[go: up one dir, main page]

US20030133937A1 - Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules - Google Patents

Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules Download PDF

Info

Publication number
US20030133937A1
US20030133937A1 US10/269,842 US26984202A US2003133937A1 US 20030133937 A1 US20030133937 A1 US 20030133937A1 US 26984202 A US26984202 A US 26984202A US 2003133937 A1 US2003133937 A1 US 2003133937A1
Authority
US
United States
Prior art keywords
sialyl
cells expressing
binding
malignant cells
elam
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/269,842
Inventor
John Magnani
Eugene Butcher
Ellen Berg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US10/269,842 priority Critical patent/US20030133937A1/en
Publication of US20030133937A1 publication Critical patent/US20030133937A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • 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
    • 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
    • C07K16/30—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

Definitions

  • the present invention is generally directed toward the inhibition of cancer metastasis mediated by endothelial adhesion molecules, and more specifically, toward such inhibition through the use of saccharides, glycoconjugates, antibodies, enzyme inhibitors, and other agents which disrupt such binding of cancer cells to endothelia.
  • cancer Despite enormous investments of financial and human resources, cancer remains one of the major causes of death. Current cancer therapies cure only about fifty percent of the patients who develop a malignant tumor. In most human malignancies, metastasis is the major cause of death.
  • Metastasis is the formation of a secondary tumor colony at a distant site. It is a multistep process of which tumor invasion is an early event. Tumor cells locally invade host tissue barriers, such as the epithelial basement membrane, to reach the interstitial stroma, where they gain access to blood vessels (“hematogenous metastasis”) or lymphatic channels for further dissemination. After invading the endothelial layer of a vessel wall, the circulating tumor cells are dislodged into the circulation and arrest in the precapillary venules of the target organ by adherence 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 it originated.
  • host tissue barriers such as the epithelial basement membrane
  • Interleukin 1 IL-1
  • tumor necrosis factor TNF
  • IL-1 Interleukin 1
  • TNF tumor necrosis factor
  • LEC-CAMs calcium-dependent cell adhesion receptors
  • GMP-140 also known as PADGEM or CD62
  • ELAM-1 on endothelial cells functions as a “homing receptor” for leukocytes.
  • ELAM-1 on endothelial cells was shown to mediate the increased adhesion of colon cancer cells to endothelium treated with cytokines (Rice and Bevilacqua, Science 246:1303-1306, 1989).
  • the present invention provides methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules.
  • the present invention provides methods for inhibiting, within a biological preparation, the binding of malignant cells expressing sialyl Le a or di-sialyl Le a , to endothelial cells.
  • the method comprises incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Le a or di-sialyl Le a , to endothelial cells expressing a LEC-CAM.
  • the method comprises incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Le a or di-sialyl Le a , to endothelial cells expressing ELAM-1.
  • the method comprises incubating the malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Le a or di-sialyl Le a by the malignant cells.
  • methods are provided for inhibiting the spread of malignant cells expressing sialyl Le a or di-sialyl Le a , to secondary sites in a warm-blooded animal.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Le a or di-sialyl Le a , to endothelial cells expressing a LEC-CAM.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Le a or di-sialyl Le a , to endothelial cells expressing ELAM-1.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Le a or di-sialyl Le a by the malignant cells.
  • the method comprises incubating a biological preparation, containing endothelial cells expressing a LEC-CAM, with at least one agent capable of reacting with both sialyl Le a and sialyl Le x .
  • the method comprises incubating a biological preparation, containing endothelial cells expressing ELAM-1, with at least one agent capable of reacting with both sialyl Le a and sialyl Le x .
  • methods for inhibiting the spread of malignant cells expressing sialyl Le a , di-sialyl Le a or dialyl Le x , to secondary sites in a warm-blooded animal.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Le a and sialyl Le x .
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Le a and sialyl Le x .
  • FIG. 1 describes pictorially a cell binding assay used to assess binding of ELAM-1 transfected cells to neoglycoproteins.
  • FIG. 2 graphically illustrates the relative binding of ELAM-1 transfected cells to certain neoglycoproteins.
  • FIG. 3 graphically illustrates the relative binding of ELAM-1 transfected cells to certain neoglycoproteins.
  • Antibody includes both monoclonal and polyclonal antibodies and may be an intact molecule, a fragment thereof, or a functional equivalent thereof.
  • the antibody may be genetically engineered. Examples of antibody fragments include F(ab′) 2 , Fab′, Fab and Fv.
  • Saccharide as used herein, includes oligosaccharides, and may be naturally derived, synthetically prepared, portions of either, and derivatives of any of the foregoing.
  • Glycoconiugate includes a saccharide which is coupled to a non-saccharide molecule, e.g., a lipid or a polypeptide.
  • the present invention is generally directed towards methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules. More specifically, the disclosure of the present invention shows that antibodies, saccharides, glycoconjugates therefrom or enzyme inhibitors may be used to inhibit the binding of malignant cells to endothelial cells for a variety of purposes in vivo and in vitro.
  • metastasis is a multistep process.
  • cancer cells circulate through the microvascular and lymph systems and then migrate through the walls of the blood or lymph vessels to establish a new and aggressive tumor at a secondary organ site.
  • a critical step in the metastasis process is the adherence of circulating cancer cells to the endothelial lining of blood vessel or lymph vessel walls.
  • the carbohydrates sialyl Le a and di-sialyl Le a which are expressed at the surface of certain cancer cells, function as a ligand (i.e., binding partner) for LEC-CAMs, such as ELAM-1, which are expressed at the surface of endothelial cells.
  • metastasis involves the adherence of cancer cells to the endothelial cells via the binding of sialyl Le a and/or di-sialyl Le a on the cancer cells to adhesion molecules on endothelial cells.
  • Other cancer cells express predominantly sialyl Le x , or sialyl Le x and sialyl Le a (and/or di-sialyl Le a ).
  • LEC-CAMs such as ELAM-1, share a carbohydrate domain common to both sialyl Le a and sialyl Le x on malignant cells, and therefore agents can be produced which are capable of binding to both.
  • Suitable blocking agents include those which inhibit the binding of malignant cells expressing sialyl Le a , di-sialyl Le a , or sialyl Le x , to endothelial cells expressing LEC-CAM adhesion molecules such as ELAM-1.
  • Representative agents include antibodies, saccharides and glycoconjugates therefrom.
