triangle is SLe x + Le x .
  • Th hollow triangle is SLe x + Le y .
  • Each point is the mean of fiv determinations.
  • the instant invention in one aspect i directed to methods and compositions for the inhibition of tumo cell metastasis potential and invasiveness.
  • Numerous tumor cell possess the ability to metastasize, i.e., to form a secondar tumor colony at a distant site.
  • Sources of malignant tumor cell include melanoma, lung, breast, colorectal and urogenita cancers, such as bladder and prostate cancers.
  • the metastasis potential of tumor cells may b inhibited through the use of (a) tumor-associated carbohydrat antigens (TACA's, as used herein TACA is meant to include LACA) (b) antibodies directed to those TACA's; (c) oligosaccharid components of those TACA's; (d) conjugates of such TACA's or o oligosaccharide components of such TACA's, such as multivalen conjugates of lysyllysine or TACA-bearing glycosphingolipid (GSL liposomes; or (e) mimetics of the TACA's.
  • TACA's tumor-associated carbohydrat antigens
  • TACA tumor-associated carbohydrat antigens
  • TACA is meant to include LACA
  • TACA antibodies directed to those TACA's
  • conjugates of such TACA's or o oligosaccharide components of such TACA's such as multival
  • TACA epitopes play essential roles in tumor cell adhesio through interaction with endothelial cells, platelets an basement membranes, whereby tumor metastasis and invasion ma 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 8 I is inhibited by monoclonal antibodies (CA19-9, CSLEA, NKH1 and KH2) directed to that epitope.
  • Colo205 tumor cells which express predominantly type 1 chain sialosyl-Le 8 and to a lesser extent sialosyl-Le x , to endothelial cells is inhibited by anti-sialosyl-Le 8 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 o endothelial cells (EC's) .
  • LECCAM major selectin
  • GMP-14 On activation of those cells, GMP-14 is redistributed rapidly to the cell surface, where it plays a important role in adhesion of platelets or EC's to certai carbohydrate epitopes expressed on blood cells or tumor cells resulting in aggregation of platelets or tumor cells, or adhesio thereof to capillary endothelia.
  • GMP-140-mediated cell adhesio is believed by the instant inventors to be involved in initiatio of metastatic deposition of tumor cells and initiation o inflammatory processes.
  • ELAM-1 is expressed on endothelial cells afte activation with interleukin-1, TGF-3, TNF- ⁇ o lipopolysaccharide.
  • ELAM-1-mediated cell adhesion also i believed to be involved in initiation of metastatic depositio of tumor cells.
  • the instant invention in another aspect is directe to inhibiting GMP-140-mediated or ELAM-1-mediated cel aggregation or adhesion, especially at tvimor cell sites.
  • GMP-140-mediated or ELAM-1-mediated cel 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 8 or sialosyl Le ; (d) antibodies that specifically bind to a hybrid sugar (a) , such as Le x /SLe x , monosialosyl-Le 8 I, Le a , Le x , monosialosyl-Le 8 II, disialosyl-Le 8 or sialosyl Le ; (e) a mixture of antibodies, particularly to the components of a hybrid sugar, such as
  • 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 directly via ELAM-1.
  • inflammation minimized by inhibiting GMP-140-mediated leukocyte aggregatio adhesion or migration at a potential site of inflammation due t (a) adhesion of leukocytes to platelets, (b) adhesion leukocytes to endothelial cells (EC) via platelets, (c) adhesi of leukocytes to EC's directly via selectin and ( transendothelial migration of leukocytes.
  • TACA's suitable for use within the instant invention a those showing differential prognostic significance (i.e., TACA that may be correlated clearly with invasive or metastat potential) .
  • su TACA's may be distinguished through a comparison of invasivenes metastasis and clinical prognosis of similar tumors showi expression vs. non-expression of such TACA's.
  • Preferred TACA for use within the present invention include H/Ley/L sialosyl-Le x (SA-Le x or SLe x ) , Le 8 , Le x , monosialosyl-Le 8 I (S or SA-Le a ) and sialosyl-Tn (SA-Tn or STn) .
  • su TACA's include hybrid sugars, such as Le x /SLe x , dimeric L sialosyl-dimeric Le x , trifuscosyl Le y , disialosyl-Le 8 a monosialosyl-Le 8 II.
  • TACA's for use within the instant inventi exhibit a differential prognostic significance.
  • a differential prognostic significance may illustrated by the fact that tumors expressing H/Le y /Le antige (as defined by monoclonal antibody MIA-15-5) showed much wor 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 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 th instant invention.
  • such antibodies include bot monoclonal and poiyclonal antibodies and maybe intact molecules, a fragment of such a molecule or a functional equivalent thereof.
  • the antibody may be engineered genetically.
  • antibod fragments include F(ab') 2 , Fab', Fab and Fv.
  • poiyclonal antibodies may be produced b immunization of an animal and subsequent collection of ser therefrom. Immunization is accomplished, for example, by systemic administration, such as by subcutaneous, intraspleni or intramuscular injection, into a rabbit, rat or mouse. It i preferred generally to follow the initial immunization with on or more booster immunizations prior to sera collection. Suc methodology is well known and described in a number o references.
  • Monoclona antibodies suitable for use within the instant invention included those of murine or human origin, or chimeric antibodies such a those which combine portions of both human and murine antibodie (i.e., antigen binding region of murine antibody plus constan regions of human antibody) .
  • Human and chimeric antibodies ma be produced using methods known by those skilled in the art Human antibodies and chimeric human-mouse antibodies ar advantageous because such antibodies are less likely than murin antibodies to cause the production of anti-antibodies whe administered clinically.
  • Monoclonal antibodies may be produced generally by th method of K ⁇ hler and Milstein (Nature 256:495, 1975; Eur. J Immunol. 6:511, 1976), as well as by various techniques whic modify the initial method of K ⁇ hler and Milstein (see Harlow an Lane (eds.), "Antibodies: A Laboratory Manual", Cold Sprin Harbor Laboratory, 1988, which is herein incorporated b reference in its entirety) . Briefly, the lymph nodes and/or spleen of an anima immunized with one of the TACA's or the oligosaccharid components thereof are fused with myeloma cells to form hybri cell lines ("hybridomas" or "clones").
  • Each hybridoma secrete a single type of im unoglobulin 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. 20 . :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 ,9-D-lactoside, lact-N-tetrose (Gal01 ⁇ 3GlcNAq9l ⁇ 3Gal01 ⁇ 4Glc) and phenyl 3-D-thiolactoside.
  • lactose and lactose derivatives such as methyl ,9-D-lactoside, lact-N-tetrose (Gal01 ⁇ 3GlcNAq9l ⁇ 3Gal01 ⁇ 4Glc) and phenyl 3-D-thiolactoside.
  • lactose derivatives also may be used, including ethyl or pheny lactoside and methyl or ethyl thiolactoside. It is believed tha such lactose derivatives block binding of melanoma cells to EC' by inhibiting melanoma cell GM 3 ganglioside interaction wit lactosy
  • oligosaccharide components suitable for inhibitin metastasis potential of cells of a particular tumor may b identified based on determining the structure of specifi carbohydrate chain(s) which are involved in the ability of th tumor to metastasize.
  • the identification o carbohydrate-containing molecules involved in the ability of tumor to metastasize may be accomplished in a variety of ways including through the use of glycosidases and inhibitors o glycosy1transferases.
  • the structure of carbohydrates bound to either lipids o proteins may be determined based on degradation, mas spectrometry, including electron-impact direct-probe (El) an fast atom bombardment (FAB) , and methylation analysis (technique described, for example, in Nudelman et al., J.
  • Degradation analysis may be accomplishe chemically and/or enzymatically, e.g., by glycosidases. Th carbohydrate sequence suggested by degradation analysis may b determined bymethylation analysis (Hakomori, J. Biochem. 55:205 1964) followed by chemical ionization mass spectrometry o permethylated sugars (Stellner et al.. Arch. Biochem. Biophys 155:464, 1974; Levery et al., Meth. Enzymol. 138:13, 1987).
  • E mass spectrometry may be performed on permethylated glycans after the appropriate degradation of intact glycans (Kannagi et al. , J. Biol. Chem. 259:8444, 1984; Nudel an 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 b activated. Activation of a carboxyl group includes formation o an "active ester", such as a succinimidyl ester.
  • active ester is known to refer to esters which are highl reactive in nucleophilic substitution reactions.
  • conjugates i which the components are linked non-covalently may be desirable to produce conjugates i which the components are linked non-covalently.
  • TACA's may be incorporated into the outer surface o glycosphingolipid (GSL) liposomes. It may be desirable to increase the in vivo half life of a oligosaccharide.
  • 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 a oligosaccharide-polyethyleneglycol conjugate is the reaction o an oligosaccharide, which has been derivatized to contain succinimidyl group, with a polyethyleneglycol having a termina amino group.
  • the latter compound has a general formula o NH 2 -(CH 2 CH 2 -0) n -CH 3 , where n typically averages 44. (i.e., molecular weight of about 2,000) to 112.9 (i.e., molecula weight of about 5,000).
  • the cell adhesion mediated selectins ELAM-1 or GMP-140
  • ELAM-1 or GMP-140 is based on recognition sialylated and fucosylated lactoseries type 1 and type 2 chai by a lectin sequence domain present at the N-terminal region the selectin molecules
  • any structure which may show mo effective blocking activity of the lectin domain than natural occurring epitopes are useful in the present invention.
  • Examples of useful mimetics include, but are not limited to, sialosyl-Le x or monosialosyl-Le 8 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 o disease processes involving GMP-140 or ELAM-1.
  • the instan invention provides a method for inhibiting tumor cell metastasi potential within a biologic preparation.
  • the method comprise incubating a biologic preparation with at least one agen selected from the group consisting of (a) tumor-associa carbohydrate antigens that exhibit differential prognos significance, (b) antibodies that specifically bind to th antigens, (c) oligosaccharide components of those antige (d) conjugates of those antigens or oligosaccharide compone and (e) mimetics of the tumor-associated carbohydrate antige the agent inhibiting the metastasis potential of the preparati
  • the instant invent also provides a method for inhibiting GMP-140-mediated ELAM-1-mediated cell aggregation or adhesion in a biolo preparation.
  • the method comprises incubating the biolo preparation with at least one agent selected from the gr consisting of (a) a hybrid sugar, such as Le x /SLe x ; (b) su components of a hybrid sugar (a) , such as Le x and S (c) monosialosyl-Le 8 I, Le a , Le x , monosialosyl-Le 8 disialosyl-Le 8 or sialosyl Le x ; (d) antibodies that specifica bind to a hybrid sugar, such as Le x /SLe or to the compon sugars thereof, monosialosyl-Le 8 I, Le a , Le x , monosialosyl-Le a disialosyl-Le 8 or sialosyl Le x ; (e) oligosaccharide compone of a hybrid sugar, such as Le x /SLe x , monosialosyl-Le 8 I, Le a
  • Suitable biologic preparations include cell cultures a cell suspensions in biologic fluids, such as blood, urine, lymp synovial and cerebrospinal fluid.
  • TACA's, oligosaccharides conjugates thereof generally will be incubated at a fin concentration of about 0.1 to IM, and typically at about 0.2 0.5 M. Incubation is performed typically for 5 to 15 minutes 37°C.
  • the preparati may be injected or implanted in an animal, e.g., to confi effectiveness of the inhibition of metastasis potential.
  • the instant invention also provides a method for inhibiti tumor cell metastasis potential in a warm-blooded animal, su as a human.
  • the method comprises administering to a warm-blood animal an effective amount of at least one agent selected fr the group consisting of (a) tumor-associated carbohydra antigens that exhibit differential prognostic significanc
  • oligosaccharide components of those antigens (c) oligosaccharide components of those antigens, (d) conjugat of those antigens or the oligosaccharide components a (e) mimetics of monosialosyl-Le a I, Le a , Le x , monosialosyl-Le 8 I disialosyl-Le a or sialosyl Le x , the agent inhibiting t metastasis potential of the preparation.
  • the instant invention also provides a method f inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregati or adhesion at a tumor cell site in a warm-blooded animal.
  • T method comprises administering to a warm-blooded animal effective amount of at least one agent selected from the gro consisting of (a) a hybrid sugar, such as Le x /SLe (b) component sugars of a hybrid sugar (a) , such as Le x a SLe x ; (c) monosialosyl-Le a I, Le a , Le x , monosialosyl-Le 8 II, disialosyl-Le 8 or sialosyl Le x ; (d) antibodies that specificall bind to a hybrid sugar, such as Le x /SLe x , monosialosyl-Le 8 I, Le 8 , Le x , monosialosyl-Le 8 II, disialosyl-Le
  • 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 8 I, Le 8 , Le x , monosialosyl-Le 8 II, disialosyl-Le 8 or sialosyl Le x ; (d) antibodies that specificall bind to a hybrid sugar, such as Le x /SLe x , monosialosyl-Le 8 I, Le a , Le x , monosialosyl-Le 8 II, disialosyl-Le 8 or sialosyl Le x (e) oligosaccharide components of a hybrid sugar, such a Le x /SLe x , monosialosyl
  • 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 b performed prior to and subsequent to administration to confir the effectiveness of the inhibition of metastatic potential o inflammatory potential.
  • One in vitro system for measuring adhesion or aggregatio of tumor cells to other cells (e.g. EC's), or for determinin successful inhibition of adhesion or aggregation is a dynami flow system similar to that described by M. B. Lawrence et al (Blood 70:1284, 1987) and which is shown in Figures 11A, 11B, 11 and 11D.
  • a parallel-plate laminar flow chamber (1) (shown upside dow for convenience) connected to a pressure pump (2) via tubing (18 is used to simulate the flow shear stresses present i physiological microvascular environments.
  • the flow chambe consists of a plastic or glass cover slip (3) resting on chamber body (16) on which a parallel, transparent plasti surface (4) is attached with a rubber or silicone gasket (5) there is a 114 ⁇ m gap between the two surfaces, and this gap i 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 shea stress is achieved by manipulation of the pressure pump (2) which is connected to the inlet manifold (8) of the flow chambe via tubing (18) .
  • Figure 11B depicts the configuration of a 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) , maintaine in a vessel in a water bath (17), is passed through the chambe via tubing (18) .
  • Cell movements are observed under an inverte phase-contrast microscope (10) and recorded by time-laps videocassette recorder (11) using a video camera (12) and digital image processor (13) .
  • Adhesion is observed as rollin followed by stopping of cells. Number of cells bound during set time, e.g.
  • Figure 11D schematically shows laminar flow of tumor cel suspension (14) through a chamber in which one surface is coate with endothelial cells (9) .
  • Rolling or stopped cells (15) a observed under an inverted microscope and recorded on videotape as described above.
  • the arrows indicate the direction of fl of the tumor cell suspension (14) .
  • the instant invention also provides method for identifying a TACA epitope to which lectin activi of a selectin, such as GMP-140, is directed.
  • the TACA epitopes were studied based on t inhibitory effect of various glycosphingolipids (GSL's), G oligosaccharides or GSL-containing liposomes on adhesion of blo cells or tumor cells to a solid phase (e.g., a plastic surfac coated with activated platelets.
  • GSL's glycosphingolipids
  • G oligosaccharides or GSL-containing liposomes e.g., a plastic surfac coated with activated platelets.
  • coati a solid phase with gelatin which was in turn was coated wi activated platelets; platelets bind readily to a gelatin-coat solid phase via GpIIb/IIIa, the major platelet integrin recepto
  • 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 8 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 8 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 t sialosyl-Le a -coated beads.
  • th primary epitope structure defined by GMP-140 is sialosyl-Le rather than sialosyl-Le x .
  • other epitope structures defined by a selectin such as GMP-140 can be identified using the instant inventiv method.
  • ELAM-1 (E-selectin) is expressed on the surface of activate endothelial cells.
  • ELAM-1 has a carbohydrate-binding domain a the amino terminal region and indeed ELAM-1 is known to bind SL and SLe 8 .
  • the instant invention is a result of systematic studies o selectin-dependent adhesion under static and dynami 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 particulat solid supports coated with glycoliposomes with activate platelets or HUVECs 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 t assess the effect on adhesion of various mAb's directed to SLe x , SLe a I, SLe 8 II, Le x , Le a and related structures; combinations o such mAb's; sialidases with various substrate specificities; o combinations of various sialidases and mAb's.
  • the method of (iv) was used to compare the intensity of adhesion under dynami conditions.
  • the instant invention relates to carbohydrate defined by formulae (I) , (II) and (III) below which ar characterized by internal sialosyl residues or a branche structure.
  • Formula (I) relates to a type 1 or extended type 1 chai with internal ⁇ 2 ⁇ 6 sialosyl substitutions and an ⁇ l ⁇ 4 fucosy substitution .
  • R. is H or a sialic acid residue in ⁇ 2 linkage
  • R 2 is H or a sialic acid residue in ⁇ 2 ⁇ 6 linkage
  • n equal to or greater than 0
  • R 3 is H or a fucosyl residue ⁇ -l ⁇ 4 linkage.
  • Formula (II) relates to a type 2 chain structure wi internal sialosyl and fucosyl substitutions.
  • R 2 is as defined for formula I, R 4 is H a fucosyl residue in ⁇ l ⁇ 3 linkage and R g is H, a sialic ac residue in ⁇ 2 ⁇ 3 linkage, NeuAc ⁇ 2 ⁇ 8NeuAc in ⁇ 2 ⁇ 3 linkage
  • R 6 ⁇ NeuAc in ⁇ 2 ⁇ 3 linkage, wherein R 6 is one or more sugars oth than a sialic acid residue and n is equal to or greater than
  • Formula (III) relates to a type 2 chain structure which a hybrid molecule comprising a branch wherein each bran comprises an epitope of a single carbohydrate antigen disclosed herein.
  • a hybrid molecule do not necessarily comprise the entirety of the two component suga that comprise the hybrid. Instead, the hybrid comprises t epitopes of the component sugars.
  • structure 1 comprises the epitopes of Le x and SLe x , however will be noted that with reference to the diagrammatic structur of the various sugars set forth hereinbelow, not all of the L 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 comprises galactose, Gal3l ⁇ 4GlcNAc or Gal01-3GlcNAc;
  • R g comprises Gal or GalNAc;
  • R comprises lactosyl ceramide or an O-linked sugar.
  • R 10 and R 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 , 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., Gal31-*3GlcNAc31 ⁇ 3Gal, repeats thereof and substitutions thereof.
  • E-selectin-dependent adhesion of Colo201 cells was inhibited only minimally by mAb CA19-9 (directed to SLe 8 ) 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 8 or by a combination of CA19-9 plus CA3F4.
  • mAb CA3F4 directed to monosialosyl Le a II and Le 8 or by a combination of CA19-9 plus CA3F4.
  • Specific reactivities of FH7 with disialosyl Le 8 and monosialosyl Le 8 II, and of CA3F4 with monosialosyl Le 8 II, were describe previously (Nudelman et al., J. Biol. Chem., 261: 5487, 1986).
  • NDV sialidase which cleave NeuAco2 ⁇ 3Gal (R, in Formula I) only slighted inhibite E-selectin-dependent adhesion, but treatment with Arthrobacte ureafaciens (AU or often denoted as AV in the Figures) or Vibri cholerae (VC) sialidases, both which cleave NeuAc ⁇ 2-+6 linkage t GlcNAc or Gal (i.e., R 2 in Formula I), completely inhibited suc adhesion.
  • NDV sialidase i combination with mAb's CA19-9 or CA3F4 strongly inhibited th adhesion. The results described above were obtained in bot static and dynamic adhesion systems, described herein.
  • the binding dynamics of selectins is vibrant, a revealed in dynamic flow systems which simulate more closel physiologic conditions, that is, for example, leukocytes or tumo cells can be moving at considerable speed in large and unocclude small vessels and at a slower speed in occluded vessels and i tissue spaces.
  • cell interactions may b mediated by interaction with a first set of molecules that shar a common characteristic
  • cell interactions may be mediated by interaction with a secon set of molecules that share a common characteristic, differen from that shared by the first set of molecules.
  • the binding requirements may var depending on the speed at which the cells are moving.
