EP0366707A1 - Anticorps monoclonaux contre le carcinome des cellules renales - Google Patents

Anticorps monoclonaux contre le carcinome des cellules renales

Info

Publication number
EP0366707A1
EP0366707A1 EP19880906254 EP88906254A EP0366707A1 EP 0366707 A1 EP0366707 A1 EP 0366707A1 EP 19880906254 EP19880906254 EP 19880906254 EP 88906254 A EP88906254 A EP 88906254A EP 0366707 A1 EP0366707 A1 EP 0366707A1
Authority
EP
European Patent Office
Prior art keywords
antigen
antibody
rcc
specific
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19880906254
Other languages
German (de)
English (en)
Inventor
Egbert Oosterwijk
Sven O Warnaar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0366707A1 publication Critical patent/EP0366707A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K16/3038—Kidney, bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/106—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from kidney or bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Mabs recognizing such antigens can be useful in reagents in diagnostic tumor pathology, and in .serum tests for tumor associated or tumor specific anti ⁇ gens. Radiolabeled Mabs specific for these antigens can also be useful for detection, localization and treatment of these * tumors _iri i o-
  • Renal cell carcinoma is a cancer which is particularly difficult to diagnose. See Oberling et al. Nature 186, 402-403 (1960) and Holthafer et al. , Lab. Invest. 4_9, 319-326 (1983) . Mabs specific to antigens which are pre-dominantly present only on RCC tumor cells would be extremely useful in the diag ⁇ nosis and treatment of this disease. Summary of the Invention
  • This invention pertains to several new antigens associated with renal cell carcinoma (RCC) and to monoclonal antibodies reactive with the antigens.
  • RCC renal cell carcinoma
  • the invention also pertains to methods of diagnosis and method of treatment of RCC and diagnostic and therapeutic compositions useful in these methods.
  • the RCC associated antigens of this invention are designated G 250, RC 38, RC 3, RC 69 and RC 154 and are characterized by distinct patterns of tissue distribution as elaborated below.
  • Antigen (Ag) G 250 is an antigen present on RCC and absent on normal adult fetal kidney tissue. Ag G 250 is absent from all other normal adult tissue (except for the epithelium of the bile ductand stomach) and most malignancies.
  • Antigen RC 38 is an antigen which is present on primary and metastatic RCC tumors. The RC 38 anti ⁇ body reacted with 46 out of 47 primary RCC tested and with 8 out of 13 RCC metastases. No reaction was seen with 179 tumors of various origin. RC 38 is also present on the mucous cells of the stomach, on the crypts and villi of the jejunum, on the crypts of the colon, on the sinuses of the liver, on the acini of the sweat glands, and on the sinuses of the lymph node.
  • RC 38 is present on the differentiating visceral glomerular epithelial cells at the capillary loop stage and on the most proximal part of the tubules connected to these regions of the fetal metanephros.
  • Antigen RC 3 is present on primary RCC and marginally present on metastic RCC.
  • Antigen RC 69 is present on primary RCC but absent on metastic RCC. Both RC 3 and RC 69 are present on proximal tubules and the thick descending part of Henle's loop in tissue sections of normal adult kidneys. RC 3 is marginally present on the parietal epithelial cells of Bowman's capsule in the region adjoining the outgoing tubule. RC 69 is present on the fetal metanephros at the middle limb of the S-shaped stage. RC 3 and RC 69 were not present on any non-renal tissues which were tested.
  • Antigen RC 154 is present on primary RCC and absent on metastic RCC. RC 154 is marginally present on the proximal tubular epithelium and on the distal tubules and small collecting ducts of the normal adult kidney. It is present on the fetal metane ⁇ phros, on the ducts of adult breast glands and on the follicles of the adult thyroid gland.
  • Antibodies reactive with the RCC antigens G 250, RC 38, RC 3, RC 69 and RC 154 can be used in the diagnosis and treatment of RCC.
  • Figure 1 shows immunoperoxidase/(DAB) staining of RCC adjacent to uninvolved kidney tissue with Mab G 250. (Magnification 64x.) Diffuse staining of RCC tumor cells (bottom) , uninvolved kidney tissue negative.
  • Figure 2 shows immunoperoxidase/DAB staining of normal liver with G 250 (magnification 160x; Magnifi ⁇ cation inset 880x) Cytoplasmatic staining of bile canuliculi is visible. The cytoplasmatic aspect of the staining is visible in the inset.
  • Fig 3a, b Radioimmunoscintigraphy of an RCC tumor bearing mouse, 2 hours (fig 3a) , and 20 hours after injection (fig 3b), 100,000 count images per figure.
  • Fig 3c Radioimmunoscintigraphy of a melanoma tumor bearing mouse, 20 hours after in ⁇ jection, 50,000 count images. Mice were given an Q ⁇ intraveneous injection of 1.5 ug Tc labeled G 250.
  • the RCC tumor as well as the liver are visible 2 and 20 hours after injection.
  • fig 3a the injection point in the tail is also visible.
  • the melanoma bearing mouse fig 3c
