JP2004535778A - hdm2 protein-specific murine α / β T-cell receptor polypeptides, nucleic acids encoding them and uses thereof - Google Patents

Abstract

The object of the present invention is to make available murine genes of T-cell receptors α-TCR and β-TCR directed to hdm2 protein.
The present invention provides a polypeptide from a murine α / β T-cell receptor that mediates an hdm2 protein-specific T-cell receptor, or a functional variant or portion thereof, a nucleic acid encoding the same, A functional variant or part thereof. The effect of the present invention is that hdm2 protein-expressing cells are recognized by T-cells that induce the gene, cytokines are expressed, and lysis and / or apoptosis of tumor or leukemia cells is induced.

Description

【Technical field】
[0001]
The present invention provides murine α / β T-cell receptors that mediate the hdm2 protein-specific T-cell response, nucleic acids encoding them, and their use in the treatment, diagnosis and / or prevention of diseases associated with the hdm2 protein. About use.
[Background Art]
[0002]
Antigen recognition by T lymphocytes (TCL) is very important in the purification and regulation of an effective immune response. A characteristic T-cell line marker is the T-cell antigen receptor (TCR). There are two distinct types of TCRs: one is a heterodimer of two disulfide-linked polypeptides (α and β); the other has been found to be structurally similar, Consists of γ- and δ-polypeptides. Both receptors associate with a set of five polypeptides, the CD3 complex, and thus together form the T-cell receptor complex (TCR-CD3 complex). The α / β-TCR is most functionally important. Because it is expressed in 95% of all T-cells and mediates a major immune response.
[0003]
α / β T-cells can be classified into two different overlapping populations: they carry the CD4 marker and are mainly responsible for the immune response (T_H) Carrying the subgroup and the CD8 marker, which are essentially cytotoxic (T_C) Is a subgroup. CD8⁺  T-cells recognize antigen together with MHC class I molecules. Such an antigen may be, inter alia, a tumor-specific or tumor-associated peptide antigen. After recognition of the peptide antigen, the cells of interest are lysed by T-cells that lyse target cells and / or induce apoptosis of these target cells, or release cytokines (eg, IL-2, IFN-γ). Destroyed.
[0004]
Of the tumor-associated peptide antigens (TAAs) presented on the surface of tumor cells in the sense of MHC class I molecules, "universal" TAAs are of particular interest. These TAAs are weakly expressed in normal cells and are mainly derived from cellular proteins that are overexpressed in tumor cells. These proteins include, inter alia, the human homologues of the “MOUSE DOUBLE MINUTE 2” proto-antigen (mdm2), “human mdm2” or, concisely, the “hdm2” proto-oncoprotein (Roth et al., 1998), which comprises numerous Is overexpressed in hematologic hyperplasia (malignant hematological systemic disease) as well as in AML, ALL and CLL (Zhou et al., 2000).
[0005]
The oligopeptide of the hdm2 protein can be displayed on the cell-surface in the context of MHC class I molecules and represents an attractive target structure for CD8-positive T-cells. The extent of the T-cell response in this case proceeds in a well-defined dynamic window (Kersh et al., 1998). The complex of peptide MHC and TCR-CD3 multimerizes, resulting in effective signal transduction, precise stoichiometry and a range of oligomerizations still discussed.
[0006]
A requirement in the development of immunotherapeutic methods for the treatment of malignant swelling is the identification of oncoprotein-specific T-cell receptors. Using such T-cell receptors, under certain conditions, T-cells are generally designated as antigen-specific, particularly tumor-specific, with the aim of effecting the alleviation and eradication of certain tumors. Reactivity can be provided. Described methods for the identification of high-affinity peptide-specific TCRs include the "phage" or "yeast-display" method (Holler et al., 1999) or the tetramer-dependent "TCR display" method (Kessels et al., 2000). )including.
[0007]
From Theobald et al. ("Targeted p53 as a General Tumor Antigen", PNAS USA 92, pp. 1199-11997, 1995), p53-specific after injection of peptides from the sequence of p53. The preparation of cytotoxic T-cells is known. These T-cell lines allowed the selective subsequent lysis of human tumor cells. Isolation of genes for specific T-cell receptors on lysed T-cells directed against p53 has not been described. WO 97/32603 describes in general the preparation of recombinant T lymphocytes expressing a specific T-cell receptor directed at tumor tissue. In this process, HLA-transgenic mice (in this case, HLA-A2) are immunized with a tumor-associated antigen, thus producing cytotoxic T lymphocytes that express specific T-cell receptors on their surface. Bring. Peptides of various genes such as Her-2 / neu, Ras, p53, tyrosinase, MART, gp100, MAGE, BAGE and MUC-1 have been described as tumor-associated antigens. A nucleotide sequence comprising at least one variable region of the α- and β-chains of the corresponding non-human T-cell receptor is isolated from Her-2 / neu-specific T lymphocytes and modified by molecular biology Various genes (particularly "humanized") are used in the form of TCR constructs. Thus, WO97 / 32603 is a fusion protein of the ζ-region of CD3 or CD8 or CD16 and the variable region of TCR, and the amino acid sequence (GGGGS)₃The use of a flexible linker is described.
[0008]
Clay et al. ("Effective introduction of tumor antigen-reactive TCL into human peripheral blood lymphocytes confers anti-tumor reactivity"; J. Immunology, 1999, 163: 507-513) describes MART-1. The isolation of the genes for the α-TCR and β-TCR chains of the specific TCR and their expression in human peripheral blood lymphocytes (PBL) are described. The TCR is directed against amino acids 25-35 of MART-1. In addition, lysis of various metastatic melanoma cell lines and retroviral vector systems using IRES elements has been described.
[0009]
("Redirected perforin-dependent lysis of colon carcinoma by CTR engineered ex vivo", J. Immunol., 2000, 164: 3705-3712) describes scFv anti-CEA receptor. An immunotherapeutic method for colon carcinoma using transduced CTL, perforin and γ-IFN is described. The chimeric specific receptor construct is introduced into primary mouse T-lymphocytes by a retroviral vector. Mice already inoculated with a colon carcinoma cell line were injected with these cells.
[0010]
Weijtens et al. (“The retrovirus vector system“ STITCH ”combined with an optimized single-chain antibody chimeric receptor gene structure allows for efficient gene transfer and expression in human T-lymphocytes”, Gene Therapy 1998). )5, 1995-1203) and Willemsen et al. ("Granulation of primary human T lymphocytes with cancer-specific single- and double-stranded TCRs", Gene Therapy (2000)).7, 1369-1377) describe a retroviral vector system for the transfer of genes into activated T-lymphocytes. This system is used to effect expression of an antibody-based chimeric receptor on the membrane of a T-cell. Thus, these T-cells can be used against specific cancer cells. The activity of the retroviral vector system is described by the specific recognition and cytolysis of renal cell carcinoma. Similarly, Eshar et al. (“Specific activation and targeting of cytotoxic lymphocytes via a chimeric single chain consisting of the antibody-binding domain of immunoglobulins and the γ or ζ subunits and the T-cell receptor”, NAS USA, 90, pp. 720-724, 1993) have the constant region of the preparation of tumor-specific lymphocytes and the T-cell receptor ("single-chain TCR" (scTCR)). Describes its use in immunotherapy based on chimeras containing the variable region of an antibody (scVS). These chimeric genes can be expressed on the surface of cytolytic T-cell hybridomas, which resulted in secretion of interleukin-2 after contact with the antigen.
[0011]
TCRs and their use specifically directed against hdm2, however, have not been described or suggested by the aforementioned publications and other references.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0012]
Accordingly, the present invention is based on the object of making available the murine genes of the T-cell receptors α-TCR and β-TCR directed to the hdm2 protein. These allow the hdm2 protein-expressing cells of CD8-positive T-cells to be recognized, cytokines to be secreted, and T-cell-induced lysis and / or aposomesis of tumor or leukemia cells to occur. .
[Means for Solving the Problems]
[0013]
According to the present invention, the object is to provide a polypeptide of the murine α / β T-cell receptor that mediates a hdm2 protein-specific T-cell response, or a functional variant thereof, according to SEQ ID NO: 1 and SEQ ID NO: 2. This is achieved by a part, or a nucleic acid encoding it, a functional variant or part thereof.
BEST MODE FOR CARRYING OUT THE INVENTION
[0014]
Double-A2.1 transgenic mouse [huCD8α / β × A2.1 / K in C57BL / 6 gene background^b]_F1Has isolated the gene for the murine α / β T-cell receptor that mediates the hdm2 protein-specific T-cell response, which is HLA-A2.1-restricted and comprises amino acids 81-88 of the hdm2 protein. Mediates specific T-cell responses directed at the peptide. The polypeptide of this TCR was not previously known. The gene was inserted by retrovirus into human peripheral blood lymphocytes (PBL) as a wild-type (WT) or modified construct, and HLA-restricted antigen recognition was⁵¹Function was checked by CD-8-mediated cytotoxic lysis of various cell lines in a chromium-release test. The gene for the hdm2-specific TCR according to the invention can be used, for example, for diagnosis, such as adaptation, and / or treatment, eg, for modulation of diseases associated with the hdm2 protein, such that a completely new therapeutic approach comes from the invention. It is not a suitable target previously known for the identification of pharmacologically active substances.
[0015]
A further aspect of the invention relates to a polypeptide according to the invention, a functional variant or part thereof, a nucleic acid encoding it, a fusion protein comprising the functional variant or part thereof.
[0016]
The fusion protein may be characterized in that it comprises a ３-region of CD3 or CD8 or CD16 or a part thereof, in particular a ＣＤ-region of human CD3 or CD8 or CD16 or a part thereof. Fusion proteins according to the invention are described in Flexible Linker (Whitlow et al., "Improved Linkers for Single-Stranded Fvs with Reduced Aggregation and Enhanced Proteolytic Stability", Prot. Engin. 6 (8) pp. 989). -995, 1993), especially the amino acid sequence (GGGGGS)₃Preferred are those containing a linker of In particular, the fusion protein according to the invention may comprise the ITAM motif of the ζ-chain or ζ-chain or part of the CD3-complex, in particular the ＣＤ-chain of human CD3 or a part thereof. The fusion protein may be further characterized in that it comprises, in particular, the CD8α of human CD8α or the Lck-binding motif of CD8α or a part thereof.
[0017]
The fusion protein according to the invention may furthermore be a chimeric partially or fully humanized α and / or β TCR chain, in particular according to SEQ ID NO: 3 or SEQ ID NO: 4. A further aspect of the invention relates to a fusion protein 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 in that it is an α / β-TCR coupled to other functional components, in particular according to SEQ ID NO: 7 or SEQ ID NO: 8.