  • E-selectin (ELAM)-mediated adhesion of HL6 cells is dependent on different carbohydrate structures when th cells are reacted in a stationary or slow moving setting or ar reacted while the cells are in rapidly moving setting.
  • ELAM binds preferentially to ⁇ 2 ⁇ sialylated and ⁇ l ⁇ 3 fucosylated structures, such as SLe x
  • ELAM preferentially binds to othe 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 an sialidases and combinations thereof of E-selectin-dependen adhesion of HL60 tumor cells, which express only type 2 chain i.e. Gal ⁇ l ⁇ 4GlcNAc?l ⁇ 3Gal and repeats thereof, and substitution thereof.
  • Treatment of HL60 cells with NDV sialidase, whic cleaves NeuAc ⁇ 2 ⁇ 3Gal (R., in Formula II) completely abolishe reactivity of the cells with anti-SLe x mAb's, although the cell remained strongly adherent to E-selectin-coated plates and t activated EC's.
  • NDV sialidase treatme of HL-60 cells, which removes NeuAc ⁇ 2 ⁇ 6Gal, completely abolish reactivity of cells with anti-SLe x mAb although the cel remained adherent to E-selectin plates and activated endotheli cells. Adhesion was inhibited effectively with a combination mAb's directed to Le x and SLe x .
  • type chain structures whose internal sialosylated structure is known (Nudelman et al., supra) type chain structures with internally sialic acid residues we hitherto unknown. Data presented in the instant applicati indicate the natural occurrence of such epitopes.
  • the structures bindable to ELAM-1 can be synthesized usi known techniques.
  • the carbohydrates can synthesized chemically using known and commercially availab reagents or can be synthesized using known and available enzym to effect the appropriate linkage.
  • known sialos transferases and fucosyl transferases can be used to derivati the basic carbohydrate backbone.
  • the carbohydrates bindable to ELAM-1 can isolated using ELAM-1 as an absorbent.
  • ELAM-1 purifi ELAM-1, cells expressing ELAM-1 or membrane preparations of cel expressing ELAM-1 can be used.
  • the ELAM-1 can be immobilized a solid phase, such as an inert bead matrix or the inside wa of a vessel, to enhance separation.
  • suitable carbohydrate bindable to ELAM-l such as extracts of HL60 or Colo201 cell obtained by known techniques, are exposed to the ELAM-l affinit matrix.
  • the ELAM-l together wit carbohydrates bindable thereto are collected.
  • the carbohydrate bound to the ELAM-l are separated from the ELAM-l, for example by altering the salt concentration of the holding buffer, an collected.
  • the various carbohydrate species can be discriminate using known procedures, such as chromatography.
  • cells known to express predominantly type 1 chai structures or type 2 chain structures are grown and membran preparations are obtained therefrom using known techniques.
  • Th glycolipid and glycoprotein fraction of the membrane prep i obtained using known techniques and exposed to an affinity colum wherein antibodies directed to carbohydrate epitopes, such a those described herein, are affixed to a matrix, such as agaros beads, to form an affinity matrix.
  • affinity chromatograph procedure the bound materials are eluted and separated furthe by known techniques, such as HLPC and TLC.
  • the separated molecules in the separatio medium can be exposed to ELAM-1-expressing cells that ar labelled to serve as a tag, for example, the cells can b labelled metabolically with a radioisotope.
  • Th ELAM-1-expressing cells will bind to the respective sites of th separation medium where separated ELAM-l epitopes are found.
  • Th TLC matrix can be autoradiographed to locate such sites of cel binding to identify ELAM-l epitope-bearing molecules.
  • Th respective sites of the TLC matrix can be excised and t molecules extracted.
  • the carbohydrates of formulae I a II can be derivatized to provide oligosaccharides with mo desirable therapeutic properties.
  • portions of t structures comprising formula I or II can be substituted, f example, with sulfur-containing sugars or fluorine-containi sugars.
  • the oligosaccharide derivatives can be prepared usi the methods disclosed hereinabove but substituting for t naturally occurring components the appropriate reagent comprisi an altered substituent, such as 6-trifluoro-fucosyl which incorporated into either of formula I or II as the fucos residues.
  • the carbohydrates bindable to ELAM-l can be used immunogens to obtain antibodies bindable to the carbohydrat bindable to ELAM-l. Either poiyclonal or monoclonal antibodi can be generated, using methods such as those describ hereinabove, and in the references cited herein, which a incorporated by reference. Monoclonal antibodies are preferre Because ELAM-l may serve to mediate intercellul interactions, interruption of binding between ELAM-l a carbohydrates bindable thereto will be beneficial.
  • carbohydrates bindable to ELAM-l, ELAM-l, antibody to ELAM-l antibody to carbohydrates bindable to ELAM-l for example, c be used to interrupt binding between ELAM-l and carbohydrat bindable thereto.
  • the carbohydrates bindable to ELAM-l, ELAM- antibody to ELAM-l or antibody to carbohydrates bindable ELAM-l are administered in therapeutically effective amounts a via routes that are determinable readily and routinely practicin settled methods of the pharmaceutic arts.
  • the terminal sialic aci is not essential in a carbohydrate bindable to ELAM-l.
  • K elements held in common are the terminal galactose, glucosamine ⁇ 2 ⁇ 6sialic acid and fucose residues.
  • antibodies capabl of binding to such a structure are effective in inhibiti ELAM-1-mediated interactions.
  • Suitable antibodies are CA3FA a FH7.
  • Compounds of formula (III) for example, Le x /SLe x , where relevant epitopes comprised the branched chain structure we identified clearly as comprising high affinity binding sites f ELAM-l under high shear stress conditions.
  • su structures can show less binding ability than simple SLe x ELAM-l at low shear stress conditions or under static conditions Using that hybrid it is noted that the terminal galactose ⁇ l linked fucose to GlcNAc at one branch and an ⁇ 2 ⁇ 3 linked siali acid and ⁇ l ⁇ 3 linked fucose at the other branch are critic sites on that hybrid structure.
  • antibodies bindable Le x such as, SH-1 and FH-2
  • SLe x such as FH-6, SNH-4 a SNH-3, are effective cooperatively in inhibiting ELAM-1-mediat adhesion at high shear stress conditions.
  • T carbohydrates or antibodies are related to ELAM-l carbohydrates bindable thereto or in certain circumstances be carbohydrates or antibodies that are not specifically tho carbohydrates believed to bind ELAM-l.
  • combination of antibodies directed to SLe x and Le x is effecti in inhibiting ELAM-l interaction.
  • Suitable SLe x antibodies a SNH3 and SNH4; and suitable Le x antibodies are SHI and FH2.
  • T skilled artisan can determine other suitable combinati practicing the methods taught herein using reagents disclos herein, with particular attention drawn to the working exampl set forth hereinbelow. The following examples are offered by way of illustrat and not by way of limitation.
  • Heptaacetyllactosylimidate (Zimmermann et al. , J. Carbohy Chem. 7:435, 1988) was reacted with methanol in dichloromethane containing trimethy1 s i1 trifluoromethanesulfonate according to a standard proced (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 sodiu methoxide, gave methyl j8-D-lactoside in 68% yield from th imidate: m.p.
  • Lactose octaacetate (Hudson & Kunz, J. Am. Chem Soc 47:2052, 1926) was treated with thiophenol and SnCl (Nicolaou et al., J. Am. Chem Soc. 110:7910, 1988) i dichloromethane at 0 ⁇ C to give phenyl heptaacety 3-D-thiolactoside in 80% yield.
  • the product was deacetylate with NaOMe in MeOH and neutralized with Amberlyst* 15 Purification of the product on a BioGel* P-2 column using wate as an eluent, followed by lyophilization of the sugar-containin fraction, left phenyl ,9-D-thiolactoside as a white amorphou powder.
  • the H-NMR spectrum superimposed that of the authent sample (BioCarb Chemicals, Lund, Sweden) .
  • the polyethyleneglycol derivative of 0-D-lactoside wa prepared from readily available 3-succinimidooxycarbonylpropy 0-(2, 3, 4, 6-tetra-0-acetyl-0- -D-galactopyran ⁇ syl)-(l ⁇ 4) 2,3,6-tri-0-acetyl- / 9-D-glucopyranoside 1 and polyethyleneglyco methyl ether (average M.W. 2000; Aldrich Chemical, Milwaukee, WI) having a terminal amino group 2. (Zalipsky et al., Eur. Poly . J 19_:1177, 1983). Treatment of 1 (100 mg, 0.12 mmol) an 2.
  • C57B1 mice Univ. , Bozeman, MT
  • C57B1 mice were maintained in plastic cages under filtered air atmosphere an provided with water and food pellets ad lib.
  • Cells were culture in RPMI 1640 supplemented with 2 mM glutamine and 10% fetal cal serum (FCS) , and detached with phosphate buffered saline (P containing 2 mM EDTA. Viability was inferred by a trypan bl exclusion test.
  • a suspension of BL6 cells (1-3 x 10 6 cells/ml RPMI 1 medium) was prepared and aliquots were incubated in the prese or absence of various oligosaccharides at various concentratio at 37 ⁇ C for 5-10 minutes. Following incubation, typical 3 x 10 4 or 2 x 10 4 cells (with or without oligosacchar pretreatment) per 200 ⁇ l were injected via a tail vein i 8-week-old female mice. After 18-21 days, the mice were kill the lungs were fixed in 10% formaldehyde in PBS (pH 7.4) tumor cell colonies were counted under a dissecting microsco thereby providing background values of metastatic melanoma col number in lung under those conditions. Data on the number the size of colonies were treated statistically by an analy of variance (ANOVA) procedure. Colonies with a diameter of 1 or greater were considered large-size and those with a diame less than 1 mm were considered small-size.
  • ANOVA an analy of variance
  • BL6 cells were incubated with vari concentrations of lactose, lacto-N-tetro (Gal01 ⁇ 3GlcNAcj81 ⁇ 3Gal/91 ⁇ 4GLc) , methyl ,3-D-lactoside or phe 0-D-thiolactoside for various durations.
  • lactose lacto-N-tetro
  • lacto-N-tetro Gibcos Universal
  • methyl ,3-D-lactoside methyl ,3-D-lactoside
  • phe 0-D-thiolactoside phe 0-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 injectio of cells under identical conditions.
  • Treatment of BL6 cells wit 0.1 M, 0.01 M or 0.005 M methyl /3-D-lactoside under the sam 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 3-D-lactosid was reproduced in three separate experiments and the reductio was found to be consistently between 35% and 45%.
  • treatmen with methyl ,3-D-lactoside or phenyl 0-D-thiolactoside unde different conditions also produced a significant reduction o metastatic colonization, i.e., total colony number was reduce to 35% or 50% of control values following preincubation wit methyl ?-I-lactoside or phenyl 3-D-thiolactoside, respectively Reduction of larger-size colonies was more apparent than that o smaller colonies in all experiments, particularly those wit phenyl ,9-D-thiolactoside ( Figure 1) .
  • Methyl /3-D-lactoside an phenyl S-D-thiolactoside both showed a slight in vitr stimulatory effect on cell number increase and on thymidin incorporation.
  • the inhibitory effect on tumor depositio is not related to the effect on cell growth in vitro or in vivo
  • the effect of methyl ,9-D-lactosi on melanoma cell metastasis was determined after administrati of the oligosaccharide, followed by inoculation with tumor cell Specifically, a one ml dose of methyl ,9-D-lactoside (at concentration of 0.25 M or 0.5 M) was injected intraperitoneal in mice.
  • mouse melanoma B16 varia showing different degrees of metastatic potent (BL6/F10/Fl/Wa4) showed the same order of expression of ganglioside, which was previously identified as melanoma-associated antigen (Hirabayashi et al., J. Biol. Ch 260:13328, 1985; Nores et al., J. Immunol. 139:3171, 1987). interacts with LacCer, which is highly expressed on endothel cells. The order of adhesion of the B16 variants o LacCer-coated solid phase or onto endothelial cells was also the same order as metastatic potential (MP) .
  • MP metastatic potential
  • Capillary endothelial cells are strongly reactive wit antibodies directed to H/Lev'/Leb, such as antibody MIA-15-5
  • Liposomes comprising H-l or Le y were made and exposed t plates to which various glycolipids had been affixed at a rang of concentrations.
  • H-bearing liposomes bound to H or L coated onto plates are bound to Le y -bearing liposomes bound to H-coated plates.
  • H and paragloboside ar related, the only difference being the presence of a termina fucose residue in H.
  • cells expressing H, Le y or Le can adhere t endothelial cells expressing H and possibly to Le y as well
  • KUM-LK-2 is a human non-adenocarcinoma cell li characterized by producing spontaneous lung metastasis in nu mice. After screening 35 human carcinoma cell lines grown nude mice, only that cell line produced metastatic deposits nude mouse lung. KUM-LK-2 was used as the parent cell line obtain, by limiting dilution technique, sub-cell lines produci lung metastasis on IV injection.
  • KUM-LK-2 was cultured in RPMI 1640 medium (GIBCO, Gra Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) 37'C in a 5% C0 2 /95% air atmosphere. Cells were treated brief with 2 mM EDTA solution and washed twice with RPMI 1640 to ma a single cell suspension in RPMI with 10% FCS. Cell viabil was > 98% as determined by trypan blue exclusion staining. cell suspension containing 1 cell per 100 ⁇ l was transferred each well of a 96-well microtiter plate (Corning Glass Wor Corning, NY) and cultured continuously for 24 hours. Each w 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 differ metastatic potential ("MP") were selected out of 25 clo obtained by limiting dilution technique on the basis of sta cell morphology.
  • the 25 clones were selected originally from clones showing stable morphology as well as consistent in vitr cell growth.
  • mice Fifty-six days after injection, mice were killed and metastatic nodules on lung surface were counted u dissecting microscope.
  • the cell surface expression of various carbohydrate epitop was analyzed by cytofluorometry using various monoclon antibodies (mAb's) directed to Le x (mAb SHI), sialosyl- (mAb SNH4) , sialosyl-dimeric Le x (mAb FH6) , T (mAb HH8 Tn (mAb 1E3) and sialosyl-Tn (mAb TKH2) . All antibodies us were culture supernatants from the respective hybridoma adjusted as 10 ⁇ g/ml of immunoglobulin.
  • Leb (structure 5) , H (structure 6) , SA-Lea I (structure 7 SA-Tn (structure 8), disialosyl-Le” (structure 9) monosialosyl-Le 8 II (structure 10), GM3 (structure 11) S-PG (structure 12) , Le x (structure 13) and Le a (structure 14 are shown below.
  • R represents a carrier molecule.
  • Cells were detached from culture flasks with 0.25% trypsi 2mM EDTA solution and 1 x 10 cells were prepared for each m treatment. Cells were incubated with a mAb for 1 hour at 4 ⁇ C a washed 2 times with RPMI 1640. Goat anti-mouse IgG or IgM-FI (Boehringer-Mannheim, Indianapolis, IN) , diluted 50 times wi PBS, then was added and incubated 30 minutes at 4 ⁇ C. Finall cells were washed 3 times, resuspended with PBS and analyzed an EPICS PROFILE flow cytometer (Epics, Hialeah, FL) .
  • Epics Hialeah, FL
  • sialosyl residues were assessed in t following manner.
  • Cells were detached using 2 mM EDTA in P washed and resuspended in 9 volumes of PBS.
  • One ml of c suspension was incubated 5 minutes at 37 ⁇ C with 0.2 U/ml Clostridiu perfringens sialidase (type X, Sigma Chemical C St. Louis, MO) .
  • cells were washed three tim resuspended with RPMI 1640 and investigated for MP and express of sialosyl-dimeric-Le x .
  • MP of HAL-8 and HAL-33 was inhibi completely by sialidase treatment of cells (see Table 2 belo Expression of sialosyl-dimeric-Le x appears to play an import role in blood-borne metastasis.
  • 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.
  • Platelets were isolated from "platelet-rich plasma” obtain from the Oregon Red Cross (Portland, OR) . Contaminating r blood cells were removed by centrifugation at 80 x g for 10 mi Platelets were centrifuged at 300 x g for 10 min and suspend in Tyrode's buffer (pH 6.5) containing 22 mM citrate buffer wi 0.35% bovine serum albumin (BSA). The platelet suspensi (1 x 10 /ml) was incubated (pH 7.2, 37 ⁇ C, 5 min) after additi of thrombin (final concentration 1 U/ml) . The mixture then w incubated at 37 ⁇ C for 10 min without stirring.
  • Tyrode's buffer pH 6.5
  • BSA bovine serum albumin
  • T thrombin-activated platelets were fixed with an equal volume 2% formaldehyde in phosphate-buffered saline (PBS), pH 7.2, a washed 2 x with PBS containing 1% BSA.
  • Activated platelets (b not non-activated platelets) showed strong reactivity wi 2.5 ⁇ g/ml anti-GMP-140 mAb AC1.2 (isotype IgG Beckton-Dickinson, San Jose, CA) when incubated at 37"C f 30 min. , followed by reaction with 50 ⁇ l of fluorescence-label goat anti-mouse Ig (Tago, Burlingame, CA) .
  • Flow cytometr profiles of activated vs. non-activated platelets with mab AC1 are shown in Figures 8A-8D.
  • Activated and non-activated platelets were fixed wi paraformaldehyde in Ca 2+ -free PBS, pH 7.2, washed 2 wi Ca ⁇ -containing PBS with 1% BSA, resuspended in CA * -PBS wi 1% BSA and 0.1% azide and the number of platelets adjusted «lxl0/ml.
  • the cell suspension was stored at 4'C and the bindi assay performed within 24 hr.
  • Fluorescent polystyrene latex beads were obtained fr Molecular Probe, Inc., Eugene, OR.
  • the beads were yellow-gre fluorescent beads with a sulfate group at the surface, diamet « 0.5 ⁇ m (actually 0.486 ⁇ m) .
  • the binding index (BI) was calculated as me fluorescence intensity (MFI) of platelets incubated wi fluorescent GSL-coated beads divided by MFI of platele incubated with fluorescent non-GSL-coated (control) beads.
  • MFI me fluorescence intensity
  • B values for various GSL's are shown in Table 3 and in Figure 9.
  • the hatched bars represent non-activated platelet and the open bars represent activated platelets.
  • the ratio o the binding index (BI) of activated/non-activated platelets fo
  • SA-Le x SA-Le a , SPG, GM3 and Le x also is shown in the "Ratio A/NA column.
  • mAb's affected platelet binding to fluorescent GSL-coate beads. Platelets were incubated with anti-GMP-140 mAb IOP6 (Immunotech, Marseille, France) at 37°C for 30 min and a bindin assay was performed using GSL-coated beads, as describe hereinabove. Non-specific mouse IgG (10 ⁇ g/ml) was used in control binding assay. Also, 10 ⁇ l of SA-Le a -coated beads (2 x 10 7 ) were incubat with 20 ⁇ l of anti-SA-Le 8 mAb CA19-9 (20 ⁇ g/ml) (mouse Ig
  • Adhesion was measured using the dynamic flow experimenta system shown in Figures 11A to 11D.
  • the coefficient of viscosity was 1.0 P, the hal channel height was 5.7 X 10 " cm and the channel width wa 1.3 cm.
  • HUVECs (Cell Systems, Kirkland, WA) were cultured confluency in 48-well plates (Costar, Cambridge, MA) stimulated with 1 U/ml IL-1 for 4 hr.
  • Non-simulated HUVEC's w used as a control.
  • Expression of E-selectin (ELAM-l) IL-1-stimulated HUVEC's was confirmed by reactivity w anti-E-selectin mAb 3B7 (IgG 2a ) (Graber et al. J. Imm. 145:8 1990) .
  • HL60 and Colo201 cells were labeled metabolically culture in the presence of [ H]-thymidine after pretreatment w glycosylation modifier and added to HUVEC-coated plates.
  • Platelets bound on plates were incubated wit anti-P-selectin mAb IOP-62 (1:2, 1:6 dilution) (Immunotech Marseille, France) at room temp for 30 min, followed by additio of HL60 cells, to evaluate dependence of adhesion on P-selecti expression.
  • Non-specific mouse IgG was used as control.
