WO1999052943A1 - T-zellrezeptor-expressionskassette - Google Patents
T-zellrezeptor-expressionskassette Download PDFInfo
- Publication number
- WO1999052943A1 WO1999052943A1 PCT/EP1999/002171 EP9902171W WO9952943A1 WO 1999052943 A1 WO1999052943 A1 WO 1999052943A1 EP 9902171 W EP9902171 W EP 9902171W WO 9952943 A1 WO9952943 A1 WO 9952943A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- region
- cell
- expression
- regions
- tcr
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
Definitions
- the invention relates to a method for the expression of T-cell receptors and "therefor suitable vectors. Further disclosed with the vectors transfected cells which are capable of respectively desired to express T-cell receptors.
- T lymphocytes are responsible for the cellular immune response.
- Diseased body cells or tumor cells are identified by the so-called T cell receptor (TCR), which binds an antigen specific for the diseased cell in the form of short peptide fragments. These peptide fragments are presented by MHC molecules on the cell surface.
- TCR T cell receptor
- T cell receptors consist of two different polypeptide subunits, usually the so-called T cell receptor ⁇ or ß chains, which are connected to each other by a disulfide bridge.
- the a and ß chains are composed of variable and constant regions.
- the variable regions of the ⁇ chain include V and J gene segments, while the variable regions of the ⁇ chain include V, D and J gene segments.
- the chromosomal TCR ⁇ chain gene consists of approx. 50 to 60 variable segments, each of which contains an exon for a V ⁇ gene segment, which is preceded by another exon, which codes for a leather sequence that imports the protein into the endoplasmic reticulum and the Allows transport to the cell surface.
- a group of 61 J segments is located a considerable distance from the V ⁇ segments. The J segments are in turn followed by a single C ⁇ segment for the constant range, the - 2 - again contains separate exons for the constant region and the hinge region and one exon for the transmembrane and cytoplasmic regions.
- the chromosomal TCRß chain locus contains a group of approx. 65 Vß gene segments, which are at a distance from two separate clusters, each containing a single Dß segment and 6 or 7 Jß segments and a single Cß segment.
- Each constant segment of the ß chain has separate exons for the constant region, the hinge, the transmembrane and the cytoplasmic region.
- a V ⁇ gene segment comes next to a J ⁇ gene segment. This creates a functional exon.
- the mRNA is formed by transcription and splicing of the VJ ⁇ exon to the constant region, which is translated into the TCR ⁇ chain.
- the rearrangement of the V ⁇ , Dß and Jß gene segments coding for the variable domain of the ⁇ chain creates a functional exon which is transcribed and added to Cß by splicing.
- the resulting mRNA is translated into the TCRß chain.
- the a and ß chains combine to form the a: ß TCR heterodimer.
- the hypervariable region of the TCR which is responsible for the specificity of antigen recognition and which lies in the region of the linking of V, (D) and J gene segments is referred to as CDR3.
- T cell receptors The limited availability of native T cells frequently represents a considerable restriction for immunological studies of the T cell receptors expressed by these cells on a functional and molecular level.
- the complex structure of the T cell receptor described above it can be composed of two genes, which in turn consist of one Numerous segments are composed, complete T cell receptors in foreign cells cannot easily be produced recombinantly.
- the object on which the present invention is based was therefore to provide a new system for the recombinant expression of T cell receptors which allows the expression of defined MHC-restricted T cell receptors and thus their investigation at a functional and molecular level.
- an expression unit which contains expression cassettes for TCR ⁇ or TCR ⁇ chains.
- These expression cassettes each contain at least the 3 'side section of the constant portions of the TCR ⁇ or ⁇ chain genes, with artificially inserted restriction sites, in particular multiple cloning sites with several restriction sites, being present in the 5' area of these sections, preferably at least one of the sites is a singular interface. If there are multiple interfaces, the most 3'-sided interface is of particular importance, since the 5'-side interfaces are omitted when the V-gene is cloned. By exploiting the degeneration of the genetic code, the introduction of these interfaces does not result in an amino acid exchange in the TCR chains.
