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EP1098960A2 - Agents for the immunotherapy of tumoral diseases - Google Patents

Agents for the immunotherapy of tumoral diseases

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Publication number
EP1098960A2
EP1098960A2 EP99948682A EP99948682A EP1098960A2 EP 1098960 A2 EP1098960 A2 EP 1098960A2 EP 99948682 A EP99948682 A EP 99948682A EP 99948682 A EP99948682 A EP 99948682A EP 1098960 A2 EP1098960 A2 EP 1098960A2
Authority
EP
European Patent Office
Prior art keywords
tumor cells
genes
mhc
tumor
cells according
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.)
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Application number
EP99948682A
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German (de)
French (fr)
Inventor
Aida Kerkmann-Tucek
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KERKMANN TUCEK AIDA
Original Assignee
KERKMANN TUCEK AIDA
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Publication of EP1098960A2 publication Critical patent/EP1098960A2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001136—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons

Definitions

  • the present invention relates to agents which are suitable for the immunotherapy of tumor diseases.
  • agents are tumor cells, a tumor cell library containing them and the vaccine comprising the tumor cells.
  • the invention relates to a method for producing the tumor cells and the use of these as well as the vaccine and the tumor cell library.
  • a wide variety of methods are used to treat tumor diseases.
  • Primary tumors are often surgically removed and patients undergo post-treatment in the form of chemotherapy and / or radiation therapy.
  • the aim of the aftertreatment is to destroy the remaining tumor cells.
  • Immunotherapy methods are also attempted in which tumor cells obtained from the primary tumors are manipulated and returned to the patient. This is intended to sensitize the immune system to the tumor cells, thereby preventing later metastasis.
  • the immunotherapy procedures are not yet showing the desired results.
  • the sensitization of the immune system is not sufficient, which means that tumor cells remain undetected and can develop into metastases.
  • Immunotherapy procedures also require that patient's own tumor cells be used for treatment. This means a great expense and time. In many cases, immunotherapy cannot be carried out at all because insufficient tumor cells are obtained from the individual patient.
  • the present invention is therefore based on the object of providing an agent with which immunotherapy of tumor diseases can be carried out, the the above disadvantages can be avoided.
  • MHC major histocompatibility complex
  • HLA-A HLA-A
  • HLA-B HLA-C
  • HLA-Dr HLA-Dr
  • HLA-DQ HLA-DP genes.
  • the applicant has recognized that the expression of MHC I and / or MHC II genes is disturbed in tumor cells. In particular, he found that many tumor cells do not express MHC II genes.
  • tumor cells which have a combination of MHC I and MHC II genes which are present in a human and also express them have a high immunogenicity. Such combinations are particularly those given in Table I.
  • the immunogenicity of such tumor cells can be increased if they also express costimulatory molecules and / or cytokines.
  • the applicant's knowledge is used to provide tumor cells with a combination of MHC I and MHC II genes present in a human, the genes being expressed.
  • tumor cells includes tumor cells from any human tumor.
  • tumors include mom carcinoma, anogenital carcinoma, lung carcinoma, colon carcinoma, brain tumor, gastric carcinoma, bladder carcinoma, liver cell carcinoma and melanoma.
  • the tumor cells can be freshly isolated or in culture. They can also be present as such or in a cell assembly, for example a (primary) tumor or metastasis.
  • combination of MHC I and MHC II genes includes any combination of MHC I and MHC II genes that may be present in a human. In particular, the combination is selected from the combinations given in Table I.
  • the expression "expressed genes” indicates that the combination of MHC I and MHC II genes is expressed. This can be achieved using standard methods. It is favorable to remove the tumor cells and possibly other cells, e.g. Lymphocytes, from the same patient, are first subjected to tissue typing in order to determine which of the MHC I and / or MHC II genes have an impaired expression. The tissue typing can e.g. by means of serological methods as are known from the "llth International Histocompatibility Workshop". The disturbed expression of the MHC I and / or MHC II genes can then be compensated for by transfection into the tumor cells of corresponding exogenous genes which may be present on expression vectors.
  • transfected MHC I and / or MHC II genes can be stable or transient, with stable being preferred.
  • the detection of expression can be carried out by conventional methods, e.g. serological procedures, cf. above.
  • the above tumor cells also have one or more genes coding for costimulatory molecules and / or cytokines which are expressed.
  • costimulatory molecules include B7, such as B7-1 or B7-2, and CD44.
  • cytokines include interleukins such as IL-2, GM-CSF, TNF- ⁇ and interferon- ⁇ .
  • Another object of the present invention is a method for producing the above tumor cells.
  • Such a process comprises the following process steps:
  • tissue typing of tumor cells encompasses any method by which the expression of MHC I and MHC II genes can be determined. Reference is made to the above explanations. It may be beneficial for the same patient from whom the tumor cells originate to have other cells, e.g. Lymphocytes undergo tissue typing. This makes it even easier to determine the combination of MHC I and MHC II genes suitable for this patient.
  • transfection of tumor cells encompasses any method by which MHC I and / or MHC II genes can be transfected in tumor cells. Reference is made to the above explanations.
  • selection for tumor cells encompasses any method by which selection can be made for tumor cells which express the MHC I and MHC II genes. Reference is made to the above explanations.
  • the tumor cells are also transfected with one or more genes coding for costimulatory molecules and / or cytokines and for expression selected these genes.
  • Another object of the present invention is a tumor cell library comprising the above tumor cells. It is preferred if the tumor cells originate from any human tumor and comprise any combination of MHC I and MHC II genes present in a human. The tumor cells particularly preferably comprise the combinations of MHC I and MHC II genes given in Table I.
  • Another object of the present invention is a vaccine containing the above tumor cells and common adjuvants, e.g. Buffers, carriers and diluents. It is preferred if the vaccine comprises tumor cells from different tumors, each with the same combination of MHC I and MHC II genes.
  • common adjuvants e.g. Buffers, carriers and diluents. It is preferred if the vaccine comprises tumor cells from different tumors, each with the same combination of MHC I and MHC II genes.
  • the present invention provides tumor cells in which a combination of MHC I and MHC II genes is expressed, the combination being in a human.
  • the combination is one that many people have.
  • the tumor cells according to the invention thus represent an agent which can be administered not only to a specific person, but to many people.
  • the tumor cells come from any human tumor.
  • the present invention is not restricted to the treatment of a specific tumor, but can be used for any spectrum of tumors.
  • suitable tumor cells according to the invention for example from the tumor cell library, can be selected and administered to the patient. It is favorable if before Administration of the tumor cells prevents replication by measures such as radiation.
