EP0906444A1 - Genetically transfected human dendritic cells, their production and use, preferably as vaccines - Google Patents

Genetically transfected human dendritic cells, their production and use, preferably as vaccines

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Publication number
EP0906444A1
EP0906444A1 EP97922844A EP97922844A EP0906444A1 EP 0906444 A1 EP0906444 A1 EP 0906444A1 EP 97922844 A EP97922844 A EP 97922844A EP 97922844 A EP97922844 A EP 97922844A EP 0906444 A1 EP0906444 A1 EP 0906444A1
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Prior art keywords
dendritic cells
human
gene
mucin
cells
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German (de)
French (fr)
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Gabriele Pecher
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Priority claimed from DE19617837A external-priority patent/DE19617837A1/en
Priority claimed from DE1996117846 external-priority patent/DE19617846A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • 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
    • A61K39/46—Cellular immunotherapy
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464469—Tumor associated carbohydrates
    • A61K39/46447—Mucins, e.g. MUC-1
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to gene-transfected human dendritic cells.
  • Gene-transfected dendritic cells can be used in basic research as well as in the construction of vaccines, e.g. B. tumor vaccines, find application. An efficient gene transfer to human dendritic cells has not been described so far.
  • the invention further relates to a method for producing these cells and their use, preferably as a vaccine.
  • the glycoprotein mucin encoded by the MUC1 gene, is expressed on the surface of pancreatic, Mamraa, colon, Parotis and ovarian carcinomas as well as on the corresponding healthy cells.
  • Muzin encoded by MUC1 consists of two thirds of 20 to 100 "tandem nucleotide repeats".
  • a "tandem nucleotide repeat” consists of 60 nucleotides that encode a polypeptide of 20 amino acids (see Fig. 2).
  • peptide epitopes are exposed on tumor cells and can be recognized as foreign by the immune system, in particular T cells (these peptide epitopes are "hidden” by healthy carbohydrates on cells and therefore dissolve normal cells no immune response). These mucin epitopes are suitable for stimulating the immune system to protect the body against a tumor.
  • Electroporation was used as the transfection method, a method which is not always successful and in which a large number of cells die.
  • EBV-immortalized B cells have been used as immune cells and transfected with MUCl vectors and used to stimulate the immune system (described in Jerome KR, N. Domenech, and OJ Finn. 1993. Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epitheal mucin complementary DNA. J. of Immunol. 151: 1654-1662 and in Pecher G. and Finn O. j. 1996. Induction of cellular immunity in chimpanzees to tumor-associated antigen mucin by vaccination with MUC1 cDNA-transfected EBV-immortalized autologous B-cells.
  • EBV Epstein-Barr virus
  • the aim of the invention is to provide gene-transfected human dendritic cells.
  • These cells are also to be genetically engineered to develop a vaccine which specifically stimulates the immune system against tumor cells already present in the body and which is intended to reduce or eliminate the tumor.
  • "professional” immune cells for the expression of tumor-associated epitopes are to be used to construct a vaccine.
  • Certain "professional” immune cells, in contrast to tumor cells, express the costimulatory ligands necessary for optimal T cell activation, such as CD80 and CD86.
  • the essential feature of the manufacturing process is the transfection of the foreign gene into the dendrial cells using liposomes.
  • the method according to the invention is efficient, simple to carry out, safe to use and, in comparison to, for example, retroviral gene transfer, inexpensive.
  • the vaccine consists of human autologous dendritic cells which are transfected with a partial sequence of the human mucin MUCl gene, which contains several "tandem repeat nucleotide sequences" from the MUCl (FIG. 2), by means of lipofectin using the plasmid, and which are by Treatment with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide express tumor-associated epitopes.
  • the MUCl transfected cells are treated with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide for 24 to 36 hours so that the immunogenic, tumor-associated mucin epitopes are formed.
  • the Expression can be checked by a FACS analysis using mucin-epitope-specific antibodies.
  • the invention also relates to the vector pCMV / MUCl according to FIG. 1 for transfection of the dendritic cells, which consists of the following essential components:
  • CMV cytomegalovirus
  • the vaccine according to the invention has the following advantages / innovations compared to previous tumor vaccines:
  • the vaccine does not contain tumor cells, but a clearly defined antigen (MUCl).
  • Immune cells used to construct the vaccine are dendritic cells.
  • dendritic cells do not produce any immunosuppressive substances such as interleukin 10.
  • Human dendritic cells are produced from peripheral blood of patients or healthy people using interleukin 4 and granulocyte macrophage colony stimulating factor. This is a simple and easy to practice procedure.
  • the transfection method is lipofection. A high gene transfer rate in dendritic cells is achieved. The method is easy to carry out and reproducible.
  • PCMV / MUCl according to Fig.l is used as the vector for gene transfer.
  • the corresponding mucin cDNA was cloned into the vector under the immediate early promoter of CMV.
  • the vector contains no cDNA for resistance to antibiotics or the like. The vector thus fulfills high requirements Safety requirements for human use.
  • Another advantage of the vaccine according to the invention is that the recognition of the mucin-peptide epitopes by cytotoxic T cells does not follow the previously known classic way of recognizing short peptide epitopes in connection with the HLA complex.
  • the mucin-peptide epitopes are recognized by the T cells without "help" from the HLA complex.
  • This peculiarity in the detection of tumor-associated mucin epitopes can be explained by the above-mentioned special "tandem repeat" structure of the molecule and the high density of the antigen on the presenting cell.
  • the repeated repetition of the immunogenic peptide-epitope motif leads to an activation of the T cells by "crosslinking" the T cell receptor without the HLA complex having to be present.
  • peptide epitopes are exposed on tumor cells that can be recognized as foreign by the immune system.
