WO2023216440A1 - Tcr specifically recognizing prame antigen peptide and use thereof - Google Patents

Abstract

Provided are a TCR specifically recognizing a PRAME antigen peptide and the use thereof. The CDR1, CDR2 and CDR3 of an α chain variable region of the TCR are respectively as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the CDR1, CDR2 and CDR3 of a β chain variable region of the TCR are respectively as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. The affinity K D value of the TCR to pMHC (HLA-A*02:01/VLDGLDVLL) can reach 1.3E-05M, and the TCR has a specific killing effect on HLA-A*02:01 +/PRAME + target cells (K562-A0201-3G5) in vitro and in vivo, and has no significant effect on other non-double-positive tumor cells or healthy human PBMCs.

Description

A TCR that specifically recognizes PRAME antigen peptide and its application

This application claims the priority of Chinese patent application 202210495457.9 with a filing date of May 7, 2022. This application cites the full text of the above-mentioned Chinese patent application.

Technical field

The invention belongs to the field of biotechnology and relates to a TCR that specifically recognizes PRAME antigen peptide and its application, specifically a TCR that recognizes HLA-A*02:01/PRAME _100-108 and its application.

Background technique

Melanoma-specific antigen (Preferentially Expressed Antigen of Melanoma, PRAME) is a member of the tumor testis antigen family. It was originally discovered in melanoma. It is immunogenic and can be specifically targeted by cytotoxic T lymphocytes (referred to as T cells). Surface antigens that recognize and trigger tumor lysis (Immunity, 1997, 6, 199; Br J Haematol, 1998, 102, 1376; J Exp Med, 2001, 193, 73; Clin Cancer Res, 2006, 12, 3130; Blood, 2008, 112 , 1876; Clin Cancer Res, 2011, 17, 5615; Oncol Rep, 2014, 31, 384; Cancer Res, 2018, 78, 3337). PRAME is a tumor-associated antigen that is highly expressed in many different cancers, such as acute and chronic leukemia, lymphoma, melanoma, non-small cell lung cancer, cervical cancer, sarcoma, head and neck cancer, renal cell carcinoma, breast cancer, etc. (Cancer Res, 1998,58,4090;Cell,2005,122,835;Blood,2009,114,15;Oral Oncol,2013,49,144;Clin Cancer Res,2013,19,2562;Oncol Rep,2014,31,384;Immunol Invest,2016,45,61 9 ; Mol Ther Methods Clin Dev, 2021, 21, 492), while it is hardly expressed or has very low expression in normal tissues (such as testis, adrenal gland, ovary and endometrium). Therefore, PRAME is an important potential target in tumor immunotherapy. (Biomark Med, 2012, 6, 629; Cancers (Basel), 2019, 11, 984). The VLDGLDVLL peptide (SEQ ID NO:1) is located at 100-108 on PRAME and can be presented through HLA-A*02:01 molecules and recognized by cytotoxic T lymphocytes to produce effects. This peptide is closely related to HLA-A *02:01 The binding affinity of the molecule is 5.2μM, and the half-decay period of the complex at 37°C is 2.5h (J.Exp.Med., 2001, 193,73).

T cell receptor gene engineered T cells (TCR-T) therapy transduces tumor antigen-specific TCR genes into normal T cells, which can enhance or re-endow the T cells with recognition The ability of tumor antigens to specifically target and kill tumor cells is an important treatment method in current adoptive cell therapy (Immunol Rev, 2014, 257, 56). Many clinical trials have proven the effectiveness and safety of TCR-T cell therapy in the treatment of melanoma, synovial cell sarcoma, myeloma and other malignant tumors (Science, 2006, 314, 126; J Clin Oncol, 2011, 29, 917; Nat Med ,2015,21,914; Blood,2017,130,1985; Cancer Discov, 2018,8,944). As of February 2022, the clinicaltrials.gov website shows that 10 clinical trials have been carried out around the world for PRAME targets, including 5 TCR-related therapies (NCT02743611, NCT03503968, NCT03318900, NCT04262466, NCT03686124). The Phase Ia clinical data of PRAME TCR-T therapy developed by Immatics N.V. has verified the effectiveness and safety of the therapy (NCT03686124, J Immunother Cancer, 2021, 9 (Suppl 2), A1009), providing information for the treatment of PRAME-related cancers. A new way. With the development of relevant technologies and the continuous deepening of research, TCR-T therapy will gradually develop in the direction of high efficiency, low toxicity and controllability, which will not only improve the efficacy and safety, but also bring convenience to clinical application, thereby providing More cancer patients bring hope of cure.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the shortcoming of the limited number of TCRs that recognize and bind to the PRAME antigen peptide in the prior art. Therefore, the present invention provides a TCR that specifically recognizes the PRAME antigen peptide. T cells modified using this TCR pass through Efficiently recognizes HLA-A*02:01/PRAME _100-108 targets to specifically kill tumor cells.

Major histocompatibility complex (MHC) molecules are members of the immunoglobulin superfamily and can be class I or class II MHC molecules. It is specific for the presentation of antigens. Different individuals have different MHCs and can present different short peptides in a protein antigen to the surface of their respective antigen-presenting cells (APC). Human MHC is often called HLA genes or HLA complexes.

T cell receptor (TCR) is the only receptor for specific antigens presented on MHC. In the immune system, the combination of antigen-specific TCR and pMHC complex triggers direct physical contact between T cells and APC, and then other cell membrane surface molecules of T cells and APC interact, causing a series of subsequent cell signals. transmission and other physiological responses, allowing T cells with different antigen specificities to exert immune effects on their target cells.