  • the present invention enables prophylactic measures against all possible tumors. For this it is only necessary to carry out a tissue typing of cells of the person to be treated and then to administer a suitable vaccine according to the invention.
  • the present invention thus represents a breakthrough in the field of immunotherapy for tumor diseases.
  • the tumor is removed from a melanoma patient, crushed and placed in several cell culture bottles with DMEM medium. After 48 hours of cultivation (37 ° C, 5% CO 2 ), the medium is changed. After 1-2 weeks, five cell culture bottles are selected, which are treated independently.
  • Blood is taken from melanoma patients of (A). This is provided with an anti-coagulant and diluted with the same volume of HBSS solution. The blood is given in tubes in which Fikol is presented. The tubes are centrifuged at 800 g for 20 min. The intermediate phase obtained is removed, resuspended in HBSS solution and centrifuged for 5 min at 500 g. The lymphocytes obtained are counted and used for Tissue typing standardized as a solution with a concentration of 1-2 x 10 6 / ml.
  • the established tumor cells of (A) are standardized in the same concentration.
  • Polystyrene plates are used which are coated with antisera against HLA-A, HLA-C, HLA-B, HLA-DR, HLA-DQ or HLA-DP.
  • 1 ⁇ l of the lymphocyte solution or tumor cell solution of (B) 1 is added to each of these plates.
  • the plates are incubated at 22 ° C. for 30 min before 5 ⁇ l of fresh complement are added in each case.
  • the plates are then incubated at 22 ° C. for 60 min, before 1 ⁇ l of an acridine orange / ethidium bromide cocktail and 1 ⁇ l Quentscher solution are added.
  • the plates are left at room temperature for 4 hours. Positive samples are identified by the development of the fluorescent stain.
  • lymphocytes have the following HLA molecules:
  • the tumor cells only have the following HLA molecules: A * 01; Cw * 07; B * 08.
  • RNA is isolated from the lymphocytes of (B) 1. and subjected to reverse transcription.
  • the cDNA obtained is subjected to a PCR method in which primer groups are used which are selected in accordance with the HLA molecules of (B) 2.
  • primer groups are used in particular:
  • DQA1 * 02 forward: CGA GTT TTA CGG TCC CTC TGG C reverse: CTC ATT GGT AGC AGC GGT AGA GTT GG
  • DQB1 * 02 forward: GTG CGT CTT GTG AGC AGA AG reverse: CGT GCG GAG CTC CAA CTG
  • DPA1 * 02 forward: CCC GCT CTG GTT TGA
  • DPB1 * 02 forward: AGG ACA GAA CTC GGT ACT AGG
  • a reverse TGA ATC CCC AAC CCA AAG TCC CC
  • the PCR conditions are as follows: 24 cycles: 1 min, 94 ° C; 45 sec, 65 ° C; 2 min, 72 ° C. End cycle: 1 min, 94 ° C; 45 sec, 65 ° C; 10 min, 72 ° C.
  • the amplified DNA is cleaved with the restriction enzymes Sall and Hindlll and inserted into the correspondingly cleaved vector M13 mpl8 or Ml3 mpl9. Recombinant DNA molecules are used to transform E. coli. JM109 used. Clones obtained are subjected to a screening process by means of hybridization with the amplified DNA. Positive clones are subjected to sequencing.
  • HLA molecules of (B) 2. are encoded by the following HLA genes: A * 0101; Cw * 0701; B * 0801; DRB1 * 0301; DQA1 * 0201; DQB1 * 0201; DPA1 * 0201; DPB1 * 0201.
  • Blood is drawn from the melanoma patient of (A). This is provided with an anti-coagulant and diluted with an eight-fold excess of RCL buffer. The blood is centrifuged in a microcentrifuge for 30 seconds. The pellet is taken up in RCL buffer and centrifuged. After repeating this step several times, the pellet is dissolved in NLB buffer and incubated with Proteinase K for 1 h at 63-65 ° C and 10 min at 95 ° C. The solution is centrifuged in a microcentrifuge for 60 seconds and the pellet is discarded. The supernatant contains the DNA from the lymphocytes.
  • This DNA is subjected to a PCR method in which those primers are used which are used in (B) 2.
  • DRB1 * 03, DQA1 * 02, DQB1 * 02, DPA1 * 02 or DPBl * 02 have been.
  • the PCR conditions are as follows: 30 cycles; 30 sec, 98 ° C; 60 sec, 55 ° C; 105 sec, 72 ° C. End cycle: 7 min, 72 ° C.
  • Samples of the amplified DNA are electrophoresed on a 1% agarose gel. Fragments of 216 bp for DRB1 * 03, of 219 bp for DQAl * 02 / DQBl * 02, and of 245 bp for DPA * 02 / DPB1 * 02 are obtained.
  • the amplified DNA is cleaved with the restriction enzymes Sall and Hindlll and inserted into the appropriately cleaved expression vector B45-neo.
  • Recombinant DNA molecules are used to transform E. coli JM109 or DH5F '.
  • Clones obtained are subjected to a screening process by means of hybridization with the amplified DNA. Positive clones are confirmed by sequencing. These clones are used to transfect the tumor cells from (A).
  • the tumor cells are trypsinized and electroporation is carried out at 400 V and 490 ⁇ FD.
  • the tumor cells are selected with G418 (400-10000 ⁇ g / ml) for 4 weeks before they are subjected to a "Fluorescence Activating Cell Sorting” (FACS). Tumor cells are obtained which express the following HLA molecules:
  • cDNAs coding for CD44, IFN- ⁇ and GM-CSF are obtained from Invitrogen.
  • the cDNAs are inserted into the expression vectors RSV.5 hygro (blunt / BamHI), pUHDl0-l (XmnI) or pBSK (BamHI).
  • Recombinant DNA molecules are used to transform E. coli JM109 or DH5'cc. Clones obtained are subjected to a screening process by means of hybridization with the cDNAs. Positive clones are confirmed by sequencing.
  • clones are used to transfect the tumor cells obtained in (B) 4.
  • the transfection is carried out with DOTAP liposomes according to the instructions of the manufacturer Boehringer Mannheim.
  • the transfected tumor cells are screened by FACS or RT-PCR. Tumor cells are obtained which express the following HLA molecules and costimulatory molecules as well as cytokines:

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Abstract

The invention relates to agents suitable for the immunotherapy of tumoral diseases. These agents are tumour cells with a combination of MHC I and MHC II genes occurring in humans, which genes are expressed. The invention further relates to a tumour cell library comprising the above tumour cells and to vaccines containing said tumour cells. The invention also relates to a method for producing the tumour cells and to the use of said cells and of the vaccines and the tumour cell library.