  • the flow chamber consists of a glas plate on which a parallel, transparent plastic surface i attached with a Silastic rubber gasket; there is a 114 ⁇ m ga between the two surfaces and the gap is connected to an inlet outlet.
  • EC's are grown as a monolay or adhesion molecules are coated, on the glass plate, and laminar flow of a cell suspension is passed through the chamb Cell movements are observed under inverted phase-contr microscope (Diaphot-TMD Nikon) and recorded by time-la videocassette recorder. Adhesion is observed as rolling follo by stopping of cells. Number of cells bound during 3 min different shear stresses from, for example, 0.4 to 4.8 dynes/ or 0.76 to 15.5 dynes/cm were counted from several fie recorded on videotape.
  • Wall shear stress (T) was calculated the equation of Lawrence et al. (Blood 75:227, 1990):
  • ⁇ coefficient of viscosity (1.0 cP)
  • Q volumetric f rate (cm/sec)
  • a half channel height (for the experime reported herein, 5.7 x 10 3 cm)
  • b channel width (1.3 cm).
  • Promyelocytic leukemia cell line HL60 has been shown express only type 2 chain and sialosylated/fucosyla derivatives as probed by specific mAb's (Symington et a J. Immunol. 134:2498, 1985) and has been extensively used a model of leukocyte adhesion mediated by E-selectin and P-selecti
  • NDV sialidase eliminates only the ⁇ 2 ⁇ 3 sialosyl resid linked to the terminal Gal whereas both Vibrio and Arthroact sialidase completely eliminate terminal and internal sialic aci residues, notably, ⁇ 2 ⁇ 6 linked sialic acid residues.
  • T indings indicate that SLe x and SLe 8 are not the sole epitopes o E-selectin and P-selectin.
  • anti-SLe x mAb's e.g., SNH3 and SNH4
  • SNH3 a SNH3
  • Anti-Le x mAb's SHI and FH2 showed consistent stronger (compared to SNH3 or SNH4) inhibition of HL60-HUVEC adhesion, as did a combination of SNH4 plus SHI or FH2.
  • NDV sialidase did not reduce significantly HL60-HUVEC adhesion, but Vibrio sialidase almost abolished reactivity completely.
  • fibronectin (FN) fibronectin (FN)
  • laminin (LN) laminin (LN)
  • truncated E-selectin and GSL's 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 x 75 mm; Corning Glassworks, Corning, NY) and dried in a refrigerator at 4*C. Dried plates were immersed in PBS at 37"C for 1 hr and washed extensively with several changes of PBS.
  • GSL-liposomes were prepared from 200 ⁇ g GSL, 200 ⁇ g cholesterol and 400 ⁇ phosphatidylcholine in 1 ml PBS. Ten ⁇ l of GSL-liposome solution was placed on a glass plate, dried at 4 ⁇ C and the plates were washed with PBS, as described above.
  • the quantity of adsorbed molecules was determined using 125I labeling for lectins, FN or LN, or [ H]cholesterol labeling fo GSL-liposomes. Under those conditions, almost the entir quantity of protein, regardless of whether FN r LN or lectin, wa adsorbed on the glass plate. For example, when 100 ⁇ g/ml FN wa applied, 12.5 + 1.8 ng/mm was adsorbed. Likewise, almost al GLS-liposome dried on the glass plate was adsorbed; e.g., whe 200 ⁇ g/ml GLS-liposome was applied, 31.3 + 5.2 ng GSL/mm wa adsorbed. EC's were coated by placing 100 ⁇ l of a suspens containing 2 x 10 5 mouse or human EC's on glass plates culturing in a C0 2 incubator at 37 ° C until confluency achieved.
  • Plates coated with adhesion molecules or EC's were affi in a flow chamber, and a suspension of B16 melanoma cells passed through the chamber as described hereinabove.
  • B16 ce were harvested from culture using 0.02% EDTA in PBS, suspended in PBS at a concentration of 1 x 10 /ml.
  • NDV sialidase had an inhibito effect only at low shear stress whereas VC or AU sialidas significantly reduced adhesion even at high shear stress
  • Anti-Le x IgG mAb SHI strongly inhibited adhesion even at hi shear stress, whereas the effect of anti-SLe x IgG 3 mAb SNH4 w minimal.
  • Strongest inhibition was produced by a combination o NDV sialidase plus anti-Le x mAb SHI.
  • a mixture of anti-Le x pl anti-SLe x mAb's produced stronger inhibitory effect than eith mAb alone.
  • Le x as well as SLe x may be ⁇ 2 ⁇ 6 sialylated at t internal Gal or GlcNAc within the same CHO chain, or ⁇ 2 sialylation may be present at an adjacent branched structur "6-C ganglioside," which is an ⁇ 2 ⁇ 6 sialylated type 2 cha structure with internal ⁇ l ⁇ 3 fucosylation (Hakomori et al Biochem. Biophys. Res. Commun. 113:791, 1983), failed to bind E-selectin. Thus, such a structure can be excluded as a possib ELAM epitope.
  • Colo201 cells In contrast to HL60 cells (which express predominan type 2 chain structure) , Colo201 cells express mainly type chain, and E-selectin-dependent Colo201 adhesion is thro type 1 chain epitopes. Colo201 cells were treated with vari mAb's following exposure to various sialidases and were asses for residual binding. Colo201 reactivity with mAb CA1 (directed to SLe a I) was inhibited almost completely by Vib sialidase, and to a lesser extent by Arthrobacter and sialidases.
  • Colo201 reactivity with mAb FH7 was reduced by Arthrobacter sialidase minimally affected by Vibrio or NDV sialidases.
  • Colo reactivity with mAb CA3F4 was enhanced by sialidase treatment.
  • mAb CA1 inhibited Colo201 adhesion slightly and was influenced onl minimally by Vibrio sialidase ( Figure 16) .
  • the SI-E 8 epitope present at the surfac of Colo201 cells is organized in such as way that it is (i) no susceptible to CA19-9 for E-selectin-dependent adhesion an (ii) not sensitive to sialidase treatment.
  • the inhibitor 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 Colo20 adhesion. See Figure 17.
  • NDV sialidase had no effect o Colo201 adhesion, particularly at high shear stresses
  • mAb CA3F4 which i directed to Le 8 with an ⁇ 2 ⁇ 6 sialosyl substitution at th penultimate GlcNAc.
  • Vibrio sialidase which efficiently cleave terminal ⁇ 2 ⁇ 3 sialosyl linkages but is less effective at removin internal sialic acid residues, reduced adhesion to some exten at high shear stress, but less so at low shear stress.
  • mAb CA3F4 inhibited adhesion strongly at hi shear stress but much less at low shear stress.
  • Truncated, recombinant ELAM-l lacking the transmembrane an cytoplasmic domains is used to coat beads, for example, capabl of packing into a standard chromatography columns.
  • the ELAM- at a concentration of 0.1-1 ⁇ g/ml is mixed with the beads and th mixture is incubated to allow binding of ELAM-l to the bea matrix.
  • a suitable incubation period is 12-24 hours a ⁇ C - room temperature.
  • the beads are washed to remove unboun ELAM-l, optionally can be blocked with an inert carrier, such a BSA, and washed again.
  • the ELAM-l coated beads can be used in a batch process packed into a suitably-sized column.
  • Cells known to carry carbohydrates bindable to ELAM-l, su as HL60 and Colo201, are obtained.
  • the cells are lysed to obta a membrane fraction using known methods, such as repeat freeze-thaw cycles.
  • the membrane fraction is obtained, f example, by centrifugation.
  • the membrane prep may be suitable source without further purification.
  • the membrane prep is treated using known methods to obta a membrane component preparation, and in particular, a fracti that contains cell surface carbohydrate.
  • the carbohydrate-ri fraction is mixed with or passed over the ELAM-l affinity matri depending on the format, the exposed matrix is washed and carbohydrates bound to the matrix are eluted, for example, exposing the matrix to a high salt buffer.
  • the resultant preparation comprises carbohydrate binda to ELAM-l and the various species are separable using -kn techniques, such as TLC or HPLC.
  • Carbohydrates bindable to ELAM-l either prepared chemica using known reagents and methods, see, for example, Exampl hereinabove, prepared enzymatically or obtained from suita cells, see, for example.
  • Example 8 hereinabove, or whole ce 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-l 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 8 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-l 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) a maintained in RPMI 1640:Dulbecco's MEM (1:1) supplemented wi 10% HI FCS. Fifty ⁇ g of a plasmid comprising cDNA of E-select in vector pCDM8 (R & D Systems, Minneapolis, MN) and 5 ⁇ g pSV2-neo (ATCC) were co-transfected into NS-1 cells (1 x 10 ) electroporation. After 48 hours in culture, the cells we transferred to medium containing 650 ⁇ g/ml G418 (Gibco, Gra Island, NY) .

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Abstract

Many tumor-associated and leukocyte-associated carbohydrate antigens function as adhesion molecules, recognized by lectins (carbohydrate-protein interaction) or complementary carbohydrates (carbohydrate-carbohydrate interaction). Common structures are found in the tumor-associated and leukocyte-associated antigens. Metastatic potential of tumor cells as well as transendothelial migration of leukocytes was suppressed by agents, or combinations of agents of the groups: (a) carbohydrate antigens; (b) antibodies directed to those antigens; (c) oligosaccharide components of those antigens; (d) conjugates of the antigens or oligosaccharides; and (e) mimetics of the antigens or oligosaccharides. Disclosed are oligosaccharides and derivatives thereof which inhibit cell adhesion and aggregation mediated by P-selectin (GMP-140) and E-selectin (ELAM-1). Effective agents for those purposes include hybrid sugars comprising multiple epitopes, such as Lex/SLex, combination of individual sugars that comprise a hybrid sugar, which may be presented on liposomes, and antibodies or combinations of antibodies directed thereto.

Description

Inhibition of Cell Adhesion by Chemically-Defined Oligosaccharides, Their Derivative Mimetics, and Antibodies Directed Thereto
Cross-Reference To Related Applications
This application is a continuation-in-part of pending U.S. Application Ser. No. 07/950720 filed 25 September, 1992; which is a continuation-in-part of pending U.S. Application Ser. No. 07/836978 filed 19 February 1992; which is a continuation-in-part of pending U.S. Application Ser. No. 07/789969 filed 12 November 1991; which is a continuation-in-part of pending U.S. Application Ser. No. 07/724983 filed 2 July 1991; which is a continuation-in-part of U.S. application Serial No. 07/575539 filed 30 August 1990 (abandoned) .
All five applications expressly are incorporated herein by reference.
Technical Field
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.
Background of the Invention
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. In most human malignancies, distant metastases often are to small to be detected at the time the primary tumor is treated Furthermore, widespread initiation of metastatic colonies usuall occurs before clinical symptoms of metastatic disease ar 5 evident. The size of the metastases, age of the patient *% dispersed anatomic location and heterogeneous composition all ar factors that hinder surgical removal of tumors and limit th concentration of anticancer drugs that can be delivered to t metastatic colonies.
10 Due to difficulties in the current approaches for treatin and preventing metastases, there is a need in the art fo improved methods and compositions capable of inhibiting t metastasis potential of tumor cells. The present invention fill those needs and further provides other related advantages.
15 On the other hand, there are common mechanisms between t initiation of the inflammatory process and metastasis. F example, both processes are triggered by adhesion of cell leukocytes in the former case and tumor cells in the latter, microvascular endothelial cells followed by transendotheli
20 migration of the leukocytes or tumor cells into the tiss spaces. Both processes are enhanced by activating platelet
Both processes are mediated strongly by specific types carbohydrates, such as tumor associated carbohydrate antige
(TACA) or leukocyte associated carbohydrate antigens (LACA) ■
25 Some TACA's share structures with LACA's.
* The instant invention is directed to and based on t inhibition of cell adhesion, for example, through TACA's
LACA's, using, for example, antibody thereto. Summary of the Invention
Briefly stated, 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. For example:
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, Fl or a4. Adhesion of high metastatic variants to endothelial cells is greater than with low metastatic variants and the adhesion is inhibited by Me-jS-lactoside, GM3 or LacCer (each within liposomes) or other lactoside derivations. The sugars and derivatives also inhibit B16 melanoma metastatic potential. Such is an example of (a) above, that is, interfering with a carbohydrate to carbohydrate interaction. ii) As to human cancer, patients whose primary tumor express defined tumor-associated carbohydrat antigens, such as H/Ley'/Leb (defined by monoclona antibody MIA-15-5) , sialosyl Tn (defined by monoclona antibody TKH2) or sialosyl-Lex (defined by monoclona antibody FH6, SNH3 or SNH4) , had a much shorte survival rate than those patients whose primary tumor do not express or which weakly express those antigens
iii) Those tumor-associated carbohydrate antigens (GM in the mouse melanoma model and H/Le /Ley, sialosyl-L or sialosyl-Tn in human tumors) are essentiall adhesion molecules which are recognized by targe cells, particularly platelets or endothelial cells Such is an example of a combination approach, that is interfering with (a) and (b) .
iv) Interaction of tumor cells with endothelial cell and platelets is mediated by LECCAM (or selectin)
ELAM-1 or GMP-140, which are expressed on activate endothelial cells and activated platelets Sialosyl-Lex antigen has been known to be recognized b those LECCAM1s. Such is an example of (b) , affectin a carbohydrate to selectin interaction.
v) GMP-140, whose expression on platelet o endothelial cells is induced by thrombin, ADP or (AMP phorbol ester, may play an important role i platelet-tumor cell interaction and mediate tumor cell metastases. While the epitope recognized by that selectin was identified previously as sialosyl-Lex (Polley et al., Proc. Natl. Acad. Sci. 88:6224, 1991), it has been found that sialosyl-Lea (also known as monosialosyl-Lea I) , monosialosyl-Lea II (a positional iso er of sialosyl-Lea) and disialosyl-Lea also are recognized by GMP-140. GMP-140 binds to sialosyl Lea better than to sialosyl-Lex. Such is another example of process (b) .
ELAM-1, whose expression on endothelial cells is induced by interleukin-1, TGF-β, TNF-α 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-Lex and sialosyl-Lea (Phillips et al.. Science 250:1130,
1990; Berg et al., J. Biol. Chem. 266:14869, 1991; Takada et al., Biochem. Biophys. Res. Co mun. 179:713, 1991) , it has been found that the selectin epitopes also are internally sialylated, penultimate fucosylated type 1 or type 2 chains, such as monosialosyl-Lea II and disialosyl-Leβ, particularly in a dynamic flow system. But the binding phenomenon is vibrant. Under static or low shear stress dynami conditions, ELAM-1 (also known as E-selectin recognizes primarily α2→3 sialylated and αl→3 or αl→ fucosylated carbohydrates, such as SLex and SLea However, under middle to high shear stress dynami conditions, molecules having formulae (I) or (II) see, for example, Figure 20, such as Lex/SLex, play a important role in providing high affinity bindin sites to E-selectin. That role is particularl evident under high shear stress conditions.
vii) Human colon tumor cells showing differentia expression of metastatic potential in nude mice showe a close correlation with the expression o sialosyl-Lex, i.e., cells with high metastati potential expressed high levels of sialosyl-Lex an vice versa.
viii) Adhesion of E-selectin-expressing cells to SLex i enhanced greatly when SLex is mixed in liposomes wit various quantities of Le . Hence, greatly enhance adhesion was observed not only with hybrid SLe/Le but also with mixed glyco-liposomes with SLex and Lex
ix) Human endothelial cells are characterized by hig expression of H (Fucαl→2Gal) and many types of huma cancers are characterized by expression of Ley, H o Le defined by monoclonal antibody MIA-15-5 Interaction of H with Ley or H with H has been established clearly, therefore, those human tumors expressing H/Ley/Leb may adhere to H-expressing endothelial cells which are mediated by Ley-H or H-H interaction. Such is an example of process (a) , that is affecting a carbohydrate to carbohydrate interaction.
x) Monoclonal antibody MIA-15-5 directed to H/Ley/Leb
inhibited lung metastasis of highly metastatic F10 and BL6 variant cells in the mouse. Furthermore, monoclonal antibody FH7 directed to disialosyl-Lea and monosialosyl-Lea II inhibited adhesion of human cancer cells expressing those antigens in a dynamic flow system.
Based on those and other various observations and considerations, 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, Ley, Leb, monosialosyl-Lex (SLex) ,
Lea, Lex, hybrid sugars, such as, Lex/SLex hybrids (Structure 1 in Figure 20) , monosialosyl-Lea I (SLea) , monosialosyl-Lea II, sialosyl Tn, lactosyl and other structures as depicted in structures 1-14, in Example 3. b) Compounds, such as those set forth in Figure 20, which can be Lex/SLex hybrids, or an appropriat mixture of the relevant components, such as Lex an SLex, provide high affinity adhesion binding sites, particularly under high shear stress conditions in dynamic flow system. Hence, such compounds bloc E-selectin-mediated adhesion of tumor cells o leukocytes to endothelial cells.
c) Oligosaccharide derivatives based on thos structures and linked to an appropriate carrier.
d) Oligosaccharide derivatives whose suga structures are modified appropriately showing bette blocking activity of tumor cell adhesion based o oligosaccharide-lectin (selectin; LECCAM) o oligosaccharide-oligosaccharide interaction.
e) Utilization of antibodies recognizing thos oligosaccharides comprising and representin tumor-associated carbohydrate antigens also ma inhibit tumor cell adhesion to endothelial cells, platelets or target cells, and may inhibit metastasis.
e) Utilization of combinations of antibodie recognizing those oligosaccharides involved in cel adhesion and representing tumor-associated antigens. Thus, in one aspect of the instant invention, a method for inhibiting tumor cell metastasis potential or inflammation within a biologic preparation is provided. The method 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,
(b) antibodies that specifically bind to those antigens,
(c) oligosaccharide components of those antigens, (d) conjugates of those antigens or oligosaccharides and (e) mimetics of the tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigens) , the agent inhibiting the metastasis potential of the preparation. Suitable biologic preparations 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,
(d) conjugates of those antigens or oligosaccharide components and (e) mimetics of the tumor-associated carbohydrate antigens
(or leukocyte-associated carbohydrate antigens) , the agent inhibiting tumor cell metastasis potential or inflammation potential. Within a related aspect, the instant invention provides variety of glycoconjugates useful for prolonging the in viv half-life of oligosaccharide components. The conjugates compris an oligosaccharide coupled to polyethyleneglycol. Additional oligosaccharide components for use within th methods and compositions of the instant invention includ lactose, lacto-N-tetrose, methyl 0-D-lactoside and pheny ϊ-D-thiolactoside. Oligosaccharide components may be use individually or in combination with one another. The instant invention further provides a variety of method for inhibiting GMP-140-mediated or ELAM-1-mediated cel aggregation or adhesion causing metastasis at a tumor site an inflammatory responses at a site.
One suchmethod inhibits GMP-140-mediated or ELAM-1-mediate cell aggregation or adhesion within a biologic preparation an comprises incubating the biologic preparation with at least on agent selected from the group consisting of: (a) a hybrid suga molecule, such as one comprising Lex and SLex (Structure 1 o Figure 20, a branched type II chain); (b) a mixture of th components of the hybrid sugar of (a) , such as, Lex and SLex (c) monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II disialosyl-Lea or sialosyl Lex; (d) antibodies that specificall bind to a hybrid sugar, such as Lex/SLex, or to the component thereof; (e) antibodies that specifically bind t monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Le or sialosyl Lex; (f) oligosaccharide components of hybri sugars, such as Lex/SLex, monosialosyl-Lea I, Lea, Lex monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex; (g conjugates of hybrid sugars, such as SLex/Lex, monosialosyl-Le I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex or of the oligosaccharide components; and (h) mimetics of hybrid sugars, such as Lex/SLe, monosialosyl-Lea I, Le", Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex, said agent inhibiting the cell aggregation or adhesion.
