DE10109854A1 - Polypeptides of an hdm2 protein-specific murine alpha / beta T cell receptor, these coding nucleic acids and their use - Google Patents

Abstract

The invention relates to polypeptides from a murine alpha / beta T-cell receptor, mediating a hdm2 protein-specific T-cell response, or functional variants or parts thereof, or nucleic acids coding for the above, functional variants, or parts thereof. The effect of the above is that hdm2 protein-expressing cells are recognised by t-cells provided with said genes, cytokines are expressed and a t-cell-induced lysis and/or apoptosis of tumour- or leukaemic-cells is induced.

Description

The invention relates to polypeptides of an hdm2 protein-specific T cell response averaging murine α / β T cell receptor, these coding nucleic acids and their ver used in the therapy, diagnosis and / or prevention of the hdm2 protein associated ized diseases.

Antigen recognition by T lymphocytes (TZL) is crucial for generation and Regulation of an effective immune response. The characteristic T cell line marker is the T cell antigen receptor (TCR). There are two defined types of TZR: One is a hete rodimer of two disulfide-linked polypeptides (α and β); the other one is structured rell similar, but consists of γ and δ polypeptides. Both receptors are in one set of five polypeptides, associated with the CD3 complex, and together form the T cell Receptor complex (TZR-CD3 complex). The α / β-TZR is the most functionally significant because it is expressed in over 95% of all T cells and mediates the supporting immune response.

α / β-T cells can be divided into two different overlapping populations: a subgroup that bears the CD4 marker and mainly supports the immune response (T _H ) and a subgroup that bears the CD8 marker and essentially is cytotoxic (T _C ). CD8 ⁺ T cells recognize antigens in association with MHC class I molecules. Such antigens can include tumor-specific or tumor-associated peptide antigens. After recognition of the peptide antigens, the cell in question is killed by the T cell lysing the target cell and / or inducing apoptosis of these target cells or releasing cytokines (e.g. IL-2, IFN-γ).

Among the tumor-associated peptide antigens (TAA) that are in the context of MHC class I Molecules presented on the surface of tumor cells are the so-called "universal" TAA of particular interest. These TAA are mainly derived from zel lular proteins that are weakly expressed in normal cells and overex in tumor cells be primed. These proteins include the human homologue of  "Mouse-Double-Minute-2" proto-antigen (mdm2), the so-called "human mdm2" or abbreviated "hdm2" proto-oncoprotein (Roth et al. 1998), which is not only more solid in a number Tumors, but also in haematological neoplasia (malignant haematological sy stem diseases) AML, ALL and CHL is overexpressed (Zhou et al. 2000).

Oligopeptides of the hdm2 protein can be used in the context of MHC class I molecules on the Cell surface are presented and represent attractive target structures for CD8 positive T cells. The extent of the T cell response runs in a defined kine table window (Kersh et al., 1998). The complex of peptide-MHC and TZR-CD3 multimeri siert to effect an efficient signal transduction, whereby the exact stoichiometry and the extent of oligomerization is still controversial.

Prerequisite for the development of immunotherapeutic methods for the treatment of Malignant tumor disease is the identification of oncoprotein-specific T cells Receptors. With such T cell receptors, T- Provide cells with antigen specificity in general and tumor reactivity in particular with the aim that the T cells reflect and eradicate a particular Induce tumor. Described methods for the identification of high affinity peptide specific TZRs include the "phage" or "yeast display" methodology (Holler et al., 1999) and a tetramer-dependent "TCR display" methodology (Kessels et al., 2000).

From Theobald et al. ("Targeting p53 as a general tumor antigen", PNAS USA 92, pp. 11993-11997, 1995), the production of p53-specific cytotoxic T cells after the injection of peptides from the sequence of p53 is known. A selection of human tumor cells could then be lysed using these T cell lines. The isolation of genes of specific T cell receptors of the anti-p53 lytic T cells is not described. WO 97/32603 generally describes a process for the production of recombining T lymphocytes which express specific T cell receptors directed against tumor tissue. An HLA-transgenic mouse (in this case HLA-A2) is immunized with tumor-associated antigen in order to produce cytotoxic T lymphocytes that express specific T cell receptors on their surface. As tumor-associated antigens peptides of various genes such as Her-2 / neu, Ras, p53, tyrosinase, MART, gp100, MAGE, BAGE and MUC-1 are described. The nucleotide sequence which contains at least one variable region of the .alpha. And .beta. Chain of the corresponding non-human T-cell receptor is then isolated from the Her-2 / neu-specific T lymphocytes and is modified in the form of various molecular biologically modified genetic (including "hu man ") TZR constructs used. WO 97/32603 describes fusion proteins of variable regions of TZR with the ζ region of D3 or CD8 or CD16, as well as the use of flexible linkers of the amino acid sequence (GGGGS) ₃ .

Clay et al. ("Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity "; J. Immunology, 1999, 163: 507-513) the isolation of genes of the α-TZR and β-TZR chains of a MART-1 specific TZR and its expression in human peripheral blood lymphocytes (PBLs). The TZR are ge directed to amino acids 25-35 of MART-1. Furthermore, the lysis is different metastatic melanoma cell lines and a retroviral vector system described by IRES elements.

Darcy et al. ("Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL ", J. Immunol., 2000, 164: 3705-3712) describe an immunotherapeutic Procedure for colon carcinomas using scFv anti-CEA receptor transdu adorned ZTL, Perforin and γ-IFN. The chimeric specific receptor construct is via a retroviral vector transduced into primary mouse T lymphocytes. These cells were in Mice injected with colon carcinoma cell lines previously inoculated.

Weijtens et al. ("A retroviral vector system, 'STITCH'; in combination with an optimized single-chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T-lymphocytes ", Gene Therapy (1998) 5, 1995-1203) and Willemsen et al. "Grafting primary human T lymphocytes with cancer-specific single-chain and two chain TCR "Gene Therapy, 2000 7, 1369-1377) describe retroviral vector systems for trans genes into activated T lymphocytes. This system is used to control the Ex pression of antibody-based chimeric receptors in the membrane of T cells cause. These T cells can then be used against specific cancer cells. The The effectiveness of the retroviral vector system is based on the specific recognition and Cytolysis of renal cell carcinoma has been described. Eshar describes similarly. et al. ( "Specific activation and targeting of cytotoxic lymphocytes through chimeric single-chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell re ceptors "P.N.A.S. USA, 90, pp. 720-724, 1993) the production of tumor-specific lymphocytes  and their use in chimeric immunotherapy, which the variable regions of an antibody (scFV's) with the constant region of the T cell recipe tors include (so-called "single-chain TCR" (scTCR)). These chimeric genes were able to the surface of cytolytic T cell hybridomas are expressed and caused the Release of interleukin-2 after contact with the antigen.

A TZR specifically directed against hdm2 and its use is however by the Publications mentioned above and in the other literature neither mentioned nor suggested.

The present invention is therefore based on the object of opposing the murine genes to provide the hdm2 protein-directed T cell receptors α-TZR and β-TZR. These cause hdm2 protein-expressing cells to be recognized by CD8-positive T cells , cytokines are released, and T cell-induced lysis and / or apoptosis is brought about by tumor or leukemia cells.

According to the invention, this object is achieved by the polypeptide of an hdm2 protein specific T cell response mediating murine α / β T cell receptor according to SEQ ID No. 1 or SEQ ID No. 2 or functional variants or parts thereof or nu coding this Small acids, functional variants or parts of them solved.

From a double A2.1 transgenic mouse [huCD8α / β × A2.1 / K ^b ] _F1 in C57BL / 6 genetic background, the genes of an hdm2 protein-specific T cell response were mediated by the murine α / β T -Cell receptor isolated, the HLA-A2.1 restricted and mediated against the peptide of amino acids 81-88 of the hdm2 protein mediate specific T cell response. The polypeptides of this TCR were previously unknown. The genes were introduced as wild type (WT) or modified constructs retrovirally into human peripheral blood lymphocytes (PBLs) and the HLA-restricted antigen recognition was checked functionally by CD8-mediated cytotoxic lysis of different cell lines in the ⁵¹ chromium release test. The genes of the hdm2-specific TZR according to the invention do not belong to the hitherto known suitable targets for diagnosis - such as indication - and / or treatment - such as modulation - of diseases related to hdm2 protein or for identification of pharmacologically active substances, so that completely new therapeutic approaches result from this invention.

Another aspect of the invention relates to a fusion protein comprising the inventive appropriate polypeptide or functional variants or parts thereof or nu encoding this small acids, functional variants or parts thereof.

The fusion protein can be characterized in that it comprises the ζ region of CD3 or CD8 or CD 16 or parts thereof, in particular the ζ region of human CD3 or CD8 or CD16 or parts thereof. A fusion protein according to the invention which comprises a flexible linker is preferred (Whitlow et al., "An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability", Prot. Engin. 6 (8), pp. 989-995, 1993), in particular a linker of the amino acid sequence (GGGGS) ₃ . In particular, the fusion protein according to the invention can comprise the ζ chain of the CD3 complex or ITAM motifs of the ζ pin or parts thereof, in particular the ζ chain of human CD3 or parts thereof. The fusion protein can further be characterized in that it comprises CD8α or the Lck binding motif of CD8α or parts thereof, in particular human CD8α.

The fusion protein according to the invention can furthermore be a chimeric partial or fully humanized α and / or β TCR chain act, in particular according to SEQ ID No. 3 or SEQ ID No. 4. Another aspect of the invention relates to a fusion protein, at which is a single chain T cell receptor, in particular according to SEQ ID no. 5 or SEQ ID No. 6. However, the fusion protein according to the invention can also be characterized thereby is characterized by the fact that it is an α / β- coupled to other functional components TZR acts, in particular according to SEQ ID No. 7 or SEQ ID No. 8.

Another object of the invention is a method for producing an Invention fusion protein for diagnosis and / or treatment of with hdm2 protein in Zu related diseases or for the identification of pharmacologically active Substances, e.g. B. in a suitable host cell in which a nucleic acid according to the invention is used.

Fusion proteins are produced here which contain the above-described invention Contain polypeptides, the fusion proteins themselves already having the function of a polypep have tids of the invention or only after the fusion portion has been split off the specific one Function is active. Above all, this includes fusion proteins with a share of approx. 1-200,  preferably about 1-150, in particular about 1-100, especially about 1-50 foreign amino acids. Examples of such peptide sequences are prokaryotic peptide sequences which e.g. B. from the Galactosidase can be derived from E. coli. Viral peptide sequences can also be used zen, such as used by bacteriophage M13, so as fusion proteins for the "phage display" method known to the person skilled in the art.

