EP0705436A1 - Procede pour le diagnostic et l'analyse du carcinome mammair - Google Patents

Procede pour le diagnostic et l'analyse du carcinome mammair

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Publication number
EP0705436A1
EP0705436A1 EP94919633A EP94919633A EP0705436A1 EP 0705436 A1 EP0705436 A1 EP 0705436A1 EP 94919633 A EP94919633 A EP 94919633A EP 94919633 A EP94919633 A EP 94919633A EP 0705436 A1 EP0705436 A1 EP 0705436A1
Authority
EP
European Patent Office
Prior art keywords
exon
aca
epitope
antibody
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP94919633A
Other languages
German (de)
English (en)
Inventor
Helmut Ponta
Karl-Heinz Heider
Peter Herrlich
Steven T. Pals
Peter Dall
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.)
Boehringer Ingelheim International GmbH
Karlsruher Institut fuer Technologie KIT
Original Assignee
Boehringer Ingelheim International GmbH
Forschungszentrum Karlsruhe GmbH
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Filing date
Publication date
Priority claimed from DE4320624A external-priority patent/DE4320624A1/de
Priority claimed from DE4320623A external-priority patent/DE4320623C2/de
Priority claimed from DE4321944A external-priority patent/DE4321944A1/de
Priority claimed from DE4414787A external-priority patent/DE4414787A1/de
Application filed by Boehringer Ingelheim International GmbH, Forschungszentrum Karlsruhe GmbH filed Critical Boehringer Ingelheim International GmbH
Priority to EP96113181A priority Critical patent/EP0767380A1/fr
Priority to EP96113180A priority patent/EP0767379A1/fr
Priority to EP96113179A priority patent/EP0767378A1/fr
Publication of EP0705436A1 publication Critical patent/EP0705436A1/fr
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5753—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the stomach or small intestine
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57535—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the large intestine, e.g. colon, rectum or anus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5755—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the uterine cervix, uterine corpus or endometrium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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 invention relates to methods for diagnosing and / or analyzing tumors by determining the expression of variable exons of the CD44 gene, agents for such methods and their use.
  • the hematogenous spread of breast cancer occurs very early in the course of the disease and is related to the later appearance of distant metastases (Diel et al, 1992).
  • the molecular mechanisms of the metastatic spread are still unknown.
  • the prognostic factors for the prediction of the risk of metastasis are currently based mainly on pathological criteria, the main factors being tumor stage, differentiation (grading) and lymph node metastasis (Fisher et al, 1990).
  • grade differentiation
  • lymph node metastasis Fisher et al, 1990.
  • lymph node metastases are missing despite advanced tumor size or low differentiation (high grade).
  • a subset of patients with lymph node-negative breast cancer who later develop distant metastases is poorly studied.
  • stomach tumors are often divided into two broad histological categories, the "intestinal” and the "diffuse” type (Lauren, 1965). Tumors of the intestinal, but not of the diffuse type, are often accompanied by chronic gastritis B and especially by intestinal metaplasias, which are thought to be precursors of dysplastic changes and adenocarcinomas of the intestinal type (Jida et Kusama, 1982; Jass, 1983; Kato et al, 1981; Sipponen et al, 1983; Sirula et al, 1974; Strickland et Mackay, 1973).
  • Pathogenetic differences between these two types of adenocarcinoma are also reflected in the observation that patients with tumors of the diffuse type often belong to blood group A, which indicates a possible influence of genetic factors on the risk of cancer (Piper, 1978), while environmental factors , eg infections with Helicobacter pylori, may be important for the development of tumors of the intestinal type (Parsonnet et al, 1991; Nomura et al, 1991).
  • environmental factors eg infections with Helicobacter pylori
  • molecular markers to distinguish between tumors of the intestinal type and those of the diffuse type.
  • the third example is cervical cancer of the uterus.
  • CD44 splice variants of CD44
  • the CD44 isoforms are generated by alternative splicing in such a way that the sequences of 10 exons (vl-vlO) are completely cut out in CD44s, but can occur in different combinations in the larger variants (Screaton et al, 1992, Heider et al, 1993, Hofmann et al, 1991)
  • the variants differ in that at a
  • CD44 variants could be detected in different human tumor cells and in human tumor tissue.
