WO1989001629A1 - Antigene associe a des tumeurs des ovaires humains specifique de l'anticorps monoclonal ov-tl3 - Google Patents

Antigene associe a des tumeurs des ovaires humains specifique de l'anticorps monoclonal ov-tl3 Download PDF

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WO1989001629A1
WO1989001629A1 PCT/US1988/002831 US8802831W WO8901629A1 WO 1989001629 A1 WO1989001629 A1 WO 1989001629A1 US 8802831 W US8802831 W US 8802831W WO 8901629 A1 WO8901629 A1 WO 8901629A1
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antigen
antibody
cells
tumor
monoclonal antibody
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PCT/US1988/002831
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Vincent R. Zurawski
Hidde J. Haisma
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Centocor, Inc.
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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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • 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
    • A61K51/1072—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 the reproductive system, e.g. ovaria, uterus, testes or prostate
    • 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

  • Ovarian cancer is the most lethal of all gynaecological cancers. Most of the ovarian tumors are of the epithelial type, of which the serous and mucinous cystadenocarcinomas predominate. The detection of ovarian tumors is often fatally delayed because of their location and the lack of diagnostic methods for early detection. By the time most patients are diagnosed they have advanced ovarian carcinoma (FIGO Stage III or IV) , and their prog ⁇ nosis is poor. Attempts have been made to identify tumor markers which would allow both an early diagnosis and treatment, thus improving the prog- nosis for patients. See e.g., Lloyd, K.O.: Human ovarian tumor antigens.
  • This invention relates to a cell surface antigen (designated CA-TL3 ) associated with primary and metastatic human ovarian carcinoma of the serous, mucinous, endo etrioid and clear cell types which demonstrates specific reactivity with the 0V-TL3 monoclonal antibody.
  • the invention also relates to methods of diagnosis and therapy of ovarian carcinoma which are based upon the CA-TL3 antigen as a distinctive marker of the tumor.
  • the CA-TL3 antigen is a cell surface antigen expressed by the major histological types of ovarian carcinoma.
  • the antigen is not expressed by non- gynaecological tumors and is only minimally ex ⁇ pressed by normal epithelium of the female genital tract. Because of its association with ovarian car ⁇ cinoma, the CA-TL3 antigen can be used to diagnose and treat ovarian carcinoma.
  • mono ⁇ clonal antibodies which are specific for the CA-TL3 antigen can be produced and used for the detection, diagnosis and treatment of ovarian carcinoma based upon their reactivity with CA-TL3.
  • These monoclonal antibodies can be used to image primary and/or metastatic ovarian carcinoma Ln vivo by immunoscin- tigraphy, thereby allowing tumor localization. In therapy, these antibodies may be used for passive immunotherapy or as a targeting agent for selective delivery of cytotoxic agents to ovarian carcinoma in vivo.
  • Figure 1A is a photograph of an immunoblot from a 10% SDS-polyacrylamide gel of OVCAR-3 cell lysates probed with radioiodinated OC125.
  • Figure IB is a photograph of an immunoblot from a 7.5% SDS-polyacrylamide gel of OVCAR-3 cell lysates probed with radioiodinated 0V-TL3.
  • FIG. 3 shows the binding of the antibodies to
  • Figure 3A depicts the binding of 125I-OC125 to glutaraldehyde fixed cells (.o) or non-fixed cells
  • Figure 3B depicts the binding of 125I-OV-TL3 to glutaraldehyde fixed cells (o) or non-fixed cells
  • Figure 4 shows the dissociation of antibodies from OVCAR-3 cells over time.
  • Figure 4A depicts the binding of 125I-OC125 to glutaraldehyde fixed cells (o) or non-fixed cells
  • Figure 6 shows the cross inhibition of the antibodies.
  • Figure 6A depicts the binding of 12 *" 5I-OC125 with different concentrations of inhibitor OC125 (o) and with different concentrations of inhibitor
  • FIG. 6B depicts the binding of 125I-OV-TL3 with different concentrations of inhibitor OC125 (o) and with different concentrations of inhibitor
  • This invention relates to a cell surface antigen (designated CA-TL3) associated with primary and metastatic human ovarian carcinoma of the serous, mucinous , endometrioid and clear cell types (i.e. the major histological types of ovarian carcinoma) .
