CN115975050A

CN115975050A - Chimeric human T cell receptors, nucleic acids, vectors, cells and pharmaceutical compositions

Info

Publication number: CN115975050A
Application number: CN202211422139.6A
Authority: CN
Inventors: 谢飞
Original assignee: Jin Qiaojiyin
Current assignee: Jin Qiaojiyin
Priority date: 2022-11-14
Filing date: 2022-11-14
Publication date: 2023-04-18

Abstract

The present invention relates to chimeric human T cell receptors, nucleic acids, vectors, cells and pharmaceutical compositions. The chimeric human T cell receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first antigen binding region and a first conserved region, the second polypeptide chain comprises a second antigen binding region and a second conserved region, the first antigen binding region and the second antigen binding region form a first binding domain which is specifically bound with a target spot, the first antigen binding region and the first conserved region are directly connected or connected through a first connecting fragment, and the second antigen binding region and the second conserved region are directly connected or connected through a second connecting fragment.

Description

Chimeric human T cell receptors, nucleic acids, vectors, cells and pharmaceutical compositions

Technical Field

The present invention relates to the field of immunotherapy, and in particular to chimeric human T cell receptors, nucleic acids, vectors, cells and pharmaceutical compositions.

Background

Worldwide cancer morbidity and mortality rates have increased. The fedback number of cancer cases worldwide in 2018 is reported to be 18.1 million, i.e., about 4.95 million new cancer cases are newly diagnosed on average each day, or 34 per minute. It is expected that 24.1 million new cancer cases will occur in 2030, with a compound annual growth rate of 2.4% between 2018 and 2030. Cancer is the leading cause of death worldwide, accounting for 16.7% of all causes of death. About 9.6 million people are expected to die of cancer in 2018 globally, i.e., on average there are nearly 2.62 million cancer-related deaths per day, or 18 per minute. The ten cancers with the largest number of deaths are respectively: lung cancer, colorectal cancer, gastric cancer, liver cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer and leukemia.

Tumor vaccines, cellular immunotherapy, T cell-targeting immunomodulatory drugs, ICIs, and the like are gradually applied to various stages of tumors, wherein ICIs single drug or combination therapy represented by programmed death receptor (ligand) 1 (pd-L1) inhibitors has been observed as sustained remission and significant survival advantage in various solid tumors and is approved for second-line or first-line therapy of various tumors. In terms of tumor immunotherapy approaches, novel anti-tumor immunotherapies aiming at multiple targets and mechanisms are actively being developed, such as lymphocyte activation gene-3 (lag-3) antagonists, CD3 immunomodulators, bispecific antibodies, chimeric Antigen Receptor (CAR) -T cell strategies, and personalized neo-antigen/cancer testis antigen nano-vaccines, etc., all promoting the leap of the tumor immunotherapy era.

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), a Chimeric Antigen Receptor T-Cell Immunotherapy.

The key to this new therapeutic strategy is the recognition of an artificial receptor called a Chimeric Antigen Receptor (CAR) for the target cell, and the ability of patient T cells to express this CAR after genetic modification. In human clinical trials, scientists have extracted some of the T cells from patients through a dialysis-like process and then genetically modified in the laboratory to introduce genes encoding the CAR so that the T cells can express the novel receptor. These genetically modified T cells are propagated in the laboratory and subsequently perfused back into the patient. These T cells bind to the molecule on the surface of the target cell using the CAR receptor they express, and this binding triggers an internal signal generation which then activates the T cells so strongly that they rapidly destroy the target cell.

In recent years, CAR-T immunotherapy has been developed to treat solid tumors, autoimmune diseases, HIV infection, and heart disease, in addition to acute leukemia and non-hodgkin lymphoma, and has a wider application scope.

Although CAR T sings all the way into hematological tumors, it is limited to only B cell lineage associated hematological tumors, and CAR T therapy is slow in advancing in more than 90% of other hematological and solid tumor areas. The following challenges are mainly faced: CAR immunogenicity, cytokine storm, lack of specific targets and target escape for blood cell types outside the B cell lineage, and the like. In addition, the problems of target point reduction of immune system evasion in solid tumors, immunosuppression environment, poor T cell transfer and infiltration and the like are difficult to overcome by the existing T cell technical medicines. To address these challenges, current CAR T therapies are difficult to achieve with good efficacy, and achieving sustained remission remains a small challenge, and patients often experience disease recurrence. In addition, most of the T cell products which are batched at home and abroad or in clinical trial are autologous therapy, a patient needs several worship time from diagnosis to treatment, and the cancer of the patient is very likely to lose the treatment window due to rapid progress; in addition, patients who have entered clinical trials via a variety of treatment regimens have T cells that are not very healthy and are not effectively used to reprogram tumors. Finally, autologous therapy often costs hundreds of thousands of dollars and is a difficult pressure to withstand in the average patient's home. Some current CAR T-based universal T cell therapies of some companies in the market partially solve the problems of long time, high cost and the like of autologous T cells, mainly focus on mature targets of B cells, have unsatisfactory clinical effects, and have the problem of target escape similar to autologous T cell therapy.

Disclosure of Invention

In view of this, there is a need to provide chimeric human T cell receptors that are less prone to immune escape than CARs and that are effective in suppressing the growth of solid tumors.

In addition, a nucleic acid encoding the T cell receptor, a vector, a cell and a pharmaceutical composition containing the nucleic acid, and a preparation method of the cell containing the nucleic acid are also provided.

A chimeric human T cell receptor, comprising:

a first polypeptide chain comprising a first antigen binding region and a first conserved region;

a second polypeptide chain comprising a second antigen binding region and a second conserved region;

the first antigen binding region and the second antigen binding region form a first binding domain for specifically binding to a target, the first antigen binding region is directly connected or connected through a first connecting fragment to the first conserved region, and the second antigen binding region is directly connected or connected through a second connecting fragment to the second conserved region.

In one embodiment, the target that specifically binds to the first binding domain comprises one of FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRINB2, CTLX, E13YIL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF;

the first binding domain is a receptor comprising one of B7H6, DNAM-1, NKG2D, CD, CD4 and CD 16.

