DE4224542A1 - Monoclonal antibodies for diagnosis or therapy - directed against conjugate of MHC class I mol and peptide antigen - Google Patents

Monoclonal antibodies for diagnosis or therapy - directed against conjugate of MHC class I mol and peptide antigen

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Publication number
DE4224542A1
DE4224542A1 DE19924224542 DE4224542A DE4224542A1 DE 4224542 A1 DE4224542 A1 DE 4224542A1 DE 19924224542 DE19924224542 DE 19924224542 DE 4224542 A DE4224542 A DE 4224542A DE 4224542 A1 DE4224542 A1 DE 4224542A1
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Prior art keywords
mhc class
mouse
antigen
cells
monoclonal antibodies
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DE19924224542
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German (de)
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Guenter Prof Dr Haemmerling
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Priority to DE19924224542 priority Critical patent/DE4224542A1/en
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Producing monoclonal antibodies directed against a conjugate of a major histocompatibility complex class I mol. (I) and a peptide antigen (II) comprises (a) isolating (I); (b) inserting a (I)-encoding gene into the genome of a mouse to permit expression of the gene; (c) conjugating (I) with (II); (d) immunising the transformed mouse with the conjugate; (e) isolating spleen cells from the mouse; and (f) producing and opt. humanising monoclonal antibodies in known manner. Process (I) may be isolated from CS7 AL/6 mouse RMA-S tumour cells or human EBV transformed cells, or may be isolated from tissue, or may be produced by recombinant DNA techniques. Step (b) may be omitted if (I) was isolated from the same mouse strain as that to be immunised. (II) may be a viral or tumour antigen, e.g, the human melanoma antigen MAGE-1 or the tumour antigen produced by the HPV E6 or E7 oncogene. USE - The antibodies are useful for diagnosis and therapy of tumours and infections, e.g, as a substitute for tumour-specific cytotoxic T cells.

Description

Die Erfindung betrifft die Herstellung von monoklonalen MHC Klasse I-plus-Peptid-restringierten Antikörpern und deren Verwendung.The invention relates to the production of monoclonal MHC Class I plus peptide restricted antibodies and their Use.

Es ist bekannt, daß zytotoxische T Lymphozyten Peptide erkennen, die von MHC (Major Histocompatibility Complex) Klasse I Molekülen auf der Zelloberfläche präsentiert werden. Diese Peptide stammen von intrazellulär synthetisierten Proteinen, z. B. von Tumorantigenen oder viralen Proteinen. Damit ist gewährleistet, daß zytotoxische T Lymphozyten körperfremde Substanzen erkennen können, die ausschließlich intrazellulär exprimiert werden und sich somit der Entdeckung durch Antikörper entziehen.It is known that cytotoxic T lymphocyte peptides recognize that from MHC (Major Histocompatibility Complex) Class I molecules presented on the cell surface become. These peptides are from intracellular synthesized proteins, e.g. B. of tumor antigens or viral proteins. This ensures that cytotoxic T lymphocytes can recognize foreign substances that are expressed only intracellularly and themselves thus elude detection by antibodies.

Auf dieser Erkenntnis beruht ein immunologischer Ansatz der Tumortherapie. Es wird versucht, tumorspezifische- zytotoxische T Lymphozyten eines Patienten in vitro herzustellen oder aus dem Tumor zu isolieren, diese in vitro in großem Maßstab zu vermehren, um dann den Patienten mit seinen eigenen tumorspezifischen-zytotoxischen T Lymphozyten zu behandeln.An immunological approach of the Tumor therapy. An attempt is made to a patient's cytotoxic T lymphocytes in vitro to produce or isolate from the tumor, this in vitro multiply on a large scale, and then the patient with its own tumor-specific cytotoxic T lymphocytes to treat.

Dieses Verfahren ist jedoch technisch äußerst aufwendig und kann daher nur in ganz begrenztem Umfang durchgeführt werden. Wünschenswert wären Antikörper, die die gleiche Spezifität wie tumorspezifische-zytotoxische T Lymphozyten aufweisen, d. h. syngenes MHC Klasse I-plus-Peptid erkennen.However, this process is technically extremely complex and can therefore only be carried out to a very limited extent become. Antibodies that are the same would be desirable Specificity like tumor-specific-cytotoxic T lymphocytes have d. H. Recognize syngeneic MHC class I plus peptide.

Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren bereitzustellen, mit dem solche Antikörper hergestellt werden können.The present invention is therefore based on the object to provide a method by which such antibodies can be produced.

Erfindungsgemäß wird dies durch folgende Schritte erreicht:According to the invention, this is achieved by the following steps:

  • a) Isolierung von unbeladenen MHC Klasse I-Molekülen, a) isolation of unloaded MHC class I molecules,  
  • b) Einführung eines ein solches Molekül von (a) codierendes Gen in das Genom einer Maus zur Expression des Gens,b) Introduction of one encoding such a molecule of (a) Gene in the genome of a mouse to express the gene,
  • c) Beladung eines Moleküls von (a) mit einem Antigen zur Ausbildung eines MHC Klasse I-plus-Peptid-Moleküls,c) loading a molecule of (a) with an antigen for Formation of an MHC class I plus peptide molecule,
  • d) Immunisierung der transformierten Maus von (b) mit dem beladenen Molekül von (c),d) immunization of the transformed mouse from (b) with the loaded molecule of (c),
  • e) Isolierung von Milzzellen aus der immunisierten Maus von (d) und Herstellung von monoklonalen MHC Klasse I-plus- Peptid-restringierten Antikörpern sowie gegebenenfalls deren Humanisierung nach üblichen Verfahren.e) isolation of spleen cells from the immunized mouse of (d) and manufacture of MHC class I-plus monoclonal Peptide-restricted antibodies and, if appropriate their humanization according to customary procedures.

Der Ausdruck "MHC" umfaßt den Ausdruck "HLA".The term "MHC" includes the term "HLA".

Das erfindungsgemäße Verfahren beruht auf dem Prinzip, daß ein Organismus in der Regel nur gegen fremde Antigene spezifische Antikörper produziert.The method according to the invention is based on the principle that an organism usually only against foreign antigens produces specific antibodies.

Erfindungsgemäß werden unbeladene MHC Klasse I-Moleküle isoliert. Dies können Zellen selbst sein, die einen Defekt in den MHC codierten Transportern haben und daher ihre MHC-Moleküle nicht mit Peptid beladen können. Solche Zellen sind beispielsweise die RMA-S Tumorzellen der CS7 BL/6 Maus oder die T2 genannten EBV transformierten Zellen des Menschen.According to the invention, unloaded MHC class I molecules isolated. These can be cells themselves that have a defect have in the MHC coded transporters and therefore theirs MHC molecules cannot be loaded with peptide. Such cells are, for example, the RMA-S tumor cells of the CS7 BL / 6 mouse or the EBV transformed cells called T2 of the People.

Unbeladene MHC Klasse I-Moleküle können ferner über bekannte biochemische Verfahren aus Geweben isoliert werden. Sie können aber auch mittels DNA-Rekombinationstechniken erhalten werden. Ihre Gene können beispielsweise in E. coli oder im Baculovirus-System exprimiert werden. Unloaded MHC class I molecules can also be known biochemical processes are isolated from tissues. they can also use recombinant DNA techniques be preserved. For example, your genes can be found in E. coli or expressed in the baculovirus system.  

Erfindungsgemäß werden die Gene von MHC Klasse I-Molekülen auch in das Genom einer Maus zur Expression der Gene eingeführt. Hierfür können übliche, dem Fachmann bekannte Verfahren verwendet werden. Durch die Transformation einer Maus mit einem MHC Klasse I-Gen und dessen Expression wird erreicht, daß diese Maus das spezielle MHC Klasse I-Molekül nicht als fremd ansieht und somit auch keine dagegen gerichtete Antikörperproduktion in Gang setzt.According to the invention, the genes of MHC class I molecules also in the genome of a mouse to express the genes introduced. Usual, known to the person skilled in the art can do this Procedures are used. By transforming a Mouse with an MHC class I gene and its expression achieved that mouse the special MHC class I molecule does not regard them as foreign and therefore does not oppose them directed antibody production starts.

Eine solche Transformation kann unterbleiben, wenn das MHC Klasse I-Molekül ursprünglich aus der zu transformierenden Maus, wie im Falle der RMA-S Tumorzellen aus der CS7 BL/6 Maus, vgl. vorstehend, oder einer syngenen Maus stammt. Eine Antikörperproduktion gegen das Molekül würde dann nicht stattfinden.Such a transformation can be omitted if the MHC Class I molecule originally derived from the one to be transformed Mouse, as in the case of the RMA-S tumor cells from the CS7 BL / 6 Mouse, cf. above, or a syngeneic mouse. A Then antibody production against the molecule would not occur.

