EP0713398A1 - POLYPEPTIDES CODED BY EXON v5 OF THE CD44 GENE AS TARGETS FOR IMMUNOTHERAPY AND IMMUNOSCINTIGRAPHY OF TUMOURS - Google Patents

POLYPEPTIDES CODED BY EXON v5 OF THE CD44 GENE AS TARGETS FOR IMMUNOTHERAPY AND IMMUNOSCINTIGRAPHY OF TUMOURS

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Publication number
EP0713398A1
EP0713398A1 EP94924268A EP94924268A EP0713398A1 EP 0713398 A1 EP0713398 A1 EP 0713398A1 EP 94924268 A EP94924268 A EP 94924268A EP 94924268 A EP94924268 A EP 94924268A EP 0713398 A1 EP0713398 A1 EP 0713398A1
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EP
European Patent Office
Prior art keywords
antibody
antiköφer
exon
variant
tumors
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
EP94924268A
Other languages
German (de)
French (fr)
Inventor
Günther Adolf
Helmut Ponta
Karl-Heinz Heider
Peter Herrlich
Steven T. Pals
Erik Patzelt
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
Forschungszentrum Karlsruhe GmbH
Original Assignee
Kernforschungszentrum Karlsruhe GmbH
Boehringer Ingelheim International GmbH
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Publication of EP0713398A1 publication Critical patent/EP0713398A1/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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

Definitions

  • the invention relates to the use of antibodies against epitopes, which are encoded by variant exons of the CD44 gene, for the treatment or / / - v / vo diagnosis of tumors, agents for the treatment or / wv / vo diagnosis of tumors are suitable, and also the use of polypeptides which contain sequences encoded by the variant exon v5 of the CD44 gene, or v5-specific anti-idiotypic antibodies as tumor vaccines.
  • tumor-associated or specific antigens are expressed in a specific malignant disease, while it is absent in other types of tumor as well as in normal adult and fetal tissues.
  • the second aspect, the absence in normal tissue, is decisive for the use of tumor-specific antibodies in tumor therapy and for the imaging of tumors. To date, no tumor-specific marker has been found that
  • agents linked to the antibody can themselves be cytotoxic, for example radioactive isotopes, bacterial or plant toxins or cytostatics, have an immunomodulatory effect, e.g. Cytokines, or for example, enzymes that convert a precursor molecule (prodrug) into a cytotoxic agent
  • Radiolabelled antibodies can be used for the visual display of tumors in vivo (imaging);
  • nuclear magnetic resonance methods have also been developed in which, for example, antibodies linked to chelated trivalent cations are used as contrast agents (Brasch, 1992; Hider et Hall, 1991; Saccavini et al, 1988).
  • no pair of antigens / antibodies has been found that allows wide use in tumor therapy and wv / ' vo diagnostics. The search for tumor-associated antigens as suitable targets for antibody-based methods of tumor therapy and diagnosis therefore continues unabated.
  • the CD44 variants are generated by alternative splicing in such a way that the sequences of 10 exons (vl-vlO) are completely cut out in CD44s, but the larger variants can occur in different combinations (Screaton et al., 1992; Tölg et al. , 1993; Hofmann et al., 1991).
  • the variants differ in that different amino acid sequences are inserted at a certain point in the extracellular part of the protein. Such variants could be detected in various human tumor cells and in human tumor tissue.
  • the expression of CD44 variants in the course of colorectal carcinogenesis was recently examined (Heider et al, 1993).
  • CD44 variants are absent in normal human colonic epithelium and only weak expression is detectable in the proliferating cells of the crypts. In later stages of tumor progression, for example in adenocarcinomas, all malignancies express variants of CD44. Tissue expression of variant CD44 at a high level could also be demonstrated in aggressive non-Hodgkin's lymphomas (Koop an et al., 1993).
  • the object of the present invention was to provide new agents for the treatment or / w-v / vo diagnosis of cancer which are based on the principle of immunological recognition of tumor-associated antigens.
  • the agents according to the invention and their use are based on antibodies against epitopes which are encoded by variant exons of the CD44 gene, in particular on antibodies against epitopes which are encoded by variant exon v5 of the human CD44 gene, and on v5 -specific anti-idiotypic antibodies.
  • the antibodies can be polyclonal or monoclonal, complete immunoglobulins, Fab or F (ab ') 2 fragments of immunoglobulins or other derivatives, bipecific, chimeric or humanized antibodies or recombinantly produced antibodies, for example single-chain antibodies (scFv) , Fab fragments, other fragments or complete immunoglobulins.
  • the antibodies can be applied without an additional agent or can be linked to a radioactive substance, a cytotoxic agent, an immunomodulating agent, a substance with the aid of which a cytotoxic agent can be generated locally, a magnetic resonance contrast agent or another detectable label.
  • the invention further relates to the use of polypeptides which contain sequences which are encoded by the variant exon v5 of the CD44 gene and v5-specific anti-idiotypic antibodies as tumor vaccines. If the antibody bears a detectable label, the label can be detected for diagnostic purposes, for example visualization of the tumor in vivo (imaging), or for example for radio-assisted surgery (radioguided surger).
  • the nucleic acid and amino acid sequence of the variant part 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.
  • the sequence of exon v5 is not important for the implementation of the invention; such variants are therefore expressly included.
  • Antibodies can be used to carry out the invention, in particular those which are directed against epitopes within the sequence of exon v5, or v5-specific anti-idiotypic antibodies. Monoclonal antibodies are particularly preferred. However, polyclonal antibodies, Fab or F (ab ') 2 fragments of immunoglobulins, recombinantly produced antibodies or fragments, for example single-chain antibodies (scFv), bispecific, chimeric or humanized antibodies or equivalent molecules can also be used for the process according to the invention serve to specifically bind exon v5-encoded epitopes. Antibodies against known amino acid sequences can be prepared by methods known per se (Catty, 1989).
  • a peptide of this sequence can be produced synthetically 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 adding a nucleic acid (which can be prepared synthetically or, for example, by a polymerase chain reaction (PCR) from a suitable sample) which codes for this sequence integrates 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, hybridids which express insert-specific antibodies can be selected using suitable methods (Wunderlich et al, 1992).
  • the entire CD44 gene or a fragment thereof can also be expressed in a suitable system without the expressed sequences being fused to other peptides, then isolated and used as antigens in immunization protocols.
  • Such methods are state of the art. Heider ei al. (1993) and Koopman et al (1993) describe the production of antibodies against variant epitopes of CD44. Instead of an intact immunoglobulin molecule, Fab or F (ab ') 2 fragments or other fragments can also be used (Kreitman et al, 1993).
  • Chimeric antibodies can also be used, for example humanized mouse antibodies (Shin et al, 1989; Güssow et Seemann, 1991), bispecific antibodies (Weiner et al, 1993; Goodwin, s 1989) or s / «# / e-cA ⁇ / w-antibody / toxin fusion proteins (Friedman et al, 1993).
  • the antibodies can be isolated from sera or from hybridoma supernatants or obtained by recombinant expression as single-chain antibodies (scFv, Johnson et Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al, 1992; Nesbit et al, 1992 ; Barbas et al, 1992).
  • the antibodies can be used alone or linked to an agent w. If the antibody is to be used therapeutically or for immunoscintigraphy, it can be linked to a suitable radioactive isotope, for example 131 I, 125 I, ul In, 186 Re, 90 Y, 99m Tc or 211 At. This linkage can take place directly or via a linker molecule, for example a chelating agent.
  • a linker molecule for example a chelating agent.
  • the antibody can also be linked to a non-radioactive cytotoxic agent.
  • a non-radioactive cytotoxic agent can be a cytostatic (Schrappe et al, 1992) or a cytotoxic polypeptide, e.g. a bacterial or vegetable toxin (Vitetta et al, 1991; Kreitman et al, 1993).
  • a cytotoxic polypeptide can be covalently, e.g. over disulfide bridges with which
  • antibodies can be linked (Theuer et al, 1993), or linked to an antibody in the form of a fusion protein in a single-chain immunotoxin (Chaudhary et al, 1990; Friedman et al, 1993).
  • the antibody can also be linked to a cytokine or another immunomodulatory polypeptide, e.g. with tumor necrosis factor or interleukin-2.
  • the antibody may also be linked to an agent which, while not itself 5, is cytotoxic, but can produce a cytotoxic substance, e.g. an enzyme that catalyzes the conversion of an inactive precursor molecule (prodrug) into a cytostatic (Wang et al, 1992; Senter et ⁇ /., 1989).
  • the antibodies according to the invention against variant CD44 epitopes can be used by systemic or topical application, for example by intravenous se (as a bolus or continuous infusion), intraperitoneal, intramuscular, subcutaneous or injection or infusion. Individual organs or limbs can also be perfused.
  • Protocols for the administration of conjugated or non-conjugated antibodies are state of the art (Mulshine et al, 1991; Larson et al, 1991; Vitetta et Thorpe, 1991; Vitetta et al, 1991; Breitz et al, 1992; Press et al, 1989; Weiner et al, 1989; Chatal et al, 1989; Sears et al, 1982).
  • the agents according to the invention are suitable for the tn-v / vo diagnosis of tumors.
  • antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging ) there are also a number of protocols on the basis of which the person skilled in the art can carry out the invention (Siccardi et al, 1989; Keenan et al, 1987; Perkins et Pimm, 1992; Colcher et al, 1987; Thompson et al, 1984).
  • Recombinant polypeptides which contain amino acid sequences encoded by exon v5, and v5-specific anti-idiotypic antibodies can be used as tumor vaccines.
  • Antiidiotypic antibodies recognize epitopes within the variable regions of immunoglobulins (idiotopes).
  • V5 -specific antiidiotypic antibodies are understood to be antibodies which are directed against idiotopes of antibodies which recognize v5-encoded amino acid sequences. If v5-specific anti-idiotypic antibodies are used as antigens in an immunization protocol, antibodies are formed against these antigens, most of which also bind to v5-coded epitopes.
  • Polypeptides containing v5-encoded sequences and v5-specific anti-idiotypic antibodies can therefore be used to immunize tumor patients.
  • the antibodies which are formed in the patient's organism against v5-coded epitopes recognize tumor cells which express these epitopes and thus support the immune defense of the tumor by the organism.
  • Variant CD44 molecules are tumor-associated antigens which are very suitable as targets for the immunotherapy and scintigraphy of cancer.
  • Variant CD44 molecules which contain the amino acid sequence encoded by the variable exon v5, an allelic variant or a fragment of this sequence and v5-specific anti-idiotypic antibodies are particularly suitable.
