WO1992019634A1 - Glycosphingolipides a chaine de type 1 prolongee utilises comme antigenes associes a des tumeurs - Google Patents

Glycosphingolipides a chaine de type 1 prolongee utilises comme antigenes associes a des tumeurs Download PDF

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Publication number
WO1992019634A1
WO1992019634A1 PCT/US1992/003842 US9203842W WO9219634A1 WO 1992019634 A1 WO1992019634 A1 WO 1992019634A1 US 9203842 W US9203842 W US 9203842W WO 9219634 A1 WO9219634 A1 WO 9219634A1
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mab
imh2
cells
chain
cancer
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PCT/US1992/003842
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Sen-Itiroh Hakomori
Steven B. Levery
Mark R. Stroud
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The Biomembrane Institute
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Publication of WO1992019634A1 publication Critical patent/WO1992019634A1/fr
Priority to US08/174,166 priority Critical patent/US6083929A/en
Priority to US09/272,755 priority patent/US6294523B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention relates generally to new human tumorassociated 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. For example, 2-3 sialylated Le a 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:
  • Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ [3Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ ] n 3GalB1 ⁇ 1Ceramide wherein n is at least 1, and when n 1, there are at least two fucosyl or two sialyl residues.
  • the present invention provides an isolated compound having the formula:
  • the present invention provides an isolated compound having the formula:
  • the present invention provides an isolated compound comprising:
  • the present invention provides an isolated compound comprising:
  • any of the compounds of the present invention may be used as an immunogen for the production of polyclonal or monoclonal antibodies, or used in the manufacture of a medicament.
  • the method comprises testing a biological sample from a warm-blooded animal for the presence or amount of a compound according to formula I. In another embodiment, the method comprises testing a biological sample from a warm-blooded animal for the presence or amount of a compound containing a structure according to formula II. In another embodiment, the method comprises testing a biological sample from a warm-blooded animal for the presence or amount of a compound according to formula III. In another embodiment, the method comprises testing a biological sample from a warm-blooded animal for the presence or amount of a compound containing a structure according to formula IV.
  • the present invention provides the cell line IMH2, as designated by ATCC No. HB 11026, and the monoclonal antibody produced by the cell line (MAb IMH2).
  • the monoclonal antibody produced by the cell line IMH2 may be used in the manufacture of a medicament or in methods.
  • MAb IMH2 is used in a diagnostic method.
  • MAb IMH2 is used in a therapeutic method.
  • Figure 1 shows the results of HPTLC immunostaining of upper neutral glycolipids with MAb NCC-ST-421.
  • Panels A and B neutral glycolipids from various tumors. Lanes 1-20 and 26-28, glycolipids were obtained from liver adenocarcinoma originating from the primary lesion indicated. Lanes 1-5 and 8- 11, colon; lanes 6, 7, 12-14, 26-28, lung; lane 15, breast; lane 16, Hodgkin's disease; lanes 17-19, gall bladder; lane 20, prostate; lane 21, renal cell carcinoma; lane 22, leiomyosarcoma; lane 23, embryonal rhabdomyosarcoma; lane 24, breast; lane 25, colon.
  • Panel C neutral glycolipids obtained from normal tissues. Lanes 1, 6, and 9, liver; lane 2, small intestine; lane 3, spleen; lanes 4-5, kidney; lane 7, pancreas; lane 8, placenta; lane 10, lung.
  • Figure 2 shows the results of TLC immunostaining of dimeric Le a after successive enzymatic degradation.
  • Panel A TLC immunostaining pattern with anti-Le a MAb
  • Panel B TLC immunostaining with MAb MNH-1
  • Panel C TLC immunostaining with MAb 1B2.
  • Lane 1 type "O" erythrocyte upper neutral fraction; lane 2, Colo205 upper neutral glycolipid fraction; lane 3, dimeric Le a ; lane 4, slow-migrating band "b” after ⁇ -fucosidase treatment of dimeric Le a ; lane 5, fast-migrating band "a” after continued ⁇ -fucosidase treatment of dimeric Le a ; lane 6, product formed after ⁇ -N-acetylhexosaminidase treatment of lane 5 compound; lane 7, product formed after ⁇ -galactosidase treatment of lane 6 compound.
