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WO1992021767A1 - Me20: anticorps monoclonaux et antigene contre le melanome humain - Google Patents

Me20: anticorps monoclonaux et antigene contre le melanome humain Download PDF

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Publication number
WO1992021767A1
WO1992021767A1 PCT/US1992/004451 US9204451W WO9221767A1 WO 1992021767 A1 WO1992021767 A1 WO 1992021767A1 US 9204451 W US9204451 W US 9204451W WO 9221767 A1 WO9221767 A1 WO 9221767A1
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antibody
surface protein
cells
accession number
atcc accession
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PCT/US1992/004451
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English (en)
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Ingegerd Hellstrom
Karl Erik Hellstrom
Hans Marquardt
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Bristol-Myers Squibb Company
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Publication of WO1992021767A1 publication Critical patent/WO1992021767A1/fr

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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
    • C07K16/3053—Skin, nerves, brain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention is directed to the discovery of a new cell surface protein designated as antigen ME20 (AgME20) which is present on human melanoma cells and of antibodies, such as mAb ME20 and antibody fragments, which have been developed and specifically immunoreact with this cell surface protein.
  • AgME20 antigen ME20
  • New peptides that contain regions in their amino acid residue sequences which substantially correspond to domains of AgME20 are also disclosed herein.
  • Tumor cells express certain antigens which are absent from, or present in small amounts on, their normal cellular counterparts. Many of these are differentiation antigens, shared by the tumor and certain embryonic cells. Some of the antigens appear with sufficient selectivity in tumors to serve as possible targets for therapeutic agents. This possibility has been reviewed for malignant melanoma (Hellström and Hellström, in Accomplishment in Cancer Research - 1984 Prize Year, General Motors Cancer Research Foundation, J.G. Fornter & J.E. Rhoads, eds., J.B. Lippincott Company, Philadelphia, (1985) pp.
  • Antibodies such as those directed to tumor-associated antigens can kill human tumor cells in the presence of human effector cells (Hellström et al. , (1981) Int. J. Cancer 27:281-285) in the presence of human serum as a source of complement (Hellström et al., (1985) Proc. Natl. Acad. Sci. 82:1499-1502; Hellström et al., (1985) Monoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular and Cellular Biology, Vol. 27, pps. 149-164, Alan R. Liss, Inc., NY).
  • Anti-tumor antibodies have been utilized in immunohistology (Garrigues et al., (1982) Int. J. Cancer 29 :511-515; Natali et al., in Cardnogenesis - A Comprehensive Survey, C.J. Conti, T.J. Slaga and A.J.P. Klein-Szanto, eds., Raven Press, Ltd. New York, (1989) Vol. 11. pps. 133-164) and immunotherapy (Hellström and Hellström (1988) Pigment Cell Res. S.l: 180-184; Neuwelt et al., (1987) Neurosurgery 20:885-895), as well as for diagnostic imaging (Larson et al., (1983) J. Clin. Invest. 72:2101-2114; Murray et al., (1988) in Malignant Melanoma: Biology, Diagnosis and Therapy, L. Nathanson, ed., Khiwer Academic Publishers, Boston, pps. 123-149).
  • Some approaches for preparing anti-cancer agents involve labelling antibodies with radioactive isotopes (Larson et al., (1983) Clin. Invest. 72:2101-2114; Order, (1984) Compr. Ther. 10:9-18; Carrasquillo et al., (1984) Cancer Treatment Reports 68:317-328; de Nardo et al., (1985) Int. J. Radiation Oncology Biol. Phys. 11:335- 345), or conjugating antibodies to toxins (Jansen et al., (1982) Immunol. Rev. 62:185-216; Vitetta and Uhr (1984) Transplant.
  • the antibody provides the specificity of targeting the agent to the appropriate tumor cell and the isotope or drug provides the ability to destroy the tumor.
  • a disadvantage of this approach until the present invention, has been the lack of high specificity of the antibody, which in many instances could bind to non-cancerous tissue as well. Because both anti-cancer drugs and radioisotopes have a high level of toxicity to normal tissues, this nonspecific uptake in various organs such as kidney, liver, or bone-marrow could lead to substantial side-effects.
  • the present invention is directed to a monoclonal antibody, designated mAb ME20, which is immunospecific for a cell surface protein that is found on human melanoma cells and on cells from a fraction of dysplastic nevi but not significantly on other cells. Both melanoma and nevi are believed to be embryologically derived from the neural crest.