  • Table 1 shows an investigation of gastric tumors from a total of 42 patients. In 42/42 cases, expression of variants CD44 molecules can be detected (polyclonal antiserum against the amino acid sequences of the exons v3-vl0). Examinations with exon-specific monoclonal antibodies then show that exons v3 / v4, v7, v8-vl0 are not and v6 is only expressed in 26/42 tumors (62%). v5, on the other hand, is expressed in 39/42 tumors (93%). In contrast to v6, both gastric tumors of the diffuse (14/17) and also of the intestinal type (25/25). In tumors that express both exons, the expression of v5 is usually stronger than that of v6 (Table 2).
  • Fig. 3 shows the results of a study of 39 colorectal carcinomas.
  • v5 is not expressed in normal tissue at all, whereas in tumor tissue it is expressed in over 80% of the tumors in the earliest stage of the tumor (early adenoma).
  • v3 expression was not detected at all, v6 expression only increased significantly in late tumor stages and v8-10 was already expressed in normal tissue.
  • v5 is expressed more frequently and to a greater extent than v6.
  • tumor-associated antigen which is the target of the antibody
  • the tumor-associated antigen is expressed in tumor tissue, but not in normal tissue, but on the other hand in the largest possible number of tumors as early as possible Tumor stage is expressed as high as possible to allow wide and reliable use.
  • CD44 variants that contain amino acid sequences encoded by exon v5 meet these requirements to a high degree.
  • the agents and uses according to the invention are thus outstandingly suitable for immunotherapy and v / vo diagnosis / immunoscintigraphy of tumors, in particular carcinomas.
  • 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.
  • VFF8 (v5) 4 (100%) 51 (82%) 8 (89%) 16 (100%)
  • Fig. 1 Schematic representation of a CD44 splice variant. This exemplary
  • Variant carries all variants of exon sequences at the single insertion site. Dark gray
  • VFF18 is indicated by bars. All monoclonal antibodies are exon-specific.
  • VFF8, 80x e: VFF4, 210x; f: VFF8, 210x; Counterstaining hematoxylin).
  • Fig. 3 Expression of variant CD44 exons in different stages of the colorectal tumor progression. Results from immunohistochemical staining of tissue sections (Example 2). Dark gray bars indicate the percentage of positive tumors. Light gray bars show samples with only focal staining.
  • 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.
  • the resulting construct (pGEX CD44v HPKII, v3-vl0) codes for a fusion protein of ⁇ 70 kD.
  • the fusion protein was expressed in E. coli and then affinity purified using glutathione agarose (Smith et al, 1988). To obtain subclones of the regions that are used for affinity purification and
  • Fusion protein Du / in contains the variant sequence from position 290-460, fusion protein DIII the variant sequence from position 378-638 (Hofmann et al, 1991).
  • the fragments containing DI and Dm were cloned into the pGEX vector system, the DII / ÜI fragment into the pATH vector (Angel et al, 1988).
  • mice Female BALB / c mice were immunized with affinity-purified fusion protein which was obtained from pGEX CD44v HPKII (exons v3-vl0) as described above. Spleen cells from an animal with a high antibody titer were fused with P3X63Ag8.653 myeloma cells using polyethylene glycol 4000. Hybridomas were selected in HAT-35 medium (Kearney et al, 1979). Determination of the antibody titer in the serum and the antibody screening were carried out by means of ELISA.
  • the microtitre plates were coated with fusion protein, with serial dilutions of serum samples or Hybridoma supernatants were incubated, and specific antibodies were detected with peroxidase-coupled antibodies against mouse IgG. Hybridomas that reacted with glutathione transferase were eliminated. The remaining antibodies were further characterized by ELISA tests, fusion proteins of the variable domains DI (exon v3), DII III (exons v5, v6) ,. DIII (exons v6, v7), DI-IV (exons v3-v8), DIII-VI (exons v7-vl0) and v6 (exon v6) [v5 v6 v7] were used. The reactivity of the antibodies with human skin keratinocytes was examined immunohistochemically.
  • Frozen sections were frozen in ice-cold methanol for 10 min. fixed, in PBS (8 g / 1 NaCl, 0.2 g / 1 KCl, 1.44 g / 1 0.24 g / 1 KH 2 PO 4 , pH 7.4) and preincubated with normal goat serum (10% in PBS). Then they were washed 3 times with PBS and incubated for 1 hour with the primary antibody (in PBS, 1% BSA).
  • Endogenous peroxidase was blocked with 0.3% H ⁇ in methanol and the sections with biotinylated second antibody (either anti-mouse or anti-rabbit F (ab ') 2 , DAKO Co ⁇ ., Santa Barbara, CA, USA, depending on the primary antibody used ) incubated.
  • biotinylated second antibody either anti-mouse or anti-rabbit F (ab ') 2 , DAKO Co ⁇ ., Santa Barbara, CA, USA, depending on the primary antibody used
  • the immune complex was visualized with horseradish peroxidase, which was coupled to biotin as a streptavidin-biotin-peroxidase complex (DAKO).
  • Tumors were said to be “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”. literature
  • Senter P D Schreiber G J, Hirschberg D L, Ashe S A, Hellström K E, Hellström I. Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomyein C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates. Cancer Res. 49, 5789-5792 (1989).
  • lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family. Cell 56, 1057-1062 (1989).

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Abstract

The present invention relates to agents for the treatment and in vivo diagnosis of tumours based on the selective bonding of antibodies to epitopes coded by variant exons of the CD44 gene. Special preference is hereby given to exon v5 of the human CD44 gene. Exon v5 is expressed in normal tissues only very slightly or not at all, whereas even at early stages of tumour progression it is expressed in large quantities. Therefore v5-coded epitopes are very suitable as targets for the immunotherapy and immunoscintigraphy of tumours.

Description

Durch Exon v5 des CD44-Gens kodierte Polypeptide als Targets für Im- muntherapie und Immunszintigraphie von Tumoren Polypeptides encoded by exon v5 of the CD44 gene as targets for immunotherapy and immunoscintigraphy of tumors
Die Erfindung betrifft die Verwendung von Antikörpern gegen Epitope, die durch Variante Exons des CD44-Gens kodiert werden, zur Behandlung oder zur /«-v/vo-Diagno- stik von Tumoren, Mittel, die zur Behandlung oder /w-v/vo-Diagnostik von Tumoren ge- s eignet sind, sowie die Verwendung von Polypeptiden, die durch das Variante Exon v5 des CD44-Gens kodierte Sequenzen enthalten, oder v5-spezifischen anti-idiotypischen Antikör¬ pern als Tumorvakzine.The invention relates to the use of antibodies against epitopes, which are encoded by variant exons of the CD44 gene, for the treatment or / / - v / vo diagnosis of tumors, agents for the treatment or / wv / vo diagnosis of tumors are suitable, and also the use of polypeptides which contain sequences encoded by the variant exon v5 of the CD44 gene, or v5-specific anti-idiotypic antibodies as tumor vaccines.
Seit der Einfuhrung der Hybridomatechnologie im Jahre 1975 (Köhler et Milstein) wurde intensiv nach tumorassoziierten bzw. -spezifischen Antigenen gesucht. Per definitio- w nem wird ein tumorspezifisches Antigen in einer bestimmten malignen Erkrankung expri¬ miert, während es in anderen Tumortypen sowie in normalen adulten und fötalen Geweben abwesend ist. Für den Einsatz tumorspezifischer Antikörper in der Tumortherapie sowie zum Imaging von Tumoren ist dabei der zweite Aspekt, die Abwesenheit im Normalgewe¬ be, entscheidend. Bis heute wurde kein tumorspezifischer Marker gefunden, der die Defi-Since the introduction of hybridoma technology in 1975 (Köhler et Milstein), intensive searches have been made for tumor-associated or specific antigens. By definition, a tumor-specific antigen is expressed in a specific malignant disease, while it is absent in other types of tumor as well as in normal adult and fetal tissues. The second aspect, the absence in normal tissue, is decisive for the use of tumor-specific antibodies in tumor therapy and for the imaging of tumors. To date, no tumor-specific marker has been found that
IS nition voll erfüllt. Eine ganze Reihe von Antikörpern gegen tumorassoziierte Antigene wur¬ de bis jetzt entwickelt und in klinischen Studien zur Behandlung von Tumoren oder zum Imaging von Tumoren eingesetzt (Mulshine etal, 1991; Vitetta et Thorpe, 1991; Larson et ed., 1991). Das Prinzip dabei ist, daß der Antikörper als Sonde spezifisch an Tumorzellen bindet und entweder direkt oder über ein an ihn gekoppeltes Agens die Tumorzelle zerstörtIS nition fully met. A whole series of antibodies against tumor-associated antigens have hitherto been developed and used in clinical studies for the treatment of tumors or for the imaging of tumors (Mulshine et al, 1991; Vitetta et Thorpe, 1991; Larson et ed., 1991). The principle here is that the antibody binds specifically to tumor cells as a probe and destroys the tumor cell either directly or via an agent coupled to it
20 oder über den Nachweis eines mit dem Antikörper verknüpften Labels zur Darstellung des Tumors genutzt werden kann. Die mit dem Antikörper verknüpften Agentien können selbst zytotoxisch sein, beispielsweise radioaktive Isotope, bakterielle oder pflanzliche Toxine oder Zytostatika, immunmodulatorisch wirken wie z.B. Zytokine, oder auch beispielsweise Enzyme sein, die ein Vorläufermolekül (Prodrug) in ein zytotoxisches Agens umwandeln20 or by detecting a label linked to the antibody to represent the tumor. The agents linked to the antibody can themselves be cytotoxic, for example radioactive isotopes, bacterial or plant toxins or cytostatics, have an immunomodulatory effect, e.g. Cytokines, or for example, enzymes that convert a precursor molecule (prodrug) into a cytotoxic agent
25 können. Bei der visuellen Darstellung von Tumoren in vivo (Imaging) können radioaktiv markierte Antikörper verwendet werden; seit einiger Zeit werden auch Kernspinresonanz¬ methoden entwickelt, bei denen z.B. mit chelatisierten dreiwertigen Kationen verknüpfte Antikörper als Kontrastmittel verwendet werden (Brasch, 1992; Hider et Hall, 1991; Saccavini et al, 1988). Bis heute konnte allerdings kein Antigen/Antikörperpaar gefunden 0 werden, das eine breite Anwendung in der Tumortherapie und w-v/'vo-Diagnostik erlaubt. Die Suche nach tumorassoziierten Antigenen als geeignete Targets für Antikörper-gestützte Methoden der Tumortherapie und -diagnostik hält daher unvermindert an.25 can. Radiolabelled antibodies can be used for the visual display of tumors in vivo (imaging); For some time now, nuclear magnetic resonance methods have also been developed in which, for example, antibodies linked to chelated trivalent cations are used as contrast agents (Brasch, 1992; Hider et Hall, 1991; Saccavini et al, 1988). To date, however, no pair of antigens / antibodies has been found that allows wide use in tumor therapy and wv / ' vo diagnostics. The search for tumor-associated antigens as suitable targets for antibody-based methods of tumor therapy and diagnosis therefore continues unabated.