  • the meta- stases were: nine mammary tumors (one with less than 1% tumor cells positive, eight negative) , four pulmonary tumors (all negative) and four colonic tumors (one with more than 50% positive tumor cells, onw with less than 1% tumor cells positive, two negative) . Percentages in bars represent percentage of tumors with staining characteristics corresponding to bar color.
  • the antibodies of this invention react with antigens present on RCC.
  • the antigens are designated G 250, RC 38, RC 3, RC 69 and RC 154.
  • RC 38 reacts with 95% of primary and 67% of meta- static RCC and did not react with other tumors tested (but did react with some normal tissue) .
  • G 250 reacts with primary and metastatic RCC but not with normal tissue.
  • RC 4 reacted with RCC and a wide spectrum of tumors.
  • G 250 The presence of Ag G 250 was determined by the staining reaction of the antigen with Mab G 250.
  • G 250 is present in a high percentage of cells in most RCC tumors and is absent from the cells of normal kidneys. With respect to the normal tissues tested G 250 is present on normal bile duct epithe ⁇ lium, mucous cells in the stomach and is marginally present on epithelial cells in the jejunum. No reaction was found with other adult tissues tested nor with any fetal tissue tested.
  • G 250 antigen was found in a few benign tumors and premalignant lesions including the two renal adenomas tested.
  • the G 250 antigen appears to be absent from the normal adult kidney as evi ⁇ denced by the data obtained in ELISA and immuno- histochemistry.
  • the G 250 appearance in renal adenoma and the general occurrence of this antigen in primary RCC suggest that induction of G 250 antigen synthesis is inherently related to tumor development, possibly due to a common initiating event such as activation of a cellular oncogene product.
  • the G 250 determinant was also found in nonRCC tumors, mainly in carcinomas but occasionally also in sarcomas. In these cases the corresponding normal tissues were negative.
  • G 250 antigen is expressed relatively frequently in colonic carcinomas.
  • G 250 stains a variety of other tumors, although with low incidence, makes it less suitable for establishing a differential diagnosis of RCC.
  • the strong staining of cell membranes in the majority of RCC tumor cells in most RCC suggests that this Mab can be useful for tumor scintigraphy.
  • RCC tumors from a cell line with moderate expression of G 250 antigen (as estimated from the i munofluorescence data) were visualized. Clear tumor resolution was obtained with RCC tumors with a diameter ranging from 5-7mm without using subtraction techniques. Tumors were also visualized with labeled G 250 in tumor bearing kidneys ex vivo.
  • Antigens RC 3, RC 69, RC 154 and RC 38 were also discovered on the cells of renal cell carcinoma tumors. These antigens were discovered by their reaction with a series of monoclonal antibodies. RC 3 antigen reacted with Mab RC 3, RC 69 antigen with Mab RC 69, RC 154 antigen with Mab RC 154 and RC 38 antigen with Mab RC 38. These monoclonal antibodies and the cell lines which produced them are specific embodiments of the monoclonal antibodies and cell lines of this invention.
  • the expression of the antigens RC 3, RC 69, RC 154 and RC 38 was found to be variable both with respect to numbers of antigen expressed and to the percentage of antigen-positive RCC tumor cells.
  • the combina ⁇ tions of antigen expression RC 3+/RC 69+/RC 154+ and RC 3+/RC 69+/RC 1-54- were frequently observed in * primary RCC whereas no tumors were found to have a combination of antigen expression RC 3-/R C69+/RC 154+ or RC 3-/RC 69-/154+.
  • the data indicates that RCC Cells with metasta- sizing capacity have lost the antigens corresponding to RC 3, RC 69 and RC 154.
  • Renal adenomas are often considered precursor lesions of RCC and the main distinguishing point between adenoma and carcinoma is considered to be the size of the lesion, diameter 3 cm constituting the borderline between these lesions See Bennington, "Histopathology of Renal Tumors" in: Renal Adenocarcinoma (UICC Technical Reporting Series No. 49) Geneva, UICC, 1980, Eds. Sufrin, G. and Buckley, A.
  • RC 38 antigen is not present on tumor cells of breast tumors, carcinomas of the gastrointestinal tract or angiosarcomas. In most tumors, RC 38 antigen is present on the endothelium of small vessels whereas it is absent in capillary endothelium of normal tissues except in the liver and lymph nodes. Therefore RC 38 might be useful in studies on angiogenesis or vascularisation in human tumors.
  • Antibodies against the enumerated RCC antigens can be used to detect RCC antigen in samples of bodily fluids (e.g. serum or plasma) .
  • bodily fluids e.g. serum or plasma
  • serological tests for circulating antigen may have diagnostic or prognostic value.
  • Immunoassays for detection of RCC antigens can be performed in any of the standard formats such as competitive or immuno- etric formats.
  • Human tissue specimens can be tested for the presence of the RCC antigens by immunohistochemical techniques such as immunoperoxi ⁇ dase staining.
  • immunofluorescent techniques can be used to examine human tissue specimens with the anti-RCC antibodies.
