WO1997010258A2 - Anticorps agissant a l'encontre d'epitopes de cadherines desmosomales, exposes a la surface de cellules cancereuses situees en dehors de desmosomes - Google Patents

Anticorps agissant a l'encontre d'epitopes de cadherines desmosomales, exposes a la surface de cellules cancereuses situees en dehors de desmosomes Download PDF

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Publication number
WO1997010258A2
WO1997010258A2 PCT/DE1996/001567 DE9601567W WO9710258A2 WO 1997010258 A2 WO1997010258 A2 WO 1997010258A2 DE 9601567 W DE9601567 W DE 9601567W WO 9710258 A2 WO9710258 A2 WO 9710258A2
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WO
WIPO (PCT)
Prior art keywords
antibodies
cells
dsg2
dsm acc
antibody according
Prior art date
Application number
PCT/DE1996/001567
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German (de)
English (en)
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WO1997010258A3 (fr
Inventor
Werner Franke
Stephan Schäfer
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Progen Biotechnik Gmbh
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Publication date
Application filed by Progen Biotechnik Gmbh filed Critical Progen Biotechnik Gmbh
Priority to EP96943842A priority Critical patent/EP0952987A2/fr
Publication of WO1997010258A2 publication Critical patent/WO1997010258A2/fr
Publication of WO1997010258A3 publication Critical patent/WO1997010258A3/fr

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    • 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
    • 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

