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  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
• • 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
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan

Definitions

• the present invention relates generally to new human tumor-associated antigens.
• This invention is more particularly related to extended type 1 chain glycosphingolipids and their uses, e.g., as immunogens and as tumor markers.
• cancer Despite enormous investments of financial and human resources, cancer remains one of the major causes of death. Current cancer therapies cure only about 50% 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.
• distant metastases are often too small to be detected at the time the primary tumor is treated.
• 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. Therefore, detection of malignancies prior to dissemination of the tumor cells from the primary site is needed to enhance the effectiveness of current cancer therapies.
• Type 1 chain antigens are abundant in normal cells and tissues, and also are cancer-associated.
• 2 ⁇ 3 sialylated Le antigen (the CA 19-9 antigen defined by the N19-9 antibody) is a cancer- associated type 1 chain-antigen.
• cancer diagnostic methods based on the detection of these known antigens have been hampered by high false positive and/or high false negative incidences.
• the present invention provides isolated compounds and methods of screening for cancers by detecting such compounds.
• the present invention provides an isolated compound, with or without fucosyl and/or sialyl residues, having the formula:
• n is 0 or an integer of 1 or more, there are at least two fucosyl and/or one or more sialyl residues, Gal represents galactose, Glc represents glucose, GlcAc represents N- acetylglucosamine, Cer represents a ceramide, and wherein said at least two fucosyl residues are linked to the GlcNAc residues via an ⁇ l ⁇ 4 linkage and/or to the terminal Gal residue via an ⁇ l ⁇ 2 linkage and said one or more sialyl residues are linked to the terminal Gal residue via an ⁇ 2 ⁇ 3 linkage and/or to one or more of the subterminal GlcNAc residues via an a2 ⁇ 6 linkage.
• the present invention provides the above-described isolated compound having the formula: ⁇ NeuAc ⁇ 2 ⁇ NeuAc ⁇ 2 ⁇ NeuAc ⁇ 2
• Fuc represents fucose and NeuAc represents N-acetylneuraminic acid.
• the invention provides the first-described compound having the formula:
• Fuc represents fucose and NeuAc represents N-acetylneuraminic acid.
• NeuAc represents N-acetylneuraminic acid.
• the present invention provides an isolated compound having the formula:
• the present invention provides an isolated compound comprising an epitope having the formula:
• any of the compounds of the present invention may be used as an immunogen for the production of polyclonal or monoclonal antibodies.
• methods for screening for cancer comprise (a) isolating a biological sample from a warm-blooded animal; and (b) testing the sample for the presence or amount of a compound.
• Figure 1 is the H-NM spectrum of extended sialyl Le from chemical shift at 4.20 ppm to 5.60 ppm covering sugar I(Glc), II(Gal), III(GlcNAc), IV(Gal), V(GlcNAc) and NI(Gal as well as fucose linked to III Glc Ac identified as F ⁇ j and fucose linked to N
• FJJJ are indicated as F v -1 and Fni-1.
• spectrum C5 proton of fucoses are
• Cis is a Cis double bond of sphingosine and R-5 and R-4 indicate spectrum of sphingosine.
• the present invention is generally directed towards compounds and methods relating to the detection of cancers. More specifically, the disclosure of the present invention shows that lacto-series type 1 chain occurs in extended forms in cancer tissues.
• type 1 chain lactosamine (Gal ⁇ l- ⁇ Glc ⁇ Ac) is known to be abundant in normal cells and tissues. Although polylactosamine antigens having an extended type 2 chain (i.e., Gal ⁇ l— ⁇ 4Glc ⁇ Ac core structure is repeated) have been detected, those with an extended type 1 chain have not been detected. Thus, lacto-series type 1 chain has traditionally been considered not to occur in extended form.
• a slow-migrating sialyl-Lewis (sLe ) active glycosphingolipid (GSL) was purified to homogeneity from the monosialyl ganglioside fraction of the colonic adenocarcinoma cell line Colo205. This compound was purified by HPLC and preparative HPTLC in two
• Le epitopes may be present as extended type 1 chains with additional
• lacto-series type 1 chain compounds may be isolated from biological starting materials, such as cancer tissue, or synthesized chemically (and/or enzymatically) following structural identification.