Es wurde kürzlich gezeigt, daß die Expression von Varianten des Oberflächen-Gly- koproteins CD44 notwendig und hinreichend ist, um sogenanntes spontanes metastatischesIt has recently been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to avoid so-called spontaneous metastatic
35 Verhalten sowohl in einer nicht-metastasierenden Pankreas-Adenokarzinom-Zellinie der Ratte als auch in einer nicht-metastasierenden Fibrosarkom-Zellinie der Ratte auszulösen (Günthert et ed., 1991). Während die kleinste CD44-Isoform, die Standardform CD44s, in einer Reihe verschiedener Gewebe, darunter Epithelzellen, ubiquitär exprimiert wird, wer¬ den bestimmte Spleißvarianten von CD44 (CD44v) nur auf einer Untergruppe von Epithel¬ zellen exprimiert. Die CD44- Varianten werden durch alternatives Spleißen so erzeugt, daß die Sequenzen von 10 Exons (vl-vlO) in CD44s komplett ausgeschnitten werden, jedoch in den größeren Varianten in verschiedenen Kombinationen vorkommen können (Screaton et al., 1992; Tölg et al., 1993; Hofmann et al., 1991). Die Varianten unterscheiden sich dadurch, daß an einer bestimmten Stelle des extrazellulären Teils des Proteins unterschied¬ liche A inosäuresequenzen inseriert sind. Solche Varianten konnten in verschiedenen menschlichen Tumorzellen und in menschlichem Tumorgewebe nachgewiesen werden. So wurde kürzlich die Expression von CD44- Varianten im Verlauf der kolorektalen Karzinoge- nese untersucht (Heider et al, 1993). Die Expression von CD44- Varianten fehlt in norma¬ lem menschlichem Kolonepithel und nur eine schwache Expression ist in den proliferieren- den Zellen der Krypten nachweisbar. In späteren Stadien der Tumorprogression, z.B. in Adenokarzinomen, exprimieren alle malignen Entartungen Varianten von CD44. Gewebs¬ expression von variantem CD44 auf hohem Niveau konnte auch in aggressiven Non- Hodgkin-Lymphomen gezeigt werden (Koop an et al., 1993).35 Behavior both in a non-metastatic pancreatic adenocarcinoma cell line in the rat and in a non-metastatic fibrosarcoma cell line in the rat (Günthert et ed., 1991). While the smallest CD44 isoform, the standard form CD44s, is ubiquitously expressed in a number of different tissues, including epithelial cells, certain splice variants of CD44 (CD44v) are only expressed on a subset of epithelial cells. The CD44 variants are generated by alternative splicing in such a way that the sequences of 10 exons (vl-vlO) are completely cut out in CD44s, but the larger variants can occur in different combinations (Screaton et al., 1992; Tölg et al. , 1993; Hofmann et al., 1991). The variants differ in that different amino acid sequences are inserted at a certain point in the extracellular part of the protein. Such variants could be detected in various human tumor cells and in human tumor tissue. The expression of CD44 variants in the course of colorectal carcinogenesis was recently examined (Heider et al, 1993). The expression of CD44 variants is absent in normal human colonic epithelium and only weak expression is detectable in the proliferating cells of the crypts. In later stages of tumor progression, for example in adenocarcinomas, all malignancies express variants of CD44. Tissue expression of variant CD44 at a high level could also be demonstrated in aggressive non-Hodgkin's lymphomas (Koop an et al., 1993).
Aufgabe der vorliegenden Erfindung war es, neue Mittel zur Behandlung oder zur /w-v/vo-Diagnostik von Krebserkrankungen bereitzustellen, die auf dem Prinzip der immu- nologischen Erkennung tumorassoziierter Antigene beruhen.The object of the present invention was to provide new agents for the treatment or / w-v / vo diagnosis of cancer which are based on the principle of immunological recognition of tumor-associated antigens.
Diese Aufgabe konnte mit der vorliegenden Erfindung gelöst werden. Die erfin¬ dungsgemäßen Mittel sowie deren Verwendung beruhen auf Antikörpern gegen Epitope, die durch Variante Exons des CD44-Gens kodiert werden, insbesondere von Antikörpern gegen Epitope, die durch das Variante Exon v5 des menschlichen CD44-Gens kodiert wer- den, sowie auf v5-spezifischen antiidiotypischen Antikörpern. Die Antikörper können polyklonal oder monoklonal sein, komplette Immunglobuline, Fab- oder F(ab')2-Fragmente von Immunglobulinen oder andere Derivate, bipezifische, chimäre oder humanisierte Anti¬ körper oder rekombinant hergestellte Antikörper, z.B. single-chain-Antikörper (scFv), Fab- Fragmente, andere Fragmente oder komplette Immunglobuline. Die Antikörper können ohne zusätzliches Agens appliziert werden oder mit einer radioaktiven Substanz, einem zytotoxischen Agens, einem immunmodulierenden Agens, einer Substanz, mit deren Hilfe ein zytotoxisches Agens lokal erzeugt werden kann, einem Kernspinresonanz-Kontrastmittel oder einem anderen detektierbaren Label verknüpft sein. Die Erfindung betrifft ferner die Verwendung von Polypeptiden, die Sequenzen enthalten, die durch das Variante Exon v5 des CD44-Gens kodiert werden, und v5-spezifischen antiidiotypischen Antikörpern als Tumorvakzine. Trägt der Antikörper ein detektierbares Label, kann eine Detektion des Labels zu diagnostischen Zwecken, z.B. Visualisierung des Tumores in vivo (Imaging^, oder bei¬ spielsweise zur radiounterstützen Chirurgie (radioguided surger ) erfolgen.This object was achieved with the present invention. The agents according to the invention and their use are based on antibodies against epitopes which are encoded by variant exons of the CD44 gene, in particular on antibodies against epitopes which are encoded by variant exon v5 of the human CD44 gene, and on v5 -specific anti-idiotypic antibodies. The antibodies can be polyclonal or monoclonal, complete immunoglobulins, Fab or F (ab ') 2 fragments of immunoglobulins or other derivatives, bipecific, chimeric or humanized antibodies or recombinantly produced antibodies, for example single-chain antibodies (scFv) , Fab fragments, other fragments or complete immunoglobulins. The antibodies can be applied without an additional agent or can be linked to a radioactive substance, a cytotoxic agent, an immunomodulating agent, a substance with the aid of which a cytotoxic agent can be generated locally, a magnetic resonance contrast agent or another detectable label. The invention further relates to the use of polypeptides which contain sequences which are encoded by the variant exon v5 of the CD44 gene and v5-specific anti-idiotypic antibodies as tumor vaccines. If the antibody bears a detectable label, the label can be detected for diagnostic purposes, for example visualization of the tumor in vivo (imaging), or for example for radio-assisted surgery (radioguided surger).
Die Nuklein- und Aminosäuresequenz des Varianten Teils des CD44-Gens ist be- kannt (Hofmann et al, 1991; Screaton et al, 1992; Tölg et al, 1993). Die Existenz dege¬ nerierter oder alleler Varianten ist für die Ausführung der Erfindung nicht von Bedeutung; solche Varianten sind daher ausdrücklich mit eingeschlossen. Die Sequenz von Exon v5The nucleic acid and amino acid sequence of the variant part 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. The sequence of exon v5
D V D R N G T T A Y E G N N GAT GTA GAC AGA AAT GGC ACC ACT GCT TAT GAA GGA AAC TGG AACD V D R N G T T A Y E G N N GAT GTA GAC AGA AAT GGC ACC ACT GCT TAT GAA GGA AAC TGG AAC
P E A H P P L I H H E H H E E CCA GAA GCA CAC CCT CCC CTC ATT CAC CAT GAG CAT CAT GAG GAA E E T P H S T S TP E A H P P L I H H E H H E E CCA GAA GCA CAC CCT CCC CTC ATT CAC CAT GAG CAT CAT GAG GAA E E T P H S T S T
GAA GAG ACC CCA CAT TCT ACA AGC ACA AGAA GAG ACC CCA CAT TCT ACA AGC ACA A
ist besonders bevorzugt. Als Mittel, um die Erfindung auszufuhren, können Antikörper dienen, insbesondere solche, die gegen Epitope innerhalb der Sequenz von Exon v5 gerich- tet sind, oder v5-spezifische antiidiotypische Antikörper. Besonders bevorzugt sind mono- klonale Antikörper. Für das erfindungsgemäße Verfahren können jedoch auch polyklonale Antikörper, Fab- oder F(ab')2-Fragmente von Immunglobulinen, rekombinant hergestellte Antikörper oder -fragmente, z.B. single-chain- Antikörper (scFv), bispezifische, chimäre oder humanisierte Antikörper oder äquivalente Moleküle dienen, die Exon-v5-kodierte Epitope spezifisch binden. Die Herstellung von Antikörpern gegen bekannte Aminosäurese¬ quenzen kann nach an sich bekannten Methoden erfolgen (Catty, 1989). Beispielsweise kann ein Peptid dieser Sequenz synthetisch hergestellt und als Antigen in einem Immunisie¬ rungsprotokoll eingesetzt werden. Ein anderer Weg ist die Herstellung eines Fusionspro¬ teins, das die gewünschte Aminosäuresequenz enthält, indem eine Nukleinsäure (die syn- thetisch oder z.B. durch Polymerase-Kettenreaktion (PCR) aus einer geeigneten Probe hergestellt werden kann), die für diese Sequenz kodiert, in einen Expressionsvektor inte¬ griert und das Fusionsprotein in einem Wirtsorganismus exprimiert wird. Das gegebenen¬ falls gereinigte Fusionsprotein kann dann als Antigen in einem Immunisierungsprotokoll eingesetzt und Insert-spezifische Antikörper oder, im Falle monoklonaler Antikörper, Hy- bridome, die insertspezifische Antikörper exprimieren, mit geeigneten Verfahren selektiert werden (Wunderlich et al, 1992). Das komplette CD44-Gen oder ein Fragment davon (z.B. der extrazelluläre Anteil) können auch in einem geeigneten System exprimiert werden, ohne daß die exprimierten Sequenzen mit anderen Peptiden fusioniert sind, dann isoliert und als Antigene in Immunisierungsprotokollen eingesetzt werden. Solche Verfahren sind Stand der Technik. Heider ei al. (1993) und Koopman et al (1993) beschreiben die Herstellung von Antikörpern gegen Variante Epitope von CD44. Statt eines intakten Immunglobulinmoleküls.können auch Fab- oder F(ab')2-Frag- mente oder andere Fragmente verwendet werden (Kreitman et al, 1993). Weiter können chimäre Antikörper verwendet werden, z.B. humanisierte Maus- Antikörper (Shin et al, 1989; Güssow et Seemann, 1991), bispezifische Antikörper (Weiner et al, 1993; Goodwin, s 1989) oder s/«#/e-cAα/w-Antiköφer/Toxin-Fusionsproteine (Friedman et al, 1993). Die Antikörper können aus Seren oder aus Hybridomaüberständen isoliert oder durch rekombi- nante Expression als single-chain-Anύköper (scFv, Johnson et Bird, 1991), komplette oder fragmentarische Immunglobuline, gewonnen werden (Coloma et al, 1992; Nesbit et al, 1992; Barbas et al, 1992). Die Antikörper können allein oder verknüpft mit einem Agens w eingesetzt werden. Soll der Antikörper therapeutisch oder für die Immunszintigraphie verwendet werden kann er mit einem geeigneten radioaktiven Isotop z.B. 131I, 125I, ulIn, 186Re, 90Y, 99mTc oder 211At verknüpft sein. Diese Verknüpfung kann direkt oder über ein Linker-Molekül, z.B. einen Chelatbildner, erfolgen. Methoden der Radiomarkierung von Antikörpern sind Stand der Technik (Larson et al, 1991; Thomas et al, 1989; Greiner etis particularly preferred. Antibodies can be used to carry out the invention, in particular those which are directed against epitopes within the sequence of exon v5, or v5-specific anti-idiotypic antibodies. Monoclonal antibodies are particularly preferred. However, polyclonal antibodies, Fab or F (ab ') 2 fragments of immunoglobulins, recombinantly produced antibodies or fragments, for example single-chain antibodies (scFv), bispecific, chimeric or humanized antibodies or equivalent molecules can also be used for the process according to the invention serve to specifically bind exon v5-encoded epitopes. 