EP0672130A1 - Peptide correspondant a l'exon 6 du gene cd44, anticorps specifiques dudit peptide et utilisation de ces anticorps dans le diagnostic des tumeurs - Google Patents

Peptide correspondant a l'exon 6 du gene cd44, anticorps specifiques dudit peptide et utilisation de ces anticorps dans le diagnostic des tumeurs

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Publication number
EP0672130A1
EP0672130A1 EP94900245A EP94900245A EP0672130A1 EP 0672130 A1 EP0672130 A1 EP 0672130A1 EP 94900245 A EP94900245 A EP 94900245A EP 94900245 A EP94900245 A EP 94900245A EP 0672130 A1 EP0672130 A1 EP 0672130A1
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EP
European Patent Office
Prior art keywords
exon
antibody
peptide
mak
antibody according
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.)
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Application number
EP94900245A
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German (de)
English (en)
Inventor
David Tarin
Yasuhiro Matsumura
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication date
Priority claimed from GB929224386A external-priority patent/GB9224386D0/en
Priority claimed from GB929226165A external-priority patent/GB9226165D0/en
Priority claimed from PCT/GB1993/001520 external-priority patent/WO1994002633A1/fr
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of EP0672130A1 publication Critical patent/EP0672130A1/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Withdrawn legal-status Critical Current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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70585—CD44
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70585—CD44

Definitions

  • the present invention is concerned with using expression of the CD44 gene or part of the CD44 gene to investigate neoplasia. Such investigation includes
  • the invention provides a simple method for carrying out routine screening for neoplasia using
  • the human CD44 gene codes for a family of variably glycosylated cell surface proteins of different sizes, the numerous functions of which are not yet fully established, but which share epitopes recognised by the CD44 monoclonal antibody (mAb) . It is known to consist of a standard portion which is expressed in haemopoietic cells and many other cell types and into which the products of additional exons may be spliced in various combinations to produce different proteins. This is a well recognised mechanism in eukaryotes for producing several often functionally unrelated proteins from the same gene, and is known as alternative splicing.
  • mAb monoclonal antibody
  • CD44 isoforms Two common CD44 isoforms have so far been purified and characterised ( ⁇ tamenkovic et al. 1989), namely i) a 90kD form consisting of a central 37kD core which is heavily glycosylated and ii) a 180kD form which has 135 extra amino acids inserted into the proximal extra-membrane domain and is even more heavily glycosylated.
  • Immuno-cytochemical and immuno- precipitation studies have shown that both are widely distributed in many different cells and tissues.
  • the former is known as the haemopoietic or standard form which is present on circulating leukocytes, bone marrow cells and numerous other cell types.
  • the other known as the epithelial variant, is detectable on several epithelial cell types. Both are believed to function as receptors mediating homotypic and heterotypic adhesive interactions, attaching cells to each other or to adjacent extracellular scaffolding.
  • the present invention results from a surprising discovery resulting from studies examining the expression of various parts of the CD44 gene in fresh tissue and body fluid samples from patients with tumours of the breast and colon and from their metastases.
  • the results indicate sharp and clear differences in CD44 expression between tissues from i) metastatic (malignant) tumours, ii) non- metastatic locally invasive tumours and benign tumours and iii) normal tissue.
  • the distinction between groups i) and ii) is important for judgment of therapy and that between groups ii) and iii) is important for early diagnosis and screening.
  • Part of this invention forms the subject of our International patent application PCT/GB93/01520 filed 20 July 1993, which provides in one aspect a method of diagnosis of neoplasia, which method comprises analysing the expression of the CD44 gene in a sample.
  • that application provides a method of assaying a sample for products of the CD44 gene or part thereof which method comprises making cDNA from messenger RNA (mRNA) in the sample, amplifying portions of the complementary DNA (cDNA) corresponding to the CD44 gene or part thereof and detecting the amplified cDNA, characterised in that the amplified cDNA is used in diagnosis of neoplasia.
  • mRNA messenger RNA
  • cDNA complementary DNA
  • diagnosis of neoplasia may refer to the initial detection of neoplastic tissue or it may be the step of distinguishing between metastatic and non- metastatic tumours. References to the term "diagnosis" as used herein are to be understood accordingly.
  • the method is particularly applicable to the diagnosis of solid tumours particularly malignant tumours e.g. carcinomas.
  • the sample on which the assay is performed is preferably of body tissue or body fluid; and not of cells cultured in vitro.
  • the sample may be a small piece of tissue or a fine needle aspirate (FNA) of cells from a solid tumour.
  • FNA fine needle aspirate
  • it may be a sample of blood or urine or another body fluid, a cervical scraping or a non- invasively obtained sample such as sputum, urine or stool.
  • the cDNA may be detected by use of one or more labelled specific oligonucleotide probes, the probes being chosen so as to be capable of annealing to part of the amplified cDNA sequence.
  • labelled oligonucleotide primers and/or labelled mononucleotides could be used.
  • detectable labels which can be employed, including radiolabels.
  • FIGS 1 to 5 are autoradiographs showing the results of various experiments reported below.
  • Figure 6 is a map of the CD44 gene showing exons, probes and primers. The numbering of the exons corresponds to that used by G. R. Screaton et al. 1992) ,
  • Figure 7 is the nucleic acid sequence of Exon 6 (shown in Figure 6), the corresponding amino acid sequence being also shown, and
  • Figure 8 is a set of autoradiographs showing the results of another experiment.
  • Figure 9 is the DNA sequence of HIV2(gp32)- CD44 exon 6 fusiongene.
  • Figure 10 is the protein sequence of
  • HIV2(gp32) -CD44 exon 6 fusionantigen HIV2(gp32) -CD44 exon 6 fusionantigen.
  • Figure 6 is a map of the CD44 gene showing exons 6 to 14.
  • the basic or standard protein can theoretically be modified by the insertion of transcripts from any, some, or all of these 9 extra exons.
  • Exon 6 was unknown at the priority date of this patent application, and constitutes a further aspect of the invention.
  • Exon 6 is over-expressed in tumours but not in normal tissues, and is located in the vicinity of exons 7 to 9. The sequence of exon 6 is given in
  • Figure 7 It contains 129 base pairs and is flanked on the 5 ' -side by the standard CD44 sequence, and on the 3 '-side usually by exon 7.