Description

Mittel zur Immuntherapie von Tumorerkrankungen Means for immunotherapy of tumor diseases
Die vorliegende Erfindung betrifft Mittel, die sich zur Immuntherapie von Tumorerkrankungen eignen. Solche Mittel sind Tumorzellen, eine diese enthaltende Tumorzell-Bibliothek und die Tumorzellen umfassende Vakzine. Ferner betrifft die Erfindung ein Verfahren zur Herstellung der Tumorzellen und die Verwendung dieser sowie der Vakzine und der Tumorzell- Bibliothek.The present invention relates to agents which are suitable for the immunotherapy of tumor diseases. Such agents are tumor cells, a tumor cell library containing them and the vaccine comprising the tumor cells. Furthermore, the invention relates to a method for producing the tumor cells and the use of these as well as the vaccine and the tumor cell library.
Zur Behandlung von Tumorerkrankungen werden die verschiedensten Verfahren durchgeführt. Vielfach werden Primärtumoren chirurgisch entfernt und die Patienten einer Nachbehandlung in Form einer Chemo- und/oder Strahlentherapie unterzogen. Durch die Nachbehandlung soll erreicht werden, daß restliche Tumorzellen zerstört werden. Auch werden Immuntherapie-Verfahren versucht, in denen aus den Primärtumoren erhaltene Tumorzellen manipuliert und den Patienten wieder zurückgegeben werden. Damit soll eine Sensibilisierung des Immunsystems für die Tumorzellen erreicht werden, wodurch eine spätere Metastasenbildung verhindert werden soll. Die Immuntherapie-Verfahren zeigen allerdings noch nicht die gewünschten Erfolge. Vielfach reicht die Sensibilisierung des Immunsystems nicht aus, wodurch Tumorzellen unerkannt bleiben und sich zu Metastasen ausbilden können. Auch fordern Immuntherapie-Verfahren, daß Patienteneigene Tumorzellen zur Behandlung verwendet werden. Dies bedeutet einen großen Kosten- und Zeitaufwand. Vielfach kann eine Immuntherapie überhaupt nicht durchgeführt werden, weil nicht genügend Tumorzellen aus dem einzelnen Patienten erhalten werden.A wide variety of methods are used to treat tumor diseases. Primary tumors are often surgically removed and patients undergo post-treatment in the form of chemotherapy and / or radiation therapy. The aim of the aftertreatment is to destroy the remaining tumor cells. Immunotherapy methods are also attempted in which tumor cells obtained from the primary tumors are manipulated and returned to the patient. This is intended to sensitize the immune system to the tumor cells, thereby preventing later metastasis. However, the immunotherapy procedures are not yet showing the desired results. In many cases, the sensitization of the immune system is not sufficient, which means that tumor cells remain undetected and can develop into metastases. Immunotherapy procedures also require that patient's own tumor cells be used for treatment. This means a great expense and time. In many cases, immunotherapy cannot be carried out at all because insufficient tumor cells are obtained from the individual patient.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem eine Immuntherapie von Tumorerkrankungen durchgeführt werden kann, wobei die vorstehenden Nachteile vermieden werden.The present invention is therefore based on the object of providing an agent with which immunotherapy of tumor diseases can be carried out, the the above disadvantages can be avoided.
Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the subject matter in the claims.
Die vorliegende Erfindung beruht auf den Erkenntnissen des Anmelders über den Zusammenhang zwischen der Expression von MHC ("major histocompatibility complex")I-, II-Genen in Tumorzellen und der Immunogenität dieser. MHC I-Gene werden auch mit HLA-A-, HLA-B- und HLA-C-Genen bezeichnet. Ferner werden MHC II-Gene auch mit HLA-Dr-, HLA-DQ- und HLA-DP-Genen bezeichnet. Der Anmelder hat erkannt, daß in Tumorzellen die Expression von MHC I- und/oder MHC II-Genen gestört ist. Insbesondere hat er gefunden, daß viele Tumorzellen keine MHC II-Gene exprimieren. Desweiteren hat er erkannt, daß durch die gestörte Expression der MHC I- und/oder MHC II-Gene und dem damit verbundenen Fehlen der entsprechenden Genprodukte auf der Oberfläche von Tumorzellen die dort vorliegenden Tumorantigene vom Immunsystem nicht als Fremd-Antigene erkannt und daher die Tumorzellen nicht zerstört werden. Andererseits hat der Anmelder gefunden, daß Tumorzellen, die eine in einem Menschen vorliegende Kombination von MHC I- und MHC II-Genen aufweisen und diese auch exprimieren, eine hohe Immunogenität aufweisen. Solche Kombinationen sind insbesondere jene, die in Tabelle I angegeben sind. Desweiteren hat er erkannt, daß die Immunogenität solcher Tumorzellen noch gesteigert werden kann, wenn sie ferner kostimulatorische Moleküle und/oder Zytokine exprimieren.The present invention is based on the knowledge of the applicant about the connection between the expression of MHC ("major histocompatibility complex") I, II genes in tumor cells and their immunogenicity. MHC I genes are also referred to as HLA-A, HLA-B and HLA-C genes. MHC II genes are also referred to as HLA-Dr, HLA-DQ and HLA-DP genes. The applicant has recognized that the expression of MHC I and / or MHC II genes is disturbed in tumor cells. In particular, he found that many tumor cells do not express MHC II genes. Furthermore, he recognized that due to the disturbed expression of the MHC I and / or MHC II genes and the associated lack of the corresponding gene products on the surface of tumor cells, the tumor antigens present there were not recognized by the immune system as foreign antigens and therefore the tumor cells not be destroyed. On the other hand, the applicant has found that tumor cells which have a combination of MHC I and MHC II genes which are present in a human and also express them have a high immunogenicity. Such combinations are particularly those given in Table I. Furthermore, he recognized that the immunogenicity of such tumor cells can be increased if they also express costimulatory molecules and / or cytokines.
Erfindungsgemäß werden die Erkenntnisse des Anmelders genutzt, Tumorzellen mit einer in einem Menschen vorliegenden Kombination von MHC I- und MHC II-Genen bereitzustellen, wobei die Gene exprimiert werden.According to the invention, the applicant's knowledge is used to provide tumor cells with a combination of MHC I and MHC II genes present in a human, the genes being expressed.
Der Ausdruck "Tumorzellen" umfaßt Tumorzellen jeglichen Tumors des Menschen. Beispiele von Tumoren umfassen Mamakarzinom, Anogenitalkarzinom, Lungenkarzinom, Colonkarzinom, Hirntumor, Magenkarzinom, Blasenkarzinom, Leberzellkarzinom und Melanom. Die Tumorzellen können frisch isoliert sein oder in Kultur vorliegen. Auch können sie als solche oder in einem Zellverband, z.B. (Primär) Tumor oder Metastase, vorliegen.The term "tumor cells" includes tumor cells from any human tumor. Examples of tumors include mom carcinoma, anogenital carcinoma, lung carcinoma, colon carcinoma, brain tumor, gastric carcinoma, bladder carcinoma, liver cell carcinoma and melanoma. The tumor cells can be freshly isolated or in culture. They can also be present as such or in a cell assembly, for example a (primary) tumor or metastasis.