- variable TCR domains must be cloned before the constant domains already contained in the base vector. If required for a specific V gene segment, the artificially inserted restriction sites can be inactivated using restriction endonucleases of partially identical recognition sequences when cloning the variable portion.
- the expression vectors according to the invention enable the simple expression of TCR sequences, in particular human TCR sequences, in eukaryotic cells. In particular, three advantages are achieved: - 4 -
- the expression cassettes can essentially be used for any TCR sequences and are therefore independent of the rearranged V region genes.
- the artificial restriction sites for cloning the V regions are therefore preferably in the 5 'region of the C regions.
- restriction sites inserted by mutagenesis result in neither a reading frame shift nor an amino acid exchange in the finished polypeptide. It is further preferred that there are no identical endogenous cleavage sites within the corresponding fragments, which results in a cloning
- a first aspect of the present invention relates to a base vector for producing a TCR expression vector which has an expression control sequence in operative linkage with a polycistronic, preferably bicistronic, expression unit, comprising:
- the base vector can be a prokaryotic or a eukaryotic vector. It is preferably in eukaryotic cells, in particular in - 5 -
- Mammalian cells such as human propagable vector. Examples of eukaryotic vectors are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1998), Cold Spring Harbor Laboratory Press, Chapter 16, and Winnacker, Genes and Clones, "An Introduction to Gene Technology” (1985) , VCH publishing company, especially in chapters 5, 8 and 10.
- the base vector can be a chromosomal or episomal vector.
- the vector is particularly preferably an episomally replicable vector, in particular a plasmid.
- the base vector according to the invention contains further sequence elements customary for expression vectors, e.g. one or more selection marker genes, such as antibiotic resistance genes, one or more origins of replication and the expression control sequence required for transcription of the expression cassette.
- This expression control sequence can be a prokaryotic or eukaryotic expression control sequence.
- This expression control sequence contains a promoter and optionally transcription regulation and / or activation sequences such as enhancers.
- the polycistronic expression unit according to the invention contains two nucleotide sequences, one of which codes for at least part of the C region of a TCR ⁇ chain and the second codes for at least part of the C region of a TCR ⁇ chain.
- the C region is preferably a human C ⁇ r or Cß region.
- These nucleotide sequences advantageously contain the complete 3 ′ region of the respective C regions. However, the presence of a complete 5 'region is not absolutely necessary since, as explained below, it can be cloned into the base vector together with the respective V region at a later point in time.
- At least one restriction site is located in the 5 'region of the nucleotide sequences coding for the C regions. This - 6 -
- Restriction interfaces are preferably different for the TCR ⁇ chain and for the TCR ⁇ chain. It is particularly preferred that the restriction sites are singular, i.e. restriction sites that occur only once in the entire vector. The introduction of these restriction sites may result in a mutation in the nucleotide sequence coding for the C region. However, this mutation is preferably a "silent" mutation, i.e. a mutation that does not result in an amino acid exchange in the C region. Examples of suitable restriction cleavage sites that can be inserted into the 5 ′ region of the nucleotide sequence coding for the C region of the TCR ⁇ chain are BamHI and / or Xmal cleavage sites.
- restriction sites can easily be introduced by mutations in codons 5 and 6 of exon 1 of the C region of the TCR ⁇ chain (see e.g. Fig. 6).
- the cassette interface is particularly preferably a BamHI site in codon 6 of the C ⁇ gene.
- restriction sites for the C region of the TCR ⁇ chain are Spei and / or Sall sites which are introduced by mutations in codons 28 and 29 of the nucleotide sequence coding for the C ⁇ region (cf. FIG. 7).
- the cassette interface is particularly preferably a Spel site in codon 29 of the Cß gene.
- Restriction sites are particularly preferably introduced into a region which codes for the first fifty amino acids of the respective C region of the TCR chain gene.
- the TCR ⁇ and TCR ⁇ chain genes of human origin the sequences of which are described by Yoshikai et al. (Nature 316 (1985), 837-840) and Toyonaga et al. (Proc. Natl. Acad. Sci. USA 82 (1985), 8624-8628).