  • the CA-TL3 antigen is expressed by primary and metastatic ovarian tumor cells of these histological types. This antigen is further characterized by its specific reactivity with the. OV-TL3 monoclonal antibody.
  • the invention also relates to methods which employ monoclonal antibodies to diagnose and treat ovarian carcinoma • based upon the use of CA-TL3 antigen as a distinctive marker of the tumor.
  • the monoclonal antibodies OC125 and 0V-TL3 were compared in terms of their interaction with ovarian carcinoma cells in an attempt to further characterize the antigens recognized by these antibodies.
  • the CA-TL3 antigen is not shed from ovarian cancer cells. Iodinated OV-TL3 was mixed with dilutions of serum and as ⁇ ites samples from ovarian cancer patients. The mixtures were then added to wells containing ovarian cancer cells, and the inhibition of antibody binding was determined (see exemplification below) . OV-TL3 binding was only slightly inhibited by non-diluted samples, which was the same for serum samples from healthy donors or from ovarian cancer patients. (d) The monoclonal antibodies OV-TL3 and OC125 recognize distinct antigenic determinants that are associated with human ovarian carcinomas, and the steric configuration does not hinder the antibodies when they are bound to the cells.
  • the antigen of the present invention is the specificity of monoclonal antibody OV-TL3 for at least two determinants of the antigen characterized herein. Additionally, the specific reactivity of OV-TL3 for the antigen defined by the invention provides a means for isolation and purifi- cation of the antigen from other material of human origin, and ultimately the characterization of antigenic determinants.
  • the CA-TL3 antigen can be used to construct an immunoadsorbent for purifica ⁇ tion of antibody reactive with the antigen by attaching the antigen to a solid phase.
  • the purified antigen and determinants thereof are useful in the production of monoclonal anti ⁇ bodies for diagnostic and therapeutic application using techniques well known in the art.
  • murine hybridomas producing monoclonal antibodies may be obtained.
  • the isolated im uno- reactive 20 kDa or 40 kDa species of CA-TL3 can be used as an immunogen preparation to raise anti-CA-TL3 antibody by employing the standard techniques of Kohler and Milstein Nature, 256, 495-497 (1975) .
  • a mouse or other suitable host is immunized with purified CA-TL3. Spleen cells are harvested and fused with suitable mouse myeloma cells.
  • the resulting hybridomas can be selected for anti-CA-TL3 antibody production on the basis of specific reactivity with the isolated CA-TL3 antigen.
  • Monoclonal antibodies raised against CA-TL3 are useful for the diagnosis and treatment of ovarian carcinoma. These antibodies can be used for ⁇ n vivo tumor imaging to localize primary or metastatic ovarian carcinoma.
  • immunoscintigraphy can be used for tumor imaging.
  • An antibody or antibody fragment (antigen binding fragment) spe- cific for CA-TL3 conjugated to a label which . generates a signal detectable by external scin- tigraphy (e.g., a gamma emitting radioisotope) is administered to a patient. After sufficient time to allow the labeled antibody to localize at the tumor site (or sites) , the signal generated by the label is detected by a photo-scanning device.
  • the detected signal is then converted to an image of the tumor.
  • This image makes is possible to locate the tumor iu vivo and to devise an appropriate therapeutic strategy.
  • Antibody fragments rather than whole antibody molecules, are generally preferred for use in tumor imaging. Because they are distributed more readily in the tissues than are entire antibody molecules, antibody fragments accumulate at the tumor(s) more rapidly. Thus an image can be obtained in less time than is possible using whole antibody. These fragments are also cleared more rapidly from tis ⁇ sues, resulting in a lower background signal. Haber et al. , U.S. Patent No. 4,036,945; Goldenberg e_t al. , U.S. Patent No. 4,331,647. The antigen binding fragments Fab and F(ab') 2 are preferred.
  • the Fab fragment can be prepared by digestion of the whole immunoglobulin molecule with the enzyme papain; and the E(ab l ) 2 fragment by digestion of the whole immunoglobulin molecule with pepsin according to any of- several well known protocols.
  • fragments can also be prepared by recombinant DNA techniques.