TCR is a functional unit for T lymphocytes to recognize antigens. It belongs to the immunoglobulin superfamily. Its coding chain includes four chains: α, β, γ and δ. TCR is composed of α chain/β chain or γ chain/δ chain with heterogeneous two chains. Glycoproteins on the surface of cell membranes that exist in aggregate form. 95% of TCRs in peripheral blood are heterodimers composed of two polypeptide chains, α and β. However, recombinant TCR can also be composed of a single TCRβ chain or TCRα chain, which has been shown to be able to bind antigen peptide-MHC molecules (WO 2005/113595).

Broadly speaking, each α and β chain contains a variable region, a connecting region, and a constant region. The β chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as a connecting region. part of the district. Each variable region contains three CDRs (complementary determining regions), CDR1, CDR2 and CDR3, embedded in framework regions. The CDR region determines the binding of TCR to the pMHC complex, in which CDR3 is recombined from the variable region and the connecting region and is called the hypervariable region. The α and β chains of TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of connected variable regions and connecting regions. The sequence of the TCR constant domain can be found in the public database of the International Immunogenetic Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is TRAC (also known as TRAC*01), and the constant domain sequence of the β chain of the TCR molecule The sequence is TRBC1 (also known as TRBC1*01) or TRBC2 (also known as TRBC2*01). In addition, the α and β chains of TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.

In addition, the α chain is composed of TRAV, TRAJ, and TRAC rearrangements in germline genes; the β chain is composed of TRBV, TRBD, TRBJ, and TRBC rearrangements in germline genes. After different V(D)J rearrangements, it is composed of V-J( When connecting (or V-D and D-J), different numbers of nucleotides are randomly inserted to form a variable region (N) that becomes the complementarity determining region 3 (CDR3). This random insertion makes the TCR alpha chain and beta chain sequences highly diverse; different When the TCR of clonal T lymphocytes is rearranged, the CDR3 length and base sequence are different. This is the region where the TCR specifically recognizes the antigen, which determines the specificity of the TCR.

It should be noted that in the present invention, the positions of CDR1 ~ CDR3 and FR1 ~ FR4 in the TCR full-length sequence are determined according to the IMGT nomenclature. This definition is well known and can be found in the IMGT public data, "T cell Receptor Factsbook , (2001) LeFranc and LeFanc, Acdamic Press, ISBN 0-12-441352-8" also discloses sequences defined by the IMGT nomenclature. However, it is well known to those in the art that the CDRs of a TCR can be defined in a variety of ways in the art. Therefore, when it comes to defining a TCR with a specific CDR sequence defined in the present invention, the scope of the TCR also covers such TCR, Its variable region sequence contains the specific CDR sequence, but due to the application of different schemes (such as different assignment system rules or combinations), the claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention.

One of the technical solutions of the present invention is: a TCR, the TCR includes an α chain and/or a β chain, the α chain includes an α chain variable region, the β chain includes a β chain variable region, and the α chain The CDR3 of the chain variable region includes the amino acid sequence shown in SEQ ID NO:6, and/or the CDR3 of the β chain variable region includes the amino acid sequence shown in SEQ ID NO:9.

In a preferred embodiment of the present invention, the CDR1 and CDR2 of the alpha chain variable region comprise the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:5 respectively, and/or the beta chain variable region The CDR1 and CDR2 contain the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8 respectively.

In a preferred embodiment of the present invention, the CDR1, CDR2 and CDR3 of the alpha chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the The CDR1, CDR2 and CDR3 of the β chain variable region respectively include the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In a specific embodiment of the present invention, the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, and the The amino acid sequences of CDR1, CDR2 and CDR3 of the β chain variable region are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively.

According to the present invention, the TCR of the present invention can be a variety of forms of antigen-binding structures, and can be natural or mutant, full-length, or antigen-binding fragments such as TCR single chains (α chain or β chain). chain) or scTCR (TCRα chain variable region and TCRβ chain variable region are composed of short peptide linkers), etc.

The α chain and/or β chain of the TCR in the present invention preferably further includes a framework region (FR); wherein:

The framework region of the α chain is derived from TRAV and TRAJ, wherein the TRAV is preferably germline TRAV20, and the TRAJ is preferably germline TRAJ36.

The framework region of the β chain is derived from TRBV, TRBD and TRBJ. The TRBV is preferably the germline TRBV7-8, the TRBD is preferably the germline TRBD1, and the TRBJ is preferably the germline TRBJ2-3.

In a preferred embodiment of the present invention, the alpha chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 10 or its derivative sequence.

In another preferred embodiment of the present invention, the β-chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 12 or its derivative sequence.

In a specific embodiment of the present invention, the amino acid sequence of the alpha chain variable region is as shown in SEQ ID NO: 10 or its derivative sequence, and the amino acid sequence of the beta chain variable region is as shown in SEQ ID NO: 12 or Its derivative sequence is shown.

The identity of the derived sequence to the original sequence is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, and remains identical to the original sequence Same functionality.

The α chain of the TCR in the present invention preferably further includes a constant region, and the constant region of the α chain is preferably derived from the human germline.

In a preferred embodiment of the present invention, the constant region of the α chain is derived from TRAC.

The β chain of the TCR in the present invention preferably further includes a constant region, and the constant region of the β chain is preferably derived from the human germline.

In a preferred embodiment of the present invention, the constant region of the β chain is derived from TRBC.

In a more preferred embodiment of the invention, the TRBC is germline TRBC2.

In a specific embodiment of the present invention, the amino acid sequence of the constant region of the alpha chain derived from the human germline is shown in SEQ ID NO: 11.

In a specific embodiment of the present invention, the amino acid sequence of the constant region of the beta chain derived from the human germline is shown in SEQ ID NO: 13.

In addition, the α chain described in the present invention may also include an extramembrane region and a transmembrane region; preferably, the α chain also includes an intracellular sequence.

The β chain may also include an extramembrane region and a transmembrane region; preferably, the β chain also includes an intracellular sequence.