• the structure of carbohydrates bound to either lipids or proteins may be determined based on degradation, mass spectrometry, including electron-impact direct-probe (El) and fast atom bombardment (FAB), and methylation analysis (techniques described below and, for example, in Nudelman et al., J. Biol. Chem. 261:5487-5495, 1986).
• Degradation analysis may be accomplished chemically and/or enzymatically, e.g., by glycosidases.
• the carbohydrate sequence suggested by degradation analysis may be determined by methylation analysis (e.g., Hakomori, J. Biochem.
• El mass spectrometry may be performed on permethylated glycans or after the appropriate degradation of intact glycans (e.g., Kannagi et al., J. Biol. Chem. 259:8444-8451, 1984; Nudelman et al, J. Biol. Chem. 263:13942-13951, 1988).
• 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.
• carbohydrate or derivatives thereof or non-carbohydrate functional equivalents thereof may be synthesized using techniques well known to those of ordinary skill in the art.
• the compounds of the present invention may be used as immunogens for the production of polyclonal and monoclonal antibodies (MAbs).
• Polyclonal antibodies may be produced by standard methodologies. For example, briefly, polyclonal antibodies may be produced by immunization of an animal with a compound of the present invention and subsequent collection of its sera. It is generally preferred to follow the initial immunization with one or more boosters prior to sera collection.
• MAbs may be generally produced by the method of Kohler and Milstein (Nature 256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976).
• hybridomas secretes a single type of immunoglobulin and, like the myeloma cells, has the potential for indefinite cell division.
• 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 a desired specificity may be performed in a variety of ways well known to those of ordinary skill in the art.
• Suitable carriers include inactivated bacteria, keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof.
• all or a portion of the carbohydrate residues of the GSLs Le -Le or Le -Le may be combined with a carrier.
• a compound of the present invention may be combined with a carrier by a variety of means, including adsorption and covalent attachment.
• a representative example of the use of a compound of the present invention as an inmunogen is the immunization of mice with Le /Le antigen.
• Le /Le isolated from Colo205 cells was combined with a suspension of acid-treated Salmonella minnesotae, injected via tail vein into BALB/c mice, and the injection repeated three times with 10-day intervals. Following the final injection, splenocytes of immunized mice were harvested and fused with myeloma cells.
• ATCC American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110 USA
• the hybridoma produces a MAb 1MH2 with an IgG3 isotype.
• Le and/or Le -Le antigens may be used to screen for cancers.
• NCC-ST-421 established according to Watanabe et al., Jpn. J. Cancer Res (Gann) 76:43-52, 1985), respectively, of neutral glycolipid fractions prepared from various tumor samples.
• samples include tissue from colonic cancer, breast cancer, Hodgkin's disease, a a gallbladder cancer and embryonal rhabdomyosarcoma.
• the GSL Le -Le was not detected in glycolipid fractions from normal tissue from spleen, liver, kidney, placenta and lung. Given the teachings provided herein, it would be evident to those of ordinary skill in the art that a variety of means for detecting tumor-associated extended type 1 antigens
• antibodies specific for Le -Le or Le -Le epitopes may be produced as described above, and the presence of immunocomplexes may be tested following contact (e.g., incubation) of such antibodies with a biological sample under conditions and for a time sufficient to permit the formation of immunocomplexes.
• Detection of the presence of immunocomplexes formed between an antigen described above and an antibody specific for the antigen may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).
• Suitable immunoassays include the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Patent 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods E. and S. Livingstone, Edinburgh, 1970); the "western blot" method of Gordon et al. (U.S.
• Patent 4,452,901 immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem. 255:4980-4983, 1980); enzyme-linked immunosorbent assays as described by, for example, Raines and Ross (J. Biol. Chem. 257:5154-5160, 1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol. 39:477, 1980); and neutralization of activity (Bowen-Pope et al, Prog Natl. Acad. Sci. USA 81:2396-2400, 1984).
• the antibodies may either be labeled or unlabeled.
• unlabeled antibodies find use in agglutination assays.