A further polypeptide ("tag") can be added to purify the proteins according to the invention. Protein tags according to the invention allow, for example, high absorption to a matrix, stringent washing with suitable buffers without eluting the complex to any appreciable extent, and then targeted elution of the absorbed complex. Examples of the protein tags known to the person skilled in the art are a (His) ₆ tag, a Myc tag, a FLAG tag, a Strep tag, a Strep tag II, a hemagglutinin tag, glutathione transferase (GST) - tag, intein with an affinity chitin binding tag or maltose binding protein (MBP) tag. These protein tags can be located at the N-, C-terminal and / or internally.

In addition to the natural polypeptides isolated from cells, all of the invention can Polypeptides or their parts have been produced under cell-free conditions, e.g. B. by synthesis or by in vitro translation. So all or part of the polypeptide of which, for example, synthesized using classic synthesis (Merrifield technique) become. Parts of the polypeptides according to the invention are particularly suitable for extraction of antisera, which can be used to search for suitable gene expression banks, in order to arrive at further functional variants of the polypeptide according to the invention.

The invention also relates to polypeptides, the derivatives of an antibody with specificity for hdm2-81-88 peptide, particularly presented in the context of HLA-A2.1.

The invention further includes retro-inverse peptides or pseudopeptides according to the poly peptide sequence of SEQ ID No. 1 to SEQ ID No. 8 or functional variants or parts from that. These peptides have -NH-CO bonds instead of the -CO-NH peptide bonds.

The object of the invention is further achieved by a nucleic acid according to the invention, which a DNA, RNA, PNA (peptide nucleic acid) or p-NA (pyranosyl nucleic acid) before preferably a DNA, in particular a double-stranded DNA with a length of at least  8 nucleotides, preferably with at least 18 nucleotides, in particular with min is at least 24 nucleotides. The nucleic acid can be characterized in that the Se sequence of the nucleic acid has at least one intron and / or a polyA sequence. she can also be in the form of their antisense sequence.

A double-stranded DNA is generally required for the expression of the gene in question is preferred, the DNA region coding for the polypeptide being particularly preferred. the This area begins with the first in a Kozak consensus sequence (Kozak, 1987, Nucleic. Acids Res. 15: 8125-48) start codon (ATG) to the next stop codon (TAG, TGA or TAA), which is in the same reading frame as the ATG. Another use The construction of anti-sense oli is the basis of the nucleic acid sequences according to the invention gonucleotides (Zheng and Kemeny, 1995, Clin. Exp. Immunol. 100: 380-2) and / or Ribozymes (Amarzguioui, et al. 1998, Cell. Mol. Life Sci. 54: 1175-202; Vaish, et al., 1998, Nucleic Acids Res. 26: 5237-42; Persidis, 1997, Nat. Biotechnol. 15: 921-2). With anti-sense Oligonucleotides can reduce the stability of the nucleic acid according to the invention and / or inhibit the translation of the nucleic acid according to the invention. For example indicate the expression of the corresponding genes in cells both in vivo and in vitro be wrested. Oligonucleotides can therefore be used as therapeutic agents. This strategy is also suitable for example for skin, epidermal and dermal cells, especially if the antisense oligonucleotides are complexed with liposomes (Smyth et al., 1997, J. In vest. Dermatol. 108: 523-6; White et al., 1999, J. Invest. Dermatol. 112: 699-705). For the Use as a probe or as an "antisense" oligonucleotide is a single stranded DNA or RNA preferred.

In addition to the natural nucleic acids isolated from cells, all of the invention can Nucleic acids or parts thereof have also been produced synthetically. Furthermore, for Implementation of the invention uses a nucleic acid that is synthetically produced has been. For example, the nucleic acid according to the invention can be chemically determined using the using the protein sequences described in SEQ ID No. 1 to SEQ ID No. 8 the genetic code z. B. can be synthesized according to the phosphotriester method (see e.g. Uhlmann, E. & Peyman, A. (1990) Chemical Revievs 90, 543-584).

Oligonucleotides are usually quickly removed by endo- or exonucleases, in particular degraded by DNases and RNases occurring in the cell. Therefore it is advantageous  modify the nucleic acid to stabilize it against degradation, so that over a long period a high concentration of the nucleic acid in the cell is maintained (WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116). Ty Such stabilization can typically be achieved by introducing one or more Internucleotide phosphor groups or by introducing one or more non- Phosphorus internucleotides can be obtained.

Suitable modified internucleotides are described in Uhlmann and Peymann (1990 Chem. Rev. 90, 544) (WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116). Modified internucleotide phosphate residues and / or non- Phosphorus bridges in a nucleic acid used in one of the uses according to the invention can contain, for example, methylphosphonate, phosphorothioate, Phosphoramidate, phosphorodithioate, phosphate ester, while non-phosphorus internucleotide Analogs, for example siloxane bridges, carbonate bridges, carboxymethyl esters, acetami data bridges and / or thioether bridges included. It is also intended that this Modifi ornamentation the shelf life of a pharmaceutical composition used in one of the inventions uses according to the invention can be improved.

Another aspect of the present invention relates to a vector, preferably in form a plasmid, shuttle vector, phagemid, cosmid, expression vector, adenoviral Vectors, retroviral vectors (Miller, et al. "Improved retroviral vectors for gene transfer and expression ", BioTechniques Vol. 7, No. 9, p 980, 1989) and / or gene therapy men vector, which contains a nucleic acid according to the invention.

For example, the nucleic acid according to the invention can be in a vector, preferably in an express ion vector or gene therapy vector. Preferably, the gene-therapeutic vector T-cell-specific regulatory sequences that function nell associated with the nucleic acid of the invention. The expression vectors can be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic Expression vectors are for expression in E. coli z. B. the vectors pGEM or pUC- Derivatives and for eukaryotic expression vectors for expression in Saccharomyces cerevisiae z. B. the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768), for expression in insect cells e.g. B. Baculovirus Vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells  z. B. the vectors Rc / CMV and Rc / RSV or SV40 vectors, all of them general are available.

In general, the expression vectors also contain suitable ones for the respective host cell Promoters such as B. the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the Met25, GAL1 or ADH2 promoter for expression in yeasts (Russel et al. (1983), J. Biol. Chem. 258, 2674-2682; Mumberg, supra), the baculovirus polyhedrin Promoter for expression in insect cells (see e.g. EP-B1-0 127 839). For the ex pression in mammalian cells, for example, promoters are suitable which have a constitutive, adjustable, tissue-specific, cell cycle-specific or metabolic-specific expressions allow on in eukaryotic cells. Adjustable elements according to the present invention are promoters, activator sequences, enhancers, silencers and / or repressor sequences Zen. Example of suitable regulatable elements, the constitutive expression in eukaryotes are promoters that are recognized by RNA polymerase III or viral Promoters, CMV enhancers, CMV promoters, CMV-LTR hybrids, SV40 promoters or LTR promoters e.g. B. from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232) and further viral promoter and activator sequences derived from at for example HBV, HCV, HSV, HPV, EBV, HTLV or HIV. An example of a regulierba The element that enables regulatable expression in eukaryotes is the tetracycli noperator in combination with a corresponding repressor (Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20).

Examples of controllable elements that he T-cell-specific expression in eukaryotes possible, are promoters or activator sequences from promoters or enhancers of those genes that code for proteins that are only expressed in these cell types.

Examples of regulatable elements, the cell cycle-specific expression in eukaryotes are promoters of the following genes: cdc25, cyclin A, cyclin E, edc2, E2F, B-myb or DHFR (Zwicker J. and Müller R. (1997) Trends Genet. 13, 3-6). Examples of regulated bare elements that allow metabolic-specific expression in eukaryotes Promoters caused by hypoxia, by glucose deficiency, by phosphate concentration or be regulated by heat shock.  

The vector according to the invention can be used for the transfection of a host cell which is preferably a T cell. A host cell is particularly preferred le, which is characterized in that it has an inventive poly on its surface peptide or fusion protein expressed.

In order to introduce nucleic acids according to the invention and thus the expression of the Polypeptide in a eu or prokaryotic cell by transfection, transduction, trans To enable formation or infection, the nucleic acid can act as a plasmid, as part of a viral or non-viral vector or particle. As viral vectors or particles The following are particularly suitable: Baculoviruses, vaccinia viruses, retroviruses, adenoviruses, adenoas Associated viruses and herpes viruses. The following are particularly suitable as non-viral carriers: Viro somen, liposomes, cationic lipids, or poly-lysine conjugated DNA.

Examples of vectors with gene therapy effects are virus vectors, for example Ade novirus vectors or retroviral vectors (Lindemann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58; Weijtens et al. "A retroviral vector system, 'Stitch'; in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T-lymphocytes ", genes Therapy (1998) 5, 1995-1203).

A preferred mechanism for the expression of polypeptides according to the invention in vivo bring, is the viral gene transfer, especially with the help of retroviral particles. This who the preferred target cells, preferably T-lymphocytes, used by the patient ducks ex vivo with the genes or nucleos coding for the polypeptides according to the invention to provide tide sequences by transduction. The target cells can then ei nes adoptive cell transfers can be reinfused into the patient in order to with the de novo inserted specificity to over tumorizid and / or immunomodulating effector functions to take. Recently, very good gene therapy successes in the Be Action of SCID-X1 disease characterized by immunocompetence in Neuge borenen in which hematological progenitor cells with an analogue intact trans gene in a non-functional mutant variant of γ- Chain gene that is used for differentiation into the different effector cells of the adaptive Immune system is essential, have been retrovirally provided (Cavazzana-Calvo et al., 2000).  

There is also the possibility of performing the gene transfer in vivo, on the one hand through preferentially stereotactic injection of the infectious particles, on the other hand by direct ap application of virus-producing cells (Oldfield, et al. Hum. Gen. Ther., 1993, 4: 39-69).

The viral vectors frequently used for the transfer of genes are as of today of technology predominantly retroviral, lentiviral, adenoviral and adeno-associated viral Vectors. These are circular nucleotide sequences derived from natural viruses, in de at least the genes coding for viral structural protein by the gene to be transferred Construct can be exchanged.

Retroviral vector systems create the prerequisite for long-term expression of the Transgens through the stable but undirected integration into the host genome. Vectors of younger generation do not have irrelevant and potentially immunogenic proteins further, there is no pre-existing immunity of the recipient to the vector. re Troviruses contain an RNA genome that is packaged in a lipid envelope that consists of parts of the Host cell membrane and virus proteins exist. To express viral genes, the RNA Genome reverse transcribed and integrated into the target cell DNA with the enzyme integrase. the it can then be transcribed and translated by the infected cell, whereby vi rale components arise that combine to form retroviruses. RNS is used exclusively then inserted into the newly created viruses. The retrovirus genome has three essentials Elle genes: gag, the ko for viral structural proteins, so-called group-specific antigens d, pol for enzymes such as reverse transcriptase and integrase and env for the coat protein ("envelope"), which is responsible for the binding of the host-specific receptor. The Production of replication-incompetent viruses takes place after transfection in so-called Packaging cell lines instead, which are additionally equipped with the gag / pol-coding genes and express them "in trans" and thus the formation of replication-incompetent complement (i.e., gag / pol deleted) transgenic virus particles. An alternative is Cotransfection of the essential virus genes, whereby only the vector containing the transgene carries the packaging signal.