  • Exon 6 In contrast to Exons 9 to 11, the products of Exon 6 (the newly-sequenced Exon) are only barely detectable in samples of normal tissues. This suggests that Exon 6 will be of particular value in the diagnosis of neoplasia.
  • nucleic acid sequence of Exon 6 as shown in Figure 7 provides as new compounds, the nucleic acid sequence of Exon 6 as shown in Figure 7, characteristic fragments thereof, sequences which are degenerated and/or represent allele variations, the homologous nucleic acid sequences, and probes, primers and other reagents capable of hybridising with the sequences or homologues .
  • probes, primers and other reagents capable of hybridising with the sequences or homologues .
  • the peptide sequence corresponding to CD44 exon 6 as shown in Figure 7, its allele variations and secondary modifications thereof and characteristic fragments thereof can be used the generate antibodies useful for the in vitro and in vivo diagnosis.
  • Said antibodies are specific to the peptide corresponding to CD44 exon 6 as shown in Figure 7, its allele variations and secondary modifications thereof and characteristic fragments thereof i.e. these antibodies bind to this peptide and possess a low cross-reactivity towards other related CD44 proteins and other proteins.
  • Said antibodies may be monoclonal or polyclonal.
  • the antibodies may be generated by using the entire peptide sequence corresponding to CD44 exon 6 as shown in Figure 7 as an antigen or by using short peptides preferebly of a minimum length of six amino acids encoding portions of the peptide sequence corresponding to CD44 exon 6, as antigens.
  • the peptides used as antigens can be produced recombinantely or chemically by methods known in the art.
  • the peptide antigens according to the invention can for example be synthesized according to Merryfield, JACS 85 (1964), 2146.
  • these peptides can be coupled to a carrier molecule for example keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) .
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • a biotinylation is required this can for example be carried out according to PNAS USA 80 (1983), 4045.
  • a fusion gene of CD44 exon 6 or its allele variations with a gene which possess a high expression level in this host.
  • a part of the gene encoding for the protein gp 32 of HIV 2 is suitable for E. coli.
  • a fusion protein which possesses as a part the peptide sequence according to Exon 6 or its allele variations is obtained.
  • polyclonal antibodies directed against the peptide sequence corresponding to CD44 exon 6, its allele variations and secondary modifications thereof and characteristic fragments are prepared by injection of suitable laboratory animal with an effective amount of a peptide or antigenic component, collecting serum from the animal, and isolating specific antibodies by any of the known immuno absorbent techniques. Although the polyclonal antibodies produced by this method are utilizable in any type of immunoassay, they are generally less favoured because of the potential heterogeneity.
  • the use of monoclonal antibodies in the in vitro diagnostic test is particularly preferred because large quantities of antibodies all of similar specificity may be produced.
  • the preparation of hybridoma cell lines for monoclonal antibody production is done by fusion of an immortal cell line and the antibody producing lymphocytes. This can be done by techniques which are well known in the art (see for example Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbour Press 1988, Bessler et al. Immunobiol. 170 (1985), 239 - 244, Jung et al., Angew. Chemie 97 (1985), 883 or Cianfriglia et al., Hybridoma Vol. 2, (1983), 451 - 457).
  • a synthetic peptide corresponding to amino acids 9 - 23 for MAK ⁇ CD44>M- 2.42.3 a synthetic peptide corresponding to amino acids 29 - 43 and for MAK ⁇ CD44>M-4.3.16 a synthetic peptide corresponding to amino acids 1 - 13 of the CD44 exon 6 peptide having the amino acid sequence shown in Figure 7 was used.
  • the antibody produced by the cell line MAK ⁇ CD44>M-1.1.12 shows a specificity to tumor tissue of lung, colon and bladder and for cells of the cell line ZR75-1 (human breast carcinoma - ATCC CRL 1500) as detected by immunohistochemistry. A specific reaction means that a strong reaction is observed with the tumor tissue whereas normal tissue shows only a weak reaction.
  • the antibody produced by the cell line MAK ⁇ CD44>M-4.3.16 shows specificity towards tumor tissue of colon and ZR75-1 cells.
  • the presence of the CD 44 protein or the peptide sequence according to CD44 exon 6 in a sample can be detected utilizing antibodies prepared as discribed above either monoclonal or polyclonal in virtually any type of immunoassay.
  • a wide range of immunoassay techniques are available as can be seen by reference to Harlow, et al. (Antibodies: A Laboratory Manual, Cold Spring Harbour Press 1988). This of course includes both single-site and two-site, or "sandwich" of the non-competitive types, as well as competitive binding assays.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention.
  • radio immunoassays examples include radio immunoassays, enzyme immunoassays or immunofluorescent assays such as FPIA or electrochemilumineszent assays, immunoassays using direct labels such as dye particles (e.g. gold sol particles), homogeneous immunoassays such as CEDIA or EMIT or turbidimetric and nephelometric methods such as latex particle agglutination assays.
  • dye particles e.g. gold sol particles
  • homogeneous immunoassays such as CEDIA or EMIT or turbidimetric and nephelometric methods such as latex particle agglutination assays.
  • turbidimetric and nephelometric methods such as latex particle agglutination assays.
  • antibodies could for example be prepared by using two different synthetic peptides as immunogens corresponding to different characteristic fragments of the peptide corresponding to CD44 exon 6. It is also possible to use only one antibody according to the invention in a sandwich assay. The other antibody could be an antibody to the other peptides corresponding to other CD44 exons or to the standard form of CD44. Such antibodies are known in the art.
  • the CD44 protein could be detected in its native form.
  • the CD44 protein is denatured prior to or during its detection because some of the antibodies according to the invention preferably bind to epitopes which are linear or which are hidden within the CD44 molecule in its native form.
  • a denaturation method any method known in the art such as treatment with detergent or chaotropic agents is suitable.
  • the adsorption of the CD44 molecule to a solid phase leads to a partial denaturation which is sufficient for the binding of the antibody.
  • CD44 proteins are expressed on most 5 cell types it was found that with the use of the antibodies according to the invention a differentiation between tumor tissue and normal tissue is possible in most cases.
  • the antibodies therefore could be used in cancer diagnosis.
  • the antibodies could be used in cancer diagnosis.
  • the antibodies could be used in cancer diagnosis.
  • the antibodies could be used in cancer diagnosis.