- the expression unit of the base vector according to the invention preferably contains a sequence which allows a "capping" independent translation of the polycistronic mRNA molecule resulting from transcription of the expression unit.
- a sequence which allows a "capping" independent translation of the polycistronic mRNA molecule resulting from transcription of the expression unit is an IRES sequence as used in a variety of different ways - 7 -
- Organisms for example viruses.
- IRES sequences can be found in the Picornaviridae, e.g. B. cardioviruses such as encephalomyocarditis virus, enteroviruses such as poliovirus, rhinoviruses such as human rhinovirus, hepatoviruses such as human hepatitis A virus (Fields et. Al., Virology, 3rd Edition (1996), Lipincott-Raven Publishers, Philadelphia), in Flaviridae , e.g. Pestiviruses such as Bovine Viral Diarrhea Virus (Vassiler et al., J. Virol.
- Retroviruses such as HTLV-1 (Attal et al., FEBS Lett. 392 (1996), 220-224) or Moloney-Murine Leukemia Virus (Vagner et al., J. Biol. Chem. 270 (1995), 20376-20383), Leishmania RNA Virus 1 (Maga et al., Mol. Cell. Biol. 15 (1995), 4884-4889), or in humans, e.g. in the BiP gene (Yang and Sarnow, Nucleic Acids Res.
- the expression unit contains a ribosomal translation initiation site directly on the 5 'side of each TCR chain sequence, e.g. the so-called Kozak sequence.
- the expression of the TCR ⁇ chain and the TCR ⁇ chain is preferred.
- the expression of the TCR ⁇ chain can also be limited in comparison to the expression of the TCR ⁇ chain.
- the expression cassette according to the invention can be constructed in the 5'-3 'direction as follows: TCR ⁇ chain gene; IRES sequence; TCR ⁇ chain gene.
- the ribosomal initiation sequence for the TCR ⁇ chain gene can be modified so that the translation initiation is somewhat weaker than for the TCR ⁇ chain gene.
- Another aspect of the present invention relates to a TCR expression vector that an expression control sequence in operative - 8th -
- nucleotide sequence coding for a complete TCR ⁇ chain (a) a nucleotide sequence coding for a complete TCR ⁇ chain and (b) a nucleotide sequence coding for a complete TCR ⁇ chain, the nucleotide sequences coding for the V regions and C regions of the TCR chains via restriction sites in the 5 'region the C regions are linked.
- the nucleotide sequences coding for the TCR chains preferably have at least one base exchange in the region of the restriction cleavage sites compared to the natural TCR sequence, the base exchanges being selected in the context of the degeneration of the genetic code, i.e. lead to a "silent mutation".
- the TCR expression vector according to the invention can in principle be produced by two different methods, namely by first producing a base vector and inserting therein the nucleotide sequences coding for a desired V region, or by dividing the nucleotide sequences coding for the desired V regions in two separate vectors combined with the corresponding C regions and then forms the expression vector from these two vectors.
- Another aspect of the present invention is thus a method for producing a TCR expression vector comprising the steps:
- Yet another aspect of the present invention is a cell transformed with a base vector or with a TCR expression vector as before.
- the cell can be a prokaryotic cell, e.g. be a gram-negative bacterial cell, especially E. coli.
- a cell used for TCR expression is preferably a eukaryotic cell, a mammalian cell, in particular a human cell. Examples of methods for introducing the vectors according to the invention into such cells can be found in Sambrook et al., Supra, and Winnacker, supra.
- a recipient cell for the transformation which is used to express one or more accessory molecules, i.e. capable of carrying out the T cell function of necessary molecules.
- accessory molecules are the cell surface markers
- the recipient cell is therefore a human T cell.
- suitable human T cells are the T cell clones and lines such as 234 (Prof.
- T cells that express a TCR with the desired specificity is to insert a TCR expression vector of the invention into an animal's germline and to recover the T cells from the resulting transgenic animal or progeny thereof.
- Transgenic mice are preferably produced. It is further preferred that the transgenic mice, in addition to the TCR, also express accessory molecules such as the human CD8 molecule and / or the human HLA-A * 0201 molecule.