  • the antibodies employed in the present invention may be polyclonal or monoclonal antibodies. Briefly, polyclonal antibodies may be produced by immunization of an animal and subsequent collection of its sera. Immunization is accomplished, for example, by a systemic administration, such as by subcutaneous, intrasplenic or intramuscular injection, into a rabbit, rat or mouse. It is generally preferred to follow the initial immunization with one or more booster immunizations prior to sera collection. Such methodology is well known and described in a number of references.
  • Monoclonal antibodies (MAbs) suitable within the present invention include those of murine or human origin, or chimeric antibodies such as those which combine portions of both human and murine antibodies (i.e., antigen binding region of murine antibody plus constant regions of human antibody).
  • Human and chimeric antibodies may be produced using methods known by those skilled in the art. Human antibodies and chimeric human-mouse antibodies are advantageous because they are less likely than murine antibodies to cause the production of anti-antibodies when administered clinically.
  • MAbs may be generally produced by the method of Kohler and Milstein ( Nature 256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976). Briefly, the lymph nodes and/or spleens of an animal immunized with sialyl Le a or di-sialyl Le a are fused with myeloma cells to form hybrid cell lines (“hybridomas” or “clones”). Each hybridoma secretes a single type of immunoglobulin and, like the myeloma cells, has the potential for indefinite cell division. It may be desirable to couple such molecules to a carrier to increase their immunogenicity.
  • Suitable carriers include keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof.
  • An alternative to the production of MAbs via hybridomas is the creation of MAb 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, 1989). Selection of antibodies exhibiting appropriate specificity may be performed in a variety of ways which will be evident to those skilled in the art. Typically, such antibodies will selectively bind with an affinity of about 10 7 liters/mol or higher.
  • MAbs suitable within the present invention include N-19-9 and HECA-452 for sialyl Le a , and FH-7 for di-sialyl Le a .
  • MAb N-19-9 is available from ATCC (American Type Tissue Collection, Rockville, Md.) as ATCC HB 8059 or may be produced as described in U.S. Pat. No. 4,471,057 (and Somatic Cell Genet. 5:957-971, 1979; J. Biol. Chem. 257:14365, 1982).
  • MAb HECA-452 may be produced according to Duijvestijn et al., Am. J. Path. 130:147-155, 1988.
  • FH-7 may be produced according to Nudelman et al., J. Biol. Chem. 261:5487, 1986.
  • sialyl Le a In addition to antibodies which are capable of binding to sialyl Le a , di-sialyl Le a or sialyl Le x , saccharides and glycoconjugates therefrom may also inhibit the binding of metastatic cells expressing sialyl Le a , di-sialyl Le a or sialyl Le x , to endothelia.
  • sialyl Le a and “di-sialyl Le a ” represent structures I and II, respectively, as follows:
  • Neu5Ac represents sialic acid
  • Gal represents galactose
  • GlcNAc represents N-acetyl-glucosamine
  • Fuc represents fucose and R is typically a ceramide (with a glucose residue interposed) or a protein.
  • Sialyl Le x is an isomer of sialyl Le a wherein the Gal-GlcNAc linkage is ⁇ 1-4 and the Fuc-GlcNAc linkage is ⁇ 1 ⁇ 3.
  • Saccharides suitable within the present invention include the carbohydrate portion of sialyl Le a or di-s-ialyl Le a (i.e., formula I or II minus R), and derivatives of either, including those which cross-react with both sialyl Le a and sialyl Le x .
  • Derivatives of these compounds include substitution of individual saccharide residues with other saccharide residues and/or with non-saccharide molecules such as hexyl rings without hydroxyl groups.
  • the internal GlcNAc may be replaced with another saccharide residue such as a glucose (Glc).
  • the carbohydrate portion of sialyl Le a , di-sialyl Le a , or derivatives thereof may be truncated by deletion of one or more saccharide residues.
  • a tetrasaccharide may be created with the structure:
  • a saccharide may be coupled to a non-saccharide molecule to form a glycoconjuqate.
  • a saccharide may be linked to a polyacrylamide.
  • a saccharide may be linked to a lipid.
  • Typical lipids include ceramide, i.e., sphingolipid bases which are acylated on the amine with a fatty acid.
  • sialyl Le a , di-sialyl Le a , or a saccharide cross-reaction with sialyl Le a and sialyl Le x may be linked to a ceramide.
  • a saccharide may be bonded to an amino acid or an amino acid-containing molecule, such as a peptide, a polypeptide or a protein.
  • Saccharides are naturally linked to an amino acid or amino acid-containing molecule via the hydroxyl group of a serine or threonine amino acid residue, but can also be linked through other groups such as an amino group.
  • Saccharides and glycoconjugates provided by the present invention may be represented by structures III and IV as follows:
  • R includes H, OH, lipid, ceramide, or one or more amino acids; x, y and z are independently selected from saccharides, or either y or z or both may be absent.
  • Saccharides may be prepared synthetically using chemical, and/or enzymatic, reagents and techniques.
  • sialyl Le a saccharides have been prepared by enzymatic synthesis (e.g., Palcic et al., Carbohydr. Res. 190:1-11, 1989).
  • Glycoconjugates may be prepared, for example, through reductive amination. The method of Zopf et al. ( Meth. Enzymol. 50:171-175, 1978; Jeffrey et al., Biochem. Biophys. Res. Commun.
  • a sugar is dissolved in water, and the same volume of amine (a 170-fold molar excess) is added together with sodium cyanoborohydride (a ten-fold molar excess).
  • the reduction is performed at pH 8 for 48 hours, and the product purified by gel chromatography.
  • Coupling to different molecules, such as proteins, may be performed by the isothiocyanate coupling method.
  • TFAN p-trifluoroacetamidoaniline
  • the reductive amination reaction is carried out in aqueous solution overnight at pH 5-6 with sodium cyanoborohydride as the reducing agent.
  • a 5-fold excess of TFAN is used.
  • TFAN-derivatized saccharides are generally protected from oxidation by N-acetylation, e.g., by treatment with methanolic acetic anhydride, to yield TFAc-derivatives.
  • the N-trifluoroacetamido protective group Prior to conjugation, the N-trifluoroacetamido protective group is removed by treatment of the TFAc derivative with aqueous ammonia or 0.5 M sodium hydroxide for 3 hours. Conjugation of the derivatives to molecules, for example to proteins such as bovine serum albumin (BSA), may be achieved by isothiocyanate coupling methods.
  • BSA bovine serum albumin
  • suitable reagents and reactions include p-tetradecylaniline derivatives of saccharides and the preparation of aminoalditols by oxidation of saccharide TFAN derivates with cerium ammonium sulfate (Lindenberg et al., J. Reprod. Fert. 89:431-439, 1990).
  • Sialyl Le a and di-sialyl Le a are type 1 carbohydrate chains (i.e., have a Gal ⁇ 1 ⁇ 3GlcNAc polylactosamine unit structure) and sialyl Le x is a type 2 carbohydrate chain (i.e., has a Gal ⁇ 1 ⁇ 4 GlcNAc polylactosamine unit structure.
  • a number of cancer cells such as colorectal and pancreatic, have a prevalence of type 1 carbohydrate chains including sialyl Le a and di-sialyl Le a .
  • Other cancer cells such as breast, lung and ovarian, have a prevalence of type 2 carbohydrate chains including sialyl Le x .