  • the activation of the immune system triggered by this is not sufficient in tumor patients to eliminate the tumor because (due to the lack of expression of CD80 and CD86 on tumor cells) there is no costimulation of T cells.
  • the vaccine according to the invention triggers an efficient, tumor-specific immune response, which is based on the activation of mucine epitope-specific, cytotoxic T cells. These T cells lead to downsizing or elimination of the tumor cells.
  • dendritic cells are transfected with MUCl (copies of the tandem nucleotide sequence of MUCl cloned into the vector) and, if appropriate, treated with the glycosylation inhibitor phenyl-N-acetyl- ⁇ -D-galactosaminide, these express the tumor-associated epitopes.
  • MUCl copies of the tandem nucleotide sequence of MUCl cloned into the vector
  • Dendritic cells are isolated from human peripheral blood and cultured. On day 4 of the culture of the dendritic cells, the dendritic cells are transfected. For this a vector is used which contains CMV as a promoter for the foreign gene MUCl. 15 ⁇ l lipofectin (Fig. 3) is used for 750,000 dendritic cells. The successful transfection of the foreign gene MUCl is detected by means of FACS analysis with the monoclonal antibody HMFG-2 against mucin epitopes. After transfection, 12% of the dendritic cells show an expression of mucin epitopes. By using a glycosylation inhibitor (Gl), mucine epitopes can be detected on the surface of 48% of the dendritic cells. This marks the successful gene transfer. Even without using the glycosylation inhibitor, there are already sufficient immunogenic mucin epitopes on the surface.
  • Gl glycosylation inhibitor
  • Mock (vector without foreign gene) transfected cells express mucin epitopes to a maximum of 2% (see Fig. 4)
  • Lymphocytes are obtained from human peripheral blood by Ficoll gradient centrifugation and kept in culture. Dendritic cells are selected by the addition of interleukin 4 and granulocyte-macrophage colony stimulating factor and by adherence to platik. The dendritic cells are transfected with the MUCl vector using liposomes. Mucin expression is checked using Western blot methods and FACS analysis with monoclonal mucin antibodies. Phenyl-N-acetyl- ⁇ -D-galactosarainide (concentration 5 mM) is added to the culture medium of the transfected cells for 36 hours. The expression of the tumor-associated mucin-peptide epitopes generated in this way persists for 72 hours and is checked with monoclonal mucin-peptide antibodies by means of FACS analysis. The vaccine is applied to the patient within these 72 hours.
  • the vaccine can be used for therapy in patients with mucin (MUCl) -expressing tumors.
  • MUCl mucin
  • the treatment of breast, pancreas, ovarian, colon and parotid tumors is preferred.
  • This vaccine can also be used in healthy people to prevent a tumor expressing mucine epitopes.

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Abstract

Genetically transfected human dendritic cells are disclosed, as well as their production and use, preferably as vaccines. The invention has applications in the pharmaceutical industry and in medicine. The dendritic cells are produced by transfection with a foreign gene by means of liposomes, preferably by means of lipofectin, a liposome preparation. The disclosed vaccine consists of human, autologous dendritic cells transfected with a partial sequence of the human mucine-MUC1 gene which contains several tandem repeat nucleotide sequences of MUC1. By using a glycosylation inhibitor, tumour-associated epitopes are created in these cells, preferably at their surface.

Description

Gentransfizierte humane dendritische Zellen, ihre Herstellung und ihre Verwendung, bevorzugt als VakzineGene-transfected human dendritic cells, their production and their use, preferably as vaccines
Beschreibungdescription
Die Erfindung betrifft gentransfizierte humane dendritische Zellen. Gentransfizierte dendritische Zellen können sowohl in der Grundlagenforschung als auch zur Konstruktion von Vakzinen, z. B. Tumorvakzinen, Anwendung finden. Ein effizienter Gentransfer in humane dendritische Zellen ist bisher nicht beschrieben worden.The invention relates to gene-transfected human dendritic cells. Gene-transfected dendritic cells can be used in basic research as well as in the construction of vaccines, e.g. B. tumor vaccines, find application. An efficient gene transfer to human dendritic cells has not been described so far.
Die Erfindung betrifft ferner ein Verfahren zur Herstellung dieser Zellen und ihre Verwendung, bevorzugt als Vakzine.The invention further relates to a method for producing these cells and their use, preferably as a vaccine.
Zelluläre Vakzine gegen Tumorerkrankungen sind seit längerem bekannt. Die klassische und vielfach klinisch eingesetzte Vakzine besteht aus einem Gemisch von bestrahlten Tumorzellen und Adjuvantien wie beispielsweise Lysate von Bacillus Calmette Guerin (BCG) oder Corynebacterium Parvum. Nach zwei Jahrzehnten klinischer Erprobung läßt sich zusammenfassen, daß diese Vakzine keine reproduzierbare Wirkung zeigt (s. Übersichtsartikel. Oettgen, H. und Old, L. , The History of Cancer Immunotherapy, in: Biological Therapy of Cancer, Eds. V. deVita, S. Hellmann and s. Rosenberg, J. B. Lippincott Company 1991, S. 87-199).Cellular vaccines against tumor diseases have been known for a long time. The classic and widely used vaccine consists of a mixture of irradiated tumor cells and adjuvants such as lysates from Bacillus Calmette Guerin (BCG) or Corynebacterium Parvum. After two decades of clinical testing, it can be summarized that this vaccine has no reproducible effect (see review article. Oettgen, H. and Old, L., The History of Cancer Immunotherapy, in: Biological Therapy of Cancer, Eds. V. deVita, S. Hellmann and s. Rosenberg, JB Lippincott Company 1991, pp. 87-199).