Definitions

  • the present invention relates to antibodies against epitopes of desmosomal cadherins which are exposed on the surface of cells outside of desmosomes, in particular also to carcinoma cells not connected by desmosomes, to methods for producing such antibodies and their use.
  • Desmosomes Cells from epithelial tissues and carcinomas are connected to each other by certain adhesive structures called desmosomes.
  • desmosomes contain transmembrane glycoproteins, which are referred to as desmosomal cadherins.
  • desmosomal cadherins are Desmoglein 2 (Dsg2) and Desmocollin 2 (Dsc2).
  • the invention is therefore based on the object of providing a means with that metastatic carcinomas can be detected quickly and reliably.
  • the present invention thus relates to antibodies against parts of desmosomal cadherins, these parts (epitopes) also being exposed on the cell surface of cells not connected by desmosomes.
  • the present invention is based on the knowledge of the applicant that on the surface of cells not connected by desmosomes, in particular individual epithelial and carcinoma cells as well as micrometastases, parts of des mosomal cadherins are exposed, which in cells connected by desmosomes as in normal tissues and carcinomas, are not accessible or are only accessible to a very limited extent. Against these extracellularly oriented parts, hereinafter referred to as "Cad-ex" parts, the applicant has produced antibodies.
  • the antibodies according to the invention are directed against extracellular epitopes of desmosomal cadherins, which are released in the form of stable molecular fragments in the course of invasive carcinoma growth.
  • the detection of corresponding fragments in body fluids serves as an indication of invasive and metastatic carcinoma growth.
  • Adjacent cells of normal epithelial tissues and malignant tumors (carcinomas) derived therefrom are connected to one another by adhesive structures which are referred to as desmosomes.
  • Cell-cell adhesion is mediated by desmomomal transmembrane proteins of the cadherin type.
  • Dsg Desmogleine
  • Dsc Desmocolline
  • Invasive-destructive carcinoma growth is accompanied by extensive proteolysis of the extracellular matrix on the invasion front. Usually through plasmin activated matrix metalloproteinases cause the breakdown of the extracellular matrix. Carcinoma cells leave the primary tumor to form metastases away from the organ after migration through lymph and blood vessels. Activated proteases cleave defined, stable molecular fragments of Desmogleine and Desmocolline from the cell surface, so that they can then be detected in the body fluids. This process takes place before the primary tumor and any existing metastases cause clinically diagnosable symptoms. The laboratory chemical detection of such molecular fragments in body fluids enables the introduction of further diagnostic measures for the early detection of a tumor.
  • the antibodies according to the invention can be polyclonal or monoclonal, with monoclonal antibodies being preferred.
  • the antibodies can be obtained from animals or humans, with guinea pigs and rabbits being preferred for polyclonal antibodies and mice being preferred for monoclonal antibodies.
  • the antibodies can be synthetic, parts or parts of them that are not necessary for the detection of Cad-ex parts of desmosomal cadherins may be missing in whole or in part, or these parts may be replaced by others that give the antibodies further favorable properties .
  • Antibodies against the Desmogleine Dsg1, Dsg2 and Dsg3 and Desmocolline Dsc1, Dsc2 and Dsc3 are preferably used.
  • the epitopes of the antibodies lie in the extracellular molecular domains, which preferably correspond to the amino acid sequence regions mentioned below: Dsg1: starting with the decapeptide EWIKFAAACR ending with the decapeptide AKDLLSDNVH Dsg2: starting with the decapeptide AWITAPVALR ending with the decapeptide REAQHDSYVG Dsg3: starting with the decapeptide EWVKFAKPCR ending with the decapeptide TRYGRPHSGRDPDDAP1 the Dekapeptide starting with the decapeptide RWAPIPCSML ending with the decapeptide IGGGGVQLGK Dsc3: starting with the decapeptide RWAPIPCSMQ ending with the decapeptide PTQCRATSRS
  • the antibodies are directed against Cad-ex parts of Dsg2.
  • Antibodies whose epitopes lie within the section of Dsg2 which is determined by the following amino acid sequence I are particularly preferred.
  • Antibodies whose epitopes lie within the section determined by the following amino acid sequence II of Dsg2 are very particularly preferred: INDNEPVFTQDVFVGSVEELS AAHTLVMKIN ATDADEPNTL NSKISYRIVS LEPAYPPVFY LNKDTGEIYT TSVTLDREEH SSYTLTVEAR DGNGEVTDKP VKQAQVQIRI LDVNDNIPW ENKVLEGMVE ENQVNVEVTR IKVFDADEIG SDNWLANFTF ASGNEGGYFH IETDAQTNEG IVTLIKEVDY EEMKNLDFSV IVANKAAFHK SIRSKYKPTP IPIKVKVKNV KEGIHFKSSV ISIYVSESMD RSSKGQIIGN FQ
  • the antibody Dsg2-G6 was deposited under DSM ACC 2236 on September 20, 995.
  • the antibodies are directed against Cadex parts of Dsc2.
  • Antibodies according to the invention can be produced by customary methods. If polyclonal or monoclonal antibodies are to be produced, it is expedient to immunize animals, in particular guinea pigs and rabbits, for the former and mice for the latter antibodies with a desmosomal cadherin and / or fragments thereof.
  • fragments thereof also encompasses synthetic peptides which have partial sequences of desmosomal cadherins. It can also be advantageous to immunize the animals with a mixture of desmosomal cadherins and / or fragments thereof. Further “boosters" of the animals can be carried out with the same desmosomal cadherins and / or fragments thereof.
  • Desmosomal cadherins and / or fragments thereof or a combination of these and the preceding desmosomal cadherins and / or fragments can also be used of which can be used for "boosters".
  • Polyclonal antibodies can then be obtained from the animal serum.
  • animal spleen cells are fused with myeloma cells.
  • Antibodies obtained are finally incubated with cells not connected by desmosomes, in particular individual epithelial and carcinoma cells and micrometastases, whereby those antibodies are identified which recognize Cad-ex parts of desmosomal cadherins.
