WO1992021364A1 - Proteines de liaison d'antigene carcinoembryonnaire et leurs procedes d'isolation et d'utilisation - Google Patents

Proteines de liaison d'antigene carcinoembryonnaire et leurs procedes d'isolation et d'utilisation Download PDF

Info

Publication number
WO1992021364A1
WO1992021364A1 PCT/US1992/004353 US9204353W WO9221364A1 WO 1992021364 A1 WO1992021364 A1 WO 1992021364A1 US 9204353 W US9204353 W US 9204353W WO 9221364 A1 WO9221364 A1 WO 9221364A1
Authority
WO
WIPO (PCT)
Prior art keywords
cea
cbp
carcinoma
binding
carcinoembryonic antigen
Prior art date
Application number
PCT/US1992/004353
Other languages
English (en)
Inventor
Peter Thomas
Carol A. Toth
Sibusisiwe M. Maswoswe
Joseph V. Briggman
Original Assignee
New England Deaconess Hospital Corporation
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
Priority claimed from US07/714,386 external-priority patent/US5278290A/en
Priority claimed from US07/708,885 external-priority patent/US5245017A/en
Priority claimed from US07/708,888 external-priority patent/US5281697A/en
Application filed by New England Deaconess Hospital Corporation filed Critical New England Deaconess Hospital Corporation
Publication of WO1992021364A1 publication Critical patent/WO1992021364A1/fr