  • slides containing cryostat section of frozen, unfixed tissue biopsy samples or cytological smears are air dried and incubated with the anti-RCC antibody in a humidified chamber at room temperature.
  • the slides are then layered with a preparation of antibody against the anti-RCC antibody.
  • the second antibody can be an anti-mouse antibody.
  • the second antibody is labeled with a fluorescent compound.
  • the staining pattern and intensities within the sample are then determined by fluorescent light microscopy.
  • An immunoscintigraphic image of RCC in vivo can be obtained by administering to a person suspected of having RCC, labeled antibody (or a mixture labeled of antibodies) against an RCC antigen and allowing sufficient time for the antibody to accumulate at the tumor site(s) .
  • the signal generated by the labeled antibody is then detected by an appropriate detecting device and the detected signal is converted to an image of the tumor.
  • the constructed image can be used to localize and to assess the size of the tumor in vivo.
  • antibodies against antigens G250 and RC 38 are preferred. These antigens are expressed by primary and metastatic RCC and thus can abe used to localize primary and dis ⁇ seminated tumor.
  • mixtures of antibody i.e., "cocktails"
  • ⁇ mixture of the antibody G 250 and RC 38 can provide an imaging composition having the advantages of the properties of both antibodies.
  • radio- isotopically labeled intact antibody or antigen- binding fragments of anti-RCC antibody such as ,the monovalent Fab' fragment or the divalent F(ab') fragment may be used.
  • the advantages of using antibody fragments in tumor radioimaging techniques are described by Goldenberg, D.M. and Deland, F.H. (1982) J. Biol. Response Modifiers 1, 121-136 and by Goldenberg in U.S. Patent 4,331,647.
  • the preferred label for immunoscintigraphy is a gamma-emitting radioisotope which can be detected with a conventional photoscanning device (such as a gamma camera) .
  • a conventional photoscanning device such as a gamma camera
  • Examples of gamma emitting radio- isotopes conventionally employed in _in vivo tumor radioimaging techniques including 123Iodine,
  • 125 Iodine, 131 Iodine, 99 ⁇ Technetium or li:L Indium A variety of methods exist for attaching the radio- isotopes to proteins either directly or via a che- lating agent such as diethylene triamine pentacetic acid (DTPA) ; any of these may be used to label the antibody or antibody fragment.
  • DTPA diethylene triamine pentacetic acid
  • the antibody may be labeled with Na[125I] by the chlor- amine-T method. See Hunter, W. M. and Greenwood, F.
  • Antibody may be directly labeled with 99m Technetium by the techni.que of
  • the antibody or antibody fragment is labeled td an appropriate specific activity (generally at least about 5 uCi/ug protein) .
  • the immunoscintigraphic composition is injected into the patient intravenously, intra-arterially or intraperit ⁇ neally.
  • the amount of radioactivity injected should be sufficient for detection by a standard gamma camera after the labeled antibody has distributed through the tissues of the body.
  • the anti-RCC antibodies of this invention may be provided in kits for radioimmunoscintigraphy in humans.
  • a kit includes either antibody the monovalent fragment Fab 1 , the bivalent fragment F(ab ! ) or a cocktail of antibodies or antibody fragments (e.g. G250 and RC 38) .
  • the labeling procedure will be prepared by the clinician.
  • the antibodies or fragments can be provided with a preattached chelator (e.g. DTPA) for labeling via the chelator.
  • the labeled antibody is prepared for injection in a physiologically ac ⁇ ceptable vehicle.
  • Imaging based on the detection of nuclear magnetic resonance (NMR) properties of tissues labeled with paramegnetic substance can also be employed.
  • Monoclonal antibodies may be used to deliver the paramagnetic substance to the RCC site and allow detection of tumor masses by NMR imaging.
  • the RCC-associated antigens of this invention also provide a basis for therapy of RCC.
  • Antibody targeted to RCC antigens can be administered in therapeutically effective (anti-tumor) amou'nts to patients afflicted with RCC.
  • the antibody can be given alone or as a carrier of an anti-tumor drug-
  • antimetabolites such as the anti- folate, methotrexate, or the purine or pyrimidine analogs mercaptopurine and fluorouracil.
  • antibiotics include antibiotics, lectins such as ricin and abrin, toxins such as the subunit of diphtheria toxin, radionuclides such as 211Astati.ne and 131Iodine, radiosensitizers such as misanidazole or neutron sen- sitizers such as boroncontaining organics.
  • lectins such as ricin and abrin
  • toxins such as the subunit of diphtheria toxin
  • radionuclides such as 211Astati.ne and 131Iodine
  • radiosensitizers such as misanidazole or neutron sen- sitizers such as boroncontaining organics.
  • neutron sen- sitizers such as boroncontaining organics.
  • Antibodies against the RCC antigens can be used to target cytotoxic cells (e.g. human T cells, monocytes or NK cells) .
  • Cytotoxic cells can be attached to RCC via Fc receptors on the cells (which bind the Fc portion of an anti-RCC antibody) or via a bridging antibody of dual specificty (i.e. an anti ⁇ body specific for the cytotoxic cell and for RCC) .