- the invention thus also relates to a method for expressing a TCR, in which a suitable host cell is transformed with a TCR expression vector and the cell is cultivated under conditions which lead to TCR expression, preferably to expression as a membrane-bound TCR heterodimer.
- the reagent kit for producing a TCR expression vector.
- the reagent kit contains a base vector as previously defined and primers for the amplification of V regions of the TCR ⁇ and TCRß chain genes.
- the reagent kit contains two separate vectors, which contain elements of the expression unit separated from the base vector, and suitable primers for the amplification of V regions.
- the reagent kit can also contain a recipient cell suitable for TCR expression, in particular a human T cell, as explained above. - 1 1 -
- Fig. 1 The production of the TCR ⁇ expression cassette. A TCR ⁇ -specific cDNA was generated using the primers
- 5'C ⁇ EXPs and 3'C ⁇ EXP produced an amplificate of the TCR ⁇ -C region, which ranged from the third codon to the stop codon.
- the oligonucleotide 5'C ⁇ EXPs enabled recognition sequences for the Xmal and Bam HI and the oligonucleotide 3'C ⁇ EXP an interface for the Sal I-
- SV40pA polyandenylation signal from SV40).
- Fig. 2 Cloning of the V ⁇ 20 domain of the T cell clone 26 / B into the
- RNA of the RCC-specific clone 26 / B was reverse-transcribed in a TCR-specific manner and the resulting cDNA was amplified using the oligonucleotides TCRAV20EXP and 5'C ⁇ EXPas.
- the amplificate comprised the entire V ⁇ 20 region (starting at the start codon) and the first nine codons of the C ⁇ region.
- TCRAV20EXP an Eco Rl interface and the optimal Kozak sequence at the 5'-end and with the oligonucleotide 5'-C ⁇ EXPas a Bam HI interface at the 3'- - 12 -
- the DNA molecules were cloned into the vector pBSCIISK + via the Eco R1 and Bam HI interfaces. After sequencing, an error-free subclone was selected and the V ⁇ 20 domain via Eco Rl and Bam Hl in the TCR ⁇
- Fig. 3 Production of the TCRß expression cassette. From a TCRß-specific cDNA using the primers 5'CßEXPs and 3'CßEXP an amplificate of the TCRß-C region was produced, which ranged from the 26th codon to the stop codon.
- the oligonucleotide 5'CßEXPs were used to add recognition sequences for Sal I and Spe I and the oligonucleotide 3'CßEXP an interface for the Hind III restriction endonuclease to the 5 'and 3' ends of the amplified DNA molecules.
- the DNA strands were cloned into pBSCIISK + via the Sal I and Hind III interfaces. After the sequencing, an error-free subclone was selected and the C ⁇ region was cloned into the expression vector pSBC I via the Sal I and Hind III interfaces.
- the vector pSBC I contains two unique restriction sites Ase I and Not I, which allow the fusion with the vector pSBC II, and also an IRES (internal ribosomal entry site) element.
- RNA of the RCC-specific clone RNA of the RCC-specific clone
- Fig. 5 Fusion of the vectors pSBC I and pSBC II to the bicistronic
- the vector pSBC I which contained the TCR ⁇ chain of clone 26 / B
- the vector pSBCII which contained the TCR ⁇ chain of clone 26 / B
- the two halves, each coding for a TCR chain, were then ligated again via the same interfaces, so that both TCR chains of clone 26 / B were coded on the fused expression vector pSBCI / II.
- Fig. 6 TCR ⁇ expression cassette.
- the cDNA sequence is listed centrally in the 5 'region of the C ⁇ region, the triplets corresponding to the actual reading frame. The triplets in bold correspond to codons 5 and 6 in exon 1.
- the primer 5'C ⁇ EXPs (shown above the cDNA sequence) leads to an Xma I in the 5 'region of the C ⁇ amplificate and, using the degenerate code at position 3 in the codon
- the nucleotide exchanges which only produce silent mutations in these two codons, and the corresponding amino acids (proline and aspartic acid) are listed at the bottom of the picture.