In jüngerer Zeit wurden Versuche unternommen, Tumorzellen genetisch zu modifizieren, mit dem Ziel eine Immunantwort gegen den Tumor auszulösen. Verwendet werden dazu vor allem Gene für Zytokine oder kostimulierende Moleküle allein oder in Kombination (Blankenstein T. , Eur. J. Cancer, 1994). Der Nachteil dieser Vakzine besteht darin, daß zu ihrer Herstellung Tumorzellen verwendet werden müssen. Diese stellen wiederum ein Potential möglicher Metastasenbildung dar, und somit kann die Vakzine selbst bei ihrer Anwendung ein Risiko für den Patienten darstellen. Tumorzellen besitzten keine kostimulierenden Liganden wie CD80 und CD86, die für eine effektive Aktivierung des Immunsystems essentiell sind. Außerdem ist nicht klar definiert, gegen welche immunogenen Strukturen auf den Tumorzellen eine Immunantwort ausgelöst wird. Erfolgt z. B. eine Reaktion gegen Autoantigene (immunogene Strukturen, die nicht nur auf Tumorzellen, sondern auch auf gesunden Zellen vorhanden sind), wäre es möglich, daß Antoimmunreaktionen durch diese Art von Vakzine ausgelöst werden.Attempts have recently been made to genetically modify tumor cells with the aim of triggering an immune response against the tumor. Genes for cytokines or costimulatory molecules are used primarily or alone or in combination (Blankenstein T., Eur. J. Cancer, 1994). The disadvantage of this vaccine is that tumor cells have to be used to produce it. These in turn represent a potential for possible metastasis, and thus the vaccine itself can pose a risk even when used for the patient. Tumor cells have no costimulatory ligands such as CD80 and CD86, which are essential for an effective activation of the immune system. In addition, it is not clearly defined against which immunogenic structures on the tumor cells an immune response is triggered. Is z. B. a reaction against autoantigens (immunogenic structures that are not only present on tumor cells, but also on healthy cells), it would be possible that this type of vaccine triggers Antoimmune reactions.
Deshalb wurde in letzter Zeit darauf fokussiert, tumorspezifisehe Strukturen zu bestimmen. Eine solche Struktur findet sich auf dem Muzinmolekül von Tumorzellen. (Finn, O.J. 1993, Tumor rejeetion antigens recognized by T lymphocytes. Current Opinion in Immunol., 5, 701-8).For this reason, the focus has recently been on determining tumor-specific structures. Such a structure is found on the mucin molecule of tumor cells. (Finn, O.J. 1993, Tumor rejeetion antigens recognized by T lymphocytes. Current Opinion in Immunol., 5, 701-8).
Das Glykoprotein Muzin, kodiert durch das Gen MUC1, ist sowohl auf der Oberfläche von Pankreas-, Mamraa-, Kolon-, Parotis- und Ovarialkarzinomen als auch auf den entsprechenden gesunden Zellen exprimiert. Muzin, kodiert durch MUC1, besteht zu zwei Dritteln aus 20 bis 100 "Tandem Nukleotid repeats". Ein "Tandem Nukleotid repeat" besteht aus 60 Nukleotiden, die ein Polypeptid von 20 Aminosäuren kodieren (s. Abb. 2).The glycoprotein mucin, encoded by the MUC1 gene, is expressed on the surface of pancreatic, Mamraa, colon, Parotis and ovarian carcinomas as well as on the corresponding healthy cells. Muzin, encoded by MUC1, consists of two thirds of 20 to 100 "tandem nucleotide repeats". A "tandem nucleotide repeat" consists of 60 nucleotides that encode a polypeptide of 20 amino acids (see Fig. 2).
Infolge einer unvollständigen Muzin-Glykosylierung im Fall der malignen Entartung liegen auf Tumorzellen Peptid-Epitope frei, die vom Immunsystem, insbesondere T Zellen, als fremd erkannt werden können (diese Peptid-Epitope sind auf gesunden Zellen von Kohlenhydraten "verdeckt" und lösen deshalb bei normalen Zellen keine Immunreaktion aus). Diese Muzin-Epitope sind für eine Stimulierung des Immunsystems zur körpereigenen Abwehr gegen einen Tumor geeignet.As a result of incomplete mucin glycosylation in the case of malignant degeneration, peptide epitopes are exposed on tumor cells and can be recognized as foreign by the immune system, in particular T cells (these peptide epitopes are "hidden" by healthy carbohydrates on cells and therefore dissolve normal cells no immune response). These mucin epitopes are suitable for stimulating the immune system to protect the body against a tumor.
Es hat bereits Versuche gegeben, Muzin-Epitope mittels Gentransfer in solchen humanen Immunzellen zur Expression zu bringen. Probleme dabei sind der Vektor, die Transfektionsmethode sowie die richtige Wahl von geeigneten Immunzellen. Der verwendete Vektor pDKOF/MUCl, (beschrieben in Jerome K. R. , N. Domenech, and O. J. Finn. 1993. Tumor- specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epithelial mucin complementary DNA. J. of Immunol. 151: 1654-1662 und in Jerome K. R. , D. Bu, and O. J. Finn. 1992. Expression of tumor-associated epitopes on Epstein-Barr Virus-immortalized B-cells and Burkitt s lymphomas transfected with epitheal mucin complementary DNA. Canc. Res. 53: 5985-5990) führt, transfiziert in humane Zellen, nicht zu einer effizienten und stabilen Muzin-Epitop- Expression und ist somit für die Verwendung in einer Vakzine nicht oder nicht so gut geeignet.Attempts have been made to express mucin epitopes by means of gene transfer in such human immune cells. Problems are the vector that Transfection method and the right choice of suitable immune cells. The vector pDKOF / MUCl used (described in Jerome KR, N. Domenech, and OJ Finn. 1993. Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epithelial mucin complementary DNA J. of Immunol. 151: 1654-1662 and in Jerome KR, D. Bu, and OJ Finn. 1992. Expression of tumor-associated epitopes on Epstein-Barr Virus-immortalized B-cells and Burkitt s lymphomas transfected with epitheal mucin complementary DNA. Canc. Res. 53: 5985-5990), transfected into human cells, does not lead to an efficient and stable mucin epitope expression and is therefore not or not so well suited for use in a vaccine.
