WO1994003629A1 - Anticorps monoclonaux specifiques contre le ganglioside 3',6'-isold1 - Google Patents

Anticorps monoclonaux specifiques contre le ganglioside 3',6'-isold1 Download PDF

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WO1994003629A1
WO1994003629A1 PCT/US1993/007302 US9307302W WO9403629A1 WO 1994003629 A1 WO1994003629 A1 WO 1994003629A1 US 9307302 W US9307302 W US 9307302W WO 9403629 A1 WO9403629 A1 WO 9403629A1
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ganglioside
isoldl
monoclonal antibody
cells
neuac
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PCT/US1993/007302
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WO1994003629A9 (fr
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Darell D. Bigner
Lars Svennerholm
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Duke University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to monoclonal antibodies directed to the lactotetraose ganglioside 3 ' ,6'-isoLDl.
  • the monoclonal antibody FH9 which binds to 3 1 ,6'-isoLDl, is described in Y. Fukushi et al., Biochemi ⁇ try 25, 2859-2866 (1986).
  • the ganglioside 3' ,6• ,8'-isoLTl is described in
  • a first aspect of the present invention is a monoclonal antibody which binds to the ganglioside IV 3 NeuAc ,III 6 NeuAc-LcOse 4 Cer (3• ,6•-isoLDl) .
  • the antibody does not bind to the ganglioside IV 3 NeuAc-LcOse 4 Cer (3'-isoLMl).
  • the antibody also does not bind to the ganglioside IV 3 NeuAc 2 -III 6 NeuAcLcOse 4 -Cer (3' ,6' ,8'-isoLTl) .
  • the antibody has the binding characteristics set forth in Figure 2 and/or Figure 5 herein.
  • the antibody binds to a 3' ,6'-isoLDl epitope selected from the group consisting of NeuAc ⁇ 2-3Gal/31-3NeuAc ⁇ 2-6GlcNAc and NeuGc ⁇ 2-3Gal/31- 3NeuGc ⁇ 2-6GlcNAc.
  • Antibodies of the invention may be coupled to a detectable group or therapeutic agent, as described in detail below. Accordingly, further aspects of the present invention is the diagnosis and treatment of diseases employing the monoclonal antibodies described herein.
  • a second aspect of the present invention is a pharmaceutical formulation comprising a monoclonal antibody as given above in a pharmaceutically acceptable carrier.
  • a third aspect of the present invention is a method for detecting the presence of cancer in a human or animal subject.
  • the method comprises contacting a sample (e.g., a tissue sample, a biological fluid) from a subject with an antibody as disclosed herein under conditions permitting the antibody to form a reaction product, and then detecting the presence or absence of the reaction product.
  • the method is particularly useful for detecting glioma ⁇ .
  • the method may be used to monitor the progression of treatment in a patient previously diagnosed as having cancer or may be used to screen patients who have not been previously diagnosed as having cancer.
  • a fourth aspect of the invention is a method of treating cancer in a human or animal subject.
  • the method comprises administering to the subject an antibody as described herein in an amount effective to combat the cancer. Any cancer for which 3' ,6'-isoLDl is a marker (e.g., where this ganglioside is expressed in greater amounts by the cancer cells than by healthy cells) may be treated by this method.
  • a fifth aspect of the invention is a method of making antibodies as described hereinabove.
  • the method comprises administering an animal xenograft cells, wherein said xenograft cells express the ganglioside 3',6'-isoLDl, for a time and in an amount effective to produce antibodies which bind to the ganglioside 3' ,6'-isoLDl.
  • the method optionally, but preferably, further includes the step of collecting spleen cells from said animal following said immunizing step, and fusing said spleen cells with a continuous cell line to produce a hybridoma cell line, which hybridoma cell line produces monoclonal antibodies as described above.
  • a sixth aspect of the invention is a monoclonal antibody-producing cell line which produces an antibody as given above. Such cell lines may, for example, be hybridoma cell lines or Escherichia coli cell lines.
  • a seventh aspect of the invention is a method of making a monoclonal antibody as given above, comprising: culturing a cell line as described above in a culture medium under conditions suitable for the production of monoclonal antibodies, and collecting the monoclonal antibody from the culture medium.