Der Ausdruck "Kombination von MHC I- und MHC II-Genen" umfaßt jegliche Kombination von MHC I- und MHC II-Genen, die in einem Menschen vorliegen kann. Insbesondere ist die Kombination aus den in Tabelle I angegebenen Kombinationen ausgewählt.The term "combination of MHC I and MHC II genes" includes any combination of MHC I and MHC II genes that may be present in a human. In particular, the combination is selected from the combinations given in Table I.
Der Ausdruck "exprimierte Gene" weist darauf hin, daß die Kombination von MHC I- und MHC II-Gene exprimiert wird. Dies kann durch übliche Verfahren erreicht werden. Günstig ist es, die Tumorzellen und ggfs. andere Zellen, z.B. Lymphozyten, des gleichen Patienten, zunächst einer Gewebetypisierung zu unterziehen um festzustellen, welche der MHC I- und/oder MHC II-Gene eine gestörte Expression aufweisen. Die Gewebetypisierung kann z.B. durch serologische Verfahren, wie sie von dem "llth International Histocompatibility Workshop" bekannt sind, erfolgen. Die gestörte Expression der MHC I- und/oder MHC II-Gene kann dann durch Transfektion von entsprechenden exogenen, ggfs. auf Expressionsvektoren vorliegenden Genen in die Tumorzellen kompensiert werden. Beispiele von Expressionsvektoren umfassen pUHD10-I, pRcRSV, pBSK, RSV.5 hygro, pBJ und B45-neo, Beispiele von Trans- fektionsverfahren umfassen Calciumphosphat-Copräzipitation, Elektroporation, Lipofektion, DOTAP-Liposome und retrovirale Transfektion. Die Expression von transfizierten MHC I- und/- oder MHC II-Genen kann stabil oder transient sein, wobei stabil bevorzugt ist. Der Nachweis der Expression kann durch übliche Verfahren, z.B. serologische Verfahren, vgl. vorstehend, erfolgen.The expression "expressed genes" indicates that the combination of MHC I and MHC II genes is expressed. This can be achieved using standard methods. It is favorable to remove the tumor cells and possibly other cells, e.g. Lymphocytes, from the same patient, are first subjected to tissue typing in order to determine which of the MHC I and / or MHC II genes have an impaired expression. The tissue typing can e.g. by means of serological methods as are known from the "llth International Histocompatibility Workshop". The disturbed expression of the MHC I and / or MHC II genes can then be compensated for by transfection into the tumor cells of corresponding exogenous genes which may be present on expression vectors. Examples of expression vectors include pUHD10-I, pRcRSV, pBSK, RSV.5 hygro, pBJ and B45-neo. Examples of transfection methods include calcium phosphate coprecipitation, electroporation, lipofection, DOTAP liposomes and retroviral transfection. The expression of transfected MHC I and / or MHC II genes can be stable or transient, with stable being preferred. The detection of expression can be carried out by conventional methods, e.g. serological procedures, cf. above.
In bevorzugter Ausführungsform weisen vorstehende Tumorzellen auch ein oder mehrere für kostimulatorische Moleküle und/oder Zytokine kodierende Gene auf, die exprimiert werden. Beispiele von kostimulatorischen Molekülen umfassen B7 , wie B7-1 oder B7-2, und CD44. Beispiele von Zytokinen umfassen Interleukine, wie IL-2, GM-CSF, TNF-α und Interferon-γ. Das Vorliegen der angesprochenen Gene in den Tumorzellen und die Expression der Gene können in üblicher Weise erreicht werden. Es wird auf vorstehende Ausführungen verwiesen.In a preferred embodiment, the above tumor cells also have one or more genes coding for costimulatory molecules and / or cytokines which are expressed. Examples of costimulatory molecules include B7, such as B7-1 or B7-2, and CD44. Examples of cytokines include interleukins such as IL-2, GM-CSF, TNF-α and interferon-γ. The presence of the addressed genes in the tumor cells and the expression of the genes can be achieved in the usual way. Reference is made to the above explanations.
Ein weiterer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung vorstehender Tumorzellen. Ein solches Verfahren umfaßt die folgenden Verfahrensschritte:Another object of the present invention is a method for producing the above tumor cells. Such a process comprises the following process steps:
(a) Gewebetypisierung von Tumorzellen,(a) tissue typing of tumor cells,
(b) Transfektion der Tumorzellen mit MHC I- und/oder MHC II- Genen, wodurch eine in einem Menschen vorliegende Kombination dieser Gene erhalten wird, und(b) transfection of the tumor cells with MHC I and / or MHC II genes, whereby a combination of these genes present in a human is obtained, and
(c) Selektion auf jene Tumorzellen, welche die MHC I- und MHC II-Gene exprimieren.(c) Selection for those tumor cells that express the MHC I and MHC II genes.
Der Ausdruck "Gewebetypisierung von Tumorzellen" umfaßt jegliches Verfahren, mit dem die Expression von MHC I- und MHC II-Genen bestimmt werden kann. Es wird auf vorstehende Ausführungen verwiesen. Günstig kann es sein, daß von dem gleichen Patienten, von dem die Tumorzellen stammen, auch andere Zellen, z.B. Lymphozyten, einer Gewebetypisierung unterzogen werden. Damit wird die Feststellung der für diesen Patienten geeigneten Kombination von MHC I- und MHC II-Genen noch erleichtert.The term "tissue typing of tumor cells" encompasses any method by which the expression of MHC I and MHC II genes can be determined. Reference is made to the above explanations. It may be beneficial for the same patient from whom the tumor cells originate to have other cells, e.g. Lymphocytes undergo tissue typing. This makes it even easier to determine the combination of MHC I and MHC II genes suitable for this patient.
Der Ausdruck "Transfektion von Tumorzellen" umfaßt jegliches Verfahren, mit dem MHC I- und/oder MHC II-Gene, in Tumorzellen transfiziert werden können. Es wird auf vorstehende Ausführungen verwiesen.The term "transfection of tumor cells" encompasses any method by which MHC I and / or MHC II genes can be transfected in tumor cells. Reference is made to the above explanations.