Als Transfektionsmethode wurde dabei die Elektroporation angewendet, eine nicht immer erfolgreiche Methode, bei der sehr viele Zellen absterben.Electroporation was used as the transfection method, a method which is not always successful and in which a large number of cells die.
Als Immunzellen wurden bisher EBV-immortalisierte B-Zellen benutzt und mit MUCl-Vektoren transfiziert und zur Stimulation des Immunsystems benutzt (beschrieben in Jerome K. R. , N. Domenech, and O. J. Finn. 1993. Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epitheal mucin complementary DNA. J. of Immunol. 151: 1654-1662 und in Pecher G. and Finn O. j. 1996. Induction of cellular immunity in chimpanzees to tumor- associated antigen mucin by vaccination with MUC1 cDNA- transfected EBV-immortalized autologous B-cells. Proc. Natl. Acad. Sei. USA 1993: 1699-1704 sowie Patentanmeldung G. Pecher: Vakzine gegen Tumorerkrankungen, DE-OS 195 166 73 vom 28.4.1995). EBV-immortalisierte B-Zellen produzieren jedoch auch immunsupprimierende Faktoren wie Interleukin 10 und zu ihrer Herstellung werden humanpathogene Viren (EBV=Epstein- Barr-Virus) verwendet.So far, EBV-immortalized B cells have been used as immune cells and transfected with MUCl vectors and used to stimulate the immune system (described in Jerome KR, N. Domenech, and OJ Finn. 1993. Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells tranfected with polymorphyc epitheal mucin complementary DNA. J. of Immunol. 151: 1654-1662 and in Pecher G. and Finn O. j. 1996. Induction of cellular immunity in chimpanzees to tumor-associated antigen mucin by vaccination with MUC1 cDNA-transfected EBV-immortalized autologous B-cells. Proc. Natl. Acad. Sei. USA 1993: 1699-1704 and patent application G. Pecher: vaccine against tumor diseases, DE-OS 195 166 73 from April 28. 1995). However, EBV-immortalized B cells also produce immunosuppressive factors such as interleukin 10 and human pathogenic viruses (EBV = Epstein-Barr virus) are used to produce them.
Die Erfindung hat das Ziel, gentransfizierte humane dendritische Zellen zur Verfügung zu stellen. Auf der Basis dieser Zellen soll ferner gentechnisch eine Vakzine entwickelt werden, die das Immunsystem gegen bereits im Körper vorhandene Tumorzellen spezifisch stimuliert und zur Verkleinerung bzw. Beseitigung des Tumors führen soll. Anstelle von Tumorzellen sollen "professionelle" Immunzellen zur Expression von tumorassoziierten Epitopen zur Konstruktion einer Vakzine verwendet werden. Bestimmte "professionelle" Immunzellen exprimieren, im Gegensatz zu Tumorzellen, die für eine optimale T-Zellaktivierung notwendigen kostimulierenden Liganden, wie Z. B. CD80 und CD86.The aim of the invention is to provide gene-transfected human dendritic cells. On the base These cells are also to be genetically engineered to develop a vaccine which specifically stimulates the immune system against tumor cells already present in the body and which is intended to reduce or eliminate the tumor. Instead of tumor cells, "professional" immune cells for the expression of tumor-associated epitopes are to be used to construct a vaccine. Certain "professional" immune cells, in contrast to tumor cells, express the costimulatory ligands necessary for optimal T cell activation, such as CD80 and CD86.
Die Erfindung wird gemäß den Ansprüchen 1, 2 und 9 realisiert, die Unteransprüche sind Vorzugsvarianten.The invention is implemented according to claims 1, 2 and 9, the subclaims are preferred variants.
Das wesentliche Merkmal des Herstellungsverfahrens ist die Transfektion des fremden Gens in die dendrischen Zellen mittels Liposomen.The essential feature of the manufacturing process is the transfection of the foreign gene into the dendrial cells using liposomes.
Das erfindungsgemäße Verfahren ist effizient, einfach durch¬ zuführen, sicher in der Anwendung, und, im Vergleich zu beispielsweise einem retroviralen Gentransfer, kostengünstig.The method according to the invention is efficient, simple to carry out, safe to use and, in comparison to, for example, retroviral gene transfer, inexpensive.
Im folgenden wird die bevorzugte Verwendung der humanen dendritischen Zellen als Vakzine näher beschrieben.The preferred use of the human dendritic cells as vaccines is described in more detail below.
Die Vakzine besteht aus humanen autologen dendritischen Zellen, die mit einer Teilsequenz des humanen Muzin-MUCl-Gens, die mehrere "Tandem Repeat Nukleotid Sequenzen" vom MUCl (Abb. 2) enthält, mittels Lipofektin unter Verwendung des Plasmids transfiziert sind, und die durch Behandlung mit dem Glykosylierungsinhibitor Phenyl-N-Acetyl-α-D-Galactosaminid tumorassoziierte Epitope exprimieren.The vaccine consists of human autologous dendritic cells which are transfected with a partial sequence of the human mucin MUCl gene, which contains several "tandem repeat nucleotide sequences" from the MUCl (FIG. 2), by means of lipofectin using the plasmid, and which are by Treatment with the glycosylation inhibitor phenyl-N-acetyl-α-D-galactosaminide express tumor-associated epitopes.