In one embodiment, the target that specifically binds to the first binding domain comprises FLT3, and the nucleic acid encoding at least one of the first antigen-binding region and the second antigen-binding region comprises a sequence as set forth in SEQ ID NO:3, or a fragment thereof.

In one embodiment, the first binding domain comprises NKG2D, and the nucleic acid encoding the first antigen-binding region comprises the amino acid sequence set forth in SEQ ID NO: 16-17, and the nucleic acid encoding the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 16-17.

In one embodiment, the first conserved region is a region of the delta chain of a human wild-type TCR other than the variable region, and the second conserved region is a region of the gamma chain of a human wild-type TCR other than the variable region;

in one embodiment, the first conserved region is a region of the α chain of a human wild-type TCR other than the variable region, and the second conserved region is a region of the β chain of a human wild-type TCR other than the variable region;

in one embodiment, the nucleic acid encoding the first conserved region is as set forth in SEQ ID NO:5, and the nucleic acid encoding the second conserved region is as shown in SEQ ID NO: and 6.

In one embodiment, the nucleic acid encoding the first linking fragment and the nucleic acid encoding the second linking fragment each independently comprise a nucleotide sequence as set forth in SEQ ID NO: 10-11, wherein the nucleic acid encoding the first linking segment is different from the nucleic acid encoding the second linking segment.

A T cell receptor, wherein said T cell receptor comprises:

a first polypeptide chain comprising a first broad spectrum binding region, a first antigen binding region and a first conserved region, said first antigen binding region being located between said first broad spectrum binding region and said first conserved region, said first antigen binding region being directly linked or linked through a first linking fragment to said first conserved region;

a second polypeptide chain comprising a second broad spectrum binding region, a second antigen binding region, and a second conserved region, said second antigen binding region being located between said second broad spectrum binding region and said second conserved region, said second antigen binding region being directly linked or linked through a second linking fragment to said second conserved region;

the first antigen binding region and the second antigen binding region form a first binding domain with tumor specificity, and the first broad spectrum binding region and the second broad spectrum binding region form a second binding domain with tumor specificity.

In one embodiment, the target that specifically binds to the first binding domain comprises one of a polypeptide presented on the cell membrane by MHC, CD19, BCMA, GPC3, claudin2, ROR1, ROR2, GPRC5D, FCRL, CEA, FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRINB2, CTLX, E13YIL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF;

in one embodiment, the first binding domain is a receptor comprising one of B7H6, DNAM-1, CD27, CD4, and CD 16;

in one embodiment, the second binding domain is a receptor comprising NKG2D.

In one embodiment, the second binding domain is NKG2D and the first binding domain targets CD19;

in one embodiment, the second binding domain comprises NKG2D1 and NKG2D2, and the nucleic acid encoding the NKG2D1 is as set forth in SEQ ID NO:16, and the nucleic acid encoding the NKG2D2 is shown as SEQ ID NO:17, the first binding domain comprises the heavy chain variable region of CD19 and the light chain variable region of CD19, and the nucleic acid encoding the heavy chain variable region of CD19 is set forth in SEQ ID NO:18, and the nucleic acid encoding the light chain variable region of the CD19 is shown in SEQ ID NO:19, respectively.

In one embodiment, the nucleic acid encoding the first linking fragment and the nucleic acid encoding the second linking fragment each independently comprise a nucleotide sequence as set forth in SEQ ID NO: 10-11, wherein the nucleic acid encoding the first junction fragment is different from the nucleic acid encoding the second junction fragment;

and/or, the first antigen binding region and the first broad spectrum binding region are linked by a third linking fragment, the second antigen binding region and the second broad spectrum binding region are linked by a fourth linking fragment, and the nucleic acid encoding the third linking fragment and the nucleic acid encoding the fourth linking fragment are each independently selected from the group consisting of SEQ ID NOs: 20-21, wherein the nucleic acid encoding the third connecting fragment is different from the nucleic acid encoding the fourth connecting fragment.

A nucleic acid comprising a nucleic acid fragment encoding the T cell receptor described above.

A vector comprising the nucleic acid described above.

A cell comprising the T cell receptor, the nucleic acid, or the vector.

In one embodiment, the cell is selected from one of a lymphocyte, a monocyte and a stem cell.

In one embodiment, the cell is a T cell.

A method for preparing the cell comprises the following steps:

introducing an expression vector for expressing the T cell receptor into a cell; and

culturing a cell containing the expression vector.

A pharmaceutical composition comprising an active agent and an excipient, wherein the active agent comprises the T cell receptor, the nucleic acid, the vector, or the cell.

Drawings

FIG. 1 is a schematic structural diagram of the complex formed on T cells by the three FLD-TCRs in example 1;

FIG. 2 is a graph of the levels of IFN γ production following T cell introduction of the three FLD-TCRs of example 1;

FIG. 3 is a schematic structural diagram of the complex formed on T cells by the three FLD-TCRs in example 5;

FIG. 4 is the results of transduction efficiencies of three FLD-TCRs in example 5;

FIG. 5 is the results of the ratio of CD4 and CD8 for the three FLD-TCRs in example 5;

FIG. 6 shows the results of three FLD-TCR differentiation states in example 5;

FIG. 7 is a graph of the level of IFN γ production following T cell introduction by incubation with target cells for the three FLD-TCRs of example 5;

FIG. 8 is the results of the lysis rate of target cells by the three FLD-TCRT cells in example 5;

FIG. 9 is a schematic structural diagram of the complex formed by the FLD-TCR and CAR on T cells in example 6;

FIG. 10 is the level of IFN γ production after incubation of FLD-TCRT cells and CART cells with target cells in example 6;

FIG. 11 is the effect of FLD-TCR and CAR on tumor size inhibition in the mouse tumor model of example 7;

FIG. 12 is the effect of FLD-TCR and CAR on survival in mouse tumor models in example 7;

FIG. 13 is a schematic structural diagram of a complex formed by NKG2D-TCR on T cells in example 8;

FIG. 14 is a partial schematic view of an expression vector for NKG2D-TCR according to example 8;

FIG. 15 shows the results of flow assay of NKG2D and EGFP co-expressing cell populations in example 8;

FIG. 16 is a graph showing the ratio of CD4 to CD8 and the T cell differentiation phenotype of NKG2D-TCR transformed into activated T cells by virus in example 8;

FIG. 17 shows the results of the killing of different cancer cells by NKG2D-TCR in example 8;

FIG. 18 shows the effect of the mouse model of NKG2D-TCR of example 8;

FIG. 19 is a schematic representation of the structure of complexes formed on T cells by the three dual target TCRs of example 9;

figure 20 is a graph of the levels of IFN γ production following incubation of the three dual-target TCRs of figure 9 with target cells.