  • the progression of CD44 variants in the course of colorectal carcinogenesis was recently investigated (Heider et al, 1993).
  • the expression of CD44 variants is missing in normal human colonic epithelium, and only weak expression is detectable in the proliferating cells of the crypts.
  • later stages of tumor progression eg in adenocarcinomas, all malignant degenerations express variants of CD44 CD44 splice variants shown in activated lymphocytes as well as in non-Hodgkin lymphomas (Koopman et al, 1993)
  • the object of the present invention was to develop new methods for diagnosing and analyzing tumors and to provide means for such methods
  • the methods according to the invention can be carried out on samples outside the human or animal body or in vivo.
  • the method according to the invention can advantageously be used to examine samples from patients who are suspected of having carcinoma of the breast, stomach, colon or cervix or who have already been diagnosed, but whose tumor is to be characterized more precisely.
  • the expression of the variant exon v6 in the case of suspected / diagnosis of breast or gastric carcinoma the expression of the exons v5 and / or v6 and in the case of suspected / diagnosis of cervical carcinoma the combined Expression of the variable exons v7 and v8 are examined.
  • Variant CD44 molecules can be detected at the protein level using antibodies or at the nucleic acid level using specific nucleic acid probes or primers for the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the invention accordingly also relates to antibodies and nucleic acids which are suitable as probes or primers for such methods, and the use of such antibodies and nucleic acids for the diagnosis and analysis of tumors, in particular carcinomas.
  • the invention preferably also relates to antibodies against epitopes which are encoded by exons v5, v6, v7 and / or v8, and nucleic acids which can hybridize with exons v5, v6, v7 and / or v8, furthermore primers which are used for RT PCR amplification of RNA containing exons v5, v6, v7 and / or v8 can be used. If the combined expression of the variants exons v7 and v8 is to be examined, this can preferably be done with an antibody which is directed against an epitope which is coded jointly by these exons (transitional epitope).
  • the CD44v-specific antibodies can be used, for example, with a detectable marker, e.g. B. a radioisotope, linked and injected into the vascular system of a patient. After the antibodies have been selectively bound to the tumor, it can be visualized using a suitable detection system. logs for such immunodiagnostic methods in vivo are state of the art (Thomas et al, 1989).
  • the nucleic acid and amino acid sequence of the variant exons of the CD44 gene is known (Hofmann et al, 1991, Screaton et al, 1992, Tölg et al, 1993).
  • the existence of degenerate or allelic variants is not important for the implementation of the invention; such variants are therefore expressly included.
  • sequence of exon v5 of the human CD44 gene is (SEQ ID NO: 1):
  • sequence of exon v6 of the human CD44 gene is (SEQ ID NO: 3):
  • sequence of exon v7 of the human CD44 gene is (SEQ ID NO: 5):
  • sequence of exon v8 of the human CD44 gene is (SEQ ID NO: 7):
  • the antibodies according to the invention are therefore preferably directed against epitopes within the amino acid sequences shown or their allelic variants.
  • An antibody against the transitional epitope v7 / v8 particularly preferably recognizes an epitope within the amino acid sequence (SEQ ID NO: 9)
  • v6-specific antibody that targets an epitope within the amino acid sequence (SEQ ID NO: 10)
  • Monoclonal antibodies are particularly preferred.
  • other antibody molecules can also be used for the method according to the invention, for example Fab or F (ab ') 2 fragments of immunoglobulins, recombinantly produced single-chain antibodies (scFv), chimeric or humanized antibodies and other molecules which bind specifically to epitopes encoded by exons v5, v6, v7 and / or v8.
  • Antibodies against known amino acid sequences can be prepared by methods known per se (Catty, 1989). For example, a peptide of this sequence can be synthesized and used as an antigen in an immunization protocol.
  • Another way is to produce a fusion protein which contains the desired amino acid sequence by using a nucleic acid (which can be prepared synthetically or, for example, by polymerase chain reaction (PCR) from a suitable sample) which codes for this sequence , integrated in an expression vector and the fusion protein is expressed in a host organism.
  • the optionally purified fusion protein can then be used as an antigen in an immunization protocol and insert-specific antibodies or, in the case of monoclonal antibodies, hybridomas which express insert-specific antibodies can be selected using suitable methods.