Der Ausdruck "Selektion auf Tumorzellen" umfaßt jegliches Verfahren, mit dem auf Tumorzellen selektioniert werden kann, die MHC I- und MHC II-Gene exprimieren. Es wird auf vorstehende Ausführungen verwiesen.The term "selection for tumor cells" encompasses any method by which selection can be made for tumor cells which express the MHC I and MHC II genes. Reference is made to the above explanations.
In bevorzugter Ausführungsform werden die Tumorzellen auch mit ein oder mehreren für kostimulatorische Moleküle und/oder Zytokine kodierenden Gene transfiziert und auf die Expression dieser Gene selektioniert. Es wird auf vorstehende Ausführungen verwiesen.In a preferred embodiment, the tumor cells are also transfected with one or more genes coding for costimulatory molecules and / or cytokines and for expression selected these genes. Reference is made to the above explanations.
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Tumorzell-Bibliothek, die vorstehende Tumorzellen umfaßt. Bevorzugt ist es, wenn die Tumorzellen von jeglichem Tumor des Menschen stammen und jegliche in einem Menschen vorliegende Kombination von MHC I- und MHC II-Genen umfassen. Besonders bevorzugt umfassen die Tumorzellen die in Tabelle I angegebenen Kombinationen von MHC I- und MHC II-Genen.Another object of the present invention is a tumor cell library comprising the above tumor cells. It is preferred if the tumor cells originate from any human tumor and comprise any combination of MHC I and MHC II genes present in a human. The tumor cells particularly preferably comprise the combinations of MHC I and MHC II genes given in Table I.
Ein weiterer Gegenstand der vorliegenden Erfindung ist eine Vakzine, die vorstehende Tumorzellen und übliche Hilfsstoffe, z.B. Puffer, Trägermittel und Verdünnungsmittel, umfaßt. Bevorzugt ist es, wenn die Vakzine, Tumorzellen verschiedener Tumoren mit jeweils gleicher Kombination von MHC I- und MHC II-Gene umfaßt.Another object of the present invention is a vaccine containing the above tumor cells and common adjuvants, e.g. Buffers, carriers and diluents. It is preferred if the vaccine comprises tumor cells from different tumors, each with the same combination of MHC I and MHC II genes.
Die vorliegende Erfindung stellt Tumorzellen bereit, in denen eine Kombination von MHC I- und MHC II-Genen exprimiert wird, wobei die Kombination in einem Menschen vorliegt. Insbesondere ist die Kombination eine solche, die bei vielen Menschen vorliegt. Somit stellen die erfindungsgemäßen Tumorzellen ein Mittel dar, das nicht nur einem bestimmten Menschen, sondern vielen Menschen verabreicht werden kann. Ferner stammen die Tumorzellen von jeglichem Tumor des Menschen. Insofern ist die vorliegende Erfindung auch nicht auf die Behandlung eines bestimmten Tumors beschränkt, sondern kann für ein beliebig großes Spektrum von Tumoren verwendet werden.The present invention provides tumor cells in which a combination of MHC I and MHC II genes is expressed, the combination being in a human. In particular, the combination is one that many people have. The tumor cells according to the invention thus represent an agent which can be administered not only to a specific person, but to many people. Furthermore, the tumor cells come from any human tumor. In this respect, the present invention is not restricted to the treatment of a specific tumor, but can be used for any spectrum of tumors.
Mit der vorliegenden Erfindung ist es möglich, Patienten, die an einer Tumorerkrankung leiden, eine Immuntherapie anzubieten. Diese hat den großen Vorteil, daß sie rasch erfolgen kann. Nach Feststellung der Tumorart und seiner Typisierung bzw. von anderen aus dem gleichen Patienten stammenden Zellen, können geeignete erfindungsgemäße Tumorzellen, z.B. aus der Tumorzell-Bibliothek, ausgewählt und dem Pateinten verabreicht werden. Günstig ist es, wenn vor Verabreichung der Tumorzellen diese durch Maßnahmen, wie Bestrahlung, an einer Replikation gehindert werden.With the present invention, it is possible to offer immunotherapy to patients suffering from a tumor disease. This has the great advantage that it can be done quickly. After determining the type of tumor and its typing or from other cells originating from the same patient, suitable tumor cells according to the invention, for example from the tumor cell library, can be selected and administered to the patient. It is favorable if before Administration of the tumor cells prevents replication by measures such as radiation.
Desweiteren ermöglicht es die vorliegende Erfindung, prophylaktisch gegen alle möglichen Tumoren vorzugehen. Hierzu ist es nur notwendig, eine Gewebetypisierung von Zellen des zu behandelnden Menschen durchzuführen und diesem dann eine geeignete erfindungsgemäße Vakzine zu verabreichen.Furthermore, the present invention enables prophylactic measures against all possible tumors. For this it is only necessary to carry out a tissue typing of cells of the person to be treated and then to administer a suitable vaccine according to the invention.
Somit stellt die vorliegende Erfindung einen Durchbruch auf dem Gebiet der Immuntherapie von Tumorerkrankungen dar.The present invention thus represents a breakthrough in the field of immunotherapy for tumor diseases.
Die Erfindung wird durch das nachstehende Beispiel erläutert.The invention is illustrated by the example below.
Beispiel: Herstellung erfindungsgemäßer TumorzellenExample: Production of Tumor Cells According to the Invention
(A) Etablierung von Tumorzellen eines Melanom-Patienten(A) Establishment of tumor cells from a melanoma patient
Aus einem Melanom-Patienten wird der Tumor entfernt, zerkleinert und in mehrere Zellkulturflaschen mit DMEM-Medium gegeben. Nach 48 Stunden Kultivierung (37°C, 5 % C02) wird das Medium gewechselt. Nach 1-2 Wochen werden fünf Zellkulturflaschen ausgewählt, die unabhängig voneinander behandelt werden.The tumor is removed from a melanoma patient, crushed and placed in several cell culture bottles with DMEM medium. After 48 hours of cultivation (37 ° C, 5% CO 2 ), the medium is changed. After 1-2 weeks, five cell culture bottles are selected, which are treated independently.
(B) Gewebetypisierung von Lymphozyten und Tumorzellen eines Melanom-Patienten(B) Tissue typing of lymphocytes and tumor cells from a melanoma patient
1. Vorbereitung von Lymphozyten und Tumorzellen für die Gewebetypisierung.1. Preparation of lymphocytes and tumor cells for tissue typing.