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
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of cancer diagnosis and treatment, and more particularly to methods of detecting and treating carcinoma that elicit carcinoembryonic antigen
  • CEA cancer-binding protein
  • this invention relates to the identification of new cancer markers, CEA binding proteins, and methods of isolating such CEA-binding proteins, antibodies specific for these proteins, as well as methods useful in the diagnosis, detection, and monitoring of carcinoma.
  • Colorectal carcinoma is a cancer which affects approximately 600,000 additional people worldwide per year.
  • the prognosis is poor in about 50% of the cases because the tumor is often not detected until the disease has spread and has reached a terminal stage. Early diagnosis is important to increase chances of arresting the carcinoma before it metastasizes, thereby leading to an improved prognosis.
  • a widely used method of the identification of cancerous tissue is to determine its structural resemblance to fetal or immature tissue.
  • tumors can be classified depending on the degree of cellular differentiation; they can be undifferen- tiated, poorly differentiated, moderately differentiated or well differentiated.
  • the behavior of a given tumor can often be related to its degree of differentiation. For example, poorly differentiated tumors tend to grow more rapidly and metastasize earlier than do differentiated tumors which more closely resemble the tissue of origin. Poorly differentiated tumors tend to have a poor prognosis and are difficult to detect.
  • One method of early tumor diagnosis is detection of the presence of a marker or antigen specific for a particular tumor.
  • These normally proteinaceous markers are synthesized by the tumor, and may be found in the serum and/or on the surface of the tumor.
  • Only a limited number of tumor markers for colorectal carcinoma have thus far been found to have clinical use. These include CEA and the sialyated Lewis a antigen (CA 19.9).
  • CEA the sialyated Lewis a antigen
  • CEA and CA 19.9 are present to a far lesser degree on poorly differentiated or undifferentiated cancer cells than on those which are more differentiated. Accordingly, many patients with undifferentiated colorectal carcinomas never develop elevated serum levels of either of these markers, even in the terminal stages of the cancer. There is also considerable overlap between the presence of CA 19.9 and CEA, the patient with a normal CEA level and an elevated level of CA 19.9 being the exception rather than the rule. Therefore, new markers suitable for identifying and monitoring undif erentiated tumors would be of great value.
  • Yet another object is to provide antibodies specific for these new markers. Still another object is to provide a method of isolating CEA-binding proteins that eliminates contamination by CEA.
  • a further object of the present invention is to provide a method and kit for the detection and monitoring of carcinoma in patients using antibodies specific for markers on carcinoma cells.
  • Yet another object of the invention is to provide a hybridoma which produces a monoclonal antibody that recognizes both undifferentiated and poorly differentiated carcinoma cells.
  • a still further object is to provide screening procedures for detecting the presence of carcinoma cells at all stages of differentiation.
  • CEA-binding proteins CBPs
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis
  • CEA-binding fragments of these tumor markers also encompasses CEA-binding fragments of these tumor markers.
  • CEA-binding protein or "CBP” is used herein to describe both proteins and fragments thereof which bind CEA.
  • the 20 kD or 21 kD CBP further includes a label, such as one selected from the group consisting of radioactive isotopes, enzymes, stable free radicals, coenzymes, fluorescent groups, chemiluminescent groups, toxins, and colorimetric groups.
  • a label such as one selected from the group consisting of radioactive isotopes, enzymes, stable free radicals, coenzymes, fluorescent groups, chemiluminescent groups, toxins, and colorimetric groups.
  • the 20 kD or 21 kD CBP is bound to a support which forms a device useful, for example, in purifying CEA.
  • antibodies and binding fragments thereof specific for a CBP are provided.
  • the antibody is a monoclonal antibody.
  • the antibody can form part of a kit for screening a patient for carcinoma.
  • This kit further includes an antibody specific for a carcinoma marker selected from the group consisting of carcinoma orosomucoid-related antigen (CORA), the CC glycoprotein, carcinoembryonic antigen (CEA), CA 19.9, non-specific cross-reacting antigen (NCA) , and alpha 1-acid glycoprotein (AGP) , serum amyloid P protein (SAP), and C-reactive protein (CRP) .
  • CORA carcinoma orosomucoid-related antigen
  • CEA carcinoembryonic antigen
  • NCA non-specific cross-reacting antigen
  • AGP alpha 1-acid glycoprotein
  • SAP serum amyloid P protein
  • CRP C-reactive protein
  • the invention also provides methods of isolating and using CBPs. More specifically, a method of isolating a CBP, such as the 20 kD, the 21 kD, or any CBP that binds CEA i vitro in the presence of a divalent cation, is provided which includes the following steps.
  • a biological sample containing CEA and a CBP is provided.
  • the biological sample may be ascites fluid, whole blood, serum, bile, saliva, sputum, lymphoid tissue, or tumor tissue obtained from a subject afflicted with carcinoma.
  • This sample is contacted with a divalent cation, such as Ca +2 , Zn +2 , or Mg +2 , at a concentration and for a time sufficient to enable the CBP to bind to CEA, thereby forming a CBP-CEA conjugate.
  • a divalent cation such as Ca +2 , Zn +2 , or Mg +2
  • the sample is contacted with an adsorbent that binds CEA, such as an immobilized immunoadsorbent including an antibody to CEA, for a time sufficient to allow the conjugate to adsorb to the adsorbent. Portions of the sample which have not adsorbed to the adsorbent are removed.
  • the CBP is then dissociated from the adsorbent-bound conjugate.
  • Dissociation can be accomplished with the use of an agent that chelates divalent cations such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[ ⁇ -aminoethyl ether]-N,N,N* ,N'-tetraacetic acid (EGTA) .
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis[ ⁇ -aminoethyl ether]-N,N,N* ,N'-tetraacetic acid
  • the dissociated CBP is then collected, for example, by executing a purification method such as high pressure liquid chromatography (HPLC) , SDS-PAGE, affinity chromatography, exclusion chromatography, ammonium sulfate precipitation, ultracentrifugation, and/or isoelectric focusing.
  • CBP can be isolated by added CEA to the sample, or by reading the sample with CEA immobilized on a solid support in the
  • a method of detecting carcinoma includes the following steps.
  • a biological sample is taken and the concentration of CEA assayed for CEA.
  • the concentration of CEA is then compared with a predetermined threshold level of CEA indicative of the presence of carcinoma.