  • suitable methods are state of the art. Heider et al. (1993) and Koopman et al. (1993) describe the production of antibodies against variant epitopes of CD44.
  • Nucleic acids with variants can also serve as a means of execution
  • Exons in particular v5, v6, v7 and / or v8, hybridize, in particular those with a homology of more than 80% to the corresponding exon sequences.
  • Such nucleic acids can be produced by methods known per se. This also applies analogously to primers which can be used in the RT-PCR of RNA to detect the expression of exons v5, v6, v7 and / or v8.
  • the method according to the invention for diagnosing and / or analyzing tumors, in particular carcinomas can expediently be carried out by examining samples taken from the body, for example from biopsies.
  • the agents according to the invention can be used advantageously.
  • tissue sections can be examined immunohistochemically with the antibodies according to the invention using methods known per se. Extracts or body fluids obtained from tissue samples can also be examined using other immunological methods using the said antibodies, for example in Western blots, enzyme-linked immunosorbent assays (ELISA, Catty et Raykundalia, 1989), radioimmunoassays (RIA, Catty et Murphy, 1989) or related immunoassays.
  • ELISA enzyme-linked immunosorbent assays
  • RIA Catty et Murphy, 1989
  • the expression of variant exons can also be detected at the nucleic acid level, for example by hybridizing RNA (Northern blot) or RT-PCR-amplified RNA obtained from tissue samples with suitable primers, or by hybridizing nucleic acids in tissue sections with suitable samples in situ Hybridization).
  • the investigations can be carried out qualitatively, semi-quantitatively or quantitatively.
  • Data obtained through detection and / or quantification of the expression of variant CD44 epitopes can thus be incorporated into the diagnosis and prognosis.
  • the combination with other prognostic parameters, such as grading, can be advantageous.
  • V5 and v6 expression are good prognostic parameters for breast cancer.
  • the comparative investigation of v5 and v6 expression makes an important contribution to the differential diagnosis of intestinal and diffuse gastric carcinoma. In colon carcinoma, v6 expression correlates very well with the tumor stage.
  • the detection of the expression of the transitional epitope encoded by v7 / v8 is an important diagnostic parameter in the diagnosis of cervical cancer.
  • Fig. 1 Schematic representation of a CD44 molecule that contains the variants exons v2 to vlO. Hatched boxes symbolize CD44 standard sequences (CD44s).
  • TM transmembrane domain. The approximate location of the epitopes recognized by different monoclonal (black bars) and polyclonal (gray bars) antibodies is indicated.
  • the antibodies VFF7, VFF8 and VFF16 are each specific for one exon, whereas mAb VFF17 reacts with an epitope that is coded together by exons v7 and v8.
  • Actuarial survival curve for breast cancer patients relating to the relationship between lymph node involvement and survival time, calculated on the basis of the Kaplan-Meier calculation and logarithmic series test.
  • Actuarial survival curve for breast cancer patients relating to the relationship between tumor grade and survival time, calculated on the basis of the Kaplan-Meier calculation and logarithmic series test.
  • Fig. 4 Actuarial survival curve for breast cancer patients relating to the relationship between exon v3 expression and survival time, calculated on the basis of the Kaplan-Meier calculation and logarithmic series test. Patients with primary invasive breast cancer expressing CD44-v3 epitope versus non-expressors. 6/28 patients with v3-negative primary tumor and 12/31 with v3 -positive tumor died in the observation period of 70 months.
  • Actuarial survival curve for breast cancer patients relating to the relationship between exon v6 expression and survival time, calculated on the basis of the Kaplan-Meier calculation and logarithmic series test.
  • Fig. 8 CD44v expression in human breast cancer. Immunohistochemistry of ductal invasive breast cancer with anti-CD44v antiserum.
  • A primary tumor.
  • B Lymph node metastases. Counterstain: hematoxylin.
  • Original magnification 20 x.
  • Fig. 9 Immunohistochemistry of normal mucosa and gastric adenocarcinomas.
  • a focal stressed anti-CD44v-positive reaction is shown in tumor cells of a moderately differentiated adenocarcinoma (intestinal type according to Lauren) of the stomach (a) and in a regional lymph node metastasis (b).