Detailed Description

The present application will be described more fully hereinafter to facilitate an understanding thereof, and may be embodied in many different forms and not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. "optionally" means by way of example and not by way of an optional meaning.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

One embodiment of the present application provides a chimeric human T cell receptor comprising a first polypeptide chain comprising a first antigen binding region and a first conserved region, and a second polypeptide chain comprising a second antigen binding region and a second conserved region, wherein the first antigen binding region and the second antigen binding region form a first binding domain that specifically binds to a target, the first antigen binding region is directly connected or connected via a first connecting fragment to the first conserved region, and the second antigen binding region is directly connected or connected via a second connecting fragment to the second conserved region.

In some embodiments, the target that specifically binds to the first binding domain comprises one of FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRIN B2, CTLX, E13Y IL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF. Alternatively, the target that specifically binds to the first binding domain comprises FLT3, and the nucleic acid fragment encoding at least one of the first antigen-binding region and the second antigen-binding region comprises a sequence as set forth in SEQ ID NO:3, or a fragment thereof. In one embodiment, the nucleic acid encoding the first antigen binding region comprises the amino acid sequence set forth in SEQ ID NO:3 and the nucleic acid encoding the second antigen binding region comprises the sequence set forth in SEQ ID NO:1, or a fragment thereof. In another embodiment, the nucleic acid encoding the first antigen binding region comprises the amino acid sequence set forth in SEQ ID NO:1, and the nucleic acid encoding the second antigen-binding region comprises the sequence set forth in SEQ ID NO:3, or a fragment thereof. In another embodiment, the nucleic acid encoding the first antigen-binding region and the nucleic acid encoding the second antigen-binding region each comprise a sequence as set forth in SEQ ID NO:3, or a fragment thereof.

In other embodiments, the first binding domain is a receptor comprising one of B7H6, DNAM-1, NKG2D, CD, CD27, and CD 16. Alternatively, the first binding domain comprises NKG2D and the nucleic acid encoding the first antigen-binding region comprises the amino acid sequence as set forth in SEQ ID NO: 16-17, and the nucleic acid encoding the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 16-17. For example, the nucleic acid encoding the first antigen-binding region is as set forth in SEQ ID NO:16, and the nucleic acid encoding the second antigen binding region is as set forth in SEQ ID NO:17 is shown; as another example, a nucleic acid encoding a first antigen-binding region is set forth in SEQ ID NO:17, and the nucleic acid encoding the second antigen binding region is as set forth in SEQ ID NO: shown at 16.

In some embodiments, the first binding domain is a natural ligand or receptor, or a natural antibody without artificial modification. Natural ligands or receptors, or natural antibodies that have not been artificially modified, are more suitable for humans and can reduce immune responses.

In some embodiments, the nucleic acid encoding the first adaptor fragment of any one of the above embodiments and the nucleic acid encoding the second adaptor fragment of any one of the above embodiments each independently comprise a sequence as set forth in SEQ ID NO: 10-11, wherein the nucleic acid encoding the first linking segment is different from the nucleic acid encoding the second linking segment. For example, the nucleic acid encoding the first linking fragment is as set forth in SEQ ID NO:10, and the nucleic acid encoding the second connecting fragment is as shown in SEQ ID NO: shown at 11. Alternatively, the nucleic acid encoding the first linking fragment is as set forth in SEQ ID NO:11, and the nucleic acid sequence encoding the second connecting fragment is shown in SEQ ID NO: shown at 12.

In some embodiments, the first conserved region of the T cell receptor of any one of the embodiments above is a region other than the variable region of the delta chain of a human wild-type TCR and the second conserved region is a region other than the variable region of the gamma chain of a human wild-type TCR. In an alternative specific example, the nucleic acid encoding the first conserved region is as set forth in SEQ ID NO:5, and the nucleic acid encoding the second conserved region is as shown in SEQ ID NO: and 6.

In other embodiments, the first conserved region of the T cell receptor of any one of the embodiments above is a region other than the variable region of the α chain of a human wild-type TCR, and the second conserved region is a region other than the variable region of the β chain of a human wild-type TCR.

In addition, another chimeric human T cell receptor is provided in an embodiment of the present application, which comprises a first polypeptide chain comprising a first pan-binding region, a first antigen-binding region and a first conserved region, wherein the first antigen-binding region is located between the first pan-binding region and the first conserved region, and the first antigen-binding region is directly connected to the first conserved region or connected thereto through a first connecting fragment; the second polypeptide chain comprises a second broad spectrum binding region, a second antigen binding region and a second conserved region, the second antigen binding region is positioned between the second broad spectrum binding region and the second conserved region, and the second antigen binding region is directly connected with the second conserved region or connected with the second conserved region through a second connecting fragment; the first antigen binding region and the second antigen binding region form a first binding domain which is specifically combined with tumor specificity, and the first broad-spectrum binding region and the second broad-spectrum binding region form a second binding domain which is specifically combined with broad-spectrum targets. The T cell receptor comprises two binding domains combined with targets, wherein the first binding domain has tumor specificity and can be combined with the specific tumor targets, and the second binding domain has broad spectrum and can be combined with the universal tumor targets, so that the immune escape is further reduced, and the treatment effect is improved.

Optionally, the target that specifically binds to the first binding domain comprises one of MHC-presented polypeptide on cell membrane, CD19, BCMA, GPC3, claudin18.2, ROR1, ROR2, GPRC5D, FCRL, CEA, FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRIN B2, CTLX, E13Y IL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF.

In some embodiments, the first binding domain is a receptor comprising one of B7H6, DNAM-1, CD27, CD4, and CD 16.