- the 3 'regions of the V ⁇ amplificates are modified identically by the 5'C ⁇ EXPas primers (shown under the cDNA sequence) and a Bam HI recognition sequence is thus created again.
- a hypothetical primer is shown, which carries a Bgl II recognition sequence and can inactivate the Bam HI site when the V ⁇ segment is cloned, as described in the text.
- Fig. 7 TCRß expression cassette.
- the cDNA sequence is central in the
- Triplets correspond to codons 28 and 29 in exon 1.
- the primer 5'CßEXPs (shown above the cDNA sequence) introduces a Sal I into the 5 'region of the Cß amplificate and, using the degenerate code at position 3 in codon 29 (G to A transition), introduces a Spe I interface ( shown at the top).
- the nucleotide exchanges, which only cause silent mutations in codons 28 and 29, and the corresponding amino acids (threonine and aspartic acid) are listed at the bottom of the picture.
- the 3 'regions of the V ⁇ amplificates are generated by the 5'CßEXPas primers
- Oligonucleotides 5'CßEXPs and 5'CßEXPas are identical in Cß1 and Cß2.
- Fig. 8 Model of the "synthetic" T cell.
- the cell line Jurkat 9-5 (middle) carries the gene for the ⁇ -galactosidase (lacZ), which is under the control of the IL-2 promoter, stably integrated into the genome.
- lacZ ⁇ -galactosidase
- the successful transfection of this cell with the pSBC l / ll fusion vector leads to the expression of the TCR chains encoded by it on the cell surface.
- the interaction of the expressed TCR with its specific ligand leads to the activation of the IL-2 promoter and thus to the intracellular production of ⁇ -galactosidase.
- the ⁇ -galactosidase is released and can convert a substrate, which leads to the coloration of the reaction solution.
- SEQ ID NO. 1 to 8 oligonucleotides for the TCR expression cassettes
- the TCR of the RCC-specific cytotoxic clone 26 / B was chosen to produce a TCR expression system.
- the ⁇ chain gene of this TCR consists of the variable region V ⁇ 20 and the constant region C ⁇ .
- the TCRß chain gene consists of the variable region Vß22 and the constant region Cß. The sequences of these and all other known V gene segments are described in Arden et al. (Immunogenetics 42 (1995), 455-500). - 16 -
- the TCR chains were cloned in two stages: In the first step, the C regions were cloned ( Figures 1 and 3), i.e. the actual cassettes are completed and in a second step the corresponding V regions are inserted into the expression cassettes ( Figures 2 and 4).
- PSBCI and pSBCII were used as base vectors. These are so-called low copy plasmids, so that all fragments in pBSC II SK + (Stratagene, catalog number 21 22 05) have been cloned in between. This vector replicated in bacteria to a high copy number and also proved to be particularly compatible with the pBSC vectors with regard to the restriction sites.
- the fragments in pBSCMSK - were sequenced and the DNA from error-free subclones was recloned into the expression vectors.
- primers were used which inserted various modifications into the corresponding DNA regions.
- the 5 'primers for the amplification of the C region (5'C ⁇ EXPs and 5'CßEXPs see Fig. 9 and SEQ ID NO. 3 and 7) each carried two recognition sequences for restriction endonucleases, the first of these recognition sequences being freely selectable and used for this purpose to clone the cassette into the expression vector.
- the second recognition sequence was the actual cassette interface and allowed later cloning of the V region (see below).
- the oligonucleotides 3'C ⁇ EXP and 3'CßEXP were used as 3 'primers.
- a PCR protocol: predaturation 94 ° C, 2 min, 1 cycle; denaturation 94 ° C, 30 sec, 30 cycles; annealing 63 ° C, 30 sec, 30 cycles; extension 72 ° C, 1 min, 30 cycles and Final polymerization 72 ° C, 10 min, 1 cycle
- the coding section of the respective C gene segment including the stop codon was amplified.
- an interface for cloning was inserted (Fig. 1 and 3). - 17 -
- the primers TCRAV20EXP and TCRBV22EXP (Fig. 9 and SEQ ID NO. 1 and 5) specific for the V gene segment were used. These primers bind to the V gene sequence in the area of the start codon and simultaneously code for the optimal Kozak sequence and a restriction site.