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, SLex/Lex; (b) a mixture of the components of a hybrid sugar (a), such as, Lex and SLex; (c) monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex; (d) antibodies that specifically bind to a hybrid sugar, such as Lex/SLex, monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex; (e) a mixture of antibodies, particularly to the components of a hybrid sugar, such as to Lex, SLex, Lea or SLea; (f) oligosaccharide components of a hybrid sugar, such as Lex/SLex, monosialosyl-Lea I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Lea or sialosyl Lex; (g) conjugates of a hybrid sugar, such as, Lex/SLe, monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex or of the oligosaccharide components; and (h) mimetics of a hybrid sugar, such as, Lex/SLex, monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex, the agent reducing the metastatic potential a the tumor cell site or inflammation in the warm-blooded animal The instant invention also provides a method of inhibitin GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesio at a site of inflammation in a warm-blooded animal thereb reducing inflammatory potential at the site and comprise administering to warm-blooded animal an effective amount of a least one agent selected from the group consisting of (a) hybrid sugar, such as, Lex/SLex; (b) an appropriate mixture o sugars which are the components of a hybrid sugar (a) , such as Lex and SLex; (c) monosialosyl-Lea I, Lea, Lex monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex (d) antibodies that specifically bind to a hybrid sugar, suc as, Lex/SLex, monosialosyl-Le8 I, Le8, Lex, monosialosyl-Lea II disialosyl-Lea or sialosyl Lex; (e) a mixture of antibodies particularly to the components of a hybrid sugar, such as to Lex SLe , Lea and SLea; (f) oligosaccharide components of a hybri sugar, such as, SLex/Lex, monosialosyl-Le8 I, Le8, Lex monosialosyl-Lea II, disialosyl-Le8 or sialosyl Lex (g) conjugates of a hybrid sugar, such as SLex/Lex monosialosyl-Lea I, Le8 Lex, monosialosyl-Le8 II, disialosyl-L or sialosyl Lex or of the oligosaccharide components; an (h) mimetics of a hybrid sugar, such as, Lex/SLex monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-L or sialosyl Lex, the agent reducing the inflammatory potential a the inflammatory site in the warm-blooded animal.
In another aspect, the instant invention provides a metho 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.
Those and other aspects of the instant invention will become evident on reference to the following detailed description and attached drawings.
Brief Description of the Drawings
Figure 1 graphically illustrates the effects of methyl jS-D-lactoside or methyl j9-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 ,3-D-lactoside ("Me-ø-lactoside") or 0.1 M phenyl 3-D-thiolactoside ("phe-/S-S-lactoside) . Twenty thousand cells were injected intravenously into C57B1 mice. Lung colony numbers were counted at 21 days and colonies were classified on the basis of diameter (> 1 mm vs. < l 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 0-D-lactoside on the number and size of lung colony deposits of BL6 cells. Methyl jS-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 represent control animals (not administered with methyl 3-D-lactoside) an groups B and C represent animals injected with 0.25 M and 0.5 methyl ,9-D-lactoside, respectively. For each group, column
5 represents the total number of colonies, column 2 the number o t colonies with diameter > 1 mm and column 3 the number of colonie with diameter < 1 mm. Number of experiments is expressed as "n"
Figure 3 graphically illustrates survival of cancer patient with or without expression of a defined tumor-associate
10 carbohydrate antigen (TACA) in the tumors. Panel 3A represent the expression of H/Ley/Le antigen in lung squamous cel carcinoma as determined by monoclonal antibody MIA-15-5
Panel 3B represents sialosyl-Lex expression in colonic cance using antibody FH6. Panel 3C represents sialosyl-Tn expressio
15 in colonic cancer using antibody TKH2. Panel 3D represent sialosyl-Tn level in sera of ovarian cancer patients.
Figure 4 graphically illustrates that melanoma cell adhesio on LacCer is based on GM3-LacCer interaction. The order o metastatic potential is BL6>F10>Fl»Wa4. Panel 4A shows th 20 order of melanoma cell adhesion on a LacCer-coated solid phase Panel 4B shows the order of melanoma cell adhesion o LacCer/Fibronectin (FN) co-coated solid phase. Panel 4C sho integrin-dependent adhesion.
Figure 5 graphically illustrates the melanoma cell (BL6 25 adhesion on LacCer (Panel 5A) and on endothelial cells (HuVEC * (Panel 5B) is inhibited by LacCer and GM3.
Figure 6 graphically illustrates the metastasis-inhibiti effect of methyl(Me)-/8-lactoside. Tumor cells were inject intravenously, followed by intraperitoneal injection of:
PBS (A); 0.25 M Me-3-lactoside (B) ; 0.5 M Me-3-lactoside (C) ;
0.5 M lactose (D) ; 0.25 M N-acetyllactosamine (E) ; or 0.5 M
Me- -galactoside (F) . Figure 7 graphically illustrates H-Ley and H-H interaction.
Panel 7A shows Hl-liposome binding to various glycolipids.
Panel 7B shows Ley-liposome binding to various glycolipids.
Figures 8A-8D are flow cyto etric profiles of non-activated
(Panels 8A and 8C) and activated (Panels 8B and 8D) platelets with anti-GMP-140 monoclonal antibody.
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-Lea-coated beads. The abscissa represents the percent inhibition. Column 1 represents anti-GMP-140-mAb, IOP62; column 2 represents anti-sialosyl-Le8 monoclonal antibody, CA19-9; column 3 represents anti-sialosyl-Lex monoclonal antibody, SNH4; and 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
(3) on which cells or adhesion molecules (9) are fixed.
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 variou monoclonal antibodies on adhesion of human colon carcinom Colo205 cells to interleukin-1-activated human umbilical vei endothelial cells in a dynamic flow system. Open circle represent a mixture of irrelevant mouse IgG plus IgM (control) the solid triangles represent monoclonal antibody CA19-9 directe to monosialosyl-Le8 I, the open triangles represent monoclona antibody SNH4 directed to sialosyl-Le , the solid circle represent monoclonal antibody FH7 directed to monosialosyl-Le8 I and disialosyl-Le8 and the solid squares represent a mixture o irrelevant mouse IgG plus IgM and non-activated endothelia cells. Figure 13 depicts binding of mAb's to HL60 cells and th effect of sialidase thereon. Binding activity was determined b flow cyto etry. Abscissa: log fluorescence intensity Ordinate: relative cell number. Panel A: Solid line, cell stained with mAb SNH4 as primary antibody. Dotted line, contro cells stained with mouse IgG plus IgM [10 μg/ml] as primar antibody. Panel B: mAb SNH3 as primary antibody; control as i Pane A. Panel C: Solid line, cells treated with Newcastl Disease Virus (NDV) sialidase and then stained with mAb SNH4 Dotted line, control cells (as in Panel A, after sialidas treatment). Panel D: NDV sialidase followed by mAb SNH3 control as in Panel C. Panel E: Vibrio cholerae (VC) sialidas followed by mAb SNH4. Panel F: VC sialidase followed by mA SNH3. Note that expression of SLex (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-Lex and anti-SLex mAb's alone and in combination (incubated 90 min at 37βC) . Panel C: effects of NDV sialidase plus mAb. Panel A: NDV sialidase (which cleaves α2-+3 sialosyl at a terminal Gal, eliminates the SLex structure and abolishes reactivity with mAb's SNH3 and SNH4, see Figure 13, but did not abolish adhesion. VC and Arthrobacter ureafaciens (AV) sialidases did abolish adhesion. Panel B: anti-SLex mAb's were less effective than anti-Lex mAb's. Combinations of both types of mAb's were most effective. Panel C: Adhesion was inhibited most effectively by NDV sialidase plus anti-Lex 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-SLexIgG3 mAb SNH4; solid circle, anti-Lex IgM mAb FH2; and hollow triangle, anti-Lex IgG, mAb SHI. Panel C: hollow circle, control; solid triangle, NDV sialidase solid circle, mAb SHI; and hollow triangle, NDV sialidase plu mAb SHI. Panel D: hollow circle, control; solid circle, mixtur (1:1) of mAb's SNH4 and FH2; and hollow triangle, mixture (1:1 of mAb's SNH4 and SHI. Cleavage of α2→3 sialosylation at terminal Gal by NDV sialidase reduced adhesion somewhat, howeve adhesion remained at low shear stress. In contrast, VC and A sialidases strongly inhibited adhesion (Panel 15A) indicating th importance of internal sialosylation (which is unaffected by ND sialidase) . That observation is substantiated by observation that (i) NDV sialidase plus mAb SHI strongly inhibited adhesio and (ii) combination of anti-SLex mAb SNH4 plus anti-Lex mAb' FH2 or SHI inhibited adhesion more strongly than SNH4 alon (Panels 15B and 15D) . Figure 16 depicts reactivity of Colo201 cells with variou mAb's, with or without sialidase treatment. Colo201 cells wer reactive strongly with anti-SLea I mAb's CA19-9 and NKH (Panel A) , anti-Le8 mAb CA3F4 (Panel B) and anti-SLe8 II mAb FH (Panel C) . Reactivity with CA19-9 was decreased by NDV sialidas (Panel D) and abolished by VC sialidase (Panel G) . Reactivit with CA3F4 was increased slightly by NDV and VC sialidase (Panels E and H) . Reactivity with FH7 was unchanged by ND sialidase (Panel F) and decreased slightly by VC sialidas (Panel I) . Figure 17 depicts adhesion of Colo201 cells t E-selectin-coated plates in a static system. Abscissa an ordinate as in Figure 14. Panel A: effects of variou sialidases (90 min. incubation, 37βC). Panel B: effects o 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 α2→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) . In 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-SLea I mAb CA19-9; hollow triangle, anti-SLea II mAb FH7; and solid triangle, anti-Lea 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 SHI on HL60 binding to ELAM-coated plates in a dynamic flow system under various shear strength conditions. The ordinat represents per cent cell binding relative to untreated contro cells. The antibodies were used at 15 μg/ml, NDV sialidase a 0.2 U/ml and VC sialidase at 0.1 U/ml. Each point represents th mean of three experiments. Number of untreated cells bound a shear stresses of 15.5, 7.75, 3.13, 1.56 and 0.78 dynes/cm2 wer 4.5, 27, 109.6 206.2 and 283.8 cells/mm2, respectively.
Figure 20 depicts various branched sugars. The hybri sugar, Lex/SLex, is depicted as structure 1. The glycolipid containing such a structure were isolated from colon carcinom or were prepared from G8 ganglioside presented in Structure originally found in human erythrocytes (Watanabe et al., J. Biol Chem., 254:8223, 1979) by enzyme catalyzed αl→3 fucosylation Structure 2 was obtained by αl→3 fucosylation of compound originally obtained from human placenta. Structure 2 however di not exhibit high affinity binding to E-selectin. Structures and 4 depict analogs with high affinity binding sites having L and sialyl-Gal31→3GalNac within the same molecule (Structure 3) or the hybrid molecule Lea/SLea, the positional isomer o structure 1.
Figure 21 depicts the relative adhesion of NS-1 cell expressing E-selectin on various "glyco-liposomes" coated on plastic surface. Panel 21A shows the result of such relativ adhesion in a dynamic flow setting under middle shear stres conditions (7.75 dynes/cm ) . The first seven bars indicat relative adhesion of NS-1 cells to SLex on each glycoliposome a indicated. Cpd I is structure 1 of Figure 20 and Cpd II i structure 2 of Figure 20. Bars 8-10 show a mixture of Lex wit different types of compounds as indicated. The value of relative adhesion is expressed in comparison with the adhesion of SLex-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/cm2) . The value is expressed in terms of the adhesion on SLex-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 SLex 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 SLex-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 μm. 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 SLex and various other glycolipids. The ordinate shows the number of cells adhered per field. The solid circle is SLex + SPG. The hollo circle is SLex + H2. The solid triangle is SLex + Lex. Th hollow triangle is SLex + Ley. Each point is the mean of fiv determinations.
Detailed Description of the Invention
As noted above, the instant invention in one aspect i directed to methods and compositions for the inhibition of tumo cell metastasis potential and invasiveness. Numerous tumor cell possess the ability to metastasize, i.e., to form a secondar tumor colony at a distant site. Sources of malignant tumor cell include melanoma, lung, breast, colorectal and urogenita cancers, such as bladder and prostate cancers. Within th instant invention, the metastasis potential of tumor cells (i.e., the ability of tumor cells to metastasize) may b inhibited through the use of (a) tumor-associated carbohydrat antigens (TACA's, as used herein TACA is meant to include LACA) (b) antibodies directed to those TACA's; (c) oligosaccharid components of those TACA's; (d) conjugates of such TACA's or o oligosaccharide components of such TACA's, such as multivalen conjugates of lysyllysine or TACA-bearing glycosphingolipid (GSL liposomes; or (e) mimetics of the TACA's. Generally, unles indicated to the contrary, tumor cells and leukocytes ar substantial equivalents inasmuch as both bind to endothelia cells by carbohydrate structures.
TACA epitopes play essential roles in tumor cell adhesio through interaction with endothelial cells, platelets an basement membranes, whereby tumor metastasis and invasion ma 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) .
Tumor cell adhesion mediated by sialosyl-Lex is inhibited by anti-sialosyl-Lex monoclonal antibodies (FH6, CSLEX, SNH3 and SNH4) and tumor cell adhesion mediated by monosialosyl-Le8 I is inhibited by monoclonal antibodies (CA19-9, CSLEA, NKH1 and KH2) directed to that epitope. Handa et al., Biochem. Biophys. Res. Commun. 181:1223, 1991; Kojima et al., Biochem. Biophys. Res. Commun. 182:1288, 1992; Hakomori, Histochem. J. , 24:771, 1992. The adhesion of Colo205 tumor cells, which express predominantly type 1 chain sialosyl-Le8 and to a lesser extent sialosyl-Lex, to endothelial cells is inhibited by anti-sialosyl-Le8 monoclonal antibody and to a lesser extent by anti-sialosyl-Lex monoclonal antibody. Those findings suggest that not only sialosyl-Lex, but also sialosyl-Lea, are the important ligands recognized by ELAM-1 and GMP-140 (previously termed CD62 or PADGEM and also known as E-selectin and P-selectin) . It is known now that adhesion of tumor cells to activate endothelial cells is based also on recognition o monosialosyl-Le8 II and disialosyl-Lea. Both monosialosyl-Le8 I and disialosyl-Le8 are defined by monoclonal antibody FH7, whic is known to inhibit strongly adhesion of various types o epithelial cancer cells (particularly colorectal gastrointestinal and pancreatic) to activated endothelial cell or platelets via selectins.
In particular, GMP-140 is the major selectin (LECCAM located on α-granules of platelets or Weibel-Pallade bodies o endothelial cells (EC's) . On activation of those cells, GMP-14 is redistributed rapidly to the cell surface, where it plays a important role in adhesion of platelets or EC's to certai carbohydrate epitopes expressed on blood cells or tumor cells resulting in aggregation of platelets or tumor cells, or adhesio thereof to capillary endothelia. GMP-140-mediated cell adhesio is believed by the instant inventors to be involved in initiatio of metastatic deposition of tumor cells and initiation o inflammatory processes. Also, ELAM-1 is expressed on endothelial cells afte activation with interleukin-1, TGF-3, TNF-α o lipopolysaccharide. ELAM-1-mediated cell adhesion also i believed to be involved in initiation of metastatic depositio of tumor cells. Thus, the instant invention in another aspect is directe to inhibiting GMP-140-mediated or ELAM-1-mediated cel aggregation or adhesion, especially at tvimor cell sites. Withi the instant invention, GMP-140-mediated or ELAM-1-mediated cel aggregation or adhesion can be inhibited through the use of (a) a hybrid sugar, such as Lex/SLex; (b) a mixture of sugars which are the components of a hybrid sugar (a) , such as Lex and SLex; (c) monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl-Le8 or sialosyl Le ; (d) antibodies that specifically bind to a hybrid sugar (a) , such as Lex/SLex, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Le ; (e) a mixture of antibodies, particularly to the components of a hybrid sugar, such as to SLex and Lex; (f) oligosaccharide components of a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex; (g) conjugates of a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Lea, Le, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex or of the oligosaccharide components; and (h) mimetics of a hybrid sugar, such as Lex/SLe , monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex.
Within the instant invention, tumor metastasis and invasion is inhibited by blocking tumor cell adhesion thereby significantly reducing or eliminating the spread of metastatic cells.
Also within the instant invention, 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 directly via ELAM-1.
Further within the instant invention, inflammation minimized by inhibiting GMP-140-mediated leukocyte aggregatio adhesion or migration at a potential site of inflammation due t (a) adhesion of leukocytes to platelets, (b) adhesion leukocytes to endothelial cells (EC) via platelets, (c) adhesi of leukocytes to EC's directly via selectin and ( transendothelial migration of leukocytes. TACA's suitable for use within the instant invention a those showing differential prognostic significance (i.e., TACA that may be correlated clearly with invasive or metastat potential) . Within the context of the instant invention, su TACA's may be distinguished through a comparison of invasivenes metastasis and clinical prognosis of similar tumors showi expression vs. non-expression of such TACA's. Preferred TACA for use within the present invention include H/Ley/L sialosyl-Lex (SA-Lex or SLex) , Le8, Lex, monosialosyl-Le8 I (S or SA-Lea) and sialosyl-Tn (SA-Tn or STn) . Derivatives of su TACA's include hybrid sugars, such as Lex/SLex, dimeric L sialosyl-dimeric Lex, trifuscosyl Ley, disialosyl-Le8 a monosialosyl-Le8 II.
As noted above, TACA's for use within the instant inventi exhibit a differential prognostic significance. By way example, such a differential prognostic significance may illustrated by the fact that tumors expressing H/Ley/Le antige (as defined by monoclonal antibody MIA-15-5) showed much wor patient prognosis than tumors not expressing those antigens. F instance, as shown in Figure 3A, patients with squamous cell lung carcinoma expressing H/Ley/Leb had only an 11% survival over a 5-year period (i.e., 89% died) whereas comparable patients not expressing H/Ley/Le had an approximately 62% survival over the same period.
Similar results were obtained for tumors showing expression vs. non-expression of sialosyl-Lex and sialosyl-Tn"antigens. More specifically, as shown in Figure 3B, patients with colonic cancer expressing sialosyl-Lex had only a 15% survival over a 5-year period, whereas comparable patients not expressing that antigen had an approximately 50% survival over that period. In a separate study, the 5-year survival of patients with early-stage colonic cancer not expressing sialosyl-Tn was 100%, as compared to 75% for patients who expressed sialosyl-Tn (se Figure 3C) . As shown in Figure 3D, similar but more obvious differences were observed in patients with ovarian cancer showin expression vs. non-expression of sialosyl-Tn antigen.
Also as noted above, antibodies or a mixture of antibodies to suitable TACA's may be employed within the context of th instant invention. As used herein, such antibodies include bot monoclonal and poiyclonal antibodies and maybe intact molecules, a fragment of such a molecule or a functional equivalent thereof. The antibody may be engineered genetically. Examples of antibod fragments include F(ab')2, Fab', Fab and Fv. Briefly, poiyclonal antibodies may be produced b immunization of an animal and subsequent collection of ser therefrom. Immunization is accomplished, for example, by systemic administration, such as by subcutaneous, intraspleni or intramuscular injection, into a rabbit, rat or mouse. It i preferred generally to follow the initial immunization with on or more booster immunizations prior to sera collection. Suc methodology is well known and described in a number o references.
While poiyclonal antibodies may be employed in the presen invention, monoclonal antibodies are preferred. Monoclona antibodies suitable for use within the instant invention includ those of murine or human origin, or chimeric antibodies such a those which combine portions of both human and murine antibodie (i.e., antigen binding region of murine antibody plus constan regions of human antibody) . Human and chimeric antibodies ma be produced using methods known by those skilled in the art Human antibodies and chimeric human-mouse antibodies ar advantageous because such antibodies are less likely than murin antibodies to cause the production of anti-antibodies whe administered clinically.
Monoclonal antibodies may be produced generally by th method of Kόhler and Milstein (Nature 256:495, 1975; Eur. J Immunol. 6:511, 1976), as well as by various techniques whic modify the initial method of Kόhler and Milstein (see Harlow an Lane (eds.), "Antibodies: A Laboratory Manual", Cold Sprin Harbor Laboratory, 1988, which is herein incorporated b reference in its entirety) . Briefly, the lymph nodes and/or spleen of an anima immunized with one of the TACA's or the oligosaccharid components thereof are fused with myeloma cells to form hybri cell lines ("hybridomas" or "clones"). Each hybridoma secrete a single type of im unoglobulin 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.