  • the foci of intestinal metaplasias react positively with mAb VFF4 (c, arrows) and with mAb VFF8 (d, arrows), accompanied by an additional reaction on the mucoid surface and the foveolar epithelium (d , Arrowheads).
  • Fig. 11 Reverse transcription / PCR amplification of CD44 transcripts in gastric carcinoma cell lines.
  • Upper part PCR amplification products after using CD44 standard primers. The first strand synthesis was carried out with polyadenylated RNA from the gastric carcinoma cell lines 2474, 2957 and 3051.
  • Lower part GADPH-specific PCR amplification products of the same RNA samples. The samples were separated in a 1.2% agarose gel, stained with ethidium bromide and visualized under UV light.
  • Fig. 14 Immunohistochemistry of normal cervical epithelium (A, C, E, G) and a squamous cell carcinoma of the cervix. (B, D, F, H). All normal cervical tissue samples showed a strong staining reaction with the polyclonal sera ⁇ nt / -CD44v3-vlO (A) anti-v3-v4 (Q, anti- ⁇ 6-vl, VFF7, VFF8 and VFF16 (not shown). The staining reactions were limited to the stratum basale and stratum spinosum, while the stratum superficiale remained unstained. The mAb VFF17 showed no staining of normal epithelial cells at all (E).
  • Frozen tissues (stored at -70 ° C) were obtained from the University Women's Clinic in Heidelberg, Germany. They were used for immunohistology using the antibodies described below. Included were 61 samples from primary breast cancer, 9 local recurrences of breast cancer, 4 cases of pure / ' «--s / ' / w carcinomas and 16 axillary lymph node metastases (from the same patients as the primary tumors). The cases were chosen at random and included a representative selection of histological tumor types, stages and gradings. is For comparison, samples of normal breast tissue, ductal hypoplastic and fibroadenomas were selected.
  • PCR 25 was amplified from human keratinocyte cDNA by polymerase chain reaction (PCR).
  • Hofmann et al. contained an EcoRI recognition site that was used to
  • ⁇ 70 kD consisting of glutathione-S-transferase from Schistosoma japonicum and the
  • CD44 exons in the vicinity of the variant exon sequences.
  • cDNAs were produced by reverse transcription and tested with glyceraldehyde-phosphate dehydrogenase-PCR before CD44s amplification in order to check the quality and amount of the cDNAs synthesized (K).
  • the PCR products obtained with CD44 standard primers were separated on 1.2% agarose (I) and transferred to a Hybond-N + membrane (Amersham). The same filter was then hybridized in succession with samples which were specific for the variants exons v3-vl0 (A-H), as indicated in the figure.
  • Lanes 1-4 normal cervical samples from 4 different patients. Exposure time: 45-60 minutes.
  • Fusion protein DI contains the CD44 sequence, which was described by Stamenkovic et al. (1989), from position 744 to position 142 of the sequence of variant CD44 as described by Hofmann et al. (1991).
  • Fusion protein Dll / ⁇ i contains the variant sequence from position 290-460
  • fusion protein Dm contains the variant sequence from position 378-638 (Hofmann et al, 1991). The fragments containing DI and DDI were cloned into the pGEX vector system, the DH / III fragment into the pATH vector (Angel et al, 1988).
  • mice Female Balb / c mice were immunized intraperitoneally with ⁇ -finite purified fusion protein (GST-CD44v3-10). In the first immunization, 90 ⁇ g of fusion protein in complete Freund's adjuvant was applied, in the second and third immunizations 50 ⁇ g of fusion protein in incomplete Freund's adjuvant. The immunizations were carried out every 4 weeks. 14 days after the last immunization, the animals were immunized for three consecutive days with 10 ⁇ g of fusion protein in PBS.
  • GST-CD44v3-10 ⁇ -finite purified fusion protein
  • spleen cells of an animal with a high antibody titer were fused with P3.X63-Ag8.653 mouse myeloma cells with the aid of polyethylene glycol 4000.
  • the hybridoma cells were then selected in microtiter plates in HAT medium (Köhler et Milstein, 1975; Kearney et al, 1979).