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 produced synthetically 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 adding a nucleic acid (which can be prepared synthetically or, for example, by a polymerase chain reaction (PCR) from a suitable sample) which codes for this sequence integrates 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, hybridids which express insert-specific antibodies can be selected using suitable methods (Wunderlich et al, 1992). The entire CD44 gene or a fragment thereof (eg the extracellular portion) can also be expressed in a suitable system without the expressed sequences being fused to other peptides, then isolated and used as antigens in immunization protocols. Such methods are state of the art. Heider ei al. (1993) and Koopman et al (1993) describe the production of antibodies against variant epitopes of CD44. Instead of an intact immunoglobulin molecule, Fab or F (ab ') 2 fragments or other fragments can also be used (Kreitman et al, 1993). Chimeric antibodies can also be used, for example humanized mouse antibodies (Shin et al, 1989; Güssow et Seemann, 1991), bispecific antibodies (Weiner et al, 1993; Goodwin, s 1989) or s / «# / e-cAα / w-antibody / toxin fusion proteins (Friedman et al, 1993). The antibodies can be isolated from sera or from hybridoma supernatants or obtained by recombinant expression as single-chain antibodies (scFv, Johnson et Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al, 1992; Nesbit et al, 1992 ; Barbas et al, 1992). The antibodies can be used alone or linked to an agent w. If the antibody is to be used therapeutically or for immunoscintigraphy, it can be linked to a suitable radioactive isotope, for example 131 I, 125 I, ul In, 186 Re, 90 Y, 99m Tc or 211 At. This linkage can take place directly or via a linker molecule, for example a chelating agent. Methods of radiolabeling antibodies are state of the art (Larson et al, 1991; Thomas et al, 1989; Greiner et
15 al, 1993; Srivastava, 1988; Rhodes et al, 1986). Für die therapeutische Verwendung kann der Antikörper auch mit einem nicht radioaktiven zytotoxischen Agens verknüpft sein. Dies kann ein Zytostatikum (Schrappe et al, 1992) oder ein zytotoxisches Polypeptid, z.B. ein bakterielles oder pflanzliches Toxin, sein (Vitetta et al, 1991; Kreitman et al, 1993). Ein solches zytotoxisches Polypeptid kann kovalent, z.B. über Disulfidbrücken, mit dem15 al, 1993; Srivastava, 1988; Rhodes et al, 1986). For therapeutic use, the antibody can also be linked to a non-radioactive cytotoxic agent. This can be a cytostatic (Schrappe et al, 1992) or a cytotoxic polypeptide, e.g. a bacterial or vegetable toxin (Vitetta et al, 1991; Kreitman et al, 1993). Such a cytotoxic polypeptide can be covalently, e.g. over disulfide bridges with which
20 Antikörper verknüpft sein (Theuer et al, 1993), oder in Form eines Fusionsproteins in einem einkettigen Immunotoxin (Chaudhary et al, 1990; Friedman et al, 1993) mit einem Antikörper verbunden sein. Der Antikörper kann ferner mit einem Zytokin oder einem ande¬ ren immunmodulatorischen Polypeptid verknüpft sein, z.B. mit Tumornekrosefaktor oder Interleukin-2. Der Antikörper kann auch mit einem Agens verknüpft sein, das zwar selbst 5 nicht zytotoxisch ist, jedoch eine zytotoxische Substanz erzeugen kann, z.B. ein Enzym, das die Umwandlung eines inaktiven Vorläufermoleküls (Prodrug) in ein Zytostatikum katalysiert (Wang et al, 1992; Senter etα/., 1989).20 antibodies can be linked (Theuer et al, 1993), or linked to an antibody in the form of a fusion protein in a single-chain immunotoxin (Chaudhary et al, 1990; Friedman et al, 1993). The antibody can also be linked to a cytokine or another immunomodulatory polypeptide, e.g. with tumor necrosis factor or interleukin-2. The antibody may also be linked to an agent which, while not itself 5, is cytotoxic, but can produce a cytotoxic substance, e.g. an enzyme that catalyzes the conversion of an inactive precursor molecule (prodrug) into a cytostatic (Wang et al, 1992; Senter etα /., 1989).
Die rekombinante Expression von Polypeptiden, die v5-kodierte Sequenzen enthal¬ ten, sowie die Herstellung von v5-spezifischen antiidiotypischen Antikörpern können mit 0 Methoden aus dem Stand der Technik durchgeführt werden (Sambrook et al, 1989; Briles et Kearney, 1985).The recombinant expression of polypeptides which contain v5-encoded sequences and the production of v5-specific antiidiotypic antibodies can be carried out using 0 methods from the prior art (Sambrook et al, 1989; Briles et Kearney, 1985).
Die erfindungsgemäße Verwendung der Antikörper gegen Variante CD44-Epitope kann durch systemische oder topische Applikation erfolgen, beispielsweise durch intravenö¬ se (als Bolus oder Dauerinfusion), intraperitoneale, intramuskuläre, subkutane o.a. Injek- 5 tion/Infüsion. Es können auch einzelne Organe oder Glieder perfundiert werden. Protokolle für die Verabreichung von konjugierten oder nichtkonjugerten Antikörpern (sei es als kom¬ plette Immunglobuline, Fragmente, rekombinante chimäre Moleküle o.a.) sind Stand der Technik (Mulshine et al, 1991; Larson et al, 1991; Vitetta et Thorpe , 1991; Vitetta et al, 1991; Breitz et al, 1992; Press et al, 1989; Weiner et al, 1989; Chatal et al, 1989; Sears et al, 1982).The antibodies according to the invention against variant CD44 epitopes can be used by systemic or topical application, for example by intravenous se (as a bolus or continuous infusion), intraperitoneal, intramuscular, subcutaneous or injection or infusion. Individual organs or limbs can also be perfused. Protocols for the administration of conjugated or non-conjugated antibodies (whether as complete immunoglobulins, fragments, recombinant chimeric molecules or the like) are state of the art (Mulshine et al, 1991; Larson et al, 1991; Vitetta et Thorpe, 1991; Vitetta et al, 1991; Breitz et al, 1992; Press et al, 1989; Weiner et al, 1989; Chatal et al, 1989; Sears et al, 1982).
Neben der therapeutischen Behandlung von Krebserkrankungen eignen sich die er¬ findungsgemäßen Mittel zur tn-v/vo-Diagnostik von Tumoren. Für die Verwendung von mit radioaktiven Isotopen konjugierten Antikörpern zur Immunszintigraphie (Imaging) gibt es ebenfalls eine Reihe von Protokollen auf deren Grundlage der Fachmann die Erfindung ausführen kann (Siccardi et al, 1989; Keenan et al, 1987; Perkins et Pimm, 1992; Colcher et al, 1987; Thompson et al, 1984).In addition to the therapeutic treatment of cancer, the agents according to the invention are suitable for the tn-v / vo diagnosis of tumors. For the use of antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging ) there are also a number of protocols on the basis of which the person skilled in the art can carry out the invention (Siccardi et al, 1989; Keenan et al, 1987; Perkins et Pimm, 1992; Colcher et al, 1987; Thompson et al, 1984).
Rekombinante Polypeptide, die durch Exon v5 kodierte Aminosäuresequenzen ent- halten, sowie v5-spezifische antiidiotypische Antikörper können als Tumorvakzine einge¬ setzt werden. Antiidiotypische Antikörper erkennen Epitope innerhalb der variablen Regio¬ nen von Immunglobulinen (Idiotope). Unter v5 -spezifischen antiidiotypischen Antikörpern sind Antikörper zu verstehen, die gegen Idiotope von Antikörpern gerichtet sind, die v5- kodierte Aminosäuresequenzen erkennen. Setzt man v5-spezifische antiidiotypische Anti- körper als Antigene in einem Immunisierungsprotokoll ein, werden gegen diese Antigene Antikörper gebildet, die zum Großteil auch an v5-kodierte Epitope binden. Polypeptide, die v5-kodierte Sequenzen enthalten, und v5-spezifische antiidiotypische Antikörper kann man also zur Immunisierung von Tumorpatienten verwenden. Die Antikörper, die dabei im Or¬ ganismus des Patienten gegen v5-kodierte Epitope gebildet werden, erkennen Tumorzellen, die diese Epitope exprimieren, und unterstützen so die immunologische Abwehr des Tumors durch den Organismus.Recombinant polypeptides, which contain amino acid sequences encoded by exon v5, and v5-specific anti-idiotypic antibodies can be used as tumor vaccines. Antiidiotypic antibodies recognize epitopes within the variable regions of immunoglobulins (idiotopes). V5 -specific antiidiotypic antibodies are understood to be antibodies which are directed against idiotopes of antibodies which recognize v5-encoded amino acid sequences. If v5-specific anti-idiotypic antibodies are used as antigens in an immunization protocol, antibodies are formed against these antigens, most of which also bind to v5-coded epitopes. Polypeptides containing v5-encoded sequences and v5-specific anti-idiotypic antibodies can therefore be used to immunize tumor patients. The antibodies which are formed in the patient's organism against v5-coded epitopes recognize tumor cells which express these epitopes and thus support the immune defense of the tumor by the organism.