  • 25 invention can also be used as a standard compound in immunological tests for the quantitative determination of CD 44.
  • the invention therefore in addition concerns the use of the peptide antigens according to the invention as a standard in an immunological test for
  • CD 44 the determination of CD 44.
  • the peptides according to the invention can be advantageous to bind several peptides according to the invention with the same or different sequences to a carrier J molecule.
  • 35 also be used as a binding partner for the antibody according to the invention in a competitive immunoassay.
  • the peptides are labeled or bound to a solid phase directly or indirectly via two specific binding partners such as (strept)avidin/biotin by methods known in the art.
  • Another aspect of the invention is a test kit containing at least one antibody which is directed against the peptide corresponding to CD44 exon 6 having the amino acid sequence as shown in Figure 7, its allele variations or secondary modifications thereof or characteristic fragments thereof among the other compounds which are necessary for the immunoassay such as buffers, detergents, stabilizers, solid phases etc. If required the peptide antigens according to the invention as a standard could also be included.
  • this invention provides a means for therapy and in vivo imaging of tumours.
  • Agents useful for this can be manufactured according to the state of the art.
  • the data obtained from studies examining the expression of various parts of the CD44 gene m samples from patients with malignant diseases surprisingly show a significant overexpression of exon 6 of the variable part of CD44.
  • the relative abundance of CD44 splice variants containing exon 6 in malignant tumours as compared to normal tissue and the increased amount of CD44 proteins containing the peptide sequence encoded by exon 6 on the surface of tumour cells as compared to normal tissue opens the possibility to use the exon 6 encoded peptide sequence as a tumour specific antigen for therapy, diagnosis both in vivo and in vitro, and in vivo imaging.
  • monoclonal antibodies (Kohler and Milstem (1975), Nature 156, 495-497) or their derivatives will be used for diagnostic and therapeutic purposes.
  • monoclonal antibodies to epitopes encoded by exon 6 of CD44 are provided.
  • data are presented, showing selective binding of these antibodies to tumour cells.
  • the antibodies according to the invention recognize the peptide corresponding to CD44 exon 6 having the ammo acid sequence shown in Fig. 7, its allel variations and phosphorylation and glycosylation products and characteristic fragments thereof.
  • Such antibodies are specific to the peptide corresponding to CD44 exon 6 also m the presence of other peptides which correspond to other CD44 exons.
  • the antibody according to the invention binds only to a little extent to proteins other than the protein encoded by exon 6. This unspecific binding must be so little as to ensure t ⁇ t no considerable damage will be caused to healthy cells when the antibodies according to the invention are used for tumour therapy or in vivo diagnosis.
  • the antibodies can be used as whole antibodies, fragments thereof (e.g. Fv, (Fv) 2 , Fab, Fab ' , F(ab) 2 , chimeric, humanized or human antibodies as long as they are binding the exon 6 protein in a suitable manner. Short-chain antibody fragments containing only the CDR regions or parts thereof conferring the specific binding to the exon 6 peptide are also suitable, especially if the antibody is a labelled one.
  • the antibodies can be used as a whole for therapy of malignant diseases (Hale et al. , Lancet 2 (1988) 1394-1399; Cobbold et al. , Prog. Clin. Biol. Res. (1990) 333,139-151).
  • the antibody or part of it is conjugated or translationally fused to a toxin molecule (immunotoxin) , thus effecting specific killing of tumour cells (Brinkmann et al.
  • bispecif c antibodies are used for tumour therapy (Bonmo et al. (1992), BFE 9, 719-723), which may be constructed by in vitro reassociation of polypeptide chains, by hybrid hybridoma generation or by construction of diabodies (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90, 6444-6448; Holliger and Winter (1993), Current Opm. Biotechnol. 4, 446-449).
  • tumours including carcinomas of the respiratory, gastrointestinal and urogemtal system as well as ocular and skin cancers (Profio M988), Proc. Soc. Photoopt. Instr. Eng. 907, 150-156; Jiang et al. (1991), J. Natl. Cancer Inst. 83, 1218-1225).
  • antibodies which resemble as closely as possible antibodies of human origin (Glassy and Dillman (1988), Mol. Biother. 1, 7-13). Such antibodies are, for example, chimeric or humanized (CDR-grafted) antibodies. Such antibodies usually are manufactured from a rodent monoclonal antibody (see e.g. for review: Morrison (1992), Annu. Rev. Immunol. 10, 239-265; Winter and Milstein (1991), Nature 349, 293-299) . In a specifically preferred embodiment of the invention, tumour specific human antibodies (Borrebaeck et al. (1988), Proc. Natl. Acad. Sci. USA 85, 3995-3999; Borrebaeck (1988), Immunol.
  • Today 9, 355-359) are used for therapeutic purposes.
  • ADCC effector functions
  • CDC Bruggemann et al. , J. Exp. Med. 166 (1987) 1357-1361.
  • a human lsotype IgG 1 antibody is used.
  • the antibody according to the invention it is preferred to couple the antibody according to the invention to a toxin, such as, for example, Pseudomonas exotoxm, Diphtheria toxin or other toxins (FitzGerald and Pastan (1989)). It is also preferred to couple the antibodies to chemotherapeutics, such as, for instance doxorubicin, or to radioactively labelled substances which have a cytotoxic effect.
  • a toxin such as, for example, Pseudomonas exotoxm, Diphtheria toxin or other toxins (FitzGerald and Pastan (1989)
  • chemotherapeutics such as, for instance doxorubicin
  • radioactively labelled substances which have a cytotoxic effect.
  • fluorescent substances are also preferred.
  • the therapeutic compounds of this invention may be administered parenterally, such as mtravascularly, intraperitoneally, subcutaneously, mtramuscuiarily, using forms known in the pharmaceutical art.
  • the active drug components of the present invention are used m liquid, powdered or lyophilized form and may be combined with a suitable diluent or carrier, such as water, a saline, aqueous dextrose, aqueous buffer, and the like. Preservatives may also be added.
  • the compounds of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
  • the compounds may also be formulated using pharmacologically acceptable acid or base addition salts.
  • the compounds or their salt may be used in a suitable hydrated form.
  • a non-toxic but therapeutically effective quantity of one or more compounds of this invention is employed in any treatment.