  • Figure 3 depicts a resolution-enhanced 1-D 1 H-NMR spectrum of dimeric Le a (downfield region).
  • Arabic numerals refer to ring protons of residues designated by roman numerals in the corresponding structure shown at top of figure.
  • R refers to protons of the sphingosine backbone only;
  • Cis refers to vinyl protons of unsaturated fatty acids. Fuc H-5/CH 3 connectivities confirmed by decoupling.
  • Figure 4 depicts chemical ionization mass chromatograms of partially methylated alditol or hexosaminitol acetates yielded from permethylated Colo205 glycolipid antigen. Separation was performed on a DB-5 bonded phase fused silica column. Peaks identified were (1) 2,3,4-tri-O-Me-Fuc-, (2) 2,3,4,6-tetra-O-Me-Gal-, (3) 2,3,6-tri-O-Me-Glc-, (4) 2,4,6-tri-O-Me-Gal-, (5) 6-mono-O-Me-GlcNAcMe. Chromatograms are summations of all relevant MH + , (MH-32) + , and (MH-60) + ions.
  • Figure 5 depicts a positive ion fast atom bombardment mass spectrum of permethylated dimeric Le a .
  • the figure is a composite of three acquisitions optimized for sensitivity under different conditions. Segment from 100-1800 a.m.u. was acquired with NBA only as matrix. Lower segment from 1800-2500 a.m.u. was acquired with addition of 15-Crown-5 to matrix. Insert segment (1900-2500 a.m.u.) was acquired with addition of sodium acetate to matrix. All assignments are nominal monoisotopic masses.
  • Figure 6 depicts a proposed scheme for fragmentation of permethylated dimeric Le a . All fragments are assigned nominal, monoisotopic masses. Pseudomolecular ions and additional fragments are listed in Table II.
  • Figure 7 graphically illustrates the reactivity of dimeric Le a , Le a - Le x and related glycolipids with MAb NCC-ST-421.
  • the reactivities of twelve glycolipids (A to L) are shown in this figure, corresponding to various structures shown in Table III.
  • A
  • FIG 8 graphically illustrates the reactivity of MAb IMH2 with various glycosphingolipids (GSLs).
  • GSLs glycosphingolipids
  • Serial double dilutions of various GSL antigens were added to 96-well flat-bottom assay plates (Probind plate, Falcon) in ethanol and dried. Initial concentration of GSL added to the first well was 100 ng/well.
  • MAb binding assay was performed by ELISA as described below. Abscissa, reciprocal of antigen dilution. Ordinate, optical density reading at 490 nm. Paragloboside is abbreviated as "PG”.
  • Panel A reactivity of type 1 chain GSLs. •, Le b /Le a .
  • FIG. 9 graphically illustrates the MAb-dependent cytotoxic effect on Colo205 cells by MAbs IMH2 and ST-421.
  • Panel A cytotoxic effects at various E:T (effector:target cell) ratios.
  • MAbs IMH2 and ST-421 were purified and applied at a concentration of about 30 ⁇ g/ ⁇ .
  • 51 Cr-labeled Colo205 cells were incubated with different ratios of human peripheral blood leukocytes (HPBL) as effectors.
  • HPBL peripheral blood leukocytes
  • lysis was more conspicuous for both IMH2 and ST-421.
  • ST-421.• IMH2. ⁇
  • control mouse IgG and D11G10 IgG 3 anti-Gg3 as non-specific MAbs.
  • Panel B dependent of cytotoxic effect on MAb concentration at constant E:T ratio 100:1. Maximal cytotoxic effect was observed at a MAb concentration of 35-70 ⁇ g/ml. Control MAb D11G10 (o) showed no cytotoxic effect.
  • Panel C cytotoxic effect with mouse splenocytes as effector cells. Experimental conditions as in Panel A. 51 Cr-labeled Colo205 cells were incubated with various ratios of mouse splenocytes as effector cells, o, ST-421. •, IMH2. ⁇ , control MAb (D11G10).