  • Dual or bi-specific antibodyes for targeting RCC can be produced by fusing an anti-RCC producing cells with a cell producing antibody against the cytotoxic cell to be targeted.
  • a cell formed by fusion of a hybridoma producing anti-RCC antibody and a hybridoma producing anti-cytotoxic cell antibody (quadroma) will produce hybrid antibody having specificity of the antibodies produced by the par ⁇ ents. See, e.g. Immunol. Rev. 1979; Cold Spring Harbor Symposium Quant. Biol. 1977: 4_1, 793.
  • a hybridoma producing an anti-T3 antibody can be fused with hybridoma producing antibody against the RCC antigen (preferably RC 38 or G250) to yield a cell line which produces T3/RCC bispecific antibody for targeting cytotoxic T cells to RCC.
  • Bispecific antibodies can also be produced as heteroantibodies by chemically coupling two antibodies of desired specificity.
  • the cytotoxic cell can be targeted by allowing the bispecific antibody to bind the cell. After targeting, the cells can be adminstered to the patient. Therapy with targeted cells can be used as an adjunct to surgical therapy, radiation therapy, or chemotherapy of RCC.
  • Monoclonal antibodies against the RCC antigens G 250, RC 38, RC 3, RC 69 and RC 154 of different immunoglobulin classes can be prepared by techniques for class switching of hybridoma antibodies. See, e.g. J. Immunol. , 1982: 128, 1271; J. Immunol. 1983:131 877; PNAS 1980 77 . ' 2909 and PNAS 1985: 8_2,8653.
  • Antibodies of the various G subclasses (1, 2a, 2b and 3) can be produced which bind to different Fc receptors on effector cells.
  • Antibody of the A, M, D and E classes can also be produced. IgE variants may interact with mast cells to provide an allergic effect at the tumor site.
  • EXAMPLE 1 Production of the G 250 Hybridoma Cell Line
  • An RBF mouse was immunized 5 times (with four week intervals) with cell homogenates from primary RCC lesions obtained from 4 different patients. Cell homogenates were diluted 1: 1 with Freund's incomplete adjuvant (Sigma, St. Louis, USA) . Three days after the last immunization the mouse was sacrificed and the spleen cells were isolated. These were fused with Sp2/0 myeloma cells essentially according to Kohler and Milstein, Nature, 256, 495-497 (1975) , and cultured in soft agar. After 10 days of growth, the agar was overlaid with nitrocellulose filters, Sharon et al., Proc. Natl. Acad.
  • Tissue Samples, Staining of Cryostat Sections and Scoring Procedure -Tissue " samples taken from surgical specimens, autopsy and abortions- were snap-frozen and stored at -70"C until used ' .
  • For-RCC at least two tissue blocks obtained fron non adjacent parts, were tested.
  • Indirect immunoperoxidase staining of air-dried and aceton fixed cryostat sections was done with hybridoma culture fluid essentially according to van Muijen et al. Am. J. Pathol. , 114 9-17 (1984) .
  • RAM-HPO was used as a second antibody and DAB/H_0 wound as substrates. Sections were counterstained with he atoxylin.
  • Cryostat sections of normal tissues were scored as negative when not a single cell was stained.
  • percentages of cells stained per cm 2 were estimated visually.
  • Four categories were arbitrarily distin ⁇ guished. These were: negative, less than 1%, between 1% and 50%, and more than 50% of tumor cells stained.
  • RCC cell lines SK-RC-1, SK-RC-6 and SK-RC-7 were obtained from Dr. L. J. Old, Memorial Sloane-Kettering Institute, New York, N. Y.
  • Single cell suspensions from fresh RCC specimen were obtained by collagenase treatment (Sigma, St. Louis, USA) .
  • Unfixed or acetone fixed cells grown on glass were examined for the presence of G 250 antigen by immunofluorescence. Undiluted culture medium was used as first step and GAM-FITC as second antibody. The cells were examined in a Leitz Orthoplan immuno- fluorescence microscope with Ploemopak.
  • G 250 a clone of hybridoma cells designated G 250 was isolated that produced antibody of the IgGl subclass reacting with cryostat but not with formalin-fixed and paraffin embedded tissue sections of RCC. G 250 did not stain any structure in cryostat sections of normal kidneys ( 50 cases) , rej cted transplant kidneys (8 cases) or kidney biopsies from 5 SLE patients. Increasing the antibody concentration 4Ox and enhancing the staining reaction with imidazole (Straus, J. Histochem. Cytochem. , 30, 491-493 (1980) ) , also did ndt lead to staining of normal kidney structures.
  • the staining reaction of RCC cryostat sections could be abolished by preabsorption with sup 2 of RCC but not with proteinase K (Sigma, St. Louis, USA) digested sup 2 RCC fractions.
  • Pretreatment of RCC tissue sections with 20mM NalO did not reduce the staining reaction.
  • the sensitivity to proteinase K suggests that G 250 recognizes a protein.
  • the antigen present in crude RCC homogenates or in sup 2 could not be characterized in immunoblotting experi ⁇ ments done essentially according to Towbin et al. , Proc. Natl. Acad. Sci., 76, 4350-4354, (1980), and could not be purified by affinity chromatography on Sepharose-G 250 columns.
  • EXAMPLE 4 Enzyme Linked Immunosorbent Assay Using G 250