The separation of these genes on the one hand enables any combination of the gal / pol- Reading frame with env reading frames obtained from different tribes, whereby Pseu prototypes with changed host tropism emerge, on the other hand this can lead to education replication-competent viruses within packaging cells can be drastically reduced.  

The coat protein derived from "gibbon ape leukemia virus" (GALV), which in the "stitch" or "bullet" vector system is used, is able to transduce human cells and is established in the packaging cell line PG13 with amphotropic host range (Miller et al., 1991). In addition, the security through selective deletion of non-essential Vi Russian sequences to prevent homologous recombination and thus production replication-competent particles increased.

New, non-viral vectors consist of autonomous, self-integrating DNA Sequences, the transposons, which by z. B. liposomal transfection into the host cell and successfully for the first time for the expression of human transgenes in mammalian cells len were used (Yant et al., 2000).

Gene therapy-effective vectors can also be obtained by inventing the nucleic acid according to the invention complexed with liposomes, since it is a very high transfecti efficiency, especially of skin cells, can be achieved (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279-85). Excipients that facilitate the transfer of nucleic acids into the Cell, for example, proteins or peptides that are bound to DNA or synthetic peptide DNA molecules that transport the nucleic acid into the nucleus of the Allow cells to be (Schwartz et al. (1999) Gene Therapy 6, 282; Brandén et al. (1999) Nature biotech. 17, 784). Excipients also include molecules that inhibit the release of Nu enable small acids into the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213) or, for example, liposomes (Uhl mann and Peymann (1990) supra). Another particularly suitable form of gentherapeuti vectors can be obtained by adding the nucleic acid of the invention Applies gold particles and these in tissue using the so-called "gene gun" before pulls into the skin or shoots cells (Wang et al., 1999, J. Invest. Dermatol., 112: 775-81).

For the gene therapy application of the nucleic acid according to the invention it is also from Advantage if the part of the nucleic acid that codes for the polypeptide is one or more non-coding sequences including intron sequences, preferably between promo tor and the start codon of the polypeptide, and / or a polyA sequence, in particular the na naturally occurring polyA sequence or an SV40 virus polyA sequence, especially on the 3'- Contains end of the gene, since this can stabilize the mRNA  (Jackson, R.J. (1993) Cell 74, 9-14 and Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478-482).

Another object of the present invention is a host cell, in particular a T- Cell with a vector according to the invention or another according to the invention Gene construct is transformed. Host cells can be both prokaryotic and eukaryotic his cells, examples of prokaryotic host cells are E. coli and eukaryotic Saccharomyces cerevisiae cells or insect cells.

A particularly preferred transformed host cell is a transgenic T precursor cell or a stem cell, which is characterized in that it constructs a gene construct according to the invention or comprises an expression cassette according to the invention. Methods for transforming or transducing host cells and / or stem cells are well known to those skilled in the art and include, for example, electroporation or microinjection. A particularly preferred transformed host cell is a patient's own T cell which, after removal, is transfected with a gene construct according to the invention. Host cells according to the invention can in particular be obtained by removing one or more cells from the patient before preferentially removing T cells, in particular CD8 ⁺ T cells, which are then transfected or transduced ex vivo with one or more genetic constructs according to the invention to obtain host cells according to the invention. The ex vivo generated specific T cells can then be re-implanted in the patient. The procedure is thus similar to that in Darcy et al. ("Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL", J. Immunol., 2000, 164: 3705-3712) described methods using scFv anti-CEA receptor-transduced CTL, perforin and γ- IFN.

Another aspect of the invention relates to a method for identifying hdm2- Protein-specific antigens, which is characterized in that hdm2-presenting Tumor cells or fractions thereof with a host cell according to the invention under certain conditions genes are brought together, in which the tumor cells or fractions thereof only then be lysed when the tumor presents the hdm2 protein-specific antigen for which ches the expressed polypeptide or fusion protein is specific.  

Another aspect of the invention relates to a method for producing an antibody, preferably a polyclonal or monoclonal antibody for diagnosis and monito ring of diseases associated with hdm2 protein or for the identification of pharma ecologically active substances in which an antibody-producing organism with a polypeptide according to the invention or functional equivalents thereof or parts thereof at least 6 amino acids, preferably with at least 8 amino acids, in particular with immunized at least 12 amino acids or with a polypeptide according to the invention coding nucleic acid is vaccinated.

The method is carried out according to methods generally known to those skilled in the art by immunization ren of a mammal, for example a rabbit, with the polypep according to the invention tid or the parts mentioned, optionally in the presence of z. B. incomplete Freund's adjuvant and / or aluminum hydroxide gels (see e.g. Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344-1349). The in the animal due to an im The polyclonal antibodies produced in the munological reaction can then be read off easily isolate well-known methods from the blood and z. B. over column chromatography clean graph. Monoclonal antibodies can, for example, according to the known metho de by Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299) become.

Another object of the present invention is an antibody for the diagnosis and / or treatment of diseases associated with hdm2 protein or for the identification of pharmacologically active substances, which is directed against a polypeptide according to the invention and reacts specifically with the polypeptides according to the invention, the above-mentioned Portions of the polypeptide are either themselves immunogenic or by coupling to suitable carriers, such as. B. bovine serum albumin can be increased in their immunogenicity. This antibody is either polyclonal or monoclonal, a monoclonal antibody is preferred. According to the present invention, the term antibody also means genetically engineered and optionally modified antibodies or anti-gene-binding parts thereof, such as. B. chimeric antibodies, humanized antibodies, multifunctional antibodies, monovalent or oligovalent antibodies, bi- or oligospecific antibodies, single-stranded antibodies, F (ab) - or F (ab) ₂ fragments (see, for example, EP-B1 -0 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213, WO 98/24884). The antibodies according to the invention can be used for the diagnosis and / or treatment of diseases associated with hdm2 protein or for the identification of pharmacologically active substances.

The present invention further relates to a method for producing a medicament tels for the treatment of diseases associated with hdm2 protein, characterized net that at least one nucleic acid, at least one polypeptide, at least one host cell or at least one antibody according to one of the preceding claims together with ge suitable additives and auxiliary substances is combined.

The present invention further relates to a medicament produced by this method agent for the treatment of diseases associated with hdm2 protein, the at least one Nucleic acid, at least one polypeptide or at least one antibody according to the ing invention, optionally together with suitable additives and adjuvants holds. The invention further relates to the use of this medicament for treatment diseases associated with hdm2 protein.

The therapy of diseases associated with hdm2 protein can be conventional Way, e.g. B. by infusions or injections, the drug of the invention medium included. The administration of the medicament according to the invention can also be used optionally in the form of liposome complexes or gold particle complexes. The Treatment by means of the medicaments according to the invention can also be administered via oral doses tion forms such. B. tablets or capsules, over the mucous membranes, for example the nose or the oral cavity, or in the form of disposers implanted under the skin. TTS are for example from EP 0 944 398 A1, EP 0 916 336 A1, EP 0 889 723 A1 or EP 0 852 493 A1 is known. The (poly) peptides according to the invention and their derivatives can also for active and / or passive immunization of patients with diseases, esp special tumor diseases associated with hdm2 are used to to achieve the induction, generation and increase of the hdm-specific ZTL and the Specifically kill tumor and leukemia cells of the patients concerned. Such a crane Examples include solid tumor diseases, lymphohematopoietic Neopla sien, malignant haematological diseases, also in the form of multiple myeloma (or Plastocytoma), histiocytic lymphoma and a CML blast flare.  

In a particularly preferred type of treatment, one or more cells, preferably T cells, in particular CD8 ⁺ T cells, are removed from the patient and are then transduced or transfected ex vivo with one or more genetic constructs according to the invention. The specific T cells generated ex vivo can then be re-implanted in the patient. The process is thus similar to that in Darcy et al. ("Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL", J. Immunol., 2000, 164: 3705-3712) described immunotherapeutic methods in colon carcinomas using scFv anti-CEA receptor-transduced CTL, Perforin and γ-IFN.

The present invention further relates to a method for producing a test for Finding functional interactors related to Erm associated with hdm2 protein diseases, which is characterized in that at least one nucleic acid, min least a polypeptide or at least one antibody according to the invention together with suitable additives and auxiliary substances is combined.

The term "functional interactors" in the sense of the present invention includes all those molecules, compounds and / or compositions and mixtures of substances understand that with the nucleic acids, polypeptides or antibodies according to the invention, if necessary together with suitable additives and auxiliary substances, under suitable conditions can interact. Possible interactors are simple chemical ones organic or inorganic molecules or compounds, but can also peptides, Pro include tones or complexes thereof. The functional interactors can because of their Interaction of the function (s) of nucleic acids, polypeptides or antibodies in vivo or influence in vitro or only to the nucleic acids according to the invention, polypepti en or bind antibodies or with them other interactions covalent or non- covalently.

The invention further comprises an identification test produced according to the invention functional interactors in connection with diseases associated with hdm2 protein gene, the at least one nucleic acid, at least one polypeptide or at least one An Antibodies according to the present invention, optionally together with suitable additives Substances and auxiliaries. Often, the pathological behavior of the cells in vitro can be imitated and substances can be sought that are normal behavior  restore the cells and have therapeutic potential. In addition, use this test system to screen substances that interact with each other inhibit the polypeptide of the invention and a functional interactor.

An object of the present invention is also a medicament for the indication and The Therapy of diseases associated with hdm2 protein, which is a nucleic acid according to the invention acid or a polypeptide according to the invention and, if appropriate, suitable additives or Contains auxiliaries, and a method for producing such a drug for loading act of diseases associated with hdm2 protein, in which an inventive Nucleic acid or a polypeptide according to the invention with a pharmaceutically acceptable its carrier is formulated.

In particular, vaccines, recombinant, come as therapeutic agents and / or prophylactic agents Particles or injections or infusion solutions into consideration which act as the active ingredient (a) TZR receptor polypeptide according to the invention and / or its derivatives and / or (b) an invented contain nucleic acid according to the invention and / or (c) T- generated in vitro or ex vivo Lymphocytes that contain a TCR specifically directed against hdm2.