  • Tlte antibodies or antibody fragments can be labelled with radioisotopes such as Indium-Ill and Technetium-9 m.
  • the gamma-emitters Indium-Ill and Technetium-99m are preferred because these radiometals are detectable with a gamma camera and have favorable half-lives for imaging in vivo.
  • Antibody can be labelled with Indium-111 or Technetium-99m via a conjugated metal chelator, such as DTPA (diethylenetriaminepentaacetic acid) .
  • DTPA diethylenetriaminepentaacetic acid
  • Krejcarek et al. Biochem. Biophys. Res. Comm. 2 :581, 1977; Khaw, B.A. et al. Science 209:295, 1980; Ganso et al., U.S. Patent No. 4,472,509; Hnatowich, U.S. Patent No. 4,479,930, the teachings of which are incorporated by reference herein.
  • a preferred method of conjugating the antibody or fragment with DTPA is by the dicyclic anhydride method. See Hnatowich et a_l. , Science 220:613-615 , 1983.
  • Human tissue specimens can be tested for expression of the CA-TL3 antigen by using monoclonal antibodies to the former in an immunohistochemical technique, such as the i muno- peroxidase staining procedure.
  • immunofluorescent techniques can be used to examine human tissue specimens.
  • slides containing cryostat sections of .frozen, unfixed tissue biopsy samples or cytological smears are air dried, and then incubated with the CA-TL3 antigen preparation in a humidified chamber at room temperature.
  • the slides are layered with a preparation of fluorescently labelled antibody directed against the monoclonal antibody.
  • the staining pattern and intensities within the sample are determined by fluorescent light microscopy.
  • the monoclonal antibodies which specifically react with the CA-TL3 antigen can be used in at least two different forms of therapy for treatment of ovarian carcinoma.
  • a physiologically acceptable vehicle e.g., normal saline
  • Intact antibody is preferred for this purpose because effector functions attributable to the F portion are retained.
  • monoclonal antibodies which specifical ⁇ ly bind to the CA-TL3 antigen can be used as tar ⁇ geting agents to deliver anti-cancer agents selec ⁇ tively to tumor cells.
  • Various pharmaceutical or cytotoxic agents can be covalently or noncovalently coupled to the antibodies.
  • useful therapeutic agents include: radioactive compounds (e.g., isotopes of Boron and Rhenium); agents which bind DNA, such as alkylating agents or various antibodies (e.g. , daunomycin, adriamycin, chlorambucil) i anti-metabolites (e.g., methotrexate) ; and inhibitors of protein synthesis (e.g., diptheria toxin and toxic plant proteins).
  • radioactive compounds e.g., isotopes of Boron and Rhenium
  • agents which bind DNA such as alkylating agents or various antibodies (e.g. , daunomycin, adriamycin, chlorambucil) i anti-metabolites (e.g., methotrexate)
  • inhibitors of protein synthesis e.g., diptheria toxin and toxic plant proteins.
  • the monoclonal antibody OV-TL3 can be administered in combination with the monoclonal antibody OC125. Because OV-TL3 and OC125 recognize distinct antigenic determinants, and the steric configuration does not hinder the antibodies when they are bound to the cells, more antibody can be bound when both antibodies are administered simultaneously.
  • Ovarian cancer cell line OVCAR-3 (Hamilton, T.C., Young, R.C, Louie, K.G., Behrens, B.C., McKoy, .M. , Grotziner, K.R. and Ozols, R.F. , Cancer Res. 4:5286-5290, 1984) was used because it can be grown in vitro as well as ⁇ n vivo in nude mice.
  • the cell line was grown in vitro in Eagle's minimal essential medium supplemented with 10% FCS (foetal calf serum), nonessential amino acids (0.1 mM) , 2 mM 1-glutamine, 1 mM sodium pyruvate, pencillin G (50 units/ml) and streptomycin (50 ug/ml) .