Die MUCl transfizierten Zellen werden mit dem Glykosylierungs-inhibitor Phenyl-N-Acetyl-α-D-Galactosaminid für 24 bis 36 Stunden behandelt, damit die immunogenen, tumorassoziierten Muzin-Epitope ausgebildet werden. Die Expression kann durch eine FACS-Analyse mittels Muzin-Epitop¬ spezifischen Antikörpern überprüft werden.The MUCl transfected cells are treated with the glycosylation inhibitor phenyl-N-acetyl-α-D-galactosaminide for 24 to 36 hours so that the immunogenic, tumor-associated mucin epitopes are formed. The Expression can be checked by a FACS analysis using mucin-epitope-specific antibodies.
Gegenstand der Erfindung ist auch der Vektor pCMV/MUCl gemäß Abb. 1 zur Transfektion der dendritischen Zellen, der aus folgenden wesentlichen Bestandteilen besteht:The invention also relates to the vector pCMV / MUCl according to FIG. 1 for transfection of the dendritic cells, which consists of the following essential components:
- immediate early Promotor des Cytomegalievirus (CMV) für Muzin-MUCl-Genabschnitte- immediate early promoter of the cytomegalovirus (CMV) for mucin-MUCl gene segments
- Teilsequenz des Muzin-MUCl-Gens.- Partial sequence of the mucin-MUCl gene.
Die erfindungsgemäße Vakzine hat folgende Vorteile/Neuerungen gegenüber bisherigen Tumorvakzinen:The vaccine according to the invention has the following advantages / innovations compared to previous tumor vaccines:
Die Vakzine enthält keine Tumorzellen, sondern ein klar definiertes Antigen (MUCl).The vaccine does not contain tumor cells, but a clearly defined antigen (MUCl).
Zur Konstruktion der Vakzine verwendete Immunzellen sind dendritische Zellen. Autologe dendritische Zellen als Immunzellen stellen, anderes als Tumorzellen oder EBV- immortalisierte Zellen, für den Patienten kein Risiko dar. Sie exprimieren in idealer Weise die für die T-Zellaktivierung notwendigen kostimulierenden Liganden wie CD89 und CD86. Sie produzieren immunstimulierende Stoffe, wie Interleukin 12. Als weiteren Vorteil produzieren dendritische Zellen im Gegensatz zu beispielsweise EBV-immortalisierten B-Zellen, keine immunsupprimierenden Stoffe wie Interleukin 10. Die Herstellung humaner dendritischer Zellen erfolgt aus peripherem Blut von Patienten oder Gesunden unter Verwendung von Interleukin 4 und Granulozyten-Makrophagen-Colonie- stimulierendem Faktor. Das ist ein einfaches und leicht zu praktizierendes Verfahren.Immune cells used to construct the vaccine are dendritic cells. Autologous dendritic cells as immune cells, other than tumor cells or EBV immortalized cells, pose no risk to the patient. They ideally express the costimulatory ligands such as CD89 and CD86 necessary for T cell activation. They produce immunostimulatory substances, such as interleukin 12. As a further advantage, in contrast to, for example, EBV-immortalized B cells, dendritic cells do not produce any immunosuppressive substances such as interleukin 10. Human dendritic cells are produced from peripheral blood of patients or healthy people using interleukin 4 and granulocyte macrophage colony stimulating factor. This is a simple and easy to practice procedure.
Transfektionsmethode ist die Lipofektion. Dabei wird eine hohe Gentransferrate in dendritischen Zellen erreicht. Die Methode ist einfach durchzuführen und reproduzierbar.The transfection method is lipofection. A high gene transfer rate in dendritic cells is achieved. The method is easy to carry out and reproducible.
Alε Vektor für den Gentransfer wird pCMV/MUCl gemäß Abb.l verwendet. In den Vektor wurde die entsprechende Muzin-cDNA unter dem immediate early Promotor von CMV kloniert. Der Vektor enthält keine cDNA für eine Resistenz gegenüber Antibiotika o.a. Der Vektor erfüllt somit hohe Sicherheitsanforderungen für die Anwendung am Menschen.PCMV / MUCl according to Fig.l is used as the vector for gene transfer. The corresponding mucin cDNA was cloned into the vector under the immediate early promoter of CMV. The vector contains no cDNA for resistance to antibiotics or the like. The vector thus fulfills high requirements Safety requirements for human use.
Ein weiterer Vorteil der erfindungsgemäßen Vakzine besteht darin, daß die Erkennung der Muzin-Peptid-Epitope durch zytotoxische T Zellen nicht dem bisher bekannten klassischen Weg der Erkennung von kurzen Peptid-Epitopen in Verbindung mit dem HLA-Komplex folgt. Die Muzin-Peptid-Epitope werden ohne "Hilfe" des HLA-Komplexes von den T-Zellen erkannt. Diese Besonderheit bei der Erkennung der tumorassoziierten Muzin- Epitope erklärt sich aus der oben genannten besonderen "Tandem repeat"-Struktur des Moleküls sowie der hohen Dichte des Antigens auf der präsentierenden Zelle. Die mehrfache Wiederholung des immunogenen Peptid-Epitop-Motivs führt zu einer Aktivierung der T Zellen durch ein "Crosslinking" des T- Zell-Rezeptors, ohne daß der HLA-Komplex vorhanden sein muß. Das ermöglicht den Einsatz der Vakzine, die die entsprechenden Muzin-Epitope enthält, bei jedem Patienten, unabhängig von HLA-Typ. Das bedeutet einen Vorteil gegenüber Vakzinen, die Epitope anderer Antigene außer Muzin enthalten, und die nur Patienten mit dem jeweiligen passenden HLA-Typ verabreicht werden können.Another advantage of the vaccine according to the invention is that the recognition of the mucin-peptide epitopes by cytotoxic T cells does not follow the previously known classic way of recognizing short peptide epitopes in connection with the HLA complex. The mucin-peptide epitopes are recognized by the T cells without "help" from the HLA complex. This peculiarity in the detection of tumor-associated mucin epitopes can be explained by the above-mentioned special "tandem repeat" structure of the molecule and the high density of the antigen on the presenting cell. The repeated repetition of the immunogenic peptide-epitope motif leads to an activation of the T cells by "crosslinking" the T cell receptor without the HLA complex having to be present. This enables the vaccine, which contains the corresponding mucin epitopes, to be used in every patient, regardless of HLA type. This means an advantage over vaccines that contain epitopes of other antigens other than mucin and that can only be administered to patients with the appropriate HLA type.