Melanom-Patienten von (A) wird Blut entnommen. Dieses wird mit einem Anti-Koagulantium versehen und mit gleichem Volumen an HBSS-Lösung verdünnt. Das Blut wird in Röhrchen gegeben, in denen Fikol vorgelegt ist. Die Röhrchen werden 20 min bei 800 g zentrifugiert . Die erhaltene Zwischenphase wird entnommen, in HBSS-Lösung resuspendiert und 5 min bei 500 g zentrifugiert. Die erhaltenen Lymphozyten werden gezählt und für die Gewebetypisierung als Lösung mit einer Konzentration von 1-2 x 106/ml standardisiert.Blood is taken from melanoma patients of (A). This is provided with an anti-coagulant and diluted with the same volume of HBSS solution. The blood is given in tubes in which Fikol is presented. The tubes are centrifuged at 800 g for 20 min. The intermediate phase obtained is removed, resuspended in HBSS solution and centrifuged for 5 min at 500 g. The lymphocytes obtained are counted and used for Tissue typing standardized as a solution with a concentration of 1-2 x 10 6 / ml.
Die etablierten Tumorzellen von (A) werden in gleicher Konzentration standardisiert.The established tumor cells of (A) are standardized in the same concentration.
2. Serologische Bestimmung von HLA-Molekülen auf Lymphozyten und Tumorzellen2. Serological determination of HLA molecules on lymphocytes and tumor cells
Es werden Polystyren-Platten verwendet, die mit Antiseren gegen HLA-A, HLA-C, HLA-B, HLA-DR, HLA-DQ bzw. HLA-DP beschichtet sind. Diesen Platten wird jeweils 1 μl der Lymphozyten-Lösung bzw. Tumorzell-Lösung von (B) 1. zugegeben. Die Platten werden 30 min bei 22°C inkubiert, bevor jeweils 5 μl frisches Komplement zugegeben werden. Anschließend werden die Platten 60 min bei 22°C inkubiert, bevor jeweils 1 μl eines Acridinorange/Ethidiumbromid-Cocktail und 1 μl Quentscher- Lösung zugegeben werden. Die Platten werden bei Raumtemperatur 4 Stunden stehengelassen. Positive Proben werden durch die Entwicklung der fluoreszierenden Färbung ausgewiesen.Polystyrene plates are used which are coated with antisera against HLA-A, HLA-C, HLA-B, HLA-DR, HLA-DQ or HLA-DP. 1 μl of the lymphocyte solution or tumor cell solution of (B) 1 is added to each of these plates. The plates are incubated at 22 ° C. for 30 min before 5 μl of fresh complement are added in each case. The plates are then incubated at 22 ° C. for 60 min, before 1 μl of an acridine orange / ethidium bromide cocktail and 1 μl Quentscher solution are added. The plates are left at room temperature for 4 hours. Positive samples are identified by the development of the fluorescent stain.
Es zeigt sich, daß die Lymphozyten folgende HLA-Moleküle aufweisen:It can be seen that the lymphocytes have the following HLA molecules:
A*01; Cw*07; B*08; DRB1*03; DQA1*02; DQB1*02; DPA1*02;A * 01; Cw * 07; B * 08; DRB1 * 03; DQA1 * 02; DQB1 * 02; DPA1 * 02;
DPB1*02.DPB1 * 02.
Die Tumorzellen weisen dagegen nur folgende HLA-Moleküle auf: A*01; Cw*07; B*08.In contrast, the tumor cells only have the following HLA molecules: A * 01; Cw * 07; B * 08.
3. Bestimmung der HLA-Gene von Lymphozyten3. Determination of the HLA genes of lymphocytes
Aus den Lymphozyten von (B) 1. wird RNA isoliert und einer reversen Transkription unterzogen. Die erhaltene cDNA wird einem PCR-Verfahren unterworfen, bei dem Primer-Gruppen verwendet werden, die entsprechend der HLA-Moleküle von (B) 2. ausgewählt sind. Insbesondere werden folgende Primer verwendet : A*01: forward: CGA CGC CGC GAG CCA GAA reverse: AGC CCG TCC ACG CAC CGRNA is isolated from the lymphocytes of (B) 1. and subjected to reverse transcription. The cDNA obtained is subjected to a PCR method in which primer groups are used which are selected in accordance with the HLA molecules of (B) 2. The following primers are used in particular: A * 01: forward: CGA CGC CGC GAG CCA GAA reverse: AGC CCG TCC ACG CAC CG
Cw*07: forward: GGA CCG GGA GAC ACA GAA C reverse: CGC ACG GGC CGC CTC CACw * 07: forward: GGA CCG GGA GAC ACA GAA C reverse: CGC ACG GGC CGC CTC CA
B*08: forward: GAC CGG AAC ACA CAG ATC TT reverse: CCG CGC GCT CCA GCG TGB * 08: forward: GAC CGG AAC ACA CAG ATC TT reverse: CCG CGC GCT CCA GCG TG
DRB1*03: forward: GAC GGA GCG GGT GCG GTA reverse: CTG CAC TGT GAA GCT CTC CADRB1 * 03: forward: GAC GGA GCG GGT GCG GTA reverse: CTG CAC TGT GAA GCT CTC CA
DQA1*02: forward: CGA GTT TTA CGG TCC CTC TGG C reverse: CTC ATT GGT AGC AGC GGT AGA GTT GGDQA1 * 02: forward: CGA GTT TTA CGG TCC CTC TGG C reverse: CTC ATT GGT AGC AGC GGT AGA GTT GG
DQB1*02: forward: GTG CGT CTT GTG AGC AGA AG reverse: CGT GCG GAG CTC CAA CTGDQB1 * 02: forward: GTG CGT CTT GTG AGC AGA AG reverse: CGT GCG GAG CTC CAA CTG
DPA1*02: forward: CCC GCT CTG GTT TGA TTT AT reverse: CAC TTC GCA TCT ATG CGADPA1 * 02: forward: CCC GCT CTG GTT TGA TTT AT reverse: CAC TTC GCA TCT ATG CGA
DPB1*02: forward: AGG ACA GAA CTC GGT ACT AGG A reverse: TGA ATC CCC AAC CCA AAG TCC CCDPB1 * 02: forward: AGG ACA GAA CTC GGT ACT AGG A reverse: TGA ATC CCC AAC CCA AAG TCC CC
Die PCR-Bedingungen sind wie folgt: 24 Zyklen: 1 min, 94°C; 45 sec, 65°C; 2 min, 72°C. Endzyklus: 1 min, 94°C; 45 sec, 65°C; 10 min, 72°C.The PCR conditions are as follows: 24 cycles: 1 min, 94 ° C; 45 sec, 65 ° C; 2 min, 72 ° C. End cycle: 1 min, 94 ° C; 45 sec, 65 ° C; 10 min, 72 ° C.