  • FCS fetal calf serum
  • nonessential amino acids 0.1 mM
  • 2 mM 1-glutamine 2 mM 1-glutamine
  • 1 mM sodium pyruvate 1 mM sodium pyruvate
  • pencillin G 50 units/ml
  • streptomycin 50 ug/ml
  • the monoclonal antibodies OC125 and OV-TL3 have been described (Bast, R.C, Freeney, M. , Lazarus, H., Nadler, L. . , Colvin, R.B. and Knapp, R.C, J. Clin. Invest. 68:1331-1337, 1981; Poels, L.G. , van Megen, Y. , Vooijs, CP., Verheyen, R.N.M. , illemen, A., van Niekerk, C.C, Jap, P.H. ., Mungyer, G. and Kenemans, P. J.N.CI_. TS i731-791, 1986).
  • Hybridomas were grown either in tissue culture or as acites in BALB/c mice. Both OC125 and OV-TL3 are of the mouse IgG-1 type.
  • the antibodies were purified as fol ⁇ lows: Mouse ascites of hybridoma tissue culture supernatant was filtered and adjusted to pH 8 with 1.0 M Tris-HCl before application onto a Protein A-Sepharose column. The unbound materials were removed from the column by washing with 0.1 M Tris-HCl until no further change in absorbance at 280 nm was seen. The antibodies were then eluted with 0.1 M citrate (pH 3.5). Fractions containing the purified antibody were pooled, concentrated and dialysed against 0.9% NaCl solution.
  • Iodination of 0C125 and OV-TL3 was performed acco ding to the one-vial method (Haisma, H.J. , Hilgers, J. and Zurawski, V.R. Jr. J. Nucl. Med. , in press). Briefly, 290 ug of antibody in 1 ml 0.1 M borate buffer (pH 8.2) was mixed with 1 mCi 125I in a vial previously coated with 1 ml iodogen (50 ug/ml) . After 10 minutes of incubation at room temperature, a sample was taken to determine the "amount of incorporated iodine.
  • ovarian carcinoma cells were added to one concentration of labeled antibody. After incubation at 4°C for 4 hours, the cells were washed. The amount of bound antibody was determined using a gamma counter. The reciprocal of the cell concentration was plotted against total input divided by bound antibody. Immunoreactivity was computed from the intercept on the y-axis. g OC125 was labeled at 1 x 10 cpm/pmol (0.4 uCi/uM) of active antibody and OV-TL3 was labeled at 0.7 x 10 cpm/pmol (0.3 uCi/uM) of active antibody.
  • OVCAR-3 cells were lysed in sample buffer before electrophoresis on a 7.5% or 10% acrylamide gel with a 3% acrylamide stacking gel (Laemmli, U.K., Nature 227:680-685, 1970).
  • the sample buffer contained 0.1% SDS.
  • the sample buffer did not contain sulfhydryl reducing agents and (b) the samples were not heated before they were loaded on the gel.
  • the gel was blotted onto nitrocellulose according to the method of Towbin (Towbin, H. , Stagheling, T. and Gordon, J. Proc. Natl. Ac. Sci. 769:4350-4354, 1979).
  • the transfer was performed at 2.0 Ampere for 2 hours with PBS as the transfer buffer. After incubation with PBS 10% FCS for 30 minutes at room temperature, the nitrocellulose sheets were overlayed with 125 I-labeled OC125 or 0V-TL3 (1 X 10 6 cpm/ml) for 2 hours. These sheets were then autoradiographed by exposure to x-ray film with the aid of a Kodak X-Omatic intensifying screen for 18-48 hours at -80°C
  • Cell monolayers were prepared by seeding 2 x I0 4 OVCAR-3 cells/well in 96-well flat bottom tissue culture plates. Before use, the cells were grown to 80-90% confluency, washed with PBS and fixed with 0.25% glutaraldehyde in PBS for 10 minutes at room temperature. The wells were then treated with 200 ug/ml pronase, trypsin, or chymo- trypsin in PBS or neuraminidase in 0.1 M acetate buffer (pH 4.5). After incubation for 1 hour at
  • Cell monolayers were prepared as for enzymatic treatments. To determine affinity constants, the wells were incubated with 50 ul labeled antibody for 4 hours at 4°C in PBS 10% FCS containing 0.1% NaN3 to prevent shedding and modulation. After three washes with PBS 10% FCS, binding was determined by counting in a gamma counter. Association and dissociation experiments were performed on both live and fixed (0.25% glutaraldehyde for 10 minutes) cells at room temperature. For association ex ⁇ periments, monolayers were incubated with 50 ul labeled antibody (100,000 cpm) in PBS 10% FCS, for different time periods. For dissociation, mono ⁇ layers were incubated for 1 hour with labeled antibody. After washing, antibody was added at 100 ug/ml. At different time intervals labeled antibody which remained bound to the cells was determined by gamma counting.
