JP2022025350A - Vector expressing protein complex and method for producing the same - Google Patents

Abstract

To provide a means for easily selecting clones in which multiple genes have been correctly inserted.SOLUTION: Provided is a method for efficiently selecting transgenic cells. The method comprises the steps of: (1) amplifying a first gene and a second gene, respectively; (2) providing an expression vector containing nucleic acids encoding (a) the amplified first gene, (b) a self-cleaving linker, (c) the amplified second gene, (d) a self-cleaving linker, and (e) a selectable marker in this order from 5' to 3' direction; and (3) transfecting an animal cell with the expression vector and culturing the transfected cell in a medium containing a selection reagent.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a cell expressing a protein complex. The present invention relates to a nucleic acid in which a plurality of nucleic acids encoding a protein and a nucleic acid encoding a selectable marker are polycistronically linked via a self-cleaving linker in the direction of 5'to 3'.

A multicistron expression vector capable of expressing a plurality of genes in one vector is utilized, for example, for gene therapy and biomedical research in which a plurality of genes are expressed. The multi-cistron expression vector has a problem that the expression levels of multiple proteins are different. However, by connecting multiple genes with a self-cleaving linker and expressing them in one vector system, multiple proteins can be expressed. It has been reported that equal amounts can be efficiently expressed (Non-Patent Document 1). As self-cleavable linkers, picornavirus 2A (2A) peptides, or 2A-like sequences of other viruses, are known to self-cleavage during translation in host cells.

Specifically, the multicistron expression vector using this self-cleavable linker functions by forming a complex of multiple gene products such as B lymphocyte antibody and T lymphocyte T cell receptor (TCR). It is used to efficiently express the protein complex responsible for.

TCR is usually a dimer composed of α and β chains. The α-chain and β-chain of the TCR are constructed by combining the V (variable) region and the C (stationary portion) region, respectively. There are about 50 types of V regions in the human body. In Patent Document 1, 80 types of primers corresponding to the leader sequence containing the translation initiation codon are prepared based on the data of the leader sequence existing on the 5'side of each V region, and using them, ordinary RT-PCR is performed. Discloses a method of amplifying TCR cDNA from a single T cell. Since the amplification product finally obtained from one T cell by the amplification method contains a leader sequence containing a translation initiation codon and a completely variable region, it can be directly inserted into an expression vector and used. Thereby, it can be expressed in T cells.

The nucleic acid sequence encoding the TCRα chain containing each V region amplified by PCR and the nucleic acid sequence encoding the TCRβ chain are linked by a self-cleavable linker and incorporated into an expression vector to efficiently express the TCRα chain and the TCRβ chain. Can be made to.

Japanese Patent Application No. 2016-523577 (Patent No. 6647197)

AL Szymczak et al, Nature Biotechnology 22: 589-594, 2004

However, when connecting multiple genes amplified by PCR, PCR errors and deletion or insertion of the base sequence at the connection site occur. Therefore, the prepared expression was prepared to verify whether the correct expression vector was prepared. It was necessary to gene transfer the vector into Escherichia coli, select clones, prepare plasmid DNA, and analyze the DNA sequence (FIGS. 1A and B). In such a conventional method, there is no problem when one or several vectors are produced, but when a large number of vectors are produced, there are problems of time, labor, and cost, and a faster and simpler method is available. It was desired.

Therefore, an object of the present invention is to provide a means for easily selecting a clone into which a plurality of genes are correctly inserted.

Under the above-mentioned problems, as a result of diligent studies, the present inventors have performed in-frame insertion of multiple genes by linking a self-cleaving linker followed by a selectable marker gene after the gene to be cloned. I found that I could easily select. The present invention has been completed based on the above findings.

According to the present invention, the following inventions are provided.
[1] 1) At least a step of amplifying at least the nucleic acid encoding the first protein and the nucleic acid encoding the second protein;
2) The nucleic acid amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the nucleic acid encoding the amplified first protein, b) self. Steps to provide an expression vector comprising a cleaving linker, c) a nucleic acid encoding the amplified second protein, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) the expression vector. Animal cells were transfected and the transfected cells were cultured in a medium containing a selection reagent, whereby cells in which all of the multiple nucleic acids amplified in step 1) were inserted in-frame were obtained. A method for producing a cell expressing a protein complex, which comprises a step of selection.
[2] 1) At least the step of amplifying the first gene and the second gene, respectively;
2) The gene amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the amplified first gene, b) self-cleavable linker, c) Steps to provide an expression vector containing the amplified second gene, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) transfect the expression vector into animal cells. Gene transfer, comprising the step of culturing the affected cells in a medium containing a selection reagent, thereby selecting cells in which all of the multiple genes amplified in step 1) were inserted in-frame. How to efficiently select cells.
[3] The method according to [1] or [2], wherein the self-cleavable linker is a nucleic acid encoding a self-cleaving 2A peptide.
[4] From [1], the selection marker is selected from the group consisting of a Zeocin ™ resistance marker, a neomycin resistance marker, a puromycin resistance marker, a blastsidedin resistance marker, and a hygromycin resistance marker. The method according to any one of [3].
[5] In the direction from 5'to 3', at least a) a nucleic acid encoding a first protein, b) a self-cleavable linker, c) a nucleic acid encoding a second protein, d) a self-cleaving linker, and e) Nucleic acid encoding a selectable marker.
[6] The first protein is the α chain of the TCR and the second protein is the β chain of the TCR, or the first protein is the β chain of the TCR and the second protein is the β chain. The method according to any one of [1] to [4], which is an α chain of TCR.
[7] In the direction from 5'to 3', a) a nucleic acid encoding a variable (V) α region of the TCR, b) a constant portion of the TCR (C) a nucleic acid encoding an α region, c) a self-cleaving linker, d) Nucleic acid encoding the Vβ region of the TCR, e) Nucleic acid encoding the Cβ region of the TCR, f) Self-cleavable linker, and g) Encoding a selection marker or in the 5'to 3'direction, a ) Nucleic acid encoding the Vβ region of the TCR, b) Nucleic acid encoding the Cβ region of the TCR, c) Self-cleaving linker, d) Nucleic acid encoding the Vα region of the TCR, e) Nucleic acid encoding the Cα region of the TCR, f) Self-cleavable linker, and g) Nucleic acid encoding a selection marker.
[8] 1) A step of amplifying a nucleic acid encoding the Vα region of the TCR and a nucleic acid encoding the Vβ region of the TCR from a biological sample derived from a patient;
2) A step of preparing a virus containing the nucleic acid according to [7] using the amplified nucleic acid;
3) Infecting animal cells with the virus and culturing the infected cells in a medium containing a selection reagent; and 4) selecting a cell line expressing TCR.
A method for producing cells for TCR gene therapy.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a means for easily selecting a clone in which a plurality of genes are correctly inserted.