[0018]
A further subject of the invention is the fusion protein of the invention, for example, in a suitable host cell, for the diagnosis and / or treatment of a disease associated with the hdm2 protein or for the identification of a pharmacologically active substance. Wherein the nucleic acid according to the present invention is used.
[0019]
Here, a fusion protein containing the polypeptide according to the present invention described above is prepared, wherein the fusion protein itself has the function of the polypeptide of the present invention itself, or has a special function of the fusion portion. Active only after removal. These include, in particular, fusion proteins having a proportion of about 1 to 200, preferably about 1 to 150, especially about 1 to 100, especially about 1 to 50 foreign amino acids. An example of such a peptide sequence is a prokaryotic peptide sequence, which is described, for example, in E. coli. E. coli galactosidase. In addition, viral peptide sequences, such as, for example, bacteriophage M13, can also be used to produce fusion proteins for the "phage display" process known to those skilled in the art.
[0020]
In purifying proteins according to the present invention, additional polypeptides ("tags") can be added. Protein tags according to the present invention can be used, for example, with high affinity absorption into the matrix, rigorous washing with suitable buffers that do not appreciably elute the complex, and subsequent controlled mode. Allows elution of the absorbed complex. An example of a protein tag known to those skilled in the art is (His)₆Tags, Myc tags, FLAG tags, Strep tags, Strep tags II, hemagglutinin tags, glutathione transferase (GST) tags, affinity chitin-binding tags or inteins with maltose-binding protein (MBP) tags. These protein tags can be located at the N-, C-terminus and / or internally.
[0021]
Apart from the native polypeptide isolated from the cells, all polypeptides or parts thereof according to the invention can be prepared under cell-free conditions, for example, by synthesis or by in vitro translation. Thus, the whole polypeptide or its peptide can be synthesized with the help of classical synthesis (Merrifield technology). Part of the polypeptide according to the invention is particularly useful for obtaining antisera, with the help of searching for its appropriate gene expression bank and thus obtaining further variants of the polypeptide according to the invention. Appropriate.
[0022]
The invention also relates in particular to polypeptides that are derivatives of antibodies with specificity for the hdm2-81-88 peptide, presented in the context of HLA-A2.1.
[0023]
The present invention further includes a retro-reverse peptide or pseudopeptide according to the polypeptide sequence of SEQ ID NO: 1 to SEQ ID NO: 8, or a functional variant or portion thereof. Instead of -CO-NH-peptide bonds, these peptides have -NH-CO- bonds.
[0024]
The object of the invention is further achieved by a nucleic acid according to the invention, which is DNA, RNA, PNA (peptide nucleic acid) or p-NA (pyranosyl nucleic acid), preferably DNA, in particular at least 8 nucleotides in length. , Preferably having at least 18 nucleotides, in particular having at least 24 nucleotides. The nucleic acid can be characterized in that the sequence of the nucleic acid has at least one intron and / or one polyA sequence. It can also be in the form of its antisense sequence.
[0025]
For expression of the gene of interest, double-stranded DNA is generally preferred, and DNA regions encoding the polypeptide are particularly preferred. This region is the first in the Kozak consensus sequence (Kozak, 1987, Nucleic Acids Res. 15: 8125-48) up to the next stop codon (TAG, TGA, eg, TAA) in the same reading frame with respect to ATG. Starts with the start codon (ATG). Further uses of the nucleic acid sequences according to the invention include antisense oligonucleotides (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). Antisense oligonucleotides can be used to reduce the stability of the nucleic acids according to the invention and / or to inhibit the translation of nucleic acids according to the invention. Thus, for example, the expression of the corresponding gene in a cell can be reduced both in vivo and in vitro. Thus, oligonucleotides may be suitable as therapeutics. This strategy is suitable, for example, in skin, epidermis and dermal cells, especially if the antisense oligonucleotide is complexed with liposomes (Smyth et al., 1997, J. Invest. Dermatol. 108: 523-6; White et al., 1999, J. Invest. Dermatol. 112: 699-705). For use as probes or as "antisense" oligonucleotides, single-stranded DNA or RNA is preferred.
[0026]
In addition to natural nucleic acids isolated from cells, all nucleic acids or parts thereof according to the invention can also be prepared synthetically. Furthermore, in practicing the present invention, nucleic acids prepared by synthesis can be used. Thus, nucleic acids according to the present invention can be prepared, for example, by the phosphotriester method (eg, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews,90, 543-584), and can be chemically synthesized using the genetic code with the help of the protein sequences set forth in SEQ ID NOs: 1-8.
[0027]
Oligonucleotides are generally rapidly degraded by endo- or exonucleases that occur in cells, especially by DNase and RNase. Therefore, it is advantageous to modify the nucleic acids in order to stabilize them against degradation so that high concentrations of the nucleic acids are maintained in the cells for an extended period of time (WO 95/11910; Macadam et al., 1998, WO 98/37240). Reese et al., 1997, WO 97/29116). Typically, such stabilization can be obtained by the introduction of one or more internucleotide phosphorus groups or by the introduction of one or more non-phosphorus internucleotides.
[0028]
Suitable modified internucleotides are summarized 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 radicals and / or non-phosphorus bridges in nucleic acids that can be used in one of the uses according to the invention include, for example, methylphosphonates, phosphorothioates, phosphoramidates, phosphorodithioates, phosphate esters While non-phosphorus internucleotide analogs include, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidodate bridges and / or thioether bridges. This modification is also intended to improve the stability of a pharmaceutical composition that can be used in one of the uses according to the invention.
[0029]
Further aspects of the invention include vectors, shuttle vectors, phagemids, cosmids, expression vectors, adenovirus vectors, retroviral vectors (preferably in the form of plasmids) comprising the nucleic acids according to the invention (Miller et al., “Gene Transfer and Expression BioTechniques Vol. 7, No. 9, P980, 1989) and / or vectors having gene therapy activity.
[0030]
Thus, the nucleic acids according to the invention can be comprised in a vector, preferably an expression vector or a vector active in gene therapy. Preferably, the vector active in gene therapy comprises a T-cell-specific regulatory sequence operably linked to a nucleic acid according to the present invention. The expression vector can be a prokaryotic or eukaryotic expression vector. Examples of prokaryotic expression vectors are described in E. coli. For expression in E. coli, for example, the vector pGEM or pUC derivative, and for eukaryotic expression vectors for expression in Saccharomyces cerevisiae, for example, the vector p426Met25 or p426GALl (Mumberg et al., (1994) Nucl. Res. 22, 67-67). 5768), and for expression in insect cells, for example, baculovirus vectors such as those disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells, for example, Vectors Rc / CMV and Rc / RSV or SV40 vectors, all of which are commonly available.
[0031]
Generally, expression vectors are, for example, E. coli. trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), Met25, GAL1 or ADH2 promoter for expression in yeast (Russel et al. (1983), J. Biol. Chem. 258, 2674-2682; Momberg, supra) and a promoter suitable for each host cell, such as the baculovirus polyhedrin promoter for expression in insect cells (see, eg, 13. EP-B1-0 127 839). For expression in mammalian cells, suitable promoters are those that allow for constitutive, regulatable, tissue-specific, cell cycle-specific or metabolically specific expression in eukaryotic cells, for example. is there. A regulatable element according to the present invention is a promoter, activator sequence, enhancer, silencer and / or repressor sequence. Examples of suitable regulatable elements that allow for constitutive expression in eukaryotes include promoters recognized by RNA polymerase III or viral promoters, CMV enhancers, CMV promoters, CMV-LTR hybrids, such as MMTV (mouse tumors). Breast cancer virus; the SV40 promoter or LTR promoter of Lee et al. (1981) Nature 214, 228-232), and also viral promoters and activator sequences derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV. . An example of a regulatable element that enables regulatable expression in eukaryotes is the tetracycline operator in combination with a suitable repressor (Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20).
[0032]
Examples of regulatable elements that allow T-cell-specific expression in eukaryotes are the promoters or enhancer promoters or activator sequences of genes encoding proteins expressed only in these cell types.
[0033]
Examples of regulatable elements that allow cell cycle-specific expression in eukaryotes are the promoters of the following genes: cdc25, cyclin A, cyclin E, cdc2, E2F, B-myb or DHFR (Zwicker J. and Muller R. (1997) Trends Genet. 13, 3-6). Examples of regulatable elements that allow for metabolically specific expression in eukaryotes are promoters that are regulated by hypoxia, by glucose deprivation, by phosphate concentrations, or by heat shock.
[0034]
The vectors according to the invention can be used for transfection of host cells which are preferentially T-cells. Particularly preferred are host cells which are characterized by expressing on their surface a polypeptide or fusion protein according to the invention.
[0035]
To enable the introduction of a nucleic acid according to the present invention, and thus the expression of a polypeptide in a eukaryotic or prokaryotic cell by transfection, transduction, transformation or infection, the nucleic acid may be a plasmid, a viral or non-viral vector or It can be present as part of a particle. Suitable viral vectors or particles in this case are, in particular, baculovirus, vaccinia virus, retrovirus, adenovirus, adeno-associated virus and herpes virus. Suitable non-viral carriers in this case are, inter alia, virosomes, liposomes, cationic lipids or poly-lysine-complex DNA.
[0036]
Examples of vectors that are active in gene therapy are viral vectors, such as adenoviral or retroviral vectors (Lindmann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell.2: 549-58; Weijtens et al., "Retroviral vector system" STITCH "combined with optimized single-chain antibody chimeric receptor gene structure enables efficient gene transfer and expression in human T-lymphocytes. ", Gene Therapy 1998)5, 1995-1203).
[0037]
A preferred mechanism for expressing the polypeptide according to the invention in vivo is, in particular, viral gene transfer with the aid of retroviral particles. These are preferably used to provide suitable target cells, preferably T-lymphocytes, of a patient ex vivo together with a gene or nucleotide sequence encoding a polypeptide according to the invention by transduction. The target cells can later be injected again into the patient, in the sense of adoptive cell transfer, to inherit tumor killing and / or immunomodulatory effector functions using the specificity inserted de novo. Recently, a similar intact transgene of a non-functional mutant variant of the γ-chain gene that occurs in children, which is essential for the differentiation of the adoptive immune system into various effector cells, has thus been obtained in hematologic precursor cells. Very good gene therapy results have been achieved with the treatment of SCID-X1 disease, which is characterized by immune incompetence in neonates provided by retroviruses (Cavazzana-Calvo et al., 2000).
[0038]
In addition, in vivo gene transfer may be achieved on the one hand by preferential stereotactic injection of infectious particles and on the other hand by direct administration of virus-producing cells (Oldfield et al., Hum. Gen. Ther. , 1993, 4: 39-69).