  • the dosage regimen for treating is selected in accordance with a variety of factors including the type, age, weight, sex and medical condition of the patient, type of tumour, the route of administration and the particular compound employed in the treatment.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the drug required regarding known antibody therapy approaches (Hale (1988), Cobbold (1990)). In so proceeding, the physician could employ relatively low doses at first, and subsequently, increased dose until a maximum response is obtained.
  • the chaotic over-expression of multiple spliced variants of the CD44 gene in tumours implies that a particular exon may or may not be over-expressed (or expressed at all) by a particular tissue sample.
  • An immunoassay using an antibody to the peptide expressed by any single exon may therefore give misleading results.
  • This invention therefore includes use, for the immunological diagnosis of neoplasia, of a mixture of antibodies to two or more, and preferably to all nine, of the CD44 exons.
  • transcripts from exon 10/11 m normal tissues indicate that this exon is not exclusively concerned with metastatic activity, even though there is marked increase in the number and signal intensity of bands hybridising with radiolabelled probe E4 in the PCR products from tumours capable of metastasis. Other supporting events are therefore believed to be required for CD44 exon 10/11 expression to result in metastatic behaviour. Nevertheless, the observation that transcripts from this exon were over-expressed in samples from metastatic tumours promises to be a very useful indicator of prognosis.
  • RNA extraction from tissue samples was performed according to the method described by Chomizynski and Sacchi (1987). Extraction from fluid samples was by use of the Microfasttrack kit marketed by Invitrogen. cDNA synthesis and subsequent amplification by the polymerase chain reaction (PCR) was performed using the SuperscriptTM preamplification system (BRL Life Technologies Inc., Middlesex, UK) with buffers and reagents supplied in this kit. In brief, this involves an initial step of first strand cDNA synthesis with reverse transcriptase, using sample RNA as the template and supplied nucleotide triphosphates.
  • PCR polymerase chain reaction
  • P4 5 ' GATGCCAAGATGATCAGCCATTCTGGAAT P1 is located with its origin 324bp upstream from the insertion site in the standard CD44 molecule (between nucleotides 782 and 783 in the sequence published by Stamenkovic et al . 1989) and P4 is 158bp downstream of this site.
  • These primers produce a PCR fragment of 482bp if a sample expresses standard CD44 (so-called haemopoietic CD44), 878bp for the epithelial form of CD44 and several other bands, if a sample contains alternatively spliced transcripts.
  • PBL peripheral blood leukocytes
  • ammonium chloride buffer (1ml packed cells to 50mls lysis buffer) and subsequent centrifugation 15 minutes later.
  • the white cell pellet was divided into 4 tubes which were seeded respectively with O ⁇ l, 1 ⁇ l, 1O ⁇ l and 100 ⁇ l of a suspension of HT29 colon carcinoma cells (5000 cells per ml) .
  • Total RNA was then extracted and each tube yielded approximately 20 ⁇ g.
  • cDNA synthesis was performed, as described above on 4 ⁇ g aliquots of the RNA obtained from each tube representing 0, 1, 10 and 100 tumour cells per aliquotted sample respectively.
  • the PCR was performed on these samples and on positive (tumour cells only) and negative (no DNA) controls using primers D1 and D5 which were designed by us to anneal specifically to exons 7 and 14 in Figure 6.
  • primers D1 and D5 were designed by us to anneal specifically to exons 7 and 14 in Figure 6.
  • primers P1 and P4 for this specific purpose because the majority of these would be soaked up by annealing to the standard portion of the gene.
  • PCR cycle parameters, blotting, probing and washing conditions were as described above.
  • the oligonucleotide sequence used for probing was ⁇ 2 P labelled E4.
  • primers (P1 and P4) amplify across the - 2 1 -
  • Primer P1 5 ' -GACACATATTGCTTCAATGCTTCAGC (458-484)
  • Primer P2 5 '-CCTGAAGAAGATTGTACATCAGTCACAGAC (488-518)
  • Primer P3 5 ' -TGGATCACCGACAGCACAGAC (746-767)
  • Primer P4 5 ' -GATGCCAAGATGATCAGCCATTCTGGAAT (912-941) for standard part (Stamenkovic 1989)
  • Primer E1 5 ' -TTGATGAGCACTAGTGCTACAGCA
  • Primer E2 5 ' -CATTTGTGTTGTTGTGTGAAGATG Primer E3-5 ' -AGCCCAGAGGACAGTTCCTGG (534-554)
  • Primer E4 5 '-TGAGATTGGGTTGAAGAAATC (558-578)
  • Primer E5 5 ' -TCCTGCTTGATGACCTCGTCCCAT (585-608)
  • E1 and E2 are on exon 6.
  • Fresh tissue samples 0.5-1 cm in diameter were obtained from surgical resection specimens or at autopsy. All samples used in this work were obtained from the residue of tissue remaining after diagnostic samples had been taken, and which would otherwise have been discarded. The samples were snap- - 25 -
  • cDNA was synthesised with viral reverse transcriptase using 5 ⁇ g of total cellular RNA as template, followed by PCR with Primer P1 and Primer P4. PCR amplification, electrophoresis and hybridisation were performed under standard conditions.
  • the oligonucleotide probe for Exon 6 products is very effective in distinguishing neoplastic from non- neoplastic samples, but not significantly more sensitive than E4, at least on samples from solid tissues, but is possibly useful for detecting organ of origin of a disseminating metastatic cell or an established metastasis.
  • the same filters were stripped and hybridised with P2 probe to show that all samples, including normal tissues, produced the standard portion of CD44. This confirmed that the differences observed between the results obtained with normal and tumour samples, probed with E2 and E4, were not due to unequal loading of PCR products.
  • the cumulative results are summarised in Table 3 which indicates that these changes are seen in a wide range of common cancers.
  • cytological examination Another 1 ml of urine was checked by Fluorescein diacetate-ethidium bromide staining to assess the viability of cells in the sample. The remainder of the urine was centrifuged at 2000rpm for 10 minutes and the cell pellet was kept at -70'C until use. mRNA extraction was performed with oligo dT cellulose tablets (invitrogen) . cDNA was synthesised with AMV reverse transcriptase (Invitrogen) .
  • the completed cDNA solution was divided equally into two tubes, one being for PCR with E1 and E5, to amplify the particular cDNA transcript, which we have found to be of diagnostic value and the other for PCR with P1 and P4 to amplify the standard form of CD44, with or without all splice variants, as an internal control.