Variante CD44-Moleküle sind tumorassoziierte Antigene, die sich sehr gut als Tar¬ gets für die Immuntherapie und -szintigraphie von -Krebserkrankungen eignen. Besonders gut eignen sich dabei Variante CD44-Moleküle, die die durch das variable Exon v5 kodierte Aminosäuresequenz, eine allele Variante oder ein Fragment dieser Sequenz enthalten, sowie v5-spezifische antiidiotypische Antikörper.Variant CD44 molecules are tumor-associated antigens which are very suitable as targets for the immunotherapy and scintigraphy of cancer. Variant CD44 molecules which contain the amino acid sequence encoded by the variable exon v5, an allelic variant or a fragment of this sequence and v5-specific anti-idiotypic antibodies are particularly suitable.
Immunhistochemische Untersuchungen mit monoklonalen Antikörpern gegen ver¬ schiedene CD44- Varianten an menschlichem Normal- und Tumorgewebe zeigen, daß viele Tumoren CD44v hoch exprimieren, während dies die entsprechenden Normalgewebe meist nur schwach oder nicht tun.Immunohistochemical investigations with monoclonal antibodies against various CD44 variants on human normal and tumor tissue show that many tumors express CD44v highly, whereas the corresponding normal tissues usually do so only weakly or not.
Die Feinanalyse der Expression verschiedener CD44- Varianten zeigt, daß nicht alle Exons gleichartig in Tumoren überexprimiert sind. Tabelle 1 zeigt eine Untersuchung an Magentumoren von insgesamt 42 Patienten. In 42/42 Fällen ist eine Expression von Varian¬ ten CD44-Molekülen nachweisbar (polyklonales Antiserum gegen die Aminosäuresequen- zen der Exons v3-vl0). Untersuchungen mit Exon-spezifischen monoklonalen Antikörpern zeigt dann, daß Exons v3/v4, v7, v8-vl0 nicht und v6 nur in 26/42 Tumoren (62%) expri¬ miert wird. v5 dagegen wird in 39/42 Tumoren (93%) exprimiert. Im Gegensatz zu v6 las¬ sen sich mit v5-spezifischen Antikörpern sowohl Magentumoren vom diffusen (14/17) als auch vom intestinalen Typ (25/25) erfassen. In Tumoren, die beide Exons exprimieren, ist die Expression von v5 meist stärker als die von v6 (Tabelle 2).The fine analysis of the expression of different CD44 variants shows that not all exons are overexpressed equally in tumors. Table 1 shows an investigation of gastric tumors from a total of 42 patients. In 42/42 cases, expression of variants CD44 molecules can be detected (polyclonal antiserum against the amino acid sequences of the exons v3-vl0). Examinations with exon-specific monoclonal antibodies then show that exons v3 / v4, v7, v8-vl0 are not and v6 is only expressed in 26/42 tumors (62%). v5, on the other hand, is expressed in 39/42 tumors (93%). In contrast to v6, both gastric tumors of the diffuse (14/17) and also of the intestinal type (25/25). In tumors that express both exons, the expression of v5 is usually stronger than that of v6 (Table 2).
Abb. 3 stellt die Ergebnisse einer Studie an 39 kolorektalen Karzinomen dar. Im Gegensatz zu allen anderen Exons wird v5 im Normalgewebe überhaupt nicht, im Tumor- gewebe dagegen bereits im frühesten Tumorstadium (frühes Adenom) in über 80% der Tumoren exprimiert. v3-Expression wurde überhaupt nicht, v6-Expression erst in späten Tumorstadien deutlich zunehmend und v8-10 bereits im Normalgewebe exprimiert nach¬ gewiesen. Auch in späteren Stadien der kolorektalen Tumorprogression wird v5 häufiger und in stärkerem Ausmaß exprimiert als v6. Eine Untersuchung von Brusttumoren (62 invasive Karzinome, 4 tw-s/tw-Karzinome,Fig. 3 shows the results of a study of 39 colorectal carcinomas. In contrast to all other exons, v5 is not expressed in normal tissue at all, whereas in tumor tissue it is expressed in over 80% of the tumors in the earliest stage of the tumor (early adenoma). v3 expression was not detected at all, v6 expression only increased significantly in late tumor stages and v8-10 was already expressed in normal tissue. Even in later stages of colorectal tumor progression, v5 is expressed more frequently and to a greater extent than v6. An examination of breast tumors (62 invasive carcinomas, 4 tw-s / tw-carcinomas,
9 lokale Rezidive, 16 Lymphknotenmetastasen) ergab ebenfalls, das v5 in diesen Tumoren sehr häufig exprimiert wird (82-100%), häufiger als v3 oder v6 (Tabelle 3).9 local recurrences, 16 lymph node metastases) also showed that v5 is expressed very frequently in these tumors (82-100%), more frequently than v3 or v6 (Table 3).
Für Verfahren der Immuntherapie und -szintigraphie ist es wichtig, daß das tumoras¬ soziierte Antigen, das das Target des Antikörpers darstellt, in Tumor-, nicht jedoch in Nor- malgewebe exprimiert, andererseits aber in einer möglichst großen Zahl von Tumoren in einem möglichst frühen Tumorstadium möglichst hoch exprimiert wird, um eine breite und zuverlässige Anwendung zu erlauben. Überraschenderweise erfüllen CD44- Varianten, die durch Exon v5 kodierte Aminosäuresequenzen enthalten, diese Voraussetzungen in hohem Maße. Die erfindungsgemäßen Mittel und Verwendungen eignen sich somit hervorragend zur Immuntherapie und -v/vo-Diagnostik/Immunszintigraphie von Tumoren, insbesondere von Karzinomen. For methods of immunotherapy and scintigraphy, it is important that the tumor-associated antigen, which is the target of the antibody, is expressed in tumor tissue, but not in normal tissue, but on the other hand in the largest possible number of tumors as early as possible Tumor stage is expressed as high as possible to allow wide and reliable use. Surprisingly, CD44 variants that contain amino acid sequences encoded by exon v5 meet these requirements to a high degree. The agents and uses according to the invention are thus outstandingly suitable for immunotherapy and v / vo diagnosis / immunoscintigraphy of tumors, in particular carcinomas.
Tabelle 1: Expression von Varianten CD44-Epitopen auf den Zelloberflächen von Magen¬ tumorenTable 1: Expression of variants CD44 epitopes on the cell surfaces of gastric tumors
Serum1 Monoklonale AntiköφerSerum 1 monoclonal antibodies
Adenokarzinome αCD44v VFF11 VFF8 VFF4 VFF9 VFF14 (v3-vl0) (v3/4) (v5) (v6) (v7) (v8-10)Adenocarcinomas αCD44v VFF11 VFF8 VFF4 VFF9 VFF14 (v3-vl0) (v3 / 4) (v5) (v6) (v7) (v8-10)
Diffuser Typ 17/172 0/17 14/17 3/17 0/17 0/17 Intestinaler Typ 25/25 0/25 25/25 23/25 0/25 0/25 Gesamt 42/42 0/42 39/42 26/42 0/42 0/42Diffuser type 17/17 2 0/17 14/17 3/17 0/17 0/17 intestinal type 25/25 0/25 25/25 23/25 0/25 0/25 total 42/42 0/42 39 / 42 26/42 0/42 0/42
1 polyklonales Antisenim 1 polyclonal antisenim
2 Zahl positiver Tumoren/Zahl der untersuchten Tumoren Tabelle 2: Expression von Varianten CD44-Epitopen in Primärtumoren des Magens und den entsprechenden Lymphknotenmetastasen 2 Number of positive tumors / number of tumors examined Table 2: Expression of variants CD44 epitopes in primary tumors of the stomach and the corresponding lymph node metastases
Antiköφerspezifität polyklonales Serum monoklonale Antiköφer anti CD44v VFF8 (v5) VFF4 (v6)Antibody specificity polyclonal serum monoclonal antibodies anti CD44v VFF8 (v5) VFF4 (v6)
Adenokarzinome3 Intensität pos. Zellen Intensität pos. Zellen Intensität pos. ZellenAdenocarcinomas 3 intensity pos. Cells intensity pos. Cells intensity pos. Cells
Diffuser TypDiffuse type
645/89 ++4 100V1006 -H- 20/90 - 0/0645/89 ++ 4 100V100 6 -H- 20/90 - 0/0
12589/89 ++ 40/50 ++ 80/90 - 0/012589/89 ++ 40/50 ++ 80/90 - 0/0
12924/89 ++ 70/80 ++ 40/30 - 0/012924/89 ++ 70/80 ++ 40/30 - 0/0
25501/89 ++ 90/70 ++ 70/70 -H- 30/3025501/89 ++ 90/70 ++ 70/70 -H- 30/30
33383/89 ++ 80/30 ++ 40/20 - 0/033383/89 ++ 80/30 ++ 40/20 - 0/0
Intestinaler TypIntestinal type
32761/88 ++ 60/10 + 10/20 + 20/9032761/88 ++ 60/10 + 10/20 + 20/90
33295/88 ++ 90/10 ++ 80/60 + 40/4033295/88 ++ 90/10 ++ 80/60 + 40/40
9891/89 +++ 100/80 ++ 70/70 -H- 20/59891/89 +++ 100/80 ++ 70/70 -H- 20/5
18352/89 +++ 90/90 -H-+ 90/80 ++ 70/5018352/89 +++ 90/90 -H- + 90/80 ++ 70/50
9069/90 ++ 90/90 ++ 60/60 + 20/809069/90 ++ 90/90 ++ 60/60 + 20/80
3 Die Zahlen beziehen sich auf Tumoren und die entsprechenden Lymphknotenmetastasen (nicht getrennt angezeigt). Die Tumoren sind in der Sammlung enthalten, die in Tabelle 1 dargestellt wurde. 3 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.
4 Intensität (weil es keinen Unterschied in der Intensität zwischen Primärtumor und Lymphknotenmetastase gab, ist hier die gemeinsame Färbung angezeigt: - negativ, + schwach, ++ mäßig, +++ stark 4 Intensity (because there was no difference in intensity between the primary tumor and lymph node metastasis, the common color is shown here: - negative, + weak, ++ moderate, +++ strong
5 Prozentsatz positiver Tumorzellen im Primärtumor 5 Percentage of positive tumor cells in the primary tumor
6 Prozentsatz positiver Tumorzellen in einer Lymphknotenmetastase des gleichen Patienten Tabelle 3: Expression von CD44- Varianten in primären in-situ- und invasiven Brusttumo¬ ren, Rezidiven und Lymphknotenmetastasen (Zahl positiver Proben). 6 Percentage of positive tumor cells in a lymph node metastasis from the same patient Table 3: Expression of CD44 variants in primary in-situ and invasive breast tumors, recurrences and lymph node metastases (number of positive samples).