FIG. 1A is a diagram showing a procedure for selecting a T cell line expressing TCR using a conventional gene cloning method. In the conventional method, the TCR gene was introduced into the IRES-GFP vector to prepare a retrovirus. IRES: Internal ribosome entry site, GFP: Green fluorescent protein. FIG. 1B is a diagram showing an analysis procedure including a step corresponding to dual β and dual α, which are problems in the conventional gene cloning method. When one T cell expresses two kinds of TCRβ or TCRα, the base sequences overlap and the correct base sequence cannot be determined. Therefore, once the cDNA is introduced into the vector, it is put into Escherichia coli to form a colony. It is necessary to prepare plasmid DNA and read the DNA sequence again. FIG. 2 is a diagram showing a procedure of a method for selecting a T cell line expressing TCR according to the present invention. A retrovirus was prepared from the TCR Vβ / Cβ / 2A / Vα / Cα / 2A / Bla ^R (Bla ^R : Blasticidin S resistance gene) vector. FIG. 3A shows the sequences (SEQ ID NOs: 5 and 6) of the hybrid 1G4 TCRβ-P2A-TCRα-P2A-Bla ^R (sequence-1). FIG. 3A shows the sequences (SEQ ID NOs: 5 and 6) of the hybrid 1G4 TCRβ-P2A-TCRα-P2A-Bla ^R (sequence-1). FIG. 3B shows the sequence (sequence-2) (SEQ ID NOs: 7 and 8) of the mCb1-P2A fragment. FIG. 4A shows the retroviral transduction of TCR into human CD8αβ ⁺ BW cells. FIG. 4B is the result of flow cytometry analysis. When cultured in the absence of Blasticidin S (BlaS), only 31% of the cells expressed TCR, whereas when cultured in the presence of Blastic, 82% of the cells expressed CD3 (TCR). Recognize. In FIG. 4C, 32 TCR retroviruses in which both β-chain and α-chain fragments of TCR were amplified were prepared, introduced into a BWT cell line, selected by Blas, and then expressed in TCR (CD3). The result of the analysis is shown. Charts 4, 5, 6, 7, 12, 13, 14, 16, 17, 20, 22, 23, 28, 31 and 32 show TCR (CD3) positive cells by sorting CD3 positive cells. It was able to be concentrated. In FIG. 4C, 32 TCR retroviruses in which both β-chain and α-chain fragments of TCR were amplified were prepared, introduced into a BWT cell line, selected by Blas, and then expressed in TCR (CD3). The result of the analysis is shown. Charts 4, 5, 6, 7, 12, 13, 14, 16, 17, 20, 22, 23, 28, 31 and 32 show TCR (CD3) positive cells by sorting CD3 positive cells. It was able to be concentrated. FIG. 5A shows the evaluation of IL-2 secretion by ELISA. A BW T cell line expressing 19 clones of TCR was co-cultured with a breast cancer cell line (MCF-7) expressing patient HLA (A * 0301, A * 2402, B * 5201), and IL was performed the next day. Analysis of -2 production by ELISA revealed that clone 24 responded. FIG. 5B shows the results of selecting a clone that is likely to react from the clones shown in FIG. 5A and analyzing it again. It was confirmed that 8 clones (1, 3, 24, 29, 4, 7, 14, 20) responded to the MCF-7 cancer cell line into which the patient HLA was introduced. In FIG. 5C, the clone 24 having the highest response among the clones of FIG. 5A (described as TCR10-59 in FIG. 5C) was gene-introduced into human peripheral blood T cells to form an MCF-7 cancer cell line. It is the result of co-culturing and measuring the production of IFN-γ by ELISA the next day. TCR2-15 is a TCR that reacts with cells into which patient HLA has been introduced into an MCF7 breast cancer cell line obtained from TIL derived from a cancer patient "patient-2" different from the patients of Examples 1 and 2. The Maximum detection limit is the upper limit of the standard curve obtained by creating a standard curve using a mouse IL2 having a known concentration and calculating the concentration. It means that the concentration cannot be calculated accurately for the concentration above the Maximum detection limit. FIG. 6 shows the results of a repertoire analysis of TCR of CD8 ⁺ T cells of tumor-infiltrating lymphocytes of a cancer patient (patient-10). The explanation of the table is as follows. Sequence ID: TCR clone name (representative) having the corresponding sequence, Functionality: In frame, TCR protein is indicated as product, TRBV: TCRβ chain V gene, TRBJ: TCRβ chain J gene, TRBD: D gene of TCRβ chain, CDR3b: Amino acid sequence of CDR3 of TCRβ chain (SEQ ID NO: 9-21 in order from the top), TIL-10_PD-1 + / CD137 + _TCRβ Factory: 90 PD-1 + CD137 + CD8 + TIL of breast cancer patient # 10 However, the number of obtained sequences having two or more of the same sequence, and Unique is the number of those having only one T cell having the TCR sequence.

The description of the present invention described below may be based on typical embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.

Further, regarding the state of cells in the present invention, the terms "single" and "one" mean that the environment does not contain cells other than the cells, unless otherwise specified. The terms "group" and "group" of cells mean that they are in an environment where multiple cells exist. Furthermore, the term "strain" for cells refers to a group of cells that can be continuously cultured in vitro without significantly changing their properties, unless otherwise specified.

(Method for producing cells expressing protein complex)
The present invention
1) At least the step of amplifying the nucleic acid encoding the first protein and the nucleic acid encoding the second protein, respectively;
2) The nucleic acid amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the nucleic acid encoding the amplified first protein, b) self. Steps to provide an expression vector comprising a cleaving linker, c) a nucleic acid encoding the amplified second protein, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) the expression vector. Cells transfected into animal cells and cultured in a medium containing a selection reagent, whereby all of the multiple nucleic acids amplified in step 1) were inserted in-frame in the cells. Provided is a method for producing a cell expressing a protein complex, which comprises a step of selecting.

In the present invention, a plurality of (for example, two or more kinds) nucleic acids such as a nucleic acid encoding a first protein and a nucleic acid encoding a second protein are connected via a self-cleavable linker, and further self-cleavage is performed on the 3'side thereof. By connecting with a nucleic acid encoding a selection marker via a sex linker and expressing it in one vector system, a plurality of proteins such as a first protein and a second protein can be efficiently expressed in equal amounts. Only animal cells into which a nucleic acid into which everything from the first protein located on the 5'side to the selection marker on the 3'side has been connected in frame can survive in the medium containing the selection reagent. Utilizing this, gene-introduced cells can be efficiently selected, whereby cells expressing a protein complex can be efficiently obtained. In one embodiment, for example, when a plurality of nucleic acids or genes amplified by RT-PCR are introduced into an expression vector by homologous recombination such as Kibson assembly, all of the plurality of nucleic acids or genes of interest are contained in the product of the homologous recombination. Some clones are introduced in-frame, while others are not well connected and cannot produce the desired protein. In the conventional method, cloning is performed, the clone is verified by gene sequence analysis, the target vector is selected, the gene is introduced into animal cells, and the target protein is expressed and verified. In the method of the present invention, a mixture of clones mixed with jadestone is gene-introduced into animal cells and cultured in a selective medium without performing such cloning or verification steps to prepare cells producing the desired protein. It is possible to do.

Efficient selection of transgenic cells has a higher probability of culturing cells transfected with a selective medium than when culturing cells transfected with a medium that does not contain a selective reagent. This means that a gene-introduced cell in which at least the nucleic acid encoding the first protein and the nucleic acid encoding the second protein are connected in frame is obtained. Whether or not at least the nucleic acid encoding the first protein and the nucleic acid encoding the second protein are connected in frame should be verified by flow cytometric analysis using an appropriate antibody as in the examples described later. Can be done.

The method for producing a cell expressing a protein complex of the present invention is particularly useful in gene therapy, drug candidate screening, vaccine production and construction of a protein complex for structural studies. In the present invention, the protein complex includes, for example, a multi-chain protein such as a B lymphocyte antibody and a T cell receptor (TCR) of a T lymphocyte, a protein complex related to gene expression such as a transcriptional regulatory factor complex, and an intranuclear protein complex. And transport complexes such as complexes involved in intracellular transport, receptor / ligand complexes, cell-cell recognition complexes, complexes involved in apoptosis, complexes involved in cell cycle control, and the like.

The protein complex is composed of a first protein and a second protein, and can further contain a third protein and a fourth protein. When the protein complex contains a third protein, in the expression vector, the nucleic acid encoding the third protein is linked to the 3'side of the nucleic acid encoding the second protein via a self-cleaving linker. Furthermore, a selection marker is connected to the 3'side of the nucleic acid encoding the third protein via a self-cleaving linker. When the protein complex further comprises a fourth protein, the nucleic acid encoding the fourth protein is linked to the 3'side of the nucleic acid encoding the third protein via a self-cleaving linker and further fourth. A selectable marker is attached to the 3'side of the nucleic acid encoding the protein of the above via a self-cleaving linker.

