Marker combination for screening target antigen-specific t cells and use thereof

Abstract

Disclosed in the present application are a marker combination for screening target antigen-specific T cells and the use thereof. The present application provides a marker combination for efficiently screening virus antigen / tumor neoantigen-specific T cells, and provides a method for in-vitro screening target antigen-specific T cells on the basis of the marker combination. Upon verification, 90% or more of the CD8+T cells screened by the method are T cells specifically targeting the virus antigen / tumor neoantigens. Compared with a previous CD137 marker used alone, the success rate of screening is significantly improved.

Description

Marker combination for screening target antigen-specific T cells and its use

[0001] Cross-reference to related applications

[0002] This patent application claims priority to the Chinese patent application filed on July 21, 2023, with application number 2023109057201 and invention name “Marker combination for screening target antigen-specific T cells and its use”. The full text of the above application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to a marker combination for screening target antigen-specific T cells and uses thereof. Background Art

[0004] In recent years, immune cell therapy technology has become a new method for tumor treatment after surgical treatment, radiotherapy, chemotherapy, small molecule targeted therapy or monoclonal antibody treatment. Among the many immune cell therapies for tumors, the mechanism of action of "Adoptive T-Cell Therapy" (ACT) is relatively clear, and the therapeutic effect is also more significant. ACT therapy is mainly divided into "Tumor Infiltrating T Lymphocytes" (TIL cell) therapy, "Chimeric Antigen Receptor T cell" (CAR-T cell) therapy, and "T Cell Receptor T cell" (TCR-T cell) therapy. TCR-T cell therapy is to transfer the TCR receptor gene that recognizes specific tumor antigens into T lymphocytes, amplify them and then infuse them back into the patient to achieve specific killing of tumor cells. TCR-T cells are HLA-restricted, so it is necessary to develop TCR receptors that match multiple HLA subtypes for tumor antigens. In the hematology field, TCR-T therapy targeting the WT1 antigen is used to prevent the recurrence of AML after hematopoietic stem cell transplantation. Because TCR-T cells can recognize tumor cell membrane, nuclear, and cytoplasmic antigens, as well as foreign protein antigens (such as viral antigens), they have unique properties that differ from those of existing small molecule targeted drugs or monoclonal antibody drugs. They are a type of "living" drug, and TCR-T has broader clinical application prospects in tumor immune cell therapy and immunotherapy for viral infections.

[0005] T cells recognize short peptide antigens on MHC proteins through their T cell receptors (TCRs) and respond to target cells in an antigen-specific manner. The TCR-peptide-MHC interaction between T cells and their target cells determines their function, thereby affecting their role in disease. The lack of methods to discover antigen-specific T cell receptors (TCRs) limits the basic understanding of the antigenic landscape of the entire T cell response. In the prior art, the marker commonly used for screening specific TCR-T is CD137+. CD137 (4-1BB) is a membrane surface glycoprotein that belongs to the tumor necrosis factor receptor family (TNFRSF9). Its expression can be induced by leukocyte activation. On T cells, T cell receptor (TCR) stimulation and subsequent CD3 signal transduction induce transient expression of CD137. However, the inventors found in their research that the screening efficiency of a single CD137 marker for specific TCR-T is low, and there is still room for improvement. For example, the Rosenbreg SA research group used flow cytometry to detect and sort CD137+CD8+T cells, and then obtained the TCR sequence of the T cells by RT-PCR. Ultimately, a total of 27 TCR sequences were identified from 6 patients, which recognized 14 new antigens expressed by autologous tumor cells. However, in further research on this method, the inventors found that although the single-cell RT-PCR amplification technology does not require complex in vitro culture and proliferation, this method cannot obtain the characteristics of cells expressing this TCR, including cell state, subpopulation, and which effector factors are expressed, so it is not conducive to evaluating the effectiveness of its TCR sequence in TCR-T.

[0006] Therefore, there is an urgent need in this field to develop a method that can efficiently screen new viral / tumor antigens to expand the clinical application scope of TCR-T.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a use of a marker combination.

[0009] Another object of the present invention is to provide a method for screening target antigen-specific T cells.

[0010] Another object of the present invention is to provide a method for constructing a lentiviral expression vector.

[0011] Another object of the present invention is to provide a method for constructing target antigen-specific TCR-T cells.