  • Figure 10 graphically illustrates the CDC (complement-dependent cytotoxicity) effect on Colo205 cells by MAb IMH2.
  • Fresh human serum was used as complement source.
  • Panel A 51 Cr-labeled Colo205 cells were incubated with about 30 ⁇ g/ml of purified IMH2 or ST-421 and various concentrations of complement (see abscissa), o, ST-421. •, IMH2. ⁇ , control mouse IgG3 with complement.
  • Panel B 51 Cr-labeled Colo205 cells were incubated with different concentrations of IMH2 (see abscissa) in the presence of 1:4 diluted human serum as a complement source.
  • IMH2. control mouse IgG 3 with complement.
  • FIG 11 graphically illustrates the inhibitory effect of MAb IMH2 on Colo205 cell growth in nude mice.
  • Colo205 cells (1 ⁇ 10 7 ) were subcutaneously injected into the backs of 6-week-old athymic Balb/c mice, followed immediately by injection of 200 ⁇ l ( ⁇ 200 ⁇ g) of purified IMH2 (1.1 mg/ml) per day for 14 days (shaded bar) (•).
  • Other mice were treated similarly with MAb ST-421 ( ⁇ ).
  • Control groups were injected with PBS containing similar quantities of non-specific mouse IgG (o).
  • Figure 12 pictorially depicts immunohistological patterns of various human carcinoma tissues stained by MAb IMH2.
  • A colonic carcinoma, ⁇ 100.
  • B colonic carcinoma, ⁇ 160.
  • C lung adenocarcinoma, ⁇ 100.
  • D liver metastasis from colonic adenocarcinoma.
  • E F, endometrial carcinoma, ⁇ 100. It is noted that in each panel adjacent normal tissues were not stained.
  • 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 ⁇ 1 ⁇ 3GlcNAc) is known to be abundant in normal cells and tissues. Although polylactosamine antigens having an extended type 2 chain (i.e., Gal ⁇ 1 ⁇ 4GlcNAc 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.
  • lactoseries type 1 chain i.e., Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ [3Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ ] n 3Gal ⁇ 1 ⁇ R, with or without sialyl and/or fucosyl residues
  • Two representative extended forms of lacto-series type 1 chain were isolated by subjecting a glycolipid fraction (extracted from tumor cells) to preparative column and thin layer chromatography.
  • GSLs glycosphingolipids
  • dimeric Le a i.e., Le a -Le a
  • Le b - Le a The GSL dimeric Le a has the structure:
  • the GSL Le b -Le a has the structure:
  • Ceramides are sphingolipid bases which are acylated on the amine with a fatty acid.
  • the Le a -Le a and Le b -Le a epitopes may be present as extended type 1 chains with additional [3Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ ] n units.
  • the Le a -Le a and Le b -Le a epitopes may be carried by glycoproteins, e.g., high molecular weight mucin-like sera glycoproteins.
  • 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.
  • biological starting materials such as cancer tissue
  • synthesized chemically (and/or enzymatically) following structural identification e.g., 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 (EI) 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).
  • EI electron-impact direct-probe
  • FAB fast atom bombardment
  • 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. 55:205-208, 1964) followed by chemical ionization mass spectrometry of permethylated sugars (e.g., Stellner et al., Arch. Biochem. Biophys. 155:464-472, 1974; Levery et al., Meth. Enzymol. 138:13-25, 1987).
  • 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). Homogeneity of the carbohydrate sequence may be demonstrated based on various chemical and physical criteria, including proton NMR spectroscopy of intact or methylated glycans and FAB mass spectrometry. Once a carbohydrate structure has been determined, the carbohydrate or derivatives thereof or non-carbohydrate functional equivalents thereof may be synthesized using techniques well known to those of normal 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,
  • 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.,
  • a compound of the present invention may be combined with a carrier in order to increase their immunogenicity.
  • 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 a - Le a or Le b - Le a 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 immunogen is the immunization of mice with Le b /Le a antigen.
  • Le b /Le a 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.
  • splenocytes of immunized mice were harvested and fused with myeloma cells.
  • a hybridoma, IMH2 which showed preferential reactivity with the immunogen, was established and deposited with ATCC (American Type Culture Collection, 12301 Parklawn Dr., Rockville, Maryland 20852 USA) as ATCC No. HB 11026.