The first, second, third and fourth proteins are arbitrary proteins and may occur naturally as a protein complex or may not naturally occur as a protein complex. Examples of the first and second proteins (complex consisting of two proteins) include TCR (α chain, β chain), CD3 (δ chain, γ chain), antibody (H chain, L chain), and CD8 (. α chain, β chain), MHC class I (H chain, β2 microglobulin), MHC class II (α chain, β chain), integrin (α chain, β chain), NK cell receptor (CD16, Fc receptor γ) Chains; CD16, ζ chains; CD16, DAP12), IL-5 receptors (α chain, β chain), but are not limited thereto. Examples of the first, second and third proteins (complex consisting of three proteins) include CD3 (δ chain, γ chain, ε chain) and IL-2 receptor (α chain, β chain, γ chain). ), Fcε receptor I (α chain, β chain, γ chain), but is not limited thereto. The first, second, third and fourth proteins (complex consisting of four proteins) include CD3 (δ chain, γ chain, ε chain, ζ chain) and B cell receptor (antibody H chain, antibody). L chain, Igα chain, Igβ chain), but are not limited thereto.

In the present invention, encoding a protein means that the ORF encoding the amino acid sequence of the protein is included in the sense or antisense so that the polynucleotide can express the protein under appropriate conditions. The nucleic acid encoding a protein in the present invention is the gene of the protein. In general, a gene can be a naturally occurring or artificially designed sequence.

Step 1) is a step of amplifying the gene of the protein complex of interest contained in cells or tissues by using the PCR method, and the nucleic acid encoding the first protein and the nucleic acid encoding the second protein are respectively. It is amplified by the primers corresponding to. If the protein complex of interest is further composed of a third protein, the nucleic acid encoding the third protein is amplified by the corresponding primer, and so is the case if it is further composed of a fourth protein. .. Although cells may be grown and then subjected to PCR, various PCR methods capable of amplifying cDNA in one or several cells are known, and such a known method is also used in the present invention. Can be applied. The nucleic acids encoding the first and second proteins may both be amplified from the same single cell or the same cell population, or may be amplified from different single cells or different cell populations, respectively. Typically, cytolysis is performed first, followed by cDNA synthesis from mRNA by a reverse transcription reaction using a dT adapter primer (RT dT Primer 2). The obtained cDNA may be amplified or used as it is as a template, or may be subjected to real-time PCR (quantitative PCR, qPCR) using appropriately designed primers.

In step 2), the nucleic acid amplified in step 1) is introduced into an expression vector by homologous recombination, and at least the nucleic acid encoding the first protein, the nucleic acid encoding the second protein, and the nucleic acid encoding the selection marker are encoded. Is a step of providing an expression vector linked and incorporated into polycistronic via a self-cleavable linker. In the present invention, it is preferable to link each gene polycistronically via a self-cleaving linker to form one expression cassette using a single promoter. The nucleic acid amplified in step 1), the self-cleaving linker and the selectable marker are used in a known seamless cloning technique, for example, a method using a Type IIS restriction enzyme, a SLIC (sequence-and ligation independent cloning) method, an overlap PCR method, and a Gibson session. It can be connected to one by the assembly method, the In-Fusion method, or the like. In the present invention, a seamless cloning method can be adopted when introducing the amplified nucleic acid into an expression vector by homologous recombination. The seamless cloning method is a technique in which two DNAs can be linked without using a special vector by having a terminal DNA homologous region of, for example, about 15 to 50 bp.

In one embodiment of the invention, the self-cleavable linker is a nucleic acid encoding a self-cleaving 2A peptide. The self-cleaving 2A peptide is a peptide sequence of about 20 amino acid residues derived from a virus, is recognized by a protease (2A peptidase) endogenous to the cell, and is cleaved at the position of 1 residue from the C-terminal. Multiple genes linked to one unit by a self-cleaving 2A peptide are transcribed and translated as one unit, and then cleaved with 2A peptidase. Examples of the self-cleaving 2A peptide of the present invention include F2A (derived from foot-and-mouth disease virus), E2A (derived from equine rhinitis A virus), T2A (derived from Thosea asigna virus), and P2A (derived from porcine teschovirus-1). Can be mentioned. The same type of self-cleaving 2A peptide may be used multiple times in one expression vector, or different types of self-cleaving 2A peptide may be used. For example, in one expression vector, a nucleic acid encoding P2A is used between the nucleic acid encoding the first protein and the nucleic acid encoding the second protein, and the nucleic acid encoding the second protein and the selection marker are used. A nucleic acid encoding P2A can also be used with the nucleic acid encoding. Furthermore, in one expression vector, a nucleic acid encoding P2A is used between the nucleic acid encoding the first protein and the nucleic acid encoding the second protein, and the nucleic acid encoding the second protein is selected. A nucleic acid encoding F2A can also be used between the nucleic acid encoding the marker. A link that can be transcribed and translated as one unit is called polycistronic.

In the self-cleaving 2A peptide, the nucleic acid encoding the two proteins linked through the peptide is once made from one mRNA, and when it becomes a protein, it is cleaved into two proteins. Therefore, since two proteins are produced in the same ribosome, those proteins are present in close proximity at the time of production, and there is a property that it is easy to form a complex. On the other hand, selectable markers have often been linked at IRES (Internal Ribosome entry site) because there is no positive reason why they must be linked with a self-cleaving 2A peptide. Furthermore, in the past, it was common to make a complete expression vector and use it, and it was rare to use an expression vector containing an incomplete one as in the present invention. Therefore, the idea that the selectable marker is used to select a gene introduced into a cell and to select a gene containing the correct gene has not been considered. Therefore, selectable markers were often connected by IRES.

There is a problem that the expression level of IRES may differ between the genes arranged before and after it. In addition, when IRES is used, even if the gene on the 5'side of the IRES is not connected in frame, the gene on the 3'side of the IRES is correctly translated, so a selectable marker is connected to the 3'side of the IRES. In some cases, this selectable marker is not an indicator of the correct insertion of the gene of interest connected to the 5'side of the IRES.

In the present invention, the selectable marker is a gene introduced as a marker for screening an organism that has succeeded in recombination, and is, for example, a drug resistance gene, a reporter marker gene, or the like. In one embodiment of the invention, the selectable marker is any one selected from the group consisting of a Zeocin ™ resistance marker, a neomycin resistance marker, a puromycin resistance marker, a blastsidedin resistance marker, and a hygromycin resistance marker. ..

Examples of the expression vector of the present invention include a plasmid vector, a binary vector, a DNA vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, an HSV vector, a transposon vector, a PAC vector, and a BAC. Examples include vectors and artificial chromosome vectors. In one embodiment of the invention, the expression vector is a viral vector, preferably a retroviral vector. A retrovirus is a particle having a (+) single-stranded RNA genome of about 100 nm, and after infection, the RNA genome is reverse transcribed into DNA and then integrated into the host chromosome. The viral genome is composed of three types of coding regions, gag, pol, and env, and LTRs (long terminal repeats) at both ends. The LTR contains elements such as promoters, enhancers, and poly A signals necessary for gene expression. It is included. Further, inside the LTR on the 5'side, there is a packaging signal (Ψ) required for the RNA virus genome to be incorporated into the virus particles. Examples of more detailed procedures for constructing the vector are described in the Examples section of this specification, Nat. Biotechnol. 2004 May; 22 (5): 589-594 and Biochem Biophys Res Commun. 474: 709-714, 2016. Can be referred to.