A drug is primarily used for gene therapy in humans and / or recombinant particles suitable that the nucleic acid according to the invention in naked Form or in the form of one of the above-described gene therapy vectors or contains in a form complexed with liposomes or gold particles. The pharmaceutical trä ger is, for example, a physiological buffer solution, preferably with a pH of approx. 6.0-8.0, preferably from approximately 6.8-7.8, in particular from approximately 7.4 and / or an osmolarity from about 200-400 milliosmol / liter, preferably from about 290-310 milliosmol / liter. additionally can the pharmaceutical carrier suitable stabilizers, such as. B. nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to the person skilled in the art substances included.

Another object of the invention relates to a method for producing a polypep tids to diagnose and / or treat hdm2 protein-related Diseases or for the identification of pharmacologically active substances in a ge suitable host cell, which is characterized in that a nucleic acid according to the invention is appropriately expressed.  

The polypeptide is thus, for example, by expression of the nucleic acid according to the invention re in a suitable expression system, as already described above, according to the subject man made known methods. Suitable host cells are, for example the E. coli strains DHS, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, In sect cell lines, e.g. B. from Spodoptera frugiperda, or the animal cells COS, Vero, 293, HaCaT and HeLa, all of which are generally available.

A diagnostic agent according to the invention for monitoring therapy contains the inventive appropriate polypeptide or the immunologically active parts thereof described in more detail above. The polypeptide or parts thereof, which are preferably attached to a solid phase, e.g. B. from nitrocellulo se or nylon, can be bound, for example, with the body fluids to be examined liquidity, e.g. As blood, are brought into contact in vitro, for example with Autoim to be able to react to munantibodies. The antibody-peptide complex can then for example using labeled anti-human IgG or anti-human IgM antibodies be directed. The label is, for example, an enzyme such as Per oxidase, which catalyzes a color reaction. The presence and the amount of people present Autoimmune antibodies can thus be detected easily and quickly via the color reaction become.

Another diagnostic for therapy monitoring contains the anti according to the invention body itself. With the help of these antibodies, for example, a tissue sample can easily and quickly examined whether the polypeptide in question in an elevated Amount is present, giving an indication of hdm2 protein to get standing diseases. In this case, the antibodies according to the invention are for example marked with an enzyme, as already described above. The specific one Antibody-peptide complex can be easily and just as quickly via an enzymatic Color reaction can be demonstrated.

Another diagnostic agent according to the invention comprises a probe, preferably a DNA probe, and / or primer. This opens up a further possibility of obtaining the nucleic acids according to the invention, for example by isolating them from a suitable gene bank using a suitable probe (see, for example, J. Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Chapter 8, pages 8.1 to 8.81, Chapter 9, pages 9.47 to 9.58 and Chapter 10, pages 10.1 to 10.67).

For example, DNA or RNA fragments with a length of approx. 100-1000 nucleotides, preferably with a length of approximately 200-500 nucleotides, in particular in particular with a length of about 300-400 nucleotides, the sequence of which consists of the polypeptides can be derived according to SEQ ID No. 1 to SEQ ID No. 8 of the sequence listing.

Alternatively, oligonucleotides can be synthesized using the derived nucleic acid sequences Siert that are suitable as primers for a polymerase chain reaction. As a primer DNA fragments with a length of approx. 10-100 nucleotides, for example preferably with a length of approximately 15 to 50 nucleotides, in particular with a length of 20-30 nucleotides, the sequence of which from the polypeptides according to SEQ ID No. 1 to SEQ ID No. 8 of the sequence listing can be derived.

The term "coding nucleic acid" refers to a DNA sequence that is iso encodable bioactive polypeptide according to the invention or a precursor. The poly peptide can be a full length sequence or any part of the coding sequence be encoded as long as the specific, for example enzymatic activity is retained.

It is known that small changes in the sequence of the nucleic acid according to the invention may be present, for example by degenerating the genetic code, or that untranslated sequences are attached to the 5 'and / or 3' end of the nucleic acid can be without significantly changing its activity. This invention encompasses therefore also so-called "functional variants" of the nucleic acids according to the invention.

The term “functional variants” denotes all DNA sequences that are complete are mentary to a DNA sequence that is under stringent conditions with a derived one Reference sequence or parts thereof, in particular the hypervariable V (D) JC region, hybridize and a similar to the corresponding polypeptide according to the invention or have identical activity.  

"Stringent hybridization conditions" mean such conditions in hybridization at 60 ° C in 2.5 × SSC buffer followed by several washes at 37 ° C in a lower buffer concentration and remains stable.

The term “functional variants” is understood in the sense of the present invention polypeptides that are functionally related to the polypeptides of the invention, d. H. Have structural features of the polypeptides. Examples of functional variants are the corresponding polypeptides, which come from organisms other than the mouse, i.e. humans, or, preferably from non-human mammals such as. B. monkeys, pigs and Rats come from. Other examples of functional variants are polypeptides by different alleles of the gene, in different individuals or in different organs organism. In particular, the present invention functional TZR variants detected that recognize the identical epitope of the hdm2 polypeptide and trigger a specific T cell response.

In a broader sense, this also includes polypeptides that have a sequence homology, ins in particular a sequence identity of approximately 70%, preferably approximately 80%, in particular approximately 90%, especially about 95% of the polypeptide with the amino acid sequence according to one of the SEQ ID No. 1 to SEQ ID No. 8 and / or to DNA sequences derived from the peptide sequences have zen. These include additions, inversions, substitutions, deletions, Insertions or chemical / physical modifications and / or exchanges or parts of the Polypeptide in the range from about 1-60, preferably from about 1-30, in particular from about 1-15, especially from about 1-5 amino acids. For example, the first amino acid can be methionine are absent without significantly changing the function of the polypeptide.

The invention will now be further explained on the basis of the attached examples and figures, without being restricted by these. It shows:
SEQ ID No. 1: polypeptide of the wild-type mouse αTZR (muvα-mucα) gene,
SEQ ID No. 2: polypeptide of the wild-type gene mouse βTZR (muvβ-mucβ),
SEQ ID No. 3: polypeptide of a chimeric partially humanized TCR gene (muvα-hucα),
SEQ ID No. 4: polypeptide of a chimeric partially humanized TCR gene (muvβ-hucβ),
SEQ ID No. 5: polypeptide of a single-chain TCR muvα-Li-muvβ-mucβ,
SEQ ID No. 6: polypeptide of a single chain TCR muvβ-Li-muvα-mucα,
SEQ ID No. 7: polypeptide of a double chain TZR in fusion with human CD3ζ chain muvα-hucα-huCD3ζ,
SEQ ID No. 8: polypeptide of a double chain TZR in fusion with human CD3ζ chain muvβ-hucβ-huCD3ζ,
SEQ ID No. 9: Primer for_bTCR_v4,
SEQ. ID No. 10: primer rev_bTCR_c4,
SEQ. ID No. 11: Primer for_Eco_bTCR_v5,
SEQ. ID No. 12: Primer rev_Asc_bTCR_c6,
SEQ. ID No. 13: Primer for_Eco_bTCR_c2,
SEQ. ID No. 14: Primer rev_Asc_bTCR_c2,
SEQ. ID No. 15: Primer rev_Asc_aTCR_c1,
SEQ. ID No. 16: Primer rev_aTCR_c5,
SEQ. ID No. 17: Primer rev_Asc_aTCR_c4,
SEQ. ID No. 18: Primer for_Eco_aTCR_c6,
SEQ. ID No. 19: Primer for_NcoI_aTCR_4,
SEQ. ID No. 20: Primer for_NcoI_aTCR_5b; phosphorylated at the 5 'end,
SEQ. ID No. 21: Primer for_NcoI_bTCR_4,
SEQ. ID No. 22: Primer for_NcoI_bTCR_5b; phosphorylated at the 5 'end,
SEQ. ID No. 23: Primer rev_aTCR_BamHI_1,
SEQ. ID No. 24: Primer rev_bTCR_BamHI_1,
SEQ. ID No. 25: Primer for_hucaTCR_2,
SEQ. ID No. 26: Primer rev_hucaTCR_2,
SEQ. ID No. 27: Primer for_hucaTCR_AlwNI_3,
SEQ. ID No. 28: Primer rev_hucaTCR_BamHI_1
SEQ. ID No. 29: Primer for_hucbTCR_3,
SEQ. ID No. 30: Primer rev_hucbTCR_2,
SEQ. ID No. 31: Primer for_hucbTCR_4; phosphorylated at the 5 'end,
SEQ. ID No. 32: Primer rev_hucbTCR_BamHI_1
SEQ. ID No. 33: Primer T7_for,
SEQ. ID No. 34: Primer T7_rev,
SEQ. ID No. 35: Primer rev_hZeta_BamHI_4,
SEQ. ID No. 36: primer rev_huca-hu_tm_Zeta,
SEQ. ID No. 37: Primer rev_hucb-hu_tm_Zeta,
SEQ. ID No. 38: polynucleotide of the wild-type mouse αTZR gene (muvα-mucα) from SEQ ID No. 1,
SEQ. ID No. 39: polynucleotide of the wild-type mouse βTZR gene (muvβ-mucβ) from SEQ ID No. 2,
SEQ. ID No. 40: polynucleotide of the chimeric partially humanized TZR gene (muvα-hucα) from SEQ ID No. 3,
SEQ. ID No. 41: polynucleotide of the chimeric partially humanized TZR gene (muvβ-hucβ) from SEQ ID No. 4,
SEQ. ID No. 42: polynucleotide of the single-chain TZR muvα-Li-muvβ-mucβ from SEQ ID No. 5,
SEQ. ID No. 43: polynucleotide of the single-chain TZR muvβ-Li-muvα-mucα from SEQ ID No. 6,
SEQ. ID No. 44: polynucleotide of the double chain TZR in fusion with human CD3ζ chain muvα-hucα-huCD3ζ from SEQ ID No. 7; and
SEQ. ID No. 45: Polynucleotide of the double chain TZR in fusion with human CD3ζ chain muvβ-hucβ-huCD3ζ from SEQ ID No. 8.

In the primer sequences, Y = C / T; R = A / G; V = A / C / G; W = A / C / T.

Fig. 1 shows a FACS analysis (see 1.4) using a vβ6-specific FITC-labeled antibody (Pharmingen). Both the murine T cell line and the resulting clone 3 were checked. Anti-TCR Vα2-FITC (BD) was used as the isotype control and the anti-pan βTZR-FITC antibody (BD) as positive control. The influenza matrix protein-specific T cell clone 58 served as a negative control.

Fig. 2 shows a vβ6-specific antibody blockade of the murine T-cell clone cytotoxic 3 in the ⁵¹ chromium release assay (see, 1.4). The blockade was evaluated depending on the dose. An anti-Vα3.2 antibody (BD) and the p53-264-272-specific T cell clone 46, which was cross-checked against its own p53 peptide to be recognized, served as a negative control. The target cell was the TAP-deficient cell line T2, which was loaded exogenously with 10 ^-8 M peptide.