In a specific embodiment of the present invention, the amino acid sequence of the α chain of the TCR containing a signal peptide is shown in SEQ ID NO: 2, wherein the signal peptide is located at positions 1 to 21 of the specific sequence; and/or, contains a signal The amino acid sequence of the β chain of the TCR of the peptide is shown in SEQ ID NO: 3, in which the signal peptide is located at positions 1 to 19 of the specific sequence.

As those skilled in the art know, the signal peptide is located at the N-terminus of the post-translational protein and guides the protein to the designated expression site. Therefore, the full-length sequence of the α chain and β chain in the present invention does not contain the signal peptide, so the above-mentioned The signal peptide sequence does not limit the full-length amino acid sequence of the above-mentioned α chain and β chain of the present invention; and in specific applications, the signal peptide can be adjusted, and the present invention is not limited to the signal peptide sequence in the above-mentioned specific embodiments.

The second technical solution of the present invention is: an isolated nucleic acid encoding the TCR as described in the first technical solution of the present invention.

The third technical solution of the present invention is: a vector containing the nucleic acid as described in the second technical solution, the vector is preferably a lentiviral vector; the nucleic acid is in a single open reading frame, or in two different open reading frames. The reading frames encode TCRα chain and TCRβ chain respectively.

As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression encapsulated in liposomes vectors as well as certain eukaryotic cells, such as producer cells. When used for prevention or treatment, viral vectors are usually used as vectors, which may include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors and simplex virus vectors. More specifically, as described in the Examples, lentiviral vectors can be used to deliver the constructs in vitro, ex vivo or in vivo.

The invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention. The host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome. Host cells are selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells, 293T cells, etc. When used for prevention or treatment, the host cells can be T lymphocytes, hematopoietic stem cells, etc. commonly used in this field.

The invention also includes isolated cells, particularly T cells, expressing the TCR of the invention. The T cells may be derived from T cells isolated from the subject, or may be part of a mixed cell population isolated from the subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC) and can be CD4 ⁺ helper T cells or CD8 ⁺ cytotoxic T cells. The cells may be in a mixed population of CD4 ⁺ helper T cells/CD8 ⁺ cytotoxic T cells. Generally, the cells can be activated with antibodies (such as anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, e.g., with a vector containing a nucleotide sequence encoding a TCR molecule of the invention. .

Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, cells are grown in culture medium and screened for appropriate activity. The methods and conditions for culturing the transfected cells produced and for recovering the TCR molecules produced are known to those skilled in the art and can be based on the present description and methods known from the prior art, depending on the specific expression vector used and Mammalian host cell changes or optimizations. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers may, for example, provide prototrophy, biocidal resistance (eg, antibiotics), or heavy metal (eg, copper) resistance to an auxotrophic host, etc. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

The fourth technical solution of the present invention is: a cell containing the nucleic acid as described in the second technical solution or the vector as described in the third technical solution; preferably, the cells are T cells or stem cells, and the The T cells are preferably CD8 ⁺ T cells.

The fifth technical solution of the present invention is: an isolated or non-naturally occurring cell that presents the TCR as described in one of the technical solutions, and the cell is preferably a T cell.

The term "cell" in the present invention may include cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of passage number. The progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations. The present invention includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transformed cells.

The sixth technical solution of the present invention is: a pharmaceutical composition, which contains the TCR as described in the first technical solution or the cells described in the fourth technical solution; preferably, the pharmaceutical composition also includes a pharmaceutical composition. Acceptable carrier.

In some embodiments, the pharmaceutical compositions of the present invention include suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers known in the art. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids such as water and oils. When administering pharmaceutical compositions intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, glycerin , propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. The compositions of the present invention can be prepared by mixing the TCR of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). The pharmaceutical preparation or pharmaceutical composition is preferably in the form of a lyophilized preparation or aqueous solution. The pharmaceutical compositions or preparations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it is ideal to also provide other anti-tumor active ingredients, such as other TCRs, antibodies, anti-tumor active agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably present in combination in amounts effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the TCR or antigen-binding fragments thereof of the invention in the form of shaped articles, such as films or microcapsules.

The seventh technical solution of the present invention is: a TCR as described in technical solution one, a cell as described in technical solution four, or a pharmaceutical composition as described in technical solution six in the preparation of drugs for preventing and treating tumors related to PRAME expression. Application; Preferably, the tumors include melanoma, acute and chronic leukemia, lymphoma, non-small cell lung cancer, cervical cancer, sarcoma, head and neck cancer, renal cell carcinoma and breast cancer.

The eighth technical solution of the present invention is: a method for treating and/or preventing PRAME expression-related tumors, which method includes administering to a patient in need a therapeutically effective amount of the TCR described in one of the technical solutions, such as the technical solution The cells described in claim 4 or the pharmaceutical composition described in claim 6.

In preferred embodiments of the invention, the tumors include melanoma, acute and chronic leukemia, lymphoma, non-small cell lung cancer, cervical cancer, sarcoma, head and neck cancer, renal cell carcinoma and breast cancer.

As used herein, the term "effective amount" means an amount of a drug or agent that elicits the biological or pharmaceutical response in a tissue, system, animal, or human, for example, that is sought by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or a reduced rate of progression of a disease or condition, as compared to a corresponding subject who does not receive such amount. amount. The term also includes within its scope amounts effective to enhance normal physiological functions.

The ninth technical solution of the present invention is: a combination therapy, which includes administering to a patient in need the TCR described in one technical solution, the cell described in technical solution four, or the drug combination described in technical solution six. substance, and a second therapeutic agent; the second therapeutic agent preferably contains other anti-tumor active ingredients, such as other TCRs, antibodies, anti-tumor active agents, small molecule drugs or immunomodulators, etc.

The tenth technical solution of the present invention is to use the TCR as described in technical solution one, the cell as described in technical solution four, or the pharmaceutical composition as described in technical solution six for the treatment and/or prevention of tumors related to PRAME expression. .

