WO2003045428A2 - Use of a technically modified cell as a vaccine for treating tumoral disease - Google Patents

Abstract

The invention relates to the use of a technically modified cell in the treatment of a tumoral disease. The technically modified cell is not derived from the respective tumoral disease. The invention also relates to a medicament for the prophylaxis, therapy and/or secondary prophylaxis of a tumoral disease, containing a technically modified cell.

Description

 Use of a technically modified cell as a vaccine for the treatment of a tumor disease

The invention relates to the use of a technically modified cell for treating a tumor disease, the technically modified cell not being derived from the respective tumor disease, and to a medicament for prophylaxis, therapy and / or secondary prophylaxis of a tumor disease containing a technically modified cell.

The activation of the body's immune system for the treatment and prevention of tumors is considered a promising approach in modern cancer therapy. Corresponding therapeutic agents are generally referred to as nakzine / tumor vaccine. The goal of a tumor vaccine is to trigger an immune response that is able to specifically destroy tumor cells in the body. These can be both tumor cells of the primary tumor and metastases of the tumor.

Some of these therapies use single or multiple antigens, which are present as proteins, peptides or DΝA expression vectors. Such naczines can be used, for example, as mixtures of individual tumor antigens, e.g. Tumor cell lysates are present.

A group of tumor vaccines are the cellular vaccines, which can be used generally as autologous and allogeneic vaccines (Pardoll DM (1998) Νat Med 4 (5 Suppl): 525-31; Wolchock JD and Livingston PO (2001) Lancet Oncol 2 (4): 205-ll; Schadendorf D et al. (2000) Immunol Lett 15; 74 (l): 67-74). In the autologous vaccine, cells from the patient's own tumor are used to produce the vaccine. The tumor cells are removed from the body, if necessary cultivated and / or modified (genetically engineered or biochemically) and made proliferation-incompetent, for example by radiation, before they are re-administered to the patient. The aim is that immune cells, in particular cytotoxic T cells and helper T cells, recognize the administered cells and thus trigger an immune response, which can then also be directed against the tumor or metastases derived therefrom.

The advantage of an autologous vaccine is that the match between the tumor antigens between the vaccine cells and the tumor cells to be controlled is as great as possible. The disadvantage is that each patient needs an individually produced vaccine and the time it takes to produce such a vaccine may be long. Since the mortality of tumor patients is very high in many tumor indications, there is a long pause between diagnosis / preparation of therapy e.g. cannot be tolerated by removing cells and subsequent vaccination. In addition, an individual vaccine is often not feasible for cost reasons.

An alternative to autologous vaccination is so-called allogeneic vaccination, ie immunization with cells that do not come from the same patient but from the same tumor type. In this form of vaccination, the vaccine cells differ from the patient's own tumor cells in two ways. On the one hand, they usually do not have the identical transplantation antigens (MHC genes), which play a role in the interaction between the vaccine cell and T cells. On the other hand, the vaccine cells and the tumor cells to be controlled differ in the pattern of the expressed tumor antigens, since tumor cells of different origins also express different tumor antigens. However, since the immune response hoped for by vaccination is directed against certain tumor antigens, the greatest possible overlap The tumor antigen pattern in allogeneic vaccines is desirable or necessary for the vaccine to work.

It has been shown in the prior art that tumor cells of the same type express similar tumor antigen patterns. For example, it is known for melanoma that melanoma cell lines mostly express the tumor antigens tyrosinase, MARTl / MelanA, Ny-ESO-1, MAGE3 and gplOO (Peter I et al. (2001) Melanoma Research 11, 21-30). This finding makes the principle of allogeneic vaccination practicable, since there is a high probability that a sufficient number of tumor antigens are identical between the vaccine cell and the tumor cell to be controlled.

Other criteria for the selection of a suitable cell line for an allogeneic vaccine are usually the expression levels of the tumor antigens and the expression of other genes, e.g. the MHC class 1 and 2 molecules, their haplotypes or the lack of immuno-inhibitory molecules (such as IL10, TGF-ß and Fas ligand).

The advantages of allogeneic vaccines are that patients can be vaccinated immediately when therapy is started, since in contrast to autologous vaccination, the vaccine does not have to be manufactured individually. In addition, every patient can potentially be vaccinated, whereas in an autologous vaccine a number of patients cannot be vaccinated, since the success rate for the cultivation of tumor cells of the patient is usually far below 100% and some of the patients do not until the vaccine is provided is more treatable.

Nevertheless, even with approaches with allogeneic vaccination, there is the difficulty that a specific allogeneic cell line is required for each tumor type in order to ensure the greatest possible overlap of the tumor antigen patterns, as explained above. The development of a specific allogeneic vaccine is however, from a commercial point of view, unattractive for various tumor types with rather low patient numbers. And even for larger indications, it would be particularly advantageous for economic reasons to have only one vaccine for as many patients or types of tumors as possible.

The object underlying the invention is therefore to develop a vaccine which is suitable for a large number of different tumor diseases and a large number of tumor patients.

According to the invention, the object is achieved by using a technically modified cell for the production of a vaccine for the treatment or prevention of a tumor disease, the technically modified cell not being derived from the respective tumor disease.

A technically modified (translated from engineered) cell according to the present invention is understood to mean a cell which has been isolated from an organism and has been adapted for cultivation in cell culture. This includes measures of subcloning, adaptation to certain growth conditions such as temperature, media or media additives such as growth factors, cytokme etc. Technically modified cells are therefore also cells which have been treated with soluble substances such as growth factors or cytokines and which then show a different expression pattern with regard to a protein.

Such technically modified cells may also have been genetically modified. Genetic engineering changes include the introduction of genetic material in the form of DNA or RNA, for example for the expression of transgenes. The technically modified cell is preferably a eukaryotic cell, particularly preferably a mammalian cell, in particular a human cell.

In particular, the term “technically modified cell” relates to cells obtained from tumors and cell lines derived from tumor cells.

For the purposes of the invention, “derived” means that one or more cell line (s) are (are) generated from an individual tumor cell or individual tumor cells. Such methods are described in the prior art.

For the purposes of the invention, “tumor cell” means both a cell obtained directly from the tumor and a cell that was derived from the cell obtained from the tumor by further cultivating the cell obtained from the tumor. Methods for culturing cells from Tumors are known in the art.

The use of technically modified cells according to the invention for the production of a vaccine has the advantage that the vaccine produced is suitable for the treatment of various tumor diseases. This novel vaccination approach makes it possible to dramatically reduce the development and manufacturing costs of vaccines, particularly in the case of rarer tumor indications, and thus makes development economically attractive.

A preferred technically modified cell according to the present invention is a cell which expresses at least one, preferably a plurality of tumor antigens, in particular endogenously (ie not introduced via a genetic modification).

According to a preferred embodiment, the cell expresses embryonic tumor antigens. According to a particularly preferred embodiment, the cell expresses at least 4, preferably at least 7, in particular at least 10 tumor antigens selected from the group comprising Ny-ESO-1, CEA1, CEA2, CEA3, α-feto protein, MAGE X2, BAGE, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE7a, GAGE8, MAGE A4, MAGE A5, MAGE A8, MAGE A9, MAGE A10, MAGE AI 1, MAGE A12, MAGE1, MAGE2, MAGE3, MAGE3b, MAGE4a, MAGE4b, MAGE5, MAGE5a, MAGE5b, MAGE6, MAGE7, MAGE8, MAGE9, PAGE1, PAGE4, CAMEL, PRAME, LAGE1, gpl00 and p53.

It has been shown that during tumor development, the characteristic genes that are expressed in the end stage of the tumor are embryonic tumor antigens such as proteins from the above-mentioned group, in particular proteins of the MAGE family, CEA, NY-ESO-1 and α-feto protein. The selection of a vaccine cell with regard to the expression of these embryonic tumor antigens thus directs the immune response specifically to tumors in the end stage (or less differentiated tumors) and / or metastases. In contrast, cellular vaccines that were produced from more differentiated tumors, ie earlier tumor stages, are only directed against such more differentiated tumors, which in particular leads to an “escape possibility” for metastases, since these are generally hardly differentiated Preferred tumor antigens of a cellular vaccine are those of the MAGE family, CEA, NY-ESO-1 and α-feto protein.

