METHOD OF DETECTING CANCER INVOLVING FRAGMENTS OF CYTOKERATIN 18 AND CORRESPONDING ANTIBODIES

Abstract: A method of detecting cancer in a patient, in which the amount of protein fragments comprising at least one amino-acid sequence selected from the group consisting of the amino-acid sequence (SEQ ID NO: 1) and homologues thereof, and the amino-acid sequence (SEQ ID NO: 2) and homologues thereof, is measured in a sample of body fluid or suspected cells from said patient, is disclosed. Also, the above polypeptides, fusion proteins, RNA and DNA sequences relevant to the polypeptides and fusion proteins, antibodies raised against the polypeptides, fusion proteins, corresponding antigen-mimicking structures or synthetic antibody-mimicking structures complementary to the pertinent epitopes, and diagnostic kits, are disclosed.

Description: Method of detecting cancer involving fragments of cyto eratin 18.and corresponding antibodies. The present invention relates to a method of detecting cancer in a patient based on detection of certain protein fragments in a sample of body fluid from said patient. Further, the invention relates to two polypeptides and their homologues, to fusion proteins comprising such polypeptides, to RNA or DNA sequences coding for the amino-acid sequence of such polypeptides, to antibodies raised against such polypeptides or fusion proteins, and to diagnostic test kits. Background It is of utmost importance to be able to diagnose cancer diseases and to predict patient survival with or without therapy. Monitoring of the efficacy of treatment with chemotherapeutics, hormones, radiotherapy etc. is important for doctor decisions. The aim of therapeutic treatment of cancer is to inhibit or eliminate uncontrolled cell growth. Failure or success of such treatment implies progressive cancer or no growth of tumors. There are several soluble serum and other body fluid markers that have been used as biological markers in the detection and the follow-up of patients with cancer, such as hormones, oncofetal antigens, enzymes, growth factors, placental proteins, glycoproteins, mucins and others. Tissue polypeptide antigen (TPA) was first isolated from various types of human cancers by Bjorklund B. and Bjδrklund V. in 1957 (Antigenicity of pooled human malignant and normal tissue by cyto-immunological technique: Presence of an insoluble heatlabile tumor antigen. Int Arch Allergy 1957; 10: 153-184). TPA is produced by both normal and malignant cells. Elevations in serum are believed to be related to cell turnover. Monoclonal antibodies directed to TPA are useful in the monitoring of patients with various malignancies. Measurements of TPA have been used in a number of human malignant tumours for follow-up of diagnosed patients, control of treatment and for the detection of recurrence. The clinical utility of measured TPA has been reported for breast cancer (Barak, M. et al, CA-15.3, TPA and MCA as markers for breast cancer. Eur J Cancer 1990; 26: 577-580; Nemoto, T. et al, Human tissue polypeptide antigen in breast cancer. J Natl Cancer Instit 1979; 63: 1247-1253), lung cancer (Buccheri, G.F. et al, Clinical value of multiple biomarker assay in patients with bronchogenic carcinoma. Cancer 1986; 57: 2389-2396; Buccheri, G.F. et al, Usefulness of tissue polypeptide antigen in staging, monitoring and prognosis of lung cancer. Chest 1988; 93: 565- 570), bladder cancer (Adolphs, H.D. et al, Significance of plasma tissue polypeptide antigen determination for diagnosis and follow-up of epithelial bladder cancer. Urol Res 1984; 12: 125-128; Carbin, B.E. et al, Urine - TPA (tissue polypeptide antigen), flow cytometry and cytology as markers for tumours invasivness in urinary bladder carcinoma. Urol Res 1989; 17: 269-272), prostate cancer (Lewenhaupt, A. et al, Tissue polypeptide antigen (TPA) as a prognostic aid in human prostatic carcinoma. Prostate 1985; 6: 285-291) and ovarian carcinoma (Crombach, G. et al, Serum levels of TPA in patients with gynaecological cancer. Protides Biol Fluids 1984; 31 : 441-444; Panza, N. et al, Cancer antigen 125, tissue polypeptide antigen, carcinoembryonic antigen and β-chain