JPH11511973A - Colon cell and colon cancer cell-related nucleic acid molecules, proteins and peptides - Google Patents

Abstract

  (57) [Summary] The present invention relates to isolated proteins and peptides found on the surface of colon cells and on the surface of colon cancer cells, and furthermore, the proteins and peptides relating to the nucleic acid molecules encoding the proteins and peptides bind to tumor-associated antibodies such as mAb A33. I do. The monomeric protein has a molecular weight of about 43 kD as determined by SDS gel electrophoresis under non-reducing conditions. Furthermore, the present invention relates to the use of the nucleic acid molecule, the isolated or multimeric form of the protein, and the antibody against the peptide in diagnosis, screening, and therapeutic methods. The invention further relates to antibodies specific for the protein and to the peptide in isolate or multimeric form.

Description

DETAILED DESCRIPTION OF THE INVENTION      Colon cell and colon cancer cell-related nucleic acid molecules, proteins and peptidesCross-references for related applications   The present application discloses colon cells and colon, both of which are incorporated herein by reference. U.S. application filed April 4, 1995 entitled Cancer Cell Associated Proteins and Peptides Published on February 2, 1996, which is a continuation-in-part of patent application Ser. No. 08 / 511,876. This is a continuation-in-part application of 08 / 597,495.Field of the invention   The present invention relates to human colon cells and colon cancer cell-related antigens, nucleic acid molecules, proteins and And peptides. Specifically, the present invention encoded by the nucleic acid molecule of the present invention Ming proteins and peptides are the surface and cells of human colon cells and human colon cancer cells And binds to colorectal cancer antibodies. The protein as an isolate form Is about 40-45 kD when subjected to SDS gel electrophoresis under non-reducing conditions. Of about 49-5 as determined by SDS gel electrophoresis under reducing conditions. It has a molecular weight of 5 kD. The protein, its peptide fragments, and The multimers can be used to develop useful reagents and methods for cancer diagnosis and treatment. Can be.Background of the Invention   Colorectal cancer is a malignant disease. In the West, especially in the United States, colorectal cancer High incidence. This type of tumor often metastasizes through lymphatics and blood vessels You. Many people with colorectal cancer eventually die from the disease. In fact, 6 2,000 Americans and 8,000 Australians die of colorectal cancer each year It is estimated that he has died.   To date, tissue therapies and chemotherapy have been developed for the treatment of colorectal cancer ing. However, the survival of colorectal cancer patients with metastatic disease There are still treatments that exhibit sufficient antitumor activity to reliably extend Absent. As a result, there is still the development of methods and products to effectively treat colorectal cancer. There is a need for departure.   Monoclonal antibody A33 is used for extensive basic medical analysis and localization in patients A mouse immunoglobulin that has been studied. (Welt et al.,J. Clin . Oncol., 8: 1894-1906 (1990), Welt et al.,J. Clin. Oncol.,12: 1561-1571 ( 1994), and Welt et al.,J. Clin. Oncol. 14: 1787-1797 (1996)). This anti The body binds to antigens found within and on the surface of normal and colon cancer cells You. This antigen is known as the A33 antigen.   In tumors originating from the colonic mucosa, A33 antigen is expressed in more than 95% of cases. It is expressed homogeneously. A33 antigen is found in many other normal tissues studied. Not. Due to its restricted expression, this system is virtually “organ features” It is defined as "unusual" (large intestine, small intestine).   By immunofluorescence experiments, mAb A33 was A33 antigen-positive in vitro Apparently internalized into cellular macropinosomes (Daghighian et al.,J. Nuc. Med.37: 1052-1057 (1996)). Mouse model MAb A33 is localized in human colon cancer xenografts in significant amounts This indicates that within 1 hour after administration, the transplanted colon cancer cells Can be identified within the quality. Rapid tumor localization and high levels of tumor Antibody incorporation is considered to be related to the following facts. (1) A3 The three antigens are not secreted, so targeting mAb A33 to tumor cells is Not disturbed by detached A33 antigen that diffuses from tumor cells into the vasculature, ( 2) When mAb A33 binds to the A33 antigen on the cell membrane, it rapidly enters the cell. The amount of antibody bound to cells And (3) some colon cancer cell lines express large amounts of the A33 antigen, Binds 800,000 mAb A33 molecules per cell. According to these characteristics , A33 antigen and, further, related proteins with similar characteristics Need to tally and sequence.   Many purification protocols usually involve the protein of interest by SDS gel electrophoresis. Utilizes a reduction step to analyze This makes proteins easier It can be identified and monitored. The inventors of the present application use the reduction conditions. In some cases, target A33 protein is I have found the surprising fact that it cannot be identified. The A33 antigen of the present invention , Identification, moni To completely eliminate the reduction step to characterize and characterize Standard technology had to change. Once the antigen is isolated, under its reducing conditions We will be able to study the behavior in   Purification of the A33 antigen requires that other proteins, including actin, be one- and two-dimensional. In gel electrophoresis, it is possible to further It was difficult. Furthermore, mAb A33 binds non-specifically to actin . The inventors of the present application have reported that this non-specific binding to actin is due to the Fc region of the antibody It turned out that it depends on the area. By removing the Fc region, The bright individuals were able to prevent actin binding. Actin is located on the cell surface of colon cancer cells It is not an antigen and is not susceptible to reduction. Thus, it is clear that actin cannot be a target for monoclonal antibody A33. Met.   Difficulties in identifying, isolating and characterizing this antigen are Despite more than 10 years since the existence of 3 antigens was known, the purification and Isolation and sequencing were supported by the fact that they were first successful with the present invention. It is.   As described herein, the inventors of the present application have identified, isolated, and characterized the A33 antigen. Lactarized. The present inventors have further found that cDNA encoding the A33 antigen is also simple. The nucleotide sequence of this cDNA was determined, and the amino acid sequence of the A33 antigen was determined. Estimated. The A33 antigen, referred to herein as the A33 protein, comprises cancer and its Foreseeing, diagnosing and diagnosing other diseases, especially cancers such as colon, rectum, gastric and And can be used to develop clinical reagents and methods useful for therapy.Summary of the Invention   The present invention is found in and on normal human colon cells and human colon cancer cells. Isolated proteins, as well as peptide fragments of said proteins. The protein and the peptide may be an A33 colon cancer antibody or the protein sequence; Are bound by a polyclonal antibody prepared against the region of SDS gel den When analyzed by electrophoresis, the isolated glycoproteins of the invention use non-reducing conditions. If used, a molecular weight of about 40-45 kD, and if reduction conditions were used, In that case, they have a molecular weight of about 49-55 kilodaltons. The present invention further provides the above-described tag. Protein Nucleic acid molecules, and cancer of said glycoproteins, peptides and nucleic acid molecules It also relates to uses in the diagnosis and treatment of.BRIEF DESCRIPTION OF THE FIGURES   The above brief description and other objects and features of the present invention will be better understood from the following description of the present invention. For a detailed description of suitable but exemplary embodiments, reference is made to the accompanying drawings. It will be more fully understood by that. here,   FIG. 1 shows LIM1215 and Hep- using A33 monoclonal antibody. Fig. 3 shows a cell fluorescence analysis of two cells.   