Technically modified cells according to the present invention are preferably tumor cells, especially testicular tumor cells (see, for example, Klade CS et al. (2002) Int J Cancer 10, 97: 217-24) or embryonic tumor cells (Rossant J. and Papioannou VE (1984 ) Cell Differ 15 (2-4): 155-61; Damjanov, I. (1990) Cancer Surv. 9 (2): 303-19), and cells derived from such tumors. Testicular tumor cells include tumor cells from geminative testicular tumors, from a seminoma, from an embryonic carcinoma or from an undifferentiated te ratom or teratocarcinoma, and chorionic carcinoma, cells from an embryonic carcinoma or a teratocarcinoma are particularly preferred.

The invention thus relates in particular to the use of a testicular tumor cell, an embroyonal tumor cell or of cells from cell lines derived from testicular tumor cells or embryonic tumor cells for producing a vaccine for the prevention or treatment of a tumor disease, the cell used and the tumor disease not originating from the same tumor type.

Surprisingly, testicular tumor cells or embryonic tumor cells are particularly suitable for the production of a medicament for the treatment of a wide variety of different tumor types, since they are able to activate a large number of different T cells. This particular suitability is due in particular to the fact that these tumor cells expressed a large number of embryonic antigens which are no longer expressed by differentiated cells. These antigens are presented by these cells and can, in a vaccination situation, trigger a specific immune reaction which is directed against all cells which express these embryonic (tumor) antigens.

As - as described above - tumors or metastases express an increasing number of embryonic (tumor) antigens during their tumor development, there is a particularly high probability that there is a certain agreement between the expressed antigens between the vaccine cell and the tumor cell and thus a specific immune response against the tumor cell is triggered.

This mechanism is based on the fact that the negative selection of the specific T cells against the "self-antigens" only takes place after the expression of the embryonic antigens has been switched off (Janeway C. et al. (1999) Immunobiology: the immune system in health and disease, 4 * ed., Current Biology Publications, New York, especially pages 227, 233, 241-257). If it weren't for that If this were the case, the body would in principle not be able to generate an immune response against an embryonic antigen.

In addition, immortal cells, which preferably express a large number of tumor antigens, can be used. Methods for immortalizing such cells are e.g. from WO 97/00946 or Katakura Y. et al. (1998, Methods Cell Biol. 57: 69-91). Stem cells, especially embryonic stem cells, can preferably be used.

According to the invention, it is possible to improve the immunogenicity of the vaccine cells by expressing cytokines, chemokines and / or costimulatory molecules.

Preferred technically modified cells are those that have been modified by genetic modification. Such a genetic modification can, for example, immortalize the cells or switch off or downregulate the expression of certain undesired genes such as e.g. immuno-inhibitory molecules (such as IL10, TGF-ß and Fas ligand). Furthermore, the cells can be modified such that they express one or more molecules from the group containing cytokines, chemokines and / or costimulatory molecules.

Methods for switching off or down-regulating the expression of genes are known to the person skilled in the art. Examples include Ribozyme (Usman N. and Bltt LM (2000) J. Clinical Investigation 106 (10): 1197-1202), antisense RNAs (Branch AD (1996) Hepatology 24: 1517-29) or RNAi technology ( Tuschl T. et al. (1999) Genes & Development 13: 3191-3197, O'Neil NJ et al. (2001) Am. J. Pharmacogenomics 1 (1): 54-53).

The cytokines and / or chemokines can be selected from the group comprising GM-CSF, G-CSF, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IFNα, IFNß, IFNγ, Flt3 L, Flt3, TNFα, RANTES, MlPlα, MlPlß, MlPlγ, MlPlδ, MIP2, MIP2α , MIP2ß, MIP3α, MIP3ß, MIP4, MIP5, MCP1, MCPlß, MCP2, MCP3, MCP4, MCP5, MCP6, όcykine, Dcckl and DCDF. Preferred cytokines / chemokines are GM-CSF, RANTES and / or MlPlα.

Costimulatory molecules can be selected from the group comprising B7.1, B7.2, CD40, Light, Ox40, 4.1.BB, Icos L, SLAM, ICAM 1, LFA-3, B7.3, CD70, HSA, CD84, CD7 , B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b, TNFR-RP, CD40L, Ox40L and Rae-1, with B7.1 and B7.2 preferred costimulatory molecules are.

Another embodiment of this invention is that such expressed cytokines, chemokines or costimulatory molecules are mutated. Such mutations include, but are not limited to, point mutations, deletions, insertions, or fusions with other peptides or proteins.

According to a further preferred embodiment, the technically modified cell can also express a fusion protein from the cytokines, chemokines or costimulatory molecules mentioned above. It is furthermore included according to the invention that the cell expresses functional variants of the above-mentioned polypeptides, functional variants being characterized in that they have essentially the same biological activity as the mentioned polypeptides. Tests are known in the prior art for the respective polypeptides, with the aid of which the polypeptides can be detected or their respective activity can be measured.

According to a particularly preferred embodiment, the technically modified cell expresses B7.2 and GM-CSF, since the combination of these two molecules leads to a surprisingly high effectiveness of the vaccine. Methods for genetically modifying cells are well known in the art. Preferred embodiments are cells which are stably transfected with at least one nucleic acid which codes for at least one cytokine, chemokine or costimulatory molecule. This means that the nucleic acid coding for such a cytokine, chemokine or costimulatory molecule is integrated into the genome of the cell and is therefore duplicated during the S-phase transition of the cell and passed on to the daughter cells as part of the normal chromatid separation. In addition, stably transfected cells can be obtained from episomal nucleic acids which are subject to replication that is independent of the cell replication machinery. Examples of such autonomous replication systems are, for example, viral replication systems containing an initiator protein such as SV40 large T antigen or EBNA1 and a start of replication such as SV40ori or EBV oriP.

Another way to obtain the expression of such cytokines, chemokines or costimulatory molecules is an episomal replicating plasmid which contains a selectable marker and the selection for this marker.

According to a preferred embodiment, the expression of such cytokines, chemokines or costimulatory molecules by a constitutive promoter, for example the CMV promoter (Vincent et al (1990) Vaccine 90, 353, the SV40 promoter (Samulski et al (1989) J Virol) 63, 3822) or the LTR promoter of retroviruses (Lipkowski et al (1988) Mol Cell Biol 8, 3988), by an inducible promoter, for example the tet promoter, and / or by a tissue-specific promoter, for example the elongation factor promoter , the Ig promoter or the IL2 / NFAT promoter.

A standard plasmid which contains a coding nucleic acid, preferably a DNA, and at least one regulatory element such as a promoter or an enhancer can be used to transfect the technically modified cells. The transfection can be carried out, for example, using calcium phosphate Transfection, electroporation or liposomal transfection can be performed. In addition, viral vectors such as the herpes simplex virus (HSV), amplicon, adeno-associated virus (AAV), an adenovirus or retrovirus can be used to transfect the cells. For example, US 6,111,591, which is hereby incorporated by reference, discloses suitable AAV vectors for the production of cells which express at least one cytokine.

AAV is preferred for transfection of the cells because this virus is particularly well suited for the integration of transgenes into the cell genome. The transgene (s) are / are preferably integrated into the AAV-SI acceptor site, in particular in the form of concatemers. An integration of concateners of the transgene has the advantage that such a multiplication of the genes leads to a stronger expression of the transgenes. If the technically modified cell cannot be infected by a naturally occurring AAV serotype, in particular AAV-2, capsid mutants can be produced with the aid of which an infection of the cell can be carried out (see WO 99/67393).