In preferred embodiments of the invention, the tumors include melanoma, acute and chronic leukemia, lymphoma, non-small cell lung cancer, cervical cancer, sarcoma, head and neck cancer, renal cell carcinoma and breast cancer.

The positive progressive effects of the present invention are:

The affinity K _D value of the TCR of the present invention and pMHC (HLA-A*02:01/VLDGLDVLL) reaches 1.3E-05M, and the affinity to HLA-A*02:01 ⁺ /PRAME ⁺ target cells (K562-A0201-3G5) is It has specific killing effects in vitro and in vivo, but has no obvious effect on other non-double-positive tumor cells or healthy human PBMC.

Description of the drawings

Figure 1 shows the sorting process of PRAME _100-108 antigen-specific double-positive monoclonal CD8 ⁺ T cells.

Figure 2A and Figure 2B show the anion exchange chromatography and SDS-PAGE electrophoresis images of PVL7 TCR after renaturation.

Figure 3A and Figure 3B show the gel filtration chromatography and SDS-PAGE electrophoresis images of PVL7 TCR after renaturation.

Figure 4A and Figure 4B show the anion exchange chromatography and SDS-PAGE electrophoresis images of HLA-A*02:01/β2M/VLDGLDVLL after renaturation; among them, the band with large molecular weight is HLA-A*02:01 and the band with small molecular weight The band is β2M, and the VLDGLDVLL polypeptide cannot see the band on SDS-PAGE because its molecular weight is too small.

Figure 5 shows the gel filtration chromatogram of HLA-A*02:01/β2M/VLDGLDVLL after renaturation.

Figure 6 shows the Gel Shift diagram after biotinylation of PRAME-pMHC.

Figure 7 shows the results of the PVL7 TCR affinity test.

Figure 8 shows the results of the positivity rate of CD8 ⁺ T cells infected with PVL7 TCR lentivirus; PVLcon is the positive TCR control group and GFP is the negative control group.

Figure 9A, Figure 9B and Figure 9C are the INF-γ release diagrams of PVL7 TCR on T ₂ cells loaded with PRAME _100-108 , NY-ESO-1 _157-165 or HPV16-E6 _29-38 polypeptides.

Figure 10 is a flow cytometry diagram of the constructed K562-A0201 monoclonal cells.

Figure 11 shows the INF-γ release graph of PVL7 TCR on different tumor cell lines.

Figure 12 shows the results of PVL7 TCR specific killing experiments on LDH in different tumor cell lines.

Figure 13A and Figure 13B show the INF-γ release diagram of PVL7 TCR on PBMC of 28 healthy people with different HLA-A types.

Figure 14 shows the tumor growth curve of PVL7 TCR-T animal experiments.

Figure 15 shows the tumor status of mice after 18 days of administration of PVL7 TCR-T cells.

Detailed ways

The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.

In addition: in the following examples, unless otherwise stated, all cell lines used were purchased from ATCC.

Example 1 Antigen-specific αβ-TCR cloning and gene sequence identification

PRAME _100-108 (VLDGLDVLL) antigen-specific CD8 ⁺ T cell TCR gene cloning methods, reagents and consumables, mainly refer to Curr.Protoc.Immunol.2002,7,1; PLoS One.2011,6,e27930; Onco Immunology .2016,5,e1175795; J Vis Exp.2011,8,3321; J Immunol Methods.2006,310,40; PLoS One.2014,9,e110741 and its citations. After CD8 ⁺ T cells were isolated from PBMC of healthy volunteers with HLA-A*02:01 genotype by immunomagnetic bead negative screening, EBV-B cells loaded with PRAME _100-108 peptide were used (J Vis Exp. 2011 , 8,3321) (EBV virus: ATCC product number VR-1492) to stimulate CD8 ⁺ T cells, and then use PE-labeled HLA-A*02:01/PRAME _100-108 tetramer (see Example 3 for the preparation method) and APC-labeled anti-CD8 antibody (Biolegend, product number 301014) to double-stain T cells, flow sort to obtain double-positive T cells, and expand and culture the T cells to a certain number before sorting again (Figure 1 ). After two rounds of stimulation culture and sorting, the double-positive T cells were cultured monoclonally using the limiting dilution method. The proliferated monoclonal T cells were flow cytometrically detected by double staining with HLA-A*02:01/PRAME _100-108 tetramer and anti-CD8 antibody and sorted to obtain PRAME _100-108 antigen-specific monoclonal T cells.

The Quick-RNA ^TM MiniPrep kit (ZYMO research, product number R1050) was used to extract the total RNA from the monoclonal T cells, and the RNA was reverse transcribed using the SMARTer RACE cDNA Amplification Kit (Clontech, product number 634923) to obtain cDNA. Then use cDNA as a template to amplify the target gene through PCR and connect it to the pUC19 vector. Transform it into E.coli-DH5α by heat shock method. After plate culture overnight, single clone colonies are picked for identification and sequencing. The gene sequence obtained by sequencing is in The IMGT database was compared and analyzed, and finally an HLA-A*02:01/PRAME _100-108 antigen-specific TCR was obtained, named PVL7 TCR. Its TCRα and β chain genotypes are shown in Table 1.

Table 1. Genotypes of TCRα and β chains

The full-length α chain sequence of PVL7 TCR (SEQ ID NO:2):

The full-length sequence of the β chain of PVL7 TCR (SEQ ID NO:3):

In the above sequence: the underlined sequence is the signal peptide region, the black bolded sequence is Vα (variable region of α chain) or Vβ (variable region of β chain), and the bolded italicized sequence is Cα (constant region of α chain). Region, SEQ ID NO: 11) or Cβ (constant region of β chain, SEQ ID NO: 13), the sequence marked in italics and underline is the transmembrane intracellular region, and the sequence marked in bold and underline is the CDR sequence.