Durch die Kombination dieser Neuerungen wird erfindungsgemäß die Konstruktion einer optimalen Vakzine erreicht.By combining these innovations, the construction of an optimal vaccine is achieved according to the invention.
Wirkprinzip der erfindungsgemaßen Vakzine:Operating principle of the vaccine according to the invention:
Infolge der unvollständigen Glykosylierung des Glykoproteins Muzin im Fall der malignen Entartung liegen auf Tumorzellen Peptid-Epitope frei, die vom Immunsystem als fremd erkannt werden können. Die dadurch ausgelöste Aktivierung des Immunsystems reicht bei Tumorpatienten nicht aus, um den Tumor zu beseitigen, weil (durch die fehlende Expression von CD80 und CD86 auf Tumorzellen) keine Kostimulation von T-Zellen erfolgt. Mit der erfindungsgemäßen Vakzine wird eine effiziente, tumorspezifische Immunantwort, die auf der Aktivierung Muzinepitop-spezifischer, zytotoxischer T-Zellen beruht, ausgelöst. Diese T-Zellen führen zur Verkleinerung bzw. Beseitigung der Tumorzellen. Werden dendritische Zellen mit MUCl (Kopien der Tandem Nukleotid-Sequenz von MUCl kloniert in den Vektor) transfiziert und gegebenenfalls mit dem Glykolysierungs-inhibitor Phenyl-N-Acetyl-α-D- Galactosaminid behandelt, exprimieren diese die tumorassoziierten Epitope. Die auf diesem Wege erreichte Kombination von Bereitstellung von kostimulierenden Liganden und einer genügenden Anzahl von tumorassoziierten Epitopen führt zu einer Aktivierung von Muzinepitop-spezifischen T- Zellen, die für eine Tumorabstoßung erforderlich sind.As a result of the incomplete glycosylation of the glycoprotein mucin in the case of malignant degeneration, peptide epitopes are exposed on tumor cells that can be recognized as foreign by the immune system. The activation of the immune system triggered by this is not sufficient in tumor patients to eliminate the tumor because (due to the lack of expression of CD80 and CD86 on tumor cells) there is no costimulation of T cells. The vaccine according to the invention triggers an efficient, tumor-specific immune response, which is based on the activation of mucine epitope-specific, cytotoxic T cells. These T cells lead to downsizing or elimination of the tumor cells. If dendritic cells are transfected with MUCl (copies of the tandem nucleotide sequence of MUCl cloned into the vector) and, if appropriate, treated with the glycosylation inhibitor phenyl-N-acetyl-α-D-galactosaminide, these express the tumor-associated epitopes. The combination of the provision of costimulatory ligands and a sufficient number of tumor-associated epitopes achieved in this way leads to an activation of mucine epitope-specific T cells which are required for tumor rejection.
Die Erfindung soll nachfolgend durch Ausführungsbeispiele näher erläutert werden.The invention will be explained in more detail below by means of exemplary embodiments.
1. Ausführungsbeispiel (Herstellung der transfizierten Zellen)1st embodiment (production of the transfected cells)
Dendritische Zellen werden aus humanem peripherem Blut isoliert und kultiviert. Am Tag 4 der Kultur der dendritischen Zellen wird die Transfektion der dendritischen Zellen vorgenommen. Dazu wird ein Vektor verwendet, der CMV als Promotor für das Fremdgen MUCl enthält. Für 750 000 dendritische Zellen werden 15 μl Lipofektin (Abb. 3) verwendet. Die erfolgreiche Transfektion des Femdgens MUCl wird mittels FACS-Analyse mit dem monoklonalen Antikörper HMFG-2 gegen Muzinepitope nachgewiesen. Nach Transfektion zeigen 12% der dendritischen Zellen eine Expression von Muzinepitopen. Durch Verwendung eines Glykosylierungs- inhibitors (Gl) sind bei 48% der dendritischen Zellen Muzinepitope auf der Oberfläche nachweisbar. Das kennzeichnet den erfolgreichen Gentransfer. Auch ohne Einsatz des Glykosylierungsinhibitors sind schon ausreichend immunogene Muzinepitope auf der Oberfläche vorhanden.Dendritic cells are isolated from human peripheral blood and cultured. On day 4 of the culture of the dendritic cells, the dendritic cells are transfected. For this a vector is used which contains CMV as a promoter for the foreign gene MUCl. 15 μl lipofectin (Fig. 3) is used for 750,000 dendritic cells. The successful transfection of the foreign gene MUCl is detected by means of FACS analysis with the monoclonal antibody HMFG-2 against mucin epitopes. After transfection, 12% of the dendritic cells show an expression of mucin epitopes. By using a glycosylation inhibitor (Gl), mucine epitopes can be detected on the surface of 48% of the dendritic cells. This marks the successful gene transfer. Even without using the glycosylation inhibitor, there are already sufficient immunogenic mucin epitopes on the surface.