Figure 2 shows undetectable after SDS gel electrophoresis using reducing conditions and non-reducing conditions. Detectable by Western blot after SDS gel electrophoresis used It shows the A33 antigen. "-B-ME" means electrophoresis using non-reducing conditions And "+ -BME" indicates electromigration using reducing conditions.   FIG. 3 shows immunization of cell lysates with or without mAb A33. Showing a sedimentation reaction,   FIG. 4 shows cell lysates incubated with or without tunica mycelis. 1 shows the immunoprecipitation reaction of the oxidization.   FIG. 5 consists of FIGS. 5A and 5B, where A3 extracted from LIM1215 cells 3 shows Western blot analysis of the three antigens under non-reducing conditions.   FIG. 6 shows actin and A33 IgG or A33 F (ab) ′._TwoFragment Figure 2 shows a biosensor analysis of the interaction between.   FIG. 7 shows the chromatographic purification protocol used for the purification of the A33 antigen. It is a flowchart shown.   FIG. 8 consists of FIG. 8A and FIG. 8B, and shows the TIM of LIM1215 colon cells, respectively. Weston of Ritton X-100 extract and Triton X-114 extract 9 shows an immunoblotting analysis.   FIG. 9 shows an anion exchange HPLC of the A33 antigen.   FIG. 10 shows size-exclusion HPLC of the A33 antigen.   FIG. 11 includes FIGS. 11A and 11B. FIG. 11A shows Superose1. 2 Fig. 7 shows micropreparative RP-HPLC information of the active fraction. FIG. 11B FIG. 4 shows a biosensor analysis of A33 antigen activity in the HPLC fraction. You.   FIG. 12 shows the amino acid sequence of the peptide fragment in the A33 antigen. .   FIG. 13 is a flow chart showing the protocol used for affinity purification of the A33 antigen. It is a chart.   FIG. 14 shows a chemical compound bound to keyhole limpet hemocyanin (KLH). The synthetic peptide SVETPQDVLRASQGKSVTLP (amino acid of SEQ ID NO: 1 2 shows Western blot analysis of sera obtained from mice and rabbits immunized in 2-21). doing.   FIG. 15 shows the use of anti-peptide IgG generated against the N-terminus of the A33 antigen. Of A33 antigen under non-reducing (panel 1) and reducing (panel 2) conditions Figure 5 shows a stunt blot analysis.   FIG. 16 consists of FIGS. 16A and 16B. FIG. 16A and FIG. 6B indicates a 2.6 kb cDNA encoding the A33 antigen.   FIG. 17 is a comparison of the deduced amino acid sequences of human and mouse A33.   FIG. 18 shows that after labeling with palmitate, mAb A33 binds to labeled A33 antigen. It indicates that it will settle.   FIG. 19 shows the inhibition of staining when using hydroxylamine.DETAILED DESCRIPTION OF THE INVENTION Example 1   Several colon cancer cell lines were obtained as listed in Table 1. LIM1215 fine Cell line is Ludwig Institute for Cancer Research, Melbourne, Aussie Obtained from Tolalia. Cell lines SK-CO-17, SK-CO-19, SK-C O-10, SK-CO-11 and SK-CO-15 are Ludwig Institute for Cancer Research, New York, and Memorial Sloan Kettering Instit ute, obtained from New York. All other cell lines are American Type Cu lture Collection, Rockville, MD         Pfreundschuh et al.,Proc. Natl. Ac ad. Sci. USA , 75: 5122-5126 (1978). American Type, based on the Budapest Agreement, Culture Collection, Rockville, Maryla nd and ATCC No. High cataloged as HB8779 Monoclonal antibody A33 (mAb) secreted by the bridoma cell line   A33) for each of these cell lines using the rosette formation assay. An experiment (rosetting assay) was performed. mAb A33 is an isotype of IgG2a A3 present within or on the surface of human colon cancer as described herein Binds to the antigen shown as 3. Some of the colorectal cancer cell lines are A33-positive by immunoassay, immunoassay and immunohistochemistry (See Table 1 below). Example 2   The LIM1215 colon cell line, which was positive in the rosette formation test of Example 1, was Grow in RPMI medium containing 10% fetal calf serum. Confluent Cells (10⁶/ Cm^Two) Using Trypsin-Versene solution I let it. Cells were treated with 10% fetal calf serum, 1 μg / ml hydrocortisone, 0% . 025 U / ml insulin and 10.82 μg / ml α-thioglycero In a tissue culture dish containing 25 ml of RPMI 1640 0 was seeded. 5 days CO2_TwoAt 37 ° C in an atmosphere of Cubbed. After removal of the medium, the cells were removed from the surface by a cell scraper. Before removal, they were washed with PBS. The cells are washed in PBS and⁹Cells / ml Was resuspended.   Next, expression of the A33 antigen on the surface of the LIM1215 colon cancer cell line was determined. And analyzed by flow cytometry according to standard techniques. Hep-2 epidermal cancer Cell lines (Boring et al.,Cancer J. Cin.Vol . 44, pp. 7-26 (1994)) was used as a negative control. . The cells are washed, containing 5 mM EDTA and 5% fetal bovine serum. 5 × 10 5 in 00 μl of PBS⁶Resuspended at a concentration of cells / ml. Cells Incubated with 5 μg of A33 mAb at 4 ° C. for 30 minutes. Buff After washing with the fluorescein-conjugated anti-mouse IgG, (Dilution 1/50). Negative control Cells were stained with an isotype-matched unrelated antibody (5 μg) followed by Performed by staining with fluorescein-conjugated anti-mouse IgG only. Flow rhino The tomometry was performed using a FACScan flow cytometer (Becton Dick). inson, San Jose, CA, U.S.A. S. A.).   FIG. 1 shows LIM1215 and Hep-2 using A33 monoclonal antibody. Figure 4 shows a cell fluorescence analysis of cells. The fluorescence obtained with the control antibody As compared to Panel A), the entire population of LIM1215 was incubated with A33 mAb. Intense and uniform fluorescence (panel B) was shown. Hep-2 cells (C and D )), The profiles shown in the panels obtained are duplicates, Cells This indicates that the matching A33 mAb was not detected. X axis is fluorescence intensity (Logarithmic unit), and the Y axis indicates the number of cells.Example 3   Cell lines that were A33 positive in the rosette formation test (Table 1) Dissolve in PBS, pH 7.4 using 0.3% Triton X-100. I let you. Other detergents known to those skilled in the art also lyse A33 positive cells Can be used to 9 A33-positive cell lines, plus 5 A The cell lysate of the 33-negative cell line (control) was purified using non-reducing conditions. A33 antigen expression was examined by Western blot analysis. About 4 Colorectal cancer in which a molecule having a molecular weight of 3 kD was A33 positive in a rosette formation test Tested by Western blot with mAb A33 in lysate from cells Was issued. This molecule was negative for A33 in the rosette formation test MAb A in lysates obtained from cell lines or containing anti-actin mAb It was not detected by any antibody other than 33. A33 antigen uses non-reducing conditions Can be detected by Western blot analysis only after analyzed SDS gel electrophoresis Noh. The A33 antigen was undetectable when using reducing conditions. Figure The Western blot shown in Fig. 2 was used for affinity purification from SW1222 cells. The obtained A33 antigen was used. The upper band (Figure 2) is the A33 tamper. The multimeric form of the protein is shown.Example 4   A33 antigen was immunoprecipitated from colorectal cancer cell lysis. To do this, Intestinal cancer cells are obtained using standard techniques known to those of skill in the art.