human chorionic gonadotropin as serum markers of epithelial ovarian carcinoma. Cancer 1988; 61 : 76-83). Bjόrklund et al published in 1987 monoclonal antibody mapping of TPA, which gave 35 different epitopes, (Biochemische und morphologische Grundlagen von TPA: Fortschritte in Richtung auf einem allegemeinen Marker fur aktive Tumoren durch monoclonale kartierung. In: Luthgens M, Schlegel G, eds. Tumormarkersystem CEA- TPA. TumorDiagnostik Verlag, 1987; 14-30). Two of these epitopes were identified to be related to human carcinoma. As a result of this monoclonal mapping of epitopes monoclonal antibodies (MAbs) were raised against these epitope structures by the application of hybridoma technology according to Kϋhler and Milstein. Monoclonal antibodies yield a highly reproducible and replenishing source of antibodies compared with a limited supply of polyclonal antibodies obtained from immunized animals. Monoclonal antibodies also provide a more specific reagent as monoclonals may react with only one specific antigenic determinant present in a given antigen molecule, whereas conventional polyclonal antisera contain a mixture of antibodies reacting with different determinants present in a single antigenic molecule. Immunoassays were developed based on one specific monoclonal antibody in combination with polyclonal horse antibody. Such immunoassays have been marketed by Beki Diagnostics AB, Sweden, under the tradename "TPS" as tumor marker for measuring TPA in serum and other body fluids. The assay measures tumor activity as opposed to such tumor marker assays which measure tumor mass. However, the amino-acid sequence of the protein fragment to which the monoclonal antibody used in the TPS assay specifically binds, has hitherto not been known. The present invention is based on the finding of two distinct amino-acid sequences on protein fragments in body fluids, to one of which the monoclonal antibody of "TPS" binds and to the other of which another of the monoclonal antibodies published in 1987 binds, respectively. Said protein fractions are, as is evident from the above cited earlier work, indicative of tumor cell activity. The above two amino-acid sequences form the bases of the different aspects of the invention. In a data base search it was revealed that the amino-acid sequences of the two polypeptides of the invention correspond to the amino acids 318 to 346 and 406 ' to 430, respectively, of human cytokeratin 18 (CK 18) [The amino-acid numbering starts with the initial methionine]. The complete amino-acid sequence of cytokeratin 18 has been published (Oshima, R.G., Millan, J.L, and Ceccena, G., Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to inplantation. Differentiation 33 (1986) 61-68). Description of the invention One aspect of the invention is directed to a method of detecting cancer in a patient, in which the amount of protein fragments comprising at least one amino-acid sequence selected from the group consisting of the amino-acid sequence (SEQ ID NO: 1 ) Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly lie Leu Leu His Leu Glu 20 25 and homologues thereof, and the amino-acid sequence (SEQ ID NO: 2) Gin Lys Thr Thr Thr Arg Arg lie Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 and homologues thereof is measured in a sample of body fluid or suspected cells from said patient, and the amount obtained is compared with a reference, whereby elevated levels indicate cancerous activity in said patient. Elevated levels may also be obtained in case the patient in question suffers from liver or kidney damage, or is subject to proliferative activity in connection with repair processes e.g. after pneumonia. However, such other causes for elevated levels are easily identified by the patient ' s doctor. In this context a homologue of said amino-acid sequence is a homologous sequence having some amino-acid substitutions, extensions and/or deletions which do not lead to the elimination of the capability of the homologous sequence to bind to the same antibodies as the amino-acid sequence SEQ ID NO: 1 Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly He Leu Leu His Leu Glu 20 25 and/or to