  • OVCAR-3 cells were prepared as for the enzymatic treatments. The cells were fixed with glutaraldehyde and incubated for 60 minutes at room temperature with different concentrations of either OC125 or OV-TL3 mixed with 125I labeled antibody or with undiluted or 5 fold diluted serum or ascites samples from patients with ovarian carcinoma,- mixed with tracer OC125 or OV-TL3.
  • mice Female athy ic Swiss-nu/nu mice (Taconic Inc., Germantown, NY) 5 to 8 weeks old were given 7 subcutaneous injections of 2.5 x 10 OVCAR-3 cells in the right flank. After 3 weeks, when tumors were approximately 1 cm in diameter, the animals were given 1 ug 125 I-labeled OC125 of OV-TL3 (2-3 uCi) in the retro orbital vein. To block uptake of free iodine by the thyroid, mice received 0.1% saturated potassium iodide in the drinking water. At 24 and 120 hour after antibody administration, mice were sacrificed (3 mice for each time point per antibody)
  • Ovarian cancer cell lysates were run on SDS 15 polyacr lamide gels and blotted on nitrocellulose.
  • nitrocellulose blots were then incubated with either 125I-labeled OC125 or OV-TL3, washed, and autoradiographed.
  • OC125 bound to a broad band with an apparent molecular weight exceeding 200,000 20 " daltons, which hardly entered the 3% stacking gel.
  • OV-TL3 reacted with two bands with apparent molecu ⁇ lar weights of 20,000 and 40,000 daltons ( Figure 1) .
  • Table I The affect on antibody binding after pretreating OVCAR-3 cells with various enzymes.
  • Affinity constants were estimated and the number of antigenic determinants was determined by measuring the binding of the 125I-labeled antibodies to the OVCAR-3 cell line. From linear double- reciprocal plots ( Figure 2) , affinity constants and the number of antigenic determinants could be calculated (Masuho, Y. , Zalutski, M. , Knapp, R.C. and Bast, R.C, Jr. Cancer Res. 4 :2813-2819 , 1984) .
  • 125 I-labeled antibodies were mixed wi.th di.lu- tions of serum and ascites samples from ovarian cancer patients. The mixtures were then added to wells containing ovarian cancer cells, and the inhibition of antibody binding was determined after 60 minutes of incubation.
  • Figure 5 shows the binding inhibition of OC125 and 0V-TL3 for serum and ascites samples diluted 5 fold. As was expected, OC125 binding was inhibited by some of the serum samples and all the ascites samples. At this dilution, inhibition of binding correlated well with CA125 values of the samples.
  • FIG. 7 indicates the uptake of the antibodies in the various tissues. No significant differences between the two antibodies were found in uptake in blood, marrow, intestine, fat, muscle and lung. At 24 hours after injection uptake of OC125 in liver and spleen was significantly higher than uptake of 0V-TL3. This difference was less apparent a.t 120 hours after injection. Kidney uptake was higher for OV-TL3 at 24 hours as well as at 120 hours after injection. Tumor uptake of OC125 and OV-TI.3 was similar at 24 hours after injection and about 5 times higher than uptake in other tissues. At 120 hours after injection of the labeled anti ⁇ body, OV-TL3 uptake in tumor was about 7 times higher than uptake in other tissues and about twice as high as OC125 uptake in tumor.
  • OC125 and OV-TL3 recognize distinct antigenic determinants that are associated with human ovarian carcinomas, and both show similar distribution patterns on tissue sec ⁇ tions of human organs and tumors.
  • OC125 binds to more than 80% of nonmucinous ovarian carcinomas including tumors of serous, endometrioid, clear cell, and undifferentiated histology (Kabawat, S.E., Bast, R.C, Bhan, A.K. , Welch, W.R. , Knapp, R.C and Colvin, R.B. Int. J. Gyn. Path. 2_:275-285, 1983).