  • 3 gram of RCC or normal kidney tissue was homogenized in 3 ml of 10 mM Tris-HCl, pH 7.4, in a Potter Elvehjem apparatus (5 strokes at 1500 rpm) .
  • Presence of relevant antigen in the respective homogenates was tested in a checkerboard assay.
  • wells in microtiter plates (Sterilin Limited, Teddington, U. K.) were filled with 100 ul of-di ⁇ lutions of sup 1 and sup 2 in 0.1 M Na CO , pH 9.5. The contents were allowed to evaporate overnight at 37°C. After blocking remaining binding sites with 3% ovalbumine in phosphate buffered saline, the wells were incubated for 2 hours at 37°C with 100 ul of G 250 culture medium. After washing and incubating with RAM-HPO, the plates were washed again and were developed with 200 ul of 0-diphenylamine and H_0 . The reactions were stopped by adding 50 ul 2.5 M H_SO and optical density readings were taken at 492 nM. As controls, Sp2/0 media, not containing antibody were used with and without RAM-HPO as second antibody.
  • G 250 antigen was present only in the sup 2 fraction of RCC.
  • the OD492 readings of wells coated with normal kidney extracts and incubat ⁇ ed with G 250 culture medium did not exceed the values of Sp2/0 or RAM-HPO incubated wells.'
  • the OD492 readings on kidney extract coated wells were in the range of 0.01-0.03, whereas the OD492 readings on RCC sup 2 coated wells were in the range of from 1.0-2.0.
  • mice bearing RCC tumors with diameters of b and 7 mm and one mouse bearing a melanoma tumor with a diameter of 6 mm were given intravenous injections
  • FIG. 3a The xenografts were distinctly visible 20 hours after injection as was the region of the liver (Fig. 3b) . After 20 hours, 7% of the total body counts were accumulated in the RCC tumors. In the melanoma bearing mouse, no tumor was visible after 2 or 20 hours after injection (Fig. 3c) . The smallest tumor visualized * weighed 60 g and measured 5x5x4 mm.
  • Tumor tissue samples excluding necrotic and haemorrhagic areas were taken from surgical specimens of various malignancies.
  • both primary and metastatic tumors were obtained at surgery.
  • autopsy material was used.
  • primary tumor and cor ⁇ responding metastases were obtained.
  • From each RCC several non adjacent tumor samples were taken. Renal adenomas were obtained at autopsy.
  • Fetal kidney tissues of 11-, 13-, 14-, 15-, 18, and 20 week gestation as estimated from bodyle gth measurements were obtained from abortions.
  • Cell homogenates were prepared from adult renal cortex or medulla and from RCC. Tissue was homoge ⁇ nized with a Potter Elvehje homogenizer (5 strokes at 1500 rpm) in three volumes of phosphate buffered . saline pH 7.4 (PBS) . After centrifugation for' 10 min. at lOOOg, the supernatant was used for immuni ⁇ zation purposes or for coating nitrocellulose fil ⁇ ters.
  • PBS phosphate buffered . saline pH 7.4
  • mice Balb/c mice were used for immunization purposes. Each animal was immunized at least three times with homogenates prepared as described above. For the immunization with RCC, homogenates from three dif ⁇ ferent patients were used. For the first injection the tissue homogenate was mixed with an equal volume of Freunds complete adjuvant (Sigma, St. Louis, USA) . Each animal was injected with 0.5 ml of the mixture of tissue homogenate and Freunds adjuvant. Booster injections with Freunds incomplete adjuvant (1:1, Sigma, St. Louis, USA) were given at two week inter ⁇ vals. Three days after the last injection the mice were killed and the spleen cells were used for fusion.
  • Freunds complete adjuvant Sigma, St. Louis, USA
  • the spleen cells of immunized mice were fused with Sp2/0 cells essentially according to Kohler and Milstein. After fusion, the cells were plated into 20 petridishes, diameter 5 cm, in soft agar (0.4%) and incubated for 10 days at 37"C in a CO incubator. Then the agar was overlaid with nitrocellulose filters saturated with a cell homogenate cor ⁇ responding to the homogenate used for immunization and a second filter, saturated with 0.75% gelatin dissolved in Hanks balanced salt solution or satu ⁇ rated with a homogenate made from a normal adult human liver, as a first screen to discrimate between kidney relevant and irrelevant antibody producing colonies.
  • These filters were prepared as follows: Cell homogenates were diluted 20 fold in Hanks balanced salt solution and sonified. The filters were soaked in Hanks balanced salt solution and put on a sintered glass funnel of diameter 47 mm. Then 10 ml of the cell homogenate was sucked through and the filters were air-dried and sterilized by UV light (30 Watt Philips TUV at 90 cm, 2 times 20 minutes each side) . To reduce toxicity of the filters, the filters were washed in sterile water. Thereafter the filters were soaked in HAT-medium containing fetal calf serum to block remaining protein binding sites.
  • the filters were removed and incubated for one hour with rabbit-anti-mouse Ig conjugated to horseradish peroxidase. After extensive washing in lOmM Tris- HCl, pH 7.4, containing 0.02% sodium-dodecyl-sulphate and 0.5% sarcosyl NL 30 (Ciba-Geigy B.V. , Arnhe , The Netherlands), the filters were developed with 0.05% di-amino-benzidine and 0.03% H_0 in 50 mM Tris-HCl, pH 7.4. The procedure followed was adapted from Sharon et al, Pro Natl Acad Sci, 76 1420-1424 (1979) .