Step 3) is a step of transfecting animal cells with the expression vector provided in step 2) and culturing the transfected cells in a medium containing a selection reagent. In the present invention, transfection can be performed by any method known in the art. For example, nucleic acids can be introduced into cells by lipofection, electroporation, microinjection, and methods using viral vectors. In one embodiment of the invention, transfection is performed by a method using a viral vector, preferably by a method using a retroviral vector. For example, when a vector plasmid prepared from the retroviral vector provided in step 2) is introduced into a packaging cell, the vector RNA genome generated by transcription becomes a virus after the vector DNA is integrated into the host chromosome. It is incorporated into the particles to generate vector viral particles. By infecting animal cells with the retrovirus released into the medium, the gene of interest (ie, at least the nucleic acid encoding the first protein and the nucleic acid encoding the second protein) can be introduced.

In the present invention, the animal cell is any animal cell, preferably a mammalian cell (human cell, monkey cell, hamster cell, rat cell, mouse cell) or an insect cell. Animal cells include bone marrow mesenchymal stromal cells, myocardial cells, Langerhans islet cells, hepatocytes, brain cells, muscle cells, skin cells, kidney cells, bone cells, stem cells (iPS, ES), hematopoietic cells, pancreatic cells, It may include thyroid cells, auditory-related cells, visual-related cells, olfactory cells, nerve cells, and other cells. In one embodiment of the invention, the animal cells are immune cells, such as T cells, B cells, NK cells, macrophages, LAK cells, monocytes or granulocytes.

The transfected cells are cultured in a medium containing a selection reagent, and cells in which all of the plurality of nucleic acids amplified in step 1) have been inserted in-frame are selected. More specifically, the cells that can grow on the selective reagent are cells that express a selectable marker (drug resistance gene), which is a cell that expresses a protein complex. Only animal cells into which a nucleic acid into which everything from the first protein located on the 5'side to the selectable marker on the 3'side has been connected in frame can survive in the medium containing the selection reagent. In the present invention, the selection reagent is a drug that acquires resistance to animal cells when the selection marker used in step 2) is expressed in animal cells. In one embodiment of the invention, the selectable reagent is the agent corresponding to the selectable marker and is selected from the group consisting of Zeocin ™, Omycin resistance, puromycin, blastsidedin, and hygromycin. Examples of the medium include RPMI-1640 medium, IMDM, MEM, DMEM medium and the like.

(Efficient selection of transgenic cells)
The present invention
1) At least the step of amplifying the first gene and the second gene, respectively;
2) The gene amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the amplified first gene, b) self-cleavable linker, c) Steps to provide an expression vector containing the amplified second gene, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) transfect the expression vector into animal cells. Genes comprising the step of culturing the infected cells in a medium containing a selection reagent, thereby selecting cells in which all of the multiple genes amplified in step 1) were inserted in-frame. It relates to a method for efficiently selecting transfectants.

In the present invention, a plurality of genes (for example, two or more types) such as the first gene and the second gene are connected via a self-cleavable linker, and a selectable marker is further attached to the 3'side thereof via the self-cleavable linker. By connecting with the encoding nucleic acid and expressing in one vector system, multiple genes such as the first gene and the second gene can be efficiently expressed in equal amounts, and the second gene arranged on the 5'side can be efficiently expressed. Only animal cells into which a nucleic acid in which everything from the gene 1 to the selection marker on the 3'side is connected in frame can survive in the medium containing the selection reagent. Utilizing this fact, transgenic cells can be efficiently selected. The method of efficiently selecting transgenic cells of the present invention is particularly useful for producing protein complexes. In one embodiment, for example, when a plurality of nucleic acids or genes amplified by RT-PCR are introduced into an expression vector by homologous recombination, the plurality of nucleic acids or genes of interest are introduced in-frame into the product of the homologous recombination. Some clones are not well connected and some clones cannot produce the desired gene product. In the conventional method, cloning is performed, the clone is verified by gene sequence analysis, the target vector is selected, the gene is introduced into animal cells, and the target gene product is expressed and verified. In the method of the present invention, a mixture of clones mixed with jadestone is gene-introduced into animal cells and cultured in a selective medium without performing such cloning or verification steps to obtain cells producing the desired gene product. It can be selected efficiently.

In the present invention, an expression vector in which at least a first gene, a second gene, and a nucleic acid encoding a selectable marker are ligated and incorporated polycistronically via a self-cleavable linker is used. The expression vector can further contain a third gene and a fourth gene in addition to the first gene and the second gene. When the third gene is contained, in the expression vector, the third gene is linked to the 3'side of the second gene via a self-cleaving linker, and further self-cleaving to the 3'side of the third gene. The selectable marker is connected via the linker. When the fourth gene is further contained, the fourth gene is linked to the 3'side of the third gene via a self-cleaving linker, and further to the 3'side of the fourth gene via a self-cleaving linker. The selection marker is connected.

(Nucleic acid)
The present invention further relates to at least a) a nucleic acid encoding a first protein, b) a self-cleaving linker, c) a nucleic acid encoding a second protein, d) self-cleaving in the 5'to 3'direction. Provided are a linker, and e) a nucleic acid encoding a selectable marker. The nucleic acids of the invention are particularly useful in the fields of gene therapy, drug candidate screening, vaccine production and construction of protein complexes for structural studies. The present invention further provides a vector containing the nucleic acid, preferably a retroviral vector. The present invention further provides a composition containing the nucleic acid, preferably a composition for preparing a protein complex.

The nucleic acid of the present invention can include a nucleic acid encoding a first protein and a nucleic acid encoding a second protein, as well as a nucleic acid encoding a third protein, and the nucleic acid encoding the third protein is a nucleic acid. A selection marker is connected to the 3'side of the nucleic acid encoding the second protein via a self-cleaving linker, and further to the 3'side of the nucleic acid encoding the third protein via a self-cleaving linker. .. The nucleic acid of the present invention can include a nucleic acid encoding a first protein, a nucleic acid encoding a second protein, a nucleic acid encoding a third protein, and a nucleic acid encoding a fourth protein. The nucleic acid encoding the protein 4 is connected to the 3'side of the nucleic acid encoding the third protein via a self-cleavable linker, and further to the 3'side of the nucleic acid encoding the fourth protein. The selection marker is connected via.

The first, second, third and fourth proteins are arbitrary proteins and may occur naturally as a protein complex or may not naturally occur as a protein complex. Examples of the first and second proteins (complex consisting of two proteins) include TCR (α chain, β chain), CD3 (δ chain, γ chain), antibody (H chain, L chain), and CD8 (. α chain, β chain), MHC class I (H chain, β2 microglobulin), MHC class II (α chain, β chain), integrin (α chain, β chain), NK cell receptor (CD16, Fc receptor γ) Chains; CD16, ζ chains; CD16, DAP12), IL-5 receptors (α chain, β chain), but are not limited thereto. Examples of the first, second and third proteins (complex consisting of three proteins) include CD3 (δ chain, γ chain, ε chain) and IL-2 receptor (α chain, β chain, γ chain). ), Fcε receptor I (α chain, β chain, γ chain), but is not limited thereto. The first, second, third and fourth proteins (complex consisting of four proteins) include CD3 (δ chain, γ chain, ε chain, ζ chain) and B cell receptor (antibody H chain, antibody). L chain, Igα chain, Igβ chain), but are not limited thereto.

(TCR α and β chains)
In a particular embodiment of the method for producing a cell expressing a protein complex according to the present invention, the first protein is an α chain of TCR and the second protein is a β chain of TCR. The first protein is the β chain of TCR and the second protein is the α chain of TCR. In this case, cells expressing TCR can be produced.

Gene therapy using cells expressing TCR is being investigated for application to specific cancers, and a cancer antigen-specific TCR gene is introduced into lymphocytes of cancer patients. In order to obtain an antigen-specific TCR gene, it is necessary to identify T cells that can recognize cancer antigens from among the T cells in peripheral blood lymphocytes (PBL) collected from patients and clone the TCR gene. .. Common approaches for this include the establishment of antigen-specific T cell clones, which usually require several months. On the other hand, the present inventors have developed a method for amplifying a gene from one cell by using the 5'-RACE method without requiring the establishment of a cell clone (Patent 4069133). Furthermore, the present inventors detect cancer-specific T cells from T lymphocytes in the peripheral blood of cancer patients, and obtain TCR cDNAs that recognize cancer from a single cancer-specific T cell. We are constructing a system that can amplify and verify its cancer specificity in a minimum of 10 days (Patent 6126804).