In some embodiments, the second binding domain is a receptor, and further, the receptor that is the second binding domain comprises NKG2D.

In some embodiments, the second binding domain is NKG2D and the target of the first binding domain is CD19. Further, the first binding domain comprises a heavy chain variable region of CD19 and a light chain variable region of CD19. In an alternative embodiment, the first binding domain comprises NKG2D1 and NKG2D2, and the nucleic acid encoding NKG2D1 is as set forth in SEQ ID NO:16, the nucleic acid encoding NKG2D2 is set forth in SEQ ID NO: shown at 17. The nucleic acid encoding the heavy chain variable region of CD19 is set forth in SEQ ID NO:18, and the nucleic acid encoding the light chain variable region of CD19 is set forth in SEQ ID NO:19, respectively.

In some embodiments, the first binding domain is a natural ligand or receptor or a natural antibody without artificial modification; the second binding domain is also a natural ligand or receptor or a natural antibody without artificial modification. Natural ligands or receptors, or natural antibodies that have not been artificially modified, are more suitable for humans and can reduce immune responses.

In some embodiments, the nucleic acid encoding the first linking fragment and the nucleic acid encoding the second linking fragment each independently comprise a sequence as set forth in SEQ id no: 10-11, the nucleic acid encoding the first linking segment is different from the nucleic acid encoding the second linking segment; the first antigen binding region and the first broad spectrum binding region are connected through a third connecting fragment, and the second antigen binding region and the second broad spectrum binding region are connected through a fourth connecting fragment. In one embodiment, the nucleic acid encoding the third linking fragment and the nucleic acid encoding the fourth linking fragment are each independently selected from the group consisting of SEQ id nos: 20-21, wherein the nucleic acid encoding the third linking segment is different from the nucleic acid encoding the fourth linking segment.

In some embodiments, the first conserved region of the T cell receptor of any one of the embodiments above is a region other than the variable region of the delta chain of a human wild-type TCR and the second conserved region is a region other than the variable region of the gamma chain of a human wild-type TCR. In an alternative embodiment, the nucleic acid encoding the first conserved region is as set forth in SEQ ID NO:5, and the nucleic acid encoding the second conserved region is as shown in SEQ ID NO: and 6.

In other embodiments, the first conserved region of the T cell receptor of any of the above embodiments is a region other than the variable region of the α chain of a human wild-type TCR and the second conserved region is a region other than the variable region of the β chain of a human wild-type TCR.

In addition, an embodiment of the present application provides a nucleic acid comprising a nucleic acid fragment encoding the chimeric human T cell receptor of any of the above embodiments.

In some embodiments, both chains of the T cell receptor are expressed on the cell by co-expression. In this case, the nucleic acid fragment for expressing the α chain and the nucleic acid fragment for expressing the β chain, or the nucleic acid fragment for expressing the δ chain and the nucleic acid fragment for expressing the γ chain are located on the same expression vector, and on the expression vector, the two recombinant chains may be linked by a sequence for expressing the 2A peptide.

In other embodiments, the nucleic acid fragments for expressing both strands of the T cell receptor are located on separate expression vectors, in which case expression of both strands of the T cell receptor on the surface of the immune cell is achieved by introducing the expression vectors into the immune cell by co-transfection or co-transduction of the different expression vectors. In this case, the nucleic acid fragments for expressing both strands of the T cell receptor are present independently.

Further, the nucleic acid may further comprise a signal peptide for directing both chains produced within the cell outside the cell, thereby forming a T cell receptor on the cell surface. In an alternative embodiment, the nucleic acid encoding the signal peptide is as set forth in SEQ ID NO:22 to 23. It is understood that, in other embodiments, the nucleotide sequence of the signal peptide is not limited to the above, but may be other.

In some embodiments, the nucleic acid further comprises a reporter gene. For example, the reporter gene is the EGFP gene. In an alternative specific example, the nucleic acid encoding the EGFP gene is as set forth in SEQ ID NO: shown at 7. It is understood that in other embodiments, the reporter gene is not limited to the above, and may be other genes.

In addition, an embodiment of the present application provides a vector comprising a nucleic acid according to any one of the above embodiments. In some embodiments, the empty vector of the above vector is an expression vector. For example, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphaviral vector, a herpes viral vector, a measles viral vector, a poxvirus vector, a vesicular stomatitis viral vector, a retroviral vector, and the like. In other embodiments, the vector is a vector (without the corresponding expression elements) for preserving the nucleic acid.

In addition, an embodiment of the present application provides a cell comprising a T cell receptor of any one of the above embodiments, a nucleic acid of any one of the above embodiments, or a vector of any one of the above embodiments.

In some embodiments, the cell is selected from one of a lymphocyte, a monocyte (e.g., PBMC), and a stem cell. Optionally, the lymphocyte comprises one of a T cell and an NK cell. Optionally, the monocytes comprise PBMCs. Optionally, the stem cells comprise induced pluripotent stem cells (ipscs). It is understood that in other embodiments, the cells are not limited to the above, but may be other cells, such as E.coli.

In addition, an embodiment of the present application provides a method for preparing the above cell, the method comprising the steps of: introducing into a cell an expression vector for expressing a T cell receptor according to any one of the embodiments above; and culturing a cell containing the expression vector.

Based on the effects of the T cell receptor, an embodiment of the present application further provides an application of the chimeric human T cell receptor, the nucleic acid, the vector and the cell of any embodiment in the preparation of a drug for treating tumor.

Furthermore, an embodiment of the present application provides a pharmaceutical composition, including an active substance and an adjuvant, where the active substance includes the chimeric T cell receptor of human origin of any embodiment described above, the nucleic acid of any embodiment described above, the vector of any embodiment described above, or the cell of any embodiment described above.

In other embodiments, the active agent further comprises a substance capable of killing tumor cells or inhibiting tumor growth.

In some embodiments, the tumor is a solid tumor.