  • Equal volumes of PCR products were loaded in each lane of a 1.2% agarose gel and stained with ethidium bromide. If the cells in the urine were to be expressing all the Exons from Exon 6 to Exon 14, it was predicted that with the current PCR protocol, using primers E1 and E4, should produce a 735 bp band. There is no band in tracks containing cDNA from normal urine or that of patients with non-neoplastic cystitis (lanes 1-8) but a clear 735 band is seen in all urine samples from patients with bladder cancer (lanes 9-16) when PCR was performed with primer E1 and E5 (upper panel) .
  • Lanes 1-4 normal urine. Lanes 5-8: cystitis urine. Lanes 9-16: from patients 1-8 with bladder cancer.
  • the inventors have explored the procedures needed to ensure the routine RT-PCR detection of abnormal CD44 gene activity in small clinical samples containing tumour cells. If a tissue sample is divided into aliquots, half of which are frozen in liquid nitrogen immediately and the remainder of which are left at ambient temperature, one can show how the ability to detect CD44 splice variants declines with time and with mode of specimen handling. Fresh samples submitted to mRNA extraction within half an hour of excision give the most reliable results and there is a gradual decline in quality over the next few hours if the fresh tissue is left at ambient temperature. If the sample is first snap frozen, the results obtained when RNA is extracted immediately after thawing are satisfactory, but decline very rapidly, beginning within 15 minutes, the larger variant transcripts being lost first and ultimately even the standard form.
  • the diagnosis method described herein can be performed in a single day, possibly in a few hours, and is capable of being automated. Use of the method has been demonstrated, on various tissue samples to detect a whole variety of cancers, and also on blood and urine samples. We therefore offer it as a convenient practical method for cancer screening and diagnosis. In principle it could also have wide general applicability to cancer detection and prevention programmes and therefore have epidemiologic and public health value. Proper application of its sensitivity, specificity and simplicity should add not only to initial cancer diagnosis but to evaluation of extent of disease in the body, to judgment of the efficacy of treatment and to early detection of tumour recurrences.
  • FIGURE LEGENDS
  • N normal
  • T primary tumour
  • M metastasis
  • Figure 1 Autoradiogram of PCR products from breast tissue samples probed with E4 (10 hours exposure of X- ray film to sample filter) .
  • Panel A malignant primary breast carcinomas with their metastases. Tracks 1, 2 and 3: patient B1 ; tracks 4, 5 and 6: patient B2; tracks 7, 8 and 9: patient B3; tracks 10 and 11: patient B4; tracks 12 and 13: patient B5. It can be seen that compared to normal breast tissue, primary breast carcinomas and their metastatic deposits overexpress several splice-variants. Note the doublet (arrows) at 1500bp and 1650bp best seen in track 5. This is present in all tumours and metastases but is - 3 1 -
  • Panel B Breast carcinomas with no clinical evidence of metastasis. Tracks 14-20 are from patients B15-B21. The tumours all overexpress several variants, but show less bands and the signal intensity is less, except track 16 (patient B17) - see text. The 1500/1650bp doublet (arrow) is easily recognisable in tracks 15, 16 and 18 at this length of exposure and became detectable in all other tumour-containing tracks on longer exposure. The illustration, however, shows only the shorter exposure, to avoid fogging the tracks which have stronger signals.
  • Panel C Fibroadenomas (FA) and fibrocystic disease of the breast (Cyst). Tracks 21 and 22, containing the benign tumour samples (samples B22 and 23), express more than the non-neoplastic sample (fibrocystic disease) in track 23 (sample B24).
  • tracks 4 and 5 were from individuals with no clinical evidence of neoplasia, as follows: track 4, breast tissue obtained at autopsy from the body of a woman who died of bacterial endocarditis; track 5, colon resected for volvulus.
  • lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell 1989; 56: 1057-062.
  • CD44 The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 1991; 10: 343-348.
  • N-Fmoc-L-norleucine N-Fmoc-L-phenylalanme
  • N-Fmoc-L-proline N-Fmoc-O-t-butyl-L-serine
  • the ammo acid derivatives Prior to coupling, the ammo acid derivatives are dissolved in DMF and activated through the addition of 1 equivalent N-hydroxybenzotriazole (HOBt) in N- methylpyrrolid one (NMP) and 1 equivalent N,N ' - dicyclocarbodnmide (DCC) m NMP.
  • HOBt N-hydroxybenzotriazole
  • NMP N- methylpyrrolid one
  • DCC N,N ' - dicyclocarbodnmide
  • the 20-mmute couplings of the HOBt-ester amino acid are carried out in DMF.
  • deprotection of the N- termini is achieved by a 3-m ⁇ nute and then a 10-m ⁇ nute treatment with 20% piperidme in DMF.
  • the peptide chain is extended through repetition of the activation/couplmg/deprotection cycles.
  • Peptides utilized later for immunogen synthesis are outfitted with an N-terminal am ocaproic acid spacer and cystein, through which the peptide is tethered to the carrier protein.
  • N-terminal am ocaproic acid spacer and cystein through which the peptide is tethered to the carrier protein.
  • a different N-terminus is synthesized and contains three gamma-aminobutyric acid moieties, lys e, and biotm (attached to the G-am o group of lys e) .
  • TFA trifluoroacetic acid
  • the peptide-beanng resin is reacted for 1 hour at room temperature (RT) with a cleavage cocktail containing 20 mL trifluoroacetic acid, 1 mL H2O, 1 mL thioanisole, 0.5 mL ethanedithiol and 1.5 g phenol. Removal of the acid-labile side-chain protecting groups, performed under Argon, is complete after an additional 2.4 h reaction time at RT in the aforementioned cocktail solution. After a brief cooling period, the deprotected peptide is precipitated through the addition of diisopropylether. The precipitate is filtered, washed with diisopropylether, dissolved in 50s acetic acid, frozen and lyophilized.
  • a cleavage cocktail containing 20 mL trifluoroacetic acid, 1 mL H2O, 1 mL thioanisole, 0.5 mL ethanedithiol and 1.5 g phenol. Removal of the acid-
  • Peptide purity is determined by reverse-phase HPLC (column - Vydac 218TP54, C 18 , 300 A, 5 ⁇ m, 4.6 x 250 mm; mobile phase - A: 0.1% TFA in H 2 0, B: 0.1% TFA in H 2 0/acetonitrile (35/65, v/v); gradient - 0-100% B in 90 min; flow rate - 1 mL/min; detection - 226 nm) .