  • This invention also includes fragments of mAb ME20 such as Fab, Fv, Fab' and (Fab') 2 fragments as well as other antibodies and fragments with similar specificity.
  • Monoclonal antibody ME20 (and its fragments) can be operatively linked, or conjugated, to another compound such as a drug, toxin, labelling agent,
  • This invention also includes diagnostic and therapeutic methods employing the antibody as well as methods and compositions for using such monoclonal antibodies.
  • the present invention is further directed to a novel cell surface antigen, designated AgME20, characteristic of human melanoma cells, as well as a substantially pure peptide that contains a region that corresponds to a domain of a mdanoma cell surface antigen such as AgME20.
  • the invention also includes diagnostic and therapeutic methods employing the peptide as well as methods and compositions using that novel peptide. DESCRIPTION OF PREFERRED EMBODIMENTS
  • the present invention is directed to compounds and methods useful in the detection and treatment of human melanoma.
  • the present invention is directed to a new monoclonal antibody, mAb ME20 and antibody fragments such as Fab, Fv, Fab' and F(ab') 2 fragments, and synthesized polypeptide chains that specifically immunoreact with a human melanoma surface protein, AgME20, to which mAb ME20 binds as described herein.
  • This surface protein is detectably present only on melanoma cells and on cells from a fraction of dysplastic nevi (2 of 6 tested). Both melanoma and nevi are believed to be embryologically derived from neural crest cells.
  • the mAb ME20, its fragments and other similarily binding antibodies and fragments can be utilized, inter alia, in the detection and/or treatment of melanoma either alone or in conjunction with another compound.
  • an antibody or fragment of the present invention is operatively linked to a compound such as a drug, toxin, labelling agent, growth factor, radionuclide or radioisotope or an enzyme.
  • the term “immunoreacts” refers to the production of a specific binding interaction similar in nature to the immunological binding that occurs between an antigen and an antibody directed to that antigen.
  • a polypeptide such as the monoclonal antibody mAb ME20, immunoreacts with a cell surface antigen, such as AgME20, on human melanoma cells.
  • the polypeptides of the present invention bind to a much lesser degree to normal human adult cells and tissue than to melanoma cells.
  • mAb ME20 binds with a high degree of specificity to a novel antigen characteristic of human melanoma.
  • MAb ME20 and similar polypeptides and fragments of the present invention, as described above, can be operatively linked to other compounds and utilized for diagnostic, localization or therapeutic purposes.
  • polypeptide As used herein, the terms “polypeptide” and “antibody” are used
  • glycosylated proteins refer to a natural or synthetic molecule composed of amino acid residues attached by peptide bonds that can specifically immunoreact with a human melanoma cell surface protein, and include glycosylated proteins called
  • immunoglobulins antibodies
  • antibody fragments synthetic proteins and protein fragments that are capable of specifically combining or immunoreacting with a melanoma surface protein.
  • the term "antigen" refers to an entity that is capable of being specifically bound by an antibody. When an antigen is capable of inducing antibody production it is also referred to as an "immunogen".
  • the term "operatively linked” refers to a linkage that does not interfere with the ability of either of the linked groups to function as described.
  • mAb ME20 is operatively linked to a biologically active compound such as a drug in a manner that permits the polypeptide to bind to a human melanoma surface protein and which does not interfere with the biological activity of the drug.
  • mAb ME20 is operatively linked to a chemotherapeutic drug, such as dacaibazine, adriamycin or mitomycin C.
  • a chemotherapeutic drug such as dacaibazine, adriamycin or mitomycin C.
  • the polypeptides of this invention that specifically immunoreact with the ME20 antigen include antibodies such as the monoclonal antibody mAb ME20.
  • the biological activity of antibodies is multi-faceted. To a large extent the Fc region of the molecule determines the ability of antibody molecules to activate complement and mediate antibody-dependent cellular cytotoxicity (ADCC) (Uanue and Benacerraf (1984) Textbook of Immunology, 2nd Edition, Williams and Wilkins, Chap. 12, pps. 218-238).
  • ADCC antibody-dependent cellular cytotoxicity
  • Antibodies are divided into various classes and subclasses. Preferred
  • monoclonal antibodies of the present invention are of the IgG1, IgG2a, and IgG3 subclasses.
  • antibodies of the IgG2a and IgG3 subclasses can often mediate ADCC and activate serum complement.