[0039]
According to the state of the art, the viral vectors often used for gene transfer are mainly retroviral, lentiviral, adenoviral and adeno-associated viral vectors. These are cyclic nucleotide sequences derived from a natural virus, wherein at least the virus structural protein-coding gene is replaced by the construct to be introduced.
[0040]
Retroviral vector systems create a requirement for long-lasting expression of the transgene by stable but undirected integration into the host genome. Younger generation vectors do not carry irrelevant and potentially immunogenic proteins and, in addition, there is no pre-existing immunity of the receptor to the vector. Retroviruses contain an RNA genome packaged in the host cell membrane and a lipid coat consisting of a portion of the viral proteins. For viral gene expression, the RNA genome is reverse transcribed and incorporated into target cell DNA by the enzyme integrase. It is later transcribed and translated by infected cells, which results in a viral construct that combines to give a retrovirus. Thus, the RNA is exclusively inserted into the newly formed virus. The retroviral genome has three essential genes: a viral structural protein, gag encoding "group-specific antigens", pol for enzymes such as reverse transcriptase and integrase, and a coat responsible for binding host-specific receptors. Carries the env for the protein ("envelope"). Production of replication-incompetent viruses additionally comprises the gag-pol-coding genes and expresses them “in trans”, thus forming replication-incompetent (ie, gag / pol-deleted) transgenic virus particles. Occurs after transfection in a "packaging cell line" supplementing An alternative is co-transfection of essential viral genes, where only the vector contains a transgene that carries the packaging signal.
[0041]
Isolation of these genes, on the one hand, allows any combination of env and gal / pol-reading frames from various strains, resulting in pseudotypes with modified host tropism. On the other hand, the formation of replication-competent virus in packaging cells can thereby be dramatically reduced. The coat protein from the gibbon ape leukemia virus (GALV) used in the "switch" or "bullet" vector systems is capable of transducing human cells and is established in the amphoteric host range within the packaging cell line PG13 (Miller et al., 1991). In addition, the prevention of homologous recombination and, thus, the production of replication-competent particles, has increased safety due to the selective deletion of non-essential viral sequences.
[0042]
Novel non-viral vectors are introduced into host cells, for example, by liposome transfection, and consist of a transposon, an autonomous self-integrating DNA sequence that was first successfully used in mammalian cells to express the human transgene. (Yant et al., 2000).
[0043]
A vector active in gene therapy can be obtained by complexing the nucleic acid according to the present invention with a liposome. Since very high transfection efficiencies can be achieved, especially for skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279-85). Excipients that enhance the introduction of nucleic acids into cells can be, for example, proteins or peptides linked to DNA or synthetic peptide-DNA molecules that allow transport of the nucleic acid to the cell nucleus (Schwartz et al. (1999) Gene Therapy). 6, 282; Branden et al. (1999) Nature Biotech. 17, 784). Excipients are molecules that allow the release of nucleic acids into the cytoplasm of cells (Planck et al. (1994) J. Biol. Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213) or, for example, Liposomes (Uhlmann and Peymann (1990) supra). Another particularly suitable form of gene therapy vector can be obtained by applying nucleic acids according to the invention to gold particles and driving them into tissue, preferably skin or cells, with the aid of a "gene gun". (Wang et al., 1999, J. Invest. Dermatol., 112: 775-81).
[0044]
In gene therapy applications of a nucleic acid according to the present invention, if a portion of the nucleic acid encoding the polypeptide is preferably an intron sequence between the promoter and initiation codon of the polypeptide, and / or especially at the 3'-end of the gene, It is also advantageous to include one or more non-coding sequences, including A-sequences, particularly naturally occurring poly-A sequences or SV40 viral poly-A sequences. This is because mRNA stabilization can be achieved by this means (Jackson, RJ (1993) Cell).74, 9-14 and Palmiter, R .; D. (1991) Proc. Natl. Acad. Sci. USA88, 478-482).
[0045]
A further subject of the invention is a host cell, in particular a T-cell, which is transformed with a vector according to the invention or another genetic construct according to the invention. The host cell can be a prokaryotic or eukaryotic cell; examples of prokaryotic host cells are E. coli. and examples of eukaryotic cells are Saccharomyces cerevisiae or insect cells.
[0046]
Particularly preferred transformed host cells are transgenic T-precursor cells or stem cells, which are characterized in that they comprise a genetic construct according to the invention or 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 the patient's own T-cell, which, after removal, is transfected with a genetic construct according to the present invention. A host cell according to the invention may be, in particular, one or more cells from a patient, preferably T-cells, especially CD8⁺-T-cells can be obtained, which are then transfected or transduced ex vivo with one or more gene constructs according to the invention and thus a host cell according to the invention Get. The specific T-cells generated ex vivo can then subsequently be re-implanted into the patient. Thus, the process is similar to that described by Darcy et al. Using CTL transduced with scFv anti-CEA receptor, perforin and γ-IFN (“engineered ex vivo”). Re-directed Perforin-Dependent Lysis of Colon Carcinoma by CTL "J. Immunol.,2000, 164: 3705-3712).
[0047]
A further aspect of the present invention is directed to conditions under which only tumor cells or a fraction thereof are lysed if the tumor presents an hdm2 protein-specific antigen specific for the expressed polypeptide or fusion protein. And a method for identifying an hdm2 protein-specific antigen, which comprises combining an hdm2-presenting tumor cell or a fraction thereof with a host cell according to the present invention.
[0048]
A further aspect of the invention relates to the production of antibodies, preferably polyclonal or monoclonal antibodies, for the diagnosis and monitoring of diseases associated with the hdm2 protein or for the identification of pharmacologically active substances, The antibody-producing organism is immunized with a polypeptide according to the invention having at least 6 amino acids, preferably having at least 8 amino acids, in particular having at least 12 amino acids or a functional equivalent thereof or a part thereof, or Vaccinated with a nucleic acid encoding a polypeptide according to the invention.
[0049]
The process generally involves immunizing a mammal, such as a rabbit, with a polypeptide according to the present invention or a portion thereof, if appropriate, for example, in the presence of incomplete Freund's adjuvant and / or aluminum hydroxide gel. (See, for example, Diamond, BA, et al. (1981) The New England Journal of Medicine, 1344-1349). The polyclonal antibody formed in the animal by the immunological reaction can then be easily isolated from blood according to generally known methods, and can be purified, for example, by column chromatography. Monoclonal antibodies are described, for example, in Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature,349, 293-299).
[0050]
A further subject of the invention is a pharmacologically directed, and specifically reactive, polypeptide of the invention for the diagnosis and / or treatment of diseases associated with the hdm2 protein. An antibody for the identification of an active substance, wherein said portion of said polypeptide is itself immunogenic or, by binding to a suitable carrier, such as, for example, bovine serum albumin, Sex can be increased. This antibody may be polyclonal or monoclonal, preferably a monoclonal antibody. The term antibody according to the present invention is used to describe, for example, chimeric, humanized, multifunctional, monovalent or oligo-, bi- or oligo-specific, single-chain, F (Ab) or F (ab)₂An antibody or an antigen-binding portion thereof optionally modified such as a fragment (for example, EP-B1-0 368 684, US 4,816,567, US 4,816,397, WO88 / 01649, WO93 / 06213). , WO 98/24884). The antibodies according to the invention can be used for the diagnosis and / or treatment of diseases associated with the hdm2 protein or for the identification of pharmacologically active substances.
[0051]
The invention furthermore comprises combining at least one nucleic acid, at least one polypeptide, at least one host cell or at least one antibody according to any one of the preceding claims with suitable additives and excipients. The present invention relates to a method for producing a medicament for treating a disease associated with the hdm2 protein.
[0052]
The present invention further relates to a disease associated with the hdm2 protein, comprising, if appropriate, at least one nucleic acid, at least one polypeptide or at least one antibody according to the invention together with suitable additives and excipients. It relates to a medicament prepared by this process for therapy. The invention further relates to the use of this medicament for the treatment of diseases associated with the hdm2 protein.
[0053]
The treatment of diseases associated with the hdm2 protein can be carried out in a customary manner, for example by infusion or injection containing the medicament according to the invention. The administration of the medicament according to the invention can, if desired, also take the form of a liposome complex or a gold particle complex. However, treatment with the medicaments according to the invention is carried out in an oral dosage form, for example via tablets or capsules, for example via the nasal or oral mucosa or in the form of a repository implanted under the skin. You can also TTS is known, for example, from EP 0 944 398 A1, EP 0 916 336 A1, EP 0 889 723 A1 or EP 0 852 493 A1. The polypeptides and derivatives thereof according to the invention can also be used for the active and / or passive immunization of sick patients, which binds hdm2 and induces hdm-specific CTL, It can also be used to achieve production and increase, specifically to destroy tumors and leukemia cells in the patient of interest. Such diseases include, for example, solid tumor symptoms, lymphohematopoietic neoplasia, malignant hematological diseases, which are in the form of multiple myeloma (or plastocyte), histiocytoma lymphoma and CML myeloblast crisis.
[0054]
In a particularly preferred type of treatment one or more cells, preferably T-cells, especially CD8⁺-T-cells are taken from the patient, which is then transduced or transfected ex vivo with one or more gene constructs according to the invention. The specific T-cells generated ex vivo can then be reimplanted into the patient. Thus, the method is similar to the immunotherapeutic method described by Darcy et al. In the case of colon carcinomas using CTL, perforin and γ-IFN transduced by the scFv anti-CEA receptor (“Ex Vivo”). Redirected porphorin-dependent lysis of colon carcinomas by CTL created by genetic engineering at J. Immunol., 2000, 164: 3705-3712).
[0055]
The present invention further relates to an hdm2 protein-related disease, characterized in that at least one nucleic acid, at least one polypeptide or at least one antibody according to the invention is combined with suitable additives and excipients. How to create tests for discovering what works functionally.
[0056]
The term "functionally acting" in the sense of the present invention interacts with a nucleic acid, polypeptide or antibody according to the invention under suitable conditions and, where appropriate, with suitable additives and excipients. It should be understood to mean all molecules, compounds and / or compositions and substance mixtures that can be used. Possible acts are simple organic or inorganic molecules or compounds, but may also include peptides, proteins or complexes thereof. In that interaction, one that acts functionally can affect the function of the nucleic acid, polypeptide or antibody in vivo, or in vitro, or can affect a nucleic acid, polypeptide or antibody according to the invention. It can only be attached, or it can have other interactions with it, either covalently or non-covalently.
[0057]
The present invention further relates to diseases associated with hdm2 proteins containing at least one nucleic acid, at least one polypeptide or at least one antibody according to the invention, together with suitable additives and excipients, if appropriate. Includes tests made in accordance with the present invention for identification of bound functionally acting agents. Thus, it is often possible to seek substances that can mimic the pathological behavior of cells in vitro, restore the normal behavior of the cells and retain therapeutic capacity. In addition, this test system can be used in screening for substances that inhibit the interaction between a polypeptide according to the invention and a functionally acting one.