^ThreeH-GlcNAc or³⁵S Labeled. Solubilization of A33-positive cells in the rosette formation test Solubilization showed a band of about 43 kD by SDS gel electrophoresis under Was immunoprecipitated with monoclonal antibody A33.   FIG. 3 shows the results of immunoprecipitation from an A33-positive lysate having a molecular weight of about 43 kD. Shows a child. This band was A33 negative in the rosette formation test It did not settle out of the lysate. Furthermore, this band was used for antibodies other than mAb A33. By (“No. 1 ° Ab” indicates that mAb A33 was not used. Indicating that).^ThreeH-GlcNAc is introduced into glycosylation site of glycoprotein These results indicate that the A33 antigen contained in the band Is a glycoprotein. Yet another testimony to this A rationale is provided in the following example.Example 5   ³⁵SW1222 cells labeled with S were combined with 5 μg / ml tunicamycin. And then incubated for 18 hours. Tunicamycin is a glycoprotein glycoprotein It is known to prevent oxidation. These cells, plus tunicamycin, The cells that were not incubated together were lysed and the A33 antibody, FB-5 antibody ( Immunoprecipitation was performed with (control) or without antibody (control). FIG. Shows the results of these immunoprecipitations.   For cells not incubated with tunicamycin, the A33 antibody Showed a band of about 43 kD. Is an isotype control Immunoprecipitation without antibody FB-5 or without antibody would produce such a 43 kD band. Not shown. In cells incubated with tunicamycin, A33 Immunoprecipitation with the antibody showed a band of 43 kD, plus three other lower molecular weights. Band and indicated. These low molecular weight bands, due to the presence of tunicamycin, This indicates the presence of A33 antigens with different degrees of glycosylation. This is the A33 antigen Provide additional evidence that they are glycoproteins and contain N-linked olive sugars. To offer.Example 6   A33 antigen was identified using two-dimensional gel electrophoresis under non-reducing conditions. First The LIM1215 colon cell line was converted to RPMI containing 10% fetal bovine serum. Grow in I medium. Confluent cells (10⁶/ Cm^Two) To Trypsi Separated from plastic Petri dishes using n-Versene solution. cell With 10% fetal bovine serum, 1 μg / ml hydrocortisone, as described above. 0.024 U / ml insulin and 10.82 μg / ml α-thioglycol Cello Culture dish containing 25 ml of RPMI 1640 (150 × 20 mm). The petri dish is kept in 5% CO for 5 days._TwoAtmosphere Incubate at 37 ° C. in an atmosphere. After removal of the medium, the cells are Washed with PBS before removal by paper. The cells are washed in PBS and⁹ Resuspended at a concentration of cells / ml.   Next, 0.3% Tri in 10 mM Tris-HCl buffer (pH 7.4) is used. 3x10 using ton X-100⁸A33 antigen from LIM1215 cells Extracted. This extract is purified with arginine / lysine containing 30% glycerol. Diluted 1: 1 with a sample buffer consisting of buffer buffer, pH 10, and non-reducing conditions. The bottom was electrophoresed on a small (8 × 8 cm) Novex two-dimensional gel electrophoresis gel.   The first dimension of the protein was determined by isoelectric focusing at pH 3.5-8.5. The second dimension was analyzed by SDS-PAGE (10% acrylamide gel). Released. A33 antigen was analyzed by immunoblot analysis using mAb A33 (FIG. 5B). Along with in a gel by staining with Coomassie Blue R-250 Position determined. For comparison, the staining pattern observed using the anti-actin mAb was The turn (FIG. 5A) is shown. Actin has similar electrophoretic properties to the A33 antigen. For comparison, they were used for comparison.Example 7   About the LIM1215 cell extract and chromatography fraction Biosensor analysis was performed. These extracts and fractions are combined with an optical instrument Osensor (BIAcore)^TM, Pharmacia Biosensor, U ppsala, Sweden) using the A33 humanized monoclonal antibody. F (ab) ’_TwoFragments were immobilized on the biosensor surface and monitored .   F (ab) '_TwoTo prepare the fragments, the A33 antibody was purified (Ki ng et al .;Br. J. Cancer, Vol. 72, pp. 1364 -1372 (1995)). F (ab) ’_TwoThe fragments were 0.1 M sodium acetate. 10 mg of A33 mAb pepsin (1% w / w) in solution (pH 3.5) Produced by digestion. Next, they were combined with 0.15 mM NaCl. Sephacryl S- equilibrated with 50 mM sodium phosphate (pH 7.4) Size exclusion chromatography on a 200 (2.8 × 60 cm) column (Pharmacia Biotech) Purified by chromatography. Elution was performed at a flow rate of 0.5 ml / min.   F (ab) ’_TwoDetection of antigen binding to the fragment can be performed on the gold sensor surface, or Surface plasmon resonance, a technique for measuring small changes in the refractive index in the vicinity Based on the phenomenon. Prior to this biosensor assay, cell extracts and chromatographic Fee fraction and BIAcore^TMBuffer (HBS); 3.4 mM ED 1 containing TA, 0.15 mM NaCl and 0.005% Tween 20 Dilute to a final volume of 100 μl in 0 mM Hepes (pH 7.4) did. Inject a sample (30 μl) onto the sensor surface at a flow rate of 5 μl / min did. After completion of the injection phase, dissociation was performed at the same flow rate for 360 seconds using BIAcore buffer. Monitored inside. The residual bound antigen was eluted and the surface was treated with 40 μl of 10 mM N It was regenerated between injections using aOH. By this processing, the sensor table The protein immobilized on the surface was not denatured. This includes the A33 antigen Injection of a sample to be performed is indicated by the equivalent signal being obtained .   FIG. 6 shows actin and complete A33 antibody or A33F (ab) '._TwoFragment 9 shows a biosensor analysis of the interaction between. Rabbit muscle actin tone The product (0.3 μg) was ligated with complete A33 (upper trace) or A33 F (ab) ′_Two 5 (on the lower trace) on the sensor surface immobilized with either Injected at a flow rate of μl / min. Protein / Protein Interaction, Surface Plasmo Monitored by resonance. At the end of the injection phase, actin binding to A33 IgG Depending on the case, a signal of 247RU was observed, whereas A33F (ab) '_Two The signal corresponding to actin binding to was only 4 RU (indicated by the arrow).Example 8   A33 antigen was purified from LIM1215 cells for sequence analysis. FIG. Flow showing the chromatographic purification protocol used for the purification of the A33 antigen It is a chart. To extract A33 antigen, LIM1215 colon cells (2 × 10⁹Cells), washed in phosphate-buffered saline (PBS), 1 mM PMS F, 1 mM pepstatin, 0.1 mM leupeptin, and 0.01 U / ml 0.3% in 15 mM Tris-HCl (pH 7.4) containing tinin (V / v) Triton X-100 or 1% (v / v) Triton X-1 Dissolution at 4 ° C. for 30 minutes (10⁸Cells / ml). The result The resulting extract was centrifuged twice at 14,000 g for 20 minutes at 4 ° C. Tr Iton-X100 supernatant was used for Green-Sepharose HE-4BD Removed directly for chromatography. On Triton X-114 extraction Qing contains 0.06% (v / v) T containing the protease inhibitors listed above. riton X-114 and 15 mM Tris-HCl (pH 7.4) Layered on 6% sucrose. Triton X-114 extract and screen The tube containing the loin was incubated at 37 ° C. for 30 minutes, then at 25 ° C. For 15 minutes at 5,000 g. Detergent, chromatograph Collected for fee purification.   