An alternative to the production of monoclonal antibodies via hybridomas is the creation of monoclonal antibodies expression libraries using bacteriophage and bacteria (e.g., Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728, 1989; Huse et al., Science 246:1275, 1289). Selection of antibodies exhibiting appropriate specificity may be performed in a variety of ways which will be evident to those skilled in the art.
Representative examples of monoclonal antibodies suitable for use within the present invention include MIA-15-5 (Miyake & Hakomori, Biochem. 20.:3328, 1991), as well as the monoclonal antibodies cited in Hakomori, Advances In Cancer Research 52:257-331, 1989.
As discussed above, oligosaccharide components of suitable TACA's also may be used in the instant invention. As used herein, the term "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 ,9-D-lactoside, lact-N-tetrose (Gal01→3GlcNAq9l→3Gal01→4Glc) and phenyl 3-D-thiolactoside. For example, both compounds were found to inhibit melanoma cell metastasis in the mouse lung. Other lactose derivatives also may be used, including ethyl or pheny lactoside and methyl or ethyl thiolactoside. It is believed tha such lactose derivatives block binding of melanoma cells to EC' by inhibiting melanoma cell GM3 ganglioside interaction wit lactosyl ceramide of the EC's.
Other oligosaccharide components suitable for inhibitin metastasis potential of cells of a particular tumor may b identified based on determining the structure of specifi carbohydrate chain(s) which are involved in the ability of th tumor to metastasize. The identification o carbohydrate-containing molecules involved in the ability of tumor to metastasize may be accomplished in a variety of ways including through the use of glycosidases and inhibitors o glycosy1transferases. The structure of carbohydrates bound to either lipids o proteins may be determined based on degradation, mas spectrometry, including electron-impact direct-probe (El) an fast atom bombardment (FAB) , and methylation analysis (technique described, for example, in Nudelman et al., J. Biol. Chem 261:5487, 1986). Degradation analysis may be accomplishe chemically and/or enzymatically, e.g., by glycosidases. Th carbohydrate sequence suggested by degradation analysis may b determined bymethylation analysis (Hakomori, J. Biochem. 55:205 1964) followed by chemical ionization mass spectrometry o permethylated sugars (Stellner et al.. Arch. Biochem. Biophys 155:464, 1974; Levery et al., Meth. Enzymol. 138:13, 1987).
Alternatively, or in conjunction with those techniques. E mass spectrometry may be performed on permethylated glycans after the appropriate degradation of intact glycans (Kannagi et al. , J. Biol. Chem. 259:8444, 1984; Nudel an 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.
As briefly discussed above, 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.
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. For example, 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.
It may be desirable to couple covalently components via a linker group. 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. For example, a carboxyl group may b activated. Activation of a carboxyl group includes formation o an "active ester", such as a succinimidyl ester. The ter "active ester" is known to refer to esters which are highl reactive in nucleophilic substitution reactions.
Alternatively, it may be desirable to produce conjugates i which the components are linked non-covalently. For example, on or more TACA's may be incorporated into the outer surface o glycosphingolipid (GSL) liposomes. It may be desirable to increase the in vivo half life of a oligosaccharide. As disclosed in the instant invention oligosaccharides may be coupled to (i.e., covalently bonded to a straight-chain amphophilic polymer, such as polyethyleneglycol A representative example of a method for producing a oligosaccharide-polyethyleneglycol conjugate is the reaction o an oligosaccharide, which has been derivatized to contain succinimidyl group, with a polyethyleneglycol having a termina amino group. The latter compound has a general formula o NH2-(CH2CH2-0)n-CH3, where n typically averages 44. (i.e., molecular weight of about 2,000) to 112.9 (i.e., molecula weight of about 5,000).
Additionally, because the cell adhesion mediated selectins, ELAM-1 or GMP-140, is based on recognition sialylated and fucosylated lactoseries type 1 and type 2 chai by a lectin sequence domain present at the N-terminal region the selectin molecules, any structure which may show mo effective blocking activity of the lectin domain than natural occurring epitopes are useful in the present invention. Su unnatural synthetic compounds, termed "mimetics", of, for example, sialosyl-Lex or sialosyl-Le8 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-Lex or monosialosyl-Le8 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-Lex, monosialosyl-Lea I and II, or the H/Ley/Leb structure having trifluoro-L-fucose, N-trifluoro-acetyl-glucosamine or sialosyl-Tn analogs containing N-trifluoro-acetyl-neuraminic acid. Thus, 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 o disease processes involving GMP-140 or ELAM-1. Regarding in vitro aspects, as noted above, the instan invention provides a method for inhibiting tumor cell metastasi potential within a biologic preparation. The method comprise incubating a biologic preparation with at least one agen selected from the group consisting of (a) tumor-associa carbohydrate antigens that exhibit differential prognos significance, (b) antibodies that specifically bind to th antigens, (c) oligosaccharide components of those antige (d) conjugates of those antigens or oligosaccharide compone and (e) mimetics of the tumor-associated carbohydrate antige the agent inhibiting the metastasis potential of the preparati Regarding further in vitro aspects, the instant invent also provides a method for inhibiting GMP-140-mediated ELAM-1-mediated cell aggregation or adhesion in a biolo preparation. The method comprises incubating the biolo preparation with at least one agent selected from the gr consisting of (a) a hybrid sugar, such as Lex/SLex; (b) su components of a hybrid sugar (a) , such as Lex and S (c) monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 disialosyl-Le8 or sialosyl Lex; (d) antibodies that specifica bind to a hybrid sugar, such as Lex/SLe or to the compon sugars thereof, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Lea disialosyl-Le8 or sialosyl Lex; (e) oligosaccharide compone of a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Lea, monosialosyl-Le8 II, disialosyl-Lea or sialosyl Lex; conjugates of a hybrid sugar, such as Lex/S monosialosyl-Le8 I, Le8, Lex, monosialosyl-Le8 II, disialosyl or sialosyl Lex or of the oligosaccharide components; (g) mimetics of a hybrid sugar, such as SLex/ monosialosyl-Lea I, Lea, Lex, monosialosyl-Lea II, disialosyl or sialosyl Lex, the agent inhibiting the cell aggregation adhesion. Suitable biologic preparations include cell cultures a cell suspensions in biologic fluids, such as blood, urine, lymp synovial and cerebrospinal fluid. TACA's, oligosaccharides conjugates thereof generally will be incubated at a fin concentration of about 0.1 to IM, and typically at about 0.2 0.5 M. Incubation is performed typically for 5 to 15 minutes 37°C. After treatment of a biologic preparation, the preparati may be injected or implanted in an animal, e.g., to confi effectiveness of the inhibition of metastasis potential. The instant invention also provides a method for inhibiti tumor cell metastasis potential in a warm-blooded animal, su as a human. The method comprises administering to a warm-blood animal an effective amount of at least one agent selected fr the group consisting of (a) tumor-associated carbohydra antigens that exhibit differential prognostic significanc
(b) antibodies that specifically bind to those antigen
(c) oligosaccharide components of those antigens, (d) conjugat of those antigens or the oligosaccharide components a (e) mimetics of monosialosyl-Lea I, Lea, Lex, monosialosyl-Le8 I disialosyl-Lea or sialosyl Lex, the agent inhibiting t metastasis potential of the preparation.
Similarly, the instant invention also provides a method f inhibiting GMP-140-mediated or ELAM-1-mediated cell aggregati or adhesion at a tumor cell site in a warm-blooded animal. T method comprises administering to a warm-blooded animal effective amount of at least one agent selected from the gro consisting of (a) a hybrid sugar, such as Lex/SLe (b) component sugars of a hybrid sugar (a) , such as Lex a SLex; (c) monosialosyl-Lea I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex; (d) antibodies that specificall bind to a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Le8, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex; (e) oligosaccharide components of a hybrid sugar, such a Le/SLex, monosialosyl-Le8 I, monosialosyl-Le8 II, Le , Lex, disialosyl-Lea or sialosyl Lex; (f) conjugates of a hybri sugar, such as Lex/SLex, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex or of th oligosaccharide components; and (g) mimetics of a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Le8, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex, said agen reducing the metastatic potential at the tumor site in th warm-blooded animal. 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 Lex/SLex; (b) component sugars of a hybrid sugar (a) , such as Lex an SLe ; (c) monosialosyl-Le8 I, Le8, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex; (d) antibodies that specificall bind to a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex (e) oligosaccharide components of a hybrid sugar, such a Lex/SLex, monosialosyl-Le8 I, Lea, Lex, monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex; (f) conjugates of a hybri sugar, such as SLex/Lex, monosialosyl-Le8 I, Le , Le , monosialosyl-Lea II, disialosyl-Lea or sialosyl Lex or of the oligosaccharide components; and (g) mimetics of a hybrid sugar, such as Lex/SLex, monosialosyl-Le8 I, Le , Le , monosialosyl-Le8 II, disialosyl-Le8 or sialosyl Lex, the agent reducing the inflammatory potential at the inflammatory site in the warm-blooded animal.
For both methods, 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. Moreover, 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.
When a combination of such an agent and a substance is desired, each compound may be administered sequentially, simultaneously or combined and administered as a single composition. Diagnostic techniques, such as CAT scans, may b performed prior to and subsequent to administration to confir the effectiveness of the inhibition of metastatic potential o inflammatory potential. One in vitro system for measuring adhesion or aggregatio of tumor cells to other cells (e.g. EC's), or for determinin successful inhibition of adhesion or aggregation is a dynami flow system similar to that described by M. B. Lawrence et al (Blood 70:1284, 1987) and which is shown in Figures 11A, 11B, 11 and 11D.
A parallel-plate laminar flow chamber (1) (shown upside dow for convenience) connected to a pressure pump (2) via tubing (18 is used to simulate the flow shear stresses present i physiological microvascular environments. The flow chambe consists of a plastic or glass cover slip (3) resting on chamber body (16) on which a parallel, transparent plasti surface (4) is attached with a rubber or silicone gasket (5) there is a 114 μm gap between the two surfaces, and this gap i 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 shea stress is achieved by manipulation of the pressure pump (2) which is connected to the inlet manifold (8) of the flow chambe via tubing (18) . Figure 11B depicts the configuration of a assembled flow chamber (1).
To fix together the cover slip (3) and the chamber body (16 very tightly, there is a continuous circular grooved space (20 on the periphery of the chamber body (16) . The circular, groove space connects to a vacuum pump by placement of the rubber or silicone rubber gasket (5) with cover slip (3) on top. Thus, by applying vacuum (21) in (20) the cover slip (3) and chamber body (16) are affixed strongly and immovable. The thin inlet and outlet slots in the chamber body (16) open to the inlet and outlet manifolds, respectively. The outlet manifold is connected to a pressure pump (2) which can be operated in either a negative or positive mode.
Cells (e.g., endothelial cells) are grown on either a glass or plastic cover slip (3) , or various adhesion molecules are affixed on (3) , and 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. Turning to Figure lie, 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) , maintaine in a vessel in a water bath (17), is passed through the chambe via tubing (18) . Cell movements are observed under an inverte phase-contrast microscope (10) and recorded by time-laps videocassette recorder (11) using a video camera (12) and digital image processor (13) . Adhesion is observed as rollin followed by stopping of cells. Number of cells bound during set time, e.g. 3 minutes, at different shear stresses, e.g., fro 0.4 to 4.8 dynes/cm , are counted from several fields recorded o videotape (Figure 11B) . Wall shear stress (T) is calculated a 3μQ/2ba2, where μ = coefficient of viscosity, e.g. 1.0 cP Q = volumetric flow rate (cm/sec) , a = half channel height, e.g 5.7 x 10" cm, and b = channel width, e.g. 1.3 cm.
Figure 11D schematically shows laminar flow of tumor cel suspension (14) through a chamber in which one surface is coate with endothelial cells (9) . Rolling or stopped cells (15) a observed under an inverted microscope and recorded on videotape as described above. The arrows indicate the direction of fl of the tumor cell suspension (14) .
As mentioned above, the instant invention also provides method for identifying a TACA epitope to which lectin activi of a selectin, such as GMP-140, is directed.
Previously, the TACA epitopes were studied based on t inhibitory effect of various glycosphingolipids (GSL's), G oligosaccharides or GSL-containing liposomes on adhesion of blo cells or tumor cells to a solid phase (e.g., a plastic surfac coated with activated platelets. In practice, that meant coati a solid phase with gelatin, which was in turn was coated wi activated platelets; platelets bind readily to a gelatin-coat solid phase via GpIIb/IIIa, the major platelet integrin recepto
In studies using that method, binding of promyelocyt leukemia HL60 cells to platelet-coated solid phase was inhibit by liposomes containing a sialosyl-Lex determinant, but not liposomes containing sialosylparagloboside (SPG) , and onlyweak by liposomes containing αl→3 fucosylated type 2 chain (Le (see Table 1 in Example 3 below) . Those results suggested that sialosyl-Lex is the carbohydrate epitope defined by GMP-140 (Polley et al. , Proc. Natl. Acad. Sci. USA 88: 6224, 1991).
However, the method described above had an important limitation: no cell line which expresses exclusively type 1 chain GSL is available. Myelogenous cell line HL60 and monocytic cell line U937 express exclusively type 2 chain and little, if any, type 1 chain. On the other hand, all known human tumor cell lines derived from colonic, gastric or lung carcinoma express both type l and type 2 chains. For those reasons, it is difficult to determine the real epitope to which GMP-140 binds. To address that problem, a new methodology was developed, as described below.
Fluorescent plastic (e.g. polystyrene) beads (diameter « 0.5 μ ) are coated with GSL. 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.
Using that method, activated platelets were found to show much stronger binding to fluorescent beads coated with monosialosyl-Le8 I (see Table 3) than to beads coated with any related GSL. The binding of platelets to sialosyl-Lea-coated beads was inhibited by anti-GMP-140 monoclonal antibody or anti-sialosyl-Le8 monoclonal antibody, but not by anti-sialosyl-Lex monoclonal antibody. Although binding of activated platelets to sialosyl-Lex-coated beads was observable. the level of binding was much lower than binding t sialosyl-Lea-coated beads. Those results indicate that th primary epitope structure defined by GMP-140 is sialosyl-Le rather than sialosyl-Lex. Of course, other epitope structures defined by a selectin such as GMP-140, can be identified using the instant inventiv method.
ELAM-1 (E-selectin) is expressed on the surface of activate endothelial cells. ELAM-1 has a carbohydrate-binding domain a the amino terminal region and indeed ELAM-1 is known to bind SL and SLe8.
Those conclusions were based on the observations tha adhesion between tumor cells or leukocytes to activated huma EC's was inhibited by SLex glycolipid or oligosaccharide, but no by other tested glycolipids or oligosaccharides available at tha time (Phillips et al.. Science 250: 1130, 1990). Later, th above-noted adhesion was found to be inhibited by SLe" I, th positional isomer of SLex glycolipid, as well (Berg et al., J Biol. Chem., 266:14869, 1991; Takada et al., Biochem. Biophys Res. Commun., 189:713, 1991).
That adhesion reaction was claimed to be inhibited by Ig anti-SLex mAb (Phillips et al., supra). However, there are othe structural variants related to SLex and SLe8 together with mAb' directed to those variants and the reported studies are based o inhibition of selectin-dependent adhesion by assumed epitop structure(s) .
The instant invention is a result of systematic studies o selectin-dependent adhesion under static and dynami circumstances. For example, 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 particulat solid supports coated with glycoliposomes with activate platelets or HUVECs 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.
Hence, the systems (i) , (ii) and (iii) were employed t assess the effect on adhesion of various mAb's directed to SLex, SLea I, SLe8 II, Lex, Lea and related structures; combinations o such mAb's; sialidases with various substrate specificities; o combinations of various sialidases and mAb's. The method of (iv) was used to compare the intensity of adhesion under dynami conditions.
Specifically, the instant inventionrelates to carbohydrate defined by formulae (I) , (II) and (III) below which ar characterized by internal sialosyl residues or a branche structure.
Formula (I) relates to a type 1 or extended type 1 chai with internal α2→6 sialosyl substitutions and an αl→4 fucosy substitution . NeuAcα2 R2 R2
4- 6 Gal/91→3GlcNAc01 [→3Gal01→3GlcNAcjBl→] n3Gal01→ (I)
4 t Fucαl In formula (I) , R. is H or a sialic acid residue in β2 linkage; R2 is H or a sialic acid residue in α2→6 linkage; n equal to or greater than 0; and R3 is H or a fucosyl residue α-l→4 linkage. Formula (II) relates to a type 2 chain structure wi internal sialosyl and fucosyl substitutions. NeuAcα2
4- J <y J\? -K->
6 | 2 | 2 f Gal01→4GlcNACj91 [→3Gal/-?l→4GlcNAc£l→3 n3Gal 3l→ (II)
I 3 I
Fucαl
In formula (II), R2 is as defined for formula I, R4 is H a fucosyl residue in αl→3 linkage and Rg is H, a sialic ac residue in α2→3 linkage, NeuAcα2→8NeuAc in α2→3 linkage
R6→NeuAc in α2→3 linkage, wherein R6 is one or more sugars oth than a sialic acid residue and n is equal to or greater than
Formula (III) relates to a type 2 chain structure which a hybrid molecule comprising a branch wherein each bran comprises an epitope of a single carbohydrate antigen disclosed herein. Hence, as used herein, a hybrid molecule do not necessarily comprise the entirety of the two component suga that comprise the hybrid. Instead, the hybrid comprises t epitopes of the component sugars. Hence, referring to Figure 2 structure 1 comprises the epitopes of Lex and SLex, however will be noted that with reference to the diagrammatic structur of the various sugars set forth hereinbelow, not all of the L or SLex molecules are found in the hybrid. As to the SL portion of the hybrid, only the terminal galactose a glucosamine, together with the attached fucosyl and sialic ac residues, of the intact SLex molecule comprise the hybrid. Similarly, for Lex, only the epitope generating terminal three sugar residues comprise the hybrid. As used herein, epitope is that portion of the sugar which interacts in the adhesion phenomenon.
R10l→6
Rg R, (III)
Rl1l→3
In formula III, each of R10 and R comprises galactose, Gal3l→4GlcNAc or Gal01-3GlcNAc; Rg comprises Gal or GalNAc; and R, comprises lactosyl ceramide or an O-linked sugar. Additionally, R10 and R 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 SLex/Le , or Lex/SLex.
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., Gal31-*3GlcNAc31→3Gal, repeats thereof and substitutions thereof. E-selectin-dependent adhesion of Colo201 cells was inhibited only minimally by mAb CA19-9 (directed to SLe8) and moderately inhibited by mAb FH7 (directed to disialosyl Lea and monosialosyl Lea II) . Colo201 adhesion was inhibited most strongly by mAb CA3F4 (directed to monosialosyl Lea II and Le8 or by a combination of CA19-9 plus CA3F4. Specific reactivities of FH7 with disialosyl Le8 and monosialosyl Le8 II, and of CA3F4 with monosialosyl Le8 II, were describe previously (Nudelman et al., J. Biol. Chem., 261: 5487, 1986).
Further evidence for the epitope structures was based on th following observations. Treatment of Colo201 cells wit Newcastle Disease Virus (NDV) sialidase, which cleave NeuAco2→3Gal (R, in Formula I) only slighted inhibite E-selectin-dependent adhesion, but treatment with Arthrobacte ureafaciens (AU or often denoted as AV in the Figures) or Vibri cholerae (VC) sialidases, both which cleave NeuAcα2-+6 linkage t GlcNAc or Gal (i.e., R2 in Formula I), completely inhibited suc adhesion. Thus, involvement of internally 2→6 sialosylate structures in the adhesion is clear. NDV sialidase i combination with mAb's CA19-9 or CA3F4 strongly inhibited th adhesion. The results described above were obtained in bot static and dynamic adhesion systems, described herein.