Preferred tumor diseases that can be treated are melanoma, egg stick cancer, breast cancer, colon cancer, leukemia, lymphoma, kidney cancer, lung cancer, prostate cancer, cervical cancer, neuroblastoma, osteosarcoma, thymoma, hepatoma, cancer of the larynx, gastric cancer, cancer of the anus Seminoma, pancreatic cancer, mesothelioma and / or brain tumor.

Such treatment can be combined with chemotherapy, radiation therapy, or surgical removal of the tumor or metastasis.

Such cells can be used as a drug used as prophylactic vaccination, as therapy for an existing tumor or as a se- Secondary prophylaxis is used, wherein the primary tumor and / or one or more metastases have been removed by surgery, chemotherapy or radiation and the risk of recurrence of a tumor is to be reduced.

According to a preferred embodiment, the cell used according to the invention is incapable of proliferation, for example by irradiation or chemical inactivation. For example, it is known that tumor cells become incapable of proliferation by gamma radiation with 25-100 Gy (see e.g. WP 97/32988). For chemical inactivation, for example, the addition of 40 μg / ml mitomycin can be used. According to the invention, “proliferation incompetent” means that the tumor cell is no longer able to proliferate.

The vaccine produced by the use according to the invention serves to trigger an immune response in the patient. In some cases, it is not necessary for the medicine to contain an adjuvant. In the context of the invention, an adjuvant is defined as a compound which can intensify the triggering of an immune response.

According to a preferred embodiment, the medicament additionally contains an adjuvant which is selected from the group comprising A1OH, CpG, double-stranded RNA; oxidized or reduced glutathione, BCG, Salmonella, complete Freund's adjuvant, incomplete Freund's adjuvant, cytokines or chemokines such as e.g. soluble proteins, iscome or specific antibodies for CD40, great receptors (stimulating) or CTLA4 (blocking).

Adjuvants which are used as toll-like receptors are preferably used.

Agonists work. These are e.g. CpG oligonucleotides. These are oligonucleotides that contain at least one CpG motif (see e.g. Wagner

H (2001) Immunity 14, 499-502). This definition also includes lipopoly saccharide (LPS) or components thereof, such as the lipid A portion or the poly or oligosaccharide portion. LPS are the main outer membrane components of almost all Gram-negative bacteria and are known to act as strong stimulators of the immune system. LPS consist of a poly- or oligosaccharide region, which are anchored by the lipid A in the outer bacterial membrane. The specific, cellular recognition of the LPS / Lipid A is mediated by the joint extracellular interaction of the LPS binding protein, the membrane-bound or the soluble form of CD 14 and the Toll-like receptor 4 * MD2 complex. This leads to a rapid activation of an intracellular signal network which leads to the signal transduction cascade homologous to that of IL-1 and IL-8 (Alexander C and Rietschel ET (2001) J Endotoxin Res 1, 3, 167-202).

The invention further includes the use of an adjuvant derived from Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS). BCG-CWS is known to be a ligand of toll-like receptors 2 and 4 and can trigger the differentiation of immune cells (Matsumoto M et al (2001) Int Immunopharmacol 1, 8, 1559-69).

The invention further comprises the use of at least one superantigen as an adjuvant. Superantigens are antigens that bind directly to T cell receptors and MHC molecules and cause direct activation of the T cells. It is known that these can also have an adjuvant effect (see e.g. Okamoto S et al (2001) Infect. Immun. 69, 11, 6633-42). Known superantigens are e.g. Staphylococcus aureus Enterotoxins A, B, C, D and E (SEA, SEB, SEC, SED, SEE), Staphylococcal aureus toxic shock syndrome toxin 1 (TSST-1), Staphylococcal exfoliating toxin or Streptococcal pyrogenic exotoxins.

Another embodiment of the invention is the use of agents that inhibit the signal transduction cascade of CTLA-4. It can are antibodies or antibody fragments that specifically bind to the extracellular domain of CTLA-4 and inhibit its signal transduction cascade. The generation or screening of such antibodies or antibody fragments is known to the person skilled in the art (see, for example, WO 0032231). Other agents which are suitable for binding CTLA-4 and inhibiting its signaling are small organic molecules, peptide analogs or soluble T cell receptors (see WO 97/20574).

The production of vaccines containing technically modified cells according to the invention or their use in the use according to the invention is carried out in the usual way using common pharmaceutical-technological processes. For this purpose, the technically modified cells are processed together with suitable, pharmaceutically acceptable auxiliaries and carriers to give the pharmaceutical forms suitable for the different indications and application sites.

Preferred auxiliaries and carriers are additives, stabilizers and / or binders. These include in particular proteins, peptides and amino acids such as e.g. human plasma proteins, especially albumin, but also recombinant serum albumin, further protease inhibitors such as e.g. Aprotinin, ε-aminocarponic acid, pepstatin A, EDTA or EGTA. This group also includes salts such as, in particular, sodium chloride, sodium octanoate, sodium caprylate and sodium acetyltryptophanate. Other suitable additives are ethanol or DMSO.

To adjust the pH, for example, osmotically active acids and alkalis, e.g. Hydrochloric acid, citric acid, sodium hydroxide solution, potassium hydroxide solution, sodium hydrogen carbonate, further buffer systems, such as e.g. Citrate, phosphate, Tris buffer, HEPES buffer, MOPS buffer or triethanolamine can be used.

A preferred formulation of a vaccine according to the invention contains approximately 10 ⁶ to 5x10 ⁸ cells in a volume of 0.1 to 10 ml, in particular 0.5 to 5 ml of a physiological saline solution with auxiliaries and carriers, for example one Infusion. Suitable infusion solutions are, for example, "Human Albumin 20% Immuno" from Baxter, Unterschleissheim (1000 ml infusion solution contain: 200 g human plasma proteins with at least 95% albumin, 3.0 g sodium chloride, 2.7 g sodium octanoate, 4.3 g sodium acetyltryptophanate ) or "Human Albumin 20% Behring" from Aventis Behring, Marburg (1000 ml infusion solution contain: 200g human plasma proteins with at least 96% albumin, 125 mmol sodium ions, max. 2 mmol potassium ions, max. 2 mmol Calcium ions, max. 100 mmol chloride ions, 16 mmol octanoate ions, 16 mmol N ^ Actyl-D-ZL-tryptophanate).

The invention further relates to a medicament for the prophylaxis, therapy and / or secondary prophylaxis of a tumor disease comprising a technically modified cell as defined above.

According to a preferred embodiment, the medicament additionally contains at least one adjuvant selected from the group comprising CpG, oxidized or reduced glutathione, BCG, Salmonella, Freund's complete adjuvant, Freund's incomplete adjuvant, cytokines or chemokines, e.g. soluble proteins, iscome or specific antibodies against CD40, Toll-like receptors (activating) or CTLA4 (blocking).

According to a further preferred embodiment, the medicament is suitable for being administered subcutaneously, intracutaneously, intravenously or intranodally.

According to a preferred embodiment of the invention, the patient is a mammal, preferably a human.

The invention further relates to a method for the treatment or prevention of a tumor disease, in which an effective amount of a technically modified cell is administered to a patient, the technically modified cell not being derived from the respective tumor disease. The above-mentioned embodiments of the use according to the invention also apply to the method according to the invention.

As explained in detail above, it has surprisingly been found in the context of the invention that cells which express embryonic tumor antigens are particularly suitable for the treatment of tumors in the end stage.

The invention therefore also relates to the use of a cell expressing at least one embryonic tumor antigen for producing a vaccine for the treatment of end-stage tumor diseases, the cell not being derived from the respective tumor disease.

According to a preferred embodiment, the cell expresses at least 3 embryonic tumor antigens.

The above-mentioned embodiments of the other use according to the invention also apply to this use according to the invention.

The following examples are intended to explain the invention in more detail without restricting it.