[0058]
The subject of the present invention is also a medicament for the identification and treatment of diseases associated with the hdm2 protein, comprising a nucleic acid according to the invention or a polypeptide according to the invention and, if appropriate, suitable additives or excipients. And a method of producing such a medicament for the treatment of a disease associated with the hdm2 protein, comprising formulating the nucleic acid according to the invention or the polypeptide according to the invention with a pharmaceutically acceptable carrier.
[0059]
Possible therapeutic and / or prophylactic agents are, as active compounds, (a) TCR-receptor polypeptides according to the invention and / or derivatives thereof and / or (b) nucleic acids according to the invention and / or (c) hdm2. Especially vaccines, recombinant particles or injection or infusion solutions containing T-lymphocytes produced in vitro or ex vivo containing a TCR specifically directed against them.
[0060]
For gene therapy applications in humans, a medicament comprising a nucleic acid according to the present invention in naked form, in one form of the above-described gene therapy active vectors, or in a complexed form with liposomes or gold particles, and // Recombinant particles are particularly suitable. The pharmaceutical carrier is, for example, preferably at a pH of about 6.0 to 8.0, preferably about 6.8 to about 7.8, especially about 7.4 and / or about 200 to 400 milliosmol / liter, preferably about Physiological buffer solution having an osmotic pressure of 290-310 milliosmol / liter. In addition, the pharmaceutical carrier can contain a suitable stabilizer, such as, for example, a nuclease inhibitor, preferably a complexing agent, such as EDTA and / or other excipients known to those skilled in the art.
[0061]
A further subject of the invention is a pharmacological agent for the diagnosis and / or treatment of diseases associated with the hdm2 protein, or in suitable host cells, characterized in that the nucleic acids according to the invention are expressed in a suitable manner. The present invention relates to a method for producing a polypeptide for the identification of a substance that is highly active.
[0062]
Said polypeptides are thus prepared, for example, by expression of a nucleic acid according to the invention in a suitable expression system already described above, according to methods generally known to those skilled in the art. Suitable host cells are described, for example, in E. coli, all commonly available. coli strains DHS, HB101 or BL21, yeast strains Saccharomyces cerevisiae, such as insect cell lines from Spodoptera frugiperda, or animal cells COS, Vero, 293, HaCaT and HeLa.
[0063]
The diagnostic agent according to the invention for therapeutic monitoring comprises a polypeptide according to the invention or an immunologically active part thereof as described in greater detail above. For example, a polypeptide or portion thereof, preferably bound to a solid phase of nitrocellulose or nylon, can be contacted in vitro with the body fluid to be tested, for example, to react blood with autoimmune antibodies. The antibody-peptide conjugate can then be detected, for example, with the aid of a labeled anti-human-IgG or anti-human-IgM antibody. The label is, for example, an enzyme such as peroxidase which catalyzes a color reaction. The presence and amount of autoimmune antibodies present can thus be easily and quickly detected by a chromogenic reaction.
[0064]
Another diagnostic agent for therapeutic monitoring comprises the antibody itself according to the present invention. With the help of these antibodies, for example, it is possible to easily and quickly examine tissue samples for the presence of increased amounts of the polypeptide of interest, thus identifying diseases associated with the hdm2 protein. it can. In this case, the antibodies according to the invention are labeled, for example, with the enzymes already mentioned above. Thereby, the specific antibody-peptide complex can be easily and similarly rapidly detected by an enzymatic color reaction.
[0065]
Further diagnostic agents according to the invention include probes, preferably DNA probes and / or primers. This opens up further possibilities for obtaining nucleic acids according to the invention, for example by isolation from a suitable gene bank with the aid of a suitable probe (see, for example, J. Sambrook et al., 1989, Molecular Cloning. A. Robotic Manual 2nd, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Chapter 8, pages 8.1-8.81, Chapter 9, pages 9.47-9.58 and Chapter 10, pages 10.1-10. 67).
[0066]
Suitable probes have a length of about 100 to 1000 nucleotides, preferably about 200 to 500 nucleotides, whose sequence can be derived from the polypeptides of SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing. DNA or RNA fragments having a length of about 300 to 400 nucleotides.
[0067]
Alternatively, with the help of the derived nucleic acid sequence, suitable oligonucleotides can be synthesized as primers for the polymerase chain reaction. Suitable primers have, for example, a length of about 10-100 nucleotides, preferably about 15-50 nucleotides, whose sequence can be derived from the polypeptides of SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing. DNA fragments having a length of 20 to 30 nucleotides.
[0068]
The term "coding nucleic acid" relates to a DNA sequence encoding an isolatable biologically active polypeptide or precursor according to the invention. The polypeptide may be encoded by the full length of the coding sequence, or any portion, as long as specificity, eg, enzymatic activity is retained.
[0069]
For example, due to the degeneracy of the genetic code, there may be small changes in the sequence of the nucleic acid according to the invention, or the untranslated sequence may cause the 5 'and / or 3' 'It is known that it can be added to the end. Accordingly, the present invention also includes "functional variants" of the nucleic acids according to the present invention.
[0070]
The term "functional variant" means that under stringent conditions, it hybridizes with the derived reference sequence or a part thereof, in particular the hypervariable V (D) JC region, and is similar or corresponding to the corresponding polypeptide according to the invention. It refers to all DNA sequences that have the same activity and are complementary to the DNA sequence.
[0071]
“Stringent hybridization conditions” means that hybridization occurs in 2.5 × SSC buffer at 60 ° C., followed by multiple washing steps at 37 ° C. with lower buffer concentration and remains stable. Should be understood to mean a condition that is
[0072]
The term “functional variant” within the meaning of the invention is understood to mean a polypeptide functionally related to the polypeptide according to the invention, ie having the structural characteristics of the polypeptide. Examples of functional variants are corresponding polypeptides from organisms other than mice, ie, humans, or preferably, from non-human mammals such as, for example, monkeys, pigs, and rats. Other examples of functional variants are polypeptides encoded by different alleles in different individuals or in different organs of an organism. The invention also covers, inter alia, functional TCR variants that recognize the same epitope of the hdm2 polypeptide and induce a specific T cell response.
[0073]
In a further sense, they may have sequence homology to polypeptides having an amino acid sequence with one of SEQ ID NO: 1 to SEQ ID NO: 8 and / or a DNA sequence derived with the aid of a peptide sequence, especially about It is understood that polypeptides having a sequence identity of 70%, preferably about 80%, especially about 90%, especially about 95%. Among these are additions, inversions, substitutions, deletions, insertions of polypeptides ranging from about 1 to 60, preferably about 1 to 30, especially about 1 to 15, especially about 1 to 5 amino acids. Or chemical / physical modifications and / or replacements or moieties. For example, the first amino acid can lack methionine without losing the function of the peptide to be significantly modified.
[0074]
The invention will now be further described, but not limited, with the help of the accompanying examples and figures. The drawings show:
SEQ ID NO: 1: wild-type gene mouse αTCR polypeptide (muvα-mucα),
SEQ ID NO: 2: polypeptide of wild-type gene mouse β TCR (muv β-muc β),
SEQ ID NO: 3: chimeric partially humanized TCR gene polypeptide (muvα-hucα),
SEQ ID NO: 4: Chimeric partially humanized TCR gene polypeptide (muvβ-hucβ)
SEQ ID NO: 5: polypeptide of single-chain TCRmuvα-Li-muvβ-mucβ,
SEQ ID NO: 6: polypeptide of single-chain TCRmuvβ-Li-muvα-mucα,
SEQ ID NO: 7: double-chain CR polypeptide fused to human CD3ζ chain muvα-hucα-huCD3ζ
SEQ ID NO: 8: double-chain TCR polypeptide fused to human CD3 {chain muvβ-hucβ-huCD3}
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; phosphorylation at the 5'-end,
SEQ ID NO: 21: primer for_NcoI_bTCR_4,
SEQ ID NO: 22: primer for_NcoI_bTCR_5b; phosphorylation 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; phosphorylation 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 wild-type mouse αTCR from SEQ ID NO: 1 (muvα-mucα),
SEQ ID NO: 39: polynucleotide of the wild-type gene mouse βTCR from SEQ ID NO: 2 (muvβ-mucβ),
SEQ ID NO: 40: polynucleotide of a chimeric partially humanized TCR gene from SEQ ID NO: 3 (muvα-hucα),
SEQ ID NO: 41: polynucleotide of a chimeric partially humanized TCR gene from SEQ ID NO: 4 (muvβ-hucβ),
SEQ ID NO: 42: single-stranded TCR muvα-Li-muvβ-mucβ polynucleotide from SEQ ID NO: 5,
SEQ ID NO: 43: single-stranded TCR muvβ-Li-muvα-mucα polynucleotide from SEQ ID NO: 6,
SEQ ID NO: 44: polynucleotide of a double-stranded TCR fused to human CD3ζ chain muvα-hucα-huCD3ζ from SEQ ID NO: 7; and
SEQ ID NO: 45: Double-stranded TCR polynucleotide fused to human CD3ＣＤ chain muvβ-hucβ-huCD3ζ from SEQ ID NO: 8.
[0075]
In the primer sequence, Y = C / T; R = A / G; V = A / C / G; W = A / C / T
【Example】
[0076]
Double-transgenic mouse huCD8 × A2K^bThe genes for the T-cell receptors αTCR and βTCR from were prepared, which mediate an HLA-A2.1-restricted and hdm2 protein / 81-88 peptide-specific T-cell response. Inserting the resulting gene as a wild-type (WT) or modified construct into human peripheral blood lymphocytes (PBL) by retrovirus,⁵¹HLA-restricted antigen recognition was functionally checked by CD8-mediated cytotoxic lysis of various cell lines in a chromium release test.
1. ) Obtaining the genes encoding the α- and β-chains of the T-cell receptor
1.1) Preparation of cytoplasmic RNA
Cytoplasmic RNA was isolated, while isolating unprocessed intron-containing RNA, DNA fragments and ribosomal RNA, located primarily in the cell nucleus, and achieving the desired "messenger" RNA enrichment. Protocols and materials were slightly modified, but mostly corresponded to the "Rneasy Mini Kit" from QIAGEN; in one preparation, hdm2 / 81-88-specific T from culture dishes of "24-well plates" -Average 50x10 of cell clone 3⁶Murine cells were combined and washed without serum in RPMI 1640 (Biowhittaker). 12.5 × 10 each per incubation tube⁶The cells were aliquoted under RNase-free conditions of the cells and incubated for 20 minutes on ice in cell membrane lysis buffer (cooled to 4 ° C.).