  • the present invention provides methods for inhibiting the binding of cancer cells to endothelia in a biological preparation.
  • biological preparations include blood vessel and/or lymph vessel endothelia in combination with a malignancy.
  • the endothelia and the malignancy may be in the form of tissue or cells removed from an organism, or cultured cells.
  • the method comprises incubating a biological preparation, which contains malignant cells expressing sialyl Le a , di-sialyl Le a or sialyl Le x and endothelial cells expressing a LEC-CAM, with an effective amount of at least one agent, such as an antibody, saccharide or glycoconjugate as described above.
  • the method comprises incubating malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Le a or di-sialyl Le a by the cells.
  • Suitable enzyme inhibitors include inhibitors of glycosyltransferases.
  • Representative examples of inhibitors for glycosyltransferases include inhibitors for fucosyltransferases (e.g., as described by Palcic et al., J. Biol. Chem. 264:17174-17181, 1989), for N-acetylglucosaminyltransferases (e.g., as described by Palcic et al., J. Biol. Chem.
  • sialyltransferases e.g., as described by Broquet et al., J. Neurochem. 54:388-394, 1990; Karaivanova et al., Cancer Biochem. Biophys. 11:311-315, 1990).
  • the present invention also provides methods for inhibiting metastasis in a warm-blooded animal such as a human.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one agent, such as an antibody, saccharide or glycoconjugate as described above.
  • the method comprises administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor (as described above) that inhibits the biosynthesis of sialyl Le a or di-sialyl Le a by malignant cells.
  • at least one enzyme inhibitor as described above
  • It will be evident to those skilled in the art how to determine the optimal effective dose for a particular agent or enzyme inhibitor, e.g., based upon in vitro and in vivo studies in non-human animals.
  • routes of administration may be used. Typically, administration will be intravenous, intracavitory (e.g., in pleural or peritoneal cavities), or in the bed of a resected tumor.
  • An agent may be administered as a composition, i.e., in combination with a pharmaceutically acceptable carrier or diluent, such as physiological saline. It will be recognized by those skilled in the art that an agent and a composition may be prepared in a sterile form. Moreover, an agent may be administered in combination with an immunotherapeutic or chemotherapeutic agent. When such a combination is desired, each substance may be administered sequentially, simultaneously, or combined and administered as a single composition. Diagnostic techniques, such as CAT scans for tumors, may be performed prior to and subsequent to administration to confirm effectiveness.
  • Neoglycoproteins were produced by BioCarb AB (Lund, Sweden) by chemically coupling 10-20 moles of a specific oligosaccharide to 1 mole of nonglycosylated albumin, bovine (BSA) or human (HSA).
  • BSA bovine
  • HSA human
  • the resulting synthetic glycoprotein (neoglycoprotein) contains multiple copies of the identical carbohydrate sequence, thereby producing a well characterized, mutivalent glycoconjugate which is extremely effective for studying carbohydrate-protein interactions.
  • oligosaccharides Depending on the size of the oligosaccharide, three different chemical spacer arms were used to couple the oligosaccharides to proteins 1) p-aminophenyl (PAP); 2) aminophenylethyl (APE); and 3) acetyl phenylene diamine were used to couple the shorter oligosaccharides to albumin since they will retain the anomeric configuration of the reducing sugars which may be involved in a potential binding site.
  • APD was used to couple the larger sugars to protein by reductive amination, which converts the reducing sugar to an aminoalditol.
  • Synthetic glycoproteins were coated onto microtiter plates by filling each well with 100 ng of the neoglycoprotein in 100 ⁇ l of 0.15 M sodium chloride, 0.01 M sodium phosphate, 0.1% sodium azide, pH 7.4, (PBS-azide) overnight at 4° C.
  • Standard enzyme-linked immunoassays ELISA were then performed on the solid phase carbohydrate structures using the appropriate antibody diluted to 10 ⁇ g/ml.
  • L1-2/pMRB107 cells were prepared by transfecting the ELAM-1 gene into the murine pre-B cell line L1-2 (Gallatin et al., Nature 304:30-34, 1983).
  • a cDNA clone encoding ELAM-1 was obtained from a cDNA library made from activated human umbilical vein endothelial cell cultures by polymerase chain reaction (PCR) amplification.
  • the ELAM-1 gene was inserted downstream of the hCMV promoter in pMRB101 [a derivative of EE6 which contains the E. coli gpt gene (Mulligan and Berg, Proc. Nat'l. Acad. Sci.
  • L1-2 cells were introduced into L1-2 cells by electroporation and the cells selected for resistance to mycophenolic acid.
  • a population of cells staining brightly for ELAM-1 were selected by FACS and cloned by limiting dilution. These cells are ELAM-1 hi LFA-1 mod CD45 hi CD44 neg LECAM-1 neg , differing from the parent cell line or control vector transfectants only in their expression of ELAM-1.
  • L1-2/pMRB101 (L1-2 vector ) cells are a similarly transformed derivative of L1-2 transfected with pMRB101 and lacking ELAM-1 expression.
  • CM Complete Medium
  • DMEM Dulbecco's Modified Eagles Medium
  • the sensitive binding assay described in Example 1 uses cells permanently transfected with ELAM-1 cDNA.
  • the mouse pre-B cell line, L1-2 transfected with ELAM-1 cDNA (L1-2 ELAM-1 ), but not vector control cDNA, L1-2 vector expresses very high levels of ELAM-1.
  • the ELAM-1 expressed by these cells is functional as L1-2 ELAM-1 cells are adhesive for neutrophils and this adhesion is blocked by anti-ELAM-1 monoclonal antibodies.
  • L1-2 ELAM-1 cells bound selectively to Sialyl Le a and Sialyl Le x neoglycoproteins, but not to a number of other glycoconjugates.
  • L1-2 ELAM-1 cells also bound, albeit more weakly, to Le a neoglycoprotein.
  • the binding to Le a is significant as L1-2 ELAM-1 cells bound poorly to Le x and not at all to the glycoconjugates prepared with the structural analogs such as LNF I. That L1-2 ELAM-1 cells did not bind other monosialylated carbohydrates, such as 3′SL, 6′SL, LSTa or LSTc demonstrates that the binding to Sialyl Le a and Sialyl Le x is not due to non-specific charge effects, but rather reflects specific structural features of these oligosaccharides.
  • the low level of binding of ELAM-1 transfectants to Le a is consistent with an essential role of fucose in recognition, but shows that neuraminic acid (also known as sialic acid) also plays a key role.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Methods and compositions are disclosed for the inhibition of cancer metastases mediated by endothelial adhesion molecules. The present invention discloses that sialyl Lea and di-sialyl Lea, which are expressed at the surface of cancer cells, function as a binding partner for LEC-CAMs, such as ELAM-1, which are expressed at the surface of endothelial cells. The present invention also discloses that LEC-CAMs, such as ELAM-1, involved in cancer metastasis share a carbohydrate domain common to both sialyl Lea and sialyl Lex. Antibodies, saccharides, glycoconjugates, enzyme inhibitors and other compounds may be used in the methods of the present invention to inhibit the binding of malignant cells to endothelial cells for a variety of purposes in vivo and in vitro.