  • Figure 1 presents a specificity analysis of antibodies of the present invention with defined gangliosides/glycolipids. Symbols are as follows: open squares — galactose; open diamonds — N-acetylglucosamine; filled squares — glucose; open circles — galactosamine; filled triangle with stem — N-acetylneuraminic acid; filled circle with stem — fucose; M ⁇ " — jSl-4 linkage.
  • ** 3'-LMl refers to IV 3 NeuAcnLc0se 4 CER, with 31-4Gal-N- Acetylglucosamine linkage ( ⁇ ) , as opposed to 3'isoLMl, or
  • Figure 2 illustrates the epitope bound by antibodies of the invention. Symbols are as given in connection with Fig. 1 above, with open triangles with stems being used to designate N-glycolylneuraminic acid.
  • Figure 3 shows the reactivity of DMAb-21 and DMAb-22 for primary CNS tumors; immunohistochemistry of acetone-fixed, frozen sections. All MAbs were tested at 5 ⁇ g/ml.
  • Figure 4 shows the reactivity of DMAb-21 and DMAb-22 for non-CNS malignancies; immunohistochemistry of acetone-fixed, frozen sections. All MAbs were used at a concentration of 5 ⁇ g/ l.
  • (a) and (b) TB 273, teratoma, 250X: (a) MOPC-104E, negative control primary antibody;
  • Mesenchymal components stain intensely, as contrasted with the total absence of staining of the epithelial component, (c) and (d) B87-321, leiomyosarcoma, 250X: (c) MOPC-104E negative control primary antibody;
  • DMAb-21 Intense staining of neoplastic cells.
  • Figure 5 provides additional information on the binding characteristics of monoclonal antibodies of the present invention. Symbols used are as given above.
  • Ganglioside designations used herein are according to IUPAC Commision on Biochemical Nomenclature recommendations and Svennerholm, (see IUPAC-IUB Commission on Biochemical Nomenclature, Eur. J. Biochem. 79, 11-21 (1977); L. Svennerholm, J. Neurochem.
  • the present invention is described primarily with respect to the diagnosis and treatment of humans subjects below, the invention can also be adapted to the diagnosis and treatment of animal subjects (e.g. , dog, cat) for veterinary purposes, and that the treatment of animal subjects is also an aspect of the present invention.
  • animal subjects e.g. , dog, cat
  • antibodies refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG are preferred, and IgM are particularly preferred.
  • the antibodies may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e. g. , M. Walker et al., Molec. Immunol . 26, 403-11 (1989) .
  • antibody as used herein includes antibody fragments which retain the capability of binding to a target antigen, for example. Fab, F(ab') 2 , and Fv fragments, and the corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques.
  • Antibodies of the invention are made by administering an animal (e.g., a mouse) xenograft cells, wherein the xenograft cells express the ganglioside 3',6'- isoLDl.
  • Xenograft cells are produced by implanting tumor cells which express the ganglioside 3',6'-isoLDl in a suitable immunocomprised host animal (e.g., a nude mouse).
  • the tumor cells may be, for example, human glioma cells or human embryonal carcinoma (teratoma) cells which express 3' ,6'-isoLDl.
  • tumor cells are routinely found, and the expression of 3' ,6'-isoLDl by such tumor cells is determined in accordance with standard techniques, such as thin layer chromatography or gas chromatography-mass spectroscopy.
  • a specific example of a suitable tumor cell is the embryonal carcinoma cell line PA1, available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland USA as ATCC CRL #1572.
  • the xenograft host is implanted with tumor cells in accordance with standard techniques. After a sufficient tumor cell mass is obtained in the xenograft host, the xenograft cells are harvested from the host and disaggregated (e.g., by trypsinization) , suspended in a suitable carrier (e.g., a tissue culture medium which maintains the cells alive) , and administered by intraperitoneal injection for a time and in an amount effective to produce antibodies which bind to the ganglioside 3',6'-isoLDl (i.e., a first injection followed by a prolonged series of separate booster injections) .
  • a suitable carrier e.g., a tissue culture medium which maintains the cells alive
  • the cells may be administered in a series of 6 to 7 separate injections, carried out separately over a period of about 200 days, with the cells being administered in an amount of from about .8 x 10 7 to 1.2 x 10 7 per injection as a suspension in from about .25 to .75 milliliters of tissue culture medium.