To perform Green-Sepharose chromatography, Trit The on-X100 extract or Triton X-114 detergent layer was added to 0.1% % Triton to a final concentration and perform high-speed protein liquid chromatography. Raffy (FPLC, Pharmacia Biotech, Uppsala, Green-Sepharose HE-4BD color connected to Sweden) (100 × 10 mm ID) at 4 ° C. The column is 0.1% C Clean with 10 mM Tris-HCl (pH 7.4) containing HAPS (w / v). Balanced. The bound protein, including actin, was purified using 1M NaCl. It eluted stepwise. The breakthrough (leakage) contains the A33 antigen This was collected for anion-exchange HPLC, described below.Example 9   In order to confirm the presence of A33 antigen, Western blot Analysis was carried out. Phasesystem separation and control unit (Phar macia Biotech) with precast Phastgels. Electrophoresis and Western blot analysis were performed. Cell extract And chromatographic fractions are described by Reid et al., Elec. troporesis, Vol. 16, pp. 1120-1130 (1995) 8-25% SDS-PAGE Ph under non-reducing conditions as described in electrophoresed on astgel or 8-25% native Pastgels, PV Transfer onto DF membrane, incubate with A33 monoclonal antibody I did it. A33 antigen purified by RP-HPLC was also used under non-reducing and reducing conditions. Using a polyclonal anti-N-terminal peptide antibody (described herein) Analyzed by blot. IgG binding to horseradish peroxidase -Labeled goat anti-mouse IgG, goat anti-human IgG or goat anti-rabbit IgG By enhanced chemiluminescence (EC L).   FIG. 8 shows Triton X-100 and Trit of LIM1215 colon cells. Figure 9 shows a Western blot analysis of on X-114 extract. Panel A It indicates the following: Lane 1: soluble in 0.3% Triton X-100 Transformed LIM1215 cells. Lane 2: Gre containing 43K A33 antigen en-Sepharose breakthrough. Lane 3: 41 kD molecular weight band -Sepharose binding protein eluted with 1M NaCl containing quality. Lane 4: rabbit muscle actin (1 μg).   Panel B shows the following. Lane 1: 1% Triton X-114 LIM1215 cells solubilized in. Lane 2: Triton X-114 aqueous phase . Lane 3: Triton X-114 detergent phase. Lane 4: Green -Sepharose breakthrough. Lane 4: eluted with 1 M NaCl Green-Sepharose binding protein.Example 10   After Green-Sepharose chromatography (described above), the anion -Exchange HPLC was performed. The Green-Sepharose breakthrough With 10 mM Tris-HCl containing 0.1% (w / v) CHAPS (p Mono Q HR 10/10 column pre-equilibrated in H7.4) Injected at 4 ° C above. Produces proteins at a flow rate of 1 ml / min for 90 minutes Eluted from the column using a linear 0-1 M NaCl gradient I let you. The fraction (1 ml) was collected by a fraction collector (FRAC100, (Pharmacia Biotech). Protein Quality was detected by absorbance at 280 nm. A33 antigen was purified under non-reducing conditions Detection was by both tan blotting and biosensor analysis.   FIG. 9 shows anion-exchange HPLC of the A33 antigen. The Green -The protein contained in the Sepharose breakthrough fraction was ono Q HR 10/10 anion-exchange column, 0-1 M NaC Elution was performed with a linear gradient indicated by 1 at a flow rate of 1 ml / min. linear Aliquots (20 μl) were taken for bioassay. I described it here And numbered by Western blot analysis under non-reducing conditions (inserted in Figure 9). In this fraction, an antigen of about 43 kD was detected.Example 11   Next, size exclusion HPLC was performed. The Mono Q column (10 ml) The active fraction eluted from the Vac was concentrated in a Speed Vac concentrator (Sav ant Instruments Inc., NY, U.S.A. S. A.) Using 1 Concentrate 0-fold and dialyze against PBS containing 0.05% CHAPS (w / v) And loaded on a Superose 12 HR10 / 30 column at 4 ° C. Ta The protein was washed with PBS containing 0.05% (w / v) CHAPS at a flow rate of 500 μl / Eluted in minutes. Fractions (0.5 ml) were collected. Protein A33 antigen was detected at 80 nm and analyzed by Western blotting and Monitored using both sensor analysis.   FIG. 10 shows a size exclusion HPLC of the A33 antigen. Protein calibration standard The elution position of the quasi (BSA dimer, BSA and trypsin inhibitor) was determined by chromatographic analysis. Shown above the topography trace. A33 antigen was also added to the fractions shown in the figure. In Western blot analysis under non-reducing conditions (insert A). 8-25% Fractions of Superose 12 activity using native gel (fraction 2 The immunoblot analysis of -5) shows that the A33 antigen was used under native conditions (without SPS). ) Showed that the sample migrated with a relative molecular weight of 180 kD (insert B, FIG. 10). .Example 12   Next, reverse phase HPLC chromatography was performed. Superose12 activity Fractions (2.5 ml) were injected multiple times at a flow rate of 1 ml / min, each 1 ml By Brownlee Aquapore RP300 micro-preparative RP- Loaded on an HPLC column (30 × 2.1 mID). The column is . Equilibrated with a starting solution consisting of 15% (v / v) trifluoroacetic acid (TFA) I left it. The protein was washed at a flow rate of 100 μl / min with 60% aqueous n-propano With a linear gradient of up to 0.125% (v / v) TFA for 60 minutes. Let out. Column temperature was 45 ° C. Perform protein detection at 215 nm Was. A33 antigen is used for both Western blotting and biosensor analysis. Used to detect. The peak containing the A33 antigen was repurified and the gradient Prior to N-terminal sequence analysis, Brownlee at a flow rate of 50 μl / min.   Aquapore RP300 micro-preparative RP-HPLC column (100 × Further concentration using 1 mm ID). Elution fractions are manually Collected.   FIG. 11 shows the micro-preparative RP-HPL of the Superose 12 active fraction. Shows C purification. Panel A, large box, absorbance at 215 nm, and Of fractions from micro-preparative RP-HPLC analyzed by osensor Figure 3 shows an elution profile. Panel A, Insert A, are SDS-PAGE (8 Aliquots (2 μl) of each fraction analyzed by -25% gel, silver stain) l) Panel A, insert B shows Western blots under non-reducing conditions. Is shown. Panel B shows individual fractions from micro-preparative RP-HPLC FIG. Aliquot of each fraction (20 μl) Was concentrated using a Speed Vac concentrator and 100 μl of BIAcor e^TMRedissolved in buffer. 30 μl aliquot using biosensor And analyzed. 46 minutes The activity was observed in the fraction eluted between and 48 minutes.Example 13   As described above, the reverse phase HPLC fraction containing the A33 antigen was subjected to the amino acid sequence Pooled for analysis. N-terminal amino acid sequence analysis of purified A33 antigen / protein To a Hewlett-Packard model G1005A protein sensor. Reid et al., Electrophoresis. Vol. 16, pp. Routine 3.0 sequence described in 1120-1130 (1995) The operation was performed according to the sensor program. The following 30 amino acid N-terminal sequence Obtained (SEQ ID NO: 1)   XSVETPQDVLRASQGKSVTLPXTYHTSXXXREGLI QWD All available proteins, DNA and expressed sequences The A33 N-terminus is also a known tag by searching the No protein and significant amino acid sequence identity was shown.   Further, Simpson was used for the reverse phase HPLC fraction containing A33 antigen. et al., Eur. J. Biochem., Vol. 183 pp. 715- Tryptic digestion was performed as described by 722 (1989). The peptide fragments T1 and T2 were obtained. These peptide fragments The amino acid sequence of is shown in FIG.Example 14   The fraction containing A33 antigen was purified using the protocol shown in FIG. Obtained from 222 cells. To perform affinity chromatography, Sch neider et al., J. Mol. Biol. Chem. Vol. 257, pp . A according to the protocol described in 10766-10768 (1982). A 33 affinity column was prepared. A33 monoclonal antibody was added to 0.1 M Dilute to 1 mg / ml in borate, pH 8.2, and add 1.5 ml protein A -Incubated with Sepharose at 4 ° C overnight. 0.1M borate After washing at pH 9.2, the protein-A-monoclonal antibody conjugate was 20 mM dimethylpimelimidate in 0.1 M borate, pH 9.2 and room temperature For 1 hour. Non-covalent antibodies were treated with 50 mM glycine, pH 2 . 5 and removed. Inert remaining dimethylpimelimidate groups by washing And the beads were incubated with 0.1 M ethanolamine pH 8.0.   Reverse phase HPLC fractions were pooled for amino acid sequence analysis. Array The analysis was performed as described in Example 13. The following A33 N-terminal sequence was obtained (located Column number 4)   ISVETPQDVLRASQGKSVTLPXTYHTSTSSREGLI QWDKL A sequence search was performed, but significant I amino acid sequence identity with known proteins There was no tea. This N-terminal sequence was used for the cDNA sequence encoding A33 antigen. Used to obtain columns (below).   