  • polypeptides of the present invention are preferably present in a
  • composition together with one or more pharmaceutically acceptable carriers.
  • the term "pharmaceutically acceptable carrier” refers to a compound which is compatible with administration to a patient and does not produce toxic or untoward effects upon such administration.
  • pharmaceutically acceptable carrier refers to a compound which is compatible with administration to a patient and does not produce toxic or untoward effects upon such administration.
  • pharmaceutically acceptable carriers are phosphate buffered saline, Ringer's solution, oils, gels and microspheres, as well as liposomes.
  • Other pharmaceutically acceptable carriers are well known in the field of pharmacy and are contemplated by the present invention.
  • labelling agent refers to a single atom or molecule that when operatively linked to a product of the present invention enables detection of its presence in an assay method.
  • Illustrative labelling agents are fluorescent dyes, radioisotopes, enzymes and antibodies that can be either independently detected or detected in conjuction with the addition of a substrate or other molecule that reacts therewith.
  • the monoclonal antibody mAb ME20 exists in two isotypic forms, IgG1 and IgG2a, respectively, and these forms have been produced from hybridomas that have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, in full compliance with the deposition requirements of the United States Patent and Trademark Office and the Budapest Treaty, on June 4, 1991 and have the Acccession numbers HB10764 and HB 10763 respectively.
  • the antibodies of the present invention are specifically immunoreactive with determinant sites on a glycoprotein antigen, referred to as AgME20, associated with human melanoma cells and which has not been found on other human tumors.
  • AgME20 glycoprotein antigen
  • Such immunoreactive specificity for these antibodies greatly diminishes the likelihood of the present antibodies binding to carcinomas of the breast, lung, colon and the like.
  • the antibodies of the present invention are useful, either alone or in
  • multimer refers to a molecule that contains several repeating units of a portion of the molecule.
  • a multimeric peptide of the present invention contains, preferably, 2 to about 10 repeating regions of a portion of an amino acid residue sequence of the peptide operatively linked together.
  • a multimeric antibody corresponds to several molecules of an antibody, such as mAb ME20, operatively linked together to result in a molecule having several functional binding sites that can each specifically immunoreact with a human mdanoma cell surface protein such as AgME20.
  • the antibodies of this invention can be further utilized in methods and tests for diagnosis, detection and treatment of human melanoma.
  • the present invention further encompasses a substantially purified peptide that contains a region which mimics, or substantially corresponds to, a domain on a human melanoma cell surface protein.
  • the peptides of the present invention are capable of specifically immunoreacting with an antibody of the present invention, such as mAb ME20.
  • substantially refers to a high levd of similarity.
  • a substantially purified peptide of the present invention refers to a preparation having less than about ten percent extraneous peptides present.
  • a substantially similar sequence in the present invention has less than about ten percent variation with the reference sequence.
  • a peptide of the present invention include
  • substantially purified natural and synthetic molecules that contain amino acid residue sequences and/or glycosylation that can immunoreact with the ME20 monoclonal antibody.
  • the ME20 human melanoma cell surface protein, AgME20 is a glycoprotein that has a molecular weight of about 80 kD to about 120 kD. In a preferred
  • the ME20 cell surface protein has a molecular weight, as determined by SDS-PAGE, of about 100 kD to about 116 kD.
  • the antibodies of the present invention can be utilized in methods of treatment and for detection of human melanoma.
  • an antibody of the present invention that is capable of binding to a human melanoma cell surface protein can be utilized to detect the presence of human melanoma cells in in vitro and in vivo assays.
  • human cells of a patient are contacted with an effective amount of the antibody to enable binding of the antibody to any melanoma cells that are present.
  • the presence of the bound antibody is then detected by any of several methods that are readily available in the art.
  • Illustrative detection methods include radiographic detection of the presence of a radioisotope operatively linked to the antibody, use of a second antibody that is detectably labelled and which second antibody immunoreacts with an exposed region of the bound antibody, and the use of an indicating means, such as a substrate which is converted to a fluorescent molecule by an enzyme operatively linked to the antibody.
  • a polypeptide of the present invention is administered at a therapeutically effective dosage to a patient together with a chemotherapeutic agent for a time period that is sufficient to result in the inhibition of further proliferation of any melanoma cells present.
  • chemotherapeutic agents are prefe ⁇ ably drugs and toxins commonly utilized in the treatment of melanoma.