  • the hybridoma produces a MAb IMH2 with an IgG 3 isotype.
  • MAb IMH2 reacts not only with the immunogen used, but also with
  • Le y /Le x antigen and to a lesser degree with short-chain Le y or Le b with hexasaccharide ceramide (i.e., IV 2 FucIII 3 FucnLc 4 Cer or IV 2 FucIII 4 FucLc 4 Cer). It showed high incidence of staining and strong reactivity with carcinomas of colon, rectum, liver, pancreas, and endometrium, but no reactivity with normal colonic mucosa at various loci, and minimal reactivity with normal liver, pancreas, or uterine endometrium. Its expression in colorectal tumors and normal cecal tissue was independent of secretor status, whereas that in normal urothelium was dependent on secretor status.
  • MAb IMH2 displayed strong lymphocyte-activated or complement-dependent killing of human colonic cancer Colo205 cells in vitro, and inhibition of Colo205 growth in vivo. Therefore, as disclosed within the present invention, a new extended type 1 chain structure, Le b /Le a , is a useful tumor marker associated with carcinomas of colon, rectum, pancreas, liver, and endometrium, and MAb IMH2 has diagnostic and therapeutic applicability for these carcinomas.
  • Methods for the detection of extended forms of type 1 chain antigen may be used to screen for cancers.
  • the GSL Le b -Le a and the GSL Le a -Le a were detected by TLC immunostaining with MAb IMH2 and MAb 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.
  • Such samples include tissue from colonic cancer, breast cancer, Hodgkin's disease, gallbladder cancer and embryonal rhabdomyosarcoma.
  • the GSL Le a -Le a was not detected in glycolipid fractions from normal tissue from spleen, liver, kidney, placenta and lung.
  • a variety of means for detecting tumor-associated extended type 1 antigens could be employed within the methods of the present invention.
  • antibodies specific for Le a -Le a or Le b -Le a 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 (BowenPope et al., Proc. 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.
  • 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 second antibody is an anti-murine antibody.
  • a 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.
  • An example of a molecule capable of binding to the immunocomplexes is protein A.
  • an immunoassay employing a labeled antigen.
  • an antigen present in a sample will compete with labeled antigen for the antibodies.
  • Reporter groups suitable for use in any of the methods include radioisotopes, fluorosphores, 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.
  • MAb ST-421 was established as previously described (Watanabe et al., Jpn. J. Cancer Res. ( Gann. 76:43-52, 1985).
  • MAb MNH-1 which defines type 1 chain N-acetyllactosamine (Gal ⁇ 1 ⁇ 3GlnNAc ⁇ 1 ⁇ R), was prepared in laboratory of the inventors;
  • MAb 1B2 which defines type 2 chain N- acetyllactosamine (Gal ⁇ 1 ⁇ 4GlcNAc ⁇ 1 ⁇ R), was established as previously described (Young et al., J. Biol. Chem. 256:10967-10972, 1981).
  • Anti-Le a MAb was obtained from Chembiomed Ltd. (Edmonton, Alberta, Canada).
  • Anti-Ley MAb AH6 was established as previously described (Abe et al., J. Biol. Chem. 258:11793- 11797, 1983), and did not show any cross-reactivity with Le .
  • Anti-Le b MAb was purchased from Chembiomed Ltd. (Edmonton, Alberta, Canada), and showed cross-reactivity with type 1 chain H.
  • Another anti-Le b MAb was purchased from Monocarb (Lund, Sweden), and showed reactivity with Le b , type 1 chain H, and Le y .
  • HPTLC immunostaining was performed using Whatman HPTLC plates (HP-KF) by a modified version (Kannagi et al., J. Biol. Chem.
  • nLc 6 and III 4 FucLc 4 were prepared by desialylation of VI 3 NeuAcnLc 6 and IV 3 NeuAcIII 4 FucLc 4 , respectively, by heating the samples at 100°C for 1 hr in 1% acetic acid.