  • monoclonal antibodies used to carry out the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265, 495-97 (1975).
  • the animal producing the antibodies is sacrificed and spleen cells obtained therefrom.
  • the spleen cells are then immortalized by fusing them with a continuous cell line (e.g., myeloma cells or lymphoma cells), typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable media and the supernatant screened for monoclonal antibodies having the desired specificity as given herein.
  • a further aspect of the present invention is to provide hybridomas which produce antibodies against an antigen found on 3' ,6'-isoLDl.
  • the antigen can be NeuAc ⁇ 2- 3Gal01-3NeuAc ⁇ 2-6GlcNAcorNeuGc ⁇ .2-3Gal/31-3NeuGc ⁇ 2-6GlcNAc, and is yet another aspect of the invention.
  • the monoclonal antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading U.S. Patent No. 4,474,893, or Cabilly et al., U.S. Patent No. 4,816,567.
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in Segel et al., U.S. Patent No. 4,676,980 (Applicants specifically intend that the disclosure of all U.S. patent references cited herein be incorporated herein by reference) .
  • Monoclonal antibodies may be chimeric antibodies produced in accordance with known techniques.
  • the monoclonal antibodies may be complementarity determining region-grafted antibodies (or "CDR-grafted antibodies”) produced in accordance with known techniques.
  • Monoclonal Fab fragments may be produced in E ⁇ cherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246, 1275- 81 (1989).
  • the method disclosed herein may be employed with subjects suspected of having cancer (e.g., cancer of the colon, breast, stomach, pnacreas, biliary tract, or ovary, or patients afflicted with teratomas or pancreatic adenocarcinomas) , particularly patients suspected of having embryonal carcinomas or gliomas of the central nervous system (CNS) .
  • the method may be employed both to monitor subjects who have been previously diagnosed as having cancer, and to screen subjects who have not been previously diagnosed as having cancer.
  • Gliomas are described in D. Russell and L. Rubinstein, Pathology of Tumor ⁇ of the Nervou ⁇ Sy ⁇ tem, pp. 83-289 (1989) (Williams and Wilkins) , and include (but are not limited to) astrocytomas and glioblastoma multiforme.
  • Embryonal carcinomas are described in Blau ⁇ tein - ⁇ Pathology of the Female Genital Tract, pp. 679-692 (R. Kurman Ed., 3d ed. 1987) (Springer Verlag, NY) .
  • the subject has been previously diagnosed as having cancer, and possibly has already undergone treatment for cancer, and the method is employed to monitor the progression of either that cancer or the treatment thereof.
  • samples may be collected from subjects who have received initial surgical treatment for ovarian cancer and subsequent treatment with antineoplastic agents for that cancer to monitor the progress of the treatment.
  • Samples taken from human subjects for use in the methods disclosed herein may be biological fluids such as serum, blood plasma, or ascites fluid. Serum is presently preferred.
  • the sample taken from the subject can be a tissue sample (e.g., biopsy tissue; scrapings; ovarian tissue removed during surgery; etc.).
  • Assays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody as disclosed herein, a labeled analyte, and the sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte.
  • Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • the reagents are usually the specimen, the antibody of the invention, and means for producing a detectable signal. Similar specimens as described above may be used.
  • the antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the specimen.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
  • Monoclonal antibodies as described herein may be used in a "two-site” or “sandwich” assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody.
  • assays are described in U.S. Patent No. 4,376,110, the disclosure of which is also incorporated herein by reference.
  • Antibodies as described herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies as described herein may likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S, 125 I, 131 I) , enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase) , and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35 S, 125 I, 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • the diagnostic kit comprises (a) an antibody of the invention conjugated to a solid support and (b) a second antibody of the invention conjugated to a detectable group.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • a second embodiment of a test kit comprises (a) an antibody as described herein, and (b) a specific binding partner for the antibody conjugated to a detectable group.
  • Ancillary agents as described above may likewise be included.