  • the chemotherapeutic agent can be added separately from the polypeptide of this invention, together with the polypeptide or operatively linked to the polypeptide, such as a polypeptide-drug conjugate.
  • the present invention also encompasses an immunogenic composition capable of engendering an immune response when administered to a patient.
  • immunogenic compositions contain a substantially purified peptide of the present invention, corresponding to a region of a human melanoma cell surface protein, and an immune response enhancing agent such as a hapten or adjuvant, such as alum.
  • This cell surface protein such as AgME20
  • vaccines for example, AgME20 may be expressed in a recombinant vaccinia virus (Estin et al., (1988) Proc. Natl. Acad. Sci.
  • an immunogenic composition containing a peptide of the present invention is administered to a patient in a sufficient dosage to elicit an immune response directed toward human mdanoma cells. It is contemplated by the present invention that the immunogenic peptide composition can engender an immune response in a patient that is specifically directed against an epitope present on a human mdanoma cell surface protein such as AgME20.
  • Kits containing antibodies and/or peptides of the present invention are also contemplated, wherein these kits further contain appropriate instructions for use of the kit.
  • the kit of the present invention contains the monoclonal antibody ME20.
  • the spleen was removed from a mouse and used in the fusion protocol.
  • the spleen was disrupted into cells by scraping through screens and rinsed with RPMI medium.
  • the spleen cells were separated from the remaining tissue by centrifugation at 200 ⁇ g for 10 minutes, xesuspended in RPMI medium and then mixed with Ag8-653 myeloma cells in a ratio of Ag8-653/spleen cells of about 1:10.
  • the mixture of Ag8-653 and immunized spleen cells was centrifuged at 200 ⁇ g for 10 minutes, the supernatant medium was removed and the cells were maintained at 37°C.
  • Polyethylene glycol (PEG, 1 ml) was added to the cells over a period of one minute with shaking for an additional one minute.
  • Iscove's Modified Dulbecco's Medium without HAT (hypoxanthine, aminopterin, thymidine) was added (1 ml over a period of 1 minute); followed by an additional 8 ml over 2 minutes. The cells were centrifuged for 10 minutes at 160 ⁇ g at room temperture. The supernatant was removed and the cells were resupsended in 25 ml of IMDM. The resuspended cells were then mixed with 2 ⁇ 10 8 mouse thymocytes obtained fresh from 3 - 4 week old Balb/C mice.
  • the mixed cell culture of Ag8-653 cells, spleen cells and thymocytes was supsended in a volume of 98 ml of IMDM medium and 2 ml of 50 ⁇ HAT.
  • the culture was maintained at 37°C/7% CO 2 for 12 to 16 hours, and the cells were then transferred to 96-well microtiter plates at 200 ⁇ l/well and maintained at 37°C/7% CO 2 . After 3 days, the plates were microscopically examined to determine fusion effiriency.
  • hybrid cells were then screened for antibody production and positive hybrids were transferred into 48-well plates with additional thymocytes.
  • the resultant antibodies produced by the hybrid clones were then screened by enzyme-linked immunosorbent assay (ELISA) upon the immunizing mdanoma cdl lines and fibroblasts (human skin fibroblasts).
  • ELISA enzyme-linked immunosorbent assay
  • the ME20 antibody was isolated based upon its property of binding to melanoma cells but not binding to fibroblasts. Further characterization of mAb ME20 was obtained by immunohistology and cell analysis on FACS.
  • H3606 melanoma cells were metabolically labelled with 35 S-methionine for 10 min followed by incubation in methionine-free RPMI-1640 medium, supplemented with 5 % dialyzed fetal bovine serum, for 0 to 10 hr at 37°C.
  • the cell pellet was extracted with phosphate-buffered saline, pH 7.4, containing either 1 % NP-40, PMSF (10 ⁇ g/ml) and aprotinin (20 ⁇ g/ml) or 0.4% Triton X-100 and proteinase inhibitors.
  • ME20 antigen was immunoprecipitated by incubating the cell lysate with mAb ME20 for 15 min at 4°C.
  • the antigen-antibody complex was precipitated with rabbit anti- mouse IgG and Protein A-Sepharose (Sigma Chemical Co., St. Louis, MO). The washed immunpprecipitate was analyzed by SDS-PAGE under reducing conditions and visualized by fluorography after impregnating the gel with AMPLIFY (Amersham, Arlington Hdghts, IL).