  • IV 3 GlcNAcnLc 4 , IV 3 Gal ⁇ 1 ⁇ 3-GlcNAcnLc 4 , IV 3 Gal ⁇ 1 ⁇ 3[Fuc1 ⁇ 4]GlcNAcnLc 4 and IV 3 Gal ⁇ 1 ⁇ 3[Fuc1 ⁇ 4]GlcNAcIII 3 FucnLc 4 (Le a -Le x ) were prepared by enzymatic synthesis.
  • Gal ⁇ 1 ⁇ 3GlcNAcIII 3 FucnLc 4 was prepared by ⁇ -fucosidase treatment of rv ⁇ 3 Gal ⁇ 1 ⁇ 3[Fuc1 ⁇ 4]GlcNAcIII 3 FucnLc 4 ; i.e., 100 ⁇ g of the glycolipid was incubated with 0.2 M citrate buffer (pH 4.5) containing 0.05 units bovine kidney ⁇ -L-fucosidase (Sigma Chemical Co., St. Louis, Mo.) for 2 hr at 37°C.
  • IV 2 III 4 Fuc2Lc 4 , V 3 III 3 Fuc 2 nLc 6 , and VI 2 V 3 Fuc 2 nLc 6 were prepared biosynthetically by ⁇ 1 ⁇ 3 fucosylation of IV 2 FucLc 4 , nLc 6 , and VI 2 FucnLc 6 (respectively) as substrates, using ⁇ 1 ⁇ 3/4 fucosyltransferase from Colo205.
  • ⁇ 1 ⁇ 3/4 fucosyltransferase was solubilized from Colo205 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 ⁇ 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 l mg glycosphingolipid (GSL) substrate, 1 mg deoxytaurocholate, 10 ⁇ mol MnCl 2 , 25 ⁇ mol Hepes buffer (pH 7.0), 5 ⁇ mol CDP-choline, 6 ⁇ mol GDP-fucose, and 500 ⁇ l enzyme preparation in a total volume of 1 ml.
  • the reaction mixture was incubated at 37°C for 16 hr, then lyophilized, extracted with isopropanol-hexane-water (IHW) (55:25:20) by sonication, and centrifuged.
  • IHW isopropanol-hexane-water
  • the supernate was subjected to HPLC on an Iatrobeads 6RS-8010 column using gradient elution of IHW from 55:40:5 to 55:25:20 over 200 min. Two ml fractions were collected and tubes containing the final product were pooled according to HPTLC migration in chloroform-methanol-water 50:40:10. GSL bands were visualized by orcinol spray reagent.
  • Each GSL with defined structure was characterized by reactivity with specific MAb(s), i.e., Le b /Le a antigen reacted with anti-Le b MAbs but not with anti-Ley MAb AH6; Le y /Le x reacted with AH6 but not with anti-Le b nor anti-Le x MAbs; Le a /Le a and Le a /Le x reacted with anti-Le a MAb as well as with MAb ST-421.
  • MAb(s) i.e., Le b /Le a antigen reacted with anti-Le b MAbs but not with anti-Ley MAb AH6
  • Le y /Le x reacted with AH6 but not with anti-Le b nor anti-Le x MAbs
  • Le a /Le a and Le a /Le x reacted with anti-Le a MAb as well as with MAb ST-421.
  • TLC immunostaining of neutral glycolipid fractions prepared from various tumor samples showed the presence of a positive band migrating slower than Le a -active ceramide pentasaccharide, and cross-reacting with anti-Le a MAb. This band was strongly stained by MAb NCC-ST-421, and was seen in the majority of tumors so far examined. Examples from colonic cancer, breast cancer, Hodgkin's disease, gallbladder cancer, and embryonal rhabdomyosarcoma are shown in Figures 1A and 1B.
  • IHW isopropanol- hexane-water
  • Folch partition DEAE-sephadex chromatography
  • HPLC HPLC on an Iatrobeads 6RS-8010 column.
  • Gradient elution of the upper-phase neutral fraction was performed in IHW from 55:40:5 to 55:25:20 over 200 minutes.
  • Two-ml fractions were collected and pooled according to HPTLC migration in chloroform-methanol-water (50:40:10).