It is noted that "active ingredient" as used herein refers to any ingredient that provides pharmacological activity or other direct effect or affects the structure or any function of the body of humans and other animals in the diagnosis, cure, mitigation, treatment, or prevention of disease. As used herein, "adjuvant" includes, but is not limited to, pharmaceutically acceptable adjuvants. Pharmaceutically acceptable excipients refer to excipients which are compatible with the other ingredients of the pharmaceutical formulation and which are suitable for use in contact with the tissue or organ of the recipient (e.g., human or animal). There are no or few complications of toxicity, irritation, allergic response, immunogenicity, or other problems with use.

In addition, an embodiment of the present application provides a method of treating a solid tumor, the method comprising administering to a patient an effective amount of an expression vector comprising a nucleic acid encoding a chimeric human T cell receptor of any of the above embodiments or an immune cell comprising a chimeric human T cell receptor of any of the above embodiments. In some embodiments, the mode of administration is intravenous.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures without specifying the specific conditions in the examples were carried out under the conventional conditions such as those described in the literature, in books, or as recommended by the manufacturer. As used herein, "IFN γ" is equivalent to "IFN- γ"; "Mock" refers to the control group.

Example 1 design of FLT3-TCR and vector construction

The TCR production is expressed in a host cell by designing the structure of each TCR and introducing the designed individual sequences into a lentiviral vector in conjunction with gene synthesis techniques.

The extracellular binding domain of TCR is the binding domain of FLT3 ligand (FLT 3 ligand, FLD) that can recognize and bind, wherein: the nucleotide sequence of the wild-type FLD (FLD 1 sequence for short) is shown as SEQ ID NO:1 is shown in the specification; FLD2 is a polynucleotide formed by codon optimization of a nucleic acid sequence of a wild-type FLD, and the sequence is shown as SEQ ID NO:2 is shown in the specification; FLD3 sequence is a polynucleotide obtained by performing synonymous mutation of a part of bases on FLD2 sequence to reduce homology with FLD1, the sequence being as shown in SEQ ID NO:3, respectively.

Referring to FIG. 1, FLD1 sequence was ligated to the sequences of the conserved regions (including constant region, transmembrane region and intracellular region) other than the variable region of the delta chain for expressing TCR and the sequences of the conserved regions (constant region, transmembrane region and intracellular region) other than the variable region of the gamma chain for expressing TCR, respectively, via a nucleic acid fragment for expressing 2A peptide (P2A, nucleotide sequence shown in SEQ ID NO: 4) to form a transfer plasmid for expressing FLD1-1-DG, wherein FLD1 was expressed by fusion with the conserved regions of the delta chain and FLD1 was expressed by fusion with the conserved regions of the gamma chain. Similarly, FLD1 sequence and FLD2 sequence are respectively connected with the sequences of the conserved regions (constant region, transmembrane region and intracellular region) except the variable region of delta chain for expressing TCR and the sequences of the conserved regions (constant region, transmembrane region and intracellular region) except the variable region of gamma chain for expressing TCR to form the transfer plasmid for expressing FLD 1-2-DG; the FLD1 sequence and the FLD3 sequence are respectively connected with the sequences of the conserved regions (constant region, transmembrane region and intracellular region) except the variable region of the delta chain for expressing TCR and the sequences of the conserved regions (constant region, transmembrane region and intracellular region) except the variable region of the gamma chain for expressing TCR to form the transfer plasmid for expressing FLD 1-3-DG. Wherein: the sequence of the delta chain conserved region of the TCR is set forth in SEQ ID NO:5, the sequence of the gamma chain conserved region of the TCR is shown as SEQ ID NO: and 6. In addition, the constructed expression vector also comprises an EGFP gene and a nucleic acid sequence for expressing a signal peptide, wherein the EGFP gene (the sequence is shown as SEQ ID NO: 7) is also connected with a sequence for expressing FLD (tumor necrosis factor D) through a 2A peptide (P2A) sequence (SEQ ID NO: 4) as a reporter gene; the delta chain signal peptide (the nucleotide sequence is shown as SEQ ID NO: 22) is positioned at the front end (N end) of the delta chain and is used for guiding the delta chain to be secreted to the outside of the cell, and the gamma chain signal peptide (the nucleotide sequence is shown as SEQ ID NO: 23) is positioned at the front end (N end) of the gamma chain and is used for guiding the gamma chain to be secreted to the outside of the cell.

Example 2 preparation of packaged TCR expressing lentivirus

1. The 293T cells were recovered and cultured for one week. 1X 10 wells were plated in 6-well plates one day before transfection ⁶ 293T cells. A1.5 mL sterile EP tube was filled with 1.5. Mu.g of the packaged mixed plasmid and 1. Mu.g of the transfer plasmid (prepared in example 1) and 250. Mu.L of serum-free medium. Mixing, incubating at 26 deg.C for 5min.

2. 1.5mL of sterilized EP tube was taken, 9. Mu.L of liposome PEI was dissolved in 250. Mu.L of serum-free medium, gently mixed, and incubated at room temperature for 5min.

3. Mixing the DNA solution and liposome solution, incubating at room temperature for 20min

4. After trypsinization and counting of 293T cells, the cells were resuspended in serum-containing medium.

5. In six well plates 1mL of growth medium containing serum was added per well, followed by addition of the DNA-liposome complex.

6. 1mL of resuspended 293T cells (1X 10) ⁶ Individual cells/mL) were added to the plate, CO at 37 ℃ ₂ Incubate overnight in the incubator.

7. The medium containing the DNA-liposome complexes was removed and replaced with DMEM (containing sodium pyruvate and optional amino acids).

8. And harvesting the supernatant containing the virus 48-72 h after transfection, centrifuging at 3000rpm for 20min, and removing the precipitate.

9. The virus supernatant was stored at-80 ℃.

EXAMPLE 3 preparation of TCR-T cells

1. Peripheral blood collection of healthy donors: the collected peripheral blood is temporarily stored in a refrigerator at 4 ℃, and is transported to a biochemical laboratory for separation and culture after 24 hours.

2. Preparation of Peripheral Blood Mononuclear Cells (PBMCs): sucking DPBS (Dulbecco's phosphor Buffered Saline) or normal Saline by a pipette, adding the DPBS or the normal Saline into the peripheral blood (volume ratio 1:1) collected in the step 1 for dilution, then slowly adding the blood cell diluent into a centrifuge tube filled with lymphocyte separation liquid (Ficoll or Histopaque-1077), centrifuging for 20min at 800g, sucking tunica albuginea cells above the lymphocyte separation liquid, transferring into a new centrifuge tube, adding a Lonza x-vivo15 culture medium, centrifuging, then discarding supernatant, and reserving cell sediment at the bottom of the centrifuge tube to obtain the peripheral blood mononuclear cells.