• unlabeled antibodies can be used in combination with labeled molecules that are reactive with immunocomplexes, or in combination with labeled antibodies (second antibodies) that are reactive with the antibody directed against the compound, such as antibodies specific for immunoglobulin.
• the antibodies can be directly labeled.
• the reporter group can include radioisotopes, fluorophores, enzymes, luminescers, or dye particles. These and other labels are well known in the art and are described, for example, in the following U.S. patents: 3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402.
• a reporter group is bound to the antibody.
• the step of detecting immunocomplexes involves removing substantially any unbound antibody and then detecting the presence of the reporter group. Unbound antibody is antibody which has not bound to the antigen.
• a reporter group is bound to a second antibody capable of binding to the antibodies specific for the antigen.
• the step of detecting immunocomplexes involves (a) removing substantially any unbound antibody (i.e., antibody not bound to the antigen), (b) adding the second antibody, (c) removing substantially any unbound second antibody and then (d) detecting the presence of the reporter group.
• the reporter group is bound to a molecule capable of binding to the immunocomplexes.
• the step of detecting involves (a) adding the molecule, (b) removing substantially any unbound molecule, and then (c) detecting the presence of the reporter group.
• a molecule capable of binding to the immunocomplexes is protein A.
• Reporter groups suitable for use in any of the methods include radioisotopes, fluorophores, enzymes, luminescers, and dye particles.
• binding partners other than antibodies
• binding partners specific for tumor-associated extended type 1 antigens of the present invention may be used to test for such antigens and that complexes formed between such binding partners and antigens may be detected by techniques analogous to those described above for immunocomplexes.
• the following examples are offered by way of illustration and not by way of limitation.
• MAb ST-421 was established as previously described (Watanabe et al., Jpn. J. Cancer
• N-acetyllactosamine (Gal ⁇ l ⁇ 3GlcNAc ⁇ l ⁇ R), was prepared in the laboratory of the inventors; MAb 1B2, which defines type 2 chain N-acetyllactosamine (Gal ⁇ l ⁇ 4GlcNAc ⁇ l ⁇ R), was established as previously described (Young et al., J. Biol.
• Anti-Le MAb was obtained from Chembiomed Ltd.
• Anti-Le y MAb AH6 was established as previously described (Abe et al, J. Biol. Chem.258:l 1793-11797, 1983), and did not show any cross-reactivity with Le .
• Anti-Le MAb was purchased from Chembiomed Ltd. (Edmonton, Alberta, b Canada), and showed cross-reactivity with type 1 chain H.
• Another anti- Le MAb was purchased from Monocarb (Lund, Sweden), and showed reactivity with Le , type 1 chain H, y and Le .
• HPTLC immunostaining was performed using Whatman HPTLC plates (HP-KF) by a modified version (Kannagi et al., J. Biol. Chem.
• NI ⁇ euAcnLcg, IN ⁇ euAcIII FucLc4, NI FucnLc ⁇ , and IN FucLc4 were isolated from human placenta, liver adenocarcinoma, human type O erythrocytes , and porcine intestine, respectively, after extraction with IHW (55:25:20) followed by Folch partition, DEAE- Sephadex chromatography, and HPTLC on an Iatrobeads 6RS-8010 column (Magnani et al., J. Biol. Chem. 257:14365-14369, 1982; Watanabe et al., J. Biol. Chem. 254:8223-8229,
• ⁇ c and III FucLc4 were prepared by desialylation of NI ⁇ euAcnLcg and
• Gal ⁇ l ⁇ 3GlcNAcIH FucnLc4 was prepared by ⁇ -fucosidase treatment of
• V Fuc2nLcg were prepared biosynthetically by ⁇ l ⁇ 3 fucosylation of IV FucLc4,
• ⁇ l ⁇ -3/4 fucosyltransferase was solubilized from Colo2O5 cells by homogenization in two volumes of 50 mM Hepes buffer (pH 7.0), 0.5 M sucrose, 1 mM EDTA, and 1% Triton CF-54 in a Potter-Elvehjem homogenizer at 4°C. The homogenate was centrifuged at 100,000 x g for 1 hr, and the supernatant was concentrated to the original volume of cells by dialysis. The enzyme preparation was stored at -80°C until needed.