Description

    CROSS REFERENCE TO RELATED APPLICATION
  • This application is a continuation-in-part to Ser. No. 688,037, filed Apr. 19, 1991, which is incorporated herein by reference.[0001]
    • TECHNICAL FIELD
  • The present invention is generally directed toward the inhibition of cancer metastasis mediated by endothelial adhesion molecules, and more specifically, toward such inhibition through the use of saccharides, glycoconjugates, antibodies, enzyme inhibitors, and other agents which disrupt such binding of cancer cells to endothelia. [0002]
    • BACKGROUND OF THE INVENTION
  • Despite enormous investments of financial and human resources, cancer remains one of the major causes of death. Current cancer therapies cure only about fifty percent of the patients who develop a malignant tumor. In most human malignancies, metastasis is the major cause of death. [0003]
  • Metastasis is the formation of a secondary tumor colony at a distant site. It is a multistep process of which tumor invasion is an early event. Tumor cells locally invade host tissue barriers, such as the epithelial basement membrane, to reach the interstitial stroma, where they gain access to blood vessels (“hematogenous metastasis”) or lymphatic channels for further dissemination. After invading the endothelial layer of a vessel wall, the circulating tumor cells are dislodged into the circulation and arrest in the precapillary venules of the target organ by adherence 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 it originated. [0004]
  • Most cancer cells fail to survive in the circulation and it appears that normally the lining of blood vessels acts as a barrier to tumor cell extravasation. Endothelial injury or perturbation increases tumor metastasis. In addition, certain factors, such as cytokines, have been shown to substantially increase the adhesion of cancer cells to treated endothelium in vitro. Interleukin 1 (IL-1) and tumor necrosis factor (TNF), which are cytokines, each stimulate the biosynthesis and expression of a cell surface receptor called ELAM-1 (endothelial leukocyte adhesion molecule) ELAM-1 is a member of a family of calcium-dependent cell adhesion receptors, known as LEC-CAMs or selectins, which includes LECAM-1 and GMP-140 (also known as PADGEM or CD62). During an inflammatory response, ELAM-1 on endothelial cells functions as a “homing receptor” for leukocytes. Recently, ELAM-1 on endothelial cells was shown to mediate the increased adhesion of colon cancer cells to endothelium treated with cytokines (Rice and Bevilacqua, [0005] Science 246:1303-1306, 1989).
  • In most human malignancies, distant metastases are often too small to be detected at the time the primary tumor is treated. Furthermore, widespread initiation of metastatic colonies usually occurs before clinical symptoms of metastatic disease are evident. The size and age variation in metastases, their dispersed anatomical location, and their heterogeneous composition are all factors that hinder surgical removal and limit the concentration of anticancer drugs that can be delivered to the metastatic colonies. It has been estimated, for example, that in 1991 there will be over 60,000 deaths and over 150,000 new cases from just colorectal cancer in the U.S. alone. [0006]
  • Due to the difficulties in the current approaches to the treatment and prevention of metastases, there is a need in the art for improved methods and compositions for inhibiting metastasis mediated by endothelial adhesion molecules. The present invention fills this need, and further provides other related advantages. [0007]
    • SUMMARY OF THE INVENTION
  • Briefly stated, the present invention provides methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules. In one aspect, the present invention provides methods for inhibiting, within a biological preparation, the binding of malignant cells expressing sialyl Le[0008] a or di-sialyl Lea, to endothelial cells. In one embodiment, the method comprises incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM. In another embodiment, the method comprises incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1. In another embodiment, the method comprises incubating the malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by the malignant cells.
  • In another aspect of the present invention, methods are provided for inhibiting the spread of malignant cells expressing sialyl Le[0009] a or di-sialyl Lea, to secondary sites in a warm-blooded animal. In one embodiment, the method comprises administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM. In another embodiment involving hematogenous metastasis, the method comprises administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1. In another embodiment, the method comprises administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by the malignant cells.
  • In a related aspect, methods are provided for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Le[0010] a, di-sialyl Lea or sialyl Lex, to endothelial cells. In one embodiment, the method comprises incubating a biological preparation, containing endothelial cells expressing a LEC-CAM, with at least one agent capable of reacting with both sialyl Lea and sialyl Lex. In another embodiment, the method comprises incubating a biological preparation, containing endothelial cells expressing ELAM-1, with at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
  • In another related aspect, methods are provided for inhibiting the spread of malignant cells expressing sialyl Le[0011] a, di-sialyl Lea or dialyl Lex, to secondary sites in a warm-blooded animal. In one embodiment, the method comprises administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Lea and sialyl Lex. In another embodiment involving hematogenous metastasis, the method comprises administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
  • These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. [0012]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 describes pictorially a cell binding assay used to assess binding of ELAM-1 transfected cells to neoglycoproteins. [0013]
  • FIG. 2 graphically illustrates the relative binding of ELAM-1 transfected cells to certain neoglycoproteins. [0014]
  • FIG. 3 graphically illustrates the relative binding of ELAM-1 transfected cells to certain neoglycoproteins.[0015]
    • DETAILED DESCRIPTION OF THE INVENTION
  • Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter. [0016]
  • Antibody—as used herein, includes both monoclonal and polyclonal antibodies and may be an intact molecule, a fragment thereof, or a functional equivalent thereof. The antibody may be genetically engineered. Examples of antibody fragments include F(ab′)[0017] 2, Fab′, Fab and Fv.
  • Saccharide—as used herein, includes oligosaccharides, and may be naturally derived, synthetically prepared, portions of either, and derivatives of any of the foregoing. [0018]
  • Glycoconiugate—as used herein, includes a saccharide which is coupled to a non-saccharide molecule, e.g., a lipid or a polypeptide. [0019]
  • As noted above, the present invention is generally directed towards methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules. More specifically, the disclosure of the present invention shows that antibodies, saccharides, glycoconjugates therefrom or enzyme inhibitors may be used to inhibit the binding of malignant cells to endothelial cells for a variety of purposes in vivo and in vitro. [0020]
  • As described above, metastasis is a multistep process. During metastasis, cancer cells circulate through the microvascular and lymph systems and then migrate through the walls of the blood or lymph vessels to establish a new and aggressive tumor at a secondary organ site. A critical step in the metastasis process is the adherence of circulating cancer cells to the endothelial lining of blood vessel or lymph vessel walls. As disclosed within the present invention, the carbohydrates sialyl Le[0021] a and di-sialyl Lea, which are expressed at the surface of certain cancer cells, function as a ligand (i.e., binding partner) for LEC-CAMs, such as ELAM-1, which are expressed at the surface of endothelial cells. Therefore, for those cancer cells, metastasis involves the adherence of cancer cells to the endothelial cells via the binding of sialyl Lea and/or di-sialyl Lea on the cancer cells to adhesion molecules on endothelial cells. Other cancer cells express predominantly sialyl Lex, or sialyl Lex and sialyl Lea (and/or di-sialyl Lea). The present invention discloses that LEC-CAMs, such as ELAM-1, share a carbohydrate domain common to both sialyl Lea and sialyl Lex on malignant cells, and therefore agents can be produced which are capable of binding to both. Inhibition of the initial binding event between LEC-CAMs and sialylated structures by the methods of the present invention prevents the adhesion of metastatic cells to the endothelial lining of blood or lymph vessel walls, thereby eliminating the spread of metastatic cells to secondary organs. Suitable blocking agents include those which inhibit the binding of malignant cells expressing sialyl Lea, di-sialyl Lea, or sialyl Lex, to endothelial cells expressing LEC-CAM adhesion molecules such as ELAM-1. Representative agents include antibodies, saccharides and glycoconjugates therefrom.