bind to the same antibodies as the amino-acid sequence SEQ ID NO: 2 Gin Lys Thr Thr Thr Arg Arg lie Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 Further, a homologue of said amino-acid sequences is any sequence which is sufficiently homologous at the nucleotide level to be recognized by a RNA or DNA sequence complementary to a RNA or DNA sequence which codes for said amino- acid sequences. Examples of homologues of the amino-acid sequence SEQ ID NO: 1 are the amino- acid sequence SEQ ID NO: 3 Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly lie 20 and the amino-acid sequence SEQ ID NO: 4 Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu Gin Met Glu Gin 1 5 10 15 Leu Asn Gly lie Leu Leu His Leu Glu 20 25 . Homologous sequences, in addition to the amino-acid sequences SEQ ID NO:3 and SEQ ID NO: 4, which with the aid of ELISA absorbance studies have been found to bind to the same antibody as the amino-acid sequence SEQ ID NO: 1 are amino- acid sequences selected from the group consisting of the amino-acid sequence SEQ ID NO:5 Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu Gin Met Glu Gin Leu Asn 1 5 10 15 Gly lie Leu Leu His, and corresponding amino-acid sequences 20 wherein Leu in position 1 is substituted by Lys, Gly, Ala or Glu, Arg in position 2 is substituted by Lys, Gly, Ala or Glu, Glu in position 3 is substituted by Lys, Gly, Ala or Tyr, Val in position 4 is substituted by Lys, Gly, Ala or Glu, Glu in position 5 is substituted by Lys, Gly, Ala or Tyr, Ala in position 6 is substituted by Lys, Gly, Tyr or Glu, Arg in position 7 is substituted by Lys, Gly, Ala or Glu, Tyr in position 8 is substituted by Lys, Gly, Ala or Glu, Ala in position 9 is substituted by Lys, Gly, Tyr or Glu, Gin in position 11 is substituted by Lys, Ala or Glu, Ala in position 12 is substituted by Lys, Ala or Glu, Asn in position 16 is substituted by Lys, Gly, Ala or Glu, Gly in position 17 is substituted by Tyr, Ala or Glu, Leu in position 19 is substituted by Lys, Gly, Ala or Glu, Leu in position 20 is substituted by Lys, Gly, Ala or Glu, and Glu in position 21 is substituted by Lys, Gly, Ala or Glu. Preferred homologues are those in which Arg in position 2 is substituted by Ala or Glu, Val in position 4 is substituted by Ala or Glu, Ala in position 6 is substituted by Glu, Arg in position 7 is substituted by Lys or Ala, Ala in position 9 is substituted by Glu, Gin in position 11 is substituted by Ala or Glu, Asn in position 16 is substituted by Lys or Ala, Leu in position 20 is substituted by Lys or Ala, and His in position 21 is substituted by Gly or Ala. The reference to be used in the comparison of this aspect of the invention is e.g. a correspondingly obtained amount in a sample of body fluid or relevant cells from healthy patients or cancer patients with no evidence of disease (NED). The method of the invention is especially useful for detecting carcinoma activity, and to a certain extent also other types of malignant tumours. Any technique which can quantify the amount of polypeptide fragments comprising at least one of the amino acid sequences SEQ ID NO: 1 , 2 and homologues thereof in a sample of body fluid such as blood, plasma, serum, urine, ascites, cerebrospinal fluid, pleura fluid, cyst fluid or suspected cells, can be used in the method of the invention. Monoclonal antibodies which specifically bind to said fragments can be used in different immunoassays, optionally labelled in accordance with the actual assay used. In one embodiment of the method of detecting cancer according to the invention the measurement is performed with the aid of an immunoassay. Also, detection systems adapted for the use of antibody-site carrying structures and/or antibodies may be used. Specific examples of the numerous immunoassays which may be used in the invention are Enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA, IRMA), Fluorescence immunoassay (FIA), Luminiscence immunoassay (LIA), Dissociation enhancement time-resolved fluoroimmunoassay (DELFIA). Another aspect of the invention is directed to a polypeptide selected from the group consisting of a polypeptide having the amino-acid sequence ( SEQ ID NO: 1 ) Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly lie Leu Leu His Leu Glu 20 25 and homologues thereof, and the amino-acid sequence (SEQ ID NO: 2) Gin Lys Thr Thr Thr Arg Arg lie Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 and homologues thereof. A further aspect of the invention is directed to a fusion protein comprising a polypeptide of the invention. A polypeptide or fusion protein according to the invention can be used i.a. as hapten or antigen to raise antibodies against such a polypeptide or fusion protein, respectively, especially monoclonal antibodies. Thus, yet another aspect of the invention is directed to an antibody raised against a polypeptide of the invention or fusion protein of the invention, corresponding synthetic antigen-mimicking structures or synthetic antibody-mimicking structures complementary to the pertinent epitopes. Still another aspect of the invention is directed to a RNA or DNA sequence coding for the amino-acid sequence of a polypeptide according to the invention or a fusion protein according to the invention. Such RNA and DNA sequences are i.a. useful in the production of the polypeptides and fusion proteins of the invention. A different aspect of the invention is directed to a RNA or DNA sequence complementary to a RNA or DNA sequence coding for an amino-acid sequence of a polypeptide according to the invention. These complementary RNA and DNA sequences may be used to locate the RNA or DNA sequences which code for the protein fractions comprising the amino-acid sequence which is to be detected in the method of detecting cancerous activity according to the invention. An additional aspect of the invention is directed to a diagnostic test kit comprising, as a diagnostic antibody, an optionally labelled antibody according to the invention, or as a gene detector, an optionally labelled RNA or DNA sequence according to the invention. Examples of such optional labels are a radioactive isotope, a lanthanide marker, a fluorescent marker, a luminescent marker or an enzyme marker. Optionally all the reagents in the diagnostic test kits of the invention are contained in separate sealed test tubes or vials marked with specific labels, and are accompanied by instructions for use. Synthesis of the polypeptides of the invention The polypeptides of the invention can be produced by any known method of producing an amino-acid sequence, such as, controlled degradation of a purified protein by proteases or other chemical methods (Allen G., Sequencing of proteins and peptides, 1989, Elsevier Science Publishers B.V.). Chemical synthesis is commonly performed by coupling of the amino acid residues or peptide fragments to one another in correct order in liquid phase to produce the desired peptide. Another common strategy is the coupling of the amino acids to one another starting with a solid phase (resin) to which the C-terminal of the first amino acid is coupled, whereupon the C-terminal of the next amino acid is coupled to the N-terminal of the first amino acid, etc, finally releasing the built-up peptide from the solid phase (so called solid-phase technique). The polypeptides of the invention were synthesized by such solid-phase technique (reviewed in Atherton E. and Sheppard R C 1989. Solid Phase Peptide Synthesis a practical approach. IRL Press at Oxford University Press ISBN 019-963066-6), and they were purified by reverse-phase high performance liquid chromatography (HPLC). The amino-acid sequences were checked by ion spray mass spectrometry (Van Dorsselear et al, 1990, Application of electrospray mass spectrometry to characterisation of recombinant proteins up to 44kDa. Biomed. Environ. Mass Spectrom. 