Antiköφer Karzinome invasive lokale Rezidive Lymphknoten¬ in situ Karzinome (n=9) metastasenAntibody carcinoma invasive local recurrence Lymph node in situ carcinoma (n = 9) metastases
(n=4) (n=62) (n=16)(n = 4) (n = 62) (n = 16)
α-CD44v (v3-vlO) 4 (100%) 57 (92%) 8 ( 89%) 16 (100%) α-DI (v3) 2 ( 50%) 33 (53%) 4 ( 44%) 16 (100%) α-DIII (v6,v7) 4 (100%) 51 (82%) 9 (100%) 16 (100%)α-CD44v (v3-vlO) 4 (100%) 57 (92%) 8 (89%) 16 (100%) α-DI (v3) 2 (50%) 33 (53%) 4 (44%) 16 (100%) α-DIII (v6, v7) 4 (100%) 51 (82%) 9 (100%) 16 (100%)
VFF8 (v5) 4 (100%) 51 (82%) 8 ( 89%) 16 (100%)VFF8 (v5) 4 (100%) 51 (82%) 8 (89%) 16 (100%)
VFF7 (v6) 4 (100%) 46 (74%) 6 ( 66%) 15 (100%)7 VFF7 (v6) 4 (100%) 46 (74%) 6 (66%) 15 (100%) 7
AbbildungenIllustrations
Fig. 1: Schematische Darstellung einer CD44-Spleißvariante. Diese beispielhafteFig. 1: Schematic representation of a CD44 splice variant. This exemplary
Variante trägt alle Varianten Exonsequenzen an der einzigen Insertionsstelle. DunkelgraueVariant carries all variants of exon sequences at the single insertion site. Dark gray
Kästen symbolisieren CD44-Standardsequenzen (CD44s). Die Lokalisierung der Epitope der monoklonalen Antiköφer VFF4, VFF7, VFF8, VFF9, VFFl l, VFF14, VFF16 undBoxes symbolize standard CD44 sequences (CD44s). The localization of the epitopes of the monoclonal antibodies VFF4, VFF7, VFF8, VFF9, VFF11, VFF14, VFF16 and
VFF18 ist durch Balken angezeigt. Alle monoklonalen Antiköφer sind exonspezifisch.VFF18 is indicated by bars. All monoclonal antibodies are exon-specific.
Fig. 2: Immunhistochemie von normaler Mucosa sowie Adenokarzinomen des Ma¬ gens. Eine fokal betonte anti-CD44v-positive Reaktion zeigt sich in Tumorzellen eines mäßig differenzierten Adenokarzinoms (intestinaler Typ nach Lauren) des Magens (a) sowie in einer regionalen Lymphknotenmetastase (b). In normaler Mucosa des Magens mit chroni¬ scher Gastritis reagieren die Foci von intestinalen Metaplasien positiv mit mAb VFF4 (c, Pfeile) sowie mit mAb VFF8 (d, Pfeile), begleitet von einer zusätzlichen Reaktion auf der mucoiden Oberfläche und dem foveolären Epithel (d, Pfeilspitzen). Fast alle Becherzellkar- zinome des Magens (diffuser Typ nach Lauren) zeigen eine negative Reaktion mit mAb VFF4 (e), und im Gegensatz zu Adenokarzinomen des intestinalen Typs ist das normale mucoide Epithel negativ (e, Pfeilspitzen). In den meisten Fällen zeigt sich in diesen Becher¬ zellkarzinomen eine positive Reaktion mit mAb VFF8 (f), und auch das verbliebene normale mucoide Epithel zeigt Immunreaktivität (f, Pfeilspitzen). (ABC-Methode a, b: anti-CD44v polyklonales Serum, 140 x; c: VFF4, 80x; d:2: Immunohistochemistry of normal mucosa and adenocarcinoma of the stomach. A focal stressed anti-CD44v-positive reaction is seen in tumor cells of a moderately differentiated adenocarcinoma (Lauren intestinal type) of the stomach (a) and in a regional lymph node metastasis (b). In normal gastric mucosa with chronic gastritis, 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). Almost all goblet carcinomas of the stomach (diffuse type according to Lauren) show a negative reaction with mAb VFF4 (e), and in contrast to adenocarcinomas of the intestinal type, the normal mucoid epithelium is negative (e, arrowheads). In most cases, these goblet cell carcinomas show a positive reaction with mAb VFF8 (f), and the remaining normal mucoid epithelium also shows immunoreactivity (f, arrowheads). (ABC method a, b: anti-CD44v polyclonal serum, 140 x; c: VFF4, 80x; d:
VFF8, 80x; e: VFF4, 210x; f: VFF8, 210x; Gegenfärbung Hämatoxylin).VFF8, 80x; e: VFF4, 210x; f: VFF8, 210x; Counterstaining hematoxylin).
Fig. 3: Expression varianter CD44-Exons in verschiedenen Stadien der kolorektalen Tumoφrogression. Ergebnisse aus immunhistochemischen Anfärbungen von Gewebs- schnitten (Beispiel 2). Dunkelgraue Balken zeigen den Prozentsatz positiver Tumoren an. Hellgraue Balken zeigen Proben mit nur fokaler Anfarbung an. Fig. 3: Expression of variant CD44 exons in different stages of the colorectal tumor progression. Results from immunohistochemical staining of tissue sections (Example 2). Dark gray bars indicate the percentage of positive tumors. Light gray bars show samples with only focal staining.
BeispieleExamples
Tumoren und GewebeTumors and tissues
Kolon: Normale und pathologische Gewebe wurden aus den Beständen der Abtei- lung für Pathologie, Academic Medical Center, Universität Amsterdam, Niederlande, ent¬ nommen. Kolorektale Karzinome (n=39) wurden nach der Klassifikation von Dukes (1937, 1980) in Stadien eingeteilt, in Dukes A (n=9), Krankheit beschränkt auf die Darmwand; Dukes B (n=14), Ausdehnung über die Muskelschicht hinaus ohne Metastasierung; Dukes C/D (n=16), Tumoren mit regionalen bzw. Fernmetastasen. Adenome wurden unterteilt in frühe Adenome (Durchmesser < 1 cm, n=l 1) und späte Adenome (Durchmesser > 1 cm, n=12) und wurden als niedrig oder hoch differenziert nach Standardkriterien gradiert.Colon: Normal and pathological tissues were taken from the stocks of the Department of Pathology, Academic Medical Center, University of Amsterdam, The Netherlands. Colorectal carcinomas (n = 39) were classified according to the classification by Dukes (1937, 1980), in Dukes A (n = 9), disease restricted to the intestinal wall; Dukes B (n = 14), extension beyond the muscle layer without metastasis; Dukes C / D (n = 16), tumors with regional or distant metastases. Adenomas were divided into early adenomas (diameter <1 cm, n = l 1) and late adenomas (diameter> 1 cm, n = 12) and were graded as low or high differentiated according to standard criteria.
Magen: Tumoφroben und Normalgewebe wurden aus den Beständen der Abteilung Pathologie der Universität Würzburg, Deutschland, ausgewählt. Die Proben waren unmit¬ telbar nach der chirurgischen Entnahme schockgefroren und bis zur Verwendung bei -80°C gelagert worden. Normalgewebe wurde von zwölf verschiedenen Tumor-Patienten sowohl aus der Koφus- als auch aus der Antrumregion des Magens entnommen. Pathologische Ge¬ webe wurden von einer Gesamtzahl von 47 Patienten mit einem Durchschnittsalter von 63 Jahren erhalten. Von den Primärkarzinomen gehörten 29 zum intestinalen und 18 zum diffu¬ sen Typ nach Lauren (1965). Die Tumorstadien reichten von lokalisiert (pTl) bis ausge- dehnt (pT4), die histologische Gradierung von gut differenzierten (Gl) bis schlecht diffe¬ renzierten (G3) Adenokarzinomen.Stomach: 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.
Mamma: Gefrorene Gewebe (aufbewahrt bei -70°C) wurden von der Universitäts¬ frauenklinik Heidelberg, Deutschland, erhalten. Eingeschlossen waren 62 Proben von primären Mammakarzinomen, 9 lokale Rezidive von Mammakarzinomen, 4 Fälle von reinen w-s/tw-Karzinomen und 16 axillare Lymphknotenmetastasen (von den gleichen Patienten wie die Primärtumoren). Die Fälle wurden zufallig ausgewählt und schlössen eine repräsen¬ tative Auswahl von histologischen Tumortypen, Stadien und Gradierungen ein. Zum Ver¬ gleich wurden Proben von normalem Brustgewebe, duktalen Hypeφlasien und Fibroade- nomen ausgewählt. Beispiel 1: Herstellung der Antikörper gegen Epitope, die von Varianten Exonsequen- zen des CD44-Gens kodiert werdenBreast: Frozen tissues (stored at -70 ° C) were obtained from the University Women's Clinic in Heidelberg, Germany. 62 samples of primary breast cancer, 9 local recurrences of breast cancer, 4 cases of pure ws / tw cancer and 16 axillary lymph node metastases (from the same patients as the primary tumors) were included. The cases were chosen randomly and included a representative selection of histological tumor types, stages and gradings. For comparison, samples of normal breast tissue, ductal hypoplastic and fibroadenoma were selected. Example 1: Production of the antibodies against epitopes which are encoded by variant exon sequences of the CD44 gene
Klonierung von pGEX-Fusionsproteinen s Die gesamte Variante Region des HPKII-Typs von CD44v (Hofmann et al, 1991) wurde aus menschlicher Keratinozyten-cDNA durch Polymerase-Kettenreaktion (PCR) amplifiziert. Die beiden PCR-Primer 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3', Positionen 25-52, und 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3', Positionen 1013-984 der LCLC97-varianten Region, wie von Hofmann et al. beschrieben, enthielten w eine EcoRI-Erkennungsstelle, die benutzt wurde, um das PCR-Produkt direkt in den Vektor pGEX-2T (Smith et al, 1988) zu klonieren. Das resultierende Konstrukt (pGEX CD44v HPKII, v3-vl0) kodiert für ein Fusionsprotein von ~70 kD. Das Fusionsprotein wurde in E. coli exprimiert und anschließend über Glutathion-Agarose affinitätsgereinigt (Smith et al, 1988). is Um Subklone der Varianten Regionen zu erhalten, die für Affinitätsreinigungen undCloning of pGEX Fusion Proteins The entire variant region of the HPKII type of CD44v (Hofmann et al, 1991) was amplified from human keratinocyte cDNA by polymerase chain reaction (PCR). The two PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3 ' , positions 25-52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3', positions 1013-984 of the LCLC97-variant region, as described by Hofmann et al. described contained an EcoRI recognition site which was used to clone the PCR product directly into the vector pGEX-2T (Smith et al, 1988). The resulting construct (pGEX CD44v HPKII, v3-vl0) codes for a fusion protein of ~ 70 kD. The fusion protein was expressed in E. coli and then affinity purified using glutathione agarose (Smith et al, 1988). To obtain subclones of the regions that are used for affinity purification and
Western-Blot-Analysen verwendet werden konnten, wurden Fragmente kloniert, die DI (v3), DII/III (v5, v6), und DIII (v6, v7) enthielten, wobei die passenden Restriktionsschnitt¬ stellen verwendet wurden. Fusionsprotein DI enthält die CD44-Sequenz, die von Stamen- kovic et al (1989) beschrieben wurde, von Position 744 bis zur Position 142 der SequenzWestern blot analyzes could be used, fragments were cloned which contained DI (v3), DII / III (v5, v6), and DIII (v6, v7), using the appropriate restriction sites. Fusion protein DI contains the CD44 sequence described by Stamenkovic et al (1989) from position 744 to position 142 of the sequence
20 von variantem CD44, wie sie von Hofinann et al (1991) beschrieben wurde. Fusionsprotein Du/in enthält die Variante Sequenz von Position 290-460, Fusionsprotein DIII die Variante Sequenz von Position 378-638 (Hofmann et al, 1991). Die DI und Dm enthaltenden Fragmente wurden in das pGEX- Vektorsystem, das DII/ÜI-Fragment in den pATH- Vektor (Angel et al, 1988) kloniert.20 of variant CD44 as described by Hofinann et al (1991). Fusion protein Du / in contains the variant sequence from position 290-460, fusion protein DIII the variant sequence from position 378-638 (Hofmann et al, 1991). The fragments containing DI and Dm were cloned into the pGEX vector system, the DII / ÜI fragment into the pATH vector (Angel et al, 1988).