However, the binding dynamics of selectins is vibrant, a revealed in dynamic flow systems which simulate more closel physiologic conditions, that is, for example, leukocytes or tumo cells can be moving at considerable speed in large and unocclude small vessels and at a slower speed in occluded vessels and i tissue spaces. Under static conditions cell interactions may b mediated by interaction with a first set of molecules that shar a common characteristic, whereas under non-static conditions cell interactions may be mediated by interaction with a secon set of molecules that share a common characteristic, differen from that shared by the first set of molecules. Furthermore under non-static conditions, the binding requirements may var depending on the speed at which the cells are moving. For example, E-selectin (ELAM)-mediated adhesion of HL6 cells is dependent on different carbohydrate structures when th cells are reacted in a stationary or slow moving setting or ar reacted while the cells are in rapidly moving setting. Unde static or low shear conditions ELAM binds preferentially to α2→ sialylated and αl→3 fucosylated structures, such as SLex, whil under high shear conditions, ELAM preferentially binds to othe structures, such as Lex, Ley, H and to various hybrid structures such as Lex/SLex. Formula II is based on inhibition by various mAB's an sialidases and combinations thereof of E-selectin-dependen adhesion of HL60 tumor cells, which express only type 2 chain i.e. Galϊl→4GlcNAc?l→3Gal and repeats thereof, and substitution thereof. Treatment of HL60 cells with NDV sialidase, whic cleaves NeuAcα2→3Gal (R., in Formula II) , completely abolishe reactivity of the cells with anti-SLex mAb's, although the cell remained strongly adherent to E-selectin-coated plates and t activated EC's. Complete inhibition of adhesion to E-selecti or EC's required treatment with AU or VC sialidase, which cleave NeuAcα2→6 linked to GlcNAc or Gal (i.e., R2 in Formula II, i addition to NeuAcα2→6 as shown in Formula II) . The R5 group i susceptible to cleavage by AU and VC sialidase but not by ND sialidase.
Further evidence for Formula II was provided by observe effects of various mAb's on E-selectin-dependent HL60 cel adhesion. The adhesion was inhibited strongly by NDV sialidas in combination with anti-Lex mAb SHI, or by anti-SLex mAb SNH4 i combination with SHI. The relevance of compounds of structure III was deduc through the use of various mAb's and sialidases E-selectin-dependent adhesion of tumor cells which express type chain sugars but also with E-selectin-transfected NS-1 ce adhesion to glycoliposomes. For example, NDV sialidase treatme of HL-60 cells, which removes NeuAcα2→6Gal, completely abolish reactivity of cells with anti-SLex mAb although the cel remained adherent to E-selectin plates and activated endotheli cells. Adhesion was inhibited effectively with a combination mAb's directed to Lex and SLex.
In contrast to type 1 chain structures whose internal sialosylated structure is known (Nudelman et al., supra) type chain structures with internally sialic acid residues we hitherto unknown. Data presented in the instant applicati indicate the natural occurrence of such epitopes.
The structures bindable to ELAM-1 can be synthesized usi known techniques. Thus, for example, the carbohydrates can synthesized chemically using known and commercially availab reagents or can be synthesized using known and available enzym to effect the appropriate linkage. For example, known sialos transferases and fucosyl transferases can be used to derivati the basic carbohydrate backbone.
Alternatively, the carbohydrates bindable to ELAM-1 can isolated using ELAM-1 as an absorbent. For example, purifi ELAM-1, cells expressing ELAM-1 or membrane preparations of cel expressing ELAM-1 can be used. The ELAM-1 can be immobilized a solid phase, such as an inert bead matrix or the inside wa of a vessel, to enhance separation. Then suitable carbohydrate bindable to ELAM-l, such as extracts of HL60 or Colo201 cell obtained by known techniques, are exposed to the ELAM-l affinit matrix. Following a washing procedure to remove unwanted an non-specifically bound components, the ELAM-l together wit carbohydrates bindable thereto are collected. The carbohydrate bound to the ELAM-l are separated from the ELAM-l, for example by altering the salt concentration of the holding buffer, an collected. The various carbohydrate species can be discriminate using known procedures, such as chromatography.
Also, cells known to express predominantly type 1 chai structures or type 2 chain structures are grown and membran preparations are obtained therefrom using known techniques. Th glycolipid and glycoprotein fraction of the membrane prep i obtained using known techniques and exposed to an affinity colum wherein antibodies directed to carbohydrate epitopes, such a those described herein, are affixed to a matrix, such as agaros beads, to form an affinity matrix. In an affinity chromatograph procedure, the bound materials are eluted and separated furthe by known techniques, such as HLPC and TLC.
When using TLC, the separated molecules in the separatio medium can be exposed to ELAM-1-expressing cells that ar labelled to serve as a tag, for example, the cells can b labelled metabolically with a radioisotope. Th ELAM-1-expressing cells will bind to the respective sites of th separation medium where separated ELAM-l epitopes are found. Th TLC matrix can be autoradiographed to locate such sites of cel binding to identify ELAM-l epitope-bearing molecules. Th respective sites of the TLC matrix can be excised and t molecules extracted.
As noted hereinabove, the carbohydrates of formulae I a II can be derivatized to provide oligosaccharides with mo desirable therapeutic properties. Thus, portions of t structures comprising formula I or II can be substituted, f example, with sulfur-containing sugars or fluorine-containi sugars. The oligosaccharide derivatives can be prepared usi the methods disclosed hereinabove but substituting for t naturally occurring components the appropriate reagent comprisi an altered substituent, such as 6-trifluoro-fucosyl which incorporated into either of formula I or II as the fucos residues.
The carbohydrates bindable to ELAM-l can be used immunogens to obtain antibodies bindable to the carbohydrat bindable to ELAM-l. Either poiyclonal or monoclonal antibodi can be generated, using methods such as those describ hereinabove, and in the references cited herein, which a incorporated by reference. Monoclonal antibodies are preferre Because ELAM-l may serve to mediate intercellul interactions, interruption of binding between ELAM-l a carbohydrates bindable thereto will be beneficial. Thu carbohydrates bindable to ELAM-l, ELAM-l, antibody to ELAM-l antibody to carbohydrates bindable to ELAM-l, for example, c be used to interrupt binding between ELAM-l and carbohydrat bindable thereto. The carbohydrates bindable to ELAM-l, ELAM- antibody to ELAM-l or antibody to carbohydrates bindable ELAM-l are administered in therapeutically effective amounts a via routes that are determinable readily and routinely practicin settled methods of the pharmaceutic arts.
As noted in formulae (I) and (II) , the terminal sialic aci is not essential in a carbohydrate bindable to ELAM-l. K elements held in common are the terminal galactose, glucosamine α2→6sialic acid and fucose residues. Thus, antibodies capabl of binding to such a structure are effective in inhibiti ELAM-1-mediated interactions. Suitable antibodies are CA3FA a FH7. Compounds of formula (III) , for example, Lex/SLex, where relevant epitopes comprised the branched chain structure we identified clearly as comprising high affinity binding sites f ELAM-l under high shear stress conditions. However, su structures can show less binding ability than simple SLex ELAM-l at low shear stress conditions or under static conditions Using that hybrid it is noted that the terminal galactose αl linked fucose to GlcNAc at one branch and an α2→3 linked siali acid and αl→3 linked fucose at the other branch are critic sites on that hybrid structure. Hence, antibodies bindable Lex, such as, SH-1 and FH-2, and to SLex, such as FH-6, SNH-4 a SNH-3, are effective cooperatively in inhibiting ELAM-1-mediat adhesion at high shear stress conditions.
Many epitopes recognized by ELAM and GMP-140 are carried 0-linked sugar chains and selectin-dependent cell adhesion c be blocked by inhibitors of O-glycosylation (Kojima et al. Biochem. Biophys. Res. Commun. 182:1288, 1992). Hence, it oft is preferable to have compounds of formulae (I) , (II) and (II carried on 0-linked carbohydrate chains. A further means of interrupting ELAM-l mediated interactio is using a combination of carbohydrates or antibodies interfere with ELAM-l binding to relevant carbohydrates. T carbohydrates or antibodies are related to ELAM-l carbohydrates bindable thereto or in certain circumstances be carbohydrates or antibodies that are not specifically tho carbohydrates believed to bind ELAM-l. For example, combination of antibodies directed to SLex and Lex is effecti in inhibiting ELAM-l interaction. Suitable SLex antibodies a SNH3 and SNH4; and suitable Lex antibodies are SHI and FH2. T skilled artisan can determine other suitable combinati practicing the methods taught herein using reagents disclos herein, with particular attention drawn to the working exampl set forth hereinbelow. The following examples are offered by way of illustrat and not by way of limitation.
EXAMPLES
Example 1 SYNTHESIS OF LACTOSE DERIVATIVES
A. Methyl ø-D-lactoside
Heptaacetyllactosylimidate (Zimmermann et al. , J. Carbohy Chem. 7:435, 1988) was reacted with methanol in dichloromethane containing trimethy1 s i1 trifluoromethanesulfonate according to a standard proced (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 sodiu methoxide, gave methyl j8-D-lactoside in 68% yield from th imidate: m.p. 211-212°C (lit. 205βC, Smith & van Cleve, J. Am Chem. Soc. 77:3159, 1955); [α]D + 1.3° (c 6.9, H20) (lit. + lβ, 5.0, H20) , ibid.
B. Phenyl /9-D-thiolactoside
Lactose octaacetate (Hudson & Kunz, J. Am. Chem Soc 47:2052, 1926) was treated with thiophenol and SnCl (Nicolaou et al., J. Am. Chem Soc. 110:7910, 1988) i dichloromethane at 0βC to give phenyl heptaacety 3-D-thiolactoside in 80% yield. The product was deacetylate with NaOMe in MeOH and neutralized with Amberlyst* 15 Purification of the product on a BioGel* P-2 column using wate as an eluent, followed by lyophilization of the sugar-containin fraction, left phenyl ,9-D-thiolactoside as a white amorphou powder.
C. Lacto-N-tetrose
The oligosaccharide (Galj3l→3GlcNAci31→3Gal/31→4Glc) wa prepared from human milk by pretreatment with ethanol an recycling BioGel P-2 column chromatography with water as eluen followed by reversed-phase (C18) high pressure liqui chromatography with water (Dua & Bush, Anal. Biochem. 133:1 1983). The H-NMR spectrum superimposed that of the authent sample (BioCarb Chemicals, Lund, Sweden) .
D. The polyethyleneglycol derivative of ,9-D-lactoside The reaction scheme is as set forth below:
T Mα -(cu u»-o-J- c:-:3
2
\
2
The polyethyleneglycol derivative of 0-D-lactoside wa prepared from readily available 3-succinimidooxycarbonylpropy 0-(2, 3, 4, 6-tetra-0-acetyl-0- -D-galactopyranσsyl)-(l→4) 2,3,6-tri-0-acetyl-/9-D-glucopyranoside 1 and polyethyleneglyco methyl ether (average M.W. 2000; Aldrich Chemical, Milwaukee, WI) having a terminal amino group 2. (Zalipsky et al., Eur. Poly . J 19_:1177, 1983). Treatment of 1 (100 mg, 0.12 mmol) an 2. (163 mg, 0.082 mmol) in dry N.N-dimethylformamide (2 ml) a room temperature for 2 hours gave, after chromatography on LH-2 with acetone as an eluent, the 9-D-lactoside heptaacetate 3. i 91% yield: [α]D-5.3° (c 0.5, chloroform). A subsequen saponi ication of 3. with 0.05 M sodium hydroxide at roo temperature for one hour, followed by lyophilization, afforde the desired lactoside 4. quantitatively: [α]D-2.4β(c 1.0 chloroform) .
Example 2
EFFECT OF LACTOSE AND LACTOSE DERIVATIVES ON METASTATIC POTENTIAL OF B16 MELANOMA CELLS
The highly metastatic BL6 clone of the B16 melanoma cel line was obtained originally from Dr. Jean Starkey (Montana Stat
Univ. , Bozeman, MT) and clones were reselected in syngeneic C57B mice according to metastatic potential. C57B1 mice wer maintained in plastic cages under filtered air atmosphere an provided with water and food pellets ad lib. Cells were culture in RPMI 1640 supplemented with 2 mM glutamine and 10% fetal cal serum (FCS) , and detached with phosphate buffered saline (P containing 2 mM EDTA. Viability was inferred by a trypan bl exclusion test.
A suspension of BL6 cells (1-3 x 106 cells/ml RPMI 1 medium) was prepared and aliquots were incubated in the prese or absence of various oligosaccharides at various concentratio at 37βC for 5-10 minutes. Following incubation, typical 3 x 104 or 2 x 104 cells (with or without oligosacchar pretreatment) per 200 μl were injected via a tail vein i 8-week-old female mice. After 18-21 days, the mice were kill the lungs were fixed in 10% formaldehyde in PBS (pH 7.4) tumor cell colonies were counted under a dissecting microsco thereby providing background values of metastatic melanoma col number in lung under those conditions. Data on the number the size of colonies were treated statistically by an analy of variance (ANOVA) procedure. Colonies with a diameter of 1 or greater were considered large-size and those with a diame less than 1 mm were considered small-size.
For one experiment, BL6 cells were incubated with vari concentrations of lactose, lacto-N-tetro (Gal01→3GlcNAcj81→3Gal/91→4GLc) , methyl ,3-D-lactoside or phe 0-D-thiolactoside for various durations. In the majority experiments, a concentration of 0.1 M was used and cells w incubated at 37βC for 10 minutes, separated from sugar-containing medium by mild centrifugation at 400 x g 10 minutes, resuspended in RPMI 1640 and injected (3 x 104 ce in 0.2 ml suspension) via a tail vein. For some experimen 2 x 10 cells were injected and colonies were counted at 21 da Viability and cell growth ability of BL6 cells after incubatio in various sugar solutions were tested by trypan blue exclusio test, by plating in RPMI 1640 culture under normal condition in vitro as well as by subcutaneous inoculation in age-matche C57B1 mice to test tumor growth.
Lactose and lacto-N-tetrose showed 26% and 36% reductions respectively, of metastatic colonies in lung when BL6 cells wer preincubated with those sugars followed by intravenous injectio of cells under identical conditions. Treatment of BL6 cells wit 0.1 M, 0.01 M or 0.005 M methyl /3-D-lactoside under the sam 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 3-D-lactosid was reproduced in three separate experiments and the reductio was found to be consistently between 35% and 45%.
In a second, independent series of experiments, treatmen with methyl ,3-D-lactoside or phenyl 0-D-thiolactoside unde different conditions also produced a significant reduction o metastatic colonization, i.e., total colony number was reduce to 35% or 50% of control values following preincubation wit methyl ?-I-lactoside or phenyl 3-D-thiolactoside, respectively Reduction of larger-size colonies was more apparent than that o smaller colonies in all experiments, particularly those wit phenyl ,9-D-thiolactoside (Figure 1) . Methyl /3-D-lactoside an phenyl S-D-thiolactoside both showed a slight in vitr stimulatory effect on cell number increase and on thymidin incorporation. Thus, the inhibitory effect on tumor depositio is not related to the effect on cell growth in vitro or in vivo In a separate experiment, the effect of methyl ,9-D-lactosi on melanoma cell metastasis was determined after administrati of the oligosaccharide, followed by inoculation with tumor cell Specifically, a one ml dose of methyl ,9-D-lactoside (at concentration of 0.25 M or 0.5 M) was injected intraperitoneal in mice. After 10 minutes, B16 melanoma cells were injec intravenously. Lung colonies were counted 19 days lat Injection of methyl ,9-D-lactoside in advance of inoculation wi tumor cells resulted in a significant reduction of l metastatic colony formation (Figure 2) .
In a separate experiment, mouse melanoma B16 varia showing different degrees of metastatic potent (BL6/F10/Fl/Wa4) showed the same order of expression of ganglioside, which was previously identified as melanoma-associated antigen (Hirabayashi et al., J. Biol. Ch 260:13328, 1985; Nores et al., J. Immunol. 139:3171, 1987). interacts with LacCer, which is highly expressed on endothel cells. The order of adhesion of the B16 variants o LacCer-coated solid phase or onto endothelial cells was also the same order as metastatic potential (MP) . In contra integrin-dependent adhesion of the B16 variants was approximat equal for BL6, F10 and Fl (see Figure 4). Those observati suggest that B16 adhesion of LacCer is based on molecu GM3-LacCer interaction. It also has been demonstrated that melanoma adhesion on endothelial cells is inhibited not only methyl-jø-lactoside but also by LacCer liposome, Gg3Cer liposo and GM3 liposome (see Figure 5) . In addition, the observations on the metastasis-inhibitor effect of methyl-/9-lactoside noted above have been extended t separate methyl-S-lactoside injection, i.e., tumor cells wer injected intravenously, followed by intraperitoneal injection o methyl-3-lactoside. In those experiments, injection o 0.25-0.5 M methyl-0-lactoside reduced lung metastatic colon number by 40%-70% (see Figure 6; A = PBS control, B = 0.25 Me-jS-lactoside; C = 0.5 M Me-^-1actoside; D = 0.5 M lactose E = 0.25 M N-acetyllactosamine; F = 0.5 M Me-0-galactoside intraperitoneal injection) .
Capillary endothelial cells are strongly reactive wit antibodies directed to H/Lev'/Leb, such as antibody MIA-15-5
That observation comports with the earlier observations that Ule
Europ. I stains endothelial cells, Holthofer et al., Lab. Invest 45:391, 1981; 47:60, 1982.
Liposomes comprising H-l or Ley were made and exposed t plates to which various glycolipids had been affixed at a rang of concentrations.
As noted in Figure 7, H-bearing liposomes bound to H or L coated onto plates. On the other hand Ley-bearing liposomes wer found to bind only to H-coated plates. H and paragloboside ar related, the only difference being the presence of a termina fucose residue in H.
Hence, cells expressing H, Ley or Le can adhere t endothelial cells expressing H and possibly to Ley as well
Those types of interactions may be the first step in tumor cel to endothelial cell adhesion. Example 3
EXPRESSION OF SIALOSYL-DIMERIC LEX ON HUMAN LUNG ADENOCARCINOMA CELL LINES AND METASTATIC POTENTIAL
KUM-LK-2 is a human non-adenocarcinoma cell li characterized by producing spontaneous lung metastasis in nu mice. After screening 35 human carcinoma cell lines grown nude mice, only that cell line produced metastatic deposits nude mouse lung. KUM-LK-2 was used as the parent cell line obtain, by limiting dilution technique, sub-cell lines produci lung metastasis on IV injection.
The procedure for the limiting dilution technique was follows. KUM-LK-2 was cultured in RPMI 1640 medium (GIBCO, Gra Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) 37'C in a 5% C02/95% air atmosphere. Cells were treated brief with 2 mM EDTA solution and washed twice with RPMI 1640 to ma a single cell suspension in RPMI with 10% FCS. Cell viabil was > 98% as determined by trypan blue exclusion staining. cell suspension containing 1 cell per 100 μl was transferred each well of a 96-well microtiter plate (Corning Glass Wor Corning, NY) and cultured continuously for 24 hours. Each w then was examined by phase contrast microscopy.
Three cell lines (HAL-8, HAL-24 and HAL-33) with differ metastatic potential ("MP") were selected out of 25 clo obtained by limiting dilution technique on the basis of sta cell morphology. The 25 clones were selected originally from clones showing stable morphology as well as consistent in vitr cell growth.
All of the clones produced spontaneous lung metastasis
However, on I.V. injection, clear differences were observed amon the clones in terms of lung metastatic deposit formation. Tw clones with high MP, five with low MP and 18 with no MP wer distinguished.
Through repeated selection by I.V. injection of the clones the most stable sub-cell lines showing consistent MP wer established. Those were HAL-8, HAL-33 and HAL-24, showing high low and no MP, respectively, to nu/nu mouse lung (see Table below) . Judging by macroscopic and microscopic examination, non of the three sub-cell lines showed metastasis in other organs o lymph nodes. The sub-cell lines represent stable variant originally present in KUM-LK-2. Based on chromosome analysis the subclones are independent.