Mock (Vektor ohne Fremdgen)-transfizierte Zellen exprimieren Muzinepitope zu höchsten 2% (s. Abb. 4)Mock (vector without foreign gene) transfected cells express mucin epitopes to a maximum of 2% (see Fig. 4)
2. Ausführungsbeispiel (Verwendung als Vakzine) 2.1. Herstellung der Vakzine2nd embodiment (use as a vaccine) 2.1. Production of the vaccine
Aus humanem peripherem Blut werden Lymphozyten per Ficoll- Gradienten-Zentrifugation gewonnen und in Kultur gehalten. Dendritische Zellen werden durch die Zugabe von Interleukin 4 und Granulozyten-Makrophagen-Colonie stimulierendem Faktor und durch die Adhärenz zu Platik selektioniert. Die dendritischen Zellen werden mittels Liposomen mit dem MUCl-Vektor transfiziert. Die Muzin-Expression wird mittels Western-Blot- Verfahren sowie FACS-Analyse mit monoklonalen Muzin- Antikörpern überprüft. In das Kulturmedium der transfizierten Zellen wird Phenyl-N-Acetyl-α-D-Galactosarainid (Konzentration 5 mM) für 36 Stunden gegeben. Die Expression der dadurch erzeugten tumorassoziierten Muzin-Peptid-Epitope hält für 72 Stunden an und wird mit monoklonalen Muzin-Peptid-Antikörpern mittels FACS-Analyse überprüft. Die Vakzine wird innerhalb dieser 72 Stunden dem Patienten appliziert.Lymphocytes are obtained from human peripheral blood by Ficoll gradient centrifugation and kept in culture. Dendritic cells are selected by the addition of interleukin 4 and granulocyte-macrophage colony stimulating factor and by adherence to platik. The dendritic cells are transfected with the MUCl vector using liposomes. Mucin expression is checked using Western blot methods and FACS analysis with monoclonal mucin antibodies. Phenyl-N-acetyl-α-D-galactosarainide (concentration 5 mM) is added to the culture medium of the transfected cells for 36 hours. The expression of the tumor-associated mucin-peptide epitopes generated in this way persists for 72 hours and is checked with monoclonal mucin-peptide antibodies by means of FACS analysis. The vaccine is applied to the patient within these 72 hours.
2.2. Verwendung der Vakzine2.2. Use of the vaccine
Die Vakzine ist für die Therapie bei Patienten mit Muzin (MUCl)-exprimierenden Tumoren einsetzbar. Bevorzugt ist die Behandlung von Mamma-, Pankreas-, Ovarial-, Kolon- und Parotistumoren. Die Anwendung dieser Vakzine kann ebenfalls bei Gesunden zur Vorbeugung eines Muzinepitope exprimierenden Tumors erfolgen. The vaccine can be used for therapy in patients with mucin (MUCl) -expressing tumors. The treatment of breast, pancreas, ovarian, colon and parotid tumors is preferred. This vaccine can also be used in healthy people to prevent a tumor expressing mucine epitopes.

Claims

Patentansprüche claims
1. Gentransfizierte humane dendritische Zellen1. Gene-transfected human dendritic cells
2. Verfahren zur Gentransfektion von humanen dendritischen Zellen, dadurch gekennzeichnet, daß die Transfektion eines fremden Gens mittels Liposomen erfolgt.2. A method for gene transfection of human dendritic cells, characterized in that the transfection of a foreign gene is carried out by means of liposomes.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß die Gentransfektion von humanen dendritischen Zellen mit der Liposoroenpräparation Lipofektin erfolgt.3. The method according to claim 2, characterized in that the gene transfection of human dendritic cells is carried out with the liposoroe preparation lipofectin.
4. Verfahren nach Anspruch 2 und 3, dadurch gekennzeichnet, daß zur liposomalen Gentransfektion von humanen dendritischen Zellen ein Vektor, der den Cytomegalievirus (CMV) als Promotor für das fremde Gen enthält, verwendet wird.4. The method according to claim 2 and 3, characterized in that a vector containing the cytomegalovirus (CMV) as a promoter for the foreign gene is used for liposomal gene transfection of human dendritic cells.
5. Verfahren nach Anspruch 2 bis 4, dadurch gekennzeichnet, daß die Konzentration von Lipofektin für die Transfektion von 750 000 dendritischen Zellen zwischen 5 bis 20 μl beträgt.5. The method according to claim 2 to 4, characterized in that the concentration of lipofectin for the transfection of 750,000 dendritic cells is between 5 to 20 ul.
6. Verfahren nach Anspruch 2 bis 5, dadurch gekennzeichnet, daß die Konzentration von Lipofektin für die Transfektion von 750 000 dendritischen Zellen 15 μl beträgt.6. The method according to claim 2 to 5, characterized in that the concentration of lipofectin for the transfection of 750,000 dendritic cells is 15 ul.
7. Verfahren nach Anspruch 2 bis 6, dadurch gekennzeichnet, daß die Transfektion an den Tagen 3 bis 5 der Kultur der dendritischen Zellen erfolgt.7. The method according to claim 2 to 6, characterized in that the transfection takes place on days 3 to 5 of the culture of the dendritic cells.
8. Verfahren nach Anspruch 2 bis 7, dadurch gekennzeichnet, daß die Transfektion am Tage 4 der Kultur der dendritischen Zellen erfolgt.8. The method according to claim 2 to 7, characterized in that the transfection takes place on day 4 of the culture of the dendritic cells.
9. Verwendung der nach Anspruch 1 bis 8 hergestellten Zellen nach Anspruch 1 als Vakzine gegen Tumorerkrankungen. 9. Use of the cells produced according to claim 1 to 8 according to claim 1 as a vaccine against tumor diseases.