In addition, a Simson e. tal., Eur. J. Biochem., Vol. 183, pp. 715-7 22 (1989), digested with Asp-N endoproteinase. Was. The peptide fragments D1, D2, D3 and D4 were obtained. These pep Tide was purified by micro-preparative RP-HPLC. These peptide flags The amino acid sequence of the comment is shown in FIG. The cycle of Edman degradation of peptide D4 In L3, one amino acid was determined to be unknown. On the side of Asp112 Has a Thr at position 114. This is a typical N-glycosylation motif Thus, evidence is provided that the A33 protein is N-glycosylated Was done.   For fractions, see Sarkar et al., Proc. Nat'l   Acad. Sci. U. S. A., Vol. 88, pp 234-238 (19 Pepsin digestion was also performed as described in 91). Peptide fragment P1 was obtained. FIG. 12 shows the amino acid sequence of the peptide fragment P1.   For RP-HPLC fragment, Thermolysin / Pepsi / Asp-N digestion was performed. Thermolysin digestion is described by Sarkar, supra. Performed as described. Peptide fragments Pc1 and Pc2 were obtained . Pepti The amino acid sequences of fragments Fc1 and Pc2 are shown in FIG.Example 15   Perform immunization studies using the immunogen derived from the amino acid sequence at the N-terminus of the A33 antigen Was. VETPQDVLRASQGKSVTL, a chemically synthesized peptide P (amino acids 2-21 of SEQ ID NO: 1) was bound to KLH and shared with an adjuvant. , Two rabbits and two rabbits were injected. Rabbits 4 times at 3 week intervals Immunized. In the first immunization, complete Freund's adjuvant (CFA) It was used. In subsequent rabbit immunizations, incomplete Freund's adjuvant ( IFA) was used. Mice are immunized 4 times at 2-week intervals using standard adjuvants It has become epidemic. Serum was obtained from these rabbits and mice. These serums are Stern blot analysis was performed.   Both rabbits and mice have the peptide and the cell line SW1221. 43 kD band when used (the same 43 kD band was used by mAb A33) (FIG. 14). I gG was purified from rabbit immune blood by protein-A affinity chromatography. Purified from Qing. Purified IgG was combined with LIM1215 cell solubilization and purified A33 antigen. The reactivity of the protein was determined by SDS-PAGE and Western blot analysis. Characterized. The IgG comprises the above-mentioned 20 amino acid peptide and the non-reduced peptide. The approximately 43 kD protein recognized by mAb A33 under native conditions It turned out to react strongly with the quality. In addition, rabbit IgG anti-serum is in reduced form. It reacted strongly with the complete A33 antigen (FIG. 15). HPLC purification A from LIM1215 A 33 antigen (0.1 μg) was added under non-reducing conditions (FIG. 15, lane 1) and under reducing conditions. (FIG. 15, lane 2), 8-25% SDS-PAGE Pastg el, and prepared for residues 2-21 of SEQ ID NO: 1 described above. Analyzed by Western blot using anti-peptide IgG. A33 anti The original N-terminal sequence and its fragment are used for the development of A33 antigen-specific antibody can do. These antibodies, in reduced or non-reduced form, are A33 Recognizes and binds to the antigen, or a fragment thereof.Example 16   Using the amino acid sequence of the A33 N-terminus of the A33 protein, The cDNA of the protein was cloned. Poly (A)⁺RNA (80 μg) as standard 2 rounds of enrichment on a column of oligo (dT) cellulose using the procedure Prepared from the interior of confluent LIM1215 cells. LIM1215 cDNA Using this mRNA, the library can be used for first-strand DNA synthesis ( Oligo (dT) and random hexamer primer to start the standard procedure) Using Clontech (Palo Alto, California, U.S.A.) . S. A.) and custom synthesis in a λZAPII expression vector.   Good screening of the library is based on the polymerase chain reaction (PCR). ) Using the DNA library prepared from the LIM1215 cDNA library. Achieved by the Six 17 mer each with only eight degeneracy An antisense oligonucleotide (R9-R14) was substituted for the A33 antigen N-terminal sequence. At amino acid residues 34-39 (LIQWDK (amino acid 34 of SEQ ID NO: 4) −39)).   These are hybridized with the sequence present in the backbone of the λZAPII vector. A33 antigen template, paired with a sense primer designed to PCR reaction with amplified LIM1215 cDNA library as source of used. Good reactions occurred when the following primers were used. PCR reaction For the template used, the template used in the amplified λZAPII vector It was a LIM1215 cDNA library. The primers used are listed below. It was right. KS primer 5'CGAGGTCGACGGTATCG (sequence No. 18) (17 mer) (in the multiple cloning site of the λZAPII vector And R10 primer (described above).   The reaction conditions were as follows.     1 μl cDNA library (10 "pfu / ml)     10 x T′aq^TM Buffer 5 μl     2.5 mM NTPs 4 μl     15 mM MgCl_Two                          5 μl     KS (50 pmoles / μl) 1 μl     R10 (50 pmoles / μl) 1 μl     Water 32.5 μl     Taq polymerase0.5 μl(Added at the end of HotStart)                                       50.0 μl   The touchdown program used for PCR was as follows.         1 95 ° C x 5 minutes         2 95 ℃ x 1 minute         3 60 ℃ x 1 minute             -2 ° C in subsequent cycles         4 72 ℃ x 2 minutes         5 Return to (2) and repeat 11 times         6 95 ℃ x 1 minute         7 37 ° C x 2 minutes         8 72 ° C x 2 minutes         9 95 ℃ x 1 minute         10 45 ℃ x 2 minutes         11 72 ° C x 2 minutes         12 Return to (9) and repeat 13 times         13 72 ° C x 5 minutes         14 Maintain 4 ℃ Three products are produced, which are 1.4 kb, 0.5 kb and 0.3 kb long. Met.   The 1.4 kb product (R10 / 1) and the 0.5 kb product (R10 / 2) and 3% agarose gel, and Bresa-clean^TMNucleic acid Uses a kit (Bresatec, Adelaide, S. Australia) And stopped. In order to produce larger quantities of these products, these purified Used as template for PCR reaction. The PCR reaction was performed with 1 μl of the LIM Instead of the 1215 cDNA library, 1 μl of the purified PCR product (R10 / 1 or R10 / 2), except that it was used as a DNA template. Performed in the same manner as described above. Two bands were generated by the R10 / 1 PCR reaction. Upper band size 1.4kb (weak) The lower band size is called 0.3 kb (strong) 10/1 300 bp, Two bands were generated by the R10 / 2 PCR reaction. Upper band size 0.5kb (strong) The lower band size is called 0.3 kb (strong) 10/2.   Said 0.3 kb fragment (10/1 300 bp and 10/2) The gel was purified as described above. Nucleotide sequencing of both fragments was performed However, the reverse complement of each sequence encodes a part of the A33 N-terminal protein sequence. I found out.   Next, a procedure for screening the LIM1215 cDNA library was performed. To amplify the correct 189 bp PCR product used as a probe, the A33 antigen The following correct primers for the cDNA sequence were synthesized. Primer # 1747 (A33 sense primer 1) 5'CCTGTCTGGA GGCTGCCAGT (20 mer) (SEQ ID NO: 19) Primer # 1748 (A33 antisense primer 1) 5 'AGGTGCA G GGCAGGGTGACA (20 mer) (SEQ ID NO: 20).   The above primers were used in the following standard PCR reaction and the expected size (1 89 bp) of the product. Standard PCR reaction conditions   10 μl to 300 bp product 1 μl   2 x 1xT'aq buffer   1.6 μl of 2.5 mM NTPs   15 mM MgCl_Two           2 μl   Primer # 1747 (50 pmoles / μl) 1 μl   Primer # 1748 (50 pmoles / μl) 1 μl   11 μl of water   Taq polymerase0.4 μl(Added last)                               20.0μl The following standard PCR program     195 ° C x 5 minutes     2 95 ° C x 1 minute     3 55 ° C x 1 minute     4 72 ° C x 1 minute     5 Return to (2) and repeat 30 times     6 72 ° C x 5 minutes     Maintain 74 ° C   The 189 bp product was separated on a 3% agarose gel and Bresa-Clea n^TMPurified using the kit. Next, the well-known random primer reaction And> 10 using the Klenow polymerase procedure⁷Specific release of dpm / μg DNA Up to the firing rate, [α³²P] ATP and [α³²P] CTP and radiolabeled with Screening 800,000 clones of LIM1215 cDNA library Used (standard procedure). After 3 rounds of screening, 13 purifications A33 antigen cDNA clones were obtained, the longest of which was about 2.8 k b. See below.   