Eine solche tritt jedoch in jedem Fall ein und ist erfindungsgemäß gewünscht, wenn das verwendete MHC Klasse I-Molekül mit einem Antigen beladen ist. Die Antikörper richten sich dann gegen das von dem MHC Klasse I-Molekül und dem Antigen gebildete Epitop, d. h. es entstehen gegen MHC Klasse I-plus-Peptid-gerichtete Antikörper.However, such occurs and is in any case desired according to the invention if the MHC class used I molecule is loaded with an antigen. The antibodies are then directed against that of the MHC class I molecule and epitope formed by the antigen, i. H. it arises against MHC Class I plus peptide-directed antibodies.

Die Beladung von MHC Klasse I-Molekülen erfolgt erfindungsgemäß nach bekannten Verfahren. Zur Beladung können Antigene unterschiedlichster Natur, insbesondere Virus- und Tumorantigene verwendet werden. Beispiele für Tumorantigene sind das aus menschlichen Melanomen isolierte Antigen (MAGE-1) und das von dem E6 bzw. E7 Onkogen des HPV produzierte Tumorantigen. Die Immunsierung der Maus mit den beladenen MHC Klasse I-Molekülen erfolgt ebenfalls nach bekannten Verfahren. MHC class I molecules are loaded according to the invention by known methods. For loading can antigens of all kinds, in particular Virus and tumor antigens can be used. examples for Tumor antigens are those isolated from human melanoma Antigen (MAGE-1) and that of the E6 and E7 oncogene of HPV produced tumor antigen. Immunization of the mouse with the loaded MHC class I molecules also follows known methods.  

Erfindungsgemäß wird der immunisierten Maus nach üblicher Zeit die Milz zur Herstellung von monoklonalen MHC Klasse I-plus-Peptid-restringierten Antikörpern entnommen. Hierfür werden übliche dem Fachmann bekannte Verfahren angewandt.According to the invention, the immunized mouse becomes more common Time the spleen to produce monoclonal MHC class I-plus peptide-restricted antibodies taken. Therefor usual methods known to the person skilled in the art are used.

Denkbar ist es allerdings auch, daß der immunisierten Maus polyklonale MHC Klasse I-plus-Peptid-restringierte Antikörper entnommen und diese gemäß bekannten Verfahren hochgereinigt werden.However, it is also conceivable that the immunized mouse polyclonal MHC class I plus peptide restricted Antibodies removed and this according to known methods be cleaned up.

Erfindungsgemäß werden also sowohl monoklonale als auch polyklonale MHC Klasse I-plus-Peptid-restringierte Antikörper bereitgestellt. Diese können gegebenenfalls durch bekannte Verfahren humanisiert werden. Sie eignen sich dann nicht nur zur Diagnose sondern auch zur Therapie von Tumoren und Infektionen.According to the invention, both monoclonal and polyclonal MHC class I plus peptide restricted Antibodies provided. This can be done if necessary known methods are humanized. Then they are suitable not only for diagnosis but also for the therapy of tumors and infections.

Das erfindungsgemäße Verfahren ermöglicht es weiterhin, MHC Klasse II-plus-Peptid-restringierte Antikörper herzustellen. Diese können ebenfalls poly- bzw. monoklonal sein. Auch können sie humanisiert werden. Sie stellen dann eine Behandlungsmöglichkeit für Autoimmunerkrankungen dar.The method according to the invention also enables MHC Produce class II plus peptide restricted antibodies. These can also be poly- or monoclonal. Also they can be humanized. Then you put one Treatment options for autoimmune diseases.

Claims (3)