19, 692-704). Construction and production of fusion proteins of the invention 800,000 phages of the human prostate cDNA library (λgtll cDNA bank from Clontech) were screened with TPS antibody and 4 positive phages were identified which were subcloned until a homogeneous population was obtained (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The bacterial expression vector pET3c (Studier et al, 1990, Methods of Enzymology 185, 60-89) was used to produce fusion proteins by cloning Bg1 ll/BamHI fragments 10 of CK 18 cDNA and PCR engineered deletions thereof in phase into the BamHI site of pET3a. The fusion proteins consisting of 260 amino acids of the T7 gene 10 protein and different parts of CK 18 were expressed. The syntheses of the fusion proteins were induced by isopropyl-β-D-thiogalactopyranoside (]PTG) and the fusion proteins were purified from sonicated cells by preparative SDS electrophoresis (Hager, D.A. and Burgess, R.R. 1980, Anal.Biochem. 190. 76) Example 1 The availability of two monoclonal antibodies each recognizing a different epitope on the same antigen, namely antibodies binding to the SEQ ID NO: 1 and 2, i.e. MAb1 and MAb2, respectively, permits the application of dual monoclonal immuno- radiometric assay. One of the monoclonal antibodies (MAb1 ) is coated on a solid phase, polystyren beads, with enhanced surface by incubation with 14 μg MAb1/ml in 0.1 M bicarbonate buffer, pH 9.6, for 24 hours at 4°C. The beads are washed twice with PBS (0.014 M Na-K phosphate, pH 7.5, containing 0.15 M NaCI) and remaining sites are blocked with PBS containing 1 % bovine serum albumin and 0.1% Tween 20. This MAb1 antibody will capture antigen when body fluids containing protein fragments comprising the epitope structure are added to the solid- phase bound antibody by binding to one of the epitope structures. The second monoclonal antibody (MAb2) is labelled with radioisotope (125-1) by the chloramine- T method (Greenwood F.C., W.M. Hunter, and j.S. Glover. 1963 The preparation of 131-1 labelled human growth hormone of high specific radioactivity. Biochem. J. 89: 114-123) and purified by size exclusion chromatography on a Superdex column. MAb2 binds to the antigen by reacting with a different epitope structure, well separated from the MAb1 binding region. Serum from blood donors or cancer patients are tested by mixing 0.100 ml of each sample with 0.100 ml of 125-1 labelled MAb2, and adding a bead-coupled antibody (MAb1 ). After incubation during agitation for 2 hours the solid phase is washed with water to wash away unbound antigen and antibody, and the amount of bound radioactivity is determined in a gamma counter. Assay of protein fragments in body fluids In order to measure the amount of protein fragments comprising both of the amino- acid sequences SEQ ID NO:1 and 2 in patient sera, the described dual monoclonal assay was applied. A serum sample from the patient is added to the reaction well, the second antibody (MAb2) labelled with isotope is also added followed by mixing. The first antibody (MAb1 ), the catching antibody bound to solid phase, is added to each well and subsequently the reaction mixture is incubated for a specified time period, washed and measured in a gamma counter. Positive samples from the patient are defined as a marker level exceeding the reference limit of 80 U/L. This cut-off value is based upon a study of 195 blood donors where the upper limit was 80 U/L (95% percentile). Table 1 illustrates the results of Example 1. In the Table the results of blood donors, patients with benign disorders and malignant diseases in various stages of development (primary diagnosis) are presented. It should be noted that elevated levels are seen in certain benign diseases in connection with increased proliferation activity due to healing of inflammatory damage, increased proliferation in connection with beningn hyperplasia (prostate hyperplasia), or simply due to decreased excretion from the liver or the kidneys , which normally remove the antigen (proliferation product) from the serum. 12 Table 1 Total num Positive Blood donors 90 0 Beniqn disorders Prostatic hyperplasia (BPH) 24 2 Renal insufficience 23 15 Liver cirrhosis 21 19 Chronic hepatitis 13 11 Pneumonia 16 4 Chronic bronchitis 17 3 Malignant disease Breast 129 116 Ovary 40 27 Cervix 32 25 Lung 70 49 Prostate 64 22 Bladder 56 22 Kidney 16 11 Gastrointestinal 40 37 Head and neck 22 11 Monitoring of a cancer patient A dual monoclonal assay was repeatedly performed during hormone treatment of a breast cancer patient with Tamoxifen. Table 2 demonstrates the monitoring of said patient with the assay . The clinical diagnosis of the patient prior to treatment was distant bone metastases and malignant pleural effusion. 