  • the slow- migrating Le a -active fraction (revealed by TLC immunostaining) was further purified by preparative TLC on Merck HPTLC plates (Silica Gel 60, Merck, Darmstadt, Germany) and used for structural characterization.
  • a positive band (by immunostaining with MAb NCC-ST-421 according to Example 1) which migrates just below dimeric Le a antigen was purified using the methods described in section B above.
  • Enzymatic degradation of 1 mg dimeric Le a 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.
  • TLC immunostaining of the purified antigen component with various MAbs was performed before and after successive enzymatic degradation.
  • Treatment of the component with bovine kidney ⁇ -fucosidase resulted in two bands: a fast-migrating band "a” ( Figure 2, lane 5), and a slow-migrating band "b” ( Figure 2, lane 4), both of which reacted with MAb MNH-1 3 ( Figure 2B) but not with anti-Le a or 1B2 ( Figures 2A, 2C).
  • Extensive treatment of the original antigen with bovine kidney ⁇ -fucosidase resulted in decreased band b and increased band a.
  • Bands a and b are therefore assumed to be Lc 6 (Table III, structure 6) and III 4 FucLc 6 (structure 13), respectively, based on their reactivity with MAbs and on further enzymatic degradation patterns. Since MNH-1 reacts specifically with unsubstituted type 1 chain structure (Gal ⁇ 1- ⁇ GlcNAcB1 ⁇ 3Gal- ⁇ 1 ⁇ R), and MAb 1B2 reacts with unsubstituted type 2 chain (Gal ⁇ 1 ⁇ 4GlcNAc ⁇ 1 ⁇ 3Gal- ⁇ 1 ⁇ R), no type 2 chain structure could be present at the terminus.
  • H-5 of the internal Fuc ⁇ 4 group is in a chemical environment similar to that in Le a -pentaglycosylceramide, while that of the outer Fuc ⁇ 1 ⁇ 4 is deshielded.
  • This is in contrast to the ⁇ -Fuc H-1 resonances, which are identical, or to the ⁇ -GlcNAc H-1 resonances, where that belonging to the innermost saccharide is shifted upfield relative to its position in Le a , while that of the outermost GlcNAc occurs at shifts identical to that in Le a .
  • Analogous examples of long-range "cross-talk" were observed in repeating type 2 Le x -hapten structures (Levery et al., Carbohydr. Res.
  • a sample of the glycolipid ( ⁇ 50 ⁇ g) was permethylated by the method of Ciucanu and Kerek (Ciucanu and Kerek, Carbohydr. Res. 131:209-217, 1984), as modified by Larson et al. (Larson et al., Carbohydr. Res. 161:281-290. 1987), except that equal volumes of Mel and DMSO were used (200 ⁇ l each). The reaction time was 60 min and Mel was removed by flushing with N2 for 25 min at 37°C prior to partitioning between CHCl 3 and H 2 O.
  • fragments at m/z 1087 and 1291 were consistent with the presence of some monofucosylated impurity. Similar fragments were observed in the spectrum of an isomeric synthetic monofucosyl type 2 chain structure (Holmes and Levery, Arch. Biochem. Biophys. 274:633-647, 1989). However, the relatively low abundance of ion at m/z 464 (Hex ⁇ HexNAc) indicates that, in this case, the single deoxyHex residue must be attached primarily to the subterminal, rather than internal, HexNAc.
  • MAb IMH2 was established after immunization of Balb/c mice with Le b /Le a antigen (Table III, structure 15) isolated from Colo205 cells (isolated as described in Example 2 above) and adsorbed on Salmonella minnesotae (adsorption according to Young et al., J. Exp. Med. 150:1008-1019, 1979). Forty ⁇ g Le b /Le a was dissolved in 100 ⁇ l ethanol and mixed with 1.6 ml PBS (pH 7.4). This solution was combined with 500 ⁇ l of a suspension containing 500 ⁇ g acid-treated S. minnesotae and incubated at 37°C for 30 min with occasional shaking.