According to the IMGT database rules, the unique key sequence of the PVL7 TCR V region and the amino acid sequence of the CDR region are shown in Table 2:

Table 2. PVL7 TCR V region α and β chain CDR region amino acid sequences

PRAME full-length sequence (SEQ ID NO:14):

In the above sequence: the underlined sequence is the PRAME _100-108 peptide.

Example 2 Expression and purification of PVL7 TCR gene

The α chain and β chain genes of PVL7 TCR were connected to the pET28a vector using Nco I/Not I enzyme cutting sites respectively, and E.coli-BL21 (DE3) was transformed by heat shock method. After plate culture overnight, single clone colonies were picked to In LB medium, culture with shaking at 37°C until OD ₆₀₀ = 0.6-0.8. Add IPTG at a final concentration of 1mM to induce the expression of the target protein. After continuing to culture at 37°C for 3 hours, centrifuge at 6000 rpm for 10 minutes to collect the cells.

Resuspend the cells in lysis buffer (1×PBS containing 0.5% Triton X-100), conduct ultrasonic disruption, and centrifuge at 12,000 rpm for 20 min. Discard the supernatant, resuspend the pellet in lysis buffer until there are no particles visible to the naked eye, centrifuge at 12,000 rpm for 10 min, repeat the above operation 2-3 times, dissolve the pellet with 6M guanidine hydrochloride solution, centrifuge at 12,000 rpm for 10 min, collect the supernatant, and the supernatant is Purified inclusion bodies. Inclusion bodies were quantified using the BCA method.

Dilute 20 mg of PVL7 TCRα chain inclusion body and 15 mg of β chain inclusion body in 5 mL of 6M guanidine hydrochloride solution, and then slowly add the TCRα chain and TCRβ chain to the pre-chilled refolding buffer (Science 1996, 274, 209; J. Mol. Biol. 1999, 285, 1831; Protein Eng. 2003, 16, 707), stirring continuously for 30 minutes at 4°C. Add the solution to the dialysis bag, place it in 10 times the volume of pre-cooled deionized water, stir and dialyze for 8-12 hours, then place it in pre-cooled dialysate (pH 8.1, 20mM Tris-HCl) and dialyze at 4°C for 8-12 hours. Repeat 2-3 times.

Take out the solution in the dialysis bag, centrifuge it at high speed for 10 minutes to remove the precipitate and bubbles, and then perform anion exchange chromatography through HiTrap Q HP (5mL), using eluent (0-2M NaCl, 20mM Tris pH 8.1) to linearly elute (Figure 2A). The elution peaks containing the target protein components were collected in sections, and after concentration, samples were taken for non-reducing SDS-PAGE electrophoresis (Figure 2B). The results showed that the purity of the target protein required further purification. Use superdex 75 10/300 to perform gel filtration chromatography on the concentrated protein sample (Figure 3A), and take samples for non-reducing and reducing SDS-PAGE electrophoresis detection (Figure 3B). The results show that the non-reducing electrophoresis lane is at 45kDa. There is a main band nearby; there are two bands in the reducing electrophoresis lane, namely the α chain and β chain of PVL7 TCR, and their purity meets the needs of subsequent experiments.

Example 3 Preparation of biotinylated antigen peptide-MHC (pMHC)

The renaturation and purification of pMHC were prepared according to the method of NIH Tetramer Core Facility. According to the online protocols, add the PRAME _100-108 peptide solution, β2M and HLA-A*02:01 inclusion body solution in sequence to the refolding buffer (0.1M Tris-HCl, 0.4M L-arginine, 2mM EDTA, 0.5mM Oxidized glutathione and 5mM reduced glutathione, 0.2mM PMSF), stir overnight at 4°C, add the same amount of HLA-A*02:01 inclusion body solution in the morning and evening the next day, 4°C After stirring for 1-3 days, dialyze three times in 10 times the volume of dialysate (pH 8.1, 20mM Tris-HCl). The dialyzed protein sample was subjected to anion exchange chromatography using HiTrap Q HP (5 mL), linear elution was performed with eluent (0-2M NaCl, 20mM Tris pH 8.1), and the elution peaks were collected and combined (Figure 4A). Samples collected from the elution peak segments (A1-A5) in Figure 4A were analyzed by reducing SDS-PAGE electrophoresis. Two bands, HLA-A*02:01 and β2M, were clearly visible (Figure 4B, A3-A5 in the figure). Corresponding to the samples collected at different elution peak nodes in Figure 4A), the purity has met the subsequent requirements, but the molecular weight of the PRAME _100-108 polypeptide is too small and no bands can be seen in the gel image. The elution peak containing the pMHC component was concentrated and purified by gel filtration chromatography (Superdex 75 10/300) (Figure 5). The pMHC complex was biotinylated with recombinant enzyme BirA (Protein Expr. Purif. 2012, 82, 162; J. Bacteriol. 2012, 194, 1113.), and then added streptavidin (SA) for reaction verification. The reaction system was prepared and Gel Shift purity identified according to the method of NIH Tetramer Core Facility. Judging from the Gel Shift electrophoresis pattern (Figure 6), the biotinylation of the PRAME-pMHC complex was successfully prepared.

Example 4 Affinity Test

Biacore is an instrument that detects affinity based on Surface Plasmon Resonance (SPR) technology. In this experiment, Biacore T200 was used to first couple biotinylated pMHC to the CM5 chip, and then detect its binding dissociation constants with different TCRs and calculate the K _D value. Based on this, the affinity of PVL7 TCR and pMHC (HLA-A*02:01/VLDGLDVLL) was tested. See Table 3 and Figure 7 for details.