  • Those peptides being less than 60% pure are purified by reverse-phase HPLC (column - Waters DeltaPak C-g, 100 A, 15 ⁇ m, 50x300 mm; mobile phase - A: 0.1% TFA in H 2 0, B: 0.1% TFA in H 2 0/acetonitrile 35/65, v/v, gradient - 0.50% B in 130 min; flow rate - 15 mL/min; detection - 226 nm) .
  • Peptide identity is verified by plasma desorption mass spectrometry. Characteristic HPLC retention times and mass spectral data for the peptides used for immunogen synthesis are listed in Table 2.
  • a carrier protein either Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA)
  • KLH Keyhole Limpet Hemocyanin
  • BSA Bovine Serum Albumin
  • MMS N-succinimidyl 3-maleimidopropionate
  • thiol- containmg peptides are conjugated with the MP- activated carrier protein.
  • BSA-MP a 74 ⁇ M BSA-Mp solution in 0.1 M KH 2 P0 4 /K 2 HP0 4 pH 7.0 is reacted with 1 equivalent (with respect to MP) of a 4 mM peptid solution in the same phosphate buffer. The solution is stirred slowly and allowed to react at RT overnight. After centrifugation, the soluble BSA-MP- peptide conjugate is separated from unbound peptide via size-exclusion chromatography (same chromatography conditions as given in section II).
  • Analyses of the protein conjugate include protein concentration determination via BCA, as well as ascertaining the remaining number of unreacted MP-groups with Ellman ' s reagent.
  • KLH-MP-peptide conjugates are synthesized similarly with the exception of activated carrier protein and peptide concentrations, which are 3 ⁇ M and 1 8 ⁇ M respectively.
  • EXAMPLE 8 Manufacture of the recombinant HIV2 (gp32)-CD44 exon 6 antigen/imraunogen
  • Exon 6 of the CD44 gene codes for a peptide of 43 amino acids as shown in Figure 7.
  • the CD44 exon 6 antigen was duplicated at the DNA level using a suitable linker (codes for the 3 C-terminal amino acids of the exon 5 of CD44) .
  • the molecular biological reagents were used according to the manufacturer's instructions.
  • HIV2 (gp32)-partial gene Plasmid pUC18_HIV2-gp32
  • the coding section of the amino acid 48-162 of the HIV2-gp32 gene was synthesized by overlapping chemical gene synthesis and subsequently subcloned in 4 4
  • a fusion gene was constructed which codes for the N- terminal of the amino acid sequence MRGSHHHHHHTDPEF (poly-His tail) and the selected HIV2-g[32 antigen.
  • the vector pQE-10 was digested with restriction endonucleases BamHI and Hindlll, and the approx. 3.4-kbp-long BamHI/HindIII-pQE-10 vector, fragment isolated by agarose gel electrophoresis.
  • the pQE-10 vector [synonym: pDS56/RBSII, 6xHis(-1)] is sourced from Diagen, Germany, and is described in Stuber, D. et al. (1990) Immunol.
  • Methods IV 121-152.
  • the plasmid pUC18_HIV2-gp32 was digested by the restriction endonucleases BamHI and Hindlll, and the ca. 400-bp-long BamHI/HindIII-HIV2-gp32 fragment isolated and ligated into the approx. 3.4-bp-long BamHI/HindIII-pQE-10 vector fragment.
  • the desired plasmid was identified by restriction mapping and designated pDS56-6HIS-HIV2-gp32.
  • a fusion gene was constructed which codes N-terminal for the amino acid sequence MRGSHHHHHHTDPEF (poly-His tail), the selected HIV2-gp 32 antigen, and 2 copies of the CD44 exon 6 antigen.
  • CD44 exon 5-6 DNA sequence from base pair position 397-538 was amplified (see Fig. 9: DNA sequence of HIV2 (gp32)-CD44 exon 6 fusion gene) and provided with suitable singular restriction endonuclease cleavage sites (BamHI and Haelll).
  • CD44 cDNA Exon 5-11
  • primer pair were used for amplification:
  • the approx. 160-bp-long PCR product was digested with BamHI and Haelll and the approx. 150-bp- long BamHI/HaeIII-CD44 exon 6 fragment isolated by agarose gel electrophoresis.
  • the CD44 exon 5-6 DNA sequence of base pair position 539-672 (see Fig. 9: DNA sequence of HIV2 (gp32) -CD44 exon 6 fusion gene) was amplified and provided with suitable singular restriction endonuclease cleavage sites (Haelll and Hindlll) using subcloned CD44 cDNA exon 5-11 as template DNA and the following primer pair:
  • Haelll Primer (3) 5'-aaaaaaCCGGCCACCACTttgatgagcactagtgctac-3 '
  • Hindlll Primer ( 4 ) 5 ' -aaaaaAA££IITTATCAagccatttgtgttgttgtgtgtg-3 - 46 -
  • the approx. 150-bp-long PCR product was digested by Haelll and Hindlll and the approx. 140-bp- long BamHI/HaeIII-CD44 (Exon 6) fragment isolated by agarose gel electrophoresis. Then the BamHI/HaeII-CD44 exon 6 fragment from the first PCR reaction and the HaeIII/HindIII-CD44 exon 6 from the 2nd PCR reaction were ligated by 3- fragment ligation into an approx. 3.8-bp-long BgIII/HindIII-pDS56-6HIS-HIV2-gp32 vector fragment. The desired plasmid was identified by restriction mapping and the PCR-synthesised DNA regions checked by DNA sequencing (construction: pDS56-HIV2-CD44 exon 6).