10. Verwendung der nach Anspruch 1 bis 8 hergestellten Zellen nach Anspruch 1 als Vakzine gegen Tumorerkrankungen, dadurch gekennzeichnet, daß als cDNA zur Transfektion der humanen dendritischen Zellen die cDNA eines humanen Tumorantigens verwendet wird.10. Use of the cells according to claim 1 to 8 as claimed in claim 1 as a vaccine against tumor diseases, characterized in that the cDNA of a human tumor antigen is used as the cDNA for the transfection of the human dendritic cells.
11. Vakzine gegen humane Tumorerkrankungen unter Verwendung der humanen cDNA des Tumorantigens Muzin, bestehend aus11. Vaccine against human tumor diseases using the human cDNA of the tumor antigen mucin, consisting of
- humanen, autologen dendritschen Zellen, die mit einer- human, autologous dendritic cells, which with a
- Teilsequenz des humanen Muzin-MUCl-Gens, die mehrere "Tandem Repeat Nukleotid Sequenzen" von MUCl enthält, transfiziert sind,Partial sequence of the human mucin-MUCl gene which contains several "tandem repeat nucleotide sequences" of MUCl are transfected,
- und bei denen durch die Anwendung eines Glykosylierungs- inhibitors tumorassoziierte Epitope, bevorzugt auf der Zelloberfläche, ausgebildet sind.- And in which tumor-associated epitopes, preferably on the cell surface, are formed through the use of a glycosylation inhibitor.
12. Vakzine nach Anspruch 11, dadurch gekennzeichnet, daß die Teilsequenz des humanen Muzin-MUCl-Gens mittels Liposomenpräparationen in die dendritischen Zellen transfiziert wird.12. Vaccine according to claim 11, characterized in that the partial sequence of the human mucin MUCl gene is transfected into the dendritic cells by means of liposome preparations.
13. Vakzine nach Anspruch 11 bis 12, dadurch gekennzeichnet, daß die Teilsequenz des humanen Muzin-MUCl-Gens mit dem Vektor pCMV/MUCl mit folgenden wesentlichen Bestandteilen transfiziert wird:13. Vaccine according to claim 11 to 12, characterized in that the partial sequence of the human mucin MUCl gene is transfected with the vector pCMV / MUCl with the following essential components:
- immediate early Promotor CMV für Muzin-MUCl-Genabschnitteimmediate CMV immediate early promoter for mucin-MUCl gene segments
- Teilsequenz des Muzin-MUCl-Gens.- Partial sequence of the mucin-MUCl gene.
14. Vakzine nach Anspruch 11 bis 13, dadurch gekennzeichnet, daß die Teilsequenz des humanen Muzin-MUCl-Gens im Vektor 12- 40 "Tandem Nukleotid-repeats" enthält, wobei ein repeat die Nukleotid-Sequenz gemäß Abb. 2 aufweist.14. Vaccine according to claim 11 to 13, characterized in that the partial sequence of the human mucin MUCl gene in the vector 12-40 contains "tandem nucleotide repeats", a repeat having the nucleotide sequence according to Fig. 2.
15. Vakzine nach Anspruch 11 bis 14, dadurch gekennzeichnet, daß die Teilsequenz des humanen Muzin-MUCl-Gens im Vektor 22 "Tandem Nukleotid-repeats" enthält. 15. Vaccine according to claim 11 to 14, characterized in that the partial sequence of the human mucin MUCl gene in the vector 22 contains "tandem nucleotide repeats".
16. Vakzine nach Anspruch 11 bis 15, dadurch gekennzeichnet, daß als Glykosylierungsinhibitor Phenyl-N-Acetyl-α-D- Galactosaminid verwendet wird.16. Vaccine according to claim 11 to 15, characterized in that phenyl-N-acetyl-α-D-galactosaminide is used as glycosylation inhibitor.
17. Vakzine nach Anspruch 11 bis 16, dadurch gekennzeichnet, daß die dendritischen Zellen aus peripherem Blut von Patienten oder gesunden Personen unter Verwendung der Zytokine Interleukin 4 und Granulozyten-Makrophagen-Colonie stumulierendem Faktor gewonnen werden, daß die so gewonnenen dendritischen Zellen die Oberflächenmarker CDla, CD80, CD86 exprimieren und, daß auf den dendritischen Zellen nach Transfektion mit MUCl tumorassoziierte immunogene Epitope auf der Oberfläche nachweisbar sind.17. Vaccine according to claim 11 to 16, characterized in that the dendritic cells from peripheral blood of patients or healthy people using the cytokines interleukin 4 and granulocyte-macrophage colony stumbling factor are obtained that the dendritic cells thus obtained have the surface marker CDla , CD80, CD86 and that tumor-associated immunogenic epitopes can be detected on the surface of the dendritic cells after transfection with MUCl.
18. Vakzine nach Anspruch 11 bis 17 zur Behandlung von Muzin exprimierenden Tumoren, wie Mamma-, Pankreas-, Ovarial-, Kolon- und Parotistumoren, bei Patienten. 18. Vaccine according to claim 11 to 17 for the treatment of mucin-expressing tumors, such as breast, pancreas, ovary, colon and parotid tumors, in patients.
EP97922844A 1996-04-19 1997-04-14 Genetically transfected human dendritic cells, their production and use, preferably as vaccines Withdrawn EP0906444A1 (en)

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US5965381A (en) 1998-03-06 1999-10-12 Ludwig Institute For Cancer Research Delivery of proteins into eukaryotic cells with recombinant yersinia
EP1068296B1 (en) 1998-03-31 2011-08-10 Geron Corporation Compositions for eliciting an immune response to a telomerase antigen
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