  • the determination of the antibody titer in the serum or the screening of the hybridoma supernatants was carried out using an ELISA.
  • microtitre plates are first coated with fusion protein (GST-CD44v3-10) or only with glutathione -S-transferase. It was then incubated with serial dilutions of serum samples or hybridoma supernatants and the specific antibodies with peroxidase-conjugated antibodies against mouse immunoglobulin were detected. Hybridomas that only reacted with glutathione S-transferase were discarded.
  • VFF7 recognizes an epitope in the amino acid sequence of exon v6.
  • VFF8 recognizes an epitope in the amino acid sequence of exon v5.
  • VFF-17 recognizes an epitope generated by exons v7 and v8 is coded together.
  • the antibodies NKI-PI and SFF2 recognize epitopes in the standard portion of the extracellular domain near the N-terminus.
  • Immunostaining was carried out according to standard protocols as previously described (Heider et al, 1993). Frozen sections (6-7 ⁇ m) were made in ice-cold methanol for 4 minutes, then in acetone for 1 minute. fixed, in PBS (8 g / 1 NaCl, 0.2 g / 1 KC1, 1.44 g / 1 0.24 g / 1 KH j PO pH 7.4) and pre-washed with normal goat serum (10% in PBS)
  • the frozen sections were coded and evaluated in various laboratories independently of one another with regard to nuclear and histological grading and immunohistological staining with antibodies against the v6 epitope.
  • the evaluation of the stained slides was carried out without knowledge of the course of the disease.
  • other CD44 epitopes were examined.
  • the coloration was weighted on a 0-4 scale. A coloration of 0-1 was rated as negative (14 patients) and a coloration of 2-4 as positive (45 patients).
  • Figure 8 shows a tumor section. All lymph node metastases (16 cases) were v6 positive, suggesting that v6 expression correlates with advanced tumor stage.
  • Table 1 Univariate analysis for comparison of total survival time (OAS) in relation to usual prognostic factors and the expression of splice variants v3 and v6 in 59 breast cancer patients using Peto's logarithmic serial test
  • Table 2 Multivariate analysis, which shows the result of a proportional risk regression analysis of the simultaneous influence of the nodal status and the surface expression of the CD44-v6 epitope on the OAS. Cox's partial nonparametric regression model was used to determine the predictive power of the factor combinations.
  • Lymph node status 0-3 versus> 4 2.45 0.12 v6, negative versus positive 5.56 0.10
  • Tumor specimens and normal tissue were selected from the inventory of the Pathology department at the University of Würzburg, Germany. The samples were snap-frozen immediately after the surgical removal and stored at -80 ° C. until use. Normal tissue was taken from twelve different tumor patients from both the body and the antral region of the stomach. Pathological tissues were obtained from a total of 47 patients with an average age of 63 years. Of the primary carcinomas, 29 belonged to the intestinal and 18 to the diffuse type according to Lauren (1965). The tumor stages ranged from localized (pT1) to extensive (pT4), the histological grading from well-differentiated (Gl) to poorly differentiated (G3) adenocarcinomas.
  • Polyclonal antisera directed against the epitopes v3-vl0 (CD44v in Fig. 1) stained 42/42 frozen sections of gastric tumors (Table 3, examples in Fig. 9a, b).
  • the coloring was heterogeneous in terms of intensity and distribution. Between 5% and 100% of the tumor cells were stained with different intensities. This confirms the finding of Heider et al. (1993) that adenocarcinomas express CD44 splice variants. Surprisingly, however, the subsequent examinations showed that the fine analysis of the expression pattern of the different variants of exons is suitable for the diagnosis and / or analysis of gastric carcinomas. Both qualitative and semi-quantitative conclusions are possible.
  • the present invention thus provides for the first time a molecular marker for distinguishing different subtypes of gastric cancer.
  • Both primary tumors and lymph node metastases were available from 10 patients. Five of these pairs were of the intestinal type and five of the diffuse type. Epitopes which were recognized by the polyclonal antiserum were present on both the primary tumors and on the metastases of all 10 tumor pairs (Table 4; FIGS. 9a and b show the primary tumor No. 9069/90 from Table 4 and the associated lymph node metastasis). All tumor samples (primary tumors and metastases) reacted with the mAb VFF8 specific for exon v5. All samples belonging to the intestinal type reacted positively after incubation with the mAb VFF4 specific for exon v6, while only one pair of the signet ring carcinomas belonged to the VFF4 + group (cf.