  • The antibodies employed in the present invention may be polyclonal or monoclonal antibodies. Briefly, polyclonal antibodies may be produced by immunization of an animal and subsequent collection of its sera. Immunization is accomplished, for example, by a systemic administration, such as by subcutaneous, intrasplenic or intramuscular injection, into a rabbit, rat or mouse. It is generally preferred to follow the initial immunization with one or more booster immunizations prior to sera collection. Such methodology is well known and described in a number of references. [0022]
  • Monoclonal antibodies (MAbs) suitable within the present invention include those of murine or human origin, or chimeric antibodies such as those which combine portions of both human and murine antibodies (i.e., antigen binding region of murine antibody plus constant regions of human antibody). Human and chimeric antibodies may be produced using methods known by those skilled in the art. Human antibodies and chimeric human-mouse antibodies are advantageous because they are less likely than murine antibodies to cause the production of anti-antibodies when administered clinically. [0023]
  • MAbs may be generally produced by the method of Kohler and Milstein ([0024] Nature 256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976). Briefly, the lymph nodes and/or spleens of an animal immunized with sialyl Lea or di-sialyl Lea are fused with myeloma cells to form hybrid cell lines (“hybridomas” or “clones”). Each hybridoma secretes a single type of immunoglobulin and, like the myeloma cells, has the potential for indefinite cell division. It may be desirable to couple such molecules to a carrier to increase their immunogenicity. Suitable carriers include keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof. An alternative to the production of MAbs via hybridomas is the creation of MAb 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, 1989). Selection of antibodies exhibiting appropriate specificity may be performed in a variety of ways which will be evident to those skilled in the art. Typically, such antibodies will selectively bind with an affinity of about 107 liters/mol or higher.
  • Representative examples of MAbs suitable within the present invention include N-19-9 and HECA-452 for sialyl Le[0025] a, and FH-7 for di-sialyl Lea. MAb N-19-9 is available from ATCC (American Type Tissue Collection, Rockville, Md.) as ATCC HB 8059 or may be produced as described in U.S. Pat. No. 4,471,057 (and Somatic Cell Genet. 5:957-971, 1979; J. Biol. Chem. 257:14365, 1982). MAb HECA-452 may be produced according to Duijvestijn et al., Am. J. Path. 130:147-155, 1988. FH-7 may be produced according to Nudelman et al., J. Biol. Chem. 261:5487, 1986.
  • In addition to antibodies which are capable of binding to sialyl Le[0026] a, di-sialyl Lea or sialyl Lex, saccharides and glycoconjugates therefrom may also inhibit the binding of metastatic cells expressing sialyl Lea, di-sialyl Lea or sialyl Lex, to endothelia. As used herein, the terms “sialyl Lea” and “di-sialyl Lea” represent structures I and II, respectively, as follows:
    Figure US20030133937A1-20030717-C00001
  • Neu5Ac represents sialic acid; Gal represents galactose; GlcNAc represents N-acetyl-glucosamine; Fuc represents fucose and R is typically a ceramide (with a glucose residue interposed) or a protein. Sialyl Le[0027] x is an isomer of sialyl Lea wherein the Gal-GlcNAc linkage is β1-4 and the Fuc-GlcNAc linkage is α1→3. Saccharides suitable within the present invention include the carbohydrate portion of sialyl Lea or di-s-ialyl Lea (i.e., formula I or II minus R), and derivatives of either, including those which cross-react with both sialyl Lea and sialyl Lex. Derivatives of these compounds include substitution of individual saccharide residues with other saccharide residues and/or with non-saccharide molecules such as hexyl rings without hydroxyl groups. For example, the internal GlcNAc may be replaced with another saccharide residue such as a glucose (Glc). Alternatively (or in addition to substitutions), the carbohydrate portion of sialyl Lea, di-sialyl Lea, or derivatives thereof, may be truncated by deletion of one or more saccharide residues. For example, a tetrasaccharide may be created with the structure:
    Figure US20030133937A1-20030717-C00002
  • Given the teachings described herein, it will be evident to those skilled in the art that other saccharides will be suitable within the present invention. [0028]
  • A saccharide may be coupled to a non-saccharide molecule to form a glycoconjuqate. For example, a saccharide may be linked to a polyacrylamide. Alternatively, a saccharide may be linked to a lipid. Typical lipids include ceramide, i.e., sphingolipid bases which are acylated on the amine with a fatty acid. For example, sialyl Le[0029] a, di-sialyl Lea, or a saccharide cross-reaction with sialyl Lea and sialyl Lex may be linked to a ceramide. Alternatively, a saccharide may be bonded to an amino acid or an amino acid-containing molecule, such as a peptide, a polypeptide or a protein. Saccharides are naturally linked to an amino acid or amino acid-containing molecule via the hydroxyl group of a serine or threonine amino acid residue, but can also be linked through other groups such as an amino group.
  • Saccharides and glycoconjugates provided by the present invention may be represented by structures III and IV as follows: [0030]
    Figure US20030133937A1-20030717-C00003
  • R includes H, OH, lipid, ceramide, or one or more amino acids; x, y and z are independently selected from saccharides, or either y or z or both may be absent. [0031]
  • Numerous methods for preparing saccharides and glycoconjugates are well known to those skilled in the art. Saccharides may be prepared synthetically using chemical, and/or enzymatic, reagents and techniques. For example, sialyl Le[0032] a saccharides have been prepared by enzymatic synthesis (e.g., Palcic et al., Carbohydr. Res. 190:1-11, 1989). Glycoconjugates may be prepared, for example, through reductive amination. The method of Zopf et al. (Meth. Enzymol. 50:171-175, 1978; Jeffrey et al., Biochem. Biophys. Res. Commun. 62:608-613, 1975) involves 4-aminophenethylamine derivatives of saccharides via reductive amination using sodium borohydride. In brief, sugars are first reacted with the amino reagent by dissolving them in the neat reagent for 15 hours. Sodium borohydride in ethanol is then added. After 5 hours, the product is separated from the reagent by gel filtration and ion exchange chromatography. The derivatives may then be coupled to a molecule containing a group which is reactive with amines. The same amine derivative may be coupled to saccharides using sodium cyanoborohydride. (Svensson et al., J. Immunol. Meth. 25:323-335, 1979). In brief, a sugar is dissolved in water, and the same volume of amine (a 170-fold molar excess) is added together with sodium cyanoborohydride (a ten-fold molar excess). The reduction is performed at pH 8 for 48 hours, and the product purified by gel chromatography. Coupling to different molecules, such as proteins, may be performed by the isothiocyanate coupling method.
  • Another example of a reagent suitable for preparing glycoconjugates by reductive amination is p-trifluoroacetamidoaniline (TFAN). The reductive amination reaction is carried out in aqueous solution overnight at pH 5-6 with sodium cyanoborohydride as the reducing agent. Typically, a 5-fold excess of TFAN is used. TFAN-derivatized saccharides are generally protected from oxidation by N-acetylation, e.g., by treatment with methanolic acetic anhydride, to yield TFAc-derivatives. Prior to conjugation, the N-trifluoroacetamido protective group is removed by treatment of the TFAc derivative with aqueous ammonia or 0.5 M sodium hydroxide for 3 hours. Conjugation of the derivatives to molecules, for example to proteins such as bovine serum albumin (BSA), may be achieved by isothiocyanate coupling methods. Other examples of suitable reagents and reactions include p-tetradecylaniline derivatives of saccharides and the preparation of aminoalditols by oxidation of saccharide TFAN derivates with cerium ammonium sulfate (Lindenberg et al., [0033] J. Reprod. Fert. 89:431-439, 1990).
  • The inhibition of the binding of cancer cells expressing sialyl Le[0034] a, di-sialyl Lea or sialyl Lex, to endothelia has a variety of in vitro and in vivo uses. Sialyl Lea and di-sialyl Lea are type 1 carbohydrate chains (i.e., have a Galβ1→3GlcNAc polylactosamine unit structure) and sialyl Lex is a type 2 carbohydrate chain (i.e., has a Galβ1→4 GlcNAc polylactosamine unit structure. A number of cancer cells, such as colorectal and pancreatic, have a prevalence of type 1 carbohydrate chains including sialyl Lea and di-sialyl Lea. Other cancer cells, such as breast, lung and ovarian, have a prevalence of type 2 carbohydrate chains including sialyl Lex.