- the 3 'primers, 5'C ⁇ EXPas and 5'CßEXPas (Fig. 9, SEQ ID NO. 2 and 6) hybridize in the 5' area of the respective C gene segment and introduced mutations according to the cassette interfaces used in each case (Fig. 2 and 4).
- the bicistronic expression unit was produced by fusing the two vectors pSBCI and pSBCII using two singular restriction sites (Ase I and Not I, Fig. 5).
- the two TCR chains flank an IRES element, which leads to "capping" -related translation initiation and thus enables efficient protein synthesis of both TCR chains.
- the capping-dependent translation of the gene located on the 5 'side of the IRES element functions somewhat more efficiently than that of the gene on the 3' side, the TCR ⁇ chain was cloned in pSBC I and the TCR ⁇ chain in pSBC II. In this way, in order to prevent the formation of TCR ⁇ homodimers, a limitation of the ⁇ chain expression compared to the ß chain expression was achieved.
- Sfil interfaces were built in before the TCRß and after the TCR ⁇ chain to allow the two chains to be recloned with the IRES element in between.
- the same Sfil interface is also located in the multiple cloning site of the vector pHEBNA-1 (Mautner et al., Oncogene 12 (1996), 1299-1307 and Mucke et al., Gene Ther. 4 (1997), - 18 -
- Fig. 6 and 7 schematically show the structure and functioning of the TCR ⁇ and ß expression cassettes.
- the 5 'region of the respective C gene segments is shown, in which the V region and C region amplificates overlap.
- the restriction sites listed were inserted by mutations in the oligonucleotides used.
- the first interface was used to clone the C gene segment into the expression vectors (TCR ⁇ : Xmal, TCRß: Sall).
- the V gene segments were cloned in via the respective second interface (TCR ⁇ : Bam HI, TCR ⁇ : Spel). Since the first cleavage sites were removed by cloning the V-gene segments, no consideration had to be given to reading frame shifts and / or amino acid changes when selecting them.
- restriction sites in the expression cassette can be inactivated if necessary, which is necessary if a V ⁇ domain is an endogenous BamHI - 19 - or a Vß domain has an endogenous Spei interface.
- any restriction sites can be inserted through the 3 'primers used for the amplification of the V region, whose core sequences, ie the central four bases of the recognition sequence, are identical to the corresponding sequences of the BamHI or Spei interface (Table 1).
- restriction endonucleases listed in the second column of Table 1 produce cohesive ends which are identical to the restriction sites of the expression cassette part i indicated in the first column.
- the cassettes for the starting enzymes can be inactivated by using these enzymes, but can be cut again by the enzymes listed in the third column.
- the enzymes listed in the brackets represent isoschizomers.
- Fig. 6 and 7 show Bgl II for the TCR ⁇ chain and Xbal for the TCRß chain. It is important that the enzymes used cut asymmetrically. The inactivation is based on the fact that the two "wrong" nucleotides have no effect on the finished polypeptide chains. For both TCR chains, the first nucleotide does not result in an amino acid exchange, since all four possible bases at these positions code for identical amino acids (Figs. 6 and 7). Due to the asymmetrical cut of the restriction - 20 - endonucleases, the second wrong nucleotide is inserted into the opposite strand and therefore has no effect on the translated mRNA.
- the cloned TCR is preferably expressed in a human T cell.
- the recipient cell should have a large proliferation potential in order to have a sufficient number of cells available for the transfection experiments in a relatively short time, but also in order to be able to greatly expand successfully transfected clones, even after a large loss of cell number due to the transfection as such.
- the recipient cell should perform a clear and measurable biological function after specific stimulation via the transfected TCR.
- T cell clones and T cell lines are briefly described below, which are suitable in principle as recipient cells due to their growth and other properties.
- T-cell con 234 An example of a suitable recipient cell is the human T-cell con 234.