13 The clinical assessment indicated progressive disease at the initiation of the hormonal treatment, and after 3 months the clinical finding was stable disease. At that time the signal had decreased significantly from an elevated value at the start of the treatment. At month 7, the clinical assessment was progressive disease, and at that occation the marker level was markedly elevated. Already 3 months prior to the clinical finding of recurrence this was indicated by the rise in the signal (lead-time). Table 2 Months Signal (U/LH Clinical assessment 0 475 Progressive disease 0.5 210 0.8 205 3.0 70 Stable disease 4.2 110 5.0 250 7.0 270 Progressive disease ♦ Normal range of marker level < 80 U/L Example 2 Design of an alternative test system Instead of using two monoclonals in the serodiagnostic test, a system was designed where MAb1 on the solid face was substituted with horse antibodies to cancer cells (HeLa). Otherwise the test was performed as described in Example 1. Out of 30 cancer patients with various gastrointestinal malignant diseases, 28 were positive when the same critera as in Example 1 were applied. Example 3 In vivo applications of antibodies directed against the seouence ID NO: 1 or 2 Labelling of antibodies directed against sequence ID NO: 1 or 2 with radioactive isotopes like 131-1, 123-1, 111-ln or 99m-Tc, and injection of these antibodies intravenously to cancer patients, make it possible to detect tumors in the body by scintigraphy without background subtraction. In an initial in vivo study 123-1 MAb1 (300 Mbq/1.5 mg MAb) was able to localize bone and lung metastases in patients suffering from breast, renal or prostatic cancer. Example 4 Production of monoclonal antibody against polypeptides SEQ. ID. NO: 1 and 2 As immunogen for production of monoclonal antibodies any type of antigen, including cancer cells (HeLa), which expose SEQ. ID. NO. 1 and/or 2 can be used provided a suitable selection system is available. Immunization with purified cytoskeleton (Achstatter et al 1986, Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol. 134 355-371), peptide-carrier protein complex (Antibodies: A laboratory manual ed: Harlow and Lane, 1988 - Cold Spring Harbor Laboratory p 77-87) or purified CK 18 fragment from cancer cells in tissue culture can be done according to methods described in (Antibodies: A laboratory manual ed: Harlow and Lane, 1988 - Cold Spring Harbor Laboratory). Balb/c mouse was injected with fusion proteins consisting of 260 amino acids of the T7 gene 10 protein and amino acids 140-430 of human CK 18. After 3-4 injections the mouse serum was tested for antibodies against human cytokeratin 18. When positive response was obtained, spleen cells were fused with a mouse myeloma cell line. Positive hybridomas were subcloned and then single-cell cloned, after which stable clones were established. 15 " " ~ The specificity of the produced antibody was then tested by immunoblotting against 8 fusion proteins with different deletions of the above described fusion protein. Table 3 Fusion proteins and reactivity of MAbs Lab. sample CK 18 aas Antibody Binding MAb1 MAb2 MAb3 MAb4 MAb5 298 140-298 - - + - + 323 140-323 - - + - + 341 140-341 + - + - + 352 140-352 + - + - + 372 140-372 + - + - + 389 140-389 + - + - + 415 140-415 + - + + + 430 140-430 + + + + + Conclusion regarding epitope localization: MAb1 MAb2 (MAb3) (MAb4) (MAb5) Epitope 318-346 406-430 140-298 381-415 140-298 region Example 5 The five monoclonals were tested against known cancer patient sera and blood donors by a two step immunoradiometric assay where the samples were incubated 2 hours with plastic beads coated with horse antibodies to cancer cells (HeLa) and the different MAbs at a concentration of 100 ng/ml. The beads were then washed to remove unbound material, and incubated with 125-1 labelled horse anti-mouse IgG for 1.5 hours. After washing unreacted radioactivity off the beads, the radioactivity bound to the beads was measured using a gamma counter. 