Example 1 Expression of tumor antigens by embryonic tumor cell lines

In order to investigate whether a testicular tumor or embryonic tumor cell line is suitable in principle, it was examined whether it actually expresses more tumor antigens than tumor cells from conventional solid tumors. The following cell lines were examined:

F9 embryo carcinoma (haplotype: H2-b) (ATCC CRL-1720) B16F10 melanoma (C57 / B16, H2-b) (ATCC CRL-6475)

CT26 Colon Carcinoma (Balb / c, H2-d) (ATCC CRC 2638)

K1735 Melanoma (C3H, H2-k) (Staroselsky AH et al. (1991) Cancer Res.

51, 6292-98)

Renca renal carcinoma (Balb / c, H2-d) (Murphy, GP and WJ Hrushesky (1973) JNatl Cancer Inst 50: 1013-25).

The analysis with regard to the expression of known tumor antigens was carried out using the array technology from Affymetrix (Santa Clara, CA, USA). For this purpose, 50 μg of total RNA of the above-mentioned cells were purified using RNeasy mini columns (Qiagen, Hilden) in accordance with the manufacturer's instructions. 5 μg of the purified total RNA was used to synthesize the first and second strand of the cDNA. This cDNA was then used to produce bio-vinylated cRNA samples as recommended by the manufacturer Affymetrix. 15 μg of the fragmented, labeled cRNA was used to hybridize Gene Chip® arrays that represent the mouse genome (U74A). The hybridization signals were then amplified with a biotin-labeled anti-streptavidin antibody and by a further SAPE staining step. The data were analyzed using Microarray Suite and Data Mining Tool Software (Affymetrix). The chip used represents U74A 12,588 transcripts of the murine genome, which consist of approx. 6000 functionally characterized sequences from the mouse UniGene database (Build 74) and a further approx. 6000 EST clusters.

The cell lines F9, B16F10, CT26, Kl 735 and Renca were compared using this technology. With regard to the expressed, known tumor antigens, it could be shown that F9 cells express almost all known tumor antigens for which the array was tested, whereas cells B16F10, CT26, Kl 735 and Renca do not express a number of important tumor antigens.

Tumor antigens expressed by all 5 cell lines tested are:

Tumor antigens that are expressed by F5 cells but not by all other 4 cell lines tested are:

• 2A -

Tumor antigens that are partially expressed by B16F16, CT26, Kl 735 or Renca cells but not expressed by F9 cell lines are:

With the exception of the "Mouse embryonic alkaline phosphatase gene", however, these are cell line-specific antigens or differentiation antigens which are not important for the effect of a vaccine.

It was therefore possible to demonstrate for the F9 embryocazinoma cell line that almost all embryonic antigens were expressed that were identified as tumor antigens of various tumor types (e.g. melanoma, colon, kidney or blood tumors) in the end stage.

It is thus clear that, when comparing the different cell lines, the F9 cells express the largest number of tumor antigens. This underlines the suitability of such a cell line as a starting point for the production of cellular vaccines for multiple tumor indications. Example 2

Cell lines such as testicular tumor or embryonic tumor cell lines that have already been cultured are available. One of these cell lines in the mouse is the F9 testicular / embryonic tumor cell line (ATCC CRL-1720). F9 cells were tested for the expression of tumor antigens (see Table 1).

According to Table 1, a variety of tumor antigens are expressed from a testicular or embryonic tumor cell line. The table shows tumor antigens that are expressed by other tumor types such as melanoma (Kl 735), kidney cancer (Renca), colon cancer (CT26), breast cancer (CaD2), B-cell plasmacytoma (J558) and granuloma (Ehrlich) , compared.

Table 1

F9 embryonic carcinoma (H2-b)

J558 B cell plasmacytoma (Balb / c, H2-d)

M3 breast adenocarcinoma (DBA mice)

CT26 colon carcinoma (Balb / c, H2-d)

CaD2 breast cancer

Renca renal carcinoma (Balb / c, H2-d)

Ehrlich plasmacytoma

K-1735 Melanoma (C3H, H2-k)

PYS teratocarcinoma (129, H2-b)

P- 19 Embryonic carcinoma (C3H / He, H2-k)

Example 3 Production of an Embryonic Tumor Cell Vaccine for the Establishment of a

mouse model

3.1 cell lines

For the cell lines PYS (DSMZ No. ACC 284, Lehman et al. (1974) J. Cell. Physiol. 84: 13-28) and P-19 (DSMZ No. ACC 316, McBurney et al. (1982) Develop. Biol. 89: 503-508; Rudnicki et al. (1990) Develop. Biol. 138: 348-358) are established syngeneic cell lines of the mouse strains C3H and 129. These were developed by the DSMZ (German Collection of Microorganisms and cell cultures; Department of Human and Animal Cell Cultures).

3.2 Plasmids

The cDNA from GMCSF and B7.2 were cloned into the vector pCI (Promega, Madison, WI, USA), which provides a CMV promoter and an SV40 3'-untranslated region. For the plasmid pAAV-mu GMCSF ₂ , two expression cassettes - which contain GMCSF with the CMV promoter and the SV40-pA site - were ligated in tandem into the plasmid pAAV base vector (see WO 00/47757, Example 4). To the ideal size a possible AAV packaging too a further 400 bp from pUC19 (bp 1516-1910) were also integrated into the vector.

For the plasmid pAAV-mu B7.2-700, the expression cassette - with B7.2, the CMV promoter and the SN40-pA site - was ligated into the plasmid pAAN base vector. Another 700 bp from pUC19 (bp 1201-1910) were necessary to generate the optimal size of the vector for possible AAV packaging.

3.3 Transfection of the Cell Lines and Preparation of the Vaccine Various transfection methods were tested for the two cell lines, it being found that calcium phosphate transfection for P-19 cells and transfection using Polyfect ™ (Qiagen, Hilden) were most suitable for PYS cells , To increase the efficiency of the transfection, the cells were additionally treated with Gene-X-press ™ (PAA ™).

a) Calcium phosphate transfection of P-19 cells

P-19 cells were harvested using PBS and trypsin-EDTA. The reaction was stopped with FCS-containing medium. The cells were washed once and the cell number was determined. Then 1.77 x 10 ⁶ P19 cells were sown in a cell culture bottle (175 cm ² ) in 22 ml medium (= day 0). The cells were incubated overnight in humidified air in an incubator (37 ° C, 5% CO ₂ ). On day 1, the cells were transfected using the following materials / method:

For each cell culture bottle, 23.3 μg of pAAV-mu B7.2-700 and 35 μg of pAAV-mu GMCSF _{2 were} mixed with 2.733 ml of the CaCl ₂ solution (270 mM, sterile filtered). The same amount of 2x BBS was then added and mixed gently. The mixture was incubated at room temperature for 15-20 minutes to form the precipitate. The cells were then removed from the incubator and the mixture was carefully distributed over the cells. Gene-X-press (PAA, cat #: U05-006) was added so that its final tration lx betmg. Then the cells were incubated overnight in a humidified incubator (35 ° C, 3% CO ₂ ). The next day, the cells were transferred to another humidified incubator (37 ° C, 5% CO ₂ ). The total volume per preparation batch was 27.5 ml.

On the second day after the transfection, the supernatant of the cells was removed and kept for the measurement of GMCSF expression. The cells were washed once with PBS and then harvested using trypsin-EDTA. The reaction was stopped with complete medium containing FCS. An aliquot was taken for the detection of B7.2 expression. The cells were then counted and irradiated with 100 Gy in a cesium source. The cell was then frozen in FCS / 10% DMSO at -80 ° C. and transferred to liquid nitrogen after two days.

b) Transfection of the PYS cells with Polyfect ™

PYS cells were harvested using PBS and trypsin-EDTA. The reaction was stopped with FCS-containing medium. The cells were washed once and the cell number was determined. Then 3.7 x ¹⁰⁶ PYS cells were sown in a cell culture bottle (175 cm ² ) in 26.8 ml of medium (= day 0). The cells were incubated overnight in humidified air in an incubator (37 ° C, 5% CO ₂ ). On day 1, the cells were transfected using the following materials / method.