  • the test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
  • Monoclonal antibodies used for therapy may be monoclonal antibodies per ⁇ e or monoclonal antibodies coupled to a therapeutic agent. Such antibodies are referred to herein as therapeutic monoclonal antibodies. Any therapeutic agent conventionally coupled to a monoclonal antibody may be employed, including (but not limited to) radioisotopes, cytotoxic agents, and chemotherapeutic agents. See generally Monoclonal Antibodies and Cancer Therapy (R. Reisfeld and S. Sell Eds. 1985) (Alan R. Liss Inc. NY). Therapeutic agents may be coupled to the antibody by direct means or indirect means (e.g. , via a chelator) .
  • radioisotopes which may be coupled to a therapeutic monoclonal antibody include, but are not limited to, 131 I, 90 Y, 211 At, 212 Bi, 67 Cu, 186 Re, 188 Re, and 212 Pb.
  • chemotherapeutic agents which may be coupled to a therapeutic monoclonal antibody include, but are not limited to, methotrexate.
  • cytotoxic agents which may be coupled to a therapeutic monoclonal antibody include, but are not limited to, ricin (or more particularly the ricin A chain) .
  • monoclonal antibodies per se which are used as therapeutic monoclonal antibodies incorporate those portions of the constant region of an antibody necessary to evoke a therapeutically useful immunological response in the subject being treated.
  • Therapeutic monoclonal antibodies may be provided in lyophylized form in a sterile aseptic container or may be provided in a pharmaceutical formulation in combination with a pharmaceutically acceptable carrier, such as sterile pyrogen-free water or sterile pyrogen-free physiological saline solution.
  • a pharmaceutically acceptable carrier such as sterile pyrogen-free water or sterile pyrogen-free physiological saline solution.
  • Subjects who may be treated with therapeutic monoclonal antibodies of the invention are, in general, subjects harboring tumors which express the ganglioside 3' ,6'-isoLDl. Examples of suitable subjects include those subjects which may be diagnosed as having cancer as set forth above, with patients afflicted with gliomas amd embryonal carcinomas again being particularly preferred.
  • the method of administration of the monoclonal antibodies of the present invention to a subject will vary with individual circumstances, e.g. the particular disease (e.g. cancer) being treated, as will the dosage and frequency of administration.
  • the antibody will be mixed, prior to administration, with a non-toxic, pharmaceutically acceptable carrier substance (e.g.
  • intravenous or intra-arterial administration injection into the cerebrospinal fluid
  • intradermal, intracavity, intrathecal or direct administration to the tumor or to an artery supplying the tumor is advantageous.
  • intrathecal administration or injection into the carotid artery are advantageous for therapy of tumors located in the brain.
  • Dosage of the antibody will depend, among other things, on the tumor being treated, the route of administration, the nature of the therapeutic agent employed, and the sensitivity of the tumor to the particular therapeutic agent. For example, the dosage will typically be about 1 to 10 micrograms per Kilogram subject body weight.
  • the dosage to the patient will typically be between about 10 to 500 mCi. Doses for other radionuclides are typically selected so that the tumoricidal dose will be equivalent to the foregoing range for 131 I.
  • the antibody can be administered to the subject in a series of more than one administration, and regular periodic administration will sometimes be required.
  • Gangliosides and neutral glycolipids used as standards and references were isolated and characterized by fast atom bombardment-mass spectrometry.
  • NeuAc-GM2 was isolated from Tay-Sachs brain in accordance with known techniques (B. Rosengren et al., J. Neurochem. 49, 834-840 (1987)) ; GM3 and GD3 were purified from metastatic melanoma tissue removed at surgery; and GD2 was prepared by bovine ,9-galactosidase treatment of GDlb purified from normal adult human brain. Purified enzyme was kindly provided by Dr. George Jourdian, University of Michigan, Ann Arbor.
  • 3'-isoLMl and GalNAc-3'-isoLMl were purified from human meconium in accordance with known techniques (P. Fredman et al., J. Biol . Chem. 264, 12122-12125 (1989)), 3 '-LM1 and 3',8'-LDl from cauda equina (18), and 3'-isoLMl and 3' ,6'- isoLDl from human glioma cell line D-54 MG-induced nude mouse and nude rat xenografts (J.-E. Mansson et al., ⁇ upra) or from brains of patients dying of polyunsaturated fatty acid lipidosis (L.