  • a splice variant of CD44 is thus expressed, which carries the exon sequence v5, and which is therefore expressed resembles the cells of diffuse-type carcinomas.
  • s PT primary tumor
  • LN lymph node metastases
  • the numbers refer to tumors and the corresponding lymph node metastases (not shown separately).
  • the tumors are included in the collection presented in Table 1. Intensity (because there was no difference in the intensity between primary tumor and lymph node metastasis, the common staining is indicated here: - negative, + weak, ++ moderate, +++ strong percentage positive tumor cells in the primary tumor percentage positive tumor cells in a lymph node metastasis of the same patient separated (Lämmli, 1970).
  • the proteins were transferred to polyvinylidene difluoride membrane (Millipore, Eschborn, Germany), using a transblot device (BIO-RAD Laboratories, Kunststoff, Germany).
  • Non-specific interactions were blocked by preincubation of the membranes with a milk powder suspension (10% dry milk powder in PBS).
  • the membranes were then incubated at room temperature with the polyclonal anti-CD44 antiserum (cf. FIG. 1), followed by a further incubation with goat anti-rabbit IgG conjugated with alkaline phosphatase (Amersham, Braunschweig, Germany). The incubation period was one hour each. After each antibody incubation, the membranes were washed with PBS containing 0.3% Tween 20 (Sigma). The binding of the antibodies was detected using an enhanced chemiluminescent system (Amersham).
  • RNA from tissues
  • polyA + RNA from cell lines
  • 5 ⁇ l of the first strand cDNA were amplified with 7 ⁇ polymerase (Amersham) in 50 ⁇ l under the buffer conditions recommended by the manufacturer.
  • polymerase Amersham
  • oligonucleotides were used which were homologous to positions 8-29 and 362-339 of the published cDNA sequence (Allen et al, 1987). After 26 rounds of amplification (95 ° C. for 1 min., 62 ° C. for 1 min., 72 ° C.
  • RNA containing exon v5 or v6 was verified by hybridization with a v5-specific or a v6-specific sample. There was a marked difference in the pattern of RNA expressed in normal tissue and in tumors. The RNA patterns also differed from tumor to tumor.
  • RNA from 10 of the 12 samples of normal gastric mucosa which had also been investigated in immunohistochemistry, resulted in two dominant bands which hybridized with exons v6 and two dominant bands of similar size which hybridized with exon v5 (four representative examples are shown in Figs. 10A and B).
  • RNA from tumor samples gave a more complex and variable pattern of splice products.
  • the tumors at least partly also contained larger splice variants up to a size which suggests the presence of all variant exon sequences (FIG. 10).
  • Both diffuse and intestinal type tumors showed strong expression of transcripts containing exon v5 ( Figure 10A, lanes 1-5).
  • Tumors were described as "positive” if more than 10% of the tumor cells were stained. If less than 10% of the tumor cells were stained, this was referred to as "focal".
  • v6 expression Information about v6 expression, e.g. can be obtained by routine immunohemochemistry, because of the surprising connection, especially of v6 expression, with the tumor stage can enable valuable knowledge for the diagnosis and prognosis of colon carcinomas.
  • N-terminal epitopes are present on the subepithelial stromal cells and in all epithelial layers (Fig. 14G). The presence of N-terminal epitopes indicates CD44 promoter activity in all stromal cells and epithelial layers. Epitopes of variant exons do not occur in stromal cells, but are strongly expressed in epithelial cells. However, these epitopes are lost in the direction of the epithelial surface and are absent in the superficial stratum (Fig. 14A, C).
  • An epitope which is formed by the transition piece from exons v7 and v8 and which is recognized by the mAb VFF17 ( ⁇ wt / ' -v7 / v8) is completely absent in the normal epithelium (FIG. 14E).
  • the pattern of immunostaining can be integrated in such a way that it indicates the surface expression of one or more CD44 isoforms with regulated access to the epitopes (for example by glycosylation) or with regulated synthesis by alternative splicing.