For each cell culture bottle, 1.8 ml of serum-free medium (SFM), 23.3 μg rAAV-mu B7.2 and 23.3 μg rAAV-mu GMCSF ₂ , 218 μl Polyfect ™ (QIAgen, Cat #: 301107) were added and mixed carefully. The mixture was incubated for 5 minutes at room temperature. Then 10.9 ml of complete medium containing Gene-X-press were added at a final concentration of 1 × (= 323.2 μl), mixed and the mixture was pipetted onto the cells. The total transfection batch was 40 ml. The cells were incubated overnight in an air-humidified incubator (37 ° C., 5% CO ₂ ). On day 3 the supernatant of the cells was removed and kept for the measurement of GMCSF expression. The cells were washed once with PBS and then harvested using trypsin-EDTA. The reaction was stopped with complete medium containing FCS. An aliquot was taken for the detection of B7.2 expression. The cells were then counted and irradiated with 100 Gy in a cesium source. The cell was then frozen in FCS / 10% DMSO at -80 ° C. and transferred to liquid nitrogen after two days.

3.4 Measurement of transgene expression (GMCSF and B7.2)

The cell supernatants from the transfection samples were kept at -20 ° C. until the measurement. The samples were diluted in stages 0, 1:10, 1:50, 1: 250, 1: 1250 and measured in the GMCSF ELISA (Pharmingen ™, Cat. No .: 555 167) according to the manufacturer's instructions. The amount of GMCSF was calculated as the total amount secreted by lxl 0 ⁶ cells in 48 hours. For optimal conditions, the amount of GMCSF secreted was approx. 300-600 ng / lxlO ⁶ cells / 48 h.

The cell aliquots were stained with anti-muCD86-PE antibodies (Pharmingen ™, Cat. No. 553692) in order to detect B7.2 expression. The antibody was used at a dilution of 1:50. The staining reaction took place in PBS / 5% FCS. The cells were then washed once with PBS, fixed in PBS / 1% formaldehyde and stored at 4 ° C. until the measurement. The B7.2 measurement was carried out using flow cytometry. An expression level> 30% was considered good.

3.5 Production of the vaccination cells

To prepare the vaccine, the frozen cells were thawed in a water bath (37 ° C.) and washed three times in PBS. The cells were each Centrifuged at 250 g for 6 minutes and resuspended in fresh PBS to remove the washing medium.

The cells were then resuspended in an appropriate volume of PBS and counted in a Neunbauer counting chamber using trypan blue exclusion staining. The number of living cells was determined and adjusted to 3 × 10 ⁶ cells per ml. 3x10 ⁵ cells (in 100 ul) per animal were injected subcutaneously into the side. After this procedure, the remaining cell suspensions were counted again to determine the survival rate of the cells throughout the procedure.

Example 4 Carrying out mouse vaccination experiments

Example 4A) Description of the tumor models

Mice should be used to examine the embryocarcinoma vaccine. Different mouse strains are required due to the different tumor models that are being examined. The following cell lines and syngeneic mouse strains are used as tumor models:

Table 2

These are syngeneic inbred strains in which the respective tumor is not rejected per se due to foreign MHC molecules. These are already established and tried animal models for the respective tumor type.

The animals are vaccinated in all cases with the tumor cell line P-19 or PYS - two embryo carcinoma cell lines with different MHC types - as a model for an embryo carcinoma cell vaccine. The tumor challenge, i.e. the injection of living tumor cells to induce tumor formation takes place with the cell line of the respective tumor model (see below). Depending on the model, either a prophylactic or therapeutic approach can be selected.

Prophylactic means that the animals are first immunized against the tumor and only then does the tumor challenge. These models all include local application of the tumor. The presence and growth of the tumor is therefore verified by palpation.

Therapeutic model means that the tumor challenge is carried out first and then an immunization is connected. It is a tumor model in which an attempt is made to imitate the situation in the tumor patient. Here too, a tumor is present before the start of therapy and does not develop after vaccination, as is the case with prophylactic models. Therefore, the therapeutic model more closely corresponds to the patient's situation.

K-1735 tumor model

K-1735 is a well-known melanoma model in the syngeneic mouse strain C3H / He. The formation of lung metastases is provoked by intravenous application of vital K-1735 melanoma cells. The injected cells settle in the final flow path of the lungs and form small metastatic nodules. The number of nodes develops in opposite proportional to the immune response of the animals to melanoma. The stronger the immune response - which is mainly induced by the subsequent vaccination - the less lung metastases grow. The time of the challenge is referred to as "day 0".

In the standard protocol, the animals are vaccinated on day 4 and boiled on day 11 after challenge (cell number: lxl 0 ⁵ ). Irradiated, no longer divisible cells are administered which are still able to secrete immunostimulatory molecules, but do not form a tumor. The carrier substance is physiological buffer (PBS). The animals are sacrificed and dissected on day 21. The lungs are removed, which are then weighed. The number of metastatic nodules in the lungs is also determined by counting. This enables a direct quantification of the therapy success.

In untreated animals (control), the development of lung metastases on day 21 has progressed to such an extent that clear differences between successfully treated and untreated animals can be measured, but not yet to the extent that a recognizable disturbance of the general condition or condition occurs ,

The great advantage of this model is the quantifiability of the therapeutic success. The success of therapy can be quantified by weighing the lungs and simply counting the lung metastases on the surface of the organ. Lung weight and number of metastases correlate very well, since the weight of the tumor mass is included in the total weight of the lungs.

B16F10 tumor model

B16F10 is another melanoma model, but in the C57BL / 6 mouse (H-2b). It is equipped with a different MHC type than the C3H mouse (H-2k). Therefore the two cell lines are allogeneic to each other. In contrast to K-1735, B16F10 only carries very small amounts of the main histocompatibility complex. Therefore, the cell is significantly less immunogenic because the presentation of antigen is inhibited.

The animals are first given two vaccinations with irradiated cells (twice every 2 weeks) and only then do they face the challenge with living tumor cells (2 weeks after vaccination). Both are applied subcutaneously, so that solid tumors under the skin develop. These can be scanned and measured to monitor tumor growth. From this, the tumor mass can be calculated, which is inversely proportional to the immunity training of the body. For this purpose, the animals are examined clinically for tumor formation every 2-3 days for several weeks. When a tumor size of »1 cm in diameter is reached, the animals are killed because rejection of the tumor is no longer to be expected, but the risk of ulceration of the tumor surface or impaired movement of the animals increases. The cell number for the tumor challenge is 1 × 10 ⁵ cells (in PBS).

In order to know the exact number of cells for the tumor challenge before starting the first immunization attempts, the minimum tumorigenic dose must first be tested (see step 1).

RenCa tumor model

RenCa is a carcinoma of the kidney and is a syngeneic model of the Balb / c mouse. It has already been used in other laboratories both as a local tumor model and for the induction of lung metastases (systemic application). Regarding the implementation of the two models and the observation of the animals, what has been said for B16F10 and K-1735 applies (see there). The exact number of cells for both methods is determined in stage 1 of the animal experiments. CT26 tumor model

CT26 is a cell line derived from Balb / c mouse colon carcinoma. This cell line has also been used by other laboratories for both local and systemic tumor models with formation of lung metastases. The procedure is the same as for Renca (see there). The minimum tumorigenic dose for the tumor challenge is also determined in stage 1.