Table 3. PRAME TCR affinity K _D values

It should be noted that: as those in the field know, SPR technology is one of the most commonly used and reliable methods for determining affinity. However, when it comes to protein quantification, chip condition, instrument status, etc., there will be differences in experiments between different batches. There is a certain error, and the error value can even reach 3 to 5 times; and the present invention uses the same batch of experiments using the same protein quantification, the same chip and the same instrument, so the data can be used to compare the affinity. However, specific numerical values do not constitute a limitation on the scope of the present invention.

Example 5 TCR lentivirus preparation and transduction of CD8 ⁺ T cells

1)TCR lentivirus packaging

The third generation lentivirus packaging system (Invitrogen, pLenti6/V5Directional TOPO ^TM Cloning Kit, product number K495510) is used to package the lentivirus containing the gene encoding the target TCR. Referring to the method provided in the kit instructions, combine the packaging plasmids pMDLg/pRRE (addgene, product number k12251), pRSV-REV (addgene, product number 12253), and pMD2.G (addgene, product number 12259) with the target gene. Shuttle plasmids such as pLenti-PVL7, pLenti-PVLcon (the TCR gene sequence is derived from TCR T4.8-1-29 in patent number US2019169261A1), pLenti-GFP (negative control), etc. are based on the mass ratio of 4:2:1:1 Mix well and use transfection reagent PEI-MAX (Polyscience, product number 23966-1) to transiently transfect 293T cells in the logarithmic growth phase. 48-50 h after transfection, the medium supernatant containing lentivirus was collected. After centrifugation and a 0.45 μm filter to remove cell debris, it was filtered with an Amicon Ultra-15 centrifugal filter equipped with Ultracel-50 filter membrane (Merck Millipore, Product No. UFC905096) and the supernatant was concentrated. The lentivirus titer was measured on the concentrated samples. The steps were described in the p24 ELISA (Clontech, Product No. 632200) kit instructions.

2) TCR lentivirus transduces CD8 ⁺ T cells

CD8 ⁺ T cells were isolated from PBMC of healthy volunteers, seeded in 48-well plates with RPMI 1640 complete medium containing 10% FBS and 100 IU/mL IL-2, with 1 × ¹⁰ cells per well, and added Anti-CD3/CD28 antibody-coupled magnetic beads were used to stimulate activated CD8 ⁺ T cells and were cultured in a cell culture incubator overnight. After stimulation overnight, add PVL7, PVLcon or GFP lentivirus at a ratio of MOI=5, and centrifuge the infection at 900g for 1 hour at 32°C. After the infection is completed, remove the lentiviral infection fluid, continue to culture the cells for 3 days, and use a magnet to remove the anti-CD3/CD28 antibody-coupled magnetic beads. Thereafter, the cells were counted every two days, and fresh complete medium was replaced or added to maintain the cell density at 1-2×10 ⁶ cells/mL. On the 9th day of cell culture, flow cytometry and positive rate analysis of T cells were performed through HLA-A*02:01/PRAME _100-108 tetramer staining. The results are shown in Figure 8. The positive rate of PVL7 TCR-T cells was 44.8%, the positive rate of PVLcon TCR-T cells was 47.1%, and the positive rate of GFP TCR-T cells was 79.3%.

Example 6 PVL7 TCR functional specificity analysis in vitro—ELISPOT method to detect INF-γ release from polypeptide-loaded _T2 cells

This example uses an ELISPOT test to analyze the release of INF-γ factors from PVL7 TCR under stimulation of T ₂ cells loaded with specific or non-specific polypeptides. The effector cells in this example are CD8 ⁺ T cells transduced by PVL7, PVLcon and GFP lentivirus in Example 5. The target cells in this example are T ₂ cells loaded with polypeptides of different concentrations. The T ₂ cells were exposed to 7 gradient concentrations (10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 M) PRAME _100-108 polypeptide, or 10 ^-6 M NY-ESO-1 _157-165 polypeptide or HPV16-E6 _29-38 polypeptide, mix well, place it in a 37°C incubator for 4 hours, and then centrifuge. Wash once with 1×PBS and resuspend the cells in RPMI 1640 medium containing 10% FBS for the next step of plating. Follow-up experimental operations were performed according to the instructions of the Human INF-γELISPOT Set kit (BD biosciences, product number 551849). Add 4×10 ³ positive effector cells/well and 4×10 ⁴ target cells/well to the ELISPOT well plate, 200 μL of culture system per well, and place the well plate in a cell culture incubator to incubate overnight. After incubation, wash according to the kit instructions, then add BCIP/NBT solution for development for 5-15 minutes, wash the well plate with deionized water, and finally invert the well plate to allow the plate to dry naturally at room temperature. Enzyme-linked immunospot analyzer (

6000Pro-Fβ, Bio-Sys) to analyze well plates.

The results are shown in Figure 9A. T ₂ cells loaded with PRAME _100-108 polypeptide strongly stimulated PVL7 TCR-T cells to release INF-γ factors in a peptide concentration-dependent manner. The trend was consistent with the positive control PVLcon. When the concentration of the loaded peptide was greater than 10 ^-7 M, the release of INF-γ from both PVL7 and PVLcon TCR-T cells reached the peak, and there was no significant difference between the two. 1G4TCR-T cells that specifically recognize NY-ESO-1 _157-165 (refer to patent CN112442118A for the preparation method) and E6con TCR-T cells that specifically recognize HPV16-E6 _29-38 (refer to patent CN113754756A for the preparation method) have significant activity ( Figure 9B & Figure 9C), while neither PVL7 nor PVLcon TCR-T cells had significant activity against T ₂ cells loaded with non-specific NY-ESO-1 _157-165 or HPV16-E6 _29-38 polypeptides. T ₂ cells loaded with PRAME/NY-ESO-1/HPV16-E6 peptide fragments were unable to stimulate GFP TCR-T cells to release INF-γ factors. In summary, the function of PVL7 TCR is close to that of the positive control PVLcon, and it is specific for the recognition of HLA-A*02:01/PRAME _100-108 .