  • Another aspect of the invention includes a method of screening for carcinoma including subjecting a biological sample from a subject to a test, such as an immunoassay, indicating the presence of a cancer marker, and screening the sample for the presence of a CBP, its presence being indicative of the presence of carcinoma.
  • a test such as an immunoassay
  • inventions include assay formats detecting CEA in a body fluid by adsorbing CBP to a support (such as a microtiter plate, for example) treating the body fluid to be screen with divalent cation, adding the treated body fluid to the CBP-bound plate, and then quantitating the bound CEA with labelled anti-CEA antibody.
  • anti-CEA antibody can be adsorbed to the support which is then treated with the body sample in the presence of divalent cation.
  • the presence of complexed CEA (or CBP to which the CEA in the body sample is complexed in the presence of divalent cation) can then be detected by treatment with labelled anti-CBP antibody.
  • CBPs free in body samples can be detected by adsorbing CEA to a support, and then treating the support with the body sample in the presence of divalent cation.
  • a method of treating carcinoma includes providing a CBP and administering it in a pharmaceutically acceptable carrier to a subject afflicted with carcinoma.
  • the CBP binds CEA present in the subject, thereby inhibiting the metastatic proliferation of carcinoma cells which occurs as a result of CEA binding to CEA receptors on various organs.
  • the provision of the CBP can be accomplished by recombinant DNA technology, automated or manual biochemical peptide synthesis, or by purification from a subject inflicted with carcinoma.
  • FIG. 1 is a diagrammatic representation of the CBP purification scheme of the invention
  • FIG. 2 is a photographic representation of a stained transfer blot of an SDS gel showing the 20 kD and 21 kD CBPs (lane 3), alpha 1-acid glycoprotein (lanes 1 and 4), column wash (lane 2), and human serum albumin (lane 5);
  • FIG. 3 is an optical scan of an HPLC of (A)
  • FIG. 4 is an optical scan of an HPLC of (A) alpha 1-acid glycoprotein (AGP) + CEA in the presence of EDTA, and (B) CEA + CBP in the presence of CaCl2,'
  • FIGs. 5A and 5B are a comparison of the amino acid sequences of C-reactive protein and the 20 kD and 21 kD CBPs.
  • FIGs. 6A and 6B are a comparison of the amino acid sequences of serum amyloid P protein and the 20 kD and 21 kD CBPs. DESCRIPTION OF THE INVENTION
  • CEA-binding proteins Proteins that influence the concentration of CEA in the blood have been isolated from patients afflicted with carcinoma. These CEA-binding proteins are also markers for carcinoma, and as such are useful in the detection and treatment of carcinoma.
  • the CBPs have been isolated using a purification method which virtually eliminates contamination by CEA.
  • This procedure is schematically represented in FIG. 1.
  • a biological sample containing CEA and CBP is provided ' .
  • the biological sample may be ascites fluid, whole blood, serum, bile, saliva, sputum, lymphoid tissue, or tumor tissue obtained from a subject afflicted with carcinoma.
  • This sample is contacted with a divalent cation, such as Ca +2 , Zn +2 f or Mg +2 , at a concentration and for a time sufficient to enable the CBP to bind to CEA, thereby forming a CBP-CEA conjugate.
  • a divalent cation such as Ca +2 , Zn +2 f or Mg +2
  • the sample is contacted with an adsorbent that binds CEA, such as an immobilized i munoadsorbent including an antibody to CEA, for a time sufficient to allow the conjugate to adsorb to the adsorbent. Portions of the sample which have not adsorbed to the adsorbent are removed.
  • the CBP is then dissociated from the adsorbent-bound conjugate. Dissociation can be accomplished with the use of an agent that chelates divalent cations such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[ ⁇ -aminoethyl ether]-N,N,N' ,N'-tetraacetic acid (EGTA) .
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis[ ⁇ -aminoethyl ether]-N,N,N' ,N'-tetraacetic acid
  • the dissociated CBP is then collected, for example, by executing a purification method such as high performance liquid chromatography (HPLC) , SDS-PAGE, affinity chromatography, exclusion chromatography, ammonium sulfate precipitation, ultracentrifugation, and/or isoelectric focusing.
  • HPLC high performance liquid chromatography
  • the instant purification procedure does not depend on the size of the binding protein, a particularly important point since CBPs isolated by this method have a molecular weight (by HPLC) similar to that of alpha-1 acid gylcoprotein (AGP) .
  • the procedure is ligand-specific, and thus any proteins that may bind to CEA nonspecifically or which require some other binding factors will not contaminate the product. Any contaminates are removed during the washing or will remain bound to CEA after the EDTA elution.
  • This method also provides an effective way to isolate CBPs from CEA to which they have bound. Because CEA is a large molecule and very immunogenic, it is important to remove it from the CBP isolate if antibodies to these CBPs are to be prepared from the isolate.
  • two CBPs were isolated, one having a molecular weight of 20 kD and one glycoprotein having a molecular weight of 21 kD, as shown in FIG. 2, lane 3, of a stained transfer blot of an SDS-PA gel.
  • These molecular weights differ from those of other known CEA binding proteins such as the 46 - 50 kD carcinoma orosomucoid-related antigen (CORA) (described in U.S. Patent No. 4,914,021), and also differ from the 41 kD alpha 1-acid glycoprotein (AGP) which does not bind CEA.
  • CORA carcinoma orosomucoid-related antigen
  • FIGs. 3A-3D show that the proteins isolated have a collective molecular weight of 41 kD (FIG. 3B) .
  • the appearance of a single peak with a retention time of about 8.5 min and the absence of a peak at about 9.4 min suggests the formation of a complex between CEA and CBP in the presence of calcium ions (FIG. 3C) .
  • a calcium chelator like EDTA for example, two peaks with retention times similar to that of CEA (FIG. 3A) and that of the uncomplexed CBPs (FIG. 3B) were observed (FIG. 3D), indicating that these CBPs do not bind CEA in the absence of calcium.
  • CBPs were further distinguished from AGP by HPLC. Like the CBPs, AGP does not bind CEA in the absence of calcium (presence of EDTA) (FIG. 4A) . However, unlike the CBPs, AGP does not bind CEA in the presence of calcium ions (FIG. 4B) . These CBPs have also been distinguished from CRP and SAP by isoelectric focusing.
  • SAP serum amyloid P protein
  • CRP C-reactive protein
  • CRP a dipentamer of 21 kD subunits
  • Both proteins undergo calcium-dependent ligand binding are composed of non-covalently linked subunits, and share a similar pentagonal disc-like molecular form (Pepys (1982) Eur. J. Rheumatol. Inflamm. 5_:386). In addition, they share at least about 60% homology of amino acid sequence.
  • CRP is an acute phase reactant in humans. Its concentration rises rapidly following acute tissue injury, infection, or inflammation, and it is often persistently elevated in cases of malignant neoplasia.