  • each vial contained approximately 5 ⁇ g of GSL antigen and 62 ⁇ g of bacteria in 250 ⁇ l PBS. Contents of each vial were injected via tail vein into 6-week-old female Balb/c mice on four occasions, with 10-day intervals. Two mice were immunized at a given time. Three days after the last injection, splenocytes of immunized mice were harvested and fused with NS/1 myeloma cells according to the procedure originally described by Young et al. (J. Exp. Med. 150:1008-1019, 1979).
  • Hybridomas were selected using 96-well plates coated with phosphatidylcholine-cholesterol-Le b /Le a 5:3:1 by weight. The quantity of Le b /Le a added was approximately 50 ng/well. Reactivity was determined by ELISA assay. Clones showing preferential reactivity with the immunogen were subcloned repeatedly until a stable clone was established. The isotype of MAb IMH2 was determined to be IgG3.
  • the binding specificity of MAb IMH2 was tested by thin-layer chromatography immunostaining of various GSL antigens by the method previously described by Magnani et al. (Anal. Biochem. 109:399-402, 1980), and modified by Kannagi et al. (Cancer Res. 43:4997-5005, 1983). IMH2 reactivity was also determined by antibody binding on solid-phase antigen as follows. Each GSL antigen, dissolved in ethanol (100 ng/50 ⁇ l), was placed in a well, serially diluted with ethanol to 0.1 ng/50 ⁇ l per well in a flat-bottom 48-well plate (Falcon, Becton-Dickinson, Lincoln Park, NJ), and dried.
  • each well was then incubated with 1% bovine serum albumin in PBS for 1 hr at room temperature, followed by reaction with IMH2 (5 ⁇ g/ml) for 18 hr at 4°C.
  • IMH2 5 ⁇ g/ml
  • Each well was washed with PBS, followed by incubation with biotinylated second antibody and avidin-peroxidase conjugate as prepared in the ELISA assay kit.
  • the orange color developed in each well was read by automated "ELISA reader” (EL312 Biokinetics Reader, Biotek Instruments, Winooski, VT), and optical density at 490 nm for each well was recorded.
  • glycosphingolipid (GSL) antigens are glycosphingolipid (GSL) antigens.
  • Colo205 was originally obtained from American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 1 mM L-glutamine, 100 IU/ml penicillin, and 10 ⁇ g/ml streptomycin.
  • Human epidermoid carcinoma A431 cell line (MacLeod et al., J. Cell. Phvsiol. 127:175-182, 1986) was originally donated by Dr. Carol MacLeod (Gildred Cancer Facility, UCSD School of Medicine, San Diego, CA).
  • This cell line expresses Le a , Le b ' Le x , Le y , and ALe b 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/l sodium pyruvate, 100 IU/ml penicillin, and 10 ⁇ g/ml streptomycin.
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • 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 ⁇ 10 6 ) were labeled by incubation with 100 ⁇ l of 51 Cr for 90 min at 37°C. After washing (3 ⁇ ) and incubation (1 hr at 37°C), cells (1 ⁇ 10 6 ml) were suspended in RPMI-1640 supplemented with 25 mM HEPES buffer and 3% bovine serum albumin. Twenty ⁇ l of labeled cells, 100 ⁇ l of IMH2 or ST-421, ad 100 ⁇ l of effector cell suspension were mixed into Microtiter U-bottom plates (Corning, NY). Non-specific mouse Ig (Sigma, St. Louis, MO) was used as a negative control.
  • Colo205 and A431 cells used for in vivo experiments were grown in vitro, washed 2 ⁇ with medium, and reconstituted at the desired cell density in PBS.
  • Cells (5 ⁇ 10 6 /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) or ST-421 in ascites fluid with corresponding concentration of IgG (1.1-1.2 ng/ml) at a dosage of 0.2 ml/animal were intraperitoneally injected lx/day for 2 weeks. Width and length of tumors were measured by the same observer 3 ⁇ /week.
  • Tumor weight was estimated as (width 2 ⁇ length)/2.
  • 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, W.A, and Ms. Debbie Bennett of The Biomembrane Institute.
  • Samples were sectioned (3 ⁇ M thickness), deparaffinized with xylene, 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% H 2 O 2 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 VI). 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.