3. Isolation and activation of T cells: the obtained peripheral blood mononuclear cells were counted, beads coupled with CD3/CD28 antibody were added in a proportion of 1:1, gently shaken for 20min, and adsorbed by a magnetic frame to obtain CD 3-positive T cells, which were activated, and then the T cells were cultured and expanded by adding complete medium (Lonza x-vivo15+10% FBS +100IU/mL IL-2).

4. Lentivirus infection: three days after activation, the lentivirus (prepared in example 2) encapsulating csTCR was added to the activated T cells at 50% volume, the other 50% volume was the T cell medium, the medium was changed 24 hours later, the culture was continued for 48 hours, the proportion of EGDP positive cells was analyzed by flow cytometry (FACS), and the transfection efficiency was calculated.

Example 4 in vitro assay validation of TCR-T cells

1. Cell flow assay

The T cells to be analyzed are washed twice by special liquid for flow dyeing, then incubated with corresponding antibodies for half an hour at normal temperature, washed twice by PBS and then subjected to cell flow analysis. All antibodies and staining fluids were from BD company,

2. cytokine secretion assay

After lentiviruses with different recombinant TCRs were introduced into activated T cells and cultured for 7 days, they were combined with MV4-11, molm-13 and K562 cells, respectively, in an effective target ratio (E: T ratio) 1:1 (number of target cells is 0.5X 10) ⁶ cells/mL, namely 0.5E6/mL, with a total volume of 1 mL) were placed in a 24-well culture plate, the supernatant was collected after 18 hours of co-culture, and the expression level of IFN-. Gamma.was measured by ELISA, with the results shown in FIG. 2.

As can be seen from FIG. 2, only the T cell vector carrying FLD1-3-DG released IFN- γ after incubation with positive cell line, and neither of the other two fusion proteins could effectively activate T cells and release IFN- γ.

Example 5 TCR after spacer sequence optimization of FLT3 Ligand (FLD) -TCR fusion protein

1. Optimized TCR and TCR-T cells are prepared. Referring to FIG. 3, FIG. 3 is a fused TCR (csTCR) based on FLT3 ligand and receptor. Fig. 3 includes: FLD1 and FLD3 are directly fused with TCR delta and TCR gamma to form FSDG, FLD1 and FLD3 are inserted with IgG1 light chain constant region (IgG 1 CL) and IgG1 heavy chain constant region (IgG 1 CH) between TCR delta and TCR gamma to form FLDG, and FLD1 and FLD3 are inserted with connecting segment (linker) between TCR delta and TCR gamma to form FSLinkDG. Wherein, the nucleotide sequences of FLD1, FLD3, TCR delta and TCR gamma are shown in example 1, and the nucleotide sequence of IgG1CL is shown in SEQ ID NO:8, the nucleotide sequence of IgG1CH is shown as SEQ ID NO:9, the nucleotide sequence of linker1 between the FLD1 sequence and TCR δ is shown in SEQ ID NO:10, the nucleotide sequence of linker2 between FLD3 sequence and TCR γ is shown in SEQ ID NO:11, respectively. The procedure for preparing TCR-T cells of this example was as described in examples 1 to 3.

2. The transfection efficiency of the optimized TCR (see example 3) was examined and the results are shown in figure 4 (transfection efficiency (%) on the ordinate of figure 4). As can be seen from FIG. 4, the transfection efficiency of lentiviruses containing expression fragments of FSDG, FLDG and FSLinkDG was high.

3. Referring to example 4, the ratio of CD4 and CD8 detected by flow cytometry using fluorescent antibody staining for three optimized TCR-T cells is shown in FIG. 5. As can be seen in fig. 5, there was no significant difference in the ratio of CD4 to CD8 in T cells transformed with the three recombinant TCRs.

4. Referring to example 4, the differentiation status of the TCR-T cells was analyzed by flow cytometry by staining the three optimized TCR-T cells with CD45RA and CD62L, and the results are shown in fig. 6. As can be seen in fig. 6, there was no significant difference in the various cell types of T cells transformed with the three recombinant TCRs.

5. The optimized TCR-T cells were tested for cytokine secretion with reference to example 4 and the results are shown in FIG. 7. As can be seen from FIG. 7, the amount of IFN γ of the T cells containing FSDG and FSLinkDG was much higher than that of the T cells containing FLDG.

6. Tumor cell killing experiment

Lentivirally transfected T cells containing expression optimized TCR elements and control T cells were compared to MV4-11 and Molm-13 and K562 cells according to E: tratio =1:1 (Target cell number: 0.5E6/mL) was placed in a 24-well plate and cultured for 20 hours in total. After centrifugation at 300. Mu.L, two washes with PBS, the cells were incubated with anti-CD3 at 1. As can be seen from FIG. 8, the killing power of the FSDG-expressing T cells and FSLinkDG-expressing T cells on MV-411 and Molm-13 was much higher than that of FLDG-expressing T cells.

Example 6 comparison of TCR to CAR

1. Preparation of TCR-T cells and CAR-T cells: referring to fig. 9, the TCR used in this example is FSLinkDG in example 5, the specific structure and sequence composition of which refers to example 5; the CAR of this example is a second generation CAR, the antigen binding sequences of the CAR are the same as FSLinkDG, and are all FLD3 (the nucleotides are shown in SEQ ID NO: 3), and the CD8 transmembrane sequence of the CAR of this example is shown in SEQ ID NO:12 and the CD8 bridging sequence is shown as SEQ ID NO:13 and the CD28 co-stimulatory sequence is shown as SEQ ID NO:14, the sequence of CD3 zeta is shown in SEQ ID NO: shown at 15.