16 The results are given in Table 4 Table 4 SAMPLE MAb1 MAb2 (MAb3) (MAb4) (MAb5) Blood donor 2955 4430 744 411 4115 (Big) cpm Cancer 12990 19942 2814 1332 8182 patients (CaPts) cpm CaPts/BIg 4.4 4.5 3.8 3.2 2.0 ratio Only hybridomas which produced antibodies which gave high signal for cancer patients resp low signal for blood donors were selected, i.e. in this example MAb1 and MAb2. Example 6 The antibodies MAb1 and MAb2 which are directed against the sequences ID. NO: 1 and 2, respectively, of the invention were compared with 4 commercially available monoclonal antibodies recognizing cytokeratin 18. These 4 monoclonal antibodies, together with MAb2, did not bind to the sequence ID. NO: 1. According to the experiments demonstrated in Table 5, which were performed in accordance with the assay format described in Exampel 5, the 4 commercially available antibodies are not suitable for the described assay (cf. column "cancer patient" in Table 5). CBL 210 (Cymbus Bioscience Ltd), an anti-cytokeratin 18 monoclonal antibody, is raised in mouse ascitic fluid recognizing adenocarcinomas, undifferentiated carcinomas and carcinomas of cervical and hepatocellular origin. MON 3006 (Sanbio bv-biological products) recognizes cytokeratin 18 (immunoblotting) and do not show crossreactivity with other cytokeratins. Monoclonal anticytokeratin peptide 18, C 1399 (Sigma Chemical Co), is raised against keratin from bovine mammary gland epithelial cell line and reacts specifically with simple epithelia. RGE 53 (EuroDiagnostics) is a monoclonal keratin antibody raised against HeLa cells and recognizing cytokeratin 18 in glandular epithelium. Cytokeratin 18 antigen, used in these experiments, was isolated from the cytoskeleton of human colon adenocarcinoma cells (WiDr) by following a procedure described by Achstaetter et al, Methods in Enzymology 134 (1984) 355-371 with minor modifications. The isolated cytokeratin fraction was further purified by preparative SDS-PAGE. The protein band corresponding to cytokeratin 18 was extracted from the gel using 0.2% SDS in water for 18 h at room temperature. The protein fraction was precipitated by aceton/water and the precipitate was dissolved in 8 M urea, 0.1 M TRIS-HCI, pH 8.0. A cytokeratin 18 fragment (14 kDa), from the cell supernatant of human colon adenocarcinoma cell line WiDr was purified using as probe monoclonal MAb1. The 14 kDa component was purified about 30000-fold in a 7% yield by a 5-step procedure; 1 ) 50% (w/v) ammonium sulfate precipitation of the cell culture medium 2)hydrophobic interaction chromatography on Phenyl Sepharose 3) Sephacryl S-300 gelfiltration chromatography in dissociating medium (8 M urea dissolved in 0.1 M TRIS-HCI, pH 8.0) 4) anion exchange chromatography on Q Sepharose and 5) reverse phase chromatography (acetonitrile gradient). SDS-gradient PAGE of the 14 kDa fraction revealed a pure protein component. The amino acid sequence analysis identified the 14 kDa component as a fragment of human cytokeratin 18 with the N- terminus starting at position 284 of the parent molecule and ended at position 410 of human cytokeratin 18. Table 5 Mab MAb1 MAb2 CBL210 MON3006 C1399 RGE53 Antigen Cytokeratin 18 45 kDa (cpm) 14751 7377 46216 7441 107229 255 Cytokeratin 18 fragment 14 kDa 90170 212 88 121 253 80 (aa 284-410) (cpm) Cancer patient (cpm) 12990 19942 1729 490 848 339 Blood donor (cpm) 2955 4430 309 645 1608 1652 19 SEQUENCE LISTING (1 ) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Beki AB (B) STREET: Fredsforsstigen 22 (C) CITY: Bromma (E) COUNTRY: Sweden (F) POSTAL CODE (ZIP): 161 70 (G) TELEPHONE: 08-290370 (H) TELEFAX: 08-288644 (ii) TITLE OF INVENTION: METHOD OF DETECTING CANCER (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly He Leu Leu His Leu Glu 20 25 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: C-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gin Lys Thr Thr Thr Arg Arg He Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly He 20 21 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu Gin Met Glu Gin 1 5 10 15 Leu Asn Gly He Leu Leu His Leu Glu 20 25 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Tyr (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 