Fig. 3 shows examples of the design-specific hdm2 constructs at the DNA level (see 2.), wherein MUV α / β murine variable α / β gene segment; muC α / β murine constant α / β gene segment; huC α / β human constant α / β gene segment; TM: gene segment coding for trans membrane; Li: gene segment coding for (GGGGS) ₃ motif and HuCD3ζ human CD3ζ chain gene segment. The restriction sites relevant for the cloning are indicated.

Fig. 4 shows the same examples of the design of hdm2-specific constructs at the protein level (see 2.), where muV α / β murine variable α / β domain; muC α / β murine constant α / β domain; huC α / β human constant α / β domain; SP: murine wild-type signal peptide; TM: transmembrane; Li Linker with (GGGGS) ₃ motif and huζ: human CD3ζ chain with 3 consecutive ITAM motifs means. The numbers derived from the bacterial clones are given for the individual chains.

Fig. 5 shows the polylinker of the construct muvα-Li-muvβ-mucβ (see 2.3). The polylinker was ultimately inserted into the resulting construct via Kpn I and Dra III.

Fig. 6 shows the polylinker of the construct muvβ-Li-muvα-mucα (see 2.3). The polylinker was ultimately inserted into the resulting construct via Sca I and Bsg I.

Fig. 7 shows the fusion site of the chimeric double chain TZR in fusion to the human CD3ζ chain muvα-hucα-huCD3ζ (see 2.4). The extracytosolic cysteine dimerizing via a disulfide bridge is indicated with a position relative to the starting methionine.

Fig. 8 shows the fusion site of the chimeric double chain TZR in fusion to the human CD3ζ chain muvβ-hucβ-huCD3ζ (see 2.4). The extracytosolic cysteine, which dimerizes via a disulfide bridge, is given with a position relative to the starting methionine.

FIG. 9 shows the "fluorescence activated cell sorting" (FACS) analysis of the constructs described under 2.) after retroviral transduction. Examples of average transduction efficiencies for OKT3-activated PBL cultures are shown 6 days after transduction. These are in the range of 10-25% of the inserted CD3 ⁺ lymphocytes. A transfection / transduction approach with the empty retroviral vector pBullet served as a negative control.

Figs. 10 to 12 show peptide titrations (see 3.4.1), where T2 target cells exogenously with the hdm2-81-88 peptide in a concentration range of 1.10 ^-6 M to 1.10 ^-12 M were loaded. The murine T cell clone 3 hdm2-81-88 (synonym: P2) of the huCD8 × A2K ^b transgenic mouse acted as a reference. The ⁵¹ chromium release tests shown were carried out with transduced PBLs which were enriched for vβ6-directed magnetic cell sorting (Miltenyi-Biotec) to over 90% vβ6 ⁺ T cells. Fig. 10: as a negative control, a construct of a non-functional single-chain T-cell receptor (nfTCR), which indeed could be expressing comparatively well on the surface of T-cells and thereby netic also ma served cell sorting accumulative was, but proved no functionality in the CTL test. Fig. 11: Peptide-titration of the murine heterodimeric wild-type construct muTZR / 8-18. Fig. 12: Peptide titration of the chimeric heterodimeric construct huTZR / 10-29.

Figs. 13 to 15 show the detection of malignant transformed cell lines. The target cells selected were HLA-A2 ⁺ . The murine T cell clone 3 hdm2- 81-88 (synonym: P2) of the huCD8 × A2K ^b transgenic mouse acted as a reference. The ⁵¹ chromium release tests shown were carried out with transduced PBLs which were enriched for vβ6-directed magnetic cell sorting (Miltenyi-Biotec) to over 90% vβ6 ⁺ T cells. Fig. 13: a construct of a non-functional single chain T cell receptor (nfTCR) was used as a negative control, which could be expressed comparatively well on the surface of the T cells and thus also enriched by magnetic cell sorting was, but proved no functionality in the CTL test. Fig. 14: murine heterodimeric wild-type construct muTZR / 8-18. Fig. 15: chimeric heterodimeric construct huTZR / 10-29.

Examples

The genes of the T cell receptors αTZR and βTZR from a double-transgenic mouse huCD8 × A2K ^{b were} prepared, which mediate an HLA-A2.1 restricted and hdm2 protein / 81-88 peptide-specific T cell response , The genes obtained were introduced as wild type (WT) or modified constructs retrovirally into human peripheral blood lymphocytes (PBLs) and the HLA-restricted antigen recognition was checked functionally by CD8-mediated cytotoxic lysis of various cell lines in the ⁵¹ chromium release test.

1. Obtaining the genes coding for the α and β chains of the T cell receptors 1.1) Preparation of the cytoplasmic RNA

The cytoplasmic RNA was isolated in order to selectively separate the still unprocessed, intron-containing RNA, DNA fragments and ribosomal RNA, which are mainly located in the cell nucleus, and to achieve an enrichment of the desired "messenger" RNA. The protocol and materials largely correspond to the "Rneasy Mini Kit" from QIAGEN with minor modifications: for one preparation, an average of 50.10 ⁶ murine cells of the hdm2 / 81-88-specific T cell clone 3 were extracted from the culture dishes of a "24-well Plate "combined and washed in RPMI 1640 (Biowhittaker) serum-free. The cells were aliquoted under RNase-free conditions of 12.5.10 ⁶ cells per incubation tube and incubated in cell membrane lysis buffer (4 ° C.-cooled) for 20 min on ice:

Cell membrane lysis buffer

50 mM: Tris-HCl / pH 8.0
140 mM: NaCl
1.5 mM: MgCl ₂

0.5%: Nonidet P40 (Roche 1 332 473)
1000 units: RNase inhibitor / ml ZML-P (Roche 799 017)
1mM: DTT

For this buffer, like all other aqueous solutions that had to be prepared, was DEPC-treated and double-autoclaved deionized (MilliQ from Millipore) water used.

The further steps were carried out according to a process familiar to the person skilled in the art using the QIAGEN protocol under nose-free conditions at room temperature and are briefly described:
The mild solubilized and aliquoted cells were centrifuged at 300 g / 2 '(minutes) / 4 ° C and the supernatant was removed. 600 ul of a β-mercaptoethanol (MSH) -containing (1.45 mM) "RLT" buffer were added, mixed well and 430 ul of 96% ETOH were added with mixing. The solution per aliquot was placed on a silica minispin column and centrifuged for 15 "(seconds) at 10,000 rpm (revolutions per minute) in an Eppendorf table joint. Washing was carried out in 700 µl" RW1 "and in 500 µl of 77% ETOH-containing "RPE" buffer, then 500 µl of the identical "RPE" buffer was added and the column was dried for a maximum of 2 'at 14,000 rpm. The RNA was eluted with 54 µl of RNase-free water at 10,000 rpm for 2 '. The yields were 30 µg RNA per 12.5.10 ⁶ murine T cells. The RNA was removed to 6 µg / 10 µl at -20 ° C.

1.2) Amplification of the β-chain gene using coupled RT-PCR

The TZR-coding RNA had to be specifically rewritten and amplified in DNA, before it could be cloned. The RT-PCR was used methodically for this purpose in the eponymous kit "Titan One Tube RT-PCR system" from Roche (1888 382) im is implemented. The "one-step protocol" was chosen, in which the reverse tran is followed by the polymerase chain reaction without further intervention. Both Enzymes used were the AMV reverse transcriptase and a Ge mixes the Thermus Aquaticus / Pwo thermostable polymerase ("Expand ™ High fidelity" Enzyme mix), which were used in a universal reaction buffer. Since the Tran the rate of description of the TCR genes and, due to the variance of the TCR genes, the conservation of gene sequences encoding the variable domains is low, a "nested" closed PCR "(polymerase chain reaction) to determine the specificity and signal strength of the amplifi graced DNS to increase. The variability of the v gene segment, whose subfamily belongs a priori, could not be predicted with a degenerate primer (for_bTCR_v4; SeqID No. 9) tries to capture that of a base sequence for the first conserved amino acids coding for the signal peptide. An artificial one Gene segment that codes for a murine TZR signal peptide should originally be synthesized chemical oligonucleotides can be connected upstream to a direction of the synthesized in vivo To ensure protein in the lumen of the endoplasmic reticulum. It became a reverse hybridizing primers within the constant gene segment (rev_bTCR_c4; Se qID No. 10) was chosen to account for the additional variance of the two possible c gene segments (c β1 or cβ2) of the β chain, which have some base differences at their 3 'end go. The oligonucleotides of the "nested PCR" were in addition to the TZR complementarity additionally at its 5 'end sequences of the restriction sites for EcoRI or AscI. The forward-hybridizing primers (for_Eco_bTCR_v5; SeqID No. 11) of the nested "PCR" was congruent in the sequence of the v gene segment with the degenerate RT-PCR Primer, whereas the one complementary to the c gene segment hybridizes in the reverse direction primers (rev_Asc_bTCR_c6; SeqID No. 12) more than 120 bases relative to the reverse 1. The primer of the TZR reading frame was offset inwards so that it was exactly a "3'-  located nested PCR "acted. This increased the specificity of the PCR, since in 2 independent dependent PCR steps at 2 different locations a sequence-specific recognition event must have taken place. The complete c gene segment was reversed degenerate primers (rev_Ase_bTCR_c2; SeqID No. 14) to detect the two potenti possible cβ1 / cβ2 gene segments and a primer corresponding to the reading direction (for_Eco_bTCR_c2; SeqID No. 13) of the cβ gene segment via RT-PCR without subsequent "Nested PCR" recorded and cloned. In the overlap area of the two amplicons in the cβ- Area was a unique NcoI interface, by means of which the truncated vβ- / cβ- and the complete cβ gene segment was combined in a subsequent subcloning could become.

The pipetting scheme and the PCR protocol of the RT-PCR corresponded in principle to that of the The method described by Roche. The subsequent "nested PCR" was carried out analogously at Replacement of the RNase inhibitor and DTT components with autoclaved water. Instead of the 2 µl RNA (0.1-1 µg) was used 2 µl of the amplificate of the RT-PCR. The technical front Exposures to the PCR device made it possible to test different hybridisies simultaneously temperatures in the 2nd step of the classic PCR cycle in a temperature gradient from 35 ° C-55 ° C in intervals for 5 selected temperatures of 36 ° C, 39 ° C, 44 ° C, 52 ° C and 56 ° C. For the higher temperatures of 44-56 ° C singular bands of the ge desired length can be achieved, the intensity of which was increased by the 2nd PCR and from were sufficient to be inserted in vectors.