- This clone has some special properties: a high growth potential, cytotoxicity and an endogenous receptor that does not recognize any determinant on the RCC-26 tumor cells. This non-recognition is particularly important since this clone is not a transformed cell and therefore requires regular restimulation via its endogenous TCR. The greatest advantage lies in the cytotoxicity of the cell, which is clear and easy to measure as proof of the specific T cell activation. - 21 -
- Established human cell lines come into consideration as further recipient cells, e.g. Molt-4, Peer, Jurkat and various Jurkat variants. These lines offer the advantage of rapid growth and can also be transfected with high efficiency. However, it is disadvantageous for certain applications that they are transformed cell lines which are unsuitable for therapeutic use and have no cytotoxicity.
- the specific activation of these T cells can be demonstrated using secreted cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF).
- IL-2 interleukin 2
- TNF tumor necrosis factor
- the detection of the activation can be carried out particularly easily.
- stimulation of the transfectant via its endogenous or transfected TCR leads to activation of the IL-2 promoter, thus to transcription of the LacZ gene and accumulation of ⁇ -galactosidase in the cytosol.
- the addition of a buffer, which contains a detergent and a substrate for the ⁇ -galactosidase, leads to the lysis of the cells and subsequently the presence of the indicator enzyme can be demonstrated by coloring the reaction buffer.
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000543499A JP2002511487A (ja) | 1998-04-09 | 1999-03-30 | T細胞レセプター発現カセット |
EP99917915A EP1068237A1 (de) | 1998-04-09 | 1999-03-30 | T-zellrezeptor-expressionskassette |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE1998116129 DE19816129A1 (de) | 1998-04-09 | 1998-04-09 | T-Zellrezeptor-Expressionskassette |
DE19816129.8 | 1998-04-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999052943A1 true WO1999052943A1 (de) | 1999-10-21 |
Family
ID=7864252
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/002171 WO1999052943A1 (de) | 1998-04-09 | 1999-03-30 | T-zellrezeptor-expressionskassette |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1068237A1 (de) |
JP (1) | JP2002511487A (de) |
DE (1) | DE19816129A1 (de) |
WO (1) | WO1999052943A1 (de) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1213288A1 (de) | 2000-11-06 | 2002-06-12 | Texas Biotechnology Corporation | Propansäurederivate die die Bindung von Integrinen an ihre Rezeptoren hemmen |
JP2005505236A (ja) * | 2000-12-19 | 2005-02-24 | アルトー バイオサイエンス コーポレイション | ヒト化免疫系を含むトランスジェニック動物 |
US10502745B2 (en) | 2008-03-05 | 2019-12-10 | Agenus Inc. | Identification of antigen- or ligand-specific binding proteins |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1985003947A1 (en) * | 1984-03-01 | 1985-09-12 | The Board Of Trustees Of The Leland Stanford Jr. U | T-cell receptor-specific for antigen polypeptides and related polynucleotides |
WO1991018019A1 (en) * | 1990-05-15 | 1991-11-28 | E.R. Squibb & Sons, Inc. | Soluble single chain t cell receptors |
WO1992006204A1 (en) * | 1990-09-28 | 1992-04-16 | Ixsys, Inc. | Surface expression libraries of heteromeric receptors |
EP0816496A2 (de) * | 1996-06-24 | 1998-01-07 | Roche Diagnostics GmbH | Nierenkarzinom-spezifische-T-Zellen |
-
1998
- 1998-04-09 DE DE1998116129 patent/DE19816129A1/de not_active Withdrawn
-
1999
- 1999-03-30 WO PCT/EP1999/002171 patent/WO1999052943A1/de not_active Application Discontinuation
- 1999-03-30 EP EP99917915A patent/EP1068237A1/de not_active Withdrawn
- 1999-03-30 JP JP2000543499A patent/JP2002511487A/ja active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1985003947A1 (en) * | 1984-03-01 | 1985-09-12 | The Board Of Trustees Of The Leland Stanford Jr. U | T-cell receptor-specific for antigen polypeptides and related polynucleotides |