4 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 5 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Tyr (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 6 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Tyr or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 8 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 9 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Tyr or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATIONS 1 (D) OTHER INFORMATION: /note = optionally Lys, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION:12 (D) OTHER INFORMATION: /note = optionally Lys, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION:16 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION:17 (D) OTHER INFORMATION: /note = optionally Tyr, Ala or Glu 23 • . - ..-, --. (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATIONS 9 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION:20 (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATIONS (D) OTHER INFORMATION: /note = optionally Lys, Gly, Ala or Glu (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu Gin Met Glu Gin Leu Asn 1 5 10 15 Gly lie Leu Leu His 20

Claims: 27947/BN/AT CLAIMS 1. A method of detecting cancer in a patient, c h a r a c t e r i s e d in that the amount of protein fragments comprising at least one amino-acid sequence selected from the group consisting of the amino-acid sequence ( SEQ ID NO: 1 ) Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly He Leu Leu His Leu Glu 20 25 and homologues thereof, and the amino-acid sequence (SEQ ID NO: 2) Gin Lys Thr Thr Thr Arg Arg He Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 and homologues thereof is measured in a sample of body fluid or suspected cells from said patient, and the amount obtained is compared with a reference, whereby elevated levels indicate cancerous activity in said patient. 25 2. A method of detecting cancer according to claim 1 , wherein said measurement is performed with the aid of an immunoassay. 3. A method of detecting cancer according to claim 2, wherein said immunoassay is selected from Enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA , IRMA), Fluorescence immunoassay (FIA), Luminiscence immunoassay (LIA), and Dissociation enhancement time-resolved fluoroimmunoassay (DELFIA). 4. A polypeptide selected from the group consisting of a polypeptide having the amino-acid sequence ( SEQ ID NO: 1) Ala Ser Leu Glu Asn Ser Leu Arg Glu Val Glu Ala Arg Tyr Ala Leu 1 5 10 15 Gin Met Glu Gin Leu Asn Gly He Leu Leu His Leu Glu 20 25 and homologues thereof, and the amino-acid sequence (SEQ ID NO: 2) Gin Lys Thr Thr Thr Arg Arg He Val Asp Gly Lys Val Val Ser Glu 1 5 10 15 Thr Asn Asp Thr Lys Val Leu Arg His 20 25 and homologues thereof. 5. A fusion protein comprising a polypeptide according to claim 4. 6. A RNA or DNA sequence coding for the amino-acid sequence of a polypeptide according to claim 4 or a fusion protein according to claim 5. 26 7. A RNA or DNA sequence complementary to a RNA or DNA sequence coding for the amino-acid sequence to be detected in the method of claim 1. 8. An antibody raised against a polypeptide according to claim 4 or a fusion protein according to claim 5, corresponding synthetic antigen-mimicking structures or synthetic antibody-mimicking structures complementary to the pertinent epitopes. 9. An antibody according to claim 8, wherein said antibody is a monoclonal antibody or an antigen-binding part thereof. 10. A diagnostic test kit comprising, as a diagnostic antibody, an optionally labelled antibody according to claim 8 or 9, or as a gene detector, an optionally labelled RNA or DNA sequence according to claim 7. 11. A diagnostic test kit according to claim 10, wherein said label is selected from a radioactive isotope, a lanthanide marker, a fluorescent marker, luminescent marker or an enzyme marker. 27

Inventors: BJOERKLUND PETER, RYDLANDER LARS, BJOERKLUND, PETER, RYDLANDER, LARS
Applicants: BEKI AB, BJOERKLUND PETER, RYDLANDER LARS, BJOERKLUND, PETER, RYDLANDER, LARS
Application year: 1995
Main classification: G01N33
IPC codes: A61K39, C07H21, C07K14, C07K16, G01N33