  • Traces of antigen can be found in the mucosa of the fallopian tube, endometrium, and endocervix. Using a radioimmunometric assay, one can measure levels of CA125 in the serum and ascites of patients with ovarian cancer. CA125 antigen can also be found in human milk, normal cervical mucus, in the central airway of the lung, and seminal plasma (Davis, H.M. , Zurawski, V.R., Bast, R.C and Klug, T.L. Cancer Res. 46:6143-6148, 1986) . OV-TL3 binds to ovarian carcinomas of serous, mucinous, endometrioid and clear cell histology.
  • OC125 recognized determinants on molecules with apparent weights of more than 200,000 daltons, as -25-
  • OV-TL3 recognized determinants on molecules * with apparent weights of 20,000 and 40,000 dalton. Enzymatic studies revealed that the OV-TL3 antigen is only slightly sensitive to proteolytic digestion and that neuraminidase treatment enhances antigen expression. This could mean that the antigen is a glycoprotein and that the antigenic determinant is on the protein backbone.
  • OV-TL3 The number of antigenic determinants per cell (OVCAR-3) was 6.0 x 10 6 for OC125 and 0.6 x 10 6 for 0V-TL3. The rate of association of OC125 was very slow (50% binding after 125 minutes) compared with that of OV-TL3 (50%.binding after 30 minutes) .
  • Dissociation rates for the two monoclonal antibodies also differed.
  • OC125 dissociated with a T, of 125 minutes on fixed cells and 85 minutes on live cells, suggesting that active shedding (or modulation) process may be involved in the dissociation of this antibody from live cells.
  • OV-TL3 showed no dif ⁇ ference in dissociation rates from live or fixed cells; however, dissociation seemed to be biphasic, with apparent T, of 60 minutes and 300 minutes, suggesting that both low and high affinity binding sites might be present on the cell surface.
  • Masuho et al. studied the interaction of OC125 using other ovarian cancer cell lines and found similar affinity constants and numbers of antigens per cell.
  • Dis- sociation of OC125 was very slow in their study ( j ⁇ exceeded 23 hours) . Since they did not include excess antibody after the initial incu ⁇ bation, rebinding of the dissociated antibodies could occur and their measurements do not reflect the dissociation rate. In this study the as ⁇ sociation rate for OC125 was slow compared with that reported by Masuho et al. , who found 50% binding after 1 hour of incubation. (They used OVCA-433 cells, on which antigen might be more accessible than on the OVCAR-3 cells.)
  • Antibody binding of OC125 was inhibited by the serum or ascites samples from ovarian cancer pa ⁇ tients. This was expected, since other studies have shown that such samples contain CA125 and that the level of antigen is an indicator of the presence and status of the disease. OV-TL3 binding was inhibited by neither of these samples nor by supernatants of ovarian cancer cells grown in tissue culture. Tissue homogenates were able to inhibit binding (data not shown) . These results suggest that OC125 antigen is shed from ovarian cancer cells and is, therefore, found in the serum and ascites samples, whereas OV-TL3 antigen is not shed from ovarian cancer cells and is, therefore, not found in these samples.
  • the biodistribution of the two antibodies was evaluated in nude mice bearing subcutaneous tumors of the OVCAR-3 cell line. Uptake in liver and spleen was higher for OC125 than for OV-TL3 at 24 hours after injection. Large amounts of CA125 antigen are shed from tumor cells and can be found in the serum and ascites from OVCAR-3 growing mice.
  • the radiolabeled OC125 can form complexes by binding to circulating antigen (Haisma et ai. , in prepara ⁇ tion) . It is likely that these complexes are cleared through liver and spleen, resulting in higher uptake in these organs. Kidney uptake was higher for OV-TL3. This could be due to reactivity of the antibody with structures in the kidney.
  • OV-TL3 is de- halogenized faster than OC125 resulting in a faster iodine excretion.
  • _in vitro experiments showed that OV-TL3 binds to fewer determinants on OVCAR-3 cells than OC125, tumor uptake was similar at 24 hours after injection. This could be because of the higher association and lower dissociation rates of OV-TL3. This would also explain the higher tumor uptake at 120 hours after injection for OV-TL3. Shedding of CA125 antigen could also attribute to the decreased binding of OC125 at later time points.