[0077]
Cell membrane lysis buffer:
50 mM Tris HCl pH 8.0
140 mM NaCl
1.5 mM MgCl₂
0.5% Nonidet P40 (Roche 1 332 473)
1000 units of Rnase inhibitor / ml ZML-P (Roche 799 017)
1 mM DTT
This buffer used DEPC-treated and dual-autoclaved deionized (MilliQ from Millipore) water, like all other aqueous solutions to be prepared.
[0078]
Further steps were carried out at room temperature under Rnase-free conditions with the aid of the QIAGEN protocol, according to the processes customary for a person skilled in the art. This step will be described briefly.
[0079]
Gently solubilized and aliquoted cells were centrifuged at 300g / 2min / 4 ° C and the supernatant was removed. 600 μl of β-mercaptoethanol (MSH) -containing (1.45 mM) “RLT” buffer was added, mixed well, and 430 μl of 96% ETOH was added with mixing. The solution per aliquot was added to a silica Minispin column and centrifuged at 10,000 rpm (rotations per minute) for 15 seconds in an Eppendorf table centrifuge. Washing in 700 μl “RW1” and 500 μl of 77% ETOH-containing “RPE” buffer was performed in each case. Finally, 500 μl of the same “RPE” buffer was added and the column was dried at a maximum of 14,000 rpm for 2 minutes. RNA was eluted for 2 minutes at 10,000 rpm using 54 μl of Rnase-free water. The yield is 12.5 × 10⁶30 μg per murine T-cell. RNA was frozen at −20 ° C. at 6 μg / 10 μl.
[0080]
1.2 Amplification of β-chain gene by coupled RT-PCR
Before it could be cloned, the TCR-coding RNA had to be specifically transcribed into DNA and amplified. For this, RT-PCR is used as a method, which is carried out in a kit of the same name from Roche, "Titanium One Tube RT-PCR System" (1888 382). A "one-step protocol" was selected, in which reverse transcription is performed without further intervention of the polymerase chain reaction. The enzymes used were AMV reverse transcriptase and Thermus Aquaticus / Pwo-thermostable polymerase (“Expand^TM  High fidelity "enzyme mix), which was used in widely used reaction buffers. Due to the transcription rate of the TCR gene and the conservation of the gene sequence encoding the variable domain due to the deviation of the TCR gene, "nested PCR" (polymerase chain reaction) was added to increase the specificity and signal of the amplified DNA. Increased strength. Using a degenerate primer (for_bTCR_v4; SEQ ID NO: 9) corresponding to the base sequence for the first conserved amino acid coding after the signal peptide, the diversity of v-gene segments whose subfamily membership is not predictable a priori Tried to determine gender. First, a synthetic oligonucleotide should add an artificial gene segment encoding the murine TCR signal peptide to ensure that the protein synthesized in vivo is directed to the lumen of the endoplasmic reticulum. Selecting the reverse-hybridizing primer (rev_bTCR_c4; SEQ ID NO: 10) within a constant gene segment, the two possible c-gene segments of the β-strand with some base differences at its 3′-end Further deviations of (c-β1 or c-β2) were avoided. The "nested PCR" oligonucleotides carried, besides TCR complementarity, the sequence of the restriction cleavage site for EcoRI or AscI at the 5'-end. The primer that hybridizes in the forward direction of the nested “PCR” (for_Eco_bTCR_v5; SEQ ID NO: 11) is identical in the sequence of the v-gene segment relative to the degenerate RT-PCR primer, while strictly located in the “3′- As per "PCR", a primer (rev_Asc_bTCR_c6; SEQ ID NO: 12) that is complementary to the c gene segment that hybridizes in the reverse direction is inserted inward by less than 120 bases relative to the reverse first primer of the TCR reading frame. Moved to. This increased the specificity of the PCR. This is because sequence-specific recognition events must occur in two independent PCR steps at two different locations. RT with the reverse degenerate primer (rev_Asc_bTCR_c2; SEQ ID NO: 14) to include the two potential cβ1 / cβ2 gene segments, including the complete c-gene segment, and without additional “nested PCR” -Cloned with the corresponding primer (for_Eco_bTCR_c2; SEQ ID NO: 13) in the reading direction of the cβ gene segment by PCR. In the overlapping region of the two amplicons in the cβ range, a unique NcoI cleavage site was located, which allowed the combined truncated vβ / cβ and the complete cβ gene segment in subsequent subcloning.
[0081]
The pipetting scheme and the PCR protocol of RT-PCR corresponded in principle to the process described by Roche. Subsequent "nested PCR" was performed similarly to the replacement of the component Rnase inhibitor and DTT with autoclaved water. Instead of 2 μl of RNA (0.1-1 μg), 2 μl of the amplicon of RT-PCR was used. The technical requirements of the PCR device are: 2 for a classical PCR cycle with a temperature gradient from 35 ° C to 55 ° C spaced at five selected temperatures of 36 ° C, 39 ° C, 44 ° C, 52 ° C and 56 ° C. Simultaneous testing of different hybridization temperatures in the second step was possible. At higher temperatures of 44-56 ° C., the intensity was increased by the second PCR and a single band of the desired length, sufficient to be inserted into the vector, could be achieved.
[0082]
Sequencing of the eight bacterial clones showed some randomized base substitutions, which were consistent with Expand to other polymerases.^TMThis was due to the relatively high error rate of the enzyme mix. In addition, all selected clones carry sequence information for the non-coding, short region of the 5-, untranslated region of the signal peptide and less than 100 bases, and thus, hence, the v-gene segment-specific shrinkage. It could be assumed that the heavy primer recognized a similar upstream sequence and gave rise to an additional sequence of the wild-type signal peptide.
[0083]
1.3 Amplification of α-chain gene by RACE-PCR
For α-chain sequencing, RACE-PCR was a targeting strategy: degenerate oligonucleotides designed in a similar manner to the batch described above are nonfunctional T-cell receptors due to a reading frameshift in the CDR3 loop. Provided exclusively the cryptographic sequence of Allele exclusion is not as effective on the β-chain of the TCR. The RACE-technique has been established in the similarly named system of Roche (1 734 792) and must be complemented with an αTCR gene-specific oligonucleotide: thus in the context of “5′-RACE-PCR” Deviations located 5 'of the coding sequence are avoided by generating a known short adenine oligonucleotide sequence at the 3'-end of the cDNA sequence reverse transcribed by terminal transferase, which results in a reading direction. In the above, it functions as a recognition sequence for the PCR primer located 5 ′. The reverse PCR primer is in the constant sequence encoding the constant domain. For reverse transcription, the primer rev_Asc_aTCR_c1 (SEQ ID NO: 15) was used, along with for_Eco_aTCR_c6 (SEQ ID NO: 18), for amplification via RT-PCR of the full length ca segment. Help for primer pairs “oligo d (T) -anchor primer” (component of RACE-PCR kit) / rev_aTCR_c5 (SEQ ID NO: 16) and “PCR anchor primer” (component of RACE-PCR kit) / rev_Asc_aTCR_c4 (SEQ ID NO: 17) The concept of a sequential two-step “nested PCR” was followed, thus subcloning the complete sequence encoding the constant domain was also added here. PCR primers had to be positioned such that a single NcoI cleavage site was present in the overlapping region of the amplified gene section. With 5'-SalI-3'-AscI, the excised PCR amplified containing the vα gene segment was cloned into pNEB193 (New England Biolabs), while the constant gene segment was inserted into pNEB193 with EcoRI and AscI. The amplicons are cloned by conventional methods by purification via LMP gel and phenol / chloroform extraction (J. Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring, ON). Subsequently, they were digested with restriction enzymes and the ends were removed with the aid of a "High Purity PCR Product Purification Kit" (Roche, 1732 668).
[0084]
The multi-step protocol mainly followed a commercial protocol with the aid of a programmable PCR device: reverse transcription, transferase reaction and a subsequent two-step “nested PCR” were performed on a PCR device “MasterCycler” from Eppendorf. In the context of specific α-chain amplification, gradient techniques were used in the hybridization steps at temperatures of 39 ° C, 44 ° C, 52 ° C and 56 ° C. Pfu polymerase (Stratagene) was selected. At this point, this was considered the enzyme with the lowest error uptake rate. The first PCR has already given a weak band of size expected at 0.9 kB in the whole temperature spectrum, the intensity of which is increased by subsequent PCR, further diluting the "primer dimer" that occurred. did it. The final PCR resulted in a truncation of the amplicon by only 70 bp, according to the theoretical position of the transferred hybridizing primer. In the agarose gel, a double band with a difference of 100 bp in length appeared, indicating the presence of functional and non-functional α-chains. Both bands could be excised on the preparative gel to include both amplicons in subsequent cloning. Seven clones encoding functional reading frames and possessing identical sequences were found.
1.4) Survey of found subfamily membership
The presence of the vβ6- or vα10-gene segment in the murine hdm2 protein / 81-88-specific T-cell clone 3 has been demonstrated in various ways:
a) RT-PCR with vβ6-specific primers or vα10-specific primers. RNA was previously purified from murine hdm2-P2-specific T-cell clone 3 by separating specific T-cells from murine "feeder" cells with an anti-huCD8 magnetically labeled antibody. The negative control used was an influenza matrix protein- (FluM158-66) -specific T-cell clone.
b) FACS analysis with vβ6-specific FITC-labeled antibody (Pharmingen) (FIG. 1). The resulting murine T-cell line and clone 3 were both investigated. The positive control used was a FITC-labeled antibody recognizing the β-chain portion independent of the subfamily.
[0085]
c) cytotoxicity⁵¹Vβ6-specific antibody block of murine T-cell clone 3 in chromium release test (FIG. 2).
[0086]
2. ) Examples of design of hdm2-specific constructs (FIG. 3 “SEQ ID NOs: 38-45” and FIG. 4 “SEQ ID NOs: 1-8”)
The retroviral vectors pBullet, a functional derivative of pStitch (Weijtens et al., 1998) belong to the "splice vector", wherein the transgene is identical to the splice acceptor and donor elements SA and SD as the env gene. It must be cloned to guarantee maximum expression. For this purpose, an NcoI cleavage site is designed within the start codon "ccATGg" with appropriate primers. This changed the second amino acid from Lys to Glu or Asn to Asp for both the wild type chains αTCR and βTCR. At the 3'-end of the reading frame, a unique BamHI-cleavage site was chosen. Restriction enzymes and modification enzymes were obtained from New England Biolabs (NEB). The thermostable polymerase was generally a natural or cloned Pfu DNA polymerase (Pyrococcus furiosus, Stratagene) or Pfx DNA polymerase (Pyrococcus sp., Life Technologies).