• Enzymatic ⁇ l ⁇ 3/4 fucosylation was performed in a reaction mixture containing 1 mg glycosphingolipid (GSL) substrate, 1 mg deoxytaurocholate, 10 ⁇ mol MnCl2, 25 ⁇ mol
• Each GSL with defined structure was characterized by reactivity with specific MAb(s), i.e., Le /Le antigen reacted with anti-Le MAbs but not with anti-Le MAb AH6;
• TLC Immunostaining TLC immunostaining of neutral glycolipid fractions prepared from various tumor samples showed the presence of a positive band migrating slower than -Le - active ceramide
• MAb NCC-ST-421 was seen in the majority of tumors so far examined. Examples from colonic cancer, breast cancer, Hodgkin's disease, gallbladder cancer, and embryonal rhabdomyosarcoma. EXAMPLE 2
• Colo205 cells (ATCC) (Semple et al., Cancer Res. 38:1345-1355, 1978) were grown in RPMI 1640 medium containing 10% fetal calf serum. Cells were harvested and passed approximately every 7 days. Cells harvested were trypsinized, centrifuged, washed twice with phosphate-buffered saline (pH 7.4) and counted using a hemocytometer. 4 x 10 cells were injected subcutaneously into each of 6 athymic (nude) mice. Tumors (approximately 2 ml each) were excised after 2 weeks and stored frozen at -80°C until needed.
• Enzymatic Degradation Enzymatic degradation of 1 mg dimeric Le was performed by sequential hydrolysis with 0.5 units of ⁇ -fucosidase (bovine kidney), 0.5 units of ⁇ -galactosidase (jackbean), and 0.5 units of ⁇ -N-acetylglucosaminidase (bovine epididymis) (Sigma Chemical Co., St. Louis, Mo.). All reactions were carried out in 0.2 M sodium citrate (pH 4.5) for 4 hr at 37°C in a water bath with shaking. Purification of each degradative product was performed by preparative HPTLC.
• Colo2O5 was originally obtained from American Type Culture Collection (ATCC) and cultured in RPMI- 1640 medium supplemented with 10% fetal calf seram, mM L- glutamine, 100 IU/ml penicillin, and 10 ⁇ g/ml streptomycin.
• ATCC American Type Culture Collection
• RPMI- 1640 medium supplemented with 10% fetal calf seram, mM L- glutamine, 100 IU/ml penicillin, and 10 ⁇ g/ml streptomycin.
• A431 cell line (MacLeod et al, J. Cell. Physiol. 127:175-182, 1986) was originally donated by Dr. Carol MacLeod (Gildred Cancer Facility, UCSD School of Medicine, San Diego, a b x y b
• This cell line expresses Le , Le , Le , Le , and ALe on the EGF receptor (Gooi. et al., Biosci. Reports 5:83-94, 1985).
• A431 cells were cultured in Dulbecco's modified Eagle's medium (Irvine Scientific, Santa Ana, CA) supplemented with 5% fetal calf serum, 1 mM glutamine, 110 mg/1 sodium pyruvate, 100 IU/ml penicillin, and 10 ⁇ g/ml streptomycin.
• ADCC Antibody-Dependent Cellular Cytotoxicity
• CDC Complement-Dependent Cvtotoxicitv
• human peripheral blood leukocytes (HPBL) (used as effector cells) were obtained from buffy coat fraction of blood from healthy volunteer donors. Briefly, mononuclear cells were separated by centrifugation through Ficoll-Hypaque gradient solution at 2000 rpm for 20 min (Mishell et al., in Mishell, B.B and Shiigi, S.M. (eds.), Selected Methods in Cellular Immunology, pp. 3-27, W.H. Freeman & Co., San Francisco, CA, 1980). Mouse splenocytes and mouse peritoneal macrophages (effector cells) were prepared as previously described by Mishell et al., with some modification as follows. Target cells (5 x 10 ) were labeled by incubation with 100 ⁇ l of Cr for 90 min at
• ADCC ADCC, except that 100 ⁇ l of diluted human serum was added as a complement source instead of effector cells.
• the serum was inactivated at 56°C for 30 min and used as a control. Percent specific lysis was calculated as described above.