Table 1 Metastatic potential of HAL-8, HAL-24 and HAL-33 in nude mice
Clone # generations #lung nodules on day 56
15.8 (8-23) 15.0 (10-22) 16.3 (11-25)
0 0 0
4.3 (3-7) 5.1 (2-8) 5.8 (3-8)
a c
Nude mice were injected (2 x 10 cells) via the tail vein at various generation times as indicat
Fifty-six days after injection, mice were killed and metastatic nodules on lung surface were counted u dissecting microscope.
T-ean of 6 animals (range in parentheses)
The cell surface expression of various carbohydrate epitop was analyzed by cytofluorometry using various monoclon antibodies (mAb's) directed to Lex (mAb SHI), sialosyl- (mAb SNH4) , sialosyl-dimeric Lex (mAb FH6) , T (mAb HH8 Tn (mAb 1E3) and sialosyl-Tn (mAb TKH2) . All antibodies us were culture supernatants from the respective hybridoma adjusted as 10 μg/ml of immunoglobulin. The structures sialosyl-Lex (structure 1), sialosyl-dimeric-Lex (structure 2 dimeric-Lex (structure 3), trifucosyl-Ley (structure 4
Leb (structure 5) , H (structure 6) , SA-Lea I (structure 7 SA-Tn (structure 8), disialosyl-Le" (structure 9) monosialosyl-Le8 II (structure 10), GM3 (structure 11) S-PG (structure 12) , Lex (structure 13) and Lea (structure 14 are shown below. R represents a carrier molecule.
Structure 1:
NeuAcα2→3Gal01→4GlcNAc01→3 [Gal01→4GlcNAc£l→3 ]nGal/31→4GLC01→R
3 t (n>0)
Fucαl
Structure 2 :
NeuAcα2→3Gal 3 l→4GlcNAc£ 1→3 Gal0 l→4GlcNAc£ 1→3 Gal01→4GLc£ l→R
3 3 t t
Fucαl Fucαl
Structure 3:
Gal£l→4GlcNAc01→3Gal01→4GlcNAc01→3Gal01→4Glc£l→R 3 3 t t
Fucαl Fucαl
Structure 4:
Gal/3l→4GlcNAc01→3 Gtnlβ1→4GlcNAc/31→3Galøl→4Glc£l→R
2 3 3 t t t (The αl→2 fucose can be
Fucαl Fucαl Fucαl replaced by H)
Structure 5 :
Fucαl→2Gal0 l→3GlcNAc£ 1→3 GaljS l→R
4 t
Fucαl Structure 6 :
Fucαl→2Galj91→3GlcNAC)01→3GaljBl→R
Structure 7 :
NeuAcα2→3GaljSl→3GlcNAc/91→3Gal/81→R
4 t
Fucαl
Structure 8 :
NeuAcα2→6GalNAcαl→0-Ser/Thr
Structure 9 :
NeuAcα2
6 NeuAcα2→3Gal/31→3GlcNAc/31→3Gal/31→R
4 t
Fucαl
Structure 10:
NeuAcα2 i
6
Gal 3 l→3GlcNAc/31→3 Gal 3 l→R
4 t
Fucαl
Structure 11:
NeuAcα2→3Gal/31→4GlcjSl→Cer
Structure 12:
NeuAcα2→3Gal31→4GlcNAc01→3GaljSl→R Structure 13 :
Gal/3 l→4GlcNAc/91→3 Gal/3 l→R 3 t Fucαl
Structure 14 :
Gal/31→3GlcNAc/31-3Gal/Sl→R 4 t Fucαl
Cells were detached from culture flasks with 0.25% trypsi 2mM EDTA solution and 1 x 10 cells were prepared for each m treatment. Cells were incubated with a mAb for 1 hour at 4βC a washed 2 times with RPMI 1640. Goat anti-mouse IgG or IgM-FI (Boehringer-Mannheim, Indianapolis, IN) , diluted 50 times wi PBS, then was added and incubated 30 minutes at 4βC. Finall cells were washed 3 times, resuspended with PBS and analyzed an EPICS PROFILE flow cytometer (Epics, Hialeah, FL) . T experiments were repeated with three different cell generation Patterns of expression of six carbohydrate epitopes (defin by the respective mAb's) on sub-cell lines HAL-8, HAL-24 a HAL-33 showed nearly identical profiles (as did the prote profiles for the three sub-cell lines) except in the case sialosyl-dimeric-Lex. In particular, HAL-8, HAL-24 and HAL- were found to express highly and equally sialosyl-Lex a sialosyl-Tn structures. Each of the three lines expressed l quantities of Lex and Tn, and did not express T. In contras expression of sialosyl-dimeric Lex was high on HAL-8, moderate HAL-33 and low on HAL-24.
The release of sialosyl residues was assessed in t following manner. Cells were detached using 2 mM EDTA in P washed and resuspended in 9 volumes of PBS. One ml of c suspension was incubated 5 minutes at 37βC with 0.2 U/ml Clostridiu perfringens sialidase (type X, Sigma Chemical C St. Louis, MO) . After incubation, cells were washed three tim resuspended with RPMI 1640 and investigated for MP and express of sialosyl-dimeric-Lex. MP of HAL-8 and HAL-33 was inhibi completely by sialidase treatment of cells (see Table 2 belo Expression of sialosyl-dimeric-Lex appears to play an import role in blood-borne metastasis.
Table 2
Effect of sialidase treatment on metastatic potential of clones HAL-8 and -33.a
Treatment Clone # lung nodules on day 56
Control (PBS) HAL-8 16.3 (9-24) HAL-33 4 . 6 (3-7) Sialidase HAL-8 0 HAL-33 0
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.
Mean of 6 animals (range in parentheses). EXAMPLE 4
IDENTIFICATION OF CARBOHYDRATE EPITOPES CAPABLE OF BINDING TO THE LECTIN DOMAIN OF GMP-140
Platelets were isolated from "platelet-rich plasma" obtain from the Oregon Red Cross (Portland, OR) . Contaminating r blood cells were removed by centrifugation at 80 x g for 10 mi Platelets were centrifuged at 300 x g for 10 min and suspend in Tyrode's buffer (pH 6.5) containing 22 mM citrate buffer wi 0.35% bovine serum albumin (BSA). The platelet suspensi (1 x 10 /ml) was incubated (pH 7.2, 37βC, 5 min) after additi of thrombin (final concentration 1 U/ml) . The mixture then w incubated at 37βC for 10 min without stirring. T thrombin-activated platelets were fixed with an equal volume 2% formaldehyde in phosphate-buffered saline (PBS), pH 7.2, a washed 2 x with PBS containing 1% BSA. Activated platelets (b not non-activated platelets) showed strong reactivity wi 2.5 μg/ml anti-GMP-140 mAb AC1.2 (isotype IgG Beckton-Dickinson, San Jose, CA) when incubated at 37"C f 30 min. , followed by reaction with 50 μl of fluorescence-label goat anti-mouse Ig (Tago, Burlingame, CA) . Flow cytometr profiles of activated vs. non-activated platelets with mab AC1 are shown in Figures 8A-8D.
Activated and non-activated platelets were fixed wi paraformaldehyde in Ca2+-free PBS, pH 7.2, washed 2 wi Ca^-containing PBS with 1% BSA, resuspended in CA*-PBS wi 1% BSA and 0.1% azide and the number of platelets adjusted «lxl0/ml. The cell suspension was stored at 4'C and the bindi assay performed within 24 hr.
Fluorescent polystyrene latex beads were obtained fr Molecular Probe, Inc., Eugene, OR. The beads were yellow-gre fluorescent beads with a sulfate group at the surface, diamet « 0.5 μm (actually 0.486 μm) . Beads (1 x 109) in 30 μl ETOH we added to 10 μg of GSL solution in 200 μl C=M, mixed well a dried under an N2 stream. The residue was resuspended in 200 ethanol, sonicated briefly and dried under a N2 stream. T dried residue was suspended in 2 ml Ca *-PBS with 3% BSA and 0. azide, sonicated for 10 min and allowed to stand at 37°C f 60 min to block the bead surface with BSA. The suspension w centrifuged at 3000 x g for 10 min, the bead pellet was wash 2 x with Ca2+-PBS containing 1% BSA and azide and final suspended in 500 μl of the same medium and stored at 4*C.
Twenty μl of platelet (non-activated or activate suspension, paraformaldehyde-fixed and containing «2 x platelets, was mixed with 10 μl of fluorescent GSL-coated bead containing «2 x 10 beads, mixed well and allowed to stand 37*C for 30 min. The platelet suspension was mixed with 200
Ca 2+-PBS and analyzed by flow cytometry (EPICS Profile, Coult Cytometry, Hialeah, FL) .
Flow cytometric analyses of platelets alone and beads alo were performed for setting a gating to include most of t signals produced by platelets and excluding signals produced free beads. The binding index (BI) was calculated as me fluorescence intensity (MFI) of platelets incubated wi fluorescent GSL-coated beads divided by MFI of platele incubated with fluorescent non-GSL-coated (control) beads. B values for various GSL's are shown in Table 3 and in Figure 9. In Figure 9, the hatched bars represent non-activated platelet and the open bars represent activated platelets. The ratio o the binding index (BI) of activated/non-activated platelets fo
SA-Lex, SA-Lea, SPG, GM3 and Lex also is shown in the "Ratio A/NA column.
Table 3
Binding index of thrombin-activated platelets to GSL-coated, sulfate-containing polystyrene beads
GSL Activated platelets Ratio (Activated/
Non-activated)
0.7 ± 0.5
4.2 ± 0.5 6.1 ± 0.2 0.7 ± 0.2 1.0 ± 0.3 a Values represent means of four separate experiments.
mAb's affected platelet binding to fluorescent GSL-coate beads. Platelets were incubated with anti-GMP-140 mAb IOP6 (Immunotech, Marseille, France) at 37°C for 30 min and a bindin assay was performed using GSL-coated beads, as describe hereinabove. Non-specific mouse IgG (10 μg/ml) was used in control binding assay. Also, 10 μl of SA-Lea-coated beads (2 x 107) were incubat with 20 μl of anti-SA-Le8 mAb CA19-9 (20 μg/ml) (mouse Ig
Signet Laboratories, Dedham, MA) at room temperature for 60 m and used for the platelet binding assay. Anti-SA-Lex mAb SN and non-specific mouse IgG were used as controls.
The results are shown in Figure 10, where the abscis represents percent inhibition and column 1 represen anti-GMP-140 mAb IOP62, column 2 represents anti-SA-Le8 m CA19-9 (alternative mAb's are NKH1 and NKH2) , column 3 represen anti-SA-Lex mAb SNH4 and column 4 represents normal mouse IgG Activated platelets showed high expression of GMP-140 evidenced by high reactivity with anti-CD62 mAb (Figures 8A-8D Activated platelets expressing GMP-140 showed strong binding wi fluorescent beads coated with SA-Lex (Figure 9) . Binding of platelets to beads coated with SA-Lex w observed but to a much lower degree than with SA-Lea (Figure 9 No binding was observed to beads coated with other GSL' Further, the binding of platelets to SA-Lea coated beads w inhibited by anti-GMP-140 mAb and anti-SA-Le8 mAb, but not anti-SA-Lex mAb (Figure 10) .
EXAMPLE 5
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
Adhesion was measured using the dynamic flow experimenta system shown in Figures 11A to 11D. The number of cells boun during 3 minutes at different shear stresses, for example, fro 0.4 to 4.8 dynes/cm , were counted from several fields recorde on videotape. The coefficient of viscosity was 1.0 P, the hal channel height was 5.7 X 10" cm and the channel width wa 1.3 cm.
Using that system, various human tumors and monoclona antibodies directed to various tumor-associated carbohydrat antigens were studied. The results of one study, adhesion o human colon carcinoma Colo205 cells to activated huma endothelial cells, is shown in Figure 12, where the absciss represents wall shear stress (dynes/cm ) and the ordinat represents cell adhesion (x 10* /field) . In Figure 12, the symbols are as follows: open circles mixture of irrelevant mouse IgG plus IgM (control) ; soli triangles, monoclonal antibody CA19-9 directed t monosialosyl-Le8 I; open triangles, monoclonal antibody SNH directed to sialosyl-Lex; solid circles, monoclonal antibody FH directed to monosialosyl-Le8 II and disialosyl-Le8; and soli squares, mixture of irrelevant mouse IgG plus IgM non-activated endothelial cells.
The results show that adhesion of Colo205 cells to activa endothelial cells was inhibited most strongly by antibody F particularly at high wall shear stress (5-10 dynes/cm 2) . contrast, antibody CA19-9 had no inhibitory effect. The findi suggest that tumor cell adhesion to endothelial cells may proc via interaction between monosialosyl-Le8 II or disialosyl-Le8 interleukin-1-activated selectin.
EXAMPLE 6
SELECTIN-DEPENDENT ADHESION OF HL60 CELLS
HUVECs (Cell Systems, Kirkland, WA) were cultured confluency in 48-well plates (Costar, Cambridge, MA) stimulated with 1 U/ml IL-1 for 4 hr. Non-simulated HUVEC's w used as a control. Expression of E-selectin (ELAM-l) IL-1-stimulated HUVEC's was confirmed by reactivity w anti-E-selectin mAb 3B7 (IgG2a) (Graber et al. J. Imm. 145:8 1990) . HL60 and Colo201 cells were labeled metabolically culture in the presence of [ H]-thymidine after pretreatment w glycosylation modifier and added to HUVEC-coated plates. Af 15 min incubation, plates were washed with PBS and adherent c number estimated by conversion from radioactivity count. another set of experiments, 96-well plates (Falcon, Lincoln, were coated with 0.1-1 μg/ml of a truncated, recombin E-selectin lacking transmembrane and cytoplasmic domains (Shim et al. Nature 349:799, 1991) for 18 hr. Plates then were coate with 1% BSA, washed with PBS and coated wit metabolically-labeled, glycosylation-modifiedcells, asdescribe above. After 60 min incubation, plates were washed with PBS an adherent cell number estimated by conversion from radioactivit count.
Assays of cell adhesion to activated or native platelet coated and fixed on 48-well plates were performed as previousl described (Handa et al.. Biochemistry 30:11682, 1991). HL6 cells were pretreated with 2mM benzyl-α-GaiNAc for 72 hr an labeled with [ H]thymidine. After washing with PBS, 1x10 cell were added to each well and plates were incubated for 30 min a room temp. After washing to remove unbound cells, bound cell were detached with trypsin and counted by liquid scintillatio counter. Platelets bound on plates were incubated wit anti-P-selectin mAb IOP-62 (1:2, 1:6 dilution) (Immunotech Marseille, France) at room temp for 30 min, followed by additio of HL60 cells, to evaluate dependence of adhesion on P-selecti expression. Non-specific mouse IgG was used as control.
Adhesion assay in a dynamic flow system
A parallel-plate laminar flow chamber connected to a infusion pump (Model 935, Harvard Apparatus, Cambridge, MA) wa used to simulate the flow shear stresses present in physiologica microvascular environments. The flow chamber consists of a glas plate on which a parallel, transparent plastic surface i attached with a Silastic rubber gasket; there is a 114 μm ga between the two surfaces and the gap is connected to an inlet outlet.
A laminar flow with defined rate and wall shear stress achieved by manipulation of the infusion pump, which is connec to the inlet of the flow chamber. EC's are grown as a monolay or adhesion molecules are coated, on the glass plate, and laminar flow of a cell suspension is passed through the chamb Cell movements are observed under inverted phase-contr microscope (Diaphot-TMD Nikon) and recorded by time-la videocassette recorder. Adhesion is observed as rolling follo by stopping of cells. Number of cells bound during 3 min different shear stresses from, for example, 0.4 to 4.8 dynes/ or 0.76 to 15.5 dynes/cm were counted from several fie recorded on videotape. Wall shear stress (T) was calculated the equation of Lawrence et al. (Blood 75:227, 1990):
T - 3μQ/2ba2
where μ= coefficient of viscosity (1.0 cP) , Q= volumetric f rate (cm/sec) , a= half channel height (for the experime reported herein, 5.7 x 103 cm) and b= channel width (1.3 cm).
HL60 adhesion to E-selectin-coated plates under static conditi
Promyelocytic leukemia cell line HL60 has been shown express only type 2 chain and sialosylated/fucosyla derivatives as probed by specific mAb's (Symington et a J. Immunol. 134:2498, 1985) and has been extensively used a model of leukocyte adhesion mediated by E-selectin and P-selecti
(Phillips et al. Science 250:1130, 1990; Polley et al. Proc
Natl. Acad. Sci. USA 88:6224, 1991; Handa et al. Bioche
Biophys. Res. Commun. 181:1223, 1991; Kojima et al. Bioche Biophys. Res. Commun. 182:1288, 1992).
When HL60 cells were treated with Newcastle Disease Viru (NDV) or Vibrio cholerae (VC) sialidase, reactivity of cells wit mAb's SNH3 and SNH4 was abolished (Figure 13) E-selectin-dependent HL60 adhesion was reduced by only abou 20-50% after treatment with NDV sialidase, whereas adhesion o the same cells treated with Vibrio or Arthrobacter ureafacie (AU) sialidase was reduced to about 5-10% of control values o essentially abolished. (NDV, VC and AU sialidase were effecti equally in eliminating SLex expression on HL60 cells.) NDV sialidase eliminates only the α2→3 sialosyl resid linked to the terminal Gal whereas both Vibrio and Arthroact sialidase completely eliminate terminal and internal sialic aci residues, notably, α2→6 linked sialic acid residues. T indings indicate that SLex and SLe8 are not the sole epitopes o E-selectin and P-selectin.
Effects of various mAb's on E-selectin-dependent HL6 adhesion were tested. Anti-SLe mAb's SNH3 and SNH4 produc strong HL60 cell aggregation, even under carefully-controll conditions. Therefore, the degree of inhibition of HL60 adhesi by SNH3 or SNH4 varied considerably since aggregated cells te to detach from E-selectin-coated plates.
In general, the degree of inhibition by those mAb's w minimal compared to the degree of inhibition previouslydescrib by Phillips et al. (supra). Anti-Lex mAb's SHI (IgG3) (Singh et al. Cancer Res. 50:1375, 1990) and FH2 (IgM) (Fukushi et a
J. Biol. Chem. 259:4681, 1984) produced a consistently high degree of inhibition than mAb's SNH3 and SNH4. A mixture of SNH3 or SNH4 with SHI or with FH2 produc stronger inhibition than any of the mAb's alone. Stronge inhibition was produced with a mixture of SNH4 and S
(Figure 14) .
If SLex is the sole epitope of HL60 cells for E-selectin a P-selectin, anti-SLex mAb's (e.g., SNH3 and SNH4) should inhib completely selectin-dependent adhesion. Treatment with N sialidase, which abolished reactivity of HL60 cells with SNH3 a
SNH4, also should inhibit E-selectin-dependent cell adhesio
However, treatment of HL60 cells with NDV sialidase followed SNH3 or SNH4 did not further reduce adhesion. Treatment with N sialidase followed by anti-Le mAb's SHI or FH2 strong inhibited E-selectin-dependent HL60 adhesion.
HL60 adhesion to activated HUVEC's in a static system
The same trends observed for HL60 adhesion E-selectin-coated plates were observed for HL60 adhesion activated HUVEC's grown in plates. Adhesion to HUVEC's w affected minimally by anti-SLex mAb's SNH2 or SNH4, in contra to the previous report by Phillips et al. (supra). Differe preparations of the mAb's varied widely in the effect HL60-HUVEC adhesion, and some mAb's caused strong aggregation HL60 cells. Anti-Lex mAb's SHI and FH2 showed consistent stronger (compared to SNH3 or SNH4) inhibition of HL60-HUVEC adhesion, as did a combination of SNH4 plus SHI or FH2. NDV sialidase did not reduce significantly HL60-HUVEC adhesion, but Vibrio sialidase almost abolished reactivity completely.