Example 7 Construction of PRAME transgenic K562 monoclonal cells

In order to construct HLA-A*02:01 ⁺ /PRAME ⁺ double-positive target cells for PVL7 TCR functional verification, the HLA-A*02:01 gene was connected to the corresponding shuttle plasmid through the method of Example 5, and lentiviral Packed and then transduced into K562 tumor cells (HLA-A*02:01 ^- /PRAME ⁺ ), K562 polyclonal cells with chromosomally integrated HLA-A*02:01 gene were initially obtained. In order to obtain K562 monoclonal cells that can stably express the HLA-A*02:01 gene, K562-A0201 polyclonal cells were isolated and cultured in a 96-well plate using the limiting dilution method, with each well containing 0.5–1 candidate cells. After the candidate cells have been amplified, use mouse anti-human HLA-A2 flow cytometry antibody (BD Pharmingen, product number 558570) to detect the expression of HLA-A*02:01 on the K562-A0201 monoclonal cell membrane surface. As shown in Figure 10, the HLA-A*02:01 expression positivity rate of K562-A0201 monoclonal cells numbered 3G5 is 99.7%. This cell will be used for in vitro functional verification and animal experiments of PVL7 TCR.

HLA-A*02:01 gene full-length sequence (SEQ ID NO:15):

Example 8 PVL7 TCR in vitro functional verification—ELISPOT method to detect INF-γ release from tumor cell lines

This example uses ELISPOT assay to analyze the release of INF-γ factor by PVL7 TCR under stimulation of different tumor cell lines. The effector cells in this example are CD8 ⁺ T cells transduced by PVL7, PVLcon and GFP lentivirus in Example 5. The tumor target cells in this example are K562, K562-A0201-3G5, Hela (Peking Union Cell Resource Center), SiHa (Peking Union Cell Resource Center), Raji (Peking Union Cell Resource Center) and HT-1080 (Peking Union Cell Resource Center). Cell Resource Center) cells. As described in Example 6, 4×10 ³ positive effector cells/well and 4×10 ⁴ tumor target cells/well were added to the ELISPOT well plate in sequence, with 200 μL of culture system per well, and the well plate was placed in a cell culture incubator for incubation. overnight. After the incubation, the ELISPOT well plate was washed and developed according to the method of Example 6, and finally the well plate was analyzed with an enzyme-linked immunospot analyzer.

The results are shown in Figure 11. PVL7 and PVLcon TCR-T cells showed strong stimulatory activity on the double-positive tumor target cell K562-A0201-3G5. The activity of PVL7 in releasing INF-γ factor was slightly higher than that of PVLcon, but there was no difference between the two. significant difference. Other tumor cells were unable to stimulate PVL7 and PVLcon TCR-T cells to release INF-γ. GFP TCR-T cells have no obvious activity against all tumor target cells.

Example 9 In vitro functional verification of PVL7 TCR—specific killing of tumor cell lines by LDH

Quantitative measurement of LDH released after target cell lysis is used to evaluate the function of effector cells in killing target cells. For specific experimental protocols, refer to Eur. J Immunol. 1993, 23, 3217. The effector cells in this example are CD8 ⁺ T cells transduced by PVL7, PVLcon and GFP lentivirus in Example 5. The tumor target cells in this example are K562 and K562-A0201-3G5 cells. Sequentially add 3×10 ⁴ positive effector cells/well and 1×10 ⁴ tumor target cells/well to a 96-well round-bottom plate, with 200 μL of culture system in each well. The cells were replaced with RPMI 1640 containing only 5% FBS when plating. culture medium, and place the well plate in a cell culture incubator for 24 h. According to CytoTox

Non-Radioactive Cytotoxicity Assay kit instructions (Promega, product number G1780), add lysis solution to the blank well of the culture medium and the maximum self-release hole of the tumor target cells, place it in a cell culture incubator and incubate for 45 minutes, take 50 μL of supernatant from each well and Mix 50 μL LDH detection solution and incubate at room temperature in the dark for 30 minutes. After the incubation, stop solution was added and the plate was read at 490 nm. Process and analyze the data according to the calculation formula: Cytotoxicity percentage %, that is, LDH release percentage % = (release amount of experimental group – self-release amount of tumor cells – self-release amount of TCR-T cells) / (maximum release amount of tumor cells – tumor cells Self-release amount)*100%. When calculating, the LDH release value of each group was subtracted from the basic light absorption value of the culture.

The results are shown in Figure 12. PVL7 and PVLcon TCR-T cells showed obvious killing effects on the double-positive tumor target cell K562-A0201-3G5. The cytotoxicity percentages of the two were close, which were 65.16±17.39% and 66.22±9.67% respectively. PVL7 and PVLcon TCR-T cells had no obvious killing effect on K562 and were close to the negative control GFP TCR-T cells.

Example 10 In vitro functional verification of PVL7 TCR—specific INF-γ release from healthy human PBMC

In order to investigate the safety of PVL7 TCR, the ELISPOT method of Example 6 was used to conduct safety investigation on PBMC of 28 healthy people. The 28 cases of healthy human PBMC included 7 cases with HLA-A*02:01 typing and 21 cases with non-HLA-A*02:01 typing (Figure 13A). The results are shown in Figure 13B. The INF-γ release results showed that PVL7 and PVLcon TCR-T cells had no obvious reaction with PBMC of healthy people.

Example 11 Animal Experimental Verification of PVL7 TCR—Xenogeneic Transplantation of PRAME Transgenic K562 Cells

The experimental animal strain in this example is B-NDG mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., SPF grade), female, 4 to 6 weeks old. During the test, the experimental animals were housed in an SPF-level animal center, and all technical indicators complied with GB14925-2010 barrier environment technical requirements. The animals in this study will eat feed whose quality meets national standards and is within the validity period. Their drinking water will be filtered and sterilized by the animal drinking pure water system or sterilized by high temperature and high pressure, and will be freely ingested from animal drinking bottles. All experimental animals were adaptively raised for 1 week before subsequent experiments were conducted.