  • the methods of detection involves the administration to the subject of a pharmaceutical formulation including the 20 kD or 21 kD CBP, or CEA-binding fragments thereof, in a physiologically acceptable carrier.
  • a biological sample is then taken. This sample may be ascites fluid, whole blood, serum, bile, saliva, sputum, lymphoid tissue, or tumor tissue. The concentration of CEA in this sample is measured.
  • This measurement can be accomplished by any number of known tests for CEA including an immunoassay (e.g., Roche ELISA, Hoffman-La Roche, Nutley, NJ) activity assay, immunoassay, quantitative electrophoresis, and the like.
  • the concentration of CEA in the biological sample is then compared with a predetermined threshold level of CEA indicative of carcinoma.
  • This threshold CEA concentration can be determined by administering a CBP to two statistically significant groups of people, one with carcinoma, and one that is disease-free.
  • the value of the threshold level may be the point at which the measured CEA concentration curves of these two groups intersect.
  • CBPs may also be used in the treatment of carcinoma as follows.
  • a CBP is administered in a pharmaceutically acceptable carrier to a subject afflicted with carcinoma.
  • the CBP binds CEA present in the subject, thereby inhibiting the metastatic proliferation of carcinoma cells which occurs as a result of CEA binding to CEA receptors on various organs (see, e.g.. Toth et al. (1985) Cancer Res. 4_5_:342-397; Toth et al. (1988) Biochem. Soc. Trans. ____.:1027-1028; Toth et al. (1989) J. Leukocyte Biol. 45_:370-376; and Hostetter et al. (1990) J. Natl. Cancer Insti. .82:380-385).
  • the concentration of calcium in various body tissues is high enough to enable efficient binding.
  • CBP CBP in both methods of use
  • purification from a subject inflicted with carcinoma can be accomplished by purification from a subject inflicted with carcinoma.
  • the CBPs may be prepared by recombinant DNA technology (see. e.g., Maniatis et al.. Molecular Cloning- A Laboratory Manual (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), or by automated or manual biochemical peptide synthesis.
  • the pharmaceutical formulation suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the formulation must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable CBPs can be brought about by the use in the compositions of agents delaying absorption.
  • Sterile injectable solutions are prepared by incorporating the CBPs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the CBP may be administered parenterally or intraperitoneally.
  • Solutions of the CBP as pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the CBP also may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or incorporated directly with the food of the diet.
  • the CBP may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspension syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of the CBP. The percentage of the compositions and preparations may, of course, be varied. The amount of CBP in such useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: excipients, such as dicalcium phosphate; a disintegrating agent; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as a sweetening agent
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • the dosage unit form may contain, in addition to materials of the above type, a liquid carrier.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets
  • a syrup or elixir may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • sucrose as a sweetening agent
  • methyl and propylparabens as preservatives
  • a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the CBP may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Antibodies to the 20 kD and 21 kD proteins are also provided by the invention. Such antibodies can be easily produced by one with ordinary skill in the art. For example, a purified CBP isolate from a subject afflicted with carcinoma as an antigen can be used as an antigen. Mice, goats, rabbits, or other animals can be challenged by injection with a solution of such an isolate emulsified in complete Freund's adjuvant at weekly intervals. After the initial injection, the booster injections can be administered without adjuvant or emulsified in incomplete Freund's adjuvant.
  • synthetic or genetically engineered analogs or fragments of the CBP produced by recombinant DNA or biochemical techniques can be used as immunogens.
  • an innoculum containing a relatively pure CBP sample isolated as described above along with Freund's adjuvent can be injected into a rabbit, mouse, rat, goat, or any mammal to produced monoclonal antibodies.
  • Monoclonal antibodies to a CBP or active binding fragments of such antibodies can be generated by applying generally known cell fusion techniques (cf. Kohler and Milstein (1976) Eur. J. Immunol. £:511-519; and M. Shulman et al. (1978) Nature 276:269-270, herein incorporated by reference) to obtain a hybridoma producing the monoclonal antibody.
  • the monoclonal antibody may be subjected to proteolysis to obtain an active Fab, F(ab')2/ or Fv fragment.
  • monoclonal antibodies are prepared by obtaining mammalian lymphocytes (preferably spleen cells), committing the lymphocytes to produce antibodies (e.g., by immunizing the mammal with the particular antigenic determinant of interest beforehand) , fusing the lymphocytes with myeloma (or other immortal) cells to form hybrid cells, and then culturing a selected hybrid cell colony in vivo or in vitro to yield antibodies which are identical in structure and specificity.
  • mammalian lymphocytes preferably spleen cells
  • committing the lymphocytes to produce antibodies e.g., by immunizing the mammal with the particular antigenic determinant of interest beforehand
  • myeloma or other immortal
  • monoclonal antibodies to a CBP can be raised by employing a purified CBP isolate from a subject afflicted with carcinoma as an antigen.
  • Mice or other animals can be challenged by injection with a solution of such whole cells emulsified in complete Freund's adjuvant at weekly intervals. After the initial injection, the booster injections can be administered without adjuvant or emulsified in incomplete Freund's adjuvant.
  • synthetic or genetically engineered analogs or fragments of the CBP produced by recombinant DNA or biochemical techniques can be used as irnmunogens.
  • Serum samples from the immunized animal can be analyzed by an enzyme linked immunoabsorbent assay ("ELISA") or the like for antibody reaction with the immunization agent.
  • Animals that exhibit antibodies titers are sacrificed and their spleens homogenized.
  • the spleen cells can be extracted and the antibody-secreting cells expanded in vitro by culturing with a nutrient medium.
  • the spleen cells are then fused with myeloma (or other immortal) cells by the above-referenced procedure of Kohler and Milstein.
  • the hybridomas so produced are screened (i.e., cloned by the limiting dilution procedure of the above-referenced Baker et al. article) to select a cell line producing antibodies which react with human ⁇ chain receptor proteins.
  • Human antibodies i.e., those obtained from human-human or human-animal hybridoma
  • animal antibodies can be used as well as animal antibodies.