Coating of adhesion molecules or EC's on glass plates in the dynamic flow system
For lectins, fibronectin (FN) , laminin (LN) , truncated E-selectin and GSL's used, 10-50 μl of a solution having a concentration of 20-200 μg/ml was placed on a marked area (0.5 cm diameter) on a glass plate (38 x 75 mm; Corning Glassworks, Corning, NY) and dried in a refrigerator at 4*C. Dried plates were immersed in PBS at 37"C for 1 hr and washed extensively with several changes of PBS. For GSL coating, GSL-liposomes wer prepared from 200 μg GSL, 200 μg cholesterol and 400 μ phosphatidylcholine in 1 ml PBS. Ten μl of GSL-liposome solution was placed on a glass plate, dried at 4βC and the plates wer washed with PBS, as described above.
The quantity of adsorbed molecules was determined using 125I labeling for lectins, FN or LN, or [ H]cholesterol labeling fo GSL-liposomes. Under those conditions, almost the entir quantity of protein, regardless of whether FNr LN or lectin, wa adsorbed on the glass plate. For example, when 100 μg/ml FN wa applied, 12.5 + 1.8 ng/mm was adsorbed. Likewise, almost al GLS-liposome dried on the glass plate was adsorbed; e.g., whe 200 μg/ml GLS-liposome was applied, 31.3 + 5.2 ng GSL/mm wa adsorbed. EC's were coated by placing 100 μl of a suspens containing 2 x 105 mouse or human EC's on glass plates culturing in a C02 incubator at 37 ° C until confluency achieved.
Plates coated with adhesion molecules or EC's were affi in a flow chamber, and a suspension of B16 melanoma cells passed through the chamber as described hereinabove. B16 ce were harvested from culture using 0.02% EDTA in PBS, suspended in PBS at a concentration of 1 x 10 /ml.
HL60 adhesion to activated HUVEC's in a dynamic flow system
The effects of various mAb's and sialidases on HL60-HU adhesion were tested also in a dynamic flow system. General the effects of the mAb's were similar to those observed wit static system. mAb SNH4 had no inhibitory effect at all un various shear stresses. mAb's SHI and FH2 showed moder inhibition. Again, strongest inhibition was obtained wit combination of SNH4 plus SHI or FH2. Adhesion was redu moderately by NDV sialidase and almost completely by Vibrio Arthrobacter sialidase. See Figure 15. The results set forth hereinabove using HL60 cells sugg that the presence of sialic acid in the carbohydrate epitope important in providing binding specificity to E-select However, the sialic residue is not required to be α2→3 linked the terminal Gal; the sialic acid residue alternatively could present at an internal location, e.g., linked to internal Gal GlcNAc. Clearly, though, αl→3 fucosylation at GlcNAc i essential.
Under dynamic conditions, NDV sialidase had an inhibito effect only at low shear stress whereas VC or AU sialidas significantly reduced adhesion even at high shear stress Anti-Lex IgG mAb SHI strongly inhibited adhesion even at hi shear stress, whereas the effect of anti-SLex IgG3 mAb SNH4 w minimal. Strongest inhibition was produced by a combination o NDV sialidase plus anti-Lex mAb SHI. A mixture of anti-Lex pl anti-SLex mAb's produced stronger inhibitory effect than eith mAb alone.
As depicted in Figure 19, at low shear stre (<4 dynes/cm ) , adhesion was inhibited significantly by N sialidase or by mAb SNH4, whereas those reagents had no effe at high shear stress (8-16 dynes/cm ) . In contrast, VC sialida completely abolished adhesion at high shear stress. mAb S inhibited adhesion more strongly at high than at low she stress, but the difference was relatively small.
Lex alone clearly is not sufficient as the E-select epitope. Rather, α2→3 plus α2→6 sialylated structures a necessary. Lex-liposomes and Le8-liposomes bind to ELAM-coat plates, and binding by SLex is stronger than by Lex or oth glycolipids. However, those epitopes must be present at the ce surface in the form of multiply O-glycosylated mucin-ty glycoproteins. Lex as well as SLex may be α2→6 sialylated at t internal Gal or GlcNAc within the same CHO chain, or α2 sialylation may be present at an adjacent branched structur "6-C ganglioside," which is an α2→6 sialylated type 2 cha structure with internal αl→3 fucosylation (Hakomori et al Biochem. Biophys. Res. Commun. 113:791, 1983), failed to bind E-selectin. Thus, such a structure can be excluded as a possib ELAM epitope.
EXAMPLE 7
COLO201 CELL ADHESION: EFFECTS OF VARIOUS SIALIDASES AND mAb's
Colo201 adhesion to E-selectin-coated plates under sta conditions
In contrast to HL60 cells (which express predominan type 2 chain structure) , Colo201 cells express mainly type chain, and E-selectin-dependent Colo201 adhesion is thro type 1 chain epitopes. Colo201 cells were treated with vari mAb's following exposure to various sialidases and were asses for residual binding. Colo201 reactivity with mAb CA1 (directed to SLeaI) was inhibited almost completely by Vib sialidase, and to a lesser extent by Arthrobacter and sialidases.
In contrast, Colo201 reactivity with mAb FH7 (directed di-SLe8 and SLe8 II) was reduced by Arthrobacter sialidase minimally affected by Vibrio or NDV sialidases. Colo reactivity with mAb CA3F4 (Nudelman et al. J. Biol. Ch 261:5487, 1986) was enhanced by sialidase treatment. mAb CA1 inhibited Colo201 adhesion slightly and was influenced onl minimally by Vibrio sialidase (Figure 16) .
It is possible that the SI-E8 epitope present at the surfac of Colo201 cells is organized in such as way that it is (i) no susceptible to CA19-9 for E-selectin-dependent adhesion an (ii) not sensitive to sialidase treatment. The inhibitor effects of mAb FH7 (Nudelman et al. supra) and, more strikingly, mAb CA3F4 on Colo201 adhesion to E-selectin-coated plates wer enhanced by pretreatment of cells with Vibrio sialidase. Arthrobacter sialidase reduced but did not abolish Colo20 adhesion. See Figure 17.
Colo201 adhesion to E-selectin-coated plates in a dynamic flo system
In the dynamic flow system, NDV sialidase had no effect o Colo201 adhesion, particularly at high shear stresses
Anti-SLe8 I mAb CA19-9 had no effect, whereas anti-SLe8 II mA
FH7 had a moderate inhibitory effect, in agreement with result from the static system.
At both low and high shear stresses, the stronges inhibition of adhesion was observed for mAb CA3F4, which i directed to Le8 with an α2→6 sialosyl substitution at th penultimate GlcNAc. Vibrio sialidase, which efficiently cleave terminal α2→3 sialosyl linkages but is less effective at removin internal sialic acid residues, reduced adhesion to some exten at high shear stress, but less so at low shear stress. Similarly, mAb CA3F4 inhibited adhesion strongly at hi shear stress but much less at low shear stress. A combinati of Vibrio sialidase plus mAb CA3F4 produced strong inhibition both high and low shear stress (Figure 18) . A similar trend was observed for NDV sialidase, whi specifically cleaves terminal α2→3 sialosyl linkages. Colo2 adhesion, at either high or low shear stress, was not affect by NDV sialidase alone, nor by NDV sialidase followed mAb CA19-9. In contrast, adhesion was inhibited strongly, both high and low shear stress, by NDV sialidase followed by m FH7 or CA3F4.
Rolling velocity (μm/sec) of Colo201 cells alo E-selectin-coated plates was evaluated after treatment wi various sialidases and mAb's. At three different shear stresse velocity was unaffected by NDV sialidases and mAb CA19-9; i.e cells once stopped (0 μm/sec) did not start rolling agai However, Vibrio or Arthrobacter sialidase, or mAb CA3F4, caus once-stopped cells to start rolling again; the resulting veloci depending on shear stress. The greatest velocity resulted fr treatment with a combination of CA3F4 plus Vibrio sialidase.
Colo201 adhesion to activated HUVEC's in a static system
As noted for Colo201 adhesion to E-selectin-coated plate mAb's CA19-9 and FH7 had negligible effect, but mAb CA3 strongly inhibited adhesion. Treatment of cells with N sialidase, which cleaves terminal α2→3 sialosyl linkage, eith briefly or for 24 hr, did not reduce adhesion significantl Treatment for 24 hr with Vibrio sialidase completely abolishe adhesion. Brief treatment with Vibrio and Arthrobacter sialidas still reduced adhesion significantly.
Colo201 adhesion to activated HUVEC's in a dynamic flow system
Inhibition of adhesion was strongest for mAb CA3F4 an non-existent for mAb CA19-9. mAb INH1 or ST421, directed t unsubstituted type 1 chain (Stroud et al. J. Biol. Chem 266:8439, 1991), also caused significant inhibition. Adhesio was unaffected by NDV sialidase but strongly inhibited by Vibri sialidase.
The observed effects of sialidases and mAb's on Colo20 adhesion to E-selectin-coated plates and to HUVEC's suggest tha the type 1 chain epitope recognized by E-selectin is internall sialosylated and fucosylated.
EXAMPLE 8
Truncated, recombinant ELAM-l lacking the transmembrane an cytoplasmic domains is used to coat beads, for example, capabl of packing into a standard chromatography columns. The ELAM- at a concentration of 0.1-1 μg/ml is mixed with the beads and th mixture is incubated to allow binding of ELAM-l to the bea matrix. A suitable incubation period is 12-24 hours a βC - room temperature. The beads are washed to remove unboun ELAM-l, optionally can be blocked with an inert carrier, such a BSA, and washed again. The ELAM-l coated beads can be used in a batch process packed into a suitably-sized column.
Cells known to carry carbohydrates bindable to ELAM-l, su as HL60 and Colo201, are obtained. The cells are lysed to obta a membrane fraction using known methods, such as repeat freeze-thaw cycles. The membrane fraction is obtained, f example, by centrifugation.
If the cell source is known to express only or predominant carbohydrates bindable to ELAM-l, the membrane prep may be suitable source without further purification.
The membrane prep is treated using known methods to obta a membrane component preparation, and in particular, a fracti that contains cell surface carbohydrate. The carbohydrate-ri fraction is mixed with or passed over the ELAM-l affinity matri depending on the format, the exposed matrix is washed and carbohydrates bound to the matrix are eluted, for example, exposing the matrix to a high salt buffer.
The resultant preparation comprises carbohydrate binda to ELAM-l and the various species are separable using -kn techniques, such as TLC or HPLC.
EXAMPLE 9
Carbohydrates bindable to ELAM-l, either prepared chemica using known reagents and methods, see, for example, Exampl hereinabove, prepared enzymatically or obtained from suita cells, see, for example. Example 8 hereinabove, or whole ce 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.
In the case of poiyclonal antisera, the blood is collected, serum separated and tested. In the case of monoclonal antibodies, 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-l 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.
Alternatively, the antisera can be made "monospecific" by absorption with cells carrying only SLex and/or SLe8 or with a solid matrix to which SLex and/or SLe8 is bound. The resultant residual activity directed to carbohydrates bindable to ELAM-l can be attributed in part to antibodies directed to carbohydrates of formula (I) , (II) or (III) . EXAMPLE 10
NS-1 cells were obtained from the ATCC (Rockville, MD) a maintained in RPMI 1640:Dulbecco's MEM (1:1) supplemented wi 10% HI FCS. Fifty μg of a plasmid comprising cDNA of E-select in vector pCDM8 (R & D Systems, Minneapolis, MN) and 5 μg pSV2-neo (ATCC) were co-transfected into NS-1 cells (1 x 10 ) electroporation. After 48 hours in culture, the cells we transferred to medium containing 650 μg/ml G418 (Gibco, Gra Island, NY) . After 15-20 days, resulting colonies were screened f E-selectin expression by staining with mAb (obtained from Newman, Maryland Research Laboratories, Otsuka Pharmaceutic Co. , Rockville, MD) . The variant expressing the highest lev of E-selectin was isolated by panning with mAb followed limiting dilution to achieve clonality.
E-selectin-dependent adhesion using transfected NS-1 cel onto SLe8, SLex, Ley, Lex, H-2, sialylparagloboside (SPG disialosyl I (Structure 6 of Figure 20) and dimeric SLex we compared at various shear stress conditions. The number of cel adhered per mm is expressed relative to adhesion on SLex-coated plates which is regarded as 100%.
Whereas adhesion to Ley and Lex slightly increased at hi shear stress, the absolute numbers of cells which adhered w much lower at both low and high shear stress conditions relati to that observed with SLex and SLe8. Adhesion with structure of Figure 20 was enhanced at middle and high shear stre conditions. The high binding capacity of structure 1 of Figure 20 wa revealed further using low concentrations of glycolipids i liposomes (glycoliposomes) . Transfected NS-1 adhesion on SLe liposomes was enhanced when Lex or Ley was added and presented a a mixed glycoliposome. The results of the experiments ar presented in Figures 21-24.
From the foregoing, it will be evident that, althoug specific embodiments of the invention have been described herei for purposes of illustration, various modifications may be mad without deviating from the spirit and scope of the invention.
All references cited herein are incorporated by reference.

Claims

WHAT IS CLAIMED IS :
1. A carbohydrate or substituted derivative thereof having the formula: ,
wherein R2 is H or an α2→6 linked sialic acid, R4 is H or an α1→3 linked fucose, R5 is H, an α2→3 linked sialic acid, an α2→3 linked NeuAcα2→8NeuAc disaccharide or an α2→3 linked R6-sialic acid carbohydrate, wherein R6 is one or more sugars, and n is equal to or greater than 0.
2. A carbohydrate or substituted derivative thereof having the formula:
,
wherein R1 is H or an α2→3 linked sialic acid, R2 is H or an α2→6 linked sialic acid, R3 is H or an αl→4 linked fucose, and n is equal to or greater than 0.
3. A carbohydrate or substituted derivative thereof having the formula:
wherein each of R10 and R11 comprises galactose, Galβ1→4GlcNAc or Galβ1→3GlcNAc; R8 comprises galactose or GalNAc; and R9 comprises lactosyl ceramide or an oxygen group of a lipid or a protein which bonds carbohydrate.
4. The carbohydrate of claim 3, wherein R10 comprises the Lex epitope and R11 comprises the SLex epitope.
5. The carbohydrate of claim 4, wherein R8 is Galβ1→4GlcNAc.
6. The carbohydrate of claim 5, wherein R9 is an oxygen group of a lipid or a protein which bonds carbohydrate.
7. The carbohydrate of claim 3, wherein R10 comprises the Lea epitope and R11 comprises the SLea epitope.
8. The carbohydrate or substituted derivative of claim 3 which is:
9. An antibody which binds specifically to a carbohydrate or substituted derivative thereof of any one of claims 1-8.
10. The antibody of claim 9, wherein said carbohydrate is the carbohydrate of claim 4.
11. The antibody of claim 9, wherein said carbohydrate is the carbohydrate of claim 7.
12. The antibody of claim 8, wherein said carbohydrate is the carbohydrate of claim 8.
13. A composition comprising at least two carbohydrates that are involved in tumor cell or leukocyte adhesion to endothelial cells.
14. The composition of claim 13, wherein one of said least two carbohydrates is SLex.
15. The composition of claim 14, which further comprises
Lex
16. The composition of claim 14, which further comprises
Ley
17. The composition of claim 13, wherein said at least two carbohydrates comprise Lea and SLea.
18. The composition of claim 13, which further comprises a liposome.
19. A composition comprising at least two antibodies, wherein each of said two antibodies specifically binds to one of said at least two carbohydrates comprising a composition of any one of claims 13-17.
20. The composition of claim 19, wherein an antibody specifically binds to SLex.
21. The composition of claim 20, which further comprises an antibody which specifically binds to Lex or Ley.
22. The composition of claim 19, wherein an antibody specifically binds to SLea.
23. The composition of claim 22, which further comprises an antibody which specifically binds to Lea or Leb.
24. A method for interrupting intercellular interactions mediated by ELAM-l with cells expressing type 1 chain comprising at the terminus: NeuAcα2
↓
6
Galβ1→3GlcNAc
4
↑
Fucα1,
comprising exposing said cells to an antibody which binds specifically to Lea.
25. The method of claim 24 wherein said antibody is CA3FA or FH7.
26. A method for interrupting ELAM-1-mediated intercellular interactions between cells comprising exposing said cells to at least one antibody which binds specifically to a carbohydrate bindable to ELAM-1.
27. The method of claim 26 wherein said carbohydrate bindable to ELAM-1 is SLex, hybrid Lex/SLex or hybrid Lea/SLea.
28. The method of claim 27 wherein said carbohydrate has the structure:
Fucα1
↓
3
Galβ1→4GlcNAcβ1→6
Galβ1→3GlcNAcβ1→R . Galβ1→4GlcNAcβ1→3
3 3
↑ ↑
SA2 Fucα1
29. The method of claim 26, which comprises an antibody which binds specifically to SLex.
30. The method of claim 29, which further comprises an antibody which binds specifically to Lex.
31. The method of claim 26, which comprises an antibody which binds specifically to SLea.
32. The method of claim 31, which further comprises an antibody which binds specifically to Lea.
33. The method of claim 26 which further comprises an antibody which binds specifically to a carbohydrate not bindable to ELAM-1.
34. The method of claim 33 wherein said carbohydrate not bindable to ELAM-1 is Lex, Ley, Lea or Leb.
35. The method of claim 30 wherein said antibody which binds specifically to SLex is SNH3 or SNH4 and said antibody which binds specifically to Lex is SHI or FH2.
36. A method for interrupting ELAM-1-mediated intercellular interactions between cells comprising exposing said cells to at least one member of the group consisting of ELAM-l, carbohydrate bindable to ELAM-1, sialidase, antibody which binds specifically to ELAM-1 and antibody which binds specifically to carbohydrate bindable to ELAM-1.
37. The method of claim 36, wherein said carbohydrate bindable to ELAM comprises an α2→3 linked sialic acid.
38. The method of claim 36, wherein said carbohydrate bindable to ELAM is Lex.
39. The method of claim 36, wherein said carbohydrate bindable to ELAM comprises an α2→6 linked sialic acid.
40. The method of claim 36, wherein said antibody which binds specifically to carbohydrate bindable to ELAM is an antibody which binds specifically to carbohydrate bindable to ELAM comprising an α2→3 linked sialic acid.
41. The method of claim 36, wherein said antibody which binds specifically to carbohydrate bindable to ELAM is an antibody which binds specifically to carbohydrate bindable to ELAM comprising an α2→6 linked sialic acid.
42. The method of claim 36, wherein said antibody which binds specifically to carbohydrate bindable to ELAM is an antibody which binds specifically to Lex.
EP93905988A 1992-02-19 1993-02-19 Inhibition of cell adhesion by chemically-defined oligosaccharides, their derivatives, mimetics, and antibodies directed thereto Withdrawn EP0638085A1 (en)

Applications Claiming Priority (7)

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US83697892A 1992-02-19 1992-02-19
US95072092A 1992-09-25 1992-09-25
US950720 1992-09-25
US99690392A 1992-12-29 1992-12-29
US996903 1992-12-29
PCT/US1993/001375 WO1993017033A1 (en) 1992-02-19 1993-02-19 Inhibition of cell adhesion by chemically-defined oligosaccharides, their derivatives, mimetics, and antibodies directed thereto
US836978 2004-04-30

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CA2227013A1 (en) * 1995-07-14 1997-02-06 Glycotech Corp. Compounds and methods for treatment of egf receptor associated cancers and purification of the egf receptor
AUPP913999A0 (en) * 1999-03-12 1999-04-01 Biota Scientific Management Pty Ltd Novel chemical compounds and their use
DE19930177B4 (en) 1999-06-30 2007-02-08 Nikolai Vladimirovich Bovin Intermolecular associating compounds and their use
DE10056136A1 (en) * 2000-11-07 2002-05-16 Nemod New Modalities Inhibiting leukocyte or tumor cell adhesion to vascular endothelial cells e.g. for combating inflammation or metastasis, using e.g. pregnancy proteins or selectin binding liposomes containing calcium-binding compound
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
FI20011664A (en) 2001-08-17 2003-02-18 Carbion Oy Cancer-specific oligosaccharide sequences and their use
JP5331293B2 (en) * 2006-04-28 2013-10-30 公益財団法人野口研究所 Oligosaccharide or its derivatives expressing diversity
JP5894732B2 (en) * 2010-11-11 2016-03-30 学校法人東京女子医科大学 Cell culture substrate evaluation method

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