  • IMH2 was reactive with rectal tumors but not with normal rectal tissue, and this reactivity was unrelated to secretor status. Conversely, IMH2 was reactive with normal cecum but less so with the single cecal tumor sample studied.
  • 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 individuals is correlated with secretor status. Genuine Lewis-negative (Le a-b - ) individuals expressed IMH2 epitope in both normal and malignant bladder tissues.

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Abstract

L'invention concerne divers composés utilisés comme immunogènes et comme marqueurs de tumeurs. L'invention concerne également des procédés relatifs à la détection du cancer. On a démontré l'existence de formes prolongées de la chaîne de type 1 de lacto-séries dans divers tissus cancéreux. L'invention concerne en outre une lignée cellulaire ainsi que l'anticorps monoclonal produit à partir de celle-ci. Ledit anticorps présente un certain nombre d'utilisations, y compris dans des procédés de diagnostic et de thérapeutique.
PCT/US1992/003842 1991-05-06 1992-05-06 Glycosphingolipides a chaine de type 1 prolongee utilises comme antigenes associes a des tumeurs WO1992019634A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002089819A1 (fr) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugues et utilisations de ceux-ci
EP1307207A1 (fr) * 2000-07-03 2003-05-07 The Biomembrane Institute Glycosphingolipides a chaine de type 1 etendue, utiles en tant qu'antigenes associes a une tumeur
JP2007191490A (ja) * 2007-04-02 2007-08-02 Biomembrane Inst 腫瘍関連抗原としての拡張ｉ型スフィンゴ糖脂質鎖

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EP0287916A1 (fr) * 1987-04-13 1988-10-26 Otsuka Pharmaceutical Co., Ltd. Essai immunologique
EP0344955A2 (fr) * 1988-05-31 1989-12-06 The Biomembrane Institute Structure glycolipidique à chaîne longue
US4939083A (en) * 1986-10-30 1990-07-03 La Jolla Cancer Research Foundation Carbohydrate specific to chronic myelogenous leukemia granulocytes

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US4939083A (en) * 1986-10-30 1990-07-03 La Jolla Cancer Research Foundation Carbohydrate specific to chronic myelogenous leukemia granulocytes
EP0287916A1 (fr) * 1987-04-13 1988-10-26 Otsuka Pharmaceutical Co., Ltd. Essai immunologique
EP0344955A2 (fr) * 1988-05-31 1989-12-06 The Biomembrane Institute Structure glycolipidique à chaîne longue

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EUROPEAN JOURNAL OF BIOCHEMISTRY vol. 203, no. 3, 1992, BERLIN DE pages 577 - 586 STROUD M.R. ET AL 'Extended Type-1 chain glycosphingolipid antigens. Isolation and characterization of trifucosyl-Leb antigen (III4V4VI2Fuc3Lc6)' *
JOURNAL OF BIOLOGICAL CHEMISTRY vol. 259, no. 11, 1984, BALTIMORE, MD US pages 7178 - 7186 WU A.M. ET AL 'Immunochemical Studies on Blood Groups' *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 256, no. 7, 1981, BALTIMORE US pages 3512 - 3524 KARLSSON K-A. AND LARSON G. 'Molecular Characterization of Cell Surface Antigens of Fetal Tissue' *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 266, no. 12, 5 May 1991, BALTIMORE US pages 8439 - 8446 STROUD M.R. ET AL 'Extended Type 1 Chain Glycosphingolipids: Dimeric Lea (III4V4Fuc2Lc6) as Human Tumor-associated Antigen' *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1307207A1 (fr) * 2000-07-03 2003-05-07 The Biomembrane Institute Glycosphingolipides a chaine de type 1 etendue, utiles en tant qu'antigenes associes a une tumeur
EP1307207A4 (fr) * 2000-07-03 2005-12-14 Biomembrane Inst Glycosphingolipides a chaine de type 1 etendue, utiles en tant qu'antigenes associes a une tumeur
WO2002089819A1 (fr) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugues et utilisations de ceux-ci
JP2007191490A (ja) * 2007-04-02 2007-08-02 Biomembrane Inst 腫瘍関連抗原としての拡張ｉ型スフィンゴ糖脂質鎖

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