  • Colonies producing antibodies that reacted with the filters coated with the relevant cell homogenate but not with the filters coated with liver cell homogenate or gelatine were picked and grown in suspension in microliter plates. Undiluted culture media of these cells were tested on cryostat sections of several tissues. Colonies producing antibodies positive on adult renal tissue sections or RCC sections and negative on liver and lung tissue sections were subcloned and further analyze'd on frozen sections of other tissues.
  • Sections of normal tissues were scored negative when not a single cell was stained. Tumors were scored as negative when no tumor cells were stained, e.g. tumor sections in which only blood vessels were stained were considered to be negative.
  • the subsite of the nephron stained by the Mabs was established by using generally accepted morpholo ⁇ gic criteria such as presence of brush-border and width of tubule lumen.
  • the position of the tissue section in the kidney was also taken into considera ⁇ tion.
  • Rabbit-anti-human Tam Horsfall protein (RAH-THP) was used to identify the ascending limb of Henle's loop and the distal convoluted tubule. Double immunofluorescence staining was performed to identify any overlap of THP containing cells and the cells stained with the Mabs.
  • EXAMPLE 8 Test Set Of Poorly Differentiated Malignant Tumors For Which The Diagnosis Of RCC Had Been Considered
  • Tumors of this test set included three cytological aspirates which present additional diagnostic difficulties, and poorly differentiated malignant tumors with a histological appearance and clinical presentation that mimicked RCC.
  • the pos ⁇ sibility of RCC had therefore been considered by the pathologist in the differential diagnosis.
  • the histological appearance of these cases included adenocarcinoma with clear cell, cribriform or acinar patterns, undifferentiated large-cell malignant tumors and spindle cell malignant tumors. None of these cases were included in the series of tumors used to test the specificity of the Mabs.
  • RC 3 subclass IgGl, was developed with spleen cells from a mouse immunized with normal adult renal cortex homogenates.
  • tissue sections of adult kidney RC 38 stained the glomerular visceral epithelium and the epithelial cells of the proximal tubules up to the thin descend ⁇ ing part of Henle's loop. Staining of the proximal tubular cells was mainly localized within the cyto ⁇ plasm. No staining of the THP containing cells— present in the distal tubules— as seen as indicated by double immunofluorescence.
  • RC 38 stained differentiating visceral glomerular epi ⁇ thelial cells at the capillary loop stage and the most proximal part of the tubules connected to these regions.
  • RC 38 stained the surface of the epithelium of the jejunum and colon, the mucous cells of the faveolar and glandular layer of the stomach ucosa and acini of sweat glands. In addition sinusoidal lining cells in the liver and lymph nodes were stained. Other tissues tested were negative (Table 3) .
  • RC 3 and RC 69 stained the proximal tubules up to the descending part of Henle's loop. No staining of more distal parts of the nephron was observed. The staining of proximal tubular cells was mainly associated with the brush-border. RC 3 faintly stained the parietal epithelial cells of Bowman's capsule in the region adjoining the outgoing tubule.
  • tumors of the urogenital tract and various tumors originating outside the urogenital tract were not stained by any of the Mabs (tables 4, 5) . These tumors included clear cell type tumors of the testis (6x) , lung (2x) , ovary (3x) and soft tissue (Ix) .
  • adenoma diameter 10 mm, a few cells were stained with RC 38 while RC 3, RC 3 and RC 154 failed to stain any cell.
  • adenoma ' diameter 4 mm, no cells were stained by any of the Mabs. Five other adenomas -four from one patient- ranging in size from 2-4 mm were tested with RC 38 only and were found to be negative.
  • Antibodies RC 38 and G 250 were used as radioi- mmunoscintigraphic (RIS) agents in nude mice bearing subcutaneous RCC and/or melanoma. Studies were done using 99mTc, 125I and 111In-labeled intact immuno- globulins (Ig) and 99mTc-labeled F(ab')2 fragments.
  • RIS radioi- mmunoscintigraphic
  • Tc G250 antibody Slow accumulation was not unexpected as first images showed that the blood supply of the tumor was poor as compared t the normal kidney tissue. No accumulation occured in normal kidney tissue.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Sont décrits des antigènes associés au carcinome des cellules rénales (RCC). Les antigènes sont présents sur les cellules du carcinome rénal et, en quantités variables, sur d'autres tumeurs bénignes et malignes et sur des cellules normales. Sont également décrites des compositions contenant un anticorps contre un antigène ou un mélange d'anticorps, ainsi que leur utilisation dans la thérapie et le diagnostic du RCC.
EP19880906254 1987-05-06 1988-05-05 Anticorps monoclonaux contre le carcinome des cellules renales Withdrawn EP0366707A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4719287A 1987-05-06 1987-05-06
US47192 1987-05-06