  • Regarding in vitro aspects, as noted above, the present invention provides methods for inhibiting the binding of cancer cells to endothelia in a biological preparation. Representative examples of biological preparations include blood vessel and/or lymph vessel endothelia in combination with a malignancy. The endothelia and the malignancy may be in the form of tissue or cells removed from an organism, or cultured cells. In one embodiment, the method comprises incubating a biological preparation, which contains malignant cells expressing sialyl Le[0035] a, di-sialyl Lea or sialyl Lex and endothelial cells expressing a LEC-CAM, with an effective amount of at least one agent, such as an antibody, saccharide or glycoconjugate as described above. In another embodiment, the method comprises incubating malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by the cells. Suitable enzyme inhibitors include inhibitors of glycosyltransferases. Representative examples of inhibitors for glycosyltransferases include inhibitors for fucosyltransferases (e.g., as described by Palcic et al., J. Biol. Chem. 264:17174-17181, 1989), for N-acetylglucosaminyltransferases (e.g., as described by Palcic et al., J. Biol. Chem. 265:6759-6769, 1990), and for sialyltransferases (e.g., as described by Broquet et al., J. Neurochem. 54:388-394, 1990; Karaivanova et al., Cancer Biochem. Biophys. 11:311-315, 1990).
  • The present invention also provides methods for inhibiting metastasis in a warm-blooded animal such as a human. In one embodiment, the method comprises administering to a warm-blooded animal an effective amount of at least one agent, such as an antibody, saccharide or glycoconjugate as described above. In another embodiment, the method comprises administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor (as described above) that inhibits the biosynthesis of sialyl Le[0036] a or di-sialyl Lea by malignant cells. It will be evident to those skilled in the art how to determine the optimal effective dose for a particular agent or enzyme inhibitor, e.g., based upon in vitro and in vivo studies in non-human animals. A variety of routes of administration may be used. Typically, administration will be intravenous, intracavitory (e.g., in pleural or peritoneal cavities), or in the bed of a resected tumor.
  • An agent may be administered as a composition, i.e., in combination with a pharmaceutically acceptable carrier or diluent, such as physiological saline. It will be recognized by those skilled in the art that an agent and a composition may be prepared in a sterile form. Moreover, an agent may be administered in combination with an immunotherapeutic or chemotherapeutic agent. When such a combination is desired, each substance may be administered sequentially, simultaneously, or combined and administered as a single composition. Diagnostic techniques, such as CAT scans for tumors, may be performed prior to and subsequent to administration to confirm effectiveness. [0037]
  • The following examples are offered by way of illustration and not by way of limitation. [0038]
    • EXAMPLES Example 1 Glycoconjugates and Assays
  • Synthetic Glycoproteins (Neoglycoproteins) [0039]
  • Neoglycoproteins were produced by BioCarb AB (Lund, Sweden) by chemically coupling 10-20 moles of a specific oligosaccharide to 1 mole of nonglycosylated albumin, bovine (BSA) or human (HSA). The resulting synthetic glycoprotein (neoglycoprotein) contains multiple copies of the identical carbohydrate sequence, thereby producing a well characterized, mutivalent glycoconjugate which is extremely effective for studying carbohydrate-protein interactions. Depending on the size of the oligosaccharide, three different chemical spacer arms were used to couple the oligosaccharides to proteins 1) p-aminophenyl (PAP); 2) aminophenylethyl (APE); and 3) acetyl phenylene diamine were used to couple the shorter oligosaccharides to albumin since they will retain the anomeric configuration of the reducing sugars which may be involved in a potential binding site. APD was used to couple the larger sugars to protein by reductive amination, which converts the reducing sugar to an aminoalditol. [0040]
  • Direct Binding of Antibodies to Synthetic Glycoproteins (Neoglycoproteins) [0041]
  • Synthetic glycoproteins were coated onto microtiter plates by filling each well with 100 ng of the neoglycoprotein in 100 μl of 0.15 M sodium chloride, 0.01 M sodium phosphate, 0.1% sodium azide, pH 7.4, (PBS-azide) overnight at 4° C. Standard enzyme-linked immunoassays (ELISA) were then performed on the solid phase carbohydrate structures using the appropriate antibody diluted to 10 μg/ml. [0042]
  • Production of ELAM-1 cDNA Transfected Cell Lines [0043]
  • L1-2/pMRB107 cells (L1-2[0044] ELAM-1) were prepared by transfecting the ELAM-1 gene into the murine pre-B cell line L1-2 (Gallatin et al., Nature 304:30-34, 1983). A cDNA clone encoding ELAM-1 was obtained from a cDNA library made from activated human umbilical vein endothelial cell cultures by polymerase chain reaction (PCR) amplification. The ELAM-1 gene was inserted downstream of the hCMV promoter in pMRB101 [a derivative of EE6 which contains the E. coli gpt gene (Mulligan and Berg, Proc. Nat'l. Acad. Sci. USA 78:2072, 1981; Stephens and Corbett, N.A.R. 17:7110, 1989)]. DNA was introduced into L1-2 cells by electroporation and the cells selected for resistance to mycophenolic acid. A population of cells staining brightly for ELAM-1 were selected by FACS and cloned by limiting dilution. These cells are ELAM-1hi LFA-1mod CD45hi CD44neg LECAM-1neg, differing from the parent cell line or control vector transfectants only in their expression of ELAM-1. L1-2/pMRB101 (L1-2vector) cells are a similarly transformed derivative of L1-2 transfected with pMRB101 and lacking ELAM-1 expression.
  • Cell Binding Assays [0045]
  • One hundred microliter samples of each synthetic glycoconjugate in phosphate buffered saline (PBS), pH 7.2, were absorbed onto glass wells of 8-chamber slides (LabTek) for two hours at RT. For some experiments glass slides were pre-coated with rabbit anti-human serum albumin (Sigma) at 200 μg/ml overnight at 4° C. and washed with PBS prior to the addition of the glycoconjugate. After blocking with 5% NBS/10 mM HEPES/Dulbecco's Modified Eagles Medium (DMEM), pH 7.0 (CM), L1-2[0046] ELAM-1 or L1-2vector cells were applied to each well (1.5×106/0.15 ml in CM). After a 25 minute incubation at RT on a rotating shaker at 50 rpm, the tops of the wells were removed and the slides washed 3× in DMEM and then fixed by incubation in 1.5% glutaraldehyde (Kodak)/DMEM. Three to six 100× fields were counted for each data point.
  • Inhibition of Binding of ELAM-1 Containing Cells by Compounds [0047]
  • One hundred and twenty nanograms of Sialyl Le[0048] a-HSA or Sialyl Lex-HSA dissolved in 100 μl of phosphate-buffered saline were absorbed per well of an 8 chambered glass (LabTek) slide for 2 hours at room temperature. During this period, L1-2ELAM-1 cells were pre-incubated for 20 minutes on ice with-- increasing concentrations of Sialyl Lea-HSA at 107 cells/ml. After washing and blocking the wells in Complete Medium (CM, 5% normal bovine serum, 10 mM HEPES, pH 7.0, DMEM), L1-2ELAM-1 cells pre-incubated with compounds were added (1×107 cells/ml) and incubated at room temperature while rotating at 50 rpm. After 25 minutes, slides were washed 3 times in Dulbecco's Modified Eagles Medium (DMEM) and then fixed in 1.5% glutaraldehyde/DMEM.
    • Example 2 Carbohydrate Structure Recognized by ELAM-1
  • The sensitive binding assay described in Example 1 uses cells permanently transfected with ELAM-1 cDNA. The mouse pre-B cell line, L1-2, transfected with ELAM-1 cDNA (L1-2[0049] ELAM-1), but not vector control cDNA, L1-2vector expresses very high levels of ELAM-1. The ELAM-1 expressed by these cells is functional as L1-2ELAM-1 cells are adhesive for neutrophils and this adhesion is blocked by anti-ELAM-1 monoclonal antibodies. When added to glass slides coated with various synthetic glycoconjugates, L1-2ELAM-1 cells bound selectively to Sialyl Lea and Sialyl Lex neoglycoproteins, but not to a number of other glycoconjugates. L1-2ELAM-1 cells also bound, albeit more weakly, to Lea neoglycoprotein. The binding to Lea is significant as L1-2ELAM-1 cells bound poorly to Lex and not at all to the glycoconjugates prepared with the structural analogs such as LNF I. That L1-2ELAM-1 cells did not bind other monosialylated carbohydrates, such as 3′SL, 6′SL, LSTa or LSTc demonstrates that the binding to Sialyl Lea and Sialyl Lex is not due to non-specific charge effects, but rather reflects specific structural features of these oligosaccharides. The low level of binding of ELAM-1 transfectants to Lea is consistent with an essential role of fucose in recognition, but shows that neuraminic acid (also known as sialic acid) also plays a key role.
  • All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0050]
  • From the foregoing, it will be evident that, although specific embodiments of the invention have been described herein for purposes of illustration, various modification may be made without deviating from the spirit and scope of the invention. [0051]