[0087]
2.1) Cloning of murine wild-type gene αTCR (muvα-mucα; SEQ ID NOs: 1-38) and βTCR (muvβ-mucβ; SEQ ID NOs: 2-39)
If the NcoI cleavage site is not internal (partially humanized strand), the gene segment is directly amplified and cloned by a primer containing the sequence for NcoI (for_NcoI_aTCR_4, SEQ ID NO: 19; for_NcoI_bTCR_4, SEQ ID NO: 21). Could be transformed.
[0088]
Since the NcoI-cleavage site for the wild-type strand occurred in the gene segment for the constant domain, it carries this type of truncated blunt-end NcoI cleavage site at the 5'-end, and thus they are T4 DNA polymerases. After blunt-end filling of the NcoI-cleaved vector DNA with the help of, the coding reading frame was amplified by an oligonucleotide (for_NcoI_aTCR_5b, SEQ ID NO: 20; for_NcoI_bTCR_5b, SEQ ID NO: 22) that complements the latter to provide a complete NcoI cleavage site. . rev_aTCR_BamHI (SEQ ID NO: 23) and rev_bTCR_BamHI_1 (SEQ ID NO: 24) were used as the final reverse primer for all murine double- and single-stranded constructs.
[0089]
2.2) Creation of chimeric, partially humanized TCR genes huαTCR (muvα-hucα; SEQ ID NO: 40) and huβTCR (muvβ-hucβ; SEQ ID NO: 41)
For functional chimerism of the murine TCR, two criteria must be satisfied:
a) The murine constant domains have to be replaced by the corresponding human domains so that these domains are completely contained in their immunoglobulin-like fold. The complete variable domain should also be included so as not to cause damage to most carboxy-terminal CDR3 loops.
[0090]
b) The fusion should have occurred such that the transposition, if possible, did not give rise to the neoantigen leading to an unintended immune response.
[0091]
In case b), in general, the following properties were obtained for both chains in comparison of the human and murine sequences: in the region of up to 8 amino acids after the last consensus motif "GxG" completing the CDR3 loop. , The position is stored relatively strongly. This region is completed by a highly conserved sequence of up to 11 amino acids. After this, the murine sequence begins to differ from the human sequence and in various cases is highly conserved. The central region is in the extension loop connecting the variable domain to the constant domain (see criterion a)). In an ideal case, chimerism within a highly conserved amino acid (AA) triplet sequence was preferred.
[0092]
In the case of αTCR, the highly conserved triplet sequence consisted of “QNP” (bases 400-408 relative to the start codon of murine αTCR). At positions 407-415, a unique AlwNI cleavage site was attached, which could be used for fusion: the 3'-shift position or AlwNI was replaced by murine instead of aspartate conserved in the human sequence. It required the presence of glutamate conserved in the sequence. This conservative amino acid substitution was not considered to be very immunogenic and thus was considered acceptable. The highly conserved region of the chimera is I¹³³-QNPEPEVAVYQLR¹⁴⁴Read, here, R¹⁴⁴Represents the first human-specific amino acid. I¹³³Is located at the ninth position after the consensus motif "GxG" that completes the CDR3 loop.
[0093]
The cloning strategy was designed to ligate the LMP-agarose-isolated EcoRI / AlwNI-cleaved vα segment to the AlwNI / BamHI-digested cαPCR amplicon, followed by cutting the mixed ligation product with EcoRI and BamHI. The ligation product, potentially prepared to the correct fragment length, was cloned into the expression vector pcDNA3.1 (-) (Invitrogen). Asymmetric test digestion yielded clones with the desired sequence of the ligation starting material.
[0094]
The human constant gene segments cα and cβ were isolated from the human leukemia cell line Jurkat by conventional RT-PCR performed as part of the Roche kit described above, followed by “nested” PCR. In the case of the human cα gene segment, an AlwNI cleavage site (T to A; see table below) must be created, on the one hand, with the help of mutagenic PCR primers at the 2 position, on the other hand, the murine sequence Compatibility had to arise. (C to a; see table below). This is because the three central positions of the AlwNI cleavage site can be degenerated.
[0095]
[Table 1]
[0096]
The naturally occurring AlwNI cleavage site in the murine sequence is shown in bold and the 3'-shift cleavage position is underlined. Degenerate triplets are typed in lower case. Base sequences required for human "primers" for mouse sequence-compatible AlwNI cleavage site extension are shown in bold. The 5 'starting base of the mutagenic primer is in italics. The resulting chimeric AA sequence is shown in bold. (See text); Highly preserved AA triplets are further underlined.
[0097]
Amplification of the cα fragment was performed by “nested” PCR. The mutation was placed on the “nested” primer (for_hucaTCR_AlwNI_3; SEQ ID NO: 27), while the RT-PCR primer (for_hucaTCR_2; SEQ ID NO: 25) still corresponded to the human wild-type sequence. The reverse "nested" primer rev_hucaTCR_BamHI_1 (SEQ ID NO: 28) carries both a stop codon and a cloneable BamHI cleavage site, whereas the reverse RT-PCR primer (rev_hucaTCR_2; SEQ ID NO: 26) has a 3'-existing non-coding. Recognized area.
[0098]
In the case of βTCR, the principle of “nested” PCR coupled with blunt end ligation was used as well (SEQ ID NOs: 29-32). The highly conserved triplet sequence consisted of "EDL" (bases 400-408 relative to the start codon of murine βTCR). The unique BstYI cleavage site, containing the variable gene segment, was equally located (bases 402-407). A primer specific for the human constant domain gene segment (for_hucbTCR_4; SEQ ID NO: 31) should be complemented via its 5 ′ end, in which phosphorylated “T” is the last of the potentially batch recognition sequence of BstYI. BstYI digestion (filled with T4-DNA polymerase to make up for the addition of dNTPs, resulting in an overhanging 5'-end that is not degraded) and 3 of the murine variable domain gene segment so that the correct reading frame meaning is retained. The '-end is blunt-ended and filled. Subsequently, the vβ fragment was cleaved with EcoRI, isolated from the LMP gel, and the cβPCR amplicon was used in ligation as a CT4 polynucleotide kinase, which was enzymatically phosphorylated, and a NEB unidirectional blunt-ended BamHI fragment. Subsequently, the combined ligation products were cut with EcoRI / BamHI and a fragment with the potentially correct fragment length was cloned into pcDNA3.1 (-). Asymmetric test digestion showed clones with the desired sequence and orientation of the ligation starting material.
[0099]
[Table 2]
[0100]
The naturally occurring BstYI cleavage site in the murine sequence is shown in bold and the 5 'shift cleavage position resulting in a 5'-overhang is underlined. The 5 'initiation and phosphorylated base "T" of the human "nested" primer, which complements the last base of BstYI in the chimeric construct, is in italics. The resulting chimeric AA sequence is shown in bold (see text); the highly conserved AA triplet is further underlined.
[0101]
The region where the chimera is highly conserved is E¹³³-D-L-N¹³⁶And here, N¹³⁶Represents the first human-specific amino acid. E¹³³Is located at the seventh position after the conserved "GxG" motif that completes the CDR3 loop.
[0102]
2.3) Creation of single-chain TCR muvα-Li-muvβ-muccβ (SEQ ID NO: 42) and muvβ-Li-muvα-muccα (SEQ ID NO: 43)
Single-chain TCRs consist of a fusion of variable domains vα and vβ and the addition of constant domains cα or cβ (Eshar et al., 1993). Such an advantage is the distinct 1: 1 stoichiometry of the two variable domains in the construct and the distinct reactivity for endogenous heterologous pairing with the exogenous double-stranded TCR to be eliminated It is in. Variable domains are redundant (GGGGS)₃The motif was linked via a flexible polylinker. The terminus of the synthetic oligonucleotide accommodates a unique cleavage site, thereby reducing the carboxy-terminus of one variable domain placed after the essential CDR3 loop to the other variable domain present after the sequence encoding the signal peptide. Could be linked with the beginning of Conceptually, five criteria were taken into account:
a) The polylinker had to be long enough to join the ends of the variable ends. For this purpose, MHC-H-2K^bThe specific murine TCR crystal structure was investigated.
[0103]
b) The polylinker should be such that the termini do not, on the one hand, adversely affect the specificity of the CDR3 loop and, on the other hand, appropriately exclude the signal peptide of an added variable domain with an extension of its functional structure. The end had to be ligated. To do this, the amino acid sequence of each variable domain was additionally included in the polylinker in cases where a unique cleavage site was not ideally present.
[0104]
c) The polylinker, on the one hand, must be flexible, this must be done with the help of a sterically favorable catenation of the glycine residues, and on the other hand, aggregate formation and precipitation It had to be sufficiently soluble to avoid: the hydroxyl group of the serine residue forms a hydrogen bond with the solvating water molecule, forming a local dipole. The coding triplet for glycine residues was changed ("GGC" and "GGA") so as not to adversely affect transcription in the correct reading frame.
[0105]
e) The polylinker cleaves the intermediate in a two-step cloning process, retaining the same internal unique cleavage site as the wild-type sequence, and wild-type at the 3'-end via these unique cleavage sites. The type array had to be almost completed.
[0106]
Under these conditions, two orientations of the variable domain were possible;
In the case of the construct muvα-Li-muvβ-mucβ (SEQ ID NO: 42; FIG. 5), the last consensus motif “GxG” of the variable vα domain and the only cleavage site KpnI after DraIII are replaced by the signal peptide in the vβ domain The coding sequence was chosen as the restriction cleavage site to complete. In a first step, the hybridized double-stranded oligonucleotide was cut on both sides with KpnI and cloned between the vα- and cα-coding sequences of the wild-type αTCR. This was followed by cleavage of the chimeric intermediate with the DraIII / BamHI and βTCR-coding sequences, which resulted in the loss of the signal peptide-coding sequence upon DraIII / BamHI digestion and cloning. The first glycine of the polylinker is at position 6 relative to the amino-terminal Gly of the "GxG" consensus motif in vα, and the blunt end ligation in between makes the last serine of the polylinker a position Asp of the vβ domain.²²Before ligating via arginine.
[0107]
In the case of the construct muvβ-Li-muvα-mucα (SEQ ID NO: 43; FIG. 6), the last consensus motif “GxG” of the variable vβ domain and the only cleavage site ScaI after BsgI are signaled in the vα domain. The peptide coding sequence was chosen as the restriction cleavage site to complete. 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 sequences of the wild-type βTCR. This was followed by cleavage of the chimeric intermediate using the BsgI / BamHI and αTCR-coding sequences, which resulted in the loss of the signal peptide-coding sequence by BsgI / BamHI digestion and cloning. The first glycine of the polylinker is at position 6 relative to the amino-terminal Gly of the "GxG" consensus motif in vβ, and the last serine of the polylinker is position Val of the vα domain.²⁴In front of
[0108]
2.4) Creation of double-stranded TCR fused with human CD3ζ-chain muvα-hucα-huCD3ζ (SEQ ID NO: 44; FIG. 7) and muvβ-hucβ-huCD3ζ (SEQ ID NO: 45; FIG. 8)
Signal transduction of the double-stranded TCR should be optimized. For this, each double-stranded α / β TCR was fused to the almost complete CD3ζ subunit of the CD3 complex. According to various publications (Eshar et al., 1994), this fusion involves the effector Lck located upstream or downstream in the cascade.⁵⁶Or results in efficient and CD3-independent coupling of the TCR to ZAP-70.