Publications (1)

Publication Number Publication Date
EP0366707A1 true EP0366707A1 (fr) 1990-05-09

Family

ID=21947563

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19880906254 Withdrawn EP0366707A1 (fr) 1987-05-06 1988-05-05 Anticorps monoclonaux contre le carcinome des cellules renales

Country Status (2)

Country Link
EP (1) EP0366707A1 (fr)
WO (1) WO1988008854A1 (fr)

Families Citing this family (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6204370B1 (en) 1992-03-11 2001-03-20 Institute Of Virology, Slovak Academy Of Sciences MN gene and protein
US6027887A (en) * 1992-03-11 2000-02-22 Institute Of Virology, Solvak Academy Of Sciences MN gene and protein
US6297041B1 (en) 1992-03-11 2001-10-02 Institute Of Virology, Slovak Academy Of Sciences MN gene and protein
US6297051B1 (en) 1997-01-24 2001-10-02 Institute Of Virology, Slovak Academy Of Sciences MN gene and protein
US6093548A (en) * 1992-03-11 2000-07-25 Institute Of Virology, Slovak Academy Of Sciences Detection and quantitation of MN-specific antibodies.
US5955075A (en) * 1992-03-11 1999-09-21 Institute Of Virology, Slovak Academy Of Sciences Method of inhibiting tumor growth using antibodies to MN protein
US5981711A (en) * 1992-03-11 1999-11-09 Institute Of Virology, Slovak Academy Of Sciences MN-specific antibodies and hybridomas
US5989838A (en) * 1992-03-11 1999-11-23 Institute Of Virology, Slovak Academy Of Sciences Immunological methods of detecting MN proteins and MN polypeptides
US5387676A (en) * 1992-03-11 1995-02-07 Ciba Corning Diagnostics Corp. MN gene and protein
US6069242A (en) * 1992-03-11 2000-05-30 Institute Of Virology, Slovak Academy Of Sciences MN gene and protein
US5972353A (en) * 1992-03-11 1999-10-26 Institute Of Virology, Slovak Academy Of Sciences MN proteins, polypeptides, fusion proteins and fusion polypeptides
US6774117B1 (en) 1992-03-11 2004-08-10 Institute Of Virology, Slovak Academy Of Sciences MN gene and protein
GB9616986D0 (en) * 1996-08-13 1996-09-25 Cancer Res Campaign Tech Materials and methods relating to the diagnosis and prophylactic and therapeutic treatment of papillary renal cell carcinoma
US7713704B1 (en) 1998-10-23 2010-05-11 Institute Of Virology Of The Slovak Academy Of Science MN gene and protein
FR2793414B1 (fr) * 1999-05-10 2003-05-23 Centre Nat Rech Scient Conjugue acide nucleique-anticorps pour delivrer un acide nucleique etranger dans les cellules
FR2793497B1 (fr) * 1999-05-10 2003-04-18 Centre Nat Rech Scient Anticorps monoclonal dirige contre les cellules de carcinome renal humain
US20080146780A1 (en) 1999-10-22 2008-06-19 Jan Zavada MN Gene and Protein
US20040077081A1 (en) 2001-02-07 2004-04-22 Egbert Oosterwijk Hybridoma cell line g250 and its use for producing monoclonal antibodies
EP1733736B1 (fr) 2001-02-07 2015-02-25 Wilex AG Procédé de production des anticorps recombinants contre des tumeurs
DK1358318T3 (da) * 2001-02-07 2007-01-02 Wilex Ag Hybridomcellelinje G250 og dens anvendelse til frembringelse af monoklonale antistoffer
JP2005538682A (ja) 2001-12-03 2005-12-22 アブジェニックス・インコーポレーテッド カルボキシックアンヒドラーゼｉｘ（ｃａｉｘ）腫瘍抗原に対する抗体
US7910549B2 (en) 2001-12-13 2011-03-22 Institute Of Virology Of The Slovak Academy Of Sciences MN gene and protein
ATE444308T1 (de) 2002-02-13 2009-10-15 Ludwig Inst Cancer Res Fusionsproteine humanisierter g250-spezifischer antikörper sowie verwendungen davon
JP5408833B2 (ja) 2002-07-01 2014-02-05 ヴィレックス アクチェンゲゼルシャフト 腎細胞腫を治療するためのＣＧ２５０およびＩＬ−２またはＩＦＮ−αを含有する抗腫瘍剤
JP2008505143A (ja) * 2004-07-02 2008-02-21 ヴィレックス アクチェンゲゼルシャフト Ｇ２５０発現腫瘍の改善されたアジュバント療法
WO2014128258A1 (fr) 2013-02-22 2014-08-28 Wilex Ag Traitement du cancer basé sur une stratification caix
WO2016199097A1 (fr) 2015-06-10 2016-12-15 National Research Council Of Canada Anticorps spécifiques de l'anhydrase carbonique ix et leurs utilisations
AU2018219887B2 (en) 2017-02-08 2024-08-15 Dragonfly Therapeutics, Inc. Multi-specific binding proteins for activation of natural killer cells and therapeutic uses thereof to treat cancer
EP4273258A3 (fr) 2017-02-20 2024-01-17 Dragonfly Therapeutics, Inc. Protéines se liant à her2, nkg2d et cd16
AU2019218136A1 (en) 2018-02-08 2020-08-13 Dragonfly Therapeutics, Inc. Antibody variable domains targeting the NKG2D receptor
CN111732660B (zh) * 2020-07-31 2020-12-29 河北佑仁生物科技有限公司 骨髓间充质干细胞和单克隆抗体联合治疗癌症