J558 tumor model

J558 is a plasmacytoma, i.e. a B cell tumor of the Balb / c mouse. The following experimental set-up acts as a model for tumor therapy: the animals are given a subcutaneous injection with live J558 tumor cells on day 0. These form a solid subcutaneous tumor that can be easily palpated and measured. The vaccination takes place on days 4 and 11. The minimum tumorigenic dose must also be determined exactly here in stage 1 of the experimental plan.

vaccine

The vaccine that is used in all tumor models is the two cell lines P-19 and PYS. These are two different embryo carcinoma cell lines that come from different mouse strains and therefore differ in the MHC type. In addition, the amount of specific tumor antigens varies. While P-19 expresses quite a large number of tumor antigens from a wide variety of tumor models, the amount is significantly lower in PYS. In the case of melanoma K-1735, P-19 can even be used as an "autologous" vaccine due to the same MHC type (= corresponds to vaccine cells that come from the same patient, or patient and vaccine do not have any different MHC- Alleles): Here the difference between autogenous and allogeneic can be examined, ie the influence of foreign MHC alleles on the development of immunity in an embryocarcinoma vaccine. In some experiments an adjuvant is used in addition to the effectiveness, which is administered together with the cells becomes. Example 4B) Information on Practical Implementation of Tumor Cell Application / Tumor Induction ("Challenge"):

Challenge is the application of living tumor cells to animals. In naive animals, the application of a challenge with a cell count that is above the minimum tumorigenic dose (MTD, see experimental part) leads to the development of local or systemic tumor growth, depending on the route of application. In this way, the immune system is "challenged" (= challenge) to reject the tumor cells completely in the optimal case - i.e. with existing or emerging immunity.

Local tumors:

To induce local tumors, animal tumor cells (K-1735, B16F10, RenCa, CT26, J558) are injected subcutaneously into the flank in a predetermined number of cells (see test section stage 1: MTD). In the absence of immunity in the animals, the tumor cells form a solid subcutaneous tumor. The tumor does not represent any particular impairment for the animals as long as there is no ulceration of the tumor surface or a restriction of the animals' movement due to excessive tumor volume. If the tumor is larger than 1 cm in diameter, the animals are killed. This prevents the end stage of the tumor with severe impairment of the general condition.

The subcutaneous injection is due to the short duration of the procedure and the low burden on the animals without anesthesia. The cells are applied in physiological buffered saline ("PBS") in a volume of 0.1 ml to a maximum of 0.2 ml. Lung metastases:

In some models, induction of lung metastases is also possible (K-1735, CT26, RenCa). For this purpose, living tumor cells are injected intravenously into the tail vein in a predetermined number of cells (see experimental section stage 1: MTD). The cells reach the lungs via the heart and thus the first final current pathway in the body. Here the tumor cells attach and form small metastasis nodes. The animals are killed at a point in time when the metastases have reached a macroscopically recognizable size (approx. 0.5-1.5 mm), but before the metastases significantly impair lung function (usually approx. Day 21).

To ensure the best possible application without severely impairing the animals, e.g. To achieve manual fixation, the animals are placed in a fixation chamber. This is usually very well tolerated by the animals. The intravenous injection is due to the short duration of the procedure and the relatively low burden on the animals without anesthesia. The cells are applied in physiological buffered saline in a volume of 0.2 ml (“PBS”).

vaccination

The animals are vaccinated with irradiated tumor cells (partly genetically modified, see experimental part). These are applied subcutaneously to the flank in physiological buffered saline (PBS) in a volume of 0.1 ml. The time for the vaccination depends on the model and the vaccination scheme. In the case of prophylactic vaccination, the animals are immunized before the challenge is applied. Application time is 4 and 2 weeks before the challenge. In the case of local tumor models, the vaccine is always placed on the opposite side, on which the challenge takes place. This ensures that only if the vaccine is systemically effective Tumor rejection occurs and not due to local immune reactions. In the case of therapeutic vaccination, the animals are immunized after the challenge. The vaccination is on days 4 and 11 after the challenge. The subcutaneous injection is also due to the short duration of the procedure and the relatively low burden on the animals without anesthesia. In some experiments, an adjuvant is also used, which is applied together with the tumor cells and does not cause any side effects or additional stress.

Observation / clinical examination of the animals

Every one to three days, especially in tumor models with local tumors, the animals are examined adspectorally and also by palpation. The development of tumor growth is determined by measuring the size of the tumor.

In tumor models with induction of lung metastases, the animals are examined with regard to their general condition in order to be able to recognize impairments caused by tumor growth in good time. Relevant parameters are the general condition of the animals, food and water intake, possibly emaciation, disturbed breathing or other impairments. If the general condition is clearly disturbed, the animals are also killed before the regular end of the experiment for animal welfare reasons.

Killing the animals

All animals are killed by breaking their necks (according to EU recommendations). In experiments with lung metastasis formation, the animals are sacrificed on day 21 after challenge and then dissected. The lung weight, the number of metastatic nodes and other deviations from the normal state are recorded. If necessary, blood or organs (eg spleen for immunological tests) are taken only after the animals have been killed. Through the exact determination of lung weight and number of metastases, the success of therapy can be quantified directly.

In experiments with local tumor formation, the animals are not killed at a defined time, but individually depending on the formation of a tumor. The killing occurs when a tumor size of approx. 1 cm in diameter is exceeded, from which rejection of the tumor is no longer to be expected. Rather, if the tumor grows, an impairment of the animals is to be expected, which should be avoided.

Example 4C) Experiment planning

Stage 1: Tumorigenicity test of the individual tumor cell lines with local application: Minimum tumorigenic dose (MTD) (experiment no. 1.1) For the following experiments in the various tumor models, the cell number must be known, which, when administered without prior immunity to the tumor, will lead to tumor formation leads. A cell number should be selected which enables tumors to develop reliably in control animals, but which is not too high to be rejected by the immune system - in the event of immunity training.

The minimum tumorigenic dose (MTD) is the minimum number of cells necessary to cause a tumor in a group of naive animals, in all animals. For vaccination experiments, a cell number that is slightly higher is usually chosen. To ensure safe tumor formation in the control animals. This applies both to local tumor models in which tumors are induced subcutaneously and to systemic models with the formation of lung metastases. The latter have the advantage of also including the immunological effect against disseminated foci of metastases. The animals receive a subcutaneous injection with the specified cell number of the respective cell line into the flank (see table). The animals are then examined and scanned every 2-3 days to determine any tumor nodules as soon as possible. The size development of the tumors is measured using a caliper and the tumor volume is calculated. When a tumor size of 1-1.5 cm in diameter is reached, rejection is no longer to be expected, which is why the animals are then killed. The minimum tumorigenic dose (MTD) is the lowest cell number that induces tumor growth in all animals in the group.

Experimental setup: Animals and model: a) Animals: Balb / c, female, 6-7 weeks model: RENCA b) Animals: Balb / c, female, 6-7 weeks model: CT26 c) Animals: Balb / c, female, 6-7 weeks model: J558 Total duration: about 4 weeks

Renca and CT26 tumorigenicity test with systemic application: Minimum tumorigenic dose in the lung metastasis model (MTD) (experiment 1.2):

For the models Renca and CT26 there are also protocols for systemic application in the tail vein, which leads to the induction of lung metastases by the settlement of the injected cells in the lungs (Yoon SS et al. (1999) Cancer Research, 59: 6251-6256, Casares N et. Al. (2001) Eur J Immunol; 31: 1780-1789). In order to determine the optimal cell number (= minimum tumorigenic dose) for this procedure, a preliminary test should also be carried out here.

