EP0765479A1 - Procede de detection du cancer a base de fragments de cytokeratine 18 et d'anticorps correspondants - Google Patents

Procede de detection du cancer a base de fragments de cytokeratine 18 et d'anticorps correspondants

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Publication number
EP0765479A1
EP0765479A1 EP95920334A EP95920334A EP0765479A1 EP 0765479 A1 EP0765479 A1 EP 0765479A1 EP 95920334 A EP95920334 A EP 95920334A EP 95920334 A EP95920334 A EP 95920334A EP 0765479 A1 EP0765479 A1 EP 0765479A1
Authority
EP
European Patent Office
Prior art keywords
glu
leu
amino
acid sequence
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95920334A
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German (de)
English (en)
Inventor
Peter BJÖRKLUND
Lars Rydlander
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BEKI AB
Original Assignee
BEKI AB
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Filing date
Publication date
Priority claimed from SE9401687A external-priority patent/SE9401687D0/xx
Application filed by BEKI AB filed Critical BEKI AB
Publication of EP0765479A1 publication Critical patent/EP0765479A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • 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.
  • 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 was first isolated from various types of human cancers by Bjorklund B. and Bj ⁇ rklund 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.
  • Tissue polypeptide antigen 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 ⁇ -chain human chorionic gonadotropin as serum markers of epithelial ovarian carcinoma. Cancer 1988; 61 : 76-83).
  • 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.
  • 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 )
  • 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.
  • 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
  • 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
  • Gin Met Glu Gin Leu Asn Gly lie 20 and the amino-acid sequence SEQ ID NO: 4
  • amino-acid sequence SEQ ID NO:5 amino-acid sequence selected from the group consisting of the amino-acid sequence SEQ ID NO:5
  • 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 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
  • 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
  • Leu in position 20 is substituted by Lys or Ala
  • 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.
  • the measurement is performed with the aid of an immunoassay.
  • 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 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.
  • 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.
  • 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.
  • optional labels are a radioactive isotope, a lanthanide marker, a fluorescent marker, a luminescent marker or an enzyme marker.
  • 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).
  • 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).
  • HPLC reverse-phase high performance liquid chromatography
  • 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).
  • phages of the human prostate cDNA library ( ⁇ gtll 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
  • CK 18 cDNA 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- ⁇ -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)
  • MAb1 and MAb2 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 ⁇ g 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
  • 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.
  • Table 1 illustrates the results of Example 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • MAb1 MAb2 MAb3
  • MAb4 MAb5
  • 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.
  • HeLa horse antibodies to cancer cells
  • 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).
  • Cytokeratin 18 45 kDa (cpm) 14751 7377 46216 7441 107229 255

Abstract

Procédé de détection du cancer chez un patient, d'après lequel on mesure dans un spécimen de fluide corporel ou de cellules suspectes la quantité de fragments de protéines comprenant au moins une séquence amino-acide sélectionnée à partir de groupe constitué par la séquence amino-acide (SEQ ID NO: 1) ainsi que ses homologues et par la séquence amino-acide (SEQ ID NO: 2) ainsi que ses homologues. L'invention concerne également lesdits polypeptides, les protéines de fusion, les séquences d'ARN et d'ADN relatives aux protéines de fusion et aux polypeptides, les anticorps contre les polypeptides et les protéines de fusion, les structures correspondantes imitant les antigènes ou les structures synthétiques imitant les anticorps, complémentaires des déterminants antigéniques correspondants, ainsi que des kits de diagnostic.
EP95920334A 1994-05-17 1995-05-15 Procede de detection du cancer a base de fragments de cytokeratine 18 et d'anticorps correspondants Withdrawn EP0765479A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
SE9401687A SE9401687D0 (sv) 1994-05-17 1994-05-17 Method of detecting cancer
SE9401687 1994-05-17
SE9500023A SE9500023L (sv) 1994-05-17 1995-01-04 Sätt att detektera cancer
SE9500023 1995-01-04
PCT/SE1995/000532 WO1995031728A1 (fr) 1994-05-17 1995-05-15 Procede de detection du cancer a base de fragments de cytokeratine 18 et d'anticorps correspondants

Publications (1)

Publication Number Publication Date
EP0765479A1 true EP0765479A1 (fr) 1997-04-02

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EP95920334A Withdrawn EP0765479A1 (fr) 1994-05-17 1995-05-15 Procede de detection du cancer a base de fragments de cytokeratine 18 et d'anticorps correspondants

Country Status (5)

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EP (1) EP0765479A1 (fr)
JP (1) JPH10500408A (fr)
AU (1) AU2582195A (fr)
SE (1) SE9500023L (fr)
WO (1) WO1995031728A1 (fr)

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US20020122791A1 (en) * 2000-12-21 2002-09-05 Nicolette Charles A. Antigenic CK-18 compounds for therapy and diagnosis and methods for using same
WO2003086456A2 (fr) * 2002-04-05 2003-10-23 Arius Research, Inc. Profilage antigenique de cellules neoplasiques, therapie oncogene faisant appel a des anticorps fonctionnels diriges contre lesdites cellules et complexes immuns cytotoxiques ainsi formes
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WO1995031728A1 (fr) 1995-11-23
SE9500023D0 (sv) 1995-01-04
AU2582195A (en) 1995-12-05
SE9500023L (sv) 1995-11-18
JPH10500408A (ja) 1998-01-13

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