WO1991018019A1 (en) * | 1990-05-15 | 1991-11-28 | E.R. Squibb & Sons, Inc. | Soluble single chain t cell receptors |
WO1992006204A1 (en) * | 1990-09-28 | 1992-04-16 | Ixsys, Inc. | Surface expression libraries of heteromeric receptors |
EP0816496A2 (de) * | 1996-06-24 | 1998-01-07 | Roche Diagnostics GmbH | Nierenkarzinom-spezifische-T-Zellen |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1213288A1 (de) | 2000-11-06 | 2002-06-12 | Texas Biotechnology Corporation | Propansäurederivate die die Bindung von Integrinen an ihre Rezeptoren hemmen |
JP2005505236A (ja) * | 2000-12-19 | 2005-02-24 | アルトー バイオサイエンス コーポレイション | ヒト化免疫系を含むトランスジェニック動物 |
JP2009034106A (ja) * | 2000-12-19 | 2009-02-19 | Altor Bioscience Corp | ヒト化免疫系を含むトランスジェニック動物 |
US10502745B2 (en) | 2008-03-05 | 2019-12-10 | Agenus Inc. | Identification of antigen- or ligand-specific binding proteins |
Also Published As
Publication number | Publication date |
---|---|
JP2002511487A (ja) | 2002-04-16 |
EP1068237A1 (de) | 2001-01-17 |
DE19816129A1 (de) | 1999-10-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE69738586T2 (de) | Hohe expressionsrate von proteinen | |
DE69034135T2 (de) | DNS-Konstrukten zur Aktivierung und Veränderung der Expression von endogenen Genen | |
DE60221801T2 (de) | Cpg-freie synthetische gene und bakterielle plasmide | |
DE69034238T2 (de) | Verfahren zur Erreichung einer Expression, oder zur Verbesserung der Expression eines Gens | |
DE3485869T2 (de) | Herstellung heterodimerer menschlicher fruchtbarkeitshormone. | |
DE3153606C2 (de) | ||
DE69420033T2 (de) | Gibbon leukemia virus retrovirale vektoren | |
EP0658198A1 (de) | Multicistronische expressionseinheiten und deren verwendung | |
WO2001016345A2 (de) | Sequenz-spezifische dna-rekombination in eukaryotischen zellen | |
DD201693A5 (de) | Verfahren zur synthetisierung von menschlichem proinsulin | |
DE69627644T2 (de) | Vektoren, die therapeutische gene fuer antimikrobielle peptide enthalten, zur verwendung in der gentherapie | |
DE69233612T2 (de) | Verfahren zur bildung einer strukturellen und funktionellen diversität in einer peptidsequenz | |
EP1068237A1 (de) | T-zellrezeptor-expressionskassette | |
DE60202196T2 (de) | Orientationsgerichtete konstruktion von plasmiden | |
EP0299303B1 (de) | Eukaryotische Expressionsvektoren mit multimeren Enhancer-Subelementen, Verfahren zu ihrer Herstellung und Verwendung | |
EP1419259B1 (de) | Verfahren zur reparatur einer mutierten rna aus einer gendefekten dna und zum gezielten abtöten von tumorzellen durch rna-transspleissen sowie verfahren zum nachweis von natürlich-transgespleisster zellulärer rna | |
DE69835369T2 (de) | Alphavirus-vektoren | |
DE3587730T2 (de) | Kontrollsysteme für rekombinant-manipulationen. | |
DE69213282T2 (de) | Antiviren-Protein aus Mirabilis jalapa | |
DE19707493C1 (de) | Nucleinsäurekonstrukte zur lang andauernden Expression von Transgenen | |
EP1000167B1 (de) | Herstellung humaner mutierter proteine in humanen zellen mittels homologer rekombination | |
DE69928960T2 (de) | Herstellung eines retrovirus, welches den mel-lokus von streptomyces enthält, sowie dessen expression in säugetierzellen | |
DE19807265A1 (de) | Adenoviraler Transfervektor für den Gentransport einer DNA-Sequenz | |
DD298269A5 (de) | Verfahren zur herstellung von fremdproteinen in tierischen zellen | |
AT389890B (de) | Verfahren zur herstellung von humaninsulin |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1999917915 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09646892 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 1999917915 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999917915 Country of ref document: EP |