  • antigenic modulation and shedding may be one of the factors that limit effective serotherapy in vivo, it would be important to choose antigenic targets that do not have this property for use in cancer therapy (Miller, R.A. et ai. , Blood 5j3_:78, 1981; Ritz, J. et ai. , J. Immunol. 125:1506-1514, 1980).
  • OV-TL3 can be exploited for use in immuno(radio) therapy or immuno-radioscintigraphy of ovarian cancer.

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Abstract

Un antigène associé au carcinome ovarien est exprimé par des carcinomes ovariens des types séreux, mucineux, endométrioïde et à cellules claires. L'invention concerne également des procédés de diagnose et de thérapie de carcinomes ovariens fondés sur le découverte et la caractérisation de l'antigène.
PCT/US1988/002831 1987-08-19 1988-08-18 Antigene associe a des tumeurs des ovaires humains specifique de l'anticorps monoclonal ov-tl3 WO1989001629A1 (fr)

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US8716487A 1987-08-19 1987-08-19
US087,164 1987-08-19

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WO1989001629A1 true WO1989001629A1 (fr) 1989-02-23

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EP (1) EP0377657A1 (fr)
JP (1) JPH02504517A (fr)
WO (1) WO1989001629A1 (fr)

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FR2635267A1 (fr) * 1988-08-09 1990-02-16 Tokuyama Soda Kk Anticorps monoclonaux et procede pour les produire
WO1991002079A1 (fr) * 1989-07-31 1991-02-21 The University Of British Columbia Anticorps monoclonaux agissant contre un antigene associe a une tumeur
EP0554356A1 (fr) * 1990-10-12 1993-08-11 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Anticorps monoclonal
WO1997025068A2 (fr) * 1996-01-05 1997-07-17 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Antigene de mesothelium, procedes et kits de ciblage de celui-ci
US5706804A (en) * 1996-10-01 1998-01-13 Minnesota Mining And Manufacturing Company Liquid resistant face mask having surface energy reducing agent on an intermediate layer therein
US6241985B1 (en) 1996-05-15 2001-06-05 Altarex Corp. Method and composition for reconforming multi-epitopic antigens to initiate an immune response
WO2002020620A2 (fr) * 2000-09-05 2002-03-14 Medical Research Council Anticorps monoclonal servant a lutter contre le carcinome ovarien
EP1384488A3 (fr) * 1992-04-07 2004-06-23 Immunomedics, Inc. Méthode et Kit pour l'imagerie des organes et des tissus, utilisant des anticorps.
US7375183B1 (en) 1996-01-05 2008-05-20 The United States Of America As Represented By The Department Of Health And Human Services Mesothelin, immunogenic peptides derived therefrom, and compositions comprising mesothelin, or immunogenic peptides thereof
US7429382B2 (en) 2002-10-16 2008-09-30 Corixa Corporation Antibodies that bind cell-associated CA 125/O772P and methods of use thereof
WO2008116299A1 (fr) * 2007-03-26 2008-10-02 F. Hoffmann-La Roche Ag Anticorps 010207-01 ayant une action sur une maladie cancereuse, produit par la lignee de cellules d'hybridome ar51a630.3
US8038994B2 (en) 1996-05-15 2011-10-18 Quest Pharmatech Inc. Combination therapy for treating disease

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DIALOG INFORMATION SERVICES, File 351: World Patent Information, Accession No. 4000925, TANABE SEIYAKU K.K., "Muoinio Ovary Tumour Cell Monoclonal Antibody Produced by Hybridoma Produced by Fusion of Myeloma Cell"; & JP,A,60 231 622 (18 November 1985). *
DIALOG INFORMATION SERVICES, File 351: World Patent Information, Accession No. 4524157, PASTAN I. et al., "Monoclonal Antibody Against Ovarian Cancer Cells Conjugated to Pseudomonas Exotoxin to Produce an Immuno-Toxin for Chemotherapeutic Treatment of Ovarian Cancer"; & US,A,6 888 960 (7 April 1987). *
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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2635267A1 (fr) * 1988-08-09 1990-02-16 Tokuyama Soda Kk Anticorps monoclonaux et procede pour les produire