  • Dsg2 The production of antibodies against Cad-ex parts of Dsg2 is described as an example.
  • isolated or recombinant e.g. Dsg2 and / or fragments thereof produced in E. coli, animal cells or yeast used for immunization of animals and for other "boosters", e.g. a polypeptide of Dsg2 which lies at least partially within the domains E1, EM, EMI, EIV and EA of Dsg2, for example the above amino acid sequence I, very particularly the above amino acid sequence II. Either glycosylated or non-glycosylated forms are used. Spleen cells are removed from the mice and fused with myeloma cells.
  • Monoclonal antibodies obtained are incubated with individual epithelial and carcinoma cells and micrometases.
  • individual epithelial and carcinoma cells and micrometases For example, the following, Cad-ex parts of Dsg2-recognizing monoclonal antibodies of the IgG 1 class, Dsg2-G6, Dsg2-G11, Dsg2-G91, Dsg2-G96 and Dsg2-129.
  • Antibodies according to the invention are distinguished by the fact that they recognize parts of desmosomal cadherins which are exposed on the free cell surface of cells which are not or only partially connected by desmosomes. Such cells are in particular individual epithelial and carcinoma cells as well as micrometastases.
  • the present invention is therefore suitable for the detection of these cells and cell groups.
  • the detection can be carried out by conventional detection methods, in particular a "western blot", an ELISA, an immunoprecipitation or by immunofluorescence microscopy.
  • antibodies according to the invention can, if appropriate, be labeled or be used in combination with labeled antibodies directed against them.
  • Antibodies according to the invention can also be used in a biosensor method.
  • the reaction of the antibodies according to the invention can take place on living or fixed cells.
  • kits which contain antibodies according to the invention together with carrier materials and customary auxiliaries such as buffers.
  • antibodies according to the invention are suitable for enriching cells which are not connected by desmosomes, in particular individual epithelial and carcinoma cells, and micrometastases, consisting of a few carcinoma cells which are partially coupled by desmosomes, in conventional cell sorting methods, in particular by FACS methods or by Conventional immunoadsorption onto suitable carriers such as coated beads, which means that these can be subjected to further diagnostic tests or used for therapeutic tests and measures.
  • suitable detection methods are e.g. Immunoblot analysis or ELISA.
  • antibodies according to the invention are suitable as mediators, further antibodies and ligands, e.g. B. toxic substances to protruding cells and Introduce cell groups.
  • the antibodies according to the invention can also be used as carriers for toxins and toxic substances.
  • they are suitable for removing tumor cells from cell suspensions.
  • Figure 1 shows an example of human carcinoma cells in immunofluorescence microscopy after they have been fixed as intact cells and have reacted with antibodies against Cad-ex parts of Dsg2, such as a single cell of a squamous epithelium in the left half.
  • Carcinoma of the vulva line A-431 of the American Type Culture Collection, catalog number ATCC CRL 1 555
  • ATCC CRL 8024 a cell pair of the line PLC of a primary liver cell carcinoma
  • the brightly lit spots in the fluorescence are the reaction sites of the externally accessible Cad-ex parts of the Dsg2 glycoprotein.
  • Figure 2 shows such a reaction site on an intact cell in immunoelectron microscopy, a human epidermis cell from the HaCaT line being used as an example and the surface-bound antibodies against Cad-ex parts of Dsg2 with a specific against the nature of these antibodies directed second antibodies were detected, which was coupled to colloidal gold particles with an average diameter of about 10 nanometers.
  • the group of six high-contrast gold particles thus marks a surface structure containing Dsg2 that does not occur in a desmosome.
  • Figure 3 shows an example of the result of the immunoblot analysis of the cell culture fluid from human HaCaT cell cultures.
  • the proteins the cell culture fluid was size fractionated electrophoretically and transferred to a nitrocellulose membrane.
  • the staining of the membrane with Ponceau reagent unspecifically shows the quantitatively most strongly represented proteins of the sample (Fig. 3, a, lane 2) and size marker proteins (Fig. 3, a, lane 2).
  • the membrane was then examined with the monoclonal antibodies Dsg2-G 1 1 (Fig. 3, b) or Dsc3-U1 14 (Fig. 3, c).
  • the specific antibodies detect a specific, approximately 90 kDa large molecular fragment of the extracellular molecular domain both for Dsg2 and for Dsg2 and for Dsc3.
  • Balb / c strain mice were used for immunization.
  • a Dsg2 polypeptide containing the above amino acid sequence II was used as the antigen.
  • Dsg2-G11, Dsg2-G91, Dsg2-G96 and Dsg2-G129 were used in the DSM under DSM ACC 2226, DSM ACC 2227, DSM ACC 2228 and DSM ACC 2229 on August 9, 1 995.
  • the antibody Dsg2-G6 was deposited with the DSM under ACC 2236 on September 20, 1995.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des anticorps agissant à l'encontre de parties de cadhérines desmosomales, lesdites parties étant exposées à la surface de cellules non liées par des desmosomes. L'invention concerne en outre des procédés de préparation desdits anticorps, ainsi que leur utilisation.
PCT/DE1996/001567 1995-08-23 1996-08-23 Anticorps agissant a l'encontre d'epitopes de cadherines desmosomales, exposes a la surface de cellules cancereuses situees en dehors de desmosomes WO1997010258A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP96943842A EP0952987A2 (fr) 1995-08-23 1996-08-23 Anticorps agissant a l'encontre d'epitopes de cadherines desmosomales, exposes a la surface de cellules cancereuses situees en dehors de desmosomes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19531033.0 1995-08-23
DE1995131033 DE19531033C2 (de) 1995-08-23 1995-08-23 Antikörper gegen an Oberflächen von Carcinom-Zellen außerhalb von Desmosomen exponierte Epitope desmosomaler Cadherine