[0109]
Modified ligation PCR-based techniques have been established, and independent of the unique cleavage site, it is possible to create any desired fusion for the preparation of chimeric proteins, and the resulting PCR The specificity of the amplificate was significantly increased compared to conventional ligation PCR. The gene fusion is defined by a reverse chimeric primer (rev_huca-hu_tm_Zeta, SEQ ID NO: 36, FIG. 7; rev_hucb-hu_tm_Zeta; SEQ ID NO: 37, FIG. 8), whereby the forward primer (T7_for; SEQ ID NO: 33) gives rise to a "mega primer": in the first conventional ligation PCR step, for the 5 'present fusion partner, it gives rise to a "mega primer" which, in addition to the sequence appendix, 3'- Responsible for short sequences specific to existing fusion partners. In order to significantly increase the specificity of the PCR signal, in the first PCR, as early as possible, the coding chimeric amplificate can be used against the first introduced "template" (T7_for; T7_rev; SEQ ID NOs: 33, 34). Externally hybridizing primers are used, then in a second PCR, internally presenting ("nested") primers are used. In this, the megaprimer is linked to a third forward primer "for_aTCR_NcoI_5b; for_bTCR_NcoI_5b; SEQ ID NOs. 20, 22" along with a reverse "nested" primer (rev_hZeta_BamHI_4; SEQ ID NO: 35) that hybridizes to the 3'-present fusion partner. Thus, an intermediate product is present that is "nested" at the 5'-end, which results in an end trimmed PCR amplicon.
[0110]
Restricting the fusion point to cysteine and forming a disulfide bridge: this causes the TCR to lose extracellular membrane sequences, transmembrane and short regions of amino acids up to dimerized cysteine, and the human α-chain to Although losing similarly short extracellular sections, the cystine-forming cysteines should exclusively encode the transmembrane and cytosolic regions, including the three bivalent "ITAM" motifs. The reverse chimeric primer encodes 21 bases at the 3'-end of each partially humanized TCR and 21 bases at the 5 'end of the human CD3' chain (Figure 7). Chimeric constructs are humanized ahead of the constant domains.
[0111]
3. ) Transduction of human peripheral blood lymphocytes (PBL)
For transduction of human peripheral T-lymphocytes, a functional derivative of the pStitch system was used (Weijtens et al., 1999). The retroviral genes required for packaging are encoded by individual plasmids during co-transfection of the packaging cell line 293T (Soneoka et al., 1995): pHit60 is a gag-pol construct from Moloney murine leukemia virus (MoMuLV). And the polymerase gene, pCOLT-Galv is the Na cell of human cells.⁺/ Encoding the env coat protein of the gibbon ape leukemia virus which can bind to the phosphate cotransporter pit and thus transduce the latter. Thus, chimeric virus particles possess the amphoteric pseudotype and can transduce a variety of mammalian cells, except for mice.
[0112]
3.1) Transfection of packing cell line 293T
The isolated bacterial clone of the T-cell receptor gene cloned into the pStitch derivative was purified by a plasmid preparation that promised to remove residual endotoxin (Qiagen, product 12362) and adjusted to 1 μg / μl. DNA was primarily inserted into the packing cell line 293T by calcium phosphate precipitation (GiboBRL-Life Technologies, product 18306-019). In this context, up to 80 μg of DNA is used in the context of wild-type or modified double-stranded T-cell receptors αTCR and βTCR:
20 μg αTCR construct
20 μg βTCR construct
20 μg of pColt-Galv
20 μg of pHit60
For single-stranded TCR, use 60 μg of DNA. 293T was cultured in modified DMEM medium (DMEM / H):
DMEM, 4.5% glucose (BioWhittaker)
10% heat inactivated FCS
2 mM glutamine
1x penicillin / streptomycin
1 x non-essential amino acids
25 mM HEPES
The day before transfection, cells 293T were transferred to T25 bins and 0.9 × 10 cells per transfection batch.⁶Cells were inoculated into 5 ml of DMEM / H. Four hours before transfection, the medium was replaced with fresh DMEM-H (3 ml) warmed to room temperature (RT). Transfections were performed according to commercial protocol instructions (Life Technologies). With careful dropwise addition, 1 ml of the transfection batch was pipetted into each bottle. DNA-Ca₃(PO₄)₂The precipitate should deposit on the adherent cells in a finely dispersed form.
[0113]
The next morning, the medium was replaced with fresh DMEM / H warmed to RT. After 6 hours, co-culture with activated PBL was performed.
[0114]
3.2) Transduction of activated PBL
3.2.1) Activation of peripheral blood lymphocytes (PBL)
Three days prior to the scheduled co-cultures, ficoll-treated PBLs were in each case 2 × 10 5 in huRPMI-P in 2 ml 24-well plates (cell tissue-treated surface).⁶Inoculated at cells / ml. Activation was performed up to 20 ng / ml with the cross-linked antibody OKT-3 (Orthoclone Diagnostics).
[0115]
huRPMI-P:
RPMI 1640 without phosphate (2 mM glutamine) (Life Tec., 11877-032)
10% human heat-inactivated AB serum (HLA-A2.1 seropositive)
25 mM HEPES
1x penicillin / streptomycin (Life Tec.)
Plate 5% CO₂Incubated at 37 ° C. in an incubator.
[0116]
3.2.2) Co-culture
For co-culture, activated PBLs from each cavity of the 24-well plate were combined and counted. The adhesive monocytes were discarded. The cells are centrifuged (1500 rpm, 5 minutes, RT) and 2.5 × 10 5 in fresh huRPMI-P.⁶Cells were harvested at a concentration of cells / ml and returned to the incubator. The medium was adjusted to 400 U / ml IL-2 (Chiron) and 5 μg / ml Polybrene (Sigma) in advance.
[0117]
Six hours after medium change, each transfection batch was sequentially trypsinized: for this, each T25 was washed with 3 ml HBSS (Life Technologies) and incubated with 1 ml trypsin-EDTA (Life Technologies) for at most 5 minutes, Lysed cells were quantitatively collected and added dropwise with stirring to the initially introduced 4 ml of huRPMI-P (RT). The 293T cells are irradiated with 2500 rad. The cells were centrifuged (1500 rpm, 5 min, RT) and resuspended in 4 ml of fresh conditioned huRPMI-P supplemented with 400 U / ml IL-2 and 5 μg / ml Polybrene. 1 ml of adjusted PBL was added to the batch and the batch (0.5 × 10⁶(PBL / ml) was incubated in an incubator for 3 days (37 ° C., 5% CO 2).₂).
[0118]
Three days after the co-culture, the suspended PBL was removed, and fresh medium huRPMI-P supplemented with IL-2 (400 U / ml) was added to 0.5 × 10 5⁶Resuspended to cells / ml. Three days later, an iterative split into fresh media was performed. Within 7 days, culture was performed up to the exchange to T75 bins. These cells were used for immunological staining (FACS analysis; Chapter 3.3, FIG. 9) or by conventional⁵¹It could be used directly in the chromium release test (chapter 3.4, FIGS. 10-15).
[0119]
3.3) Example of FACS analysis
1. After retroviral transduction in "Fluorescence activated cell sorting" (FACS). ) Were analyzed. For this, 0.25 × 10⁶Cells are stained with a fluorophore-labeled antibody under saturation conditions: heterologously expressed β-chain is detected using anti-vβ6-FITC (BD); all of the T-cells are labeled anti-CD3 -Detected by PC5 (Coulter-Beckman) (FIG. 9).
[0120]
The negative control used was a sample transduced with an empty pStitch derivative. Wild-type muvα-mucα / muvβ-mucβ (2.1) and chimeric construct muvα-hucα / muvβ-hucβ (2.2) achieve transduction efficiencies in the range of 10-25% after 6 days of co-culture. did. Expression could be reproduced in a large number of HLA-A2-positive T-cell donors. Comparison as expression of percentage detection of successfully transduced T-cells using green fluorescent protein expressed in cytosol (eGFP, recloned from eGFP-N1 from Clontech) as external standard Weak transduction efficiency was shown to be a methodological issue and not an inherent issue of the TCR transgene.
[0121]
3.3.1) Enrichment of vβ6-positive T-cells by magnetic sorting
To enrich for successfully transduced T-cells, they were separated by a magnetic field with magnetically labeled vβ6 antibody (according to the Miltenyi-Biotec procedure). For this, a magnetically labeled antibody recognizing the rat IgG component of a vβ6-specific antibody (Miltenyi-Biotec “goat anti-rat IgG MicroBeads”, product 485-01) was used. > 90% vβ6-positive CD3⁺It was possible to achieve -T-cell enrichment.
[0122]
3.4) Cytolytic activity of transduced T-cells
Idiomatic⁵¹In the chromium release test, transduced T-cells were checked for their cytotoxic specificity. In this system,⁵¹Target cells were radiolabeled by chromium incorporation. If the effector cells modified by the retrovirus recognize the target cells in a peptide-specific manner, the latter are driven to apoptosis by the effector functions of the T-cells and destroyed by lysis. The extent of chromium released is indicative of the effectiveness of cell recognition and lysis. The efficacy was tested by a wide range of the ratio of effector cells used to target cells (E: T). The reference used was murine hdm2-81-88 peptide-specific T-cell clone 3, from which the T-cell receptor gene was isolated. The target cells used were as follows:
T2: Human TAP-deficient cell line exogenously loaded with any desired peptide. The specific peptide was hdm2-81-88, an irrelevant control peptide derived from the influenza matrix protein FluM1.
[0123]
Saos-2 / 6: A transfectant of the human Saos-2 cell line that expresses hdm2 and processes it endogenously.
[0124]
Saos2 / 143: p53 mutant Val¹⁴³A transfectant of the human Saos-2 cell line carrying Ala. The P53 mutant positively regulates the expression of hdm2.
[0125]
JY: EBV-transformed LCL-B cell line
UocB11, EU-3: pre-B cell leukemia
IM-9: Plasmacytoma.