Furthermore, the present inventors prepared 80 types of primers corresponding to the leader sequence containing the translation initiation codon based on the data of the leader sequence existing on the 5'side of each V region, and used them as usual. We are developing a method for amplifying TCR cDNA from one T cell by RT-PCR (Patent Document 1). Since the amplification product finally obtained from one T cell by the amplification method contains a leader sequence containing a translation initiation codon and a completely variable region, it can be directly inserted into an expression vector and used. Thereby, it can be expressed in T cells.

The TCR is usually a dimer composed of an α chain and a β chain, and the α chain and the β chain of the TCR are constructed by combining a V (variable) region and a C (stationary part) region, respectively. There are about 50 types of V regions in the human body, and in order to comprehensively analyze them, a quicker and simpler method than the conventional method has been desired. The nucleic acid sequence encoding the TCRα chain containing each V region amplified by PCR and the nucleic acid sequence encoding the TCRβ chain are linked by a self-cleavable linker and incorporated into an expression vector to efficiently express the TCRα chain and the TCRβ chain. Can be made to. The present invention further provides a method for easily selecting a clone in which a plurality of genes have been inserted in-frame by connecting a self-cleaving linker followed by a selectable marker gene after the TCR α-chain and β-chain genes.

In certain embodiments of the invention,
1) A step of amplifying the nucleic acid encoding the variable (V) α region of the TCR and the nucleic acid encoding the Vβ region of the TCR, respectively;
2) In the direction from 5'to 3', a) the nucleic acid encoding the Vα region of the amplified TCR, b) the stationary portion of the TCR (C) the nucleic acid encoding the α region, c) the self-cleavable linker, d). An expression vector containing the nucleic acid encoding the Vβ region of the amplified TCR, e) the nucleic acid encoding the Cβ region of the TCR, f) the self-cleavable linker, and g) the nucleic acid encoding the selection marker, or 5'to 3'. In the direction, a) a nucleic acid encoding the Vβ region of the amplified TCR, b) a nucleic acid encoding the Cβ region of the TCR, c) a self-cleaving linker, d) a nucleic acid encoding the Vα region of the amplified TCR, e. ) A step of providing an expression vector containing a nucleic acid encoding the Cα region of TCR, f) a self-cleavable linker, and g) a nucleic acid encoding a selection marker; and 3) transfecting the expression vector into animal cells. Provided is a method for producing a cell expressing TCR, which comprises a step of culturing the affected cell in a medium containing a selection reagent.

In the method of the present invention, the α chain and β chain of TCR can be efficiently expressed in equal amounts, and everything from the Vα region of TCR arranged on the 5'side to the selection marker on the 3'side is in. Only animal cells into which the frame-connected nucleic acid has been introduced can survive in the medium containing the selection reagent. Utilizing this, gene-introduced cells can be efficiently selected, whereby a method for producing cells expressing TCR can be efficiently obtained. In one embodiment of the invention, the animal cells are immune cells, such as T cells, B cells, NK cells, macrophages, LAK cells, monocytes or granulocytes. Further, the immune cells may be immune cells induced to differentiate from iPS cells or ES cells. In one embodiment of the invention, the animal cells are preferably T cells, more preferably T cells that do not express the endogenous TCR.

The step of amplifying the nucleic acid encoding the variable (V) α region of the TCR and the nucleic acid encoding the Vβ region of the TCR can be carried out, for example, by adopting the method for amplifying the TCR cDNA described in Patent Document 1. .. Amplification products can be obtained by performing PCR using cDNA from one cell as a template. Immunospot-array assay on a chip using various existing means, such as flow cytometry or special chips, to identify the T cells of interest and sort one into a container for PCR. , Assay) can be carried out. Flow cytometry is typically focused on two types of surface markers and is used to classify cells that are both positive. One of the two surface markers can be CD4 or CD8, so a CD4 antibody or anti-CD8 antibody can be used for sorting one cell of interest. Sorting of one cell of interest can also be performed by the ISAAC method. This method is carried out using a microwell array (chip) having a plurality of wells on one main surface of the substrate and the wells having a size in which only one T cell can be accommodated. Regarding this method, the prior application of the present inventors (for example, Japanese Patent Application Laid-Open No. 2009-34047 (Patent 4148367)) can be referred to. For sorting, an antigen, an antigen peptide, or a multimerized complex of an MHC molecule and an antigen peptide (for example, MHC / p tetramer) or the like can also be used.

If necessary, one cell to be used may be stimulated in advance under conditions effective for TCR gene amplification. An effective condition for TCR gene amplification is a condition for processing T cells so that amplification by PCR can be effectively performed, which is usually a condition for increasing mRNA that can be a template to a sufficient extent for PCR. Is. This condition may, for example, maintain T cells in the presence of at least IL-2, preferably IL-2 and PHA, for a treatment-effective period. In addition, maintaining T cells in a population during stimulation may effectively increase mRNA that can serve as a template. Regarding stimulation under conditions effective for TCR gene amplification, the previous application by the present inventors (for example, WO2014 / 017533) can be referred to. According to the studies by the present inventors, the TCR can be amplified by the present invention regardless of whether the CD4 positive T cells or the CD8 positive T cells are stimulated.

The cDNA used as a template from one cell can be obtained by obtaining mRNA from the cell and performing a reverse transcription reaction from the obtained mRNA. In the RT-PCR (reverse transcription polymerase chain reaction) method, which amplifies cDNA from mRNA by reverse transcription, a method has been developed in which mRNA is directly separated from cells without performing a total RNA or mRNA extraction operation. For example, the method using magnetic beads coated with Oligo (dT) can complete the separation of mRNA in a short time. In the present invention, such a method can be preferably used.

The conditions for PCR amplification can be appropriately designed by those skilled in the art. PCR amplification can be performed in a system of 1 to several tens of μL, and reagents and enzymes used for general PCR such as buffers, dNTPs, and polymerases can also be used for PCR amplification of the present invention. The concentration of the primer is not particularly limited, but can be, for example, 0.01 to 5 μM. The temperature and time for PCR can be set in the same manner as for general PCR, depending on the polymerase used. In addition, existing devices for PCR can be used to carry out the PCR amplification of the present invention.

It may be confirmed by staining the cell surface with CD3 that the nucleic acid encoding the TCR is properly introduced into the cell. When the TCR gene is introduced into human primary T lymphocytes, which has been conventionally performed, the endogenous TCR may associate with CD3 and be expressed on the cell surface. It is not possible to distinguish between the combination of CD3 and the combination of intrinsic TCR and CD3. Therefore, it is necessary to use an antigen-specific MHC ttramer to confirm the target TCR. However, in the present application, since TCR is introduced into cells having no endogenous TCR, it can be confirmed only by staining the cell surface with CD3.

In the conventional method of introducing the TCR gene into the IRES-GFP vector, for example, the TCRα chain cDNA is amplified by RT-PCR from T cells expressing two types of TCRα chains, and the DNA is amplified by the direct sequence method without cloning. When reading a sequence, the problem arises that the two DNA sequences overlap and the sequence cannot be read. Therefore, it is necessary to once introduce cDNA into the vector, put it in Escherichia coli, form colonies, prepare plasmid DNA, and read the DNA sequence again (Fig. 1B). Two types of TCRα chains can be determined by this, but if these two types of TCRα chains are both functional, it is necessary to introduce two types of TCRα chains into T cells. This is because there are TCRα chains that are well expressed on the cell surface and α chains that are not well expressed due to compatibility with the TCRβ chain. Both of the two TCRα chains may be expressed. Since the expression vector is prepared assuming such a situation, it is very time-consuming and inefficient.
In comparison with this, in the method for producing a cell expressing TCR of the present invention, if the correct combination of TCRβ chain and TCRα chain is combined, it is expressed on the cell surface, so that the T cell being analyzed has two types of TCRα chains. Since it is not necessary to consider whether or not it is expressed and there is no need for analysis, it can be said that it is more efficient than the conventional method.