2. Comparison of cytokine secretion: the TCR-T cells and CAR-T cells carrying the step 1 are co-cultured with K562, MV4-11 and Molm-13 for 18 hours according to ET 1:1 (0.5E6/each target cell), then supernatants are collected, and an ELISA detection kit is used for detecting the secretion level of IFN gamma, and the result is shown in FIG. 10. As can be seen in FIG. 10, the CAR-T cells produced slightly more IFN γ than TCR-T cells (csTCR).

Example 7 animal experiments with TCR and CAR

1. Cell line (b): human lymphoma cell line MV4-11

MV4-11 cells are human myelogenous leukemia cells, a mouse human myelogenous leukemia model can be constructed in a subcutaneous injection mode, and FLT3 expression of the mouse myelogenous leukemia model is positive and can be used as TCR-T cells and target cells.

MV4-11 cell culture

The MV4-11 cell line was a suspension cell line and was rapidly grown in 1640 medium (Gibco) containing 10% FBS. The cell density is 2-3 × 10 ⁶ one/mL requires passaging. At passage, the cell suspension was centrifuged at 500g for 5 minutes in a centrifuge tube, and the supernatant was discarded. Adjusting the cell density to 0.3-0.5X 10 ⁶ Culture was continued at one/mL. Under the normal growth condition, the MV4-11 cell line is passaged for 2 to 3 days, and the cell density is maintained between 0.3 and 3 multiplied by 10 ⁶ The concentration of the active ingredients is only required to be within one/mL.

3. Cell line inoculation

Resuspending MV4-11 cells in physiological saline, and adjusting the viable cell concentration to 1.5X 10 ⁷ individuals/mL (3E 6 cells/mouse), which were mixed with Matrigel (BD, china) on ice according to 2:1, and mixing the mixture fully. The inoculation was performed by means of subcutaneous injection. To successfully grow 100mm ³ The tumor of (2) is used as a judgment standard for the success of the construction of a mouse myelogenous leukemia model. Wherein the tumor volume calculation formula is as follows: tumor volume (mm) ³ ) = tumor long diameter: (mm) x tumor minor diameter 2 (mm) ² )×0.5；

4. Mouse lymphoma model dosing

D0 was recorded on the day of dosing. Mice successfully modeled were randomly divided into three groups of 5 mice each. Cell infusion by tail vein injection: control human T cells 200. Mu.L (10X 10 in total) ⁶ One/one), CAR-T cells in example 6 200 μ L (10 × 10 total) ⁶ One/one), 200. Mu.L (10X 10 in total) of TCR-T cells in example 6 ⁶ One/one)) were injected once for all mice, and tumor measurements were performed every 3 to 5 days, with the results shown in fig. 11 to 12.

From FIGS. 11 to 12, it can be seen that the expression was comparable to the CAR-T cells in example 6. The TCR-T cells of example 6 have better in vivo anti-tumor activity in a murine xenograft model (all of example 6 are of human origin).

Example 8NKG2D-TCR

Referring to FIGS. 13 and 14, viruses for expressing a TCR with a target binding domain of NKG2D (NKG 2D-TCR) and for expressing the NKG2D-TCR were prepared according to examples 1-2, wherein NKG2D comprises a nucleotide sequence shown in SEQ ID NO:16 and the nucleotide sequence of NKG2D1 is shown in SEQ ID NO: NKG2D2, linker (abbreviated as "L" in the figure) shown in fig. 17 was also set up as in example 5, linker1 was linked to the delta chain, linker2 was linked to the gamma chain, and the nucleotide sequences of Linker1 and Linker2 were also as shown in SEQ ID NO:10 to 11.

The results of transferring a virus expressing NKG2D-TCR into Jurkat cell line, staining with NKG2D antibody, and flow-detecting NKG 2D-EGFP co-expressed cell population are shown in FIG. 15. As can be seen in fig. 15, the recombinant TCR was successfully transferred into T cells and co-expressed with the reporter EGFP in the same T cell population.

NKG2D-TCR was transferred into activated T cells (see examples 3 and 4) by virus and tested in vitro, and the results are shown in FIGS. 16 and 17. The proportion of CD4 and CD8 in part A in FIG. 16, and the proportion of T cell differentiation phenotype (naive (TN, CD62L + CD45RA +), central memory (TCM, CD62L + CD45 RA-), effector memory (TEM, CD62L-CD45 RA-), and terminally differentiated T cells (TT, CD62L-CD45RA +), in part B in FIG. 16. FIG. 17 shows the result of the killing of NKG2D-TCR on cancer cells (the specific test procedure includes incubating the NKG 2D-TCR-carrying T cells and the control cells with A549, ACHN and MV4-11 at a ratio of 1:1 to 1:16 for 20 hours, and detecting the killing effect of NKG2D-TCR-T cells on tumor cells by flow assay, wherein "ET" in FIG. 17 indicates the effective target ratio). As can be seen from FIG. 16, the T cells transformed with the recombinant TCR had a normal ratio of CD4 to CD8 and had various T cell populations such as naive, central memory cells, effector memory cells and terminally differentiated cells. As can be seen from FIG. 17, NKG2D-TCR showed strong killing effect against A549, ACHN and MV 4-11.

Animal validation of NKG2D-TCR was performed according to example 7, wherein the assay was divided into two groups, one group being control T cells (CTR, blank human T cells not expressing NKG2D-TCR, without additional transfer of any vector) and one group being assay group TS (NKG 2D-TCR, prepared as described above), each group having 8 mice. Injection via tail vein 10 days, 14 days and 21 days after tumor cell injection ⁶ T cells, tumor size was measured every 3 days or so, and tumor volume exceeded 1000mm ³ I.e., humanitarian sacrifice of mice, the results are shown in fig. 18. As can be seen from fig. 18, the TS group was able to significantly reduce the tumor size and improve the survival rate of mice compared to the control group.

Example 9 Dual target TCR

Referring to FIG. 19, the double target TCR ND19-1, ND19-2 and ND19-3 can be prepared by the method described in example 1, wherein the nucleotide sequences of NKG2D1 and NKG2D2 are the same as those of example 8, and the nucleotide sequence of CD19VH is shown in SEQ ID NO:18, and the nucleotide sequence of the CD19VL is shown as SEQ ID NO:19, respectively. NKG2D1 was linked to CD19VH via a linker fragment (Connector 1), the nucleotide sequence of which is shown in SEQ ID NO:20 is shown in the figure; NKG2D2 is linked to CD19VL via a linker fragment (Connector 2), the nucleotide sequence of which is shown in SEQ ID NO: shown at 21.