WO1991002079A1 (fr) * 1989-07-31 1991-02-21 The University Of British Columbia Anticorps monoclonaux agissant contre un antigene associe a une tumeur
EP0420394A1 (fr) * 1989-07-31 1991-04-03 University Of British Columbia Anticorps monoclonaux contre un antigène associé à une tumeur
US5650291A (en) * 1989-07-31 1997-07-22 The University Of British Columbia Monoclonal antibodies against an antigen associated with ovarian cervical and other tumors
EP0554356A1 (fr) * 1990-10-12 1993-08-11 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Anticorps monoclonal
EP0554356A4 (en) * 1990-10-12 1993-09-15 Us Health A monoclonal antibody
US5525337A (en) * 1990-10-12 1996-06-11 The United States Of America As Represented By The Department Of Health And Human Services Monoclonal antibody binding cell surface antigens for diagnosing cancer
JP2660241B2 (ja) * 1990-10-12 1997-10-08 アメリカ合衆国 モノクロナール抗体
US7811570B2 (en) 1992-04-07 2010-10-12 Immunomedics, Inc. Method of affecting a function of or ablating a non-malignant cell
EP1384488A3 (fr) * 1992-04-07 2004-06-23 Immunomedics, Inc. Méthode et Kit pour l'imagerie des organes et des tissus, utilisant des anticorps.
US6153430A (en) * 1996-01-05 2000-11-28 The United States Of America As Represented By The Department Of Health And Human Services Nucleic acid encoding mesothelin, a differentiation antigen present on mesothelium, mesotheliomas and ovarian cancers
US7375183B1 (en) 1996-01-05 2008-05-20 The United States Of America As Represented By The Department Of Health And Human Services Mesothelin, immunogenic peptides derived therefrom, and compositions comprising mesothelin, or immunogenic peptides thereof
WO1997025068A2 (fr) * 1996-01-05 1997-07-17 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Antigene de mesothelium, procedes et kits de ciblage de celui-ci
WO1997025068A3 (fr) * 1996-01-05 1997-10-23 Us Gov Health & Human Serv Antigene de mesothelium, procedes et kits de ciblage de celui-ci
US6083502A (en) * 1996-01-05 2000-07-04 The United States Of America As Represented By The Department Of Health And Human Services Mesothelium antigen and methods and kits for targeting it
US6241985B1 (en) 1996-05-15 2001-06-05 Altarex Corp. Method and composition for reconforming multi-epitopic antigens to initiate an immune response
US8038994B2 (en) 1996-05-15 2011-10-18 Quest Pharmatech Inc. Combination therapy for treating disease
US5706804A (en) * 1996-10-01 1998-01-13 Minnesota Mining And Manufacturing Company Liquid resistant face mask having surface energy reducing agent on an intermediate layer therein
US7229780B2 (en) 2000-09-05 2007-06-12 The Medical Research Council Monoclonal antibody against ovarian carcinoma
WO2002020620A3 (fr) * 2000-09-05 2003-01-03 Medical Res Council Anticorps monoclonal servant a lutter contre le carcinome ovarien
WO2002020620A2 (fr) * 2000-09-05 2002-03-14 Medical Research Council Anticorps monoclonal servant a lutter contre le carcinome ovarien
US7429382B2 (en) 2002-10-16 2008-09-30 Corixa Corporation Antibodies that bind cell-associated CA 125/O772P and methods of use thereof
US8124086B2 (en) 2002-10-16 2012-02-28 Purdue Pharma L.P. Antibodies that bind cell-associated CA 125/O772P and methods of use thereof
US8299230B2 (en) 2002-10-16 2012-10-30 Purdue Pharma L.P. Nucleic acid molecules encoding antibodies that bind cell-associated CA 125/O772P
US9676866B2 (en) 2002-10-16 2017-06-13 Purdue Pharma L.P. Antibodies that bind cell-associated CA 125/O772P
WO2008116299A1 (fr) * 2007-03-26 2008-10-02 F. Hoffmann-La Roche Ag Anticorps 010207-01 ayant une action sur une maladie cancereuse, produit par la lignee de cellules d'hybridome ar51a630.3

Also Published As

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JPH02504517A (ja) 1990-12-20
EP0377657A1 (fr) 1990-07-18

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