In the present invention, a) a nucleic acid encoding a variable (V) α region of TCR, b) a nucleic acid encoding a stationary portion (C) α region of TCR, and c) a self-cleaving linker in the direction from 5'to 3'. , D) Nucleic acid encoding the Vβ region of TCR, e) Nucleic acid encoding the Cβ region of TCR, f) Self-cleavable linker, and g) Expression vector containing nucleic acid encoding a selection marker or 5'to 3'. In the direction, a) the nucleic acid encoding the Vβ region of TCR, b) the nucleic acid encoding the Cβ region of TCR, c) the self-cleaving linker, d) the nucleic acid encoding the Vα region of TCR, and e) the Cα region of TCR. Provided are nucleic acids encoding, f) self-cleavable linkers, and g) nucleic acids encoding selection markers. The present invention further provides a vector containing the nucleic acid, preferably a retroviral vector.

(Manufacturing method of cells for tumor treatment)
The present invention
1) A step of amplifying a nucleic acid encoding the Vα region of the TCR and a nucleic acid encoding the Vβ region of the TCR from a biological sample derived from a patient;
2) A step of preparing a virus containing the nucleic acid according to the above (nucleic acid) section using the amplified nucleic acid;
3) Infecting animal cells with the virus and culturing the infected cells in a medium containing a selection reagent; and 4) selecting a cell line expressing TCR.
A method for producing a cell for TCR gene therapy is provided.

Typical examples of diseases or conditions treatable by TCR gene therapy cells produced by the methods of the invention are cancers or infectious diseases, preferably cancer. Diseases to which the present invention can be applied include cancers or infectious diseases, cancers include adult cancers and early childhood cancers, as well as gastrointestinal cancers, lung cancers, and refractory cancers. Includes esophageal cancer, head and neck cancer, ovarian cancer, multiple bone marrow species, etc. Infectious diseases include viral infections (eg, acquired immunodeficiency syndrome (AIDS, coronavirus infection), adult T-cell leukemia, Ebola hemorrhagic fever, influenza, viral hepatitis, viral meningitis, yellow fever, cold syndrome). , Mad dog disease, cytomegalovirus infection, severe acute respiratory syndrome (SARS), progressive polyfocal leukoencephalopathy, varicella, herpes zoster, limb mouth disease, dengue fever, infectious erythema, infectious mononuclear sphere, natural pox , Wind eruption, acute gray erythema myelitis (Polio), measles, pharyngeal conjunctival fever (Pool fever), Marburg hemorrhagic fever, Hantavirus renal hemorrhagic fever, Lassa fever, epidemic parotid inflammation, Westnile fever, Herpangina, Chikungunia fever) , Bacterial infections, viral infections, parasitic infections, prion diseases. "Treatment" includes reducing, preventing, treating, and controlling progression, unless otherwise stated.

In the present specification, a biological sample derived from a patient is isolated from a liquid such as blood, lymph, saliva or cerebrospinal fluid, a solid tissue piece such as a skin biopsy or excised tumor, or a solid tissue thereof collected from the patient. It is a cell. In one embodiment of the invention, the patient-derived biological sample is one cell, preferably one T cell.

In the step of selecting cells expressing TCR in the method of the present invention, cells surviving in the selective medium can be selected as cells expressing TCR cDNA. Further functional analysis can be performed on the selected cells. As a functional analysis, for example, the secretion of IL-2 can be evaluated as described in Examples described later. TCR cDNA-introduced cells (1 × 10 ⁵ ) were treated with IFNγ (100 U / mL, PeroTech Inc.) for 48 hours with a 1 × 10 ⁵ cancer cell line (eg, breast cancer cell line MCF-7). In the presence of 100 U / mL mouse IL-1α (Wako Junyaku Co., Ltd., Osaka, Japan), the cells were cultured in a 96-well plate in 0.2 mL of RPMI medium, and after overnight culture, the supernatant was collected and each of them was cultured. The amount of mouse IL-2 in the supernatant can be measured by enzyme immunoassay (ELISA) according to the manufacturer's instructions. In addition, cells into which TCR cDNA has been introduced should be co-cultured with a cancer cell line expressing patient HLA (for example, breast cancer cell line MCF-7), and IL-2 production should be analyzed by ELISA the next day. You can also. In addition, IFN-γ secretion may be analyzed.

Furthermore, it is possible to evaluate whether TCR has a sufficient ability to induce cytotoxic activity in selected cells. For example, cells in which TCR is expressed on the cell membrane are used as effector cells and are brought into contact with target cells presenting an antigen recognized by TCR. Contact between TCR-expressing cells and antigen-presenting target cells damages the target cells if the TCR has sufficient ability to induce cytotoxic activity. Contact can be performed by co-culturing both cells. The ratio of the TCR-expressing cell (effector cell) to the antigen-presenting target cell (target cell) at the time of contact (effector / target cell ratio, E / Tratio) is a ratio at which cytotoxic activity is sufficiently induced. .. Usually, it is 5 or more, preferably 10 or more. Take time to sufficiently induce cytotoxic activity. It is usually 1 to 48 hours, preferably 2 to 12 hours. Various means for assessing cytotoxicity or cytotoxicity known in the art are available. For example, the methods described in (1) to (4) below can be used. (1) By staining apoptotic cells with a polycaspase reagent, a method for measuring cytotoxicity by flow cytometry can be performed. (2) When a target cell into which a luciferase gene is introduced and expresses luciferase is used, the number of living cells can be measured as the luciferase activity. Cytotoxicity can be measured by measuring the luciferase activity and measuring the number of living cells. Further, (3) the target cell can be labeled with 51Cr and measured by the release of 51Cr from dead cells. (4) When the target cells into which the GFP gene has been introduced and the GFP is expressed are used, the number of living cells can be measured using flow cytometry. Cytotoxic activity can be measured by measuring the number of living cells.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In the present specification, unless otherwise specified, "%" and the like are based on mass, and the numerical range is described as including the end points thereof.

Comparative example (conventional method)
In the conventional method, the TCR gene was introduced into the IRES-GFP vector.
The flow of the experiment is as follows (FIGS. 1A and B):
Vβ cDNA is amplified by RT-PCR from a single T cell, and the base sequence of the amplified Vβ cDNA fragment is analyzed.
Perform a repertoire analysis of TCRβ.
Here, when one T cell expresses two types of TCRβ, the base sequences overlap and the correct base sequence cannot be determined (dual β).
In this case, the Vβ cDNA is incorporated into the vector and the gene is introduced into E. coli.
Escherichia coli is sown on an agar medium, and the next day, about 10 Escherichia coli colonies are picked up, the Vβ gene is amplified, the base sequence is determined, and functional Vβ is identified.
Next, Vα cDNA is amplified by RT-PCR from T cells for which an expression vector is prepared, and the base sequence of the amplified Vα cDNA fragment is analyzed.
Perform a repertoire analysis of TCRα.
Here, when one T cell expresses two types of TCRα, the base sequences overlap and the correct base sequence cannot be determined (dual α).
In this case, the Vα cDNA is incorporated into the vector and the gene is introduced into E. coli.
Escherichia coli is sown on an agar medium, and the next day, about 10 Escherichia coli colonies are picked up, the Vα gene is amplified, the base sequence is determined, and functional Vα is identified.
Next, an expression vector for TCR is prepared. If one T cell has two types of functional Vβ and two types of Vα, four types of expression vectors are prepared.
The TCR Vβ gene and the TCR Vα gene are introduced into the vector shown in the figure by Gibson Assembly (in vitro).
The TCR gene is introduced into E. coli and cultured overnight on an agar medium (Colony culture).
Colonies are picked up, amplified from Vβ5'upstream to BlaR3'downstream by PCR, the DNA sequence is determined, and E. coli having the plasmid DNA with the correct insert is selected.
The selected E. coli is cultured and the plasmid DNA (retroviral vector containing the TCR gene) is purified.
The retroviral vector containing the TCR gene is introduced into Phoenix A cells (packaging cells).
The produced retrovirus infects peripheral blood T cells.
Co-culture with antigen-presenting cells to confirm TCR reactivity.