  • Monosialo- and oligosialoganglioside fractions were prepared from cultured cell pellets, normal tissue, or tumor xenografts of known weight and/or cell count as previously described (C. Wikstrand et al., J. Neuropathol . Exp. Neurol . 50, 756-769 (1991); P. Fredman et al., Biochim. Biophy ⁇ . Acta 618, 42-52 (1980)).
  • Densitometric scanning of resorcinol-visualized ganglioside bands was performed at 620 nm with a CAMAG TLC Scanner II (CAMAG, Muttenz, Switzerland) . Quantitative measurement of total ganglioside sialic acid was performed by the resorcinol assay in accordance with known techniques (L. Svennerholm, Biochim. Biophy ⁇ . Acta 24, 604-611 (1957)).
  • Neuroblastoma cell lines SK-N-MC, SK-N-SH, LAN-1, and LAN-5 were kindly provided by Dr. Robert Seeger, UCLA.
  • the cell line N417D (small-cell lung carcinoma) was obtained from the National Cancer Institute; and the embryonal carcinoma cell line PA-1 was the gift of Dr.
  • tumors were separated from overlying skin with sterile scissors and the tumor placed in a Petri dish.
  • the tumor was dissected to obtain viable tissue and to remove membranes, dead cells and fat; tumor tissue was placed in a clean Petri dish, weighted, and minced with two scalpels.
  • Minced tumor material was placed in a trypsinizing flask with 0.25% trypsin (Sigma, St. Louis, MO) in Dulbecco's phosphate-buffered saline (Gibco, Grand Island, NY, D-PBS; Ca ++ and Mg ++ free, pH 7.1) at a ratio of 5 mis per gram of tissue and allowed to stir at 37° for 15-20 minutes.
  • the supernatant phase containing dissociated cells was filtered through sterile cheesecloth- covered funnels, and cells pelleted at approximately 200xg for 5 minutes. The supernatant was removed and the cell pellet resuspended in 0.83% ammonium chloride (pH 6.0) in water and allowed to stand for 3 minutes, at which point an equal volume of fetal calf serum (Gibco) was added and the cells pelleted again at 200 x g. Supernatant was removed and the cells washed once with 10 mis of D-PBS. Following an additional centrifugation of 200 x g, pellets were resuspended in 10 mis serum-free Zinc Option (ZO) tissue culture medium (Gibco) .
  • ZO Zinc Option
  • mice Female BALB/c mice, 15 weeks of age (Charles
  • Initial reactivity screen was performed against mono- and oligosialoganglioside fractions of D-54 MG xenograft cells, of which 3'-isoLMl and 3' ,6'- isoLDl are the predominant mono- and disialogangliosides, respectively, to optimize selection for the target gan ⁇ glioside 3' ,6'-isoLDl, with minimal selection of hybrids producing antibodies to other PA-1-associated gangliosides.
  • Hybrids were cloned in methylcellulose semisolid medium (C. Wikstrand et al., J. Neuroimmunol . 3, 43-62 (1982)) three times and cultured for the production of antibody-containing supernatant or cells for ascites establishment.
  • Antibodies were filtered through a 0.22- ⁇ Millipore filter, protein concentration was determined by Lowry assay, IgM concentration was determined by capture enzyme-linked immunosorbent assay (C. Wikstrand et al., ⁇ upra) , and purified antibody was stored for use at 4 ° C. Antibody purity was checked by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • Antibody binding is expressed as a binding ratio, calculated by dividing experimental cpm by negative control cpm; a binding ratio >3 is considered positive as these values exceed the mean background value by >3 standard deviations (C. Wikstrand and D. Bigner, ⁇ upra) .
  • Each neoplasm was graded with regard to percent of neoplastic cells positive on a four-tiered scheme: less than 25% cells positive (+/ ⁇ ) . 25-50% cells positive (+) , 50-75% cells positive (++) , and 75-100% (+++) cells positive. Any antigen localization in benign cells within the tissue section was noted.
  • DMAb-21 and DMAb-22 were initially selected for further analysis on the basis of positive activity for D-54 MG rat xenograft oligosialoganglioside fractions by SP-RIA and HPTLC immunostain in comparison with DMAb-14 ( Figure 1) .