  • the combination of exons v7 / v8, which is indicated by the occurrence of the transitional epitope, is excluded.
  • RNAs were analyzed by semi-quantitative RT-PCR.
  • RNA isolated from tumor or normal tissue
  • 5 ⁇ l of the first strand cDNA were 7.
  • -Polymerase (Amersham) in a volume of 50 ⁇ l in Taq l-oly- merase buffer (Amersham) amplified.
  • oligonucleotides were used which were homologous to positions 8-29 and 362-339 of the published cDNA sequence (Allen et al, 1987).
  • primers were used which were homologous to positions 513-540 and 934-958 of the published human CD44 sequence (Stamenkovic et al, 1991). After 25 (GAPDH) or 30 (CD44) amplification cycles (1 minute at 94 ° C, 1 minute at 62 ° C, 2.5 minutes at 72 ° C), 10 ⁇ l of the reaction mixture were separated in a 1.2% agarose gel. The amplification products were visualized after ethidium bromide staining and the CD44 products were then transferred to nylon membranes (Hybond-N +, Amersham) for Southern blotting.
  • Hybridization with samples for variant exons at long exposure document the presence of alternatively spliced long CD44 transcripts in the RNA samples (FIG. 15).
  • This RNA species may encode CD44 v3-v6 or v4-v7.
  • at least one other species is present, the so-called epithelial variant CD44v8-v10 (also with a transcript length of 850 bp).
  • PCR data show that almost all samples of malignant tissue contain RNA for CD44 splice variants (Fig. 16). Lanes 6 and 9 of Figure 4 represent samples with only a small number of tumor cells. Therefore the expected amount of CD44v-RNA was too small to be amplified sufficiently. However, the tumor cells showed strong expression of variant epitopes in immunostaining.
  • the ethidium bromide staining documents the relative frequency of PCR products in tumor samples in comparison to samples from normal epithelium (difference in intensity of the hybridization signals 20-fold) (FIG. 15). It should be noted that the band that represents CD44s could come from RNA from tumor cells or stromal cells. The relative frequency of different exon sequences differed from sample to sample. Interestingly, the samples with the strongest expression and the greatest number of different isoforms included those from patients who already had lymph node metastases at the time of the operation.
  • glycoprotein CD44 confers meta-static potential to rat carcinoma cells.
  • Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl. Acad Be. U.S.A. 89: 12160-12164 (1992).
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne un procédé pour le diagnostic et l'analyse de tumeurs, reposant sur la détermination de l'expression de variantes déterminées d'exon du gène CD44. Cette détermination peut se faire au niveau de la protéine ou de l'acide nucléique. Dans une forme de mise en oeuvre préférée, l'expression est déterminée dans un matériau de biopsie, au moyen d'anticorps spécifiques de l'exon. C'est ainsi, par exemple, que l'expression v6 convient comme paramètre pronostique pour le cancer du sein, l'expression d'un épitope de transition, qui est codé conjointement par les exons v7 et v8, pour le diagnostic du cancer du col de l'utérus.