In the conventional method, the base sequences of Vβ and Vα amplified by RT-PCR are analyzed, and in the case of T cells expressing dual beta and dual plasmid, the base sequences cannot be read, so once inserted into the vector, Escherichia coli. It is necessary to collect about 8 clones of the above, analyze the base sequence of the cloned plasmid DNA, and select a vector with the correct base sequence (a sequence in which a stop codon does not appear in the middle and a TCR protein is produced). In addition, after inserting the amplified Vβ and Vα into the TCR expression vector, it is necessary to analyze whether or not they are correctly inserted by a sequencer. This operation is complicated and it is difficult to handle a large number of samples. In addition, when expressing TCR in T cells, it is necessary to consider the combination of dual alpha and dual beta.

In comparison, in the method exemplified in Example 1 of the present invention shown below, the final assay is not selected (in the middle, the cells are cultured in a Blas selective medium, but humans do not have to take the trouble of selection). Therefore, it is possible to handle a large number of samples.

Example 1
In this embodiment, a method for comprehensively analyzing the function of the TCR is illustrated.
A retrovirus was prepared from the TCR Vβ / Cβ / 2A / Vα / Cα / 2A / Bla ^R (Bla ^R : Blast Saidin S resistance gene) vector and used in BW cells (T cell line lacking endogenous TCR). After infection and culture, the cells were stained with APC-anti-CD3 antibody and flow cytometric analysis was performed.

The flow of the experiment is as follows (Fig. 2):
1: Vβ cDNA is amplified by RT-PCR from a single T cell, and the base sequence of the amplified Vβ cDNA fragment is analyzed. Perform a repertoire analysis of TCRβ. If one T cell expresses two types of TCRβ and the base sequences overlap and the correct base sequence cannot be determined (dual β), continue the analysis without worrying about it.
2: Next, Vα cDNA is amplified by RT-PCR from T cells for which an expression vector is prepared.
3: The TCR Vβ gene and the TCR Vα gene are introduced into the vector shown in FIG. 2 by Gibson Assembly (in vitro).
4: Introduce the gene into E. coli and incubate overnight.
5: Purify plasmid DNA from E. coli.
6: The fragment amplified by PCR is gene-introduced into PlateE cells (packaging cells).
7: Infect the BWT cell line with the produced retrovirus.
8: Select cells expressing the Bla ^R gene in the presence of BlaS.
9: Analyze the function of TCR expressed in BW T cell line.

<Cell>
Breast cancer cells MCF-7 cells were obtained from ATCC (ATCC, HTB-22). HLA-A, B, or C cDNA from breast cancer patient "patient-10" was transduced into MCF-7 cells. (In Example 2 described later, instead of preparing a cancer cell line of a breast cancer patient "patient-10", the patient HLA molecule (A * 0301, A * 2402, B * 5201) is expressed in the breast cancer cell line. It was analyzed whether it would react.)
Since GFP is contained in the vector, GFP-positive cells were selected by FACSAria II as HLA-expressing cells of the patient.
BW cells are from Ellis L. et al., Dana-Farber Cancer Institute, Harvard Medical School. Favorably provided by Professor Reinherz (Feng Y, BrazilKN, Kobayashi E, Mallis RJ, ReinherzEL, Lang MJ. Mechanosensing drives acuity of αβ T-cell recognition. Proc NatlAcad Sci US A. 2017 Aug 15. pii: 201703559. Doi: 10.1073 / pnas.1703559114.). Human CD8α and β cDNA were transduced into BW cells using a retroviral vector (human CD8αβ ⁺ BW cells).

<Construction of hybrid 1G4 TCR vector>
The cDNA of the hybrid 1G4 TCRβ-P2A-TCRα-P2A-BlaR (sequence-1) (FIG. 3A) was artificially synthesized and cloned into the Eco RI / Sal I site in the pMXs retroviral vector (Cell Biolabs).

<Amplification of TCR cDNA derived from a single T cell>
Previous reports of TCR cDNA (Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Amplified from one T cell by one-step RT-PCR and second PCR described in Biophys Res Commun. 474: 709-714, 2016. doi: 10.1016 / j.bbrc. 2016.05.015). T cells were isolated from lymphocytes infiltrating the tumor tissue of the breast cancer patient "patient-10" by single-cell sorting CD8-positive PD-1-positive CD137-positive T cells on a 96-well PCR plate.

<Construction of TCR vector>
The third PCR was performed in the same manner as the second PCR by re-amplifying the second PCR products with Primer 1 and Primer 2 for TCRα and β, respectively. The third PCR product was ligated into a linearized vector using the Gibson Assembly method. The linearized vector was amplified by PCR with Primer 3 and Primer 4 using the Hybrid 1G4 TCR vector as a template. The primer sequences are shown in Table 1.

For plasmid construction, 1 μL of third PCR product of TCRα and TCRβ, 1 μL of mCb1-P2A fragment (10 ng / μL, sequence-2, FIG. 3B), 0.5 μL of linearized vector (50 ng / μL), and NEBuilder HiFi DNA. 3.5 μL of assembly master mix (New England Biolabs, Beverly, USA) was mixed and incubated at 50 ° C. for 20 minutes. The constructed plasmid was used as ECOS competent E.I. E. coli JM109 cells (Nippon Gene) were transformed and cultured in 2 mL of LB medium containing ampicillin for 18 to 24 hours. For plasmid DNA, E.I. Purified from colli culture. Purified plasmid concentration was measured with NanoDrop 2000 (Thermo Scientific, Rockford, USA). Note that mCb1 is part of the constant region of the TCR of the mouse.

<Retrovirus production>
First, 1.0 × 105 Plat-E cells (donated by Professor Toshio Kitamura of the University of Tokyo) were supplied with 10% FCS, ⁵⁰ mmol / L 2-mercaptoethanol, streptomycin (100 mg / mL) one day before transfection. Cultivated in 24-well plates with 0.5 mL DMEM containing penicillin (100 U / mL). The pMXs-TCRβ-P2A-TCRα-P2A-BlaS ^R vector was transfected into Plat-E cells using the FuGENE6 transfection reagent (Roche, E2692) according to the manufacturer's instructions. Cells were cultured at 37 ° C. in 5% CO ² atmosphere. The next day, the cell medium was changed. After 2 days, the culture supernatant was collected, filtered through a 0.45 mm filter (Millipore, SLHV033RS) and stored at -80 ° C until use.

<Introduction of TCR retrovirus transduction into human CD8αβ ⁺ BW cells>
To transduce TCR into human CD8αβ ⁺ BW cells with a retrovirus, wells of a 96-well plate treated with non-tissue culture were coated with 20 μL retronectin (50 mg / mL, Takara, T100B) overnight at 4 ° C. to give the TCR. The encoded retrovirus was spin-filled into wells by centrifugation at 1900 xg for 2 hours at 32 ° C. according to the manufacturer's instructions. Next, 104 human ^{CD8αβ +} BW cells in 100 μL DMEM containing 10% FCS, 50 mmol / L 2-mercaptoethanol, streptomycin (100 mg / mL), and penicillin (100 U / mL) were retrovirused. In addition to the filling wells, they were spun down at 1000 × g at 32 ° C for 10 minutes and incubated overnight at 37 ° C in 5% CO ² . The next day, TCR transduced human CD8αβ ⁺ BW cells were grown in the presence of 20 μg / mL blasticidin S (KK-400, Funakoshi, Tokyo, Japan) for an additional 2 days and used in the experiment.