• Colo205 and A431 cells used for in vivo experiments were grown in vitro, washed 2x with medium, and reconstituted at the desired cell density in PBS.
• Cells (5 x 10 /100 ⁇ l) were subcutaneously injected into the backs of 5- to 7-week-old athymic BALB/c mice, and intraperitoneal administration of MAb was started immediately after injection. Purified
• IMH2 1.1 mg/ml
• ST-421 in ascites fluid with corresponding concentration of IgG
• Control animals received ascites protein produced by mouse myeloma cell line NS1 in BALB/c mice. Seven mice per group were used for each experiment, and experiments were run in duplicate. Mean values of tumor weight based on the duplicate experiments were plotted.
• tumors and adjacent normal tissues were obtained from surgical specimens fixed with formalin and paraffin-embedded.
• normal tissues and some tumor tissues from brain, thymus, lung, liver, stomach, colon, kidney, adrenal gland, spleen, pancreas, uterus (with endometrium), and skin were obtained by fresh necropsy from accident victims. Both surgical and necropsy specimens were provided through the courtesy of the Department of Pathology, Swedish Medical Center, Seattle, WA, and Ms. Debbie Bennett of The Biomembrane Institute.
• Samples were sectioned (3 ⁇ M thickness), deparaff ⁇ nized with zylene, dehydrated in ethanol, treated with primary MAb, subsequently treated with biotinylated secondary MAb and peroxidase-conjugated avidin, and stained with 3',3'-diaminobenzidine. Endogenous peroxidase activity was blocked by treatment of sections with 0.3% H2O2 for 20 min. Some sections were incubated with mouse IgG as a negative control. Biotinylated goat anti-mouse IgM, avidin, and biotin were from Vectastain (Burlingame, CA).
• MAb IMH2 reacted strongly and with high incidence with tumors from colon, rectum, liver, pancreas, and endometrium (Table I). In contrast, it showed no reactivity with normal mucosae of distal colon and rectum, including crypt regions and goblet cells. It reacted with lung adenocarcinoma, but not with large cell or small cell carcinoma. One out of 5 cases of squamous cell carcinoma showed strong positive reactivity. MAb IMH2 did not react with tissues of normal brain, lung, spleen, skin, or with various blood cells including granulocytes.
• Observed locations of normal tissues with strong staining were as follows: Hassall's bodies and epithelial reticular cells of thymus (thymocytes were negative); mucous epithelium and secretory glands of gastric mucosa (lamina intestinal, serosa, and muscle layer were negative); both medulla and cortex of adrenal glands. Locations of normal tissues with moderate to weak positive staining were: epithelial cells of proximal and distal convolutions of kidney (other parts were negative); cells in Langerhans' islets in pancreas (other parts of pancreas were negative); cecal mucosa; urothelium. Very weak staining was observed for hepatocytes (other parts of liver, infralobular connective tissue, central vein, bile duct, and Kupffer's cells were negative). These results are summarized in Table I.
• Le and Le determinants are correlated with secretor status of the individual (Sakamoto et al., Molec. Inrmun. 21:1093-1098, 1984; ⁇ rntoft et al., J. Urol.
• IMH2 epitope is expressed in normal urothelium, but its expression is diminished to varying degrees in bladder tumors. There seems to be a correlation with grade of atypia, i.e., IMH2 epitope expression is lowest in highly invasive tumors. Again, this trend is similar to that of ABH antigen expression in normal and malignant bladder tissues. However, in contrast to colonic tissues, IMH2 epitope expression in bladder tissues from blood group A a-b- individuals is correlated with secretor status. Genuine Lewis-negative (Le ) individuals expressed IMH2 epitope in both normal and malignant bladder tissues. TABLE II. Immunohistological staining by MAb IMH2 of normal and malignant colonic tissues: Relationship with host Lewis status .
• phenotypic status was determined by ⁇ l ⁇ 4 fucosyltransferase activity in saliva, and erythrocyte reactivity a b with anti-Le and -Le MAbs. Definitions of phenotypes may be found in Holmes et al, Arch. Biochem. Biophvs. 274:14-25, 1989, and ⁇ rntoft et al, Lab. Invest. 58:576- 583, 1988.

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