  • H3606 human mdanoma cells were metabolically labdled with 3 H-glucosamine by incubation in 50% ISCOVE's DMEM, 50% glucose-free RPMI-1640, and 5% dialyzed fetal bovine serum.
  • ME20 antigen was immunppredpitated from the cell lysate with mAb ME20, rabbit anti-mouse IgG, and Protein A-Sepharose. The immunoprecipitate was analyzed by SDS-PAGE under reducing conditions and visualized by autoradiography.
  • mAb ME20 The ability of mAb ME20 to bind and internalize into tumor cells was investigated by an indirect immunotoxin method to detect the mtemalization of an antibody-toxin conjugate with subsequent lolling of cells that internalize the conjugate, according to the method of Till et al., (1988) Cancer Res. 48: 1119-1123.
  • a Fab fragment of goat anti-mouse immunoglobulin coupled to ricin A chain was reacted with cells previously exposed to 10 ⁇ g/ml of either mAb ME20 or mAb BR96.
  • MAb BR96 is a monoclonal antibody that spe ⁇ fically binds to breast, colon and lung cancer cells and is internalized by BR96 antigen-positive cells (Hellstrom et al., (1990) Cancer Res. 50:2183-2190).
  • MAb BR96 binds to breast, colon, and lung cancer cells and i s internalized by antigen positive cells.
  • the FACS analysis of mAb ME20 binding measured the binding of a fluorescein isothiocynate (F ⁇ TC)-labelled secondary antibody to tumor cells in vitro that had been treated with 10 ⁇ g/ml of tither mAb BR96 or mAb ME20.
  • F ⁇ TC fluorescein isothiocynate
  • the binding ratio represents a ratio between the brightness, designated linear fluorescence equivalent (LFE), of cells stained by the specific mAb ME20 versus the unstained cell population.
  • LFE linear fluorescence equivalent
  • H3606 human melanoma cells Confocal microscopy was carried out on H3606 human melanoma cells in order to further illustrate the internalization of mAb ME20.
  • Viable H3606 cells were stained by exposure at 4°C to mAb ME20 conjugated to phycoerythrin (mAb ME20-PE).
  • mAb ME20-PE phycoerythrin conjugated to phycoerythrin conjugated to phycoerythrin
  • the mAb ME20-PE had a diffuse cytoplasmic perinuclear localization and was largely absent from the cell surface indicating that the antibody had been internalized.
  • the avidity of mAb ME20 binding to H3606 human mdanoma cells was studied for two isotypes of mAb ME20.
  • lodinated mAb ME20 ( 125 I-mAbME20) was assayed for immunoreactivity to antigen positive cells and then utilized in binding studies on H3606 cells. Scatchard analysis was carried out and the results shown a Ka of approximately 10 -8 M for both the IgGl and IgG2a isotypes of mAb ME20.
  • ME20 antigen was isolated from human melanoma tumor cells (H3606 cells) obtained from a metastatic lesion established as a cell line by the inventors and partially purified by immunoaffinity chromatography. H3606 cells were suspended in phosphate-buffered saline (PBS). An equal volume of solubilization buffer was added to reach a final concentration of 0.4 M NaCl, 1 % Triton X-100, 10 mM EDTA in 17 mM phosphate buffer, pH 7.4.
  • PBS phosphate-buffered saline
  • the buffer contained the following protdnase inhibitors, 1 mM PMSF (phenylmethylsulfonyl flouride), 2 mM BAEE (N ⁇ -benzoyl-L-arginine ethyl ester), 1 ⁇ g/ml each of leupeptin, aprotinin, and pepstatin.
  • the cells were treated with solubilization buffer by gentle agitation for 30 min, at 4°C. Insoluble material was removed by centrifiigation at 100,000 ⁇ g for 90 min, at 4°C.
  • ME20 antigen was purified from the supernatant by immunoaffinity chromatography.
  • MAb ME20 mAb (5 mg/ml) in 0.2 M sodium phosphate buffer containing 0.5 M NaCl, pH 8.2, was added to 1 g of wet tresyl-Sepharose (Sigma). Coupling continued with gentle agitation by tumbling for 16 h, at 4°C.
  • the gel was treated with 0.2 M Tris-HCl, pH 8.5, for 5 h, at 20°C, and washed with: (1) 0.2 M sodium acetate buffer containing 0.5 M NaCl, pH 3.5; (2) 17 mM phosphate buffer containing 0.4 M NaCl and 1% Triton X-100, pH 7.4; (3) phosphate-buffered saline containing 1% Triton X-100, pH 7.4; (4) 100 mM diethylamine containing 0.2% Triton X-100, pH 11.5; and (5) phosphate-buffered saline containing 1% Triton X-100.