Referring to example 2, ND19-1, ND19-2 and ND19-3 were virus-transduced into T cells to give corresponding ND 19-1T cells, ND 19-2T cells and ND 19-3T cells, and then these three T cells were co-cultured with Raji and Raji-CD19KO cell lines at 1:1 (0.5E6/each target cell) for 18 hours, respectively, and then supernatants were collected and assayed for the secretion level of IFN γ using an IFN γ ELISA assay kit, as shown in FIG. 20. As can be seen from FIG. 20, ND19-2 has a good dual-target effect.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present application, so as to understand the technical solutions of the present application in detail and in detail, but not to be construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present application by those skilled in the art are all within the scope of the claims attached to the present application. Therefore, the protection scope of the present patent application shall be subject to the content of the appended claims, and the description and the drawings shall be used for explaining the content of the claims.

Claims

1. A chimeric human T cell receptor, comprising:

2. The T cell receptor of claim 1, wherein the target that specifically binds to the first binding domain comprises one of FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRIN B2, CTLX, E13YIL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF;

3. The T cell receptor of claim 1, wherein the target that specifically binds to the first binding domain comprises FLT3, and the nucleic acid encoding at least one of the first antigen binding region and the second antigen binding region comprises a sequence as set forth in SEQ ID NO:3, and (b) a fragment shown in (b).

4. The T-cell receptor according to claim 1, wherein said first binding domain comprises NKG2D and the nucleic acid encoding said first antigen-binding region comprises the sequence set forth in SEQ ID NO: 16-17, and the nucleic acid encoding the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 16-17, or a fragment thereof.

5. A T-cell receptor according to claim 1, wherein the first conserved region is a region of the delta chain of a human wild-type TCR other than the variable region and the second conserved region is a region of the gamma chain of a human wild-type TCR other than the variable region;

alternatively, the first conserved region is a region of the α chain of a human wild-type TCR other than the variable region and the second conserved region is a region of the β chain of a human wild-type TCR other than the variable region;

preferably, the nucleic acid encoding said first conserved region is as set forth in SEQ ID NO:5, and the nucleic acid encoding the second conserved region is as shown in SEQ ID NO: and 6.

6. The T-cell receptor according to any one of claims 1 to 5, wherein the nucleic acid encoding the first linking fragment and the nucleic acid encoding the second linking fragment each independently comprise the sequence as set forth in SEQ ID NO: 10-11, wherein the nucleic acid encoding the first linking segment is different from the nucleic acid encoding the second linking segment.

7. A chimeric human T cell receptor, wherein said T cell receptor comprises:

a first polypeptide chain comprising a first broad-spectrum binding region, a first antigen-binding region, and a first conserved region, said first antigen-binding region being located between said first broad-spectrum binding region and said first conserved region, said first antigen-binding region being linked to said first conserved region either directly or through a first linking fragment;

the first antigen-binding region and the second antigen-binding region form a first binding domain that specifically binds to a tumor-specific antigen, and the first broad-spectrum binding region and the second broad-spectrum binding region form a second binding domain that specifically binds to a broad-spectrum target.

8. The T cell receptor of claim 7, wherein the target for specific binding to the first binding domain comprises one of MHC-presented cell membrane polypeptide, CD19, BCMA, GPC3, claudin18.2, ROR1, ROR2, GPRC5D, FCRL5, CEA, FLT3, E3adnectin, IL-10, TPO, IL-11, EPHRIN B2, CTLX, E13Y IL13, T1E, APRIL, triAPRIL, LEA-1, FSH, and GM-CSF;

alternatively, the first binding domain is a receptor comprising one of B7H6, DNAM-1, CD27, CD4 and CD 16;

the second binding domain is a receptor, which includes NKG2D.

9. The T-cell receptor according to claim 8, wherein the second binding domain is NKG2D and the first binding domain targets CD19;

further, the second binding domain comprises NKG2D1 and NKG2D2, and the nucleic acid encoding the NKG2D1 is as set forth in SEQ ID NO:16, and the nucleic acid encoding the NKG2D2 is as shown in SEQ ID NO:17, the first binding domain comprises the heavy chain variable region of CD19 and the light chain variable region of CD19, and the nucleic acid encoding the heavy chain variable region of CD19 is set forth in SEQ ID NO:18, and the nucleic acid encoding the light chain variable region of CD19 is as shown in SEQ ID NO:19, respectively.

10. The T-cell receptor according to claim 7, wherein the first conserved region is a region other than the variable region of the delta chain of a human wild-type TCR and the second conserved region is a region other than the variable region of the gamma chain of a human wild-type TCR;

further, the nucleic acid encoding the first conserved region is as set forth in SEQ ID NO:5, and the nucleic acid encoding the second conserved region is as shown in SEQ ID NO: and 6, respectively.

11. The T-cell receptor according to any one of claims 7 to 10, wherein the nucleic acid encoding the first linking fragment and the nucleic acid encoding the second linking fragment each independently comprise the sequence as set forth in SEQ ID NO: 10-11, wherein the nucleic acid encoding the first junction fragment is different from the nucleic acid encoding the second junction fragment;

12. A nucleic acid comprising a nucleic acid fragment encoding the T cell receptor of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A cell comprising the T cell receptor of any one of claims 1 to 11, the nucleic acid of claim 12 or the vector of claim 13.

15. The cell of claim 14, wherein the cell is selected from one of a lymphocyte, a monocyte, and a stem cell.

16. The cell of claim 15, wherein the cell is a T cell.

17. A method for preparing a cell according to claim 14 or 15, comprising the steps of:

introducing into a cell an expression vector for expressing a T cell receptor according to any one of claims 1 to 11; and

culturing a cell containing the expression vector.

18. A pharmaceutical composition comprising an active agent comprising a T cell receptor according to any one of claims 1 to 11, a nucleic acid according to claim 12, a vector according to claim 13, or a cell according to any one of claims 14 to 15, and an excipient.