  • Gen. Genet. 150: 53-61) was transformed with the HIV2 (gp32) -CD44 exon 6 expression plasmid pDS56-HIV2-
  • CD44 exon 6 resistance ampicillin
  • IacI repressor plasmid pUHA1 resistance kanamycin
  • RM82/pUHA1/pDS56-HIV2-CD44 exon 6 cells were cultured in DYT medium (1% (w/v) yeast extract, 1% ( w / v ) Bacto Tryptone, Difco, and 0.5% NaCl) with 50 mg/1 ampicillin and 50 mg/1 kanamycin up to an optical density of 0.6-0.9 at 550 nm, and then induced with IPTG (1-5 mmol/1 end concentration). After an induction phase of 4-8 h, the cells were harvested by centrifugation, washed with 10 mmol/1 phosphate buffer, pH 6.8, and stored at -20'C until further processing. - 4 7 -
  • the cell pellet from 1 ml of culture medium (RM82/pUHA2/pDS56-HIV2-CD44 exon 6 cells) was re ⁇ suspended in 0.25 ml 10 mmol/1 phosphate buffer, pH 6.8, and 1 mmol/1 EDTA and the cells mechanically lysed by means of a French press. After centrifugation, 1/5 volumes of 5xSDS sample buffer: 50 mmol/1 Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue) was added to the supernatant. The insoluble cell debris fraction was resuspended in 0.3 ml 1xSDS sample buffer with 6-8 M urea.
  • the expression level for the HIV2 (gp32) -CD44 exon 6 antigen was 30-50% in relation to the E.coli total protein.
  • the insoluble components were then centrifuged down on a Sorvall centrifuge.
  • the pellet was resuspended in 100 ml 0.1 mol/1 phosphate buffer, pH 8.5, incubated for 30 min at 25"C, and the IB product isolated by centrifugation.
  • the 2.5 g IB pellet (wet weight) was suspended in 25 ml 6 mol/1 guanidine-HCl, 0.1 mol/1 phosphate buffer, pH 8.5, by stirring for 2 h at 25 C.
  • the insoluble components were separated off by centrifugation and the clear supernatant applied to an NTA column equilibrated with 6 mol/1 guanidine-HCl, 0.1 mol/1 phosphate buffer, pH 8.5 (column volume: 50 ml, NTA gel from the Diagen Company, Germany; Hochuli, E. et al. (1988). Bio/Technology 6: 1321-1325).
  • the column was then washed with about 5 column volumes of 8 mol/1 urea, 10 mmol/1 Tris-HCl, and 0- 1 mmol/1 phosphate buffer, pH 8.5. Subsequently, the HIV2 (gp32) -CD44 exon 6 antigen was eluted with 8 mol/1 urea and 0.1 mol/1 phosphate buffer, pH 4.0, and the HIV2(gp32)-CD44 exon 6 antigen-containing fractions pooled.
  • HIV2 (gp32) carrier antigen was produced by using the plasmid pDS56-6HIS-HIV2-gp32.
  • Biotinylation was performed with biotinoyl-*- aminocapronic acid-N-hydroxy- succinimide (Bi-X-NHS) (Boehringer Mannheim, Germany Cat. No. 1003933) in the molar ratios 1:3, 1:6, and 1:10.
  • HIV2 (gp32)-CD44 exon 6 present in a concentration of 7.1 mg/ml in 8 M urea, 0.1 M sodium phosphate buffer, 10 mM Tris, pH ca. 6, was diluted with dialysis buffer (0.1 M sodium phosphate buffer, 0.5% SDS, 10 mM DTT, pH 8.5) to 2 mg/ml and dialyzed against the aforementioned dialysis buffer at room temperature and further diluted to 1 mg/ml.
  • the appropriate quantity of Bi-X-NHS was added to the fusion protein, incubated for 2 h at room temperature, and than the reaction was stopped by addition of 1 M lysine/HCI, pH 6.5, to 2 mM. After dialysis against 0.1 M sodium phosphate buffer, 0.5% SDS, pH 6.0, the reagent HIV2 (gp32)-CD44 exon 6-Bi was stored at -20"C.
  • the peptide HIV2. (gp32) was biotinylated analogous.
  • mice Immunization of mice was performed according to Cianfriglia et al . (1983). Hybridoma, Vol. 2, No. 4: 451-457.
  • immunogen synthetic peptides corresponding to am o acid 1 - 13, 9 - 23, 19 - 33, 29 - 43 and 1 - 43 coupled to a carrier protein (see example 7) as well as the HIV2 (gp32)-CD44 exon 6 antigen were used.
  • mice were terminated by cervical dislocation, and their spleens removed under sterile conditions.
  • the spleen cells were teased out of the connective tissue in RPMI 1640 basic medium.
  • the cell suspension was then passed through a sieve and centrifuged at 200 g
  • Hybridoma cells were multiplied over a period of 7 days at an inoculation density of 1 x 10 ⁇ cells/ml in RPMI 1640 +10% FCS on a'Techne biological stirrer (THERMO- DUX, Wertheim/Main, Model MCS-104XL, Cat. No. 144-050). Mean concentrations of 100 ⁇ g MAB/ml were achieved in the culture supernatant. Purification was performed using standard protein chemistry methods.
  • HIV2(gp32) protein were obtained. These cultures were further assessed by dot-blot and imraunohistology.
  • Method A Cells from tumor cell lines (e.g. ZR-75 1 or MDA 4A4) were detached from the flask by scraping and the cell suspensions were dropped onto glass slides, dried and fixed with methanol.
  • tumor cell lines e.g. ZR-75 1 or MDA 4A4
  • Method B Freeze-dried sections of tumor and normal tissue were fixed with acetone.
  • the substrate was added and incubated with the sample (15-20 min). After washing with tap water the nuclei were stained with hematoxylin for 30 sec. The samples were dried and embeded with Cristal Mount (Kaiser ' s jelly).
  • Cristal Mount Karl ' s jelly
  • the results obtained with monoclonal antibody from cell lines MAB ⁇ CD44>M-1.1.12 and 4.3.16 are presented in Table 4.
  • Method B the MAB 1.1.12 shows high specificity for tumor tissue from the lung, colon and bladder and MAB 4.3.16 revealed specificity for tumor tissue from the colon.
  • MAB 1.1.12 and MAB 4.3.16 showed increased reactivity to - 55 -
  • the cell line ZR-75-1 (exon 6 high-producer), a human breast cancer cell line (ATCC CRL 1500) than to the cell line MDA4A4 (exon 6 low-producer) .
  • This cell iine is a subclone of cell line MDA-MB-435S (ductal carcinoma, breast, human; ATCC HTB 129; the subclone was produced according to Bao et al f Differentiation 52 (1993), 239-246; MDA4A4 is identical to MDA-MB-435-C2 of this reference) .