Sequencing 8 bacterial clones revealed some randomized base changes, the error rate of the Expand ™ enzyme which is comparable to other polymerases Mixes were based. The selected clones also all carried the sequence information for the Si signal peptide and a short region of the non-codogenic, 5'-untranslated region of less than 100 bases, so that it could be assumed that the v gene segment specific degenerate primers recognized a similar sequence upstream and the additional che sequence of the wild-type signal peptide.

1.3) Amplification of the α-chain gene using RACE-PCR

For the determination of the sequence of the α chain, RACE-PCR was the strategy that led to the goal: the degenerate oligonucleotide designed analogously to the approach described above only provided the codogenic sequence of a non-functional T cell receptor due to a reading frame shift in the CDR3 loop. The allelic exclusion is not valid to the same extent as for the β chain of the TZR. The RACE technology is established in the system of the same name from Roche (1 734 792) and must be supplemented with αTZR gene-specific oligonucleotides: for this purpose, the 5'-localized variance of the "5'-RACE-PCR" is used bypassed the codogenic sequence by generating a known, short adenine oligonucleotide sequence at the 3 'end of the reverse transcribed cDNA sequence by means of the terminal transferase, which subsequently acts as a recognition sequence for a 5' localized PCR primer in the reading direction , The reverse PCR primers lie within the invariant sequence that codes for the constant domain. For the reverse transcription, the primer rev_Asc_aTCR_c1 (SeqID No. 15) was used, which together with for_Eco_aTCR_c6 (SeqID No. 18) was also used for amplification via RT-PCR of the full-length cα segment. The concept of a consecutive 2-step "nested PCR" using the primer pairs "Oligo d (T) anchor primer" (component of the RACE-PCR kit) / rev_aTCR_c5 (SeqID No. 16) and "PCR anchor primer" ( Component of the RACE-PCR-Kit) / rev_Asc_aTCR_c4 (SeqID No. 17) was followed, so that here too the subcloning of the complete sequence, which codes for the constant domain, followed. The PCR primers had to be such that the singular NcoI site was in the overlap area of the amplified gene sections. The truncated PCR amplicon containing the vα gene segment was cloned via 5'-SalI and 3'-AscI in pNEB 193 (New England Biolabs), whereas the constant gene segment was inserted into pNEB193 via EcoRI and AscI. The amplificates were cloned in a classical manner (J. Sambrook et al., 1989, Molecular Cloning. A Labo ratory Manual 2 ^nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor) by means of cleaning with LMP gels and phenol / chloroform extraction before they were digested with restriction enzymes and the ends were separated using the "High Pure PCR Product Purification Kit" (Roche, 1 732 668).

The multi-step protocol is largely based on the commercial protocol under Zu using a programmable PCR device: reverse transcription, transferase Reaction and the subsequent 2-step "nested PCR" are in the "MasterCycler" PCR device the Eppendorf company. As part of the specific α chain The gradient technology came into amplification in the hybridization step for the temperatures 39 ° C, 44 ° C, 52 ° C and 56 ° C for use. Pfu polymerase (Stratagene) was used chooses because at this point it was considered the enzyme with the lowest rate of failure. The first PCR showed a weak band of the expected size of 0.9 kB in total  Temperature spectrum, the intensities of which are amplified by the subsequent PCR that could and also occurring "primer dimer" could be diluted. In the final PCR, the amplificate was shortened by 70 bp according to theo retic position of the offset hybridizing primers. One was found in the agarose gel Double band from 100 bp length difference depending on the presence of the functional and the non-functional α chain. The gangs were put together in the preparative Gel cut out to record both amplicons in the subsequent cloning. It 7 clones were found which code for a functional reading frame and are identical Possessed sequences.

1.4) Checking the subfamilies found

The existence of a vβ6 or vα10 gene segment in the murine hdm2 protein / 81-88 specific T cell clone 3 was verified in various ways:

• a) RT_PCR using a vβ6-specific primer or a vα10-specific primer. The RNA was previously purified from the murine hdm2-P2 specific T cell clone 3, by targeting the specific T cells via anti-huCD8 magnetically labeled antibodies the murine "feeder" cells were separated. An influenza Matrix protein (FluM1 58-66) specific T cell clone.
• b) FACS analysis ( Fig. 1) using a vβ6-specific FITC-labeled antibody (Pharmingen). Both the murine T cell line and the resulting clone 3 were checked. A FITC-labeled antibody was used as a positive control, which recognizes the β-chain portion independently of the subfamily.
• c) vβ6-specific antibody blockade of the murine T cell clone 3 in the cytotoxic ⁵¹ chromium release test ( FIG. 2).

2. Examples of the design of hdm2-specific constructs ( Fig. 3 [SEQ ID Nos. 38-45] and 4 [SEQ ID Nos. 1-8])

The retroviral vector pBullet, a functional derivative of pStitch (Weijtens et al., 1998) belongs to the so-called splice vectors, in which the transgene in the same sequence Context can be cloned relative to the splice acceptor and donor elements SA and SD must be like the env gene to ensure maximum expression. To do this, use ent speaking primer designed an NcoI interface within the start codon "ccATGg". This changed the 2nd amino acid of Lys for both wild-type chains αTZR and βTZR  Glu or Asn according to Asp. At the 3 'end of the reading frame, the singular BamHI Interface selected. The restriction enzymes and modifying enzymes were developed by New England Biolabs (NEB) related. The thermostable polymerase was mostly the native or cloned Pfu DNA polymerase (Pyrococcus furiosus, Stratagene) or Pfx DNA polymerase (Pyrococcus sp., Life Technologies)

2.1) Cloning of the Murine Wild-Type Genes αTZR (muvα-mucα; SeqID Nos. 1-38) and βTZR (muvβ-mucβ; Seq ID No. 2-39)

In the event that the NcoI interface did not exist internally (partially humanized chains), the gene segments could be amplified and cloned directly via primers which the Se contained sequences for NcoI (for_NcoI_aTCR_4, SeqID No. 19; for_NcoI_bTCR_4, SeqID No. 21).

Because the NcoI interface for the wild-type chains in the gene segment for the constant domains occurred, the codogenic reading frames via oligonucleotides (for_NcoI_aTCR_5b, SeqID No. 20; for_NcoI_bTCR_5b, SeqID No. 22) amplified, the 5'-terminal ver shortened, blunt-ended NcoI interfaces had, so that these after blunt-ended filling len NcoI-cut vector DNA using the T4 DNA polymerase is too full with the latter permanent NcoI interfaces complemented. rev_aTCR_BamHI (SeqID No. 23) and rev_bTCR_BamHI_1 (SeqID No. 24) were used as a final reverse primer for all murine double chain and single chain constructs are used.

2.2) Generation of the chimeric, partially humanized TZR genes hu αTZR (muvα- hucα; SeqID No. 40) and hu βTZR (muvβ-hucβ; SeqID No. 41)

Two criteria should be met for a functional chimerization of the murine TZRs:

• a) the murine constant domains should thus against the corresponding human Do män be exchanged so that these domains in their immunoglobulin-like Convolution structure can be fully grasped. The complete variables were also To capture domains in order not to impair the almost car in this domain causing boxyterminal lying CDR3 loop.
• b) The fusion had to take place in such a way that the transition did not generate any neoantigen that could lead to an unwanted immune response.

In the case of b), both chains were generally more human than muri the following properties: in a range of up to 8 amino acids behind The last consensus motif "G × G" that completes the CDR3 loop is the positions relatively well preserved. This area is characterized by a highly conserved sequence of up to 11 amino acids completed. After that, the murine begin from the human sequences zen, highly conserved for each species. The central area lies in the stretched loop that connects the variable domain with the constant one (see criterion a)). Ideally, the chimerization was within a highly conserved amino acid (AS) - Triplet sequence.

In the case of the αTZR, the highly conserved triplet sequence consisted of "QNP" (base 400-408 relative to the start codon of the murine αTZR). Position 407-415 was followed by a unique AlwNI interface that could be used for the fusion: the 3'-offset position of the AlwNI meant that instead of the aspartate conserved in human sequences, the glutamate conserved in murine sequences was present. This conservative amino acid exchange was considered to be less immunogenic and therefore tolerable. The chimeric, highly conserved region was I ¹³³ -QNPEPAVYQLR ¹⁴⁴ , in which R ^{144 represents} the first human-specific amino acid. I ¹³³ is at the ninth position behind the consensus motif "G × G" that completes the CDR3 loop.

The cloning strategy envisaged that the LMP-agarose-isolated EcoRI / AlwNI-cut to ligate vα segments with the AlwNI / BamHI digested cα-PCR amplificates, the ge mixed ligation products with EcoRI and BamHI and the prepared Li tion product of the potentially correct fragment length in the expression vector pcDNA3.1 (-) (Invitrogen) to clone. An asymmetric test digest resulted in the clones with the ge desired sequence of the ligation educts.

The human constant gene segments cα and cβ were by classic RT-PCR as they implemented as part of the Roche kit described above, followed by "ne sted "PCR isolated from the human leukemia cell line Jurkat.

In the case of the human cα gene segment, the AlwNI interface (T to A; see table below) had to be generated with the aid of mutagenic PCR primers at 2 positions and the compatibility with the murine sequence had to be established (c according to a ; see table below), since the middle 3 positions of the AlwNI interface may be degenerate:

The AlwNI interface naturally occurring in the murine sequence is shown in bold, the 3'-offset cutting position is underlined. The degenerate triplet is screeched ben. The required base changes for the human "primer" to maintain a Mouse sequence-compatible AlwNI interfaces are shown in bold. The 5'-initial base of the mutagenic primer is italic. The resulting chimeric AS sequence is shown in bold (see Text); the highly preserved AS triplet is also underlined.

The cα fragment was amplified by means of a "nested" PCR. The mutations were placed in the "nested" primer (for_hucaTCR_AlwNI_3; SeqID No. 27), whereas the RT-PCR primer (for_hucaTCR_2; SeqID No. 25) or the human wild-type sequence corresponded. The reverse "nested" primer rev_hucaTCR_BamHI_1 (SeqID No. 28) had this probably the stop codon as well as the clonable BamHI interface, whereas the reverse RT-PCR primer (rev_hucaTCR_2; SeqID No. 26) the 3'-lying non-codogenic Be recognized richly.

In the case of the βTZR, the "nested" PCR principle was also coupled to a blunt-ended Li application (SeqID nos. 29-32). The highly conserved triplet sequence persisted from "E-D-L" (base 400-408 relative to the start codon of the murine βTZR). Congruent be found a unique BstYI interface (base 402-407). A primer specific for the human constant domain gene segment (for_hucbTCR_4; SeqID No. 31) should have its 5'-terminal end, which has a phos "T" phorylated the last base of the potentially palindromic recognition sequence of BstYI that is digested with BstYI (produces overhanging 5 'ends that bind to the T4 DNA Complement the polymerase with the addition of dNTPs and do not digest) and bluntly filled 3 'end of the murine variable domain gene segment com implement so that the correct reading frame context was preserved. The vβ fragment was trimmed with EcoRI and isolated from an LMP gel which became cβ-PCR amplificate as an additionally enzymatically phosphorylated (T4 polynucleotide kinase, NEB) blunt ended  BamHI fragment used in the ligation. The mixed ligation products were trimmed with EcoRI / BamHI and the fragment with the potentially correct one Fragment length cloned in pcDNA3.1 (-). The clones also showed an asymmetrical test digest the desired sequence and orientation of the ligation educts.