[0126]
3.4.1) Peptide titration (FIGS. 10 to 12)
For cell line T2, 1 × 10^-6M ~ 1 × 10^-12M concentration series of hdm2-81-88 peptide were exogenously loaded. Peptide-specific and dose-dependent cytotoxic T-cell responses could be observed for both muvα-mucα / muvβ-mucβ (2.1) and muvα-hucα / muvβ-hucβ (2.2) . For the two constructs, at E: T = 20: 1, half-maximal lysis is in the subnanomolar range of the peptide used. The non-functional single-chain T-cell receptor served as a negative control, which was expressed according to 3.3.1) and could be enriched, but showed no lytic activity. The positive control used was murine hdm2-81-88 peptide-specific T-cell clone 3.
[0127]
3.4.2) Recognition of malignantly transformed cell lines (FIGS. 13-15)
The cell lines described above were able to lyse peptide-specifically. Some with pre-B-cell leukemia were able to lyse up to 100% at high E: T with the wild-type chain. Just as in the case of peptide titration, the wild-type chain (2.1) showed higher lysis efficiency compared to the partially humanized chimeric chain (2.2). The non-functional single-chain T-cell receptor served as a negative control, which was expressed according to 3.3.1) and could be enriched, but showed no lytic activity. The positive control used was murine hdm2-81-88 peptide-specific T-cell clone 3.
[Brief description of the drawings]
[0128]
FIG. 1 shows FACS analysis with vβ6-specific FITC-labeled antibody (Pharmigen) (see 1.4). The resulting murine T-cell line and clone 3 were both checked. The isotype control used was anti-TCR Vα2-FITC (BD) and the positive control was anti-pan βTCR-FITC-antibody (BD). The negative control used was influenza matrix protein-specific T-cell clone 58.
FIG. 2 shows cytotoxicity.⁵¹Shows vβ6-specific antibody block of murine T-cell clone 3 in chromium release test (see 1.4). Blocks were evaluated in a dose-dependent manner. The negative control used was, on the one hand, the anti-Vα3.2 antibody (BD) and, on the other hand, the p53-264-272-specific T-cell clone 46, which is an individual that recognizes the p53 peptide. Cross checked against. 10^-8The TAP-deficient cell line T2, which was exogenously loaded with M peptide, served as target cells.
FIG. 3 shows an example of the design of an hdm2-specific construct at the DNA level (see 2.), wherein muV α / β is a murine variable α / β gene segment; β is the murine constant α / β gene segment; huC α / β is the human constant α / β gene segment; TM is the gene segment encoding the transmembrane; Li: (GGGGGS)₃The gene segment encoding the motif and HuCD3ζ is a human CD3ζ-chain gene segment. The restriction cleavage sites associated with cloning are indicated.
FIG. 4 shows the same example of the design of an hdm2-specific construct at the protein level (see 2.), wherein muV α / β is a murine variable α / β domain; muC α / Β is a murine constant α / β domain; huC α / β is a human constant α / β domain; SP: murine wild-type signal peptide; TM: transmembrane; Li is (GGGGS)₃Linker with motif and huζ: Human CD3ζ-chain with 3 consecutive ITAM motifs. The numbers from the bacterial clones are indicated for each chain.
FIG. 5 shows the polylinker of the construct muvα-Li-muvβ-mucβ (see 2.3). The polylinker was finally inserted into the construct obtained by Kpn I and Dra III.
FIG. 6 shows the polylinker of the construct muvβ-Li-muvα-mucα (see 2.3). The polylinker was finally inserted into the construct by Sca I and Bsg I.
FIG. 7 shows the fusion site of a chimeric double-stranded TCR fused to human CD3ζ-chain muvα-hucα-huCD3ζ (see 2.4). Extracytosolic cysteines that dimerize through a disulfide bridge are indicated by their relative position to the starting methionine.
FIG. 8 shows the fusion site of a chimeric double-stranded TCR fused to human CD3ζ-chain muvβ-hucβ-huCD3ζ (see 2.4). Extracytosolic cysteines that dimerize through a disulfide bridge are indicated by their relative position to the starting methionine.
FIG. 9 shows 2. after retroviral transduction. 3) shows a “fluorescence-activated cell sorting” (FACS) analysis of the construct described in 1). An example of the average transduction efficiency for OKT3-activated PBL cultures 6 days after transduction is shown. These are the used CD3⁺It ranges from 10 to 25% of lymphocytes. The negative control used was a transfection / transduction batch containing the empty retroviral vector pBullet.
FIGS. 10-12 show peptide titrations (see 3.4.1), where T2 target cells were loaded at 1 × 10^-6M ~ 1 × 10^-12The hdm2-81-88 peptide was exogenously loaded in a concentration series of M. The reference substance used was huCD8 × A2K^bTransgenic mouse murine T-cell clone 3 hdm2-81-88 (synonym: P2). Indicated⁵¹The chromium release test was performed using transduced PBLs, which showed> 90% of vβ6 by magnetic cell-sorting (Miltenyi-Biotec) directed to vβ6.⁺It was enriched up to T-cells. FIG. 10: The negative control used is a construct of a non-functional single-chain T-cell receptor (nfTCR), which can be expressed relatively well on the surface of T-cells and thereby magnetic cell sorting , But showed no functionality in the CTL test.
FIG. 11: Peptide titration of murine heterodimer wild type construct muTCR / 8-18.
FIG. 12. Peptide titration of chimeric heterodimeric construct huTCR / 10-29.
FIGS. 13-15 show recognition of malignantly transformed cell lines. The selected target cells were HLA-A2⁺Met. The reference used was huCD8 × A2K^b-Transgenic mouse murine T-cell clone 3 hdm2-81-88 (synonym: P2). Indicated⁵¹Chromium release tests show that vβ6> 90% by magnetic cell sorting (Miltenyi-Biotec) directed to vβ6.⁺Performed with transduced PBL enriched to -T-cells. Figure 13: The negative control used is a construct of a non-functional single chain T-cell receptor (nfTCR), which can be expressed relatively well on the surface of T-cells, Although enrichment was also achieved by the treatment, the CTL test showed no functionality.
FIG. 14. Murine heterodimer wild type construct muTCR / 8-18.
FIG. 15. Chimeric heterodimeric construct huTCR / 10-29.

Claims (26)

Murine α / β T-cell receptor polypeptide that mediates an hdm2 protein-specific T-cell response according to SEQ ID NO: 1 or SEQ ID NO: 2, or a functional variant or portion thereof, or a nucleic acid encoding the same, the functional Mutants or parts. A fusion protein comprising the polypeptide according to claim 1, or a functional variant or part thereof, or a nucleic acid encoding the same, a functional variant or part thereof. 3. The fusion protein according to claim 2, comprising the ζ-region of CD3 and / or CD8, CD16 or a part thereof, particularly the ζ-region of human CD3 and / or CD8, CD16 or a part thereof. 4. The fusion protein according to claim 2, comprising a flexible linker, in particular a linker of the amino acid sequence (GGGGS) ₃ . 3. The fusion protein according to claim 2, which is a chimeric, at least partially humanized fusion protein according to SEQ ID NO: 3 or SEQ ID NO: 4. The fusion protein according to any one of claims 2 to 5, which is a single-chain T-cell receptor according to SEQ ID NO: 5 or SEQ ID NO: 6. The fusion protein according to any one of claims 2 to 4, which is a double-stranded T-cell receptor according to SEQ ID NO: 7 or 8. Α- or β-chain of a T-cell receptor comprising the antigen recognition sequence of an antibody specific for amino acids 81-88 of the protein hdm2 or of the complex of hdm2 81-88 and HLA-A2. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide is prepared by synthesis. A DNA or RNA, preferably a DNA, especially a double-stranded DNA having a length of at least 8 nucleotides, preferably having at least 18 nucleotides, in particular having at least 24 nucleotides. 3. The nucleic acid according to 2. 11. The nucleic acid according to claim 1, wherein the sequence of the nucleic acid comprises at least one intron and / or one polyA sequence. 12. A nucleic acid according to any one of claims 1, 2, 10 or 11 in the form of a complementary "antisense" sequence. The nucleic acid according to any one of claims 1, 2, or 10 to 12, wherein the nucleic acid has been prepared by synthesis. 14. A vector comprising a nucleic acid according to any one of claims 1, 2 or 10 to 13, preferably a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or particle and / or active in gene therapy. Vector in the form of a simple vector. A host cell transfected with the vector of claim 14, or infected or transduced with a particle. The host cell according to claim 15, which is a T-cell or a T-cell precursor cell or a stem cell. 17. The host cell according to claim 15, wherein the polypeptide or the fusion protein according to any one of claims 1 to 9 is expressed on the surface thereof. If the tumor expresses the hdm2 protein-specific antigen specific for the expressed polypeptide or fusion protein, it is possible to obtain the hdm2-protein under conditions where the tumor cells or fractions thereof are only lysed. A method for identifying an hdm2 protein-specific antigen, comprising combining a presenting tumor cell or a fraction thereof with the host cell according to claim 17. The polypeptide or a functional equivalent thereof, wherein the antibody-producing organism has at least 6 amino acids, preferably at least 8 amino acids, especially at least 12 amino acids, according to any one of claims 1 to 9 or For diagnosing, treating and / or monitoring the treatment of a disease associated with the hdm2 protein, characterized in that it is immunized with it or vaccinated with a nucleic acid encoding it, and / or A method for the production of an antibody, preferably a polyclonal or monoclonal antibody, directed against a polypeptide, fusion protein or nucleic acid according to any one of claims 1 to 13 for the identification of substances which are active at the same time. 20. An antibody prepared as described in claim 19 directed against the polypeptide of any one of claims 1-9. Recombination characterized in that it comprises an antigen recognition sequence of the α- or β-chain of the T-cell receptor specific for amino acids 81 to 88 of the protein hdm2 or of the complex of hdm2 81-88 and HLA-A2. antibody. Hdm2 protein, characterized in that at least one nucleic acid, at least one polypeptide, at least one host cell or at least one antibody according to any one of the preceding claims is combined with suitable additives and excipients. A method of producing a medicament for the treatment of a disease associated with. Comprising, if appropriate, at least one nucleic acid, at least one polypeptide, at least one host cell or at least one antibody according to any one of the preceding claims, together with suitable additives and excipients. A medicament for treating a disease associated with the hdm2 protein, characterized by comprising: 24. Use of a medicament according to claim 23 for treating a disease associated with the hdm2 protein. Combination of at least one nucleic acid, at least one polypeptide or at least one antibody according to any one of the preceding claims, with suitable excipients and excipients, a function relating to a disease associated with the hdm2 protein. How to create tests to find what works. Hdm2, characterized in that it comprises at least one nucleic acid, at least one polypeptide or at least one antibody according to any one of the preceding claims, if appropriate together with suitable additives and excipients. Tests to identify those that work functionally for protein-related diseases.