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Publication Number Publication Date
WO1997010258A2 true WO1997010258A2 (fr) 1997-03-20
WO1997010258A3 WO1997010258A3 (fr) 1997-07-24

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EP (1) EP0952987A2 (fr)
DE (1) DE19531033C2 (fr)
WO (1) WO1997010258A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004048411A2 (fr) * 2002-11-14 2004-06-10 Adherex Technologies, Inc. Composes et techniques destines a moduler des fonctions de cadherines non classiques
US7481999B2 (en) 1998-05-05 2009-01-27 Adherex Technologies, Inc. Compounds and methods for modulating OB-cadherin-mediated function

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIFFERENTIATION, Bd. 59, Nr. 2, September 1995, LONDON, GROSSBRITANNIEN, Seiten 113-126, XP000674532 B. PETERS ET AL.: "Maintenance of cell-type-specific cytoskeletal character in epithelial cells out of epithelial context: Cytokeratins and other cytoskeletal proteins in the rests of Malassez of the periodontal ligament." *
DIFFERENTIATION, Bd. 60, Nr. 2, Mai 1996, LONDON, GROSSBRITANNIEN, Seiten 99-108, XP000674533 S. SCH[FER ET AL.: "Immunological identification and characterization of the desmosomal cadherin Dsg2 in coupled and uncoupled epithelial cells and in human tissues." *
EXPERIMENTAL CELL RESEARCH, Bd. 211, Nr. 2, April 1994, NEW YORK, NY, VSA, Seiten 391-399, XP000674513 S. SCH[FER ET AL.: "Identification of the ubiquitous human desmoglein, Dsg2, and the expression catalogue of the desmoglein subfamily of desmosomal cadherins." *
JOURNAL OF SURGICAL ONCOLOGY, Bd. 57, Nr. 2, Oktober 1994, SAN DIEGO, CA, VSA, Seiten 105-110, XP000674529 S. NATSUGOE ET AL.: "Expression of desmoglein I in squamous cell carcinoma of the esophagus." *
MOLECULAR MEMBRANE BIOLOGY, Bd. 11, Nr. 4, Oktober 1994, WASHINGTON, DC, VSA, Seiten 229-236, XP000674534 J. LORIMER ET AL.: "Cloning, sequence analysis and expression pattern of mouse desmocollin 2 (DSC2), a cadherin-like adhesion molecule." *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7481999B2 (en) 1998-05-05 2009-01-27 Adherex Technologies, Inc. Compounds and methods for modulating OB-cadherin-mediated function
WO2004048411A2 (fr) * 2002-11-14 2004-06-10 Adherex Technologies, Inc. Composes et techniques destines a moduler des fonctions de cadherines non classiques
WO2004048411A3 (fr) * 2002-11-14 2005-03-10 Adherex Technologies Inc Composes et techniques destines a moduler des fonctions de cadherines non classiques
US7476509B2 (en) 2002-11-14 2009-01-13 Adherex Technologies Inc. Compounds and methods for modulating functions of nonclassical cadherins

Also Published As

Publication number Publication date
WO1997010258A3 (fr) 1997-07-24
DE19531033C2 (de) 1998-01-29
EP0952987A2 (fr) 1999-11-03
DE19531033A1 (de) 1997-02-27

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