The BstYI cleavage naturally occurring in the murine sequence is shown in bold, the 5 'offset cutting position, which generates 5' overhangs, is underlined. The 5 'initial and phosphorylated base "T" of the human "nested" primer, which is the last base of BstYI added to the chimeric construct is italic. The resulting chimeric AS sequence is shown in bold posed (see text); the highly preserved AS triplet is also underlined.

The chimeric, highly conserved region was E ¹³³ -DLN ¹³⁶ , in which N ^{136 represents} the first human-specific amino acid. E ¹³³ is in the seventh position behind the conserved "G × G" motif that closes the CDR3 loop.

2.3) Generation of the single-chain TZRs muvα-Li-muvβ-muccβ (SeqID No. 42) and muvβ-Li-muvα-muccα (SeqID No. 43)

Single chain TCRs consist of the fusion of the variable domains vα with vβ and an attached constant domain cα or cβ (Eshar et al., 1993). The advantage of such constructs lies in the defined 1: 1 stoichiometry of the two variable domains in one construct and the defined reactivity due to the heterogeneous pairing of endogenous with exogenous double chain TZRs that can be excluded. The variable domains were connected via a flexible polylinker of a redundant (GGGGS) ₃ motif. The ends of the synthetic oligonucleotide harbored unique interfaces with which the carboxy-terminal end of one variable domain, located behind the essential CDR3 loop, could be linked to the beginning of the other variable domain, behind the sequence encoding the signal peptide. Conceptually, five criteria were observed:

• a) the polylinker had to be long enough to connect the ends of the variable ends. For this purpose, murine TZR crystal structures with an MHC-H-2K ^b specificity were consulted.
• b) the polylinker had to connect the ends in such a way that on the one hand the specificity the CDR3 loop was not impaired, on the other hand the signal peptide downstream variable domain was sufficiently eliminated while maintaining its functional Structure. To accomplish this, amino acid sequences were added to the polylinker of the respective variable domain for the case in which the singular Interfaces were not ideal.
• c) The polylinker had to be flexible on the one hand, which with the help of a sterically favorable Lining up of glycine residues should be accomplished to the other side be sufficiently soluble to avoid aggregate formation and precipitation: the hydroxyl Group of the serine residue forms hydrogen bonds with solvating water Molecules and forms local dipoles.
• d) In order not to impair the transcription in the correct reading frame, the kodo gene triplets for the glycine residues varied ("GGC" and "GGA").
• e) The polylinker must have the same internal singular interfaces as the wild type sequences to digest the intermediates in a 2-step cloning process cut and the almost complete wild type sequences over this singular cut add 3'-terminal.

Under these conditions, it was possible to allow two orientations for the variable domain:
In the case of the construct muvα-Li-muvβ-mucβ (SeqID No. 42; Fig. 5), the unique interface KpnI behind the last consensus motif "G × G" of the variable vα domain and DraIII as the sequence encoding the signal peptide final restriction site selected in the vβ domain. In a first step, the hybridized, double-stranded oligonucleotide was cut on both sides with KpnI and cloned between the vα-coding and cα-coding sequence of the wild-type αTZR. The chimeric intermediate was then cut open with DraIII / BamHI and the βTZR coding sequence, which had lost its signal peptide coding sequence via a DraIII / BamHI digest, was cloned into it. The first glycine of the polylinker was at position 6 relative to the amino-terminal gly of the "G × G" consensus motif in vα, the last serine of the polylinker, connected by arginine due to an intermediate blunt-ended ligation, before position Asp ^{22 of} the vβ- Domain.

In the case of the construct muvβ-Li-muvα-mucα (SeqID No. 43; Fig. 6), the singular interface ScaI was behind the last consensus motif "G × G" of the variable vβ domain and BsgI as the sequence encoding the signal peptide final restriction site in the vα domain selected. In a first step, the hybridized, double-stranded oligonucleotide was cut on both sides with ScaI and cloned between the vβ-coding and cβ-coding sequence of the wild-type βTZR. The chimeric intermediate was then cut open with BsgI / BamHI and the αTZR coding sequence, which had lost its signal peptide coding sequence via a BsgI / BamHI digest, was cloned into it. The first glycine of the polylinker was at position 6 relative to the amino-terminal gly of the "G × G" consensus motif in vβ, the last serine of the polylinker before position Val ^{24 of} the vα domain.

2.4) Generation of the double chain TZRs in fusion to the human CD3ζ chain muvα-hucα-huCD3ζ (SeqID No. 44; Fig. 7) and muvβ-hucβ-huCD3ζ (SeqID No. 45; Fig. 8)

The signal transduction of the double chain TZRs should be optimized. For this purpose, the respective double chain α / βTZRs were fused to the almost complete CD3ζ subunit of the CD3 complex. According to various publications (Eshar et al., 1994), this fusion achieves an efficient and CD3-independent coupling of the TZR to the effectors Lck ⁵⁶ and ZAP-70 located upstream and downstream in the cascade.

A modified ligation-PCR-based technique was established, with the help of which one could generate any fusions for the production of chimeric proteins independently of singular interfaces and the specificity of the PCR amplificates obtained was significantly increased compared to a conventional ligation-PCR. The genetic fusion is defined via a reverse, chimeric primer (rev_huca-hu_tm_Zeta, SeqID No. 36, FIG. 7; rev_hucb-hu_tm_Zeta; SeqID No. 37, FIG. 8), which is linked to a forward primer (T7_for; SeqID No. 33) produces the "mega-primer" as a PCR amplificate: in a first, conventional ligation-PCR step, a "mega-primer" is generated for the 5'-lying fusion partner, which also serves as a short sequence for the 3 ' -lying fusion partner carries specific sequence. In order to significantly increase the specificity of the PCR signals, primers are used in the first PCR that hybridize outside the codogenic, chimeric amplifi cate to the "templates"(T7_for;T7_rev; SeqID No. 33, 34), and then to use nested primers in the second PCR. In this, the mega-primer together with a reverse "nested" primer (rev_hZeta BamHI_4; SeqID No. 35) hybridizing to the 3'-lying fusion partner provides an intermediate product which is forward with a third forward, "nested" at the 5'-end -Primer (for_aTCR_NcoI_5b; for_bTCR_NcoI_5b; SeqID No. 20, 22) delivers a PCR amplified product trimmed at the ends.

The fusion point was determined on the disulfide-bridging cysteines: this caused the TZRs to lose their cytoplasmic sequence, the transmembrane and a short range of extracytoplasmic amino acids to the dimerizing cysteine, the hu mane chain also lost a short extraycytoplasmic section, but should ex encode including their cystine-forming cysteine for the transmembrane and cytosolic region including the three bivalent "ITAM" motifs. The reverse chimeric primer codes for the 3'-terminally located 21 bases of the respective partially humanized TZR and for the 5'-terminally lying 21 bases of the human CD3ζ chain ( FIG. 7). The chimeric constructs are humanized from the constant domain.

3.) Transduction of Human Peripheral Blood Lymphocytes (PBLs)

A functional derivative of the pStitch system (Weijtens et al., 1999) was used for transduction of human peripheral T lymphocytes. The retroviral genes required for packaging are encoded via individual plasmids by cotransfection of the 293T packaging cell line (Soneoka et al., 1995): pHit60 encodes the gag-pol structure and polymerase genes from the Moloney murine leukemia virus (MoMuLV), pColt-Galv for the env-envelope protein of the "gibbon ape leukemia virus", which is able to bind to the Na ⁺ - / phosphate synporter pit of human cells and thus to transduce the latter. The chimeric virus particles thus have an amphotropic pseudotype and can transduce various mammalian cells, except mouse.

3.1) Transfection of the 293T packaging cell line

The isolated bacterial clones of the T cell receptor genes cloned into the pStitch derivative were purified via plasmid preparations which promise removal of residual endotoxins (Qiagen, product 12362) and adjusted to 1 µg / µl. The DNA was transiently introduced into the packaging cell line 293T via the calcium phosphate precipitation (GiboBRL-Life Technologies, product 18306-019). Here, up to 80 µg DNA are used in the context of the WT or modified double chain T cell receptors αTZR and βTZR:
20 µg αTZR construct
20 µg βTZR construct
20 µg pColt galv
20 µg pHit 60

In the case of single chain TZRs, 60 µg DNA is used. 293T was cultivated in a modified DMEM medium (DMEM / H):
DMEM, 4.5% glucose (organic Whittaker)
10% heat-inactivated FCS
2 mM glutamine
1 × penicillin / streptomycin
1 × non-essential amino acids
25mM HEPES

On the day before the transfection, the 293T cells were seeded onto 0.9.10 ⁶ cells per T25 bottle and transfection batch in 5 ml DMEM / H. 4 h before transfection, the medium was replaced with fresh DMEM-H (3 ml) warmed to room temperature (RT). The transfection was carried out according to the regulation of the commercial protocol (Life Technologies). 1 ml of the transfection batch was pipetted into the respective bottle with careful dropping. The DNA-Ca ₃ (PO ₄ ) ₂ precipitate should be deposited on the adherent cells in finely divided form.

The following morning the medium was against fresh DMEM / H warmed to RT replaced. The cultivation with the activated PBLs took place 6 hours later.

3.2) Transduction of the activated PBLs 3.2.1) Activation of peripheral blood lymphocytes (PBLs)

3 days before the co-cultivation was started, fikollized PBLs at 2.10 ⁶ cells / ml in huRPMI-P were each sown in 2 ml of a 24well plate (cell tissue-treated surfaces). The activation took place via the cross-linking antibody OKT-3 (Orthoclone-Diagnostics) at 20 ng / ml.
huRPMI-P:
RPMI 1640 (2 mM glutamine) without phosphate (Life Tec., 11877-032)
10% human, heat-inactivated AB serum (HLA-A2.1 seropositive)
25mM HEPES
1 × penicillin / streptomycin (Life Tec.)

The plates were incubated in an incubator at 37 ° C and 5% CO ₂ .

3.2.2) Cultivation

For cocultivation, the activated PBLs from the respective wells of a 24well plate were combined and counted. Adherent monocytes were discarded. The cells are centrifuged off (1500 rpm, 5 min, RT) and taken up in a concentration of 2.5.10 ⁶ cells / ml in fresh huRPMI-P and returned to the incubator. The medium was previously adjusted to 400 U / ml IL-2 (Chiron) and 5 µg / ml polybrene (Sigma).