  • human hybridoma production techniques see U.S. Patent No. 4,451,570 issued to Royston et al. on May 29, 1984; U.S. Patent No. 4,529,694 issued to Lazarus et al. on July 16, 1985 and Zurawski et al., "Continuously Proliferating Human Cell Lines Synthesizing Antibody of Predetermining Specificity" in Monoclonal Antibodies (Plenum Press, New York 1980), also incorporated by reference.
  • Active antibody fragments can be derived from the monoclonal antibodies disclosed herein by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin, and then subjected to HPLC gel filtration. The appropriate fraction containing Fab, F(ab)2, or Fv can then be collected and concentrated by membrane filtration or the like. For further description of general techniques for the isolation of active fragments, see for example, Khaw, et al.. Vol. 23 J. Nucl. Med.. pp. 1011-1019 (1982), incorporated by reference. The antibodies and antibody fragments used herein can be labeled preferably with radioactive labels by a variety of techniques other than the above-described Baker et al. technique.
  • the biologically active molecules can also be labeled with a radionucleotide via conjugation with the cyclic anhydride of diethylenetriamine penta-acetic acid (DTPA) or bromoacetyl aminobenzyl ethylamine diamine tetra-acidic acid (BABE) .
  • DTPA diethylenetriamine penta-acetic acid
  • BABE bromoacetyl aminobenzyl ethylamine diamine tetra-acidic acid
  • the instant invention also relates to a method for screening subjects for carcinoma. It includes subjecting a biological sample to at least one test selected from a plurality of tests, each of which is specific for a carcinoma cell.
  • Useful biological samples include ascites fluid, whole blood, serum, bile, lymphoid tissue, or tumor tissue obtained from a subject afflicted with carcinoma. The method used to obtain these samples is dependent on the nature of the sample and would be known by a medical practitioner.
  • Each test correlates the presence of a specific marker with the presence of a carcinoma cell, and in some instances, with a degree of differentiation of that cell.
  • the screening method of the present invention includes tests for tumor markers CEA, CA 19.9, NCA, AGP, CORA ( U.S. Patent Application Ser. No. 441,368), and the CC glycoprotein ( U.S. Patent o. 4,921,789), as well as any additional markers which indicate the presence of carcinoma, such as CRP or SAP.
  • the tests may be performed in a sequential manner until the presence of at least one marker has been proven.
  • the tests performed may be assays, for example, to determine enzyme-linked activity, or may be immunoassays which utilize an antibody specific for a particular marker (see, e.g., U.S. Patent No. 4,892,933).
  • an antibody specific for a particular marker see, e.g., U.S. Patent No. 4,892,933
  • antibody raised to a cancer marker can be adhered to an adsorbent via chemical modification and/or covalent linkage using a bifunctional cross-linking reagent.
  • This invention provides a convenient kit for screening biological samples for colorectal carcinoma.
  • This kit includes antisera or purified polyclonal or monoclonal antibodies specific for the 20 kD and 21 kD CBPs, and antibodies for at least one other tumor marker such as the CC glycoprotein, CORA, NCA, CA 19.9, CEA, AGP, SAP, or CRP. These antibodies can be prepared by methods well known to those skilled in the art (see description above) .
  • this kit may contain antigen binding fragments of such antibodies such as Fv, Fab, or F(ab)2 fragments obtained by known proteolytic cleavage or recombinant DNA techniques. Screening may be performed by any immunoassay procedures known in the art such as, for example, ra ioimmunoassay, Western blot analysis, or nitrocellulose "dot" analysis.
  • CBPs were isolated from ascites fluid from patients with colorectal adenocarcinoma using the following procedure. Exogenous CaCl2 was added to human ascites fluid to give a final concentration of 5 mM. The ascites was then incubated with an anti-CEA monoclonal antibody (Hybritech, San Diego, CA) coupled to an affinity gel (Affi-Gel 10; Bio-Rad) . After an overnight incubation with agitation at 4°C, the immunoadsorb ' ent plus ascites fluid was put onto a (Affigel 10, Biorad, Richmond, CA) column, and any unbound material allowed to flow into a waste tube.
  • an anti-CEA monoclonal antibody Hybritech, San Diego, CA
  • affinity gel Adsorb ' ent plus ascites fluid was put onto a (Affigel 10, Biorad, Richmond, CA) column, and any unbound material allowed to flow into a waste tube.
  • CBP was eluted from the column using elution buffer containing EDTA (0.1 M Tris, pH 7.4, 0.15 M NaCl, lOrnM EDTA).
  • CBP extracted from human ascites fluid as described above was passed through a size exclusion column (GF-250, DuPont) .
  • the eluates were analyzed with the mobile phase (0.2 M sodium phosphate, pH 7.0, 0.005% sodium azide) at a flow rate of 1 ml/min and detection at 280 nm.
  • the resulting elution profiles are shown in FIGs. 3A-3D.
  • CBP migrates with CEA in the presence of calcium (FIG. 3C) , while in the absence of calcium (presence of EDTA) , it migrates as a separate peak (FIG. 3D).
  • CBPs purified as described in EXAMPLE 1 were electrophoresed on an SDS-polyacrylamide gel (10-20% Mini-Gel, ISS, Newton, MA) according to the method of Laemmli (Nature (1970) 227:680-685).
  • the proteins on the gel were then electrotransferred to an Immobilon "P Transfer Membrane (Millipore) with transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 15% methanol v/v) according to the method of Towbin et al. (Proc. Natl. Acad. Sci. (USA) 76_:4350-4354) .
  • Affinity purified CBP was tested for the ability to bind to CEA in the presence of calcium ions as follows. Varied amounts of CBP was mixed with 5-10 ⁇ l CEA (100 ⁇ g/ml) CEA in the presence of 5 mM CaCl2- The sample was then run on an HPLC sizing column (DuPont GF-250), and the elution profile was recorded (FIG. 3C) .
  • the 20 kD CBP, the 21 kD CBP, CRP, and SAP were radioiodinated with l 2 5 ⁇ using the chloramine T procedure of Greenwood et al (Biochem. J. (1963) ££:114-123) to procure a specific activity of about 6-10 mCi/mg. Focusing was carried out in agarose gels essentially as described by Saravis et al (Immunol. Meth. (1979) 22:91-96). Radiolabelled protein was detected by audoradiography using Kodak X-OMAT film.
  • NUNC-Immuno Plates (Naperville, IL) were coated with 500 ng/well CEA mAb in carbonate buffer (0.05 of sodium carbonate, ph 9.6), and were incubated overnight (ON) at 4°C.
  • the plates were washed with phosphate buffered saline (PBS) containing 1% (wt/vol) bovine serum albumin (BSA) and 0.5% (vol/vol) Tween 20 (PBS-BSA-TWEEN) . They were then treated with 2% BSA in PBS for two hours at 4°C to block any remaining sites on the plate.
  • Antigen and high CEA serum standards were diluted and added to the appropriate wells at 50 ⁇ l/well.
  • CEA-Binding Protein (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
  • NAME Serum amyloid P component
  • x PUBLICATION INFORMATION
  • AUTHORS Mantzouranis, Evanelia C.
  • NAME C-reactive protein
  • PUBLICATION INFORMATION (A) AUTHORS: Lei, Ke-Jian Liu, Maria