The labeled PCR probe was also used for Northern analysis to determine whether LIM1215 cells Total RNA and poly (A)⁺Approximately 2.8 kb size m in concentrated RNA A strong hybridisation signal with a single species of RNA is generated, The 8 kb clone was suggested to be near full length. Deploy multiple clones As determined by sequencing, all encode the A33 antigen N-terminal protein sequence I understood. Figure 2. Complete nucleotide sequence of 2.6 kb clone (clone 11) This is shown in FIG.   One 2.6 kb cDNA was radiolabeled as described above (ie, Klen). [α] in random primer reaction with ow polymerase³²P] ATP and [α³²P] When used for Northern analysis (using CTP), the A33 antigen positive cell line ( LIM1215, LIM1899 and LIM1863) and normal human colon epithelium About 2.8 kb of strong RNA, total RNA prepared from tissues Was obtained, but A33 antigen-negative cell lines (LIM2099, LIM24 05, LIM2537).   The 319 amino acid translated protein product (A33 antigen) was Inferred from the nucleotide sequence of the loan. Protein translation is based on 5 of the cDNA sequence. It was expected to start at the second ATG from the 'end. This is a translation Kozak consensus sequence for initiation (GCCC (R) CCATGG (array No. 21)). The putative full length translated protein product is 319 And has the following amino acid sequence (SEQ ID NO: 22).   This protein is cleaved to correspond to amino acids 22-319 of SEQ ID NO: 22. 21 amino acids that produce a 298 amino acid mature protein with a known N-terminus It is proposed to contain an acidic hydrophobic leader sequence. That is,   Due to the position of the first in-frame stop codon, a po A repeptide chain is expected. In the hydrophilicity plot constructed from the amino acid sequence Based on that, the molecule appears to have three parts. That is, 213 The extracellular region of amino acids (sequence alignment of conserved residues (Presumed to have a roblin-like domain) A hydrophobic transmembrane domain and a highly polar intracellular C-terminal tail. This general structure is Suggests that the molecule is involved in signal transduction.   Starting at base pair 113, counting from the 5 'end of clone 11, the salt of clone 11 CDN encoding the 298 amino acid protein, ending in base pair 1070 The A sequence is as follows (SEQ ID NO: 23).   Comparisons with available DNA and protein databases are based on the amino acids described above. The protein consisting of the acid (SEQ ID NO: 22) was found to be novel. Only Meanwhile, the available expression sequence tag (EST Analysis of the database revealed a portion of the human A33 antigen cDNA (nucleotide 286). -529) and a murine embryonic carcinoma cell line F9 (EMBL approval number MM88A). 09; 74 between 249 base pairs EST from DDBJ approval number D28657) % Sequence similarity was revealed. This EST contains the mouse of the human A33 antigen cDNA. Since it was possible to correspond to a part of the homologue, sense and antise A PCR primer (17 mer) was hybridized to the end of the EST clone. It was designed as follows to soy.   Using these primers in the touchdown PCR program described above, Amplify a 218 bp product from a normal adult mouse colon crypt cDNA library Was. For details of the mouse cDNA library used, see, for example, Biol. Chem. 268: 27214-27225 (1993). Simple single To illustrate, cDNA was purified from poly (A) purified from adult mouse colonic crypt epithelium.⁺ Reverse transcribe from the enriched RNA and then clone it into a λgt-11 expression vector. I did. The product was gel purified and DNA sequenced. It was shown that it closely corresponds to the F9EST.   Translation of the mouse colon PCR product was performed by translating a portion of the A33 antigen sequence (truncated). Significant homology to residues 64-104 of the molecule, residues 85-125 of SEQ ID NO: 22) Revealed. Alignment between predicted human and mouse protein sequences Is shown below   The F9 PCR product was radiolabeled ([α³²P] ATP and [α³² P] CTP) to obtain multiple mouse tissue RNAs (colonic crypt, small intestinal crypt, kidney, liver , Brain, spleen, thymus, lung, pancreas, testis, heart and thigh muscles) Used as a probe in A strong band of about 2.6 kb in size Only seen in lanes containing RNA prepared from the fossa and small intestinal crypts, Very weak signals were seen when pancreatic RNA was used. This chick Close correspondence with the size of the A33 antigen mRNA and the alignment Both the F9 clone and the restricted tissue expression indicate that the F9 clone It strongly suggests that it encodes the us homolog. Furthermore, these Is that the F9 EST contains an error, and the genuine sequence is: Better described by the sequence of the PCR product described herein. And suggests.   Next, using the F9 PCR product, a full-length mouse A33 cDNA clone was prepared. The mouse colon crypt cDNA library described above to obtain did. Using standard methods, 20 clones were identified, most of which were approximately 2. It had a 2 kb A33 cDNA insert, but two had a longer 4.2k b. These two long claw using standard method And two of the 2.2 kb clones were DNA sequenced. The 3 'sequence of the 4.2 kb clone does not correspond to the A33 antigen cDNA and BLAST Publicly available library sequences using the FASTA and FASTA algorithms In the similarity search, the 3 'end is stomach, non-muscle Ca²⁺For ATP-ase cDNA It is clear that it is a response.   All 5 'ends of the four clones were recognized as A33 antigen nucleotide sequences It was possible. Therefore, the 4.2 kb clone has an A33cD NA at its 3 'end with non-stomach Ca²⁺It has ATP-ase cDNA. Sequencing of the 4.2 kb clone revealed 2202 base pairs of A33 cDNA. . The short clone contained the cDNA of 2122 base pairs of the antigen. The longest ORF translation is -NH_TwoIncomplete 318 amino acid protein Make predictions. It shows the same basic structure as the human antigen, which And highly homologous. 20 amino acid hydrophobic leader sequence (lacking initiating methionine ) Is seen (human One V-set, one C2 set immunoglobulin-like Domain, one 24 amino acid hydrophobic transmembrane domain, and 61 amino acid The intracellular domain is presented. Further, the N-terminal region comprises a human cleavage site: Consensus peptide cleavage site similar to ADA @ ISVET (SEQ ID NO: 30): ADA @ LTVET (SEQ ID NO: 29), each of which has 298 amino acids Produces acid mature protein.   The whole analysis shows a 71% similarity between the mouse and human sequences in the extracellular domain , 67% similarity in the transmembrane domain, and 5% in the intracellular domain This shows a similarity of 4%. The mouse proteins are 91, 179, and 2 02 compared to the human sequence with three potential sites, positions 78, 91, 1 79 and 202 show four potential N-linked glycosylation sites.   The nucleotide and deduced amino acid sequences of the mouse clone were set forth in SEQ ID NO: 31. And 32. Alignment of the deduced amino acid sequences of the human and mouse sequences FIG.Example 17   Further experiments were performed using the protocol described above, also encoding A33. The longest A33 antigen cDNA clone (clone 18) was found. This black The nucleotide sequence of the sequence is shown in SEQ ID NO: 33. Clone 18 is about 2.8kg Clone 11 has a base length of 2.6 kilobases, as This clone is slightly longer than clone 11 described above in that . In SEQ ID NO: 29, nucleotides 345-1302 are set forth in SEQ ID NO: 33 It is considered to encode the amino acid sequence described above.Example 18   As mentioned above, the A33 molecule is a glycoprotein with N-linked glycosylation. Considered to be quality. Further research on post-translational modifications to antigens Was.   