Die amplifizierte DNA wird mit den Restriktionsenzymen Sall und Hindlll gespalten und in den entsprechend gespaltenen Vektor M13 mpl8 bzw. Ml3 mpl9 inseriert. Rekombinante DNA- Moleküle werden zur Transformation von E. coli. JM109 verwendet. Erhaltene Klone werden einem Screening-Verfahren mittels Hybridisierung mit der amplifizierten DNA unterzogen. Positive Klone werden einer Sequenzierung unterworfen.The amplified DNA is cleaved with the restriction enzymes Sall and Hindlll and inserted into the correspondingly cleaved vector M13 mpl8 or Ml3 mpl9. Recombinant DNA molecules are used to transform E. coli. JM109 used. Clones obtained are subjected to a screening process by means of hybridization with the amplified DNA. Positive clones are subjected to sequencing.
Es zeigt sich, daß die HLA-Moleküle von (B) 2. durch folgende HLA-Gene kodiert sind: A*0101; Cw*0701; B*0801; DRB1*0301; DQA1*0201; DQB1*0201; DPA1*0201; DPB1*0201.It can be seen that the HLA molecules of (B) 2. are encoded by the following HLA genes: A * 0101; Cw * 0701; B * 0801; DRB1 * 0301; DQA1 * 0201; DQB1 * 0201; DPA1 * 0201; DPB1 * 0201.
4. Isolierung von HLA-D Genen aus Lymphozyten und Transfektion dieser in Tumorzellen4. Isolation of HLA-D genes from lymphocytes and transfection of these into tumor cells
Dem Melanom-Patienten von (A) wird Blut entnommen. Dieses wird mit einem Anti-Koagulantium versehen und mit achtfachem Überschuß an RCL-Puffer verdünnt. Das Blut wird 30 sec in einer Mikrozentrifuge zentrifugiert . Das Pellet wird mit RCL- Puffer aufgenommen und zentrifugiert. Nach mehrmaligem Wiederholen dieses Schrittes wird das Pellet in NLB-Puffer gelöst und mit Proteinase K 1 h bei 63-65°C und 10 min bei 95°C inkubiert. Die Lösung wird 60 sec in einer Mikrozentrifuge zentrifugiert und das Pellet verworfen. Der Überstand enthält die DNA aus den Lymphozyten.Blood is drawn from the melanoma patient of (A). This is provided with an anti-coagulant and diluted with an eight-fold excess of RCL buffer. The blood is centrifuged in a microcentrifuge for 30 seconds. The pellet is taken up in RCL buffer and centrifuged. After repeating this step several times, the pellet is dissolved in NLB buffer and incubated with Proteinase K for 1 h at 63-65 ° C and 10 min at 95 ° C. The solution is centrifuged in a microcentrifuge for 60 seconds and the pellet is discarded. The supernatant contains the DNA from the lymphocytes.
Diese DNA wird einem PCR-Verfahren unterzogen bei dem jene Primer verwendet werden, die in (B) 2. für die HLA-D Gene, DRB1*03, DQA1*02, DQB1*02, DPA1*02 bzw. DPBl*02 eingesetzt worden sind.This DNA is subjected to a PCR method in which those primers are used which are used in (B) 2. for the HLA-D genes, DRB1 * 03, DQA1 * 02, DQB1 * 02, DPA1 * 02 or DPBl * 02 have been.
Die PCR-Bedingungen sind wie folgt: 30 Zyklen; 30 sec, 98°C; 60 sec, 55°C; 105 sec, 72°C. Endzyklus: 7 min, 72°C.The PCR conditions are as follows: 30 cycles; 30 sec, 98 ° C; 60 sec, 55 ° C; 105 sec, 72 ° C. End cycle: 7 min, 72 ° C.
Proben der amplifizierten DNA werden auf einem 1 % Agarose-Gel elektrophoretisch aufgetrennt. Es werden Fragmente von 216 bp für DRB1*03, von 219 bp für DQAl*02/DQBl*02 , und von 245 bp für DPA*02/DPB1*02 erhalten.Samples of the amplified DNA are electrophoresed on a 1% agarose gel. Fragments of 216 bp for DRB1 * 03, of 219 bp for DQAl * 02 / DQBl * 02, and of 245 bp for DPA * 02 / DPB1 * 02 are obtained.
Die amplifizierte DNA wird mit den Restriktionsenzymen Sall und Hindlll gespalten und in den entsprechend gespaltenen Expressionsvektor B45-neo inseriert. Rekombinante DNA-Moleküle werden zur Transformation von E. coli JM109 oder DH5F' verwendet. Erhaltene Klone werden einem Screening-Verfahren mittels Hybridisierung mit der amplifizierten DNA unterzogen. Positive Klone werden durch Sequenzierung bestätigt. Diese Klone werden zur Transfektion der Tumorzellen von (A) verwendet. Die Tumorzellen werden trypsiniert und es wird eine Elektroporation bei 400 V und 490μ FD durchgeführt. Die Tumorzellen werden mit G418 (400 - lOOOμg/ml) 4 Wochen selektioniert, bevor sie einem "Fluorescence-Activating-Cell- Sorting" (FACS) unterzogen werden. Es werden Tumorzellen erhalten, die folgende HLA-Moleküle exprimieren:The amplified DNA is cleaved with the restriction enzymes Sall and Hindlll and inserted into the appropriately cleaved expression vector B45-neo. Recombinant DNA molecules are used to transform E. coli JM109 or DH5F '. Clones obtained are subjected to a screening process by means of hybridization with the amplified DNA. Positive clones are confirmed by sequencing. These clones are used to transfect the tumor cells from (A). The tumor cells are trypsinized and electroporation is carried out at 400 V and 490μ FD. The tumor cells are selected with G418 (400-10000 μg / ml) for 4 weeks before they are subjected to a "Fluorescence Activating Cell Sorting" (FACS). Tumor cells are obtained which express the following HLA molecules:
A*0101; Cw*0701; B*0801; DRB1*0301; DQA1*0201; DQB1*0201; DPA1*0201; DPB1*0201.A * 0101; Cw * 0701; B * 0801; DRB1 * 0301; DQA1 * 0201; DQB1 * 0201; DPA1 * 0201; DPB1 * 0201.
5. Transfektion von Tumorzellen mit Genen, die für kostimulatorische Moleküle bzw. Zytokine kodieren.5. Transfection of tumor cells with genes that code for costimulatory molecules or cytokines.
cDNAs, die für CD44, IFN-γ bzw. GM-CSF kodieren, werden von Invitrogen erhalten. Die cDNAs werden in die Expressionsvektoren RSV.5 hygro (blunt/BamHI) , pUHDl0-l (XmnI) bzw. pBSK (BamHI) inseriert. Rekombinante DNA-Moleküle werden zur Transformation von E. coli JM109 oder DH5'cc verwendet. Erhaltene Klone werden einem Screening-Verfahren mittels Hybridisierung mit den cDNAs unterzogen. Positive Klone werden durch Sequenzierung bestätigt.cDNAs coding for CD44, IFN-γ and GM-CSF are obtained from Invitrogen. The cDNAs are inserted into the expression vectors RSV.5 hygro (blunt / BamHI), pUHDl0-l (XmnI) or pBSK (BamHI). Recombinant DNA molecules are used to transform E. coli JM109 or DH5'cc. Clones obtained are subjected to a screening process by means of hybridization with the cDNAs. Positive clones are confirmed by sequencing.