Each transfection batch was trypsinized in succession 6 h after changing the medium: for this purpose, each T25 was washed with 3 ml HBSS (Life Technologies), incubated with 1 ml trypsin-EDTA (Life Technologies) for a maximum of 5 minutes, the dissolved cells were taken up quantitatively and placed in 4 ml huRPMI-P (RT) added dropwise with stirring. The 293T cells are irradiated with 2500 rad. These are centrifuged off (1500 rpm, 5 min, RT) and resuspended in 4 ml of fresh, adjusted huRPMI-P, supplemented with 400 U / ml IL-2 and 5 µg / ml polybrene. 1 ml of the adjusted PBLs was added to the mixture and the mixture (0.5.10 ⁶ PBLs / ml) was incubated for three days in an incubator (37 ° C., 5% CO ₂ ).

On day 3 after cocultivation, the suspended PBLs were removed and resuspended in fresh huRPMI-P medium supplemented with IL-2 (400 U / ml) at 0.5.10 ⁶ cells / ml. 3 days later there was another split into fresh medium. In these 7 days the maximum expansion took place until the transition to T75 bottles. These cells could be used directly in an immunological staining (FACS analysis; Chapter 3.3, Fig. 9) or in a classic ⁵¹ chromium release test (Chapter 3.4, Fig. 10-15).

3.3) Examples of FACS analysis

The constructs described under 2.) were analyzed after retroviral transduction in "fluo rescence activated cell sorting" (FACS). For this purpose, 0.25.10 ⁶ cells were saturated with fluorophore-labeled antibodies: the heterologously expressed β chain was detected with anti-vβ6-FITC (BD); all of the T cells via the marker anti-CD3-PC5 (Coulter-Beckman) ( FIG. 9).

A sample transduced with the empty pStitch derivative served as a negative control. The Wild-type muvα-mucα / muvβ-mucβ (2.1) and the chimeric constructs muvα-hucα / muvβ- hucβ (2.2) achieved transduction efficiencies in the range of 10-25% 6 days after Kokulti vation. Expression could be reproduced in several donors of HLA-A2 positive T cells be decorated. The cytosolic expressed green fluorescent protein (eGFP, cloned from eGFP-N1 from Clontech) served as an external standard and showed that the ver equally weak transduction efficiency as an expression of the percentage proof consequently transduced T cells is a methodological problem and not an intrinsic problem is the TZR transgene.

3.3.1) Enrichment of vβ6-positive T cells via magnetic sorting

In order to enrich the successfully transduced T cells, they were sorted in the magnetic field using magnetically marked vβ6 antibodies (according to the Miltenyi-Biotec regulation). For this purpose, a magnetically labeled antibody was used which recognizes the rat IgG portion of the vβ6-specific antibodies (Miltenyi-Biotec "Goat anti-rat IgG MicroBeads", product 485-01). Vβ6-positive CD3 ⁺ T cells could be enriched by more than 90%.

3.4) Cytolytic activity of the transduced T cells

The transduced T cells were tested in a classical ⁵¹ chromium release test for their cytotoxic specificity. In this system, target cells were radioactively labeled by incorporating ⁵¹ chromium. If the retrovirally modified effector cells recognized the target cell in a peptide-specific manner, the latter were driven into apoptosis by the effector functions of the T cell and killed by lysis. The extent of the released chromium nuclide makes a statement about the effectiveness of cell recognition and lysis. The effectiveness was tested over a wide range of the ratio of effector cells to target cells (E: T). The murine hdm2-81-88 peptide-specific T cell clone 3, from which the T cell receptor genes were isolated, served as reference. The target cells were:
T2: human TAP-deficient cell line, which was to be loaded exogenously with any peptide. The specific peptide was hdm2-81-88, an irrelevant control peptide was derived from the Influen za matrix protein FluM1.
Saos-2/6: Transfectant of the human Saos-2 cell line that expresses hdm2 and processes endogenously.
Saos2 / 143: Transfectant of the human Saos-2 cell line, which carries the p53 mutant Val ¹⁴³ Ala. The p53 mutant positively regulates the expression of hdm2.
JY: EBV-transformed LCL-B cell line
UocB11, EU-3: pre-B cell leukemia
IM-9: Plasmocytoma

3.4.1) Peptide Titration ( Fig. 10-12)

The cell line T2 was loaded exogenously with the hdm2-81-88 peptide in a concentration series from 1.10 ^-6 M to 1.10 ^-12 M. A peptide-specific and dose-dependent cytotoxic T cell response was observed for both muvα-mucα / muvβ-mucβ (2.1) and muvα-hucα / muvβ-hucβ (2.2). For the two constructs, the half-maximal lyses in the subnanomolar range were at E: T = 20: 1. A non-functional single-chain T cell receptor functioned as a negative control. It was expressed and enrichable according to 3.3.1), but showed no lysis activity. The murine hdm2-81-88 peptide-specific T cell clone 3 served as a positive control.

3.4.2) Detection of malignant transformed cell lines ( Fig. 13-15)

The cell lines described above could be lysed specifically for peptides. Some of the pre-B In the case of WT chains, cell leukaemias could be lysed 100% with high E: T's. As in the case of peptide titration, the wild-type chains (2.1) showed higher lysis Efficiency compared to the partially humanized, chimeric chains (2.2). As a negative The control was a non-functional single-chain T cell receptor, which although expri was lubricated and enrichable according to 3.3.1), but showed no lysis activity. As a positive The murine hdm2-81-88 peptide-specific T cell clone 3 served as a control.  

SEQUENCE LISTING

Claims (26)

1. Polypeptide of a murine mediating an hdm2 protein-specific T cell response α / β T cell receptor according to SEQ ID No. 1 or SEQ ID No. 2 or functional variants th or parts thereof or nucleic acids encoding it, functional variants or Parts of it. 2. Fusion protein comprising a polypeptide according to claim 1 or functional variants or parts thereof or nucleic acids encoding it, functional variants or parts from that. 3. Fusion protein according to claim 2, characterized in that it is the ζ region of CD3 and / or CD8, CD16 or parts thereof, in particular the ζ region of human CD3 and / or CD8, CD16 or parts thereof. 4. Fusion protein according to claim 2 or 3, further comprising a flexible linker, in particular a linker of the amino acid sequence (GGGGS) ₃ . 5. Fusion protein according to claim 2, characterized in that it is a chimeric, at least partially humanized fusion protein, in particular according to SEQ ID no. 3 or SEQ ID No. 4. 6. Fusion protein according to one of claims 2 to 5, characterized in that it is is a single chain T cell receptor, in particular according to SEQ ID No. 5 or SEQ ID No. 6. 7. Fusion protein according to one of claims 2 to 4, characterized in that it is is a double chain T cell receptor, in particular according to SEQ ID No. 7 or SEQ ID No. 8. 8. α- or β-chain of a T cell receptor that contains the antigen recognition sequence for the Amino acids 81-88 of the hdm2 protein or a complex of hdm281-88 and HLA- A2 specific antibody.   9. Polypeptide according to one of claims 1 to 8, characterized in that the polypep tid has been produced synthetically. 10. Nucleic acid according to claim 1 or 2, characterized in that it is a DNA or RNA, preferably a DNA, in particular a double-stranded DNA with a length of at least 8 nucleotides, preferably with at least 18 nucleotides, in particular re with at least 24 nucleotides. 11. Nucleic acid according to one of claims 1, 2 or 10, characterized in that the Sequence of the nucleic acid has at least one intron and / or a polyA sequence. 12. Nucleic acid according to one of claims 1, 2, 10 or 11 in the form of its complementary "Antisense" sequence. 13. Nucleic acid according to one of claims 1, 2 or 10 to 12, characterized in that the nucleic acid has been synthesized. 14. Vector, preferably in the form of a plasmid, shuttle vector, phagemids, cosmids, Ex pressure vector, retroviral vector, adenoviral vector or particle and / or gene therapeutic vector, comprising a nucleic acid according to one of the An sayings 1, 2 or 10 to 13. 15. Host cell transfected with a vector or infected or transduced with a parti Kel according to claim 14. 16. Host cell according to claim 15, characterized in that it is a T cell or is a T cell progenitor cell or a stem cell. 17. Host cell according to claim 15 or 16, characterized in that on its surface a polypeptide or fusion protein according to any one of claims 1 to 9 is expressed. 18. A method for the identification of hdm2 protein-specific antigens, thereby records that hdm2 presenting tumor cells or fractions thereof with a host cell according to claim 17 are brought together under conditions in which the  Tumor cells or fractions thereof can only be lysed if the tumor Protein-specific antigen for which the expressed polypeptide or Fusi protein is specific. 19. A method for producing an against a polypeptide, fusion protein or a nucleotide acid according to one of claims 1 to 13 directed antibody, preferably one polyclonal or monoclonal antibody for diagnosis, treatment and / or over monitoring the treatment of diseases and / or associated with hdm2 protein Identification of pharmacologically active substances, characterized in that an antibody producing organism with a polypeptide or functional equi valents thereof or parts thereof with at least 6 amino acids, preferably with at least at least 8 amino acids, in particular with at least 12 amino acids according to one of the types say immunized 1 to 9 or vaccinated with these coding nucleic acids. 20. Antibody, prepared according to claim 19, characterized in that it is against a Polypeptide according to one of claims 1 to 9 is directed. 21. Recombinant antibody, characterized in that it contains the antigen Recognition sequence of the α or β chain for amino acids 81-88 of the protein hdm2 or a complex of hdm281-88 and HLA-A2 specific T cell Includes receptor. 22. Process for the manufacture of a medicament for the treatment of as with hdm2 protein associated diseases, characterized in that at least one nucleic acid, at least one polypeptide, at least one host cell or at least one antibody according to one of the preceding claims together with suitable additive and auxiliary fen is combined. 23. Medicines for the treatment of diseases associated with hdm2 protein, thereby characterized in that it has at least one nucleic acid, at least one polypeptide least a host cell or at least one antibody according to one of the aforementioned An sayings, optionally together with suitable additives and auxiliaries.   24. Use of a medicament according to claim 23 for the treatment of with hdm2- Protein Associated Diseases. 25. Method of making a test for finding functional interactors in Zu connection with diseases associated with hdm2 protein, characterized in that at least one nucleic acid, at least one polypeptide or at least one antibody according to one of the preceding claims together with suitable additive and auxiliary fen is combined. 26. Test to identify functional interactors related to hdm2 protein associated diseases, characterized in that it contains at least one nucleic acid, at least one polypeptide or at least one antibody according to one of the aforementioned claims, possibly together with suitable additives and adjuvants holds.