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des protéines, ou des fragments de celles-ci, liant spécifiquement un antigène carcinoembryonnaire (CEA) en présence d'un cation divalent in vitro. L'invention concerne également des procédés d'isolation de ces protéines ou d'autres protéines liant le CEA. Ce procédé consiste à obtenir un échantillon biologique contenant un CEA et une protéine de liaison de CEA (CBP), et à le mettre en contact avec un cation divalent, à une concentration donnée et pendant une durée suffisante pour permettre la liaison dudit CBP au CEA, formant ainsi un conjugué CBP-CEA. On met ensuite en contact l'échantillon avec un adsorbant liant le CEA, pendant une durée suffisante pour permettre l'adsorption du conjugué dans l'adsorbant. Les éventuelles parties de l'échantillon non adsorbé par l'adsorbant sont éliminées. Le CBP est dissocié du conjugué lié à l'adsorbant, et il est collecté.
PCT/US1992/004353 1991-05-31 1992-05-22 Proteines de liaison d'antigene carcinoembryonnaire et leurs procedes d'isolation et d'utilisation WO1992021364A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US07/714,386 US5278290A (en) 1991-05-31 1991-05-31 Binding protein for CEA and uses thereof
US708,888 1991-05-31
US714,386 1991-05-31
US07/708,885 US5245017A (en) 1991-05-31 1991-05-31 Method for isolating CEA-binding protein
US07/708,888 US5281697A (en) 1991-05-31 1991-05-31 CEA-binding protein and uses thereof
US708,885 1991-05-31

Publications (1)

Publication Number Publication Date
WO1992021364A1 true WO1992021364A1 (fr) 1992-12-10

Family

ID=27418849

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/004353 WO1992021364A1 (fr) 1991-05-31 1992-05-22 Proteines de liaison d'antigene carcinoembryonnaire et leurs procedes d'isolation et d'utilisation

Country Status (3)

Country Link
EP (1) EP0587777A4 (fr)
AU (1) AU2158292A (fr)
WO (1) WO1992021364A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6774209B1 (en) 2000-04-03 2004-08-10 Dyax Corp. Binding peptides for carcinoembryonic antigen (CEA)
WO2009009034A2 (fr) * 2007-07-06 2009-01-15 Promedior, Inc Procédées et compositions utiles dans le traitement de la mucosite
US7632685B2 (en) 2002-11-12 2009-12-15 Becton, Dickinson And Company Method of predicting the onset of sepsis in SIRS-positive individuals using mass spectrometry
US8247370B2 (en) 2006-12-04 2012-08-21 Promedior, Inc. Conjoint therapy for treating fibrotic diseases
US8329659B2 (en) 2009-06-17 2012-12-11 Promedior, Inc. SAP variants and their use
US8497243B2 (en) 2007-07-06 2013-07-30 Promedior, Inc. Methods and compositions useful in the treatment of mucositis
US9233140B2 (en) 2009-03-11 2016-01-12 Promedior, Inc. Treatment methods for hypersensitive disorders
US9296800B2 (en) 2009-06-04 2016-03-29 Promedior, Inc. Serum amyloid P derivatives and their preparation and use
US9708661B2 (en) 2008-04-03 2017-07-18 Becton, Dickinson And Company Advanced detection of sepsis
US9884899B2 (en) 2007-07-06 2018-02-06 Promedior, Inc. Methods for treating fibrosis using CRP antagonists
US10443099B2 (en) 2005-04-15 2019-10-15 Becton, Dickinson And Company Diagnosis of sepsis
US10702583B2 (en) 2009-03-11 2020-07-07 Promedior, Inc. Treatment methods for autoimmune disorders

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4782014A (en) * 1985-06-25 1988-11-01 Ciba-Geigy Corporation Assay and purification of amyloid components, applications, and kits therefor
US4892933A (en) * 1988-04-20 1990-01-09 New England Deaconess Hospital Corporation Monoclonal antibody for colorectal carcinoma

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1174800B (it) * 1983-11-21 1987-07-01 Alberto Bartorelli Metodo per la determinazione di immunoglobuline umane specifiche per gli antigeni dei carcinomi umani e sua utilizzazione nella sierodiognosi di forme primitive tumorali
US4914021A (en) * 1988-03-04 1990-04-03 New England Deaconess Hospital Corporation Carcinoma orosomucoid-related antigen, a monoclonal antibody thereto, and their uses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4782014A (en) * 1985-06-25 1988-11-01 Ciba-Geigy Corporation Assay and purification of amyloid components, applications, and kits therefor
US4892933A (en) * 1988-04-20 1990-01-09 New England Deaconess Hospital Corporation Monoclonal antibody for colorectal carcinoma

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
European Journal of Rheumatology and Inflammation, Vol. 5, issued 1982, PEPYS, "C-Reactive Protein: A Review of its Structure and Function", pages 386-397, especially page 386. *
Scandinavian Journal of Immunology, Vol. 24, issued 1986, LEVO et al., "Serum Amyloid P-Component Levels in Patients with Malignancy", pages 147-151, especially the Abstract. *
See also references of EP0587777A4 *
The Journal of Biological Chemistry, Vol. 260, No. 22, issued 05 October 1985, POTEMPA et al., "Effect of Divalent Metal Ions and pH Upon the Binding Reactivity of Human Serum Amyloid P Component, A C-Reactive Protein Homologue, for Zymosan", pages 12142-12147, especially page 12143, column 1. *
The Journal of Biological Chemistry, Vol. 260, No. 24, issued 25 October 1985, PRELLI et al., "The Primary Structure of Human Tissue Amyloid P Component from a Patient with Primary Idiopathic Amyloidosis", pages 12895-12898, especially the Abstract. *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6774209B1 (en) 2000-04-03 2004-08-10 Dyax Corp. Binding peptides for carcinoembryonic antigen (CEA)
US6919424B2 (en) 2000-04-03 2005-07-19 Dyax Corp. Binding peptides for carcinoembryonic antigen (CEA)
EP1268524B1 (fr) * 2000-04-03 2005-11-02 Dyax Corp. Peptides de liaison a l'antigene carcino-embryonnaire (ace)
US7438890B2 (en) 2000-04-03 2008-10-21 Dyax Corp. Binding peptides for carcinoembryonic antigen (CEA)
US7632685B2 (en) 2002-11-12 2009-12-15 Becton, Dickinson And Company Method of predicting the onset of sepsis in SIRS-positive individuals using mass spectrometry
US7645613B2 (en) 2002-11-12 2010-01-12 Becton, Dickinson And Company Mass spectrometry techniques for determining the status of sepsis in an individual
US11578367B2 (en) 2005-04-15 2023-02-14 Becton, Dickinson And Company Diagnosis of sepsis
US10443099B2 (en) 2005-04-15 2019-10-15 Becton, Dickinson And Company Diagnosis of sepsis
US8247370B2 (en) 2006-12-04 2012-08-21 Promedior, Inc. Conjoint therapy for treating fibrotic diseases
US8497243B2 (en) 2007-07-06 2013-07-30 Promedior, Inc. Methods and compositions useful in the treatment of mucositis
US9884899B2 (en) 2007-07-06 2018-02-06 Promedior, Inc. Methods for treating fibrosis using CRP antagonists
WO2009009034A3 (fr) * 2007-07-06 2009-02-26 Promedior Inc Procédées et compositions utiles dans le traitement de la mucosite
WO2009009034A2 (fr) * 2007-07-06 2009-01-15 Promedior, Inc Procédées et compositions utiles dans le traitement de la mucosite
US9708661B2 (en) 2008-04-03 2017-07-18 Becton, Dickinson And Company Advanced detection of sepsis
US9885084B2 (en) 2008-04-03 2018-02-06 Becton, Dickinson And Company Advanced detection of sepsis
US10221453B2 (en) 2008-04-03 2019-03-05 Becton, Dickinson And Company Advanced detection of sepsis
US9233140B2 (en) 2009-03-11 2016-01-12 Promedior, Inc. Treatment methods for hypersensitive disorders
US10702583B2 (en) 2009-03-11 2020-07-07 Promedior, Inc. Treatment methods for autoimmune disorders
US9296800B2 (en) 2009-06-04 2016-03-29 Promedior, Inc. Serum amyloid P derivatives and their preparation and use
US8329659B2 (en) 2009-06-17 2012-12-11 Promedior, Inc. SAP variants and their use
US9556246B2 (en) 2009-06-17 2017-01-31 Promedior, Inc. SAP variants and their use

Also Published As

Publication number Publication date
AU2158292A (en) 1993-01-08
EP0587777A1 (fr) 1994-03-23
EP0587777A4 (fr) 1995-05-10

Similar Documents

Publication Publication Date Title
AU619827B2 (en) Carcinoma orosomucoid-related antigen, a monoclonal antibody thereto, and their uses
EP0167616B1 (fr) Marqueurs tumoraux de cytokeratine et analyses en vue de leur detection
EP0098162A2 (fr) Anticorps monoclonaux spécifiques de l'antigène carcinoembryonnaire
WO1992021364A1 (fr) Proteines de liaison d'antigene carcinoembryonnaire et leurs procedes d'isolation et d'utilisation
Seon et al. Monoclonal antibody that defines a unique human T-cell leukemia antigen
CA2049938C (fr) Anticorps monoclonal reconnaissant une glycoproteine specifique d'un antigene de la mucine de la membrane du globule de gras du lait humain
US5338832A (en) Antigen recognized by MCA 16-88
EP0369816A2 (fr) Anticorps monoclonaux spécifiques pour la mucine polymorphe épithéliale humaine
JPS62501563A (ja) リウマチ性関節炎の特徴である蛋白質
EP0200464B1 (fr) Antigène associé aux tumeurs
EP0454782A1 (fr) Haptoglobine apparentee au cancer (hpr).
JP2813457B2 (ja) 膀胱ガン用の特有なタンパク質マーカー
EP0345750B1 (fr) Protéase polyfonctionelle
CA2139518C (fr) Regions antigeniques des complexes de particules liberes par les tumeurs et anticorps
AU618209B2 (en) Antigen recognized by mca 16-88
US5245017A (en) Method for isolating CEA-binding protein
US5281697A (en) CEA-binding protein and uses thereof
US5278290A (en) Binding protein for CEA and uses thereof
JPH02154694A (ja) 肺癌モノクローナル抗体および細胞表面抗原
EP0345811B1 (fr) Anticorps monoclonaux spécifiques pour fibrinopeptide A humain
Velt et al. Identification of a leukemia-associated antigen of human acute lymphocytic leukemia
EP0465652A1 (fr) Anticorps contre une chaine lourde de myosine des muscles lisses
US5241052A (en) Carcinoma orosomucoid-related antigen, a monoclonal antibody thereto, and their uses
US5204450A (en) Carcinoma orosomucoid-related antigen, a monoclonal antibody thereto, and their uses
CA1335355C (fr) Anticorps monoclonal anti-cpbii

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1992913689

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1992913689

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1992913689

Country of ref document: EP