<Flow cytometry analysis>
TCR transduced human CD8αβ ⁺ BW cells were cultured in the presence of 20 μg / mL Blas and in the absence of Blas, stained with APC-anti-CD3 antibody, and flow cytometric analysis was performed. BW cells originally express GFP.

As a result of flow cytometric analysis, when cultured in the absence of Blasticidin S (BlaS), only 31% of the cells expressed TCR, but when cultured in the presence of Blastic, 82% of the cells were CD3 ( TCR) was expressed (FIG. 4B).

By the above method, 32 TCR retroviruses in which both β-chain and α-chain fragments of TCR were amplified were prepared, introduced into a BW T cell line, selected by Blas, and then TCR (CD3) was expressed. The result of the analysis is shown in FIG. 4C. Charts 4, 5, 6, 7, 12, 13, 14, 16, 17, 20, 22, 23, 28, 31 and 32 show TCR (CD3) positive cells by sorting CD3 positive cells. It was able to be concentrated. The advantage of this method is that it is not necessary to worry about dual alpha and dual beta.

Example 2
In this example, functional analysis was performed on the TCR transduced human CD8αβ ⁺ BW cells selected by Blas in Example 1.

<Evaluation of IL-2 secretion by enzyme-linked immunosorbent assay (ELISA)>
100 U / mL mouse IL- with 1 × 10 ⁵ MCF-7 cells treated with TCR cDNA-introduced human CD8αβ ⁺ BW cells (1 × 10 ⁵ ) with human IFNγ (100 U / mL, PeproTech Inc.) for 48 hours. In the presence of 1α (Wako Junyaku Co., Ltd., Osaka, Japan), the cells were cultured in a 96-well plate in 0.2 mL of RPMI medium. After culturing overnight, the supernatant was collected and the amount of mouse IL-2 in each supernatant was measured by ELISA (R & D Systems) according to the manufacturer's instructions.

The results are shown in FIG. 5A. A BW T cell line expressing 19 clones of TCR was co-cultured with a breast cancer cell line expressing patient HLA (MCF-7), and the next day, IL-2 production was analyzed by ELISA. It turned out to react. It is derived from a unique TCR sequence.

From the clones shown in FIG. 5A, clones likely to react were selected and analyzed again. As a result, as shown in FIG. 5B, 8 clones (1, 3, 24, 29, 4, 7, 14, 20) were found. It was confirmed that the patient responded to the MCF-7 cancer cell line into which HLA was introduced.

Among the clones of FIG. 5A, the clone 24 having the highest response (described as TCR10-59 in FIG. 5C) was gene-introduced into human peripheral blood T cells and co-cultured with the MCF-7 cancer cell line. The next day, IFN-γ production was measured by ELISA (FIG. 5C). It was found that when expressed in human T cells, it exhibits high reactivity.

Example 3
TCR was obtained from tumor infiltrating lymphocyte TIL (tumor infiltrating lymphocyte) using the conventional method and the method of the present invention, and its tumor reactivity was analyzed. FIG. 6 shows the results of a repertoire analysis of TCR of CD8 ⁺ T cells of tumor-infiltrating lymphocytes of a cancer patient (patient-10).

As shown in the pie chart of FIG. 6, in 82 clones, 2 to 6 T cells having the same TCR sequence are present. In addition, there are 47 T cells expressing each unique TCR. It is considered that T cells having the same sequence proliferate clonally in response to the tumor, and it is highly possible that tumor-specific TCR is present in the T cells. , I have analyzed the function of about 5 TCRs of clones growing clonally. Tumor-reactive TCRs may also be present in the remaining TCRs, but they were left unanalyzed because the conventional method is time-consuming.
On the other hand, in the method exemplified in Example 1 of the present invention, almost all the functions of the recovered TCR can be analyzed. Of the 47 clones expressing unique TCRs one by one, 15 clones can amplify the TCRβ chain but not the TCRα chain, or vice versa, or both the TCRβ chain and the TCRα chain. Even if it was amplified, it could not be analyzed because TCR was not expressed on the cell surface. However, as shown in Examples 1 and 2 above, functional analysis could be performed on 32 clones.

The present invention is useful in the field of genetic engineering. Furthermore, the present invention is useful in fields useful in the fields of life science, medical treatment, etc. related to TCR gene therapy for cancer and infectious diseases.

SEQ ID NOs: 1-4: Primer sequences SEQ ID NOs: 5 and 6: Amino acid SEQ ID NOs: 7 and 8: vector sequence and its coding region Amino acid SEQ ID NOs: 9-21: TCRβ chain CDR3 Amino acid sequence of

Claims (8)

1) At least the step of amplifying the nucleic acid encoding the first protein and the nucleic acid encoding the second protein, respectively;
2) The nucleic acid amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the nucleic acid encoding the amplified first protein, b) self. Steps to provide an expression vector comprising a cleaving linker, c) a nucleic acid encoding the amplified second protein, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) the expression vector. Animal cells were transfected and the transfected cells were cultured in a medium containing a selection reagent, whereby cells in which all of the multiple nucleic acids amplified in step 1) were inserted in-frame were obtained. A method for producing a cell expressing a protein complex, which comprises a step of selection. 1) At least the step of amplifying the first gene and the second gene, respectively;
2) The gene amplified in step 1) is introduced into the expression vector by homologous recombination, and in the direction from 5'to 3', at least a) the amplified first gene, b) self-cleavable linker, c) Steps to provide an expression vector containing the amplified second gene, d) a self-cleaving linker, and e) a nucleic acid encoding a selection marker; and 3) transfect the expression vector into animal cells. Gene transfer, comprising the step of culturing the affected cells in a medium containing a selection reagent, thereby selecting cells in which all of the multiple genes amplified in step 1) were inserted in-frame. How to efficiently select cells. The method of claim 1 or 2, wherein the self-cleavable linker is a nucleic acid encoding a self-cleaving 2A peptide. Any of claims 1 to 3, wherein the selectable marker is any one selected from the group consisting of a Zeocin ™ resistance marker, a neomycin resistance marker, a puromycin resistance marker, a blastsidedin resistance marker, and a hygromycin resistance marker. The method described in item 1. In the 5'to 3'direction, at least a) nucleic acid encoding the first protein, b) self-cleavable linker, c) nucleic acid encoding the second protein, d) self-cleavable linker, and e) selection. Nucleic acid encoding a marker. The first protein is the α chain of the TCR and the second protein is the β chain of the TCR, or the first protein is the β chain of the TCR and the second protein is the α of the TCR. The method according to any one of claims 1 to 4, which is a chain. In the direction from 5'to 3', a) a nucleic acid encoding a variable (V) α region of the TCR, b) a constant portion of the TCR (C) a nucleic acid encoding an α region, c) a self-cleaving linker, d) TCR. Nucleic acid encoding the Vβ region of, e) Nucleic acid encoding the Cβ region of the TCR, f) Self-cleavable linker, and g) Encoding a selection marker or in the direction from 5'to 3', a) TCR. Nucleic acid encoding Vβ region, b) Nucleic acid encoding Cβ region of TCR, c) Self-cleavable linker, d) Nucleic acid encoding Vα region of TCR, e) Nucleic acid encoding Cα region of TCR, f) Self Cleavable linker, and g) Nucleic acid encoding a selection marker. 1) A step of amplifying a nucleic acid encoding the Vα region of the TCR and a nucleic acid encoding the Vβ region of the TCR from a biological sample derived from a patient;
2) A step of preparing a virus containing the nucleic acid according to claim 7 using the amplified nucleic acid;
3) Infecting animal cells with the virus and culturing the infected cells in a medium containing a selection reagent; and 4) selecting a cell line expressing TCR.
A method for producing cells for TCR gene therapy.

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