As reported in the above references, cell line SW1222, LIM 1215, CO LO205 were all positive for A33, but SW620 and MF-SH were A3 positive. Shade about 3 Gender. All these cell lines^Three500μCi / m with H palmitate l is metabolically labeled and then lysed by detergent. Lysates were sedimented with A33 and FB5 as described. Here again, FB 5 functions as a negative control. The sediment is then SDS-PAGE Analyzed and analyzed by autoradiography.   The results are shown in FIGS. In FIG. 18, the reduction conditions and the non-reduction The molecular weight under^ThreeWe can see that H palmitate labels A33 antigen You. Labeled sediment was found in all three positive cells, but negative cells Not found in the vesicles. FB5 was negative in all tests .   The SDS gel was run on 1M hydroxylase prior to autoradiography. When treated with min (pH 7.5), no staining was observed, as shown in FIG. Was. This is because the palmitate group (acyl) is linked by a thioester. And (Cys)_FourThe domain is present in the molecule and palmitate is linked to it. It seems to have been.   The isolated, characterized and sequenced A33 antigen is useful for cancer, especially It can be used for diagnosis of colorectal cancer characterized by the expression of A33 antigen. For example, large A sample suspected of containing intestinal cancer cells was subjected to A33 antigen or its fragment. A33 protein / antibody complex is formed upon contact with an antibody specific for the antibody. If such a complex is present, it may be positive in colon cancer diagnosis. Is shown.   Further, the A33 antigen identifies a ligand (binding partner) linked thereto. Available for The A33 antigen can be isolated and expressed as a recombinant. Biological sources, including tissue culture media, tissue extracts, and cell lysates. Can be used to screen for the presence of a partner. Join partner If found, it is isolated, purified, and sequenced. This is a biosensor Can be used in combination with affinity and other chromatography techniques . Furthermore, the A33 antigen is tagged, and the antigen is applied to the biosensor surface and affinity. It can help in immobilizing in a particular direction relative to the body. Known to those skilled in the art Binding partners can be identified using known techniques. For example, Stitt e t al.,Cell, Vol. 80, pp. 661-670 (1995), Nice et al.,J. Chromatography A. , Vol. 660, pp. 169-185 (1994) and Bartley et al.,Nature, Vol. 368, p. 558 (1994); Lachmann et al., Proc. Natl. Acad. Sci. USA 93: 2523-2527 (1993).   In addition, a cDNA encoding the A33 antigen is described herein. 5 'end or This cDNA, including the untranslated portion at the 3 'end, expresses the A33 antigen Production of A33 antigen double-stranded cDNA molecules from existing tissues and cell lines, It is possible to facilitate the production of genomic clones of A33 antigen from DNA . To do this, the cDNA for A33 is converted from the mRNA template to double-stranded c RT-PCR (anti-transcriptase-PCR), a general process for producing DNA molecules This technique is used to design complementary primers used in the technique described above. Furthermore, A33 cDNA is obtained by extracting the A33 antigen gene part from the genomic DNA template. Design complementary primers used in standard PCR reactions for amplification Especially available.   It is possible that the A33 antigen belongs to a new family of related proteins. Write here The A33 cDNA sequence listed contains c encoding a protein related to the A33 antigen. Under low stringency conditions to amplify DNA and portions of genomic DNA molecules Of specific, degenerate oligonucleotides used in PCR reactions It can be used to design limers. In addition, low stringency A33 antigen gene family was analyzed by Southern analysis of genomic DNA under transient conditions. Specific degenerate oligonucleotide probes to identify members of A33 cDNA can be used to design probes.   These steps utilizing the A33 cDNA are well known to those skilled in molecular biology. This is a standard process. For example, Molecular Cloning: A Laboratory man ual, 2_ndedition, 1989 (edited by Sambrook J, Fritsch EF & Maniatis T) Co ld Spring Harbor Laboratory Press, U.S.A. and Current Protocols in Molecular Biology Volumes I & II, 1989 (edited by Ausubel, FM) Grne Pub See lishing Associates and Willy-Interscience, USA.   Although the present invention has been described with reference to preferred embodiments, these embodiments are only illustrative. It is intended to illustrate a number of features of the invention, and within the spirit and scope of the invention, Various modifications and the like can be made to these embodiments.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] August 19, 1997 [Content of Amendment] Claims : 1. An isolated protein-containing molecule, the protein portion of which has a molecular weight of 40-45 kD as determined by SDS-PAGE under non-reducing conditions, wherein said molecule binds to monoclonal antibody A33. 2. 2. The isolated protein-containing molecule of claim 1, wherein said molecule is recombinant. 3. An isolated protein-containing molecule consisting of the amino acid sequence shown in SEQ ID NO: 22 or amino acids 22-319 of SEQ ID NO: 22. 4. 4. The isolated protein-containing molecule of claim 3, wherein cys43 and cys117 are linked by a disulfide bond. 5. An isolated peptide molecule comprising an antigenic fragment of the amino acid sequence of SEQ ID NO: 22, wherein the peptide binds to monoclonal antibody A33. 6. 6. The isolated peptide of claim 5, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. 7. 6. The isolated peptide of claim 5, wherein said peptide is recombinant. 8. An isolated nucleic acid molecule encoding the protein portion of the protein-containing molecule of claim 1. 9. 9. The isolated nucleic acid molecule of claim 8, wherein said molecule has the nucleotide sequence set forth in SEQ ID NO: 23. 10. 9. The isolated nucleic acid molecule of claim 8, wherein said A33 protein has the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence consisting of amino acids 22-319 of SEQ ID NO: 22. 11. An expression vector comprising the isolated nucleic acid molecule of claim 8 operably linked to a promoter. 12. A host cell transformed or transfected with the nucleic acid molecule of claim 8. 13. A host cell transformed or transfected with the expression vector of claim 11. 14． (a) isolating or expressing a molecule containing an amino acid sequence corresponding to the linear sequence of amino acids of SEQ ID NO: 22, (b) screening a biological source using the molecule, (c) Isolating a ligand that binds to the molecule from a biological source. 15. 15. The method of claim 14, wherein said molecule has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 22. 16. (a) a step of developing a primer pair complementary to the nucleotide sequence shown in SEQ ID NO: 29; and (b) an organism expressing the A33 antigen by performing a reverse transcriptase polymerase chain reaction using the primer pair. Obtaining an A33 antigen double-stranded cDNA from a source. 17． (a) developing a primer pair complementary to the nucleotide sequence shown in SEQ ID NO: 29; and (b) conducting a polymerase chain reaction using the primer pair to convert the A33 antigen genomic DNA template into the A33 antigen gene. A method of obtaining an A33 antigen gene fragment comprising: amplifying the fragment. 18. (a) developing a specific degenerate oligonucleotide primer complementary to the portion of the nucleotide sequence shown in SEQ ID NO: 29, and (b) performing a low stringency polymerase chain reaction using the primer, Amplifying a portion of cDNA and genomic DNA encoding a protein associated with the A33 antigen, the method comprising: identifying a nucleic acid molecule encoding a protein associated with the A33 antigen. 