The animals receive an intravenous injection with different times (see table) of the two cell lines. Subsequently, the animals are observed every 1-2 days to determine whether there are any disturbances in their general well-being: eg disturbed breathing, emaciation, etc. If this is the case, the animals in the group concerned will also be killed prematurely. On day 21 after application of the tumor cells, all animals are dissected and the lungs weighed to quantify the tumor load. A group of animals without injection serves as a negative control for determining the pure lung weight. Then the number of lung metastases on the lung surface is determined and related to the lung weight. The number of cells leading to mean numbers of lung metastases, i.e. allowing the number to vary up and down (as a sign of a therapeutic effect), is defined as the tumor dose for subsequent experiments. Experimental setup: Animals and model: a) Animals: Balb / c, female, 6-7 weeks model: RENCA b) Animals: Balb / c, female, 6-7 weeks model: CT26 Total duration: about 4 weeks

Stage 2: Prophylactic vaccination with transgenic embryocarcinoma cells in various tumor models

In this part of the experiment, the effect of prophylactic immunization with genetically engineered embryo carcinoma cells is to be investigated in some of the tumor models. Can transgenes B7.2 and GMCSF or RANTES induce immunity to tumor antigens which leads to rejection of living tumor cells that are subsequently administered? Prophylactic vaccination with embryo carcinoma cells in the melanoma model (B16F10) (experiment no. 2.1)

The effect of the embryo carcinoma cells against local tumors of melanoma B16F10 is to be investigated. On day 0 and 14, the animals received a vaccine with irradiated P-19 or PYS vaccine cells (3 × 10 ⁵ cells) which were transfected with various transgenes (B7.2-GMCSF or B7.2-RANTES). Cells without transgene are carried as transgene controls. As a control for the immunity of the animals, one group is vaccinated with autologous cells (B16F10), which also carry the two transgene combinations. A buffer control ("PBS") is used to control the influence that the manipulation of the animals has. On day 28, the animals are administered a subcutaneous challenge (lxlO ⁵ B16F10) in the flank on the opposite side of the challenge to induce tumor growth. The animals are then regularly examined for tumor growth over a period of several weeks.

Experimental setup: Animals and model: Animals: C57BL / 6, female, 6-7 weeks Model: B16F10 Total duration: about 3 months

Prophylactic vaccination with embryo carcinoma cells in the local renal carcinoma model (RenCa) (experiment no. 2.2)

The effect of the embryo carcinoma cells against local tumors of renal carcinoma Renca will be examined. The procedure corresponds exactly to that in experiment no. 2.1. . described However, transfected RenCa cells (RenCa-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. The challenge cell number (RenCa) is based on the results of the MTD study (see experiment 1.1).

Experimental setup:

Animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: RenCa

Total duration: about 3 months

Prophylactic vaccination with embryo carcinoma cells in the colon carcinoma model (CT26) (experiment no.2.3)

The effect of the embryo carcinoma cells against local tumors of the colon carcinoma CT26 is to be examined. The procedure corresponds exactly to that in experiment no. 2.1. . described However, transfected CT26 cells (CT26-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. The cell number of the Challenge (CT26) is based on the results of the MTD study (see experiment 1.1).

Experimental setup:

Animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: CT26

Total duration: about 3 months

Prophylactic vaccination with embryo carcinoma cells in the plasmacytoma model (J558) (experiment no. 2.4)

The effect of the embryo carcinoma cells against local tumors of the plasmacytoma J558 is to be investigated. The procedure corresponds exactly to that in experiment no. 2.1. . described However, transfected J558 cells (J558-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. The challenge cell number (J558) is based on the results of the MTD study (see experiment 1.1).

Experimental setup: Animals and model: Animals: Balb / c, female, 6-7 weeks model: J558 Total duration: about 3 months

Stage 3: Therapeutic vaccination with transgenic embryocarcinoma cells in various tumor models

In this part of the trial application, the effect of therapeutic immunization with genetically engineered embryo carcinoma cells is to be investigated in some of the tumor models. There are established protocols for the induction of lung metastases for the models K-1735, Renca and CT26, which can then be treated by immunization. The model comes close to the situation in the patient, where only therapeutic intervention and no prophylactic one is possible. The lung metastasis model also has the advantage of being able to test the aspect of immunity to metastases distributed in the body. General immunity is required.

Therapeutic vaccination with embryo carcinoma cells in the lung metastasis model of melanoma (experiment no. 3.1)

The effect of embryocarcinoma cells against lung metastases from melanoma K-1735 is to be investigated. On day 0, the animals received a challenge with lxlO ⁵ K-1735 cells intravenously into the tail vein. On days 4 and 11, vaccination is carried out with irradiated P-19 or PYS vaccine cells (3 × 10 ⁵ cells, subcutaneously), which were transfected with various transgenes (B7.2-GMCSF or B7.2-RANTES). Cells without are used as transgenic controls Carried transgene. As a control for the immunity of the animals, one group is vaccinated with autologous cells (K-1735), which also carry the two transgene combinations. A buffer control ("PBS") serves to control the influence that the manipulation of the animals exerts. On day 21 all animals are dissected, the lungs removed to determine the lung weight and number of metastases.

Experimental setup:

Animals and model: Animals: C3H / He, female, 6-7 weeks Model: K-1735-M2 Total duration: about 5 weeks (including adaptation)

Therapeutic vaccination with embryo carcinoma cells in the lung metastasis model of renal carcinoma (experiment no. 3.2)

The effect of the embryo carcinoma cells against lung metastases of renal carcinoma RenCa is to be examined. The procedure corresponds exactly to that in experiment no.3.1. . described However, transfected RenCa cells (RenCa-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. The challenge cell number (RenCa) is based on the results of the MTD study (see experiment 1.2). In experiments 3.2 and 3.3, 6 indicator animals are to be carried in addition to the listed experimental animals. These only receive a challenge and no vaccination and serve to determine the optimal time for the section. Since there is no experience with these two models so far, this secures the section at the right time. Since there is no immunization, it can be assumed that the greatest number of metastases occurs in these animals. 3 animals should be killed 3 days before and on the day of the planned section to determine whether the metastasis is already sufficient for an evaluation. If this is the case, the section should be brought forward. If this is not the case, depending on the findings of the second group of indicator animals, the section takes place on the day of the planned section, or is pushed back again by a few days. This serves to prevent the animals from being killed too early if no statement can be made, or to avoid excessive metastasis.

Experimental setup: Animals and model: Animals: Balb / c, female, 6-7 weeks model: Renca

Total duration: about 5 weeks (including adaptation)

In order to be able to carry out this experiment, a good formation of metastases in the lungs - and only there - is a prerequisite for the evaluation of the results. Therefore, as described above, the number of cells required for the challenge in experiment 1.2 must be determined. Instead, the experiment can also be carried out in the local tumor model. The experimental groups do not change, only the treatment of the animals: In this case, the challenge is carried out subcutaneously on the flank instead of intravenously. The subsequent therapeutic vaccination takes place as in the lung metastasis model. However, the animals are then regularly examined for the detection of emerging local tumors (cf. experiments in stage 2) and killed when a tumor size of> 1 cm is reached.

Therapeutic vaccination with embryo carcinoma cells in the lung metastasis model of colon carcinoma (experiment no. 3.3)

The effect of the embryo carcinoma cells against lung metastases of the colon carcinoma CT26 is to be examined. The procedure corresponds exactly to that in experiment no.3.1. . described However, transfected CT26 cells (CT26-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. The challenge cell count (CT26) is based on the results of the MTD study (see experiment 1.2). In this experiment, indicator animals should also be used to ensure the correct time of section (see under 3.2).

Experimental setup:

Animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: CT26

Total duration: about 5 weeks (including adaptation)

In order to be able to carry out this experiment, a good formation of metastases in the lungs - and only there - is a prerequisite for evaluating the results. Therefore, as described above, the number of cells required for the challenge in experiment 1.2 must be determined. Instead, the experiment can also be carried out in the local tumor model. The experimental groups do not change, only the treatment of the animals: In this case, the challenge is carried out subcutaneously on the flank instead of intravenously. The subsequent therapeutic vaccination takes place as in the lung metastasis model. However, the animals are then regularly examined for the detection of emerging local tumors (cf. experiments in stage 2) and killed when a tumor size of> 1 cm is reached.