2525
Polyklonales AntiserumPolyclonal antiserum
Die Herstellung und Reinigung des polyklonalen Antiserums gegen die Variante Re¬ gion des CD44-Moleküls ist in der Literatur beschrieben (Heider et al, 1993).The preparation and purification of the polyclonal antiserum against the variant region of the CD44 molecule has been described in the literature (Heider et al, 1993).
0 Monoklonale Antikörper0 monoclonal antibodies
Weibliche BALB/c-Mäuse wurden mit affinitätsgereinigtem Fusionsprotein immuni¬ siert, das aus pGEX CD44v HPKII (Exons v3-vl0) wie oben beschrieben erhalten wurde. Milzzellen eine Tieres mit hohem Antiköφertiter wurde mit P3X63Ag8.653-Myelomzellen unter Verwendung von Polyethylenglykol 4000 fusioniert. Hybridome wurden in HAT- 35 Medium selektiert (Kearney et al, 1979). Bestimmung der Antiköφertiter im Serum sowie das Antiköφerscreening wurden mittels ELISA durchgeführt. Die Microtiteφlatten wurden mit Fusionsprotein beschichtet, mit seriellen Verdünnungen von Serumproben oder Hybridomaüberständen inkubiert, und spezifische Antiköφer wurden mit Peroxidase- gekoppelten Antiköφern gegen Maus-IgG detektiert. Hybridome, die mit Glutathion- Transferase reagierten, wurden eliminiert. Die verbleibenden Antiköφer wurden mit ELISA-Tests weiter charakterisiert, wobei Fusionsproteine der variablen Domänen DI (Exon v3), DII III (Exons v5, v6),. DIII (Exons v6, v7), DI-IV (Exons v3-v8), DIII- VI (Exons v7-vl0) bzw. v6 (Exon v6) [v5 v6 v7] verwendet wurden. Die Reaktivität der Anti¬ köφer mit menschlichen Hautkeratinozyten wurde immunhistochemisch untersucht.Female BALB / c mice were immunized with affinity-purified fusion protein which was obtained from pGEX CD44v HPKII (exons v3-vl0) as described above. Spleen cells from an animal with a high antibody titer were fused with P3X63Ag8.653 myeloma cells using polyethylene glycol 4000. Hybridomas were selected in HAT-35 medium (Kearney et al, 1979). Determination of the antibody titer in the serum and the antibody screening were carried out by means of ELISA. The microtitre plates were coated with fusion protein, with serial dilutions of serum samples or Hybridoma supernatants were incubated, and specific antibodies were detected with peroxidase-coupled antibodies against mouse IgG. Hybridomas that reacted with glutathione transferase were eliminated. The remaining antibodies were further characterized by ELISA tests, fusion proteins of the variable domains DI (exon v3), DII III (exons v5, v6) ,. DIII (exons v6, v7), DI-IV (exons v3-v8), DIII-VI (exons v7-vl0) and v6 (exon v6) [v5 v6 v7] were used. The reactivity of the antibodies with human skin keratinocytes was examined immunohistochemically.
Die Exonspezifität verschiedener verwendeter monoklonaler Antiköφer (VFF4, VFF7, VFF8, VFF9 VFFl l, VFF14, VFF 16 und VFF18) ist in Fig.l dargestellt.The exon specificity of various monoclonal antibodies used (VFF4, VFF7, VFF8, VFF9, VFFl l, VFF14, VFF 16 and VFF18) is shown in Fig.l.
Beispiel 2: ImmunhistochemieExample 2: Immunohistochemistry
Gefrierschnitte wurden in eisgekühltem Methanol 10 min. fixiert, in PBS (8 g/1 NaCl, 0.2 g/1 KCl, 1.44 g/1 0.24 g/1 KH2PO4, pH 7.4) gewaschen und mit norma¬ lem Ziegenserum (10% in PBS) präinkubiert. Dann wurden sie 3x mit PBS gewaschen und für 1 Stunde mit dem Primärantiköφer (in PBS, 1% BSA) inkubiert. Endogene Peroxidase wurde mit 0.3% H^ in Methanol blockiert und die Schnitte mit biotinyliertem Zweitanti- köφer (entweder anti-Maus oder anti-Kaninchen F(ab')2, DAKO Coφ., Santa Barbara, CA, USA, abhängig vom verwendeten Primärantiköφer) inkubiert. Der Immunkomplex wurde mit Meerrettich-Peroxidase visualisiert, die als Streptavidin-Biotin-Peroxidasekomplex an Biotin gekoppelt wurde (DAKO). Nach dreißigminütiger Inkubation mit dem Streptavidin- Biotin-Peroxidase-Komplex wurden die Schnitte mit 3,3-Amino-9-ethyl-carbazol (Sigma Chemicals, Deisenhofen, Deutschland) für 5 bis 10 min. entwickelt und die Reaktion mit HjO abgestoppt. Die Zellen wurden mit Hämatoxylin gegengefarbt, mit Glyzerin-Gelatine eingedeckelt und mikroskopisch untersucht.Frozen sections were frozen in ice-cold methanol for 10 min. fixed, in PBS (8 g / 1 NaCl, 0.2 g / 1 KCl, 1.44 g / 1 0.24 g / 1 KH 2 PO 4 , pH 7.4) and preincubated with normal goat serum (10% in PBS). Then they were washed 3 times with PBS and incubated for 1 hour with the primary antibody (in PBS, 1% BSA). Endogenous peroxidase was blocked with 0.3% H ^ in methanol and the sections with biotinylated second antibody (either anti-mouse or anti-rabbit F (ab ') 2 , DAKO Coφ., Santa Barbara, CA, USA, depending on the primary antibody used ) incubated. The immune complex was visualized with horseradish peroxidase, which was coupled to biotin as a streptavidin-biotin-peroxidase complex (DAKO). After 30 minutes of incubation with the streptavidin-biotin-peroxidase complex, the sections were treated with 3,3-amino-9-ethyl-carbazole (Sigma Chemicals, Deisenhofen, Germany) for 5 to 10 min. developed and the reaction with HjO stopped. The cells were counterstained with hematoxylin, capped with glycerin gelatin and examined microscopically.
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Claims

Patentansprüche claims
1. Verwendung eines Antiköφers gegen ein Epitop, das durch mindestens ein vari- antes Exon des CD44-Gens kodiert wird, zur selektiven Bindung an Tumoren in vivo.1. Use of an antibody against an epitope, which is encoded by at least one variant exon of the CD44 gene, for selective binding to tumors in vivo.
2. Verwendung eines Antiköφers nach Anspruch 1, dadurch gekennzeichnet, daß das Variante Exon Exon v5 ist.2. Use of an Antiköφers according to claim 1, characterized in that the variant is exon exon v5.
3. Verwendung eines -Antiköφers nach Anspruch 2, dadurch gekennzeichnet, daß das Exon v5 für die Aminosäuresequenz3. Use of a -Antiköφers according to claim 2, characterized in that the exon v5 for the amino acid sequence
DVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEETPHSTSTDVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEETPHSTST
oder eine allele Variante oder ein Fragment dieser Sequenz kodiert.or encodes an allelic variant or a fragment of this sequence.
4. Verwendung eines Antiköφers nach Anspruch 3, dadurch gekennzeichnet, daß das Exon v5 die Nukleinsäuresequenz4. Use of an Antiköφers according to claim 3, characterized in that the exon v5 the nucleic acid sequence
GATGTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACACCCTC CCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAAGATGTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACACCCTC CCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAA
oder eine degenerierte oder allele Variante oder ein Fragment dieser Sequenz enthält.or contains a degenerate or allelic variant or a fragment of this sequence.
5. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 4, dadurch gekenn- zeichnet, daß der Antiköφer monoklonal ist.5. Use of an Antiköφer according to claims 1 to 4, characterized in that the Antiköφer is monoclonal.
6. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 4, dadurch gekenn¬ zeichnet, daß der Antiköφer das Fab- oder F(ab '^-Fragment eines Immunglobulins, ein rekombinant hergestellter Antiköφer oder ein rekombinant hergestelltes Antiköφerfrag- ment, ein rekombinant hergestellter single-chain-Anύkörpeτ (scFv), ein chimärer, bispezifi¬ scher oder humanisierter Antiköφer ist.6. Use of an Antiköφer according to claims 1 to 4, characterized gekenn¬ characterized in that the Antiköφer the Fab or F (from '^ fragment of an immunoglobulin, a recombinantly produced Antiköφer or a recombinantly produced Antiköφerfragment, a recombinantly produced single -chain-Anύkörpeτ (scFv), is a chimeric, bispecific or humanized antibody.
7. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 6, dadurch gekenn¬ zeichnet, daß der Antiköφer mit einem zytotoxischen Agens verknüpft ist.7. Use of an Antiköφer according to claims 1 to 6, characterized gekenn¬ characterized in that the Antiköφer is linked to a cytotoxic agent.