19. (a) developing a specific degenerate oligonucleotide primer complementary to the portion of the nucleotide sequence set forth in SEQ ID NO: 29; (b) utilizing the primer to generate genomic DNA under low stringency conditions. Performing a Southern analysis to obtain the nucleic acid molecule encoding a protein associated with the A33 antigen, the method comprising: identifying a nucleic acid molecule encoding a protein associated with the A33 antigen.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/08 A61K 39/395 E // A61K 31/00 635 T 39/395 C12N 15/00 C 5/00 A (C12N 15 / 09 ZNA C12R 1:91) (71) Applicant Ritter, Gerd New York 10105 New York Avenue of the Americas 1345 (71) Applicant Simpson, Richard, Jay Victoria, Australia 3050 Melbourne P.A. O Royal Melbourne Hospital (71) Applicant Nice, Edward Victoria, Australia 3050 Melbourne (unassigned) P O Royal Melbourne Hospital (71) Applicant Moritz, Earl, El O Victoria 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (71) Applicant Catimmel, Bruno Victoria 3050 Melbourne (no address) P.O.L. Melbourne Hospital (71) Person J, Hun Victoria 3050 Melbourne, Melbourne (no address) POH Royal Melbourne Hospital (71) Applicant Burgess, Anthony Victoria 3050 Melbourne, Australia (no address) POH Royal Le Melbourne Hospital (71) Applicant Heath, Joan Victoria, Australia 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (71) Applicant White, Sara Victoria 3050 Melbourne, Australia (no address) P.O.L. Royal Melbourne Hospital (71) Applicant Johnston, Cameron Victoria, Australia 3050 Melbourne, P.O.L.Le. Melbourne Hospital (72) Inventor Old, Lloyd, Jay United States New York 10105 New York Avenue of the Americas 1345 (72) Inventor Welt, Sydney United States of America New York 10105 New York Avenue of the Americas 1345 (72) Inventor Ritter, Gerd United States of America New York 10105 New York Avenue of the Americas Mericaz 1345 (72) Inventor Simpson, Richard, Jay Victoria, Australia 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (72) Inventor Nice, Ed Australia Australia Victoria 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (72) Inventor Moritz, Earl, El Victoria 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (72) Inventor Catimel, Bruno Victoria 3050 Melbourne, Australia (no address) P.O. Royal Melbourne Hospital (72) Inventor, H. Victoria 3050 Melbourne, Australia (no address) P.O.・ Melbourne Hospital (72) Inventor Burgess, Anthony Victoria, Australia 3050 Melbourne (no address) P.O. Royal Melbourne Hospital (72) Inventor Heath, Joan Victoria, Australia 3050 Melbourne (no address) P.O.Loya Le Melbourne Hospital (72) inventor White, Sarah Victoria 3050 Melbourne (no address) P.O.Loya Le Melbourne Hospital (72) Inventor Johnston, Cameron Victoria, Australia 3050 Melbourne (no address) P.O. Royal Melbourne Hospital

Claims (1)

[Claims] 1. Contains isolated proteins found inside and on the surface of colon cells and colon cancer cells A molecule,   The isolated protein-containing molecule is measured by SDS gel electrophoresis under non-reducing conditions. At a molecular weight of about 40-45 kD, and SDS gel electrophoresis under reducing conditions. An isolated protein-containing molecule having a molecular weight of about 49-50 kD in the determination of 2. 2. The isolated glycosylation-containing molecule of claim 1, wherein said molecule is a monoclonal antibody. Isolated glycosylation-containing molecule that binds to antibody A33. 3. 2. The isolated protein-containing molecule of claim 1, wherein said molecule is recombinant. 4. A protein comprising the amino acid sequence of SEQ ID NO: 22 and the amino acid of SEQ ID NO: 22 An isolated tan selected from the group consisting of proteins consisting of acids 22-319 Park quality. 5. Cys43 and cys117 of SEQ ID NO: 22 are linked by a disulfide bond 5. The isolated protein-containing molecule of claim 4, wherein said molecule is bound. 6. Linear sequence of amino acids SEQ ID NO: 22 which binds to monoclonal antibody A33 An isolated peptide consisting of 7. A group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 7. The isolated peptide according to claim 6, which is a peptide having an amino acid sequence selected from a loop. Chid. 8. An isolated nucleic acid molecule encoding an A33 protein. 9. 9. The isolation of claim 8, wherein said molecule has the nucleotide sequence set forth in SEQ ID NO: 23. Nuclear Acid molecule. 10. The amino acid sequence represented by SEQ ID NO: 22, 9. The isolated nucleus of claim 8, which has an amino acid sequence consisting of amino acids 22-314 of No. 22. Acid molecule. 11. A source having the isolated nucleic acid molecule of claim 8 operably linked to a promoter. Current vector. 12. Transformation or transfection with the nucleic acid molecule of claim 8. Host cells. 13. Transformation or transfer with the expression vector of claim 11. Host cells 14． Protein-containing molecules found inside and on the surface of colon cells and colon cancer cells Wherein the antibody is not A33 and the molecule is non- Approximately 40-45 kD molecular weight as determined by SDS gel electrophoresis under reducing conditions , And about 49-55 kD in the SDS-PAGE measurement under reducing conditions. Proteins found inside and on the surface of colon cells and colon cancer cells with abundance Antibodies specific for contained molecules. 15. Characterized by the presence of A33 antigen inside and on the surface of colon cancer cells A method for diagnosing colorectal cancer,   (A) containing the colon cancer cells so that a protein / antibody complex is formed; The sample considered to be, and the protein-containing molecule of claim 1, or a derivative thereof. Contacting with a specific antibody to the peptide fragment to be,   (B) the complex, wherein the presence of the complex indicates a diagnosis that colon cancer is positive; Deciding whether or not is present. 16. (a) a fragment containing an amino acid sequence corresponding to the linear sequence of amino acids of SEQ ID NO: 22 Isolating or expressing the offspring;   (B) screening a biological source using the molecule;   (C) isolating a ligand that binds to the molecule from the biological source; A method for obtaining a ligand that binds to the A33 antigen, comprising: 17． The molecule is SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 2 17. The method of claim 16, having an amino acid sequence selected from the group consisting of: 18. (a) Step of developing a primer complementary to the sequence shown in SEQ ID NO: 29 When,   (B) performing a reverse transcriptase polymerase chain reaction using the primer, Obtaining an A33 antigen double-stranded cDNA from a biological source expressing the A33 antigen. A method for obtaining a nucleic acid molecule encoding the A33 antigen. 19. A method for obtaining an A33 antigen gene fragment, comprising:   (A) developing a primer complementary to the sequence shown in SEQ ID NO: 29; ,   (B) Performing a polymerase chain reaction using the primers to obtain an A33 antigen Amplifying A33 antigen gene fragment from genomic DNA template When, A method for obtaining an A33 antigen gene fragment comprising: 20. (a) a specific degenerate sequence complementary to the sequence shown in SEQ ID NO: 29 A step of developing a oligonucleotide primer,   (B) low stringency polymerase chain using the primers The reaction is carried out to obtain a cDNA encoding a protein related to the A33 antigen and a gene. Amplifying a portion of the A33 antigen How to identify 21. (a) a specific degenerate sequence complementary to the sequence shown in SEQ ID NO: 29 Re Developing a nucleotide primer,   (B) Genome D under low stringency conditions using the primers Performing a Southern analysis of NA to obtain a protein related to the A33 antigen. A method for identifying a protein related to the A33 antigen.