  • the cell lysate was applied to a 1 ml column of (mAb ME20)-Sepharose and recirculated for 12 to 16 hours, at 4°C.
  • the affinity support was washed with PBS containing 0.2% Triton X-100 and the antigen was eluted with 100 mM diethylamine containing 0.2% Triton X-100, pH 11.5.
  • the duate was neutralized with 2M Tris-HCl buffer, pH 6.8.
  • the partially purified AgME20 was futher purified by SDS-PAGE.
  • the column eluate was dialyzed against 0.1 % SDS.
  • SDS-PAGE (10% acrylamide) was performed according to the Laemmli method using minislab gels with 0.75 mm-thick spacers (BioRad).
  • ME20 antigen was also recovered from SDS-polyacrylamide gels by
  • PTH amino add derivatives were analyzed by reverse phase high performance liquid chromatography (rpHPLC) using a model 120A on-line HPLC unit (Applied Biosystems, Inc.) with PTH C18 column (2.1 ⁇ 220 mm, Applied Biosystems, Inc.) and a sodium acetate/tetrahydrofuran/acetonitrile gradient for elution. Data reduction and quantitation were performed by using a Ndson 760 interface, a Hewlett Packard 9816 computer, and modd 900A/model 475A
  • Sequence I.D. No. 1 The ammoterminal sequence, designated as Sequence I.D. No. 1, is as follows:

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Abstract

Sont décrits une nouvelle catégorie de molécules polyopeptidiques, notamment des anticorps, qui entrent en immunoréaction spécifique avec une protéine de surface cellulaire détectable sur les cellules tumorales d'un mélanome humain, ainsi que des peptides spécifiques correspondant aux domaines immunoréactifs de la protéine de surface cellulaire. Sont également décrites des méthodes de détection et de traitement du mélanome humain.
PCT/US1992/004451 1991-06-05 1992-05-27 Me20: anticorps monoclonaux et antigene contre le melanome humain WO1992021767A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5844075A (en) * 1994-04-22 1998-12-01 The United States Of America As Represented By The Department Of Health And Human Services Melanoma antigens and their use in diagnostic and therapeutic methods
US6537560B1 (en) * 1994-04-22 2003-03-25 The United States Of America As Represented By The Department Of Health And Human Services Melanoma antigens and their use in diagnostic and therapeutic methods
WO2006119527A2 (fr) 2005-05-11 2006-11-16 Avir Green Hills Biotechnology Research Development Trade Ag Diagnostic du melanome
US7846450B2 (en) 1996-07-11 2010-12-07 United States Of America, As Represented By The Secretary, Department Of Health And Human Services Melanoma associated peptide analogues and vaccines against melanoma
US20110300067A1 (en) * 2008-09-09 2011-12-08 Ekaterina Dadachova Methods for increasing efficacy of radioimmunotherapy of melanoma

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0189849A2 (fr) * 1985-02-01 1986-08-06 Sloan-Kettering Institute For Cancer Research Méthode de traitement de maladies neuro-ectodermales et de carcinomes épithéliaux chez les humains
GB2188637A (en) * 1986-02-07 1987-10-07 Oncogen Vaccines against melanoma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0189849A2 (fr) * 1985-02-01 1986-08-06 Sloan-Kettering Institute For Cancer Research Méthode de traitement de maladies neuro-ectodermales et de carcinomes épithéliaux chez les humains
GB2188637A (en) * 1986-02-07 1987-10-07 Oncogen Vaccines against melanoma

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF NUCLEAR MEDICINE vol. 26, no. 10, October 1985, NEW YORK, US pages 1172 - 1179; P. BEAUMIER ET AL.: 'Melanoma localization in nude mice with monoclonal Fab against p97.' *
MOLECULAR IMMUNOLOGY vol. 18, no. 3, March 1981, OXFORD, GB pages 207 - 218; K. MITCHELL ET AL.: 'Structural characterization of the "melanoma-specific" antigen detected by monoclonal antibody 691I5Nu-4-B.' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 88, October 1991, WASHINGTON DC, US pages 9228 - 9232; B. KWON ET AL.: 'A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12.' *

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