Select B-NDG mice that have passed the adaptability observation and inoculate K562-A0201-3G5 tumor cells subcutaneously, adjust the cell density to 2×10 ⁷ cells/mL, and inoculate 0.2 mL into each mouse. Observations were conducted every day after inoculation. When the average tumor volume was ^about 100 mm, they were divided into three groups, namely Model Control group, PVL7 TCR-T group and GFP TCR-T group, with 7 animals in each group. The PVL7 TCR-T group was given a dose of 4×10 ⁸ positive cells/kg, the GFP TCR-T group was given the same dose of T cells, and the animals in the model control group were given an equal volume of vehicle. The mice in each group were given tail vein injection. After administration, each group was intraperitoneally injected with IL-2, 50,000 IU/animal, for 5 consecutive days. The tumor diameter was measured on the day of grouping, and the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every 3 days. The tumor volume was calculated according to the formula 1/2 × a × b ² , and the tumor growth curve was drawn. The animals were euthanized 18 days after administration, and the tumors were taken for photography.

The tumor growth curves and tumor bodies of mice in each group are detailed in Figures 14 and 15. PVL7 TCR-T cells can effectively kill tumor cells K562-A0201-3G5 and inhibit tumor growth. The tumor growth curve of the GFP TCR-T group did not change significantly compared with the Model Control group.

Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these embodiments without departing from the principles and essence of the present invention. Revise. Accordingly, the scope of the present invention is defined by the appended claims.

Claims (13)

1. A TCR, the TCR includes an α chain and/or a β chain, the α chain includes an α chain variable region, and the β chain includes a β chain variable region, characterized in that, the α chain variable region CDR3 includes the amino acid sequence shown in SEQ ID NO:6, and/or the CDR3 of the β chain variable region includes the amino acid sequence shown in SEQ ID NO:9.
2. The TCR of claim 1, wherein CDR1 and CDR2 of the α chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:5, and/or the β CDR1 and CDR2 of the chain variable region include the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8 respectively.
3. The TCR of claim 2, wherein CDR1, CDR2 and CDR3 of the alpha chain variable region are the amino acids shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively. sequence, and the CDR1, CDR2 and CDR3 of the β chain variable region are the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively.
4. The TCR of claim 1, wherein the α chain and/or β chain of the TCR further includes a framework region;

   The framework region of the alpha chain is derived from TRAV and TRAJ, wherein the TRAV is preferably germline TRAV20, and the TRAJ is preferably germline TRAJ36; and/or,

   The framework region of the β chain is derived from TRBV, TRBD and TRBJ. The TRBV is preferably the germline TRBV7-8, the TRBD is preferably the germline TRBD1, and the TRBJ is preferably the germline TRBJ2-3.
5. The TCR of claim 4, wherein the alpha chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 10 or its derivative sequence, and/or the beta chain of the TCR can The variable region contains the amino acid sequence shown in SEQ ID NO: 12 or its derivative sequence;

   Preferably, the amino acid sequence of the α chain variable region is as shown in SEQ ID NO: 10 or its derivative sequence, and/or the amino acid sequence of the β chain variable region is as shown in SEQ ID NO: 12 or its derivative sequence. Show;

   The identity of the derived sequence to the original sequence is 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, and remains identical to the original sequence Same functionality.
6. The TCR according to any one of claims 1 to 5, wherein the α chain and/or β chain of the TCR further includes a constant region, and the constant region of the α chain and/or the β chain is preferably derived from in the human germline;

   Preferably, the constant region of the α chain is derived from TRAC, and/or, the constant region of the β chain is derived from TRBC, and the TRBC is preferably germline TRBC2;

   More preferably, the amino acid sequence of the constant region of the α chain derived from the human germline is as shown in SEQ ID NO: 11, and/or the amino acid sequence of the constant region of the β chain derived from the human germline is as shown in SEQ ID NO: Shown in 13.
7. The TCR according to claim 6, wherein the α chain and/or β chain of the TCR further includes an extramembrane region and a transmembrane region;

   Preferably, the α chain and/or β chain of the TCR also includes intracellular sequences;

   More preferably, the amino acid sequence of the α chain of the TCR including the signal peptide is as shown in SEQ ID NO: 2, wherein the signal peptide is located at positions 1 to 21 of SEQ ID NO: 2; and/or, the amino acid sequence including the signal peptide The amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 3, in which the signal peptide is located at positions 1 to 19 of SEQ ID NO: 3.
8. An isolated nucleic acid, characterized in that it encodes the TCR according to any one of claims 1 to 7.
9. A vector comprising the nucleic acid as claimed in claim 8, the vector is preferably a lentiviral vector; the nucleic acid encodes TCRα chain and TCRβ respectively in a single open reading frame, or in two different open reading frames. chain.
10. A cell containing the nucleic acid of claim 8 or the vector of claim 9; preferably, the cells are T cells or stem cells, and the T cells are preferably CD8 ⁺ T cells.
11. An isolated or non-naturally occurring cell presenting the TCR according to any one of claims 1 to 7, preferably a T cell.
12. A pharmaceutical composition, characterized in that it contains the TCR according to any one of claims 1 to 7 or the cell according to claims 10 or 11; preferably, the pharmaceutical composition also contains a drug acceptable carrier.
13. The application of a TCR according to any one of claims 1 to 7, a cell according to claims 10 or 11, or a pharmaceutical composition according to claim 12 in the preparation of drugs for preventing and treating tumors related to PRAME expression;

    Preferably, the tumors include melanoma, acute and chronic leukemia, lymphoma, non-small cell lung cancer, cervical cancer, sarcoma, head and neck cancer, renal cell carcinoma and breast cancer.

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