Diese Klone werden zur Transfektion der in (B) 4. erhaltenen Tumorzellen verwendet. Die Transfektion wird mit DOTAP- Liposomen nach Anleitung des Herstellers Boehringer Mannheim durchgeführt. Die transfizierten Tumorzellen werden durch FACS bzw. RT-PCR gescreent. Es werden Tumorzellen erhalten, die folgende HLA-Moleküle und kostimulatorische Moleküle sowie Zytokine exprimieren:These clones are used to transfect the tumor cells obtained in (B) 4. The transfection is carried out with DOTAP liposomes according to the instructions of the manufacturer Boehringer Mannheim. The transfected tumor cells are screened by FACS or RT-PCR. Tumor cells are obtained which express the following HLA molecules and costimulatory molecules as well as cytokines:
A*0101; Cw*0701; B*0801; DKB1*0301; DQA1*0201; DQBl* 0201; DPA1*0201; DPB1*0201; IFN-γ; CD44; GM-CSF. Tabelle I Häufige HLA KombinationenA * 0101; Cw * 0701; B * 0801; DKB1 * 0301; DQA1 * 0201; DQBl * 0201; DPA1 * 0201; DPB1 * 0201; IFN-γ; CD44; GM-CSF. Table I Common HLA combinations

Claims

Patentansprüche claims
1. Tumorzellen mit einer in einem Menschen vorliegenden Kombination von MHC I- und MHC II-Genen, wobei die Gene exprimiert werden.1. Tumor cells with a combination of MHC I and MHC II genes present in a human, the genes being expressed.
2. Tumorzellen nach Anspruch 1 , wobei ferner ein oder mehrere Gene für kostimulatorische Moleküle exprimiert werden.2. Tumor cells according to claim 1, wherein further one or more genes for costimulatory molecules are expressed.
3. Tumorzellen nach Anspruch 2, wobei die kostimulatorischen Moleküle B7 und CD44 umfassen.3. Tumor cells according to claim 2, wherein the costimulatory molecules comprise B7 and CD44.
Tumorzellen nach einem der Ansprüche 1-3, wobei ferner ein oder mehrere Gene für Zytokine exprimiert werden.Tumor cells according to one of claims 1-3, wherein further one or more genes for cytokines are expressed.
Tumorzellen nach Anspruch 4, wobei die Zytokine Interleukine, GM-CSF, TNF-σ und Interferon-γ umfassen.Tumor cells according to claim 4, wherein the cytokines comprise interleukins, GM-CSF, TNF-σ and interferon-γ.
Tumorzellen nach einem der Ansprüche 1-5, wobei die Kombination von MHC I- und MHC II-Genen ausgewählt ist aus:Tumor cells according to any one of claims 1-5, wherein the combination of MHC I and MHC II genes is selected from:
7. Tumorzellen nach einem der Ansprüche 1 - 6, umfassend die folgende Kombination von MHC I- und MHC II-Genen:7. Tumor cells according to one of claims 1-6, comprising the following combination of MHC I and MHC II genes:
A*0101; Cw*0701; B*0801; DRB1*0301; DQA1*0201; DQB1*0201; DPA1- *0201; DPB1*0201. A * 0101; Cw * 0701; B * 0801; DRB1 * 0301; DQA1 * 0201; DQB1 * 0201; DPA1- * 0201; DPB1 * 0201.
8. Tumorzellen nach einem der Ansprüche 1 - 7, umfassend die folgende Kombination von MHC I-/Il-Genen und Genen für IFN-γ, CD44 sowie GM- CSF:8. Tumor cells according to any one of claims 1-7, comprising the following combination of MHC I / Il genes and genes for IFN-γ, CD44 and GM-CSF:
A*010ϊ ; Cw*0701 ; B*0801 ; DRB1*0301 ; DQA1*0201 ; DQB1*0201 ; DPA1- *0201 ; DPB1*0201 ; IFN-γ; CD44; GM-CSF.A * 010ϊ; Cw * 0701; B * 0801; DRB1 * 0301; DQA1 * 0201; DQB1 * 0201; DPA1- * 0201; DPB1 * 0201; IFN-γ; CD44; GM-CSF.
9. Verfahren zur Herstellung der Tumorzellen nach Anspruch 1 , umfassend die folgenden Verfahrensschritte:9. A method for producing the tumor cells according to claim 1, comprising the following process steps:
(a) Gewebetypisierung von Tumorzellen,(a) tissue typing of tumor cells,
(b) Transfektion der Tumorzellen mit MHC I- und/oder MHC II-Genen, wodurch eine in einem Menschen vorliegende Kombination dieser Gene erhalten wird, und(b) transfection of the tumor cells with MHC I and / or MHC II genes, whereby a combination of these genes present in a human is obtained, and
(c) Selektion auf jene Tumorzellen, welche die MHC I- und MHC II-Gene exprimieren.(c) Selection for those tumor cells that express the MHC I and MHC II genes.
10. Verfahren nach Anspruch 9, wobei ferner die Tumorzellen mit ein oder mehreren für kostimulatorische Moleküle und/oder Zytokine kodierenden Genen transfiziert und auf die Expression dieser Gene selektioniert werden.10. The method according to claim 9, further comprising transfecting the tumor cells with one or more genes coding for costimulatory molecules and / or cytokines and selecting them for the expression of these genes.
11. Tumorzell-Bibliothek, umfassend die Tumorzellen nach einem der Ansprüche 1-8.11. Tumor cell library comprising the tumor cells according to any one of claims 1-8.
12. Vakzine, umfassend die Tumorzellen nach einem der Ansprüche 1-8 und übliche Hilfsstoffe.12. Vaccine comprising the tumor cells according to any one of claims 1-8 and customary auxiliaries.
13. Verwendung der Tumorzellen nach einem der Ansprüche 1-8, der Tumorzell-Bibliothek nach Anspruch 11 oder der Vakzine nach Anspruch 12 zur Prophylaxe und/oder Behandlung von Tumorerkrankungen. 13. Use of the tumor cells according to any one of claims 1-8, the tumor cell library according to claim 11 or the vaccine according to claim 12 for the prophylaxis and / or treatment of tumor diseases.
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