8. Verwendung eines Antiköφers nach Anspruch 7, dadurch gekennzeichnet, daß der Antiköφer mit einem radioaktiven Isotop verknüpft ist. 8. Use of an Antiköφer according to claim 7, characterized in that the Antiköφer is linked to a radioactive isotope.
9. Verwendung eines Antiköφers nach Anspruch 7, dadurch gekennzeichnet, daß der Antiköφer mit einem Toxin verknüpft ist.9. Use of an Antiköφer according to claim 7, characterized in that the Antiköφer is linked to a toxin.
10. Verwendung eines Antiköφers nach Anspruch 7, dadurch gekennzeichnet, daß s der Antiköφer mit einem Zytostatikum verknüpft ist.10. Use of an Antiköφer according to claim 7, characterized in that s the Antiköφer is linked to a cytostatic.
11. Verwendung eines Antiköφers nach Anspruch 7, dadurch gekennzeichnet, daß der Antiköφer mit einem Zytokin verknüpft ist.11. Use of an Antiköφer according to claim 7, characterized in that the Antiköφer is linked to a cytokine.
w 12. Verwendung eines Antiköφers nach Anspruch 7, dadurch gekennzeichnet, daß der Antiköφer mit einem Enzym verknüpft ist, das die Umwandlung eines Vorläufermo¬ leküls (Prodrug) in ein zytotoxisches Agens katalysieren kann.12. Use of an antibody according to claim 7, characterized in that the antibody is linked to an enzyme which can catalyze the conversion of a precursor molecule (prodrug) into a cytotoxic agent.
13. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 12 zur Immunthera- 15 pie von Tumoren.13. Use of an antibody according to claims 1 to 12 for immunotherapy of tumors.
14. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 6 oder 8 zur in-vivo- Diagnostik von Tumoren.14. Use of an Antiköφers according to claims 1 to 6 or 8 for in-vivo diagnosis of tumors.
20 15. Verwendung eines Antiköφers nach den Ansprüchen 1 bis 14, dadurch gekenn¬ zeichnet, daß die Tumoren Karzinome sind.15. Use of an antibody according to claims 1 to 14, characterized in that the tumors are carcinomas.
16. Verwendung eines Antiköφers nach Anspruch 15, dadurch gekennzeichnet, daß die Karzinome Adenokarzinome sind.16. Use of an Antiköφers according to claim 15, characterized in that the carcinomas are adenocarcinomas.
2525
17. Verwendung eines Antiköφers nach Anspruch 15, dadurch gekennzeichnet, daß die Karzinome Magenkarzinome sind.17. Use of an Antiköφers according to claim 15, characterized in that the carcinomas are gastric carcinomas.
18. Verwendung eines Antiköφers nach Anspruch 15, dadurch gekennzeichnet, daß 30 die Karzinome Kolonkarzinome sind.18. Use of an Antiköφers according to claim 15, characterized in that 30 the carcinomas are colon carcinomas.
19. Verwendung eines Antiköφers nach Anspruch 15, dadurch gekennzeichnet, daß die Karzinome Mammakarzinome sind.19. Use of an Antiköφers according to claim 15, characterized in that the carcinomas are breast carcinomas.
35 20. Verwendung eines Antiköφers gegen ein Epitop, das durch das Variante Exon v5 des CD44-Gens kodiert wird, zur Herstellung eines Arzneimittels zur Behandlung von Tumoren. 35 20. Use of an antibody against an epitope, which is encoded by the variant exon v5 of the CD44 gene, for the manufacture of a medicament for the treatment of tumors.
21. Verwendung eines Antiköφers gegen ein Epitop, daß durch das Variante Exon v5 des CD44-Gens kodiert wird, zur Herstellung eines /«-v/'vo-Tumordiagnostikums.21. Use of an antibody against an epitope, which is encoded by the variant exon v5 of the CD44 gene, for the production of a / «- v / ' vo tumor diagnostic.
22. Verwendung eines Polypeptides, das durch Exon v5 des CD44-Gens kodierte Sequenzen enthält, oder eines v5-spezifischen antiidiotypischen Antiköφers als Tumor- vakzin.22. Use of a polypeptide which contains sequences encoded by exon v5 of the CD44 gene or a v5-specific anti-idiotypic antibody as a tumor vaccine.
23. Mittel zur Behandlung von Tumoren, dadurch gekennzeichnet, daß es einen Antiköφer gegen ein Epitop enthält, das durch das Variante Exon v5 des CD44-Gens ko- diert wird.23. Agent for the treatment of tumors, characterized in that it contains an antibody against an epitope which is encoded by the variant exon v5 of the CD44 gene.
24. Mittel zur /w-v/vo-Diagnostik von Tumoren, dadurch gekennzeichnet, daß es einen Antiköφer gegen ein Epitop enthält, das durch das Variante Exon v5 des CD44-Gens kodiert wird.24. Means for / w-v / vo diagnosis of tumors, characterized in that it contains an antibody against an epitope which is encoded by the variant exon v5 of the CD44 gene.
25. Mittel zur Erzeugung von Antiköφern gegen Variante CD44-Moleküle in vivo, dadurch gekennzeichnet, daß es einen oder mehrere v5-spezifische antiidiotypische An¬ tiköφer enthält.25. Means for generating antibodies against variant CD44 molecules in vivo, characterized in that it contains one or more v5-specific anti-idiotypic antibodies.
26. Mittel nach den Ansprüchen 20 bis 25, dadurch gekennzeichnet, daß der Anti¬ köφer monoklonal ist.26. Composition according to claims 20 to 25, characterized in that the anti-body is monoclonal.
27. Mittel nach den Ansprüchen 20 bis 25, dadurch gekennzeichnet, daß der Anti¬ köφer das Fab- oder F(ab')2-Fragment eines Immunglobulins, ein rekombinant hergestellter Antiköφer oder ein rekombinant hergestelltes Antiköφerfragment, ein rekombinant herge¬ stellter single-chain-Anύkörper (scFv), ein chimärer, bispezifischer oder humanisierter Antiköφer ist.27. Composition according to claims 20 to 25, characterized in that the anti-köφer the Fab or F (ab ') 2 fragment of an immunoglobulin, a recombinantly produced antibody or a recombinantly produced antibody fragment, a recombinantly produced single chain antibody (scFv), a chimeric, bispecific or humanized antibody.
28. Mittel nach den Ansprüchen 20 bis 23 oder Anspruch 26, dadurch gekennzeich- net, daß der Antiköφer mit einem radioaktiven Isotop, einem Toxin, einem Zytostatikum, einem Zytokin oder einem anderen zytotoxischen oder immunmodulatorischen Agens oder mit einem Enzym, das die Umwandlung eines Vorläufermoleküls in ein zytotoxisches Agens katalysieren kann, verknüpft ist.28. Agent according to claims 20 to 23 or claim 26, characterized in that the antibody with a radioactive isotope, a toxin, a cytostatic agent, a cytokine or another cytotoxic or immunomodulatory agent or with an enzyme which converts a Can catalyze precursor molecule in a cytotoxic agent, is linked.
29. Mittel nach Anspruch 21 oder 24, dadurch gekennzeichnet, daß der Antiköφer mit einem radioaktiven Isotop oder mit einem Kontrastmittel für die Kernspinresonanz¬ spektroskopie verknüpft ist. 29. A composition according to claim 21 or 24, characterized in that the Antiköφer is linked to a radioactive isotope or to a contrast agent for nuclear magnetic resonance spectroscopy.
30. Antiidiotypischer Antiköφer, dadurch gekennzeichnet, daß er ein Idiotop eines Antiköφers bindet, der Variante CD44-Moleküle bindet, die durch Exon v5 kodierte Se¬ quenzen enthalten. 30. Anti-idiotypic antibody, characterized in that it binds an idiotope of an antibody, the variant binds CD44 molecules which contain sequences encoded by exon v5.
EP94924268A 1993-08-07 1994-07-21 POLYPEPTIDES CODED BY EXON v5 OF THE CD44 GENE AS TARGETS FOR IMMUNOTHERAPY AND IMMUNOSCINTIGRAPHY OF TUMOURS Ceased EP0713398A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4326573A DE4326573A1 (en) 1993-08-07 1993-08-07 Polypeptides encoded by exon v5 of the CD44 gene as targets for immunotherapy and immunoscintigraphy of tumors
DE4326573 1993-08-07
PCT/EP1994/002398 WO1995004547A1 (en) 1993-08-07 1994-07-21 POLYPEPTIDES CODED BY EXON v5 OF THE CD44 GENE AS TARGETS FOR IMMUNOTHERAPY AND IMMUNOSCINTIGRAPHY OF TUMOURS

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DE19540515C1 (en) * 1995-10-31 1997-02-06 Boehringer Ingelheim Int Tumor therapy through adoptive transfer of CD44v-specific cytotoxic T lymphocytes
UY24389A1 (en) * 1995-12-06 2001-10-25 Karlsruhe Forschzent PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF FLAT EPITHELIUM CARCINOMA
DE19648209A1 (en) * 1996-11-21 1998-05-28 Boehringer Ingelheim Int Procedure for tumor cell depletion of CD34-positive cells
DE19708713C2 (en) * 1997-03-04 2002-11-28 Boehringer Ingelheim Int Use of preparations containing anti-CD44 antibodies for the treatment of certain tumors and for the suppression of immune reactions
DE19911329A1 (en) * 1998-03-27 2000-09-21 Benes Ivan Friedrich Radioimmunoconjugate which can be used in human therapy and process for its preparation
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
EP1401472A4 (en) * 2001-05-25 2005-04-06 Univ Jefferson Alternative splice forms of proteins as basis for multiple therapeutic modalities
PT2886126T (en) 2013-12-23 2017-09-13 Exchange Imaging Tech Gmbh Nanoparticle conjugated to cd44 binding peptides
BR112017000710B1 (en) 2014-07-15 2024-02-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem ISOLATED POLYPEPTIDE, COMPOSITION OF MATTER, AND, USE OF THE ISOLATED POLYPEPTIDE OR COMPOSITION OF MATTER
CN116496398B (en) * 2022-10-31 2023-10-31 南京元迈细胞生物科技有限公司 Antibody specifically binding to v5 exon of CD44 and application thereof

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DE4014510A1 (en) * 1990-05-07 1991-11-14 Kernforschungsz Karlsruhe VARIANT CD44 SURFACE PROTEINS, THESE ENCODING C-DNA SEQUENCES, ANTIBODIES AGAINST THESE PROTEINS AND THEIR USE IN DIAGNOSTICS AND THERAPY
DE4134982A1 (en) * 1991-10-23 1993-04-29 Kernforschungsz Karlsruhe USE OF ANTIBODY-CONTAINING PREPARATIONS FOR IMMUNE SUPPRESSION

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