  • Figure 1 As revealed by the titration in SP-RIA of purified MAbs against the total oligosialoganglioside fraction of D-54 MG rat xenograft, no difference in the activity of the three MAbs is apparent.
  • Oligosialoganglioside extracts from human cauda equina, human 20-week gestation fetal brain, and brain tissue from a case of polyunsaturated fatty acid lipidosis also contained 3' ,6'-isoLDl, but in much lower concentrations than in D-54 MG xenografts (2X, 5X, and 10X concentrations, respectively, of extract required for detection).
  • DMAb-22 does not react with purified 3',8*-LDl from human red blood cells, the oligosialoganglioside fraction of normal adult brain cerebral cortex, or purified fuc-3'-isoLMl (the CA-50 antigen).
  • Oligosialoganglioside fractions were prepared from packed cell pellets or xenograft tissue as described above. Human cell lines examined included 14 glioma, 5 medulloblastoma, 3 teratoma, and 5 lines of various origins: 1 pancreatic carcinoma (HPAF) , 1 small-cell carcinoma of the lung (N417) , 1 melanoma (SK-MEL-28) , 1 rhabdomyosarcoma (TE-671) , and 1 neuroblastoma (LAN-1) (Table 2) .
  • HPAF pancreatic carcinoma
  • N41-7 small-cell carcinoma of the lung
  • SK-MEL-28 1 melanoma
  • TE-671 1 rhabdomyosarcoma
  • LAN-1 neuroblastoma
  • Both DMAb-21 and DMAb-22 were screened against the panel of frozen, acetone-fixed tissues listed in Table 3; again, results with the two antibodies were completely concordant; 15/30 of the glial tumor blocks were tested repetitively on consecutive sections with no significant discrepancies noted in the antigen localization patterns.
  • Immunohistochemical staining with both DMAb-21 and DMAb-22 revealed 14/23 gliomas to exhibit localization in the neoplastic cell population (+/ - to +++) • Reactivity with the MAbs roughly correlated with glioma cell size; cells with abundant cytoplasm tended to exhibit strong cytoplasmic immunoreactivity and cells with scant cytoplasm tended to be nonreactive (Figure 3A) .
  • Fibrillar glioblastomas multiforme exhibited moderate, diffuse cytoplasmic reactivity (+++) of neoplastic cells.
  • the non-small cell component was prominently stained as compared to the negative small cell component (Figure 3C) .
  • Cytoplasmic reactivity was the predominant reactivity pattern in the glioblastoma multiforme; membranous localization was never observed without cytoplasmic localization, and was never stronger than the cytoplasmic pattern. Nuclear reactivity was evident in three glioblastomas multiforme.

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Abstract

L'invention se rapporte à des anticorps monoclonaux qui se lient au ganglioside IV3NeuAc, III6NeuAc-LcOse¿4?Cer(3',6'-isolD1) ainsi qu'à leurs procédés de fabrication et à des procédés diagnostiques et thérapeutiques d'utilisation de ceux-ci. L'invention se rapporte également à des anticorps monoclonaux préférés qui se lient à un épitope de 3',6'-isolD1 sélectionné dans le groupe se composant de NeuAcα2-3Galβ1-3NeuAcα2-6GlcNAc et NeuGcα2-3Galβ1-3NeuGcα2-6GlcNAc.
PCT/US1993/007302 1992-07-31 1993-07-29 Anticorps monoclonaux specifiques contre le ganglioside 3',6'-isold1 WO1994003629A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012071216A2 (fr) * 2010-11-22 2012-05-31 Duke University Anticorps dirigés contre des gangliosides tumoraux
WO2012071216A3 (fr) * 2010-11-22 2012-07-12 Duke University Anticorps dirigés contre des gangliosides tumoraux
US9441048B2 (en) 2010-11-22 2016-09-13 The United States Of America As Represented By The Secretary Of Health And Human Services Antibody for 3′-isoLM1 and 3′,6′-iso-LD1 gangliosides
US10052382B2 (en) 2010-11-22 2018-08-21 Duke University Methods for using antibodies for 3′ iso-LM1 and 3′, 6′-iso-LD1tumor gangliosides

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