1. Verfahren zur Herstellung von monoklonalen MHC Klasse I-plus-Peptid-restringierten Antikörpern, gekennzeichnet durch folgende Schritte:
  • a) Isolierung von unbeladenen MHC Klasse I-Molekülen,
  • b) Einführung eines ein solches Molekül von (a) codierendes Gen in das Genom einer Maus zur Expression des Gens,
  • c) Beladung eines Moleküls von (a) mit einem Antigen zur Ausbildung eines MHC Klasse I-plus-Peptid-Moleküls,
  • d) Immunisierung der transformierten Maus von (b) mit dem beladenen Molekül von (c),
  • e) Isolierung von Milzzellen aus der immunisierten Maus von (d) und Herstellung von monoklonalen MHC Klasse I-plus-Peptid-restringierten Antikörpern sowie gegebenenfalls deren Humanisierung nach üblichen Verfahren.
1. A method for producing monoclonal MHC class I plus peptide-restricted antibodies, characterized by the following steps:
  • a) isolation of unloaded MHC class I molecules,
  • b) introduction of a gene encoding such a molecule from (a) into the genome of a mouse for expression of the gene,
  • c) loading a molecule of (a) with an antigen to form an MHC class I plus peptide molecule,
  • d) immunizing the transformed mouse from (b) with the loaded molecule from (c),
  • e) isolation of spleen cells from the immunized mouse of (d) and production of monoclonal MHC class I plus peptide-restricted antibodies and, if appropriate, their humanization by customary methods.
2. Monoklonale MHC Klasse I-plus-Peptid-restringierte Antikörper nach dem Verfahren nach Anspruch 1.2. MHC class I plus peptide restricted monoclonal Antibodies according to the method of claim 1. 3. Verwendung der monoklonalen MHC Klasse I-plus-Peptid­ restringierten Antikörper nach dem Verfahren nach Anspruch 1 zur Diagnose und Therapie von Tumoren und Infektionen.3. Use of the MHC class I-plus peptide restricted antibodies after the procedure Claim 1 for diagnosis and therapy of tumors and Infections.
DE19924224542 1992-07-24 1992-07-24 Monoclonal antibodies for diagnosis or therapy - directed against conjugate of MHC class I mol and peptide antigen Withdrawn DE4224542A1 (en)

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DE19924224542 DE4224542A1 (en) 1992-07-24 1992-07-24 Monoclonal antibodies for diagnosis or therapy - directed against conjugate of MHC class I mol and peptide antigen

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DE19924224542 DE4224542A1 (en) 1992-07-24 1992-07-24 Monoclonal antibodies for diagnosis or therapy - directed against conjugate of MHC class I mol and peptide antigen

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0752876A1 (en) * 1994-02-01 1997-01-15 Ludwig Institute For Cancer Research Monoclonal anbibodies which bind to tumor rejection antigen precursor mage-1, recombinant mage-1, and mage-1 derived immunogenic peptides
WO1999058693A1 (en) * 1998-05-14 1999-11-18 Centre National De La Recherche Scientifique - Cnrs Complex formed by a peptide and a major histocompatibility complex at the surface of phages
WO2005028510A2 (en) * 2003-09-25 2005-03-31 Vsevolod Ivanovich Kiselev Methods, kits, and compositions for the development and use of monoclonal antibodies specific to antigens of low immunogenicity

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0752876A1 (en) * 1994-02-01 1997-01-15 Ludwig Institute For Cancer Research Monoclonal anbibodies which bind to tumor rejection antigen precursor mage-1, recombinant mage-1, and mage-1 derived immunogenic peptides
EP0752876A4 (en) * 1994-02-01 1999-07-07 Ludwig Inst Cancer Res Monoclonal anbibodies which bind to tumor rejection antigen precursor mage-1, recombinant mage-1, and mage-1 derived immunogenic peptides
WO1999058693A1 (en) * 1998-05-14 1999-11-18 Centre National De La Recherche Scientifique - Cnrs Complex formed by a peptide and a major histocompatibility complex at the surface of phages
FR2778669A1 (en) * 1998-05-14 1999-11-19 Centre Nat Rech Scient Genetically modified microorganism, especially phage, useful for screening for antigenic peptides recognized by T cells
WO2005028510A2 (en) * 2003-09-25 2005-03-31 Vsevolod Ivanovich Kiselev Methods, kits, and compositions for the development and use of monoclonal antibodies specific to antigens of low immunogenicity
WO2005028510A3 (en) * 2003-09-25 2005-08-25 Vsevolod Ivanovich Kiselev Methods, kits, and compositions for the development and use of monoclonal antibodies specific to antigens of low immunogenicity
EA010506B1 (en) * 2003-09-25 2008-10-30 Всеволод Иванович КИСЕЛЕВ Methods, kits and compositions for the development and use of monoclonal antibodies specific to antigenes of low immunogenicity
AU2004274368B2 (en) * 2003-09-25 2010-06-24 Vsevolod Ivanovich Kiselev Methods, kits, and compositions for the development and use of monoclonal antibodies specific to antigens of low immunogenicity

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