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4713352A (en) * 1981-08-31 1987-12-15 Sloan-Kettering Institute For Cancer Reseach Monoclonal antibody panel for early diagnosis and therapy of renal carcinoma
DE3485712D1 (de) * 1983-03-11 1992-06-17 Sloan Kettering Inst Cancer Monoklonale antikoerper gegen menschliche nierenkrebsantigene und verfahren.
AU6032786A (en) * 1985-07-25 1987-01-29 University Of Minnesota Detection, imaging and therapy of renal cell carcinoma with monoclonal antibodies in vivo

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8808854A1 *

Also Published As

Publication number Publication date
WO1988008854A1 (fr) 1988-11-17

Similar Documents

Publication Publication Date Title
EP0366707A1 (fr) Anticorps monoclonaux contre le carcinome des cellules renales
CA1338706C (fr) Anticorps monoclonaux anti-cancer du sein chez la femme; leur utilisation pour visualiser ce type de cancer
Welt et al. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33.
Oosterwdk et al. Monoclonal antibody G 250 recognizes a determinant present in renal‐cell carcinoma and absent from normal kidney
JP3095752B2 (ja) Ｔａｇ‐７２及びヒト癌腫に対して結合特異性を有する第二世代モノクローナル抗体及びその使用方法
US4724213A (en) Murine hybridoma Lym-1 and diagnostic antibody produced thereby
JP2810230B2 (ja) 感染性及び炎症性病変の検出及び治療のためのキメラ抗体
US5153118A (en) Monoclonal antibodies having binding specificity to human prostate tumor-associated antigens and methods for employing the same
Stein et al. Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and tumor targeting
JPH0564130B2 (fr)
EP0210970A2 (fr) Détection, visualisation et thérapie de carcinomes de cellules rénales utilisant des anticorps monoclonaux
EP1871810A2 (fr) Anticorps anti-psma conjugués
JP2574269B2 (ja) ヒト前立腺腫瘍関連抗原に対する結合特異性を有するモノクロ−ナル抗体類およびそれらを使用する方法
Babaian et al. Radioimmunoscintigraphy of prostate cancer
Feng et al. New anti-Cu-TETA and anti-Y-DOTA monoclonal antibodies for potential use in the pre-targeted delivery of radiopharmaceuticals to tumor
US5084266A (en) Method for tumor imaging utilizing a labelled tumor specific antibody and a non-tumor reactive antibody
Gasparini et al. Tumor imaging of colo‐rectal carcinoma with an anti‐CEA monoclonal antibody
US5250297A (en) Tumor-associated antigen, antibodies, compositions and uses therefor
AU4415100A (en) Monoclonal antibody directed against cells of human renal cell carcinoma
Dienhart et al. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen
Britton et al. Radioimmunoscintigraphy
JPH03188099A (ja) 新規な腫瘍関連抗原
EP0473651A1 (fr) Anticorps monoclonaux reconnaissant les cellules malignes provenant de carcinomes cystiques
AU625856B2 (en) Second generation monoclonal antibodies having binding specificity to tag-72 and human carcinomas and methods for employing the same
Hanabata et al. Immunohistochemical study on hepatic tumors--KM01 stains compared with AFP, CEA, CA19-9 and RAS P21.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19891121

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17Q First examination report despatched

Effective date: 19911220

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19920701