Therapeutic vaccination with embryo carcinoma cells in the lung metastasis model of the plasmacytoma (experiment no. 3.4)

The effect of the embryo carcinoma cells against local tumors of the plasmacytoma J558 is to be investigated. Since there is no lung metastasis model for J558, therapeutic vaccination against local tumors is used here. For this, the animals receive a challenge with a predetermined cell number on day 0 (see experiment 1.1) subcutaneously in the flank of J558 cells. On days 4 and 11, the vaccination is carried out with irradiated P-19 or PYS vaccine cells (3 × 10 ⁵ cells, subcutaneously) or with the controls. The animals are then examined for tumor growth over several weeks and the resulting tumor is measured. In this case, transfected J558 cells (J558-B7.2-GMCSF, or -B7.2-RANTES) serve as an autologous control. Experimental setup:

Animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: J558

Total duration: about 3 months (including adaptation)

Stage 4: Therapeutic vaccination with transgenic embryo carcinoma cells together with an adjuvant in various tumor models:

In this part of the experimental application, the additional effect of an adjuvant in connection with an embryocarcinoma vaccine is to be investigated. This should be done again in the therapeutic model, since this is where the effects can be examined most clearly. This is the best place to test the potency of such a combined vaccine, since the model comes closest to the patient's situation. In addition, an adjuvant is to be used here.

Therapeutic vaccination with embryo carcinoma cells and adjuvant in the lung metastasis model of melanoma (experiment no. 4.1)

The effect of embryocarcinoma cells against lung metastases from melanoma K-1735 is to be investigated. The procedure corresponds to that in experiment 3.1. The only change is the application of the adjuvant together with the vacuum zine in a syringe. Therefore, the treatment of the animals does not change. The embryo carcinoma line is used as the vaccine. turned out to be optimal.

Experimental setup:

Animals and model:

Animals: C3H / He, female, 6-7 weeks

Model: K-1735-M2

Therapeutic vaccination with embryo carcinoma cells and adjuvant in the lung metastasis model of renal carcinoma (trial no. 4.2)

The effect of the embryo carcinoma cells against lung metastases of renal carcinoma RenCa is to be examined. The procedure corresponds to that in experiment 3.2. The only change is the application of the adjuvant together with the vaccine in a syringe. Therefore, the treatment of the animals does not change. The embryo carcinoma cell line is used as the vaccine. turned out to be optimal. Indicator animals are also carried here (see Experiment 3.2). Experiment setup: animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: RenCa Total duration: about 5 weeks (including adaptation)

Therapeutic vaccination with embryo carcinoma cells and adjuvant in the lung metastasis model of the colon carcinoma (experiment no. 4.3) The effect of the embryo carcinoma cells against lung metastases of the colon carcinoma CT26 is to be investigated. The procedure corresponds to that in experiment 3.3. The only change is the application of the adjuvant together with the vaccine in a syringe. Therefore, the treatment of the animals does not change. The embryo carcinoma cell line is used as the vaccine. turned out to be optimal. Indicator animals are also carried here (see Experiment 3.2). Experiment setup: animals and model:

Animals: Balb / c, female, 6-7 weeks

Model: CT26 Total duration: about 5 weeks (including adaptation)

Claims

claims

1. Use of a technically modified cell for the production of a vaccine for the treatment or prevention of a tumor disease, the technically modified cell not being derived from the respective tumor disease.

2. Use according to approach 1, wherein the technically modified cell expresses several tumor antigens, preferably embryonic tumor antigens, in particular containing at least 4, especially at least 10, from the group

Ny-ESO-1, CEA1, CEA2, CEA3, α-feto protein, MAGE X2, BAGE, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE7a, GAGE8, MAGE A4, MAGE A5, MAGE A8, MAGE A9, MAGE A10, MAGE Al l, MAGE A12, MAGE1, MAGE2, MAGE3, MAGE3b, MAGE4a, MAGE4b, MAGE5, MAGE5a, MAGE5b, MAGE6, MAGE7, MAGE8,

MAGE9, PAGE1, PAGE4, CAMEL, PRAME, LAGE1, gplOO and p53.

3. Use according to one of claims 1 or 2, wherein the technically modified cell is a tumor cell, preferably a testicular tumor cell or an embryonic tumor cell.

4. Use according to spoke 1 to 2, wherein the technically modified cell is an immortal or an immortalized cell, preferably a stem cell, in particular an embryonic stem cell.

5. Use according to one of claims 1 to 4, wherein the technically modified cell has been genetically modified in order to express one or more molecules from the group comprising cytokines, chemokines and / or costimulatory molecules.

6. Use according to claim 5, wherein the cytokine and / or chemokine are selected from the group comprising GM-CSF, G-CSF, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL 20, IL21, IL22, IFNα, IFNß, IFNγ, Flt3 L, Flt3, TNFα, RANTES, MlPlα, MlPlß, MlPlγ, MlPlδ, MIP2, MIP2α, MIP2ß, MIP3α, MIP3ß,

MIP4, MIP5, MCP1, MCPlß, MCP2, MCP3, MCP4, MCP5, MCP6, 6cykine, Dcckl and DCDF.

7. Use according to one of claims 5 to 6, wherein the costimulatory molecule is selected from the group comprising B7.1, B7.2, CD40, Light, Ox40, 4.1.BB, Icos L, SLAM; ICAM 1, LFA-3, B7.3, CD70, HSA, CD84,

CD7, B7 RP-1 L, MAdCAM-1, VCAM-1, CS-1, CD82, CD30, CD120a, CD120b, TNFR-RP, CD40L, Ox40L and Rael.

8. Use according to any one of claims 5 to 7, wherein the cytokine, chemokine and / or costimulatory molecule at least one point mutation, insertion, deletion or a fusion with at least one other peptide or

Is protein.

9. Use according to one of claims 1 to 8, wherein the tumor disease is melanoma, ovarian cancer, breast cancer, colon cancer, leukemia, lymphoma, kidney cancer, lung cancer, prostate cancer, cervical cancer, neuroblastoma, osteosarcoma, thymoma, hepatoma, larynx

Anal cancer, stomach cancer, thyroid cancer, seminoma, pancreatic cancer, mesenthelioma and / or brain tumor.

10. Use according to one of claims 1 to 9, wherein the treatment is combined with chemotherapy, radiation therapy or the surgical removal of the tumor or the metastasis.

11. Use according to one of claims 1 to 10, wherein the vaccine is suitable, in combination with an adjuvant selected from the group comprising CpG, oxidized or reduced glutathione, BCG, Salmonella, Freund's complete adjuvant, Freund's incomplete adjuvant, cytokines or chemokines eg soluble proteins, iscome or specific antibodies against CD40, Toll-like receptors (activating) or CTLA4 (blocking).

12. Use of a cell expressing at least one embryonic tumor antigen for the production of a vaccine for the treatment of end-stage tumor diseases, the cell not being derived from the respective tumor disease.

13. Use according to claim 12, wherein the cell expresses at least 3 tumor antigens.

14. Use according to claims 12 or 13, wherein the cell at least one tumor antigen as defined in claim 2 and / or at least one cytokine,

Chemokine and / or costimulatory molecule as defined in claims 5 to 8 expressed.

15. Use according to claims 12 to 14, wherein the cell is a cell as defined in claims 3 or 4.

16. Use according to claims 12 to 15, wherein the tumor disease is as defined in spoke 9.

17. Use according to claims 12 to 16, wherein the treatment is as defined in claim 10.

18. Use according to claims 12 to 17, wherein the vaccine is suitable, in combination with an adjuvant selected from the group comprising CpG, oxidized or reduced glutathione, BCG, Salmonella, complete Freund's adjuvant, incomplete Freund's adjuvant, cytokines or chemokines e.g. soluble proteins, iscome or specific antibodies against CD40, Toll-like receptors (activating) or CTLA4 (blocking).

19. Medicament for the prophylaxis, therapy and / or secondary prophylaxis of a tumor disease containing a technically modified cell according to one of claims 1 to 8.

20. Medicament according to spoke 19, the medicament additionally comprising at least one adjuvant selected from the group comprising CpG, oxidized or reduced glutathione, BCG, Salmonella, Freund's complete adjuvant, Freund's incomplete adjuvant, cytokines or chemokines e.g. contains soluble proteins, iscome or specific antibodies against CD40, Toll-like receptors (activating) or CTLA4 (blocking).

21. Medicament according to one of claims 19 or 20, wherein the medicament is suitable for being administered subcutaneously, intracutaneously, intravenously or intranodally.