EP94919633A 1993-06-22 1994-06-15 Procede pour le diagnostic et l'analyse du carcinome mammair Ceased EP0705436A1 (fr)

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EP96113181A EP0767380A1 (fr) 1993-06-22 1994-06-15 Procédé pour le diagnostic et l'analyse de carcinomes du col de l'utérus
EP96113180A EP0767379A1 (fr) 1993-06-22 1994-06-15 Procédé pour le diagnostic et l'analyse de carcinomes de colon
EP96113179A EP0767378A1 (fr) 1993-06-22 1994-06-15 Procédé pour le diagnostic et l'analyse du tumeurs de l'estomac

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DE4320624 1993-06-22
DE4320623 1993-06-22
DE4320624A DE4320624A1 (de) 1993-06-22 1993-06-22 Verfahren zur Diagnose und Analyse von Mammakarzinomen
DE4320623A DE4320623C2 (de) 1993-06-22 1993-06-22 Verfahren zur Diagnose und Analyse von Adenokarzinomen
DE4321944 1993-07-02
DE4321944A DE4321944A1 (de) 1993-07-02 1993-07-02 Verfahren zur Diagnose und Analyse von Kolonkarzinomen
DE4414787 1994-04-28
DE4414787A DE4414787A1 (de) 1994-04-28 1994-04-28 Verfahren zur Diagnose und Analyse von Zervixkarzinomen
PCT/EP1994/001952 WO1995000851A1 (fr) 1993-06-22 1994-06-15 Procede pour le diagnostic et l'analyse de tumeurs

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EP94919633A Ceased EP0705436A1 (fr) 1993-06-22 1994-06-15 Procede pour le diagnostic et l'analyse du carcinome mammair
EP96113181A Ceased EP0767380A1 (fr) 1993-06-22 1994-06-15 Procédé pour le diagnostic et l'analyse de carcinomes du col de l'utérus
EP96113179A Ceased EP0767378A1 (fr) 1993-06-22 1994-06-15 Procédé pour le diagnostic et l'analyse du tumeurs de l'estomac

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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
US5861494A (en) 1995-06-06 1999-01-19 Human Genome Sciences, Inc. Colon specific gene and protein
DE19540515C1 (de) * 1995-10-31 1997-02-06 Boehringer Ingelheim Int Tumortherapie durch adoptiven Transfer CD44v-spezifischer zytotoxischer T-Lymphozyten
UY24389A1 (es) * 1995-12-06 2001-10-25 Karlsruhe Forschzent Composición farmacéutica para el tratamiento de carcinoma de epitelio plano
DE19545472A1 (de) * 1995-12-06 1997-06-12 Boehringer Ingelheim Int Verfahren zur Diagnose und Therapie von Plattenepithelkarzinomen
DE19653607A1 (de) 1996-12-20 1998-06-25 Boehringer Ingelheim Int Verfahren zur Diagnose und Therapie von Hodgkin-Lymphomen
IL133647A0 (en) * 1999-06-08 2001-04-30 Yissum Res Dev Co Novel cd44 variant
US7534605B2 (en) 1999-06-08 2009-05-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem CD44 polypeptides, polynucleotides encoding same, antibodies directed thereagainst and method of using same for diagnosing and treating inflammatory diseases
RU2164349C1 (ru) * 1999-07-27 2001-03-20 Нижегородская государственная медицинская академия Способ определения стоматологического статуса
US6972324B2 (en) 2001-05-18 2005-12-06 Boehringer Ingelheim Pharmaceuticals, Inc. Antibodies specific for CD44v6
CN1561223A (zh) * 2001-08-24 2005-01-05 斯塔凡·斯特伦布拉德 新型药物
US7460960B2 (en) 2002-05-10 2008-12-02 Epitome Biosystems, Inc. Proteome epitope tags and methods of use thereof in protein modification analysis
US7618788B2 (en) 2002-05-10 2009-11-17 Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
EP1539235A2 (fr) 2002-07-01 2005-06-15 Human Genome Sciences, Inc. Anticorps qui se lient specifiquement a reg iv
US20050009110A1 (en) * 2003-07-08 2005-01-13 Xiao-Jia Chang Methods of producing antibodies for diagnostics and therapeutics
US7855057B2 (en) 2006-03-23 2010-12-21 Millipore Corporation Protein splice variant/isoform discrimination and quantitative measurements thereof
MX2009010400A (es) 2007-03-30 2010-02-18 Chu Sainte Justine Método para la determinacion de riesgo de escoliosis.
RU2428100C2 (ru) * 2009-03-20 2011-09-10 Государственное образовательное учреждение высшего профессионального образования Читинская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию Способ определения интенсивности кариеса постоянных зубов у детей в период сменного прикуса
CA2914213A1 (fr) * 2013-06-17 2014-12-24 Alain Moreau Methode d'augmentation de signalisation de gipcr dans les cellules d'un sujet scoliotique
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EP3169343B1 (fr) 2014-07-15 2020-03-25 Yissum Research and Development Company of the Hebrew University of Jerusalem Ltd. Polypeptides isolés de cd44 et utilisations associées
AU2020216780B2 (en) * 2019-01-28 2025-05-08 Multitude Therapeutics Inc. Antibodies specific to CD44
CN116496398B (zh) * 2022-10-31 2023-10-31 南京元迈细胞生物科技有限公司 一种特异性结合CD44的v5外显子的抗体及其用途

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