Claims (18)

1. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM, comprising:
incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM.
2. The method of claim 1 wherein the agent is a saccharide, a glycoconjugate or an antibody that inhibits the binding of sialyl Lea or di-sialyl Lea to a LEC-CAM.
3. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1, comprising:
incubating the biological preparation with at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1.
4. The method of claim 3 wherein the agent is a saccharide, a glycoconjugate or an antibody that inhibits the binding of sialyl Lea or di-sialyl Lea to ELAM-1.
5. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM, comprising:
incubating said malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by said malignant cells.
6. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1, comprising:
incubating said malignant cells with at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by said malignant cells.
7. A compound having the formula:
Figure US20030133937A1-20030717-C00004
wherein x, y and z are independently selected from saccharides or y or z or both are not present, and R is H, OH, lipid, ceramide, or one or more amino acids, with the proviso that x, y and z are not present in the combination wherein x is GlcNAc, y is Gal and z is Glc.
8. A compound having the formula:
Figure US20030133937A1-20030717-C00005
wherein x, y and z are independently selected from saccharides or y or z or both are not present, and R is H, OH, lipid, ceramide, or one or more amino acids, with the proviso that x, y and z are not present in the combination wherein x is GlcNAc, y is Gal and z is Glc.
9. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea or di-sialyl Lea, to secondary sites, comprising:
administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing a LEC-CAM.
10. The method of claim 9 wherein the agent is a saccharide, a glycoconjugate or an antibody that inhibits the binding of sialyl Lea or di-sialyl Lea to a LEC-CAM.
11. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea or di-sialyl Lea, to secondary sites by hematogenous metastases, comprising:
administering to a warm-blooded animal an effective amount of at least one agent that inhibits the binding of malignant cells expressing sialyl Lea or di-sialyl Lea, to endothelial cells expressing ELAM-1.
12. The method of claim 11 wherein the agent is a saccharide, a glycoconjugate or an antibody that inhibits the binding of sialyl Lea or di-sialyl Lea to ELAM-1.
13. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea or di-sialyl Lea, to secondary sites, comprising:
administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by said malignant cells.
14. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea or di-sialyl Lea, to secondary sites by hematogenous metastases, comprising:
administering to a warm-blooded animal an effective amount of at least one enzyme inhibitor that inhibits the biosynthesis of sialyl Lea or di-sialyl Lea by said malignant cells.
15. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea, di-sialyl Lea or sialyl Lex, to endothelial cells expressing a LEC-CAM, comprising:
incubating the biological preparation with at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
16. A method for inhibiting within a biological preparation the binding of malignant cells expressing sialyl Lea, di-sialyl Lea or sialyl Lex, to endothelial cells expressing ELAM-1, comprising:
incubating the biological preparation with at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
17. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea, di-sialyl Lea or sialyl Lex, to secondary sites, comprising:
administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
18. A method for inhibiting in a warm-blooded animal the spread of malignant cells expressing sialyl Lea, di-sialyl Lea or sialyl Lex, to secondary sites by hematogenous metastases, comprising:
administering to a warm-blooded animal an effective amount of at least one agent capable of reacting with both sialyl Lea and sialyl Lex.
US10/269,842 1991-04-19 2002-10-10 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules Abandoned US20030133937A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/269,842 US20030133937A1 (en) 1991-04-19 2002-10-10 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US68803791A 1991-04-19 1991-04-19
US72177191A 1991-06-25 1991-06-25
US08/238,684 US6121233A (en) 1991-04-19 1994-05-05 Methods for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
US09/447,532 US6465434B1 (en) 1991-04-19 1999-11-23 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
US10/269,842 US20030133937A1 (en) 1991-04-19 2002-10-10 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/447,532 Continuation US6465434B1 (en) 1991-04-19 1999-11-23 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules

Publications (1)

Publication Number Publication Date
US20030133937A1 true US20030133937A1 (en) 2003-07-17

Family

ID=27104142

Family Applications (3)

Application Number Title Priority Date Filing Date
US08/238,684 Expired - Lifetime US6121233A (en) 1991-04-19 1994-05-05 Methods for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
US09/447,532 Expired - Fee Related US6465434B1 (en) 1991-04-19 1999-11-23 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
US10/269,842 Abandoned US20030133937A1 (en) 1991-04-19 2002-10-10 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US08/238,684 Expired - Lifetime US6121233A (en) 1991-04-19 1994-05-05 Methods for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
US09/447,532 Expired - Fee Related US6465434B1 (en) 1991-04-19 1999-11-23 Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules

Country Status (1)

Country Link
US (3) US6121233A (en)

Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003249641B2 (en) * 2002-05-16 2009-08-20 Glycomimetics, Inc. Compounds and methods for inhibiting selectin-mediated function
AT413486B (en) 2002-07-03 2006-03-15 Igeneon Krebs Immuntherapie USE OF AN ANTIBODY DIRECTED AGAINST LEWIS ANTIGENE
US20040096396A1 (en) * 2002-07-03 2004-05-20 Glycomimetics, Inc. Compositions and methods for diagnosis and therapy of medical conditions involving angiogenesis
US20040219158A1 (en) * 2003-05-02 2004-11-04 Glycomimetics, Inc. Compositions and methods for diagnosis and therapy of medical conditions involving infection with pseudomonas bacteria
CA2546784A1 (en) * 2003-11-19 2005-06-16 Glycomimetics, Inc. Glycomimetic antagonists for both e- and p-selectins
US7361644B2 (en) * 2003-11-19 2008-04-22 Glycomimetics, Inc. Specific antagonist for both E- and P-selectins
US20090036386A1 (en) * 2005-05-25 2009-02-05 Glycomimetics, Inc Heterobifunctional compounds for selectin inhibition
ATE528311T1 (en) * 2005-08-09 2011-10-15 Glycomimetics Inc GLYCOMIMETIC INHIBITORS OF PA-IL-LECTIN, PA-IIL-LECTIN OR BOTH LECTINS FROM PSEUDOMONAS
EP2264043B1 (en) 2005-09-02 2017-11-08 GlycoMimetics, Inc. Heterobifunctional pan-selectin inhibitors
US20090176717A1 (en) * 2006-06-01 2009-07-09 Glycomimetics, Inc. Galactosides and thiodigalactosides as inhibitors of pa-il lectin from pseudomonas
JP5298020B2 (en) 2006-10-12 2013-09-25 グリコミメティクス， インコーポレイテッド Glucose mimic substitution of hexose and N-acetylhexosamine
NZ598863A (en) * 2007-02-09 2013-11-29 Glycomimetics Inc Methods of use of glycomimetics with replacements for hexoses and n-acetyl hexosamines
US8039442B2 (en) * 2007-07-18 2011-10-18 Glycomimetics, Inc. Compounds and methods for treatment of sickle cell disease or complications associated therewith
WO2009073911A1 (en) * 2007-12-10 2009-06-18 Mater Medical Research Institute Treatment and prophylaxis
US8895510B2 (en) * 2008-04-08 2014-11-25 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
WO2009152245A1 (en) * 2008-06-13 2009-12-17 Glycomimetics, Inc. Treatment of cancers of the blood using selected glycomimetic compounds
JP5726171B2 (en) * 2009-05-01 2015-05-27 グリコミメティックス インコーポレイテッド Heterobifunctional inhibitors of E-selectin and CXCR4 chemokine receptors
US8921328B2 (en) 2010-09-14 2014-12-30 Glycomimetics, Inc. E-selectin antagonists
WO2013096926A1 (en) 2011-12-22 2013-06-27 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
AU2013355238B2 (en) 2012-12-07 2017-12-14 Glycomimetics, Inc. Compounds, compositions and methods using E-selectin antagonists for mobilization of hematopoietic cells
EP3227310B1 (en) 2014-12-03 2019-07-31 GlycoMimetics, Inc. Heterobifunctional inhibitors of e-selectins and cxcr4 chemokine receptors
CA3009836A1 (en) 2016-01-22 2017-07-27 Glycomimetics, Inc. Glycomimetic inhibitors of pa-il and pa-iil lectins
WO2017151708A1 (en) 2016-03-02 2017-09-08 Glycomimetics, Inc. Methods for the treatment and/or prevention of cardiovescular disease by inhibition of e-selectin
EP3497131B1 (en) 2016-08-08 2022-03-09 GlycoMimetics, Inc. Combination of t-cell checkpoint inhibitors with inhibitors of e-selectin or cxcr4, or with heterobifunctional inhibitors of both e-selectin and cxcr4.
CA3037850A1 (en) 2016-10-07 2018-04-12 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
WO2019108750A1 (en) 2017-11-30 2019-06-06 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
CN111566117A (en) 2017-12-29 2020-08-21 糖模拟物有限公司 Heterobifunctional inhibitors of E-selectin and galectin-3
CA3091454A1 (en) 2018-03-05 2019-09-12 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4471057A (en) * 1981-06-30 1984-09-11 The Wistar Institute Detection of colorectal carcinoma
DK17885D0 (en) * 1985-01-14 1985-01-14 Karlsson Karl Anders ANTIVIRAL AGENT
US4876199A (en) * 1985-04-04 1989-10-24 Fred Hutchinson Cancer Research Center Hybridomas producing monoclonal antibodies to mono-, di-, and trifucosylated type 2 chain
US4851511A (en) * 1986-01-30 1989-07-25 Fred Hutchinson Cancer Research Center Monoclonal antibody that specifically binds to disialosyl Lea
DE3787403D1 (en) * 1986-05-09 1993-10-21 Pulverer Gerhard Use of specific monosaccharides for the manufacture of a medicament for the prevention of metastases of malignant tumors.
EP0381310A1 (en) * 1989-01-30 1990-08-08 The Biomembrane Institute Monoclonal antibodies directed to tumor-associated gangliosides and fucogangliosides and method for production thereof
US5272263A (en) * 1989-04-28 1993-12-21 Biogen, Inc. DNA sequences encoding vascular cell adhesion molecules (VCAMS)
EP0408859B1 (en) * 1989-05-23 1995-08-09 Otsuka Pharmaceutical Co., Ltd. Monoclonal antibodies to activated endothelial cells
WO1991019501A1 (en) * 1990-06-15 1991-12-26 Cytel Corporation Intercellular adhesion mediators
JP3096305B2 (en) * 1990-07-17 2000-10-10 ボード オブ リージェンツ オブ ザ ユニバーシティ オブ オクラホマ Glycoprotein ligands for GMP-140
US5211937A (en) * 1990-07-30 1993-05-18 Glycomed Incorporated Method of determining a site of inflammation utilizing elam-1 ligands
US5143712A (en) * 1990-07-30 1992-09-01 Glycomed Incorporated Method of determining a site of inflammation utilizing elam-1 ligands
NZ240316A (en) * 1990-10-25 1996-12-20 Univ Michigan Compound for treating disease mediated by the elaboration of elam-1 on endothelial cells
HUT64476A (en) * 1990-11-23 1994-01-28 Gen Hospital Corp Process for inhibiting interacton between proteins and carbohydrate causing cell-adhesion

Also Published As

Publication number Publication date
US6121233A (en) 2000-09-19
US6465434B1 (en) 2002-10-15

Similar Documents

Publication Publication Date Title
US6465434B1 (en) Methods and compositions for the inhibition of cancer metastasis mediated by endothelial adhesion molecules
EP0580763B1 (en) Compositions and methods for endothelial binding
Brooks et al. Altered glycosylation of proteins in cancer: what is the potential for new anti-tumour strategies
US6124267A (en) O-glycan inhibitors of selectin mediated inflammation derived from PSGL-1
EP0730608B1 (en) Compositions comprising complement related proteins and carbohydrates, and methods for producing and using said compositions
Kobata et al. Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours
ES2208759T3 (en) O-GLICANS INHIBITORS OF THE INFLAMMATION MEDIATED BY SELECTINE.
US5801044A (en) Nucleic acid encoding an antibody that inhibits cell adhesion protein-carbohydrate interactions
US20100249383A1 (en) Novel Ligand Involved In The Transmigration Of Leukocytes Across the Endothelium and Uses Therefor
EP0972782B1 (en) Monoclonal antibody which recognizes the oligosaccharide n-glycolylated-galactose-glucose sialic acid in malignant tumors, and composition containing it
Kunzendorf et al. A sialyl-Lex-negative melanoma cell line binds to E-selectin but not to P-selectin
WO1993017033A1 (en) Inhibition of cell adhesion by chemically-defined oligosaccharides, their derivatives, mimetics, and antibodies directed thereto
US6096725A (en) Methods of using αGal oligosaccharides as immune system targeting agents
AU659808B2 (en) Inhibition of metastasis potential and invasiveness by oligosaccharides or oligosaccharide antigens or antibodies
US6294523B1 (en) Extended type 1 chain glycosphingolipids as tumor-associated antigens
WO1995020400A1 (en) Anti-inflammatory containing monoclonal antibodies having reactivity with sialyl-lewis x sugar chains originating in hemangioendothelial cell membrane
JPH06145190A (en) Chemically defined oligosaccharide, its derivative or analogue, and inhibition of metastatic potential and infiltration of tumoral cell by antibodies against them
Lowe The carbohydrate components of selectin ligands
KR100463567B1 (en) Peptides and Zero-Glycan Inhibitors of Selectin-Mediated Inflammation
EP1428832A2 (en) Peptide and O-glycan inhibitors of selectin mediated inflammation
Kim Pathophysiological roles of selectins in colon carcinoma biology
HK1066813A (en) Peptide and o-glycan inhibitors of selectin mediated inflammation

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION