[go: up one dir, main page]

WO1989004841A1 - Glycoproteine associee a une tumeur gastrointestinale, exprimant le determinant antigenique ca 19-9 - Google Patents

Glycoproteine associee a une tumeur gastrointestinale, exprimant le determinant antigenique ca 19-9 Download PDF

Info

Publication number
WO1989004841A1
WO1989004841A1 PCT/US1988/003955 US8803955W WO8904841A1 WO 1989004841 A1 WO1989004841 A1 WO 1989004841A1 US 8803955 W US8803955 W US 8803955W WO 8904841 A1 WO8904841 A1 WO 8904841A1
Authority
WO
WIPO (PCT)
Prior art keywords
glycoprotein
population
kda
cells
human epithelial
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.)
Ceased
Application number
PCT/US1988/003955
Other languages
English (en)
Inventor
Thomas Leo Klug
Norman Cipriano Ledonne, Jr.
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.)
Janssen Biotech Inc
Original Assignee
Centocor Inc
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 Centocor Inc filed Critical Centocor Inc
Publication of WO1989004841A1 publication Critical patent/WO1989004841A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines

Definitions

  • the molecular antigenic determinant, CA 19-9 is the basis of an important diagnostic tool as its concentration level is usually elevated in the serum of patients with colorectal and/or pancreatic cancers. As a result, measurements of CA 19-9 serum levels are useful in the differential diagnosis of patients suspected of having one of these cancers. Measurement of serum CA 19-9 levels is achieved by using antbodies that specifically bind to it. These antibodies are produced in the laboratory by hybridomas, cloned cell lines in which each cell is derived from a hybrid of a mouse myeloma cell and a spleen cell from a mouse that had been immunized with human colon carcinoma cell line, that produces the CA 19-9 determinant.
  • a purified molecular species carrying the CA 19-9 determinant could be isolated, then one would have the option of immunizing mice with that species instead of with entire cells. Furthermore, one could use that molecular species as a standard in the immunoassay used to detect the CA 19-9 determinant in sera. Indeed, one could also use it as a probe in a test that is the converse of the one for CA 19-9 antigen : a test for the presence of anti-CA 19-9 antibody in humans .
  • human carcinoma cells shed relatively large molecular entities , with a size distribution in the range of about 200 , 000 daltons ( 200 kDa) to 2000 kDa .
  • These entities were shown to contain both protein and the carbohydrate group , sialylated lacto-N-fucopentaose II , whose sial ic acid and fucose moieties are both required for antibody binding .
  • sialylated lacto-N-fucopentaose II whose sial ic acid and fucose moieties are both required for antibody binding .
  • Ovarian epithelial tumor-associated high molecular entities can be dissociated into one antigenic determinantcarrying glycoprotein species of 200 kDa (H. M. Davis et al . Cancer Research. 46 , 6143-6148 , 1986) . That appears to be the situation most similar to the present invention. Even in that case , however , there are apparently fundamental differences in the structures of the aggregates, as compared to the present case : For one thing , the ovarian antigenic determinant is protein only.
  • destabilization o f natural ly o c cur ing aggegates to produce the 2 10 kDa glycoprotein in either an unaggregated state or as a detergentdissociable aggregate requires reduction and carboxymethylation in the presence of a chaotropic agent.
  • destabilization of the naturally occuring aggregates could be achieved by exposing them to a chaotropic agent without concomitant reduction and carboxymethylation.
  • reduction and alkylation in the presence of a chaotropic agent had been reported previously, it had not been reported as a means of producing a tumor-associated antigen.
  • glycoprotein population of the present invention is a human epithelial glycoprotein population in which:
  • each glycoprotein contains the CA 19-9 antigenic determinant;
  • the average mobility is that of a protein of about 210 kDa and the mobilities vary from that of a protein of about 150 kDa to one of about 250 kDa;
  • (iv) has an amino acid composition, quantitated as phenyl isothiocyanate derivatives in mole % in parentheses, as follows: asp (5.1), glu (9.8), ser (19.7). Gly (8.4), his (1.5), arg (5.5) thr(8.8), ala (7.5), pro (7.3), tyr (2.5), val (5.1), met (0.9), ile (3.4), leu (5.6), phe (2.7), lys (4.1); and
  • (v) has a carbohydrate composition, quantitated as trimethylsilyl derivatives in molar ratios in parentheses, as follows: fucose (4.1), mannose (1.0), galactose (11.8), N-acetylgalactosamine (2.5) , N-acetylglucosamine (4.9) , N-acetylneuraminic acid (5.1); and each glycoprotein is either in an unaggregated state or a zwitterionic detergent dissociable state.
  • the invention is a process for facilitating the production of a glycoprotein population of the present invention which comprises the steps of:
  • the glycoprotein population of the present invention is useful as a reagent for immunizing mice in order to generate mouse spleen cells that can be used to make hybridomas that produce antibodies against the CA 19-9 determinant. Furthermore, one can use it as a standard in the immunoassay used to detect the CA 19-9 determinant in suspected colorectal and pancreatic cancer patients. In addition, it can be used to test for the presence of anti-CA 19-9 antibodies in humans.
  • FIG. l Sizing of glycoproteins expressing CA 19-9 activity by gel filtration on Sepharose 4B-CL.
  • the perchloric acid soluble fraction of SW1116 supernatants was chroraatographed in 50 mM Tris-HCl, pH 8.0, containing 10 mM dithiothreitol, 0.1% SDS, 150 mM sodium chloride, and 25 mM EDTA.
  • CA 19-9 activity as determined by the CA 19-9 IRMA (solid circles) and optical density at 280 nm (open circles) are presented for each column fraction. Pooled fractions are indicated by Roman numerals I, II, and III.
  • exclusion volume of the column [A] blue dextran) and the elution positions of murine immunoglobulins M ([B] 800 kDa) and G ([C] 160 kDa) and the inclusion volume (D) in the same column buffer but without dithiothreitol are indicated by arrow.
  • the molecular mass markers are myosin heavy chain (200 kDa) , beta-galactosidase (116 kDa), phosphorylase b (92 kDa), albumin (66 kDa), and ovalbumin (45 kDa).
  • FIG. 3 Autoradiograph of an SDS polyacrylamide 3-12% gradient gel of purified radioiodinated 19-9cGP antigen (lane B) after exhaustive digestion with pronase (lane A) or neuraminidase (lane C). Radioiodinated 19-9cGP isolated from 40 ml of serum from a patient with colorectal cancer by the same procedure as 19-9cGP in lane A is shown in lane D. Approximately 5000 dpm of radiolabelled glycoproteins were applied to each lane. Sample treatment and molecular mass markers as described in Figure 2.
  • Figure 4 Sizing of purified radioiodinated 19-9cGP (solid line) and asialo-19-9cGP (dotted line) by size-exclusion chromatography on a TSK-4000 SW HPLC column equilibrated with 0.2 M phosphate, pH 6.8.
  • the exclusion volume of the column [A] blue dextran) and elution times of thyroglobulin ([B] 660 kDa), beta-galactosidase ([B] 464 kDa), immunoglobulln G ([C] 160 kDa), ovalbumin ([E] 45 kDa), and salts (F) are indicated by arrows.
  • Mab 19-9 monoclonal antibody 1116NS-19-9; CA 19-9, the carbohyhdrate determinant recognized by Mab 19-9; 19-9cGP, CA 19-9 containing glycoprotein; 19-9cGP (Y kDa) , a 19-9 cGP of molecular mass Y kDa; IRMA, immunoradiometric assay; Gdn-HCl, guanidine hydr ochloride; TMS, trimethylsilyl ; PITC, phenylisothiocyanate; Fuc, fucose; Man, mannose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; NeuAc, N-acetylneuraminic acid. All sugars are assumed to be in the D-configuration, except fucose which is L.
  • Murine hybridoma 1116NS-19-9 (H. Koprowski et al. Somatic Cell Genet., 5, 957-972, 1979; ATCC No. HB 8059, American Type Culture Collection, Rockville, Maryland) was obtained from Dr. Zenon Steplewski of the Wistar Institute, Philadelphia, PA.
  • Sepharose 4B-CL and Protein A-Sepharose 4B were purchased from Pharmacia.
  • Trimethylsilylating reagent (TriSilZ) and amino acid standards (Standard H) were purchased from Pierce.
  • the fused silica cross-linked methyl gas chromatography column (Ultra-1) was purchased from Hewlett Packard. Highly purified carbohydrate reference standards were obtained from Pfanstiehl, Waukegan, IL.
  • the Micropak TSK 4000 SW HPLC column was from Varian Instruments. Fish gelatin was purchased from Norland Industries, Inc., New Brunswick, NJ. All other reagents were from standard supply houses, and were of the highest purity commercially available. Neuraminidase was purchased from Calbiochem, alpha-fucosidase from Sigma Chemical Co.
  • Glycoproteins expressing the CA 19-9 determinant were obtained from the cell culture supernatants of the SW1116 human colorectal carcinoma cell line (A. Leibovitz et al, Cancer Res., 36, 4562-4569, 1976; ATCC No. CCL 233, American Type Culture Collection, Rockville, Maryland). SW1116 cells were grown as previously described (10) and culture supernatants were collected seven to ten days after cells had reached confluence.
  • Solid phase immunoradlometric assays for the detection of CA 19-9 activity were performed as described previously (R.E. Ritts et al, Int. J. Cancer, 33, 339-345, 1984) using kits manufactured by Centocor, Inc., Malvern, Pennsylvania. Briefly, the IRMA utilizes a two-step homogeneous "sandwich" configuration in which purified Mab 19-9 antibody adsorbed to polystyrene beads binds the glycoprotein antigen complex in the first step, and the amount of antigen bound to the bead is determined by binding of radioiodinated Mab 19-9 in the second incubation step.
  • This particular assay configuration is effective since more than one CA 19-9 determinant is associated with each antigen molecule or antigen complex.
  • the amount of antigen is expressed in units/ml or units/mg of protein as determined by reference to a CA 19-9 solution stored at -80 °C. The value of one unit was arbitrary but the same for all experiments reported here, thereby allowing an accurate index of the amounts of purification achieved by the procedures described.
  • Reactivity of purified 19-9cGP with polyclonal anti-carcinoembryonic antigen (anti-CEA) antibodies was determined using a radioimmunoassay to CEA (Abbot Labs, N. Chicago, IL).
  • Immunoglobulin 1116NS 19-9 was purified from ascites by affinity chromatography on Protein A-Sepharose 4B (P. L. Ey et al, Immunochemistry. 15, 429-436, 1978). Purified antibody was bound and covalently cross-linked to a Protein A-Sepharose 4B column by the procedure of C. Schneider et al ( J. Biol . Chem.. 257, 10766-10769, 1982) using 20 mM dimethylpemilimidate as the bifunctional cross-linker. Approximately 4 mg of 19-9 antibody bound per ml of Protein A-Sepharose 4B.
  • Typical individual lots of pooled SW1116 supernatants contained one to eight liters.
  • the perchloric acid soluble material was fractionated by gel chromatography on a Sepharose 4B-CL column equilibrated with 50 mM Tris , 150 mM sodium chloride, 10 mM dithiothreitol , 0.1% SDS, at pH 8.0.
  • Human cells of epithelial origin are a likely source for the CA 19-9 antigen .
  • the antigen may be found in normal and carcinoma cells of pancreatic, colorectal, stomach, bile duct or lung origin. There is at least a 10-fold greater expression of the antigen in the cancer cells.
  • the buffer for immunoadsorption and washing of the column was 50 mM sodium citrate, pH 4.5, the pH optimal for Mab 19-9 affinity
  • the affinity column was washed with alternating cycles of acidic buffer (0.1 M sodium acetate with 0.5 M sodium chloride, pH 4.0), and basic buffer (0.1 M sodium bicarbonate with 0.5 M sodium chloride, pH 10.0) prior to each affinity procedure to remove nonspecifically bound proteins from the Mab 19-9 affinity column.
  • acidic buffer 0.1 M sodium acetate with 0.5 M sodium chloride, pH 4.0
  • basic buffer 0.1 M sodium bicarbonate with 0.5 M sodium chloride, pH 10.0
  • Step. #4 proved to be essential for obtaining pure 19-9cGP. All affinity steps were done at room temperature (20-25 °C).
  • the proteins in the first Mab 19-9 affinity eluate were reduced with 50 mM dithiothreitol in the presence of 6M guanidine hydrochloride (6 M Gdn-HCl) for 4 h at 45 C and subsequently carboxymethylated with 75 mM iodoacetic acid.
  • 6M guanidine hydrochloride (6 M Gdn-HCl)
  • the alkylated proteins were exhaustively dialyzed against cold water, and 19-9cGP was repurified by a second Mab 19-9 affinity chromatography procedure as described above.
  • the diethylamine/taurodeoxycholate eluate was neutralized and dialyzed against 20 mM Tris-HCl, pH 6.8.
  • the dialysate was then applied to a 2.0 x 1.0 cm column of DEAE TrisAcryl column equilibrated with 20 mM Tris-HCl, pH 6.8, and the column was eluted sequentially with ten column volumes each of 0, 50, 100, 150, 200, 300 and 1000 mM sodium chloride in 20 nM Tris-HCl, pH 6.8.
  • the majority of the CA 19-9 activity usually greater than 90S. of the total activity, eluted with 100 and 150 mM chloride.
  • the purity of the isolated 19-9cGP was determined by SDS polyacrylamide gel electrophoresis (SDS:PAGE) and autoradiography after radloiodination of the antigen by the Bolton-Hunter method (A. E.
  • Exoglycosidase digestions were performed in 0.1 M acetate buffer, ph 4.5, for 24 hours at 37 C. Unit values of exoglycosidases were chosen in order to ensure complete digestion of appropriate oligosaccharide substrates as monitored by thin layer chromatography. Pronase digestion of 19-9cGP was done in 0.1 M Tris-HCl, 10 mM calcium chloride, 2% (w/v) pronase (solution pretreated at 60 C for 1 h) , pH 8.0, for 72 h at 60 C. Pronase digestions were terminated by heating the solution at 100 C for three minutes.
  • SDS polyacrylamide slab gels were run using the buffer system of Laemmli (U. K. Laemmli. Nature. 227. 680-685, 1970) with 3-12% gradient gels run to their pore-size limit (2000 volt-hours).
  • Gels for autoradiography were dried and exposed to X-Omat AR film with a Cronex (Dupont) Quanta-Ill intensifying screen for 12-24 hours at -80 C.
  • Monoclonal antibody immunoblotting analysis of 19-9cGPs after separation by electrophoresis was performed by the procedure of H. Towbin et al (Proc. Natl. Acad. Scl. U.S.A.. 76, 4350-4354, 1979).
  • HPLC HPLC was used to determine the apparent purity and molecular weight of the purified protein under non-reducing, non-denaturing conditions.
  • Molecular weight estimates were obtained by sizeexclusion chromatography on a system consisting of a Waters SM-6000 pump connected to a 7.5 x 300 mm Micropak TSK 4000 SW column equilibrated with 0.2 M potassium phosphate, pH 6.8. The molecular weight was estimated by comparing the retention time of radiolabelled proteins to the retention times of molecular weight standards which were monitored by optical density at 214 nm.
  • the amount of serine in 19-9cGP was determined by extrapolation of picomoles of serine to 0 hours hydrolysis time with normalization to an internal standard of 250 picomoles of aminobutyric acid.
  • Carbohydrate composition was determined from gas chromatographic analysis of triraethylisilyl (TMS) derivatives after HCl-methanolysis (R. A. Laine et al. Methods Enzymol., 28, 159-167, 1972).
  • TMS-methylglycosides were separated and identified using a Hewlett-Packard Model 5790 gas chromatograph interfaced to a Hewlett Packard Model 5970 mass selective detector (MSD) equipped with a 25 m Ultra-1 capillary column. The column was developed at 3 °C/min from 125 to 160 °C, then 10 °C/min from 160 to 235 °C.
  • the flow rate was 2.91 ml/min; split ratio, 1:14; carrier gas. Helium.
  • chaotropic agents in decreasing order of preference are guanidine hydrochloride, urea, alkaline salts of bromine (e.g., LiBr ⁇ , ammonium thiocyanate, perchlorates, sodium or ammonium sulfate and polyethylene glycols.] All other treatments had no effect on the aggregated material in Fraction I, Figure 1 and hence the results of these negative experiments are not shown. This glycoprotein is apparently identical to that found in Fraction III, Figure 1, and identified in Figure 2, Lane C.
  • D CA19-9 activity determined by the CA 19-9 immunoradlometric assay. cFold-purification relative to starting material in units CA 19-9/mg. 100 percent of the CA 19-9 activity and 33 percent of the protein is retained relative to the starting material. The majority of the fetal calf serum proteins in the initial tissue culture media passes through the 100 kDa cutoff hollow fiber filter. The perchloric acid extraction step results in less than 5 percent loss of CA 19-9 activity, but if fold-purification is expressed as CA 19-9 units/mg of protein, removal of acid insoluble proteins results in a net 59-fold purification. As expected the Mab 19-9 affinity steps result in the greatest fold-purification and are primarily responsible for the eventual production of homogeneous 19-9CGP antigen preparations.
  • the initial antibody affinity step results in an apparent 50 percent loss of CA 19-9 activity, but gives an antigen preparation with 34,000 times the specific activity of the starting material.
  • the loss in activity could not be attributed to incomplete binding of loaded antigen, as all affinity fractions were assayed for CA 19-9 activity, and no activity was ever observed in any of the column flow-throughs or washings. This apparent loss in activity is probably due to a decrease in antigen valency due to disaggregation.
  • the reduction and alkylation step in the presence of 6 M Gdn-HCl was found to be indispenslble in the purification scheme.
  • CA 19-9 activity remained partially associated with high molecular weight aggregates after Mab 19-9 affinity chromatography, and radiolabelled material subsequently demonstrated many contaminating low molecular weight proteins by SDS:PAGE after reduction in the presence of 6 M Gdn-HCl (data not shown). Reduction, alkylation, and Mab 19-9 reaffinity results in a further 40 percent loss of CA 19-9 activity, but gives a substantially purer antigen preparation of 7.5 x 10 6 units/mg.
  • a final DEAE ion-exchange step serves to remove some persistent non-immunoreactive low molecular weight contaminants.
  • the reduction conditions are more critical than the alkylation conditions. Reduction must be done at temperatures greater than room temperature if complete reduction is to take place within a reasonable time, i.e., less than 8 hours. Temperatures greater than 60 °C may give side reactions harming protein. The higher the temperature, the shorter the time needed for reduction. For a range of times, 1-8 hours, the range of temperatures is 30 to 60°C. Most organic reducing agents would be somewhat effective, but only dithiothreitol (DTT) and beta-mercaptoethanol (BME) would be able to completely disrupt the aggregates at a reasonable concentration of reducing agent.
  • DTT dithiothreitol
  • BME beta-mercaptoethanol
  • the alkylation conditions are not important within broad limits: for times of 0.5 to 2 hours, temperatures of 15 to 40 °C are used.
  • concentration of the alkylating agent should be kept as low as possible, however, to avoid agent reactions with other side chains on the protein (methionines); Use molar excess (1.5 x the reducing agent concentration).
  • the nature of the alkylating agent is probably not important within broad limits. Any radical alkylating substituent capable of blocking the the sulfhydryl group would work. A preferably small group was used in order to avoid changing the biochemical behavior beyond blocking the sulfhydryls. Larger alkylating moities such as carbobenzoxy- radicals would increase the hydrophobic (non-polar) nature of the antigen.
  • the protein was estimated to be greater than 95% pure as determined by SDS: PAGE and autoradiography of radiolabelled 19-9cGP, an indication that the preparation was substantially free of other proteins.
  • the purified proteins were radiodinated and analyzed by gradient SDS: PAGE.
  • the efficacy of the purification scheme can be seen in Figure 3, lane B, in which only a single band with an average molecular mass of 210 kDa is visible.
  • that band contains a relatively homogeneous population of glycoproteins, within a size range of about 150 kDa to 250 kDa.
  • the size distribution within the population was probably due to variations in the amount of carbohydrate per glycoprotein molecule; the protein moiety was probably the same in all molecules within the population. It is important to point out that this glycoprotein antigen, in our experience, iodinated very poorly; thus, contaminant proteins showed up readily by this type of analysis.
  • the radioiodinated protein was analyzed by HPLC on a TSK 4000 sizing column equilibrated with 0.2 M phosphate, pH 6.8 ( Figure 4). Only one rather broad peak of radioactivity with a mean retention time of 8.87 minutes is found for the intact antigen.
  • asialo-19-9cGP also elutes as a broad peak, but in contrast to the results seen with gel electrophoresis (Figure 3, lane C), has a retention time of 9.30 minutes indicating a lower molecular mass of approximately 800 kDa.
  • SDS or some similar zwitterionic detergent is needed to keep the antigen unaggregated.
  • Othe detergents that may be used include deoxycholate or its derivatives and CHAPS.
  • composition determined on TMS derivatives bA11 values are molar ratios with Man set to 1.0. II. In the intact antigen, sugars were found in the ratios of Fuc: Man: Gal: GalNAc: GlcNAc: NeuAc as 4.1: 1.0: 11.8: 2.5: 4.9: 5.1. The presence of both Man and GalNAc suggest that both N-and O-linked oligosaccharides are found on 19-9cGP. The amino acid composition of purified 19-9cGP is presented in Table III.
  • the most notable aspect of the composition was that serine , proline , and threonine together accounted for greater than 35% of the amino acids which is consistent with a mucin-like protein structure .
  • the extremely high mole percent of serine ( 19.7% ) is unusual .
  • the relatively high amount of acidic and acid amide amino acids and no detectable cysteine in 19-9cGP is not typical of a mucin.
  • carbohydrate and amino acid analyses of the same antigen preparation indicated that the antigen was as much as 85% carbohydrate by weight .
  • Treatment of the purified 19-9cGP with neuraminidase and afucosidase resulted in complete loss of CA 19-9 activity.
  • the purified 19-9cGP also did not react with polyclonal antisera to CEA (data not shown) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Est décrite une population de glycoprotéines de cellules épithéliales humaines d'un poids moléculaire moyen d'environ 210 KDa et ayant une répartition de taille relativement homogène, chaque glycoprotéine dans la population contenant le déterminant antigénique CA 19-9. On peut générer la population à partir de plus grandes entités moléculaires en les exposant, en présence d'un agent chaotropique, à un agent de réduction ainsi qu'à un agent d'alkylation.
PCT/US1988/003955 1987-11-25 1988-11-04 Glycoproteine associee a une tumeur gastrointestinale, exprimant le determinant antigenique ca 19-9 Ceased WO1989004841A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US125,195 1980-02-27
US12519587A 1987-11-25 1987-11-25

Publications (1)

Publication Number Publication Date
WO1989004841A1 true WO1989004841A1 (fr) 1989-06-01

Family

ID=22418610

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1988/003955 Ceased WO1989004841A1 (fr) 1987-11-25 1988-11-04 Glycoproteine associee a une tumeur gastrointestinale, exprimant le determinant antigenique ca 19-9

Country Status (1)

Country Link
WO (1) WO1989004841A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0478146A1 (fr) * 1990-08-31 1992-04-01 The Wistar Institute ADN et polypeptide de l'antigène CO-029 associé à une tumeur
US6225049B1 (en) 1992-06-17 2001-05-01 The United States Of America As Represented By The Department Of Health And Human Services Human insulinoma-associated cDNA

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, (Washington D.C., USA), Volume 18, issued 1979, ROSE et al., "Resolution of the Major components of human lung mucosal gel and their capabilities for Reaggregation and gel formation", pages 4030-4037. See entire document. *
CANCER RESEARCH (Baltimore, Maryland, USA), Volume 46, issued 1986, DAVIS et al., "Characterization of the CA 125 antigen associated with human epithelial ovarian carcinomas", pages 6143-6148, especially pages 6145, 6147. *
CANCER RESEARCH, (Baltimore, Maryland, USA), Volume 44, issued 1984, KLUG et al., "Tumorigenicity in athymic nude mice of the human colon carcinoma cell line SW 1116 expressing the tumor associated antigenic determinant Ca 19-9", pages 5212-5218, especially pages 5212 and 5216. *
CANCER RESEARCH, (Baltimore, Maryland, USA), Volume 48, issued 1988, KLUG et al., "Purification and Composition of a novel gastrointestinal tumorassociated glycoprotein expressing lacto-N-fuco pentaose II (Ca 19-9)", pages 1505-1511, see entire document. *
EUROPEAN JOURNAL OF BIOCHEMISTRY, (Berlin, Germany), Volume 149, issued 1985, HANISCH et al., "Ca 125 and Ca 19-9: two cancer-associated Sialylsaccharide antigens on a mucus glycoprotein from human milk", page 323-330, especially pages 328 and 330. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0478146A1 (fr) * 1990-08-31 1992-04-01 The Wistar Institute ADN et polypeptide de l'antigène CO-029 associé à une tumeur
US5668002A (en) * 1990-08-31 1997-09-16 The Wistar Institute DNA and polypeptide for tumor-associated antigen CO-029
US6225049B1 (en) 1992-06-17 2001-05-01 The United States Of America As Represented By The Department Of Health And Human Services Human insulinoma-associated cDNA

Similar Documents

Publication Publication Date Title
Goetzl et al. Affinity labeling of a mouse myeloma protein which binds nitrophenyl ligands. Kinetics of labeling and isolation of a labeled peptide
Buchegger et al. Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA‐95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD
Mage [6] Preparation of Fab fragments from IgGs of different animal species
Peterson et al. Highly purified papain-solubilized HL-A antigens contain β2-microglobulin
Fuks et al. Carcinoembryonic antigen (CEA): Molecular biology and clinical significance
CA1183451A (fr) Methode de production d'un peptide tryptique de csap et d'anticorps anticsap
Freedman et al. The effects of complete modification of amino groups on the antibody activity of antihapten antibodies. Reversible inactivation with maleic anhydride
Borel et al. IgG asymmetric molecules with antipaternal activity isolated from sera and placenta of pregnant human
US4544640A (en) Anti immune complex antibody for determining SLE, rheumatoid arthritis or tetanus
EP0332651B1 (fr) Procede d'isolation de l'antigene ca 125
Klug et al. Purification and composition of a novel gastrointestinal tumor-associated glycoprotein expressing sialylated lacto-N-fucopentaose II (CA 19-9)
Shimano et al. Isolation, characterization and clinical evaluation of a pancreas cancer‐associated antigen
Iacobelli et al. Purification and characterization of a 90 kDa protein released from human tumors and tumor cell lines
Tragardh et al. Chemical, physical-chemical, and immunological properties of papain-solubilized human transplantation antigens
EP0454782B1 (fr) Haptoglobine apparentee au cancer (hpr)
CA1338324C (fr) Anticorps monoclonal pour l'analyse immunologique selective d'un procollagene intact (type iii) et d'un procollagene (type iii) dans les liquides organiques
Pande et al. Comparative structural studies of human plasma and amniotic fluid fibronectins
Janatova [46] C3, C5 components and C3a, C4a, and C5a fragments of the complement system
Givol et al. Isolation and fragmentation of antibodies to polytyrosyl gelatin
Eggertsen et al. Characterization of tryptic fragments of human complement factor C3
US5132287A (en) Methods of treating disorders which cause conglutinin deficiency
WO1989004841A1 (fr) Glycoproteine associee a une tumeur gastrointestinale, exprimant le determinant antigenique ca 19-9
EP0175310A1 (fr) Une protéine essentiellement pure spécifique de l'arthrite rhumatoide et un anticorps contre cette protéine
Hannestad et al. Multiple M-components in a single individual—I: The structural relationship of two serum γMκ M-components as revealed by combining specificity and individual antigenic specificity
Kim et al. Molecular characterization of the epitope in prostate and breast tumor-associated PR92 antigen

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

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