  • the culture PK 9.00.22 showed a high specificity to tumor tissue of colon with method B. With method A this cultured cell line showed also a marked specificity for the ,cell line ZR 75-1.
  • Cells of lines HT29 (ATCC HTB 38 - colon adenocarcinoma) and MDA4A4 were cultured in a medium according to ATCC catalogue and were harvested with or without protease additive.
  • the cells harvested without protease additive were centrifuged, added to double the volume of lysis buffer (50 mM potassium phosphate buffer, 150 mM NaCl, pH 8.0), homogenised for 5 min in a Dounce homogenizer and the quantity of protein determined. On the basis of this protein value, the cellular suspension was adjusted to a protein concentration of 1-2 mg/ml using lysis buffer with or without detergent [1% Triton X-100 - 57 -
  • the supernatant obtained after centrifuging off the membranes contained sufficient CD44 (standard form) and CD44v (CD44 with additional exons) for antibody assessment. Because of the mRNA concentration m the cells, it is assumed that MDA4A4 contains predominantly CD44-standard form and hardly any exon 6- containing CD44v. HT29 cells, on the other hand, should contain mainly exon 6- containing CD44v.
  • CD44 or CD44v can be obtained is to harvest the cells with trypsin instead of the aforementioned cell harvest with subsequent cellular separation.
  • the supernatant obtained after addition of trypsin inhibitor and centrifuging off the cells also contains sufficient CD44 and CD44v for antibody assessment.
  • the specificity of the reaction can be shown by addition of free Exon6 peptide to the Ab before incubation of the nitrocellulose. If the reaction is specific for Exon6 or CD44v, either no or only very slight binding of the AB to the nitrocellulose takes place after addition of the free peptide. Best results were obtained with the following clones: * MAB ⁇ CD44>M-1.1.12 * MAB ⁇ CD44>M-2.42.3
  • the antibodies produced by the cell lines MAK ⁇ CD44>M-1.1.12 and MAK ⁇ CD44>M-2.42.3 are able to bind to dotted CD44 exon 6 peptide and to an extract of 59
  • HT29 cells but not to an extract of MDA4A4 cells. Binding of the monoclonal antibody to dotted CD44 exon 6 peptide and to an extract of HT29 ceils is specific for the tumorspecific variant of CD44v because preincubation of both antibodies with synthetic CD44 exon 6 inhibits the binding of the antibodies to nitrocellulose (Table 5) .

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Abstract

On constate une surexpression prononcée des variantes épissées multiples du gène CD44 tumoral par rapport au tissu normal équivalent. Cette constatation est le point de départ d'un procédé de diagnostic de néoplasies par l'analyse d'un échantillon de tissu ou de liquide de l'organisme ou de déchets. On a localisé et séquencé un nouveau exon 6 de 129bp. On a préparé des anticorps spécifiques de cet exon et on les revendique à titre de nouveaux composés utilisables dans la détection de protéines CD44 et dans le traitement et l'imagerie in vivo des tumeurs.
EP94900245A 1992-11-20 1993-11-22 Peptide correspondant a l'exon 6 du gene cd44, anticorps specifiques dudit peptide et utilisation de ces anticorps dans le diagnostic des tumeurs Withdrawn EP0672130A1 (fr)

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GB929224386A GB9224386D0 (en) 1992-11-20 1992-11-20 Nucleic acid sequence
GB9224386 1992-11-20
GB9226165 1992-12-16
GB929226165A GB9226165D0 (en) 1992-12-16 1992-12-16 Nucleic acid sequence
WOPCT/GB93/01520 1993-07-20
PCT/GB1993/001520 WO1994002633A1 (fr) 1992-07-21 1993-07-20 Methode de diagnostic
PCT/GB1993/002394 WO1994012631A1 (fr) 1992-11-20 1993-11-22 Peptide correspondant a l'exon 6 du gene cd44, anticorps specifiques dudit peptide et utilisation de ces anticorps dans le diagnostic des tumeurs

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UA58482C2 (uk) * 1994-06-08 2003-08-15 Бьорінгер Інгельхайм Інтернаціональ Гмбх Моноклональне антитіло vff-18 проти сd44v6 і його фрагменти
DE19540515C1 (de) * 1995-10-31 1997-02-06 Boehringer Ingelheim Int Tumortherapie durch adoptiven Transfer CD44v-spezifischer zytotoxischer T-Lymphozyten
DE19545472A1 (de) * 1995-12-06 1997-06-12 Boehringer Ingelheim Int Verfahren zur Diagnose und Therapie von Plattenepithelkarzinomen
WO1997030173A1 (fr) 1996-02-15 1997-08-21 Chugai Seiyaku Kabushiki Kaisha Anticorps monoclonal reconnaissant les antigenes presents a la surface des cellules endotheliales d'un vaisseau tumoral
FR2775984B1 (fr) * 1998-03-11 2006-09-15 Bioscreen Therapeutics Sa Criblage differentiel qualitatif
US6881571B1 (en) 1998-03-11 2005-04-19 Exonhit Therapeutics S.A. Qualitative differential screening
DE19653607A1 (de) 1996-12-20 1998-06-25 Boehringer Ingelheim Int Verfahren zur Diagnose und Therapie von Hodgkin-Lymphomen
WO1999004036A1 (fr) * 1997-07-14 1999-01-28 Isis Innovation Limited Detection de cancer a base de cd44
US7189397B2 (en) * 1999-10-08 2007-03-13 Arius Research Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US8048416B2 (en) 1999-10-08 2011-11-01 Hoffmann-La Roche Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US20050100542A1 (en) 1999-10-08 2005-05-12 Young David S. Cytotoxicity mediation of cells evidencing surface expression of CD44
US7947496B2 (en) 1999-10-08 2011-05-24 Hoffmann-La Roche Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US8071072B2 (en) 1999-10-08 2011-12-06 Hoffmann-La Roche Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
PE20021098A1 (es) * 2001-05-18 2003-02-11 Boehringer Ingelheim Int ANTICUERPOS ESPECIFICOS DE CD44v6
US6972324B2 (en) 2001-05-18 2005-12-06 Boehringer Ingelheim Pharmaceuticals, Inc. Antibodies specific for CD44v6
EP2611921A2 (fr) * 2010-08-30 2013-07-10 F.Hoffmann-La Roche Ag Construction d'expression procaryote

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