[0012] In order to solve the above technical problems, the present invention provides a marker combination for screening target antigen-specific T cells in the first aspect, wherein the marker combination includes CD137 + and CD200 + .

[0013] A second aspect of the present invention provides a method for screening target antigen-specific T cells, the method comprising the steps of:

[0014] S1, obtaining antigen-presenting cells loaded with target antigens, and culturing the antigen-presenting cells with T cells for co-stimulation; and

[0015] S2, CD8 cultured from step S1 + CD137 sorting in T cells + and CD200 + CD8 + T cells, thereby obtaining the target antigen-specific T cells.

[0016] In some preferred embodiments, the target antigen is a viral antigen, a tumor-specific antigen (TSA) and / or a tumor-associated antigen (TAAs).

[0017] In some preferred embodiments, the target antigen and the antigen-presenting cell are combined to obtain an antigen-presenting cell complexed with the target antigen.

[0018] In some preferred embodiments, target antigens are added to antigen-presenting cells for incubation, and then irradiated to obtain antigen-presenting cells complexed with target antigens.

[0019] In some preferred embodiments, the co-stimulation culture system includes IL-21, IL-2, IL-7 and IL-15.

[0020] In some preferred embodiments, the viral antigen is selected from at least one of CMV, HPV and EBV.

[0021] In some preferred embodiments, the tumor-specific antigen is selected from at least one of point-mutated TP53 and KRAS.

[0022] In some preferred embodiments, the tumor-associated antigen is selected from at least one of NY-ESO-1 and WT1. In some preferred embodiments, the target antigen is a peptide segment derived from EBV-LMP1.

[0023] In some preferred embodiments, the antigen-presenting cells are selected from at least one of the virus-infected target cells of monocytes, dendritic cells, B cells, Langerhans cells and tumor cells; preferably dendritic cells.

[0024] In some preferred embodiments, the sorting is performed by density gradient centrifugation, immunodensity centrifugation, immunomagnetic bead method or flow cytometry; more preferably, flow cytometry is used.

[0025] In some preferred embodiments, the CD8 + T cells were sorted.

[0026] In some preferred embodiments, the fluorescent dye is Cy5, FITC, APC, PI, 7-AAD, PE, PerCP or a composite dye formed by a combination thereof.

[0027] In some preferred embodiments, the fluorescent dye is a composite dye, such as Percep / Cy5-5 or CD137-PE-Cy7.

[0028] A third aspect of the present invention provides a method for constructing a lentiviral vector, comprising the steps of:

[0029] SA, screening to obtain target antigen-specific T cells using the method described in the second aspect of the present invention, analyzing their TCR sequences to obtain target gene sequences encoding the TCR sequences; and

[0030] SB, constructing a lentiviral vector containing an exogenous gene sequence, wherein the exogenous gene sequence includes the target gene sequence.

[0031] In some preferred embodiments, the target gene sequence includes a first sequence and a second sequence, wherein the first sequence encodes TCRα protein and the second sequence encodes TCRβ protein.

[0032] In some preferred embodiments, the exogenous gene sequence comprises a first sequence, a sequence encoding a linker, and a second sequence, connected in sequence; wherein the linker element is selected from at least one of P2A, T2A, E2A, and F2A. In some preferred embodiments, the first sequence and / or the second sequence are codon-optimized.

[0033] A fourth aspect of the present invention provides a method for constructing a target antigen-specific TCR-T cell, the method comprising the steps of:

[0034] Sa, packaging the lentiviral vector obtained by the method according to the third aspect of the present invention using packaging cells to obtain packaging cells for producing lentiviral particles;

[0035] Sb, culturing the packaging cells to express the lentiviral particles; and

[0036] Sc, use the lentiviral particles obtained in step Sb to infect CD8 + T cells, thereby obtaining the target antigen-specific TCR-T cells.

[0037] In some preferred embodiments, the packaging cell is a HEK293 cell line, a HEK293T cell line, a HEK 293E cell line, a HEK293FT cell line, a 293Vec cell line, a TE671 cell line, a HT1080 cell line or a HeLa cell line; more preferably, a HEK293T cell line.

[0038] Compared with the prior art, the present invention has at least the following advantages:

[0039] (1) The present invention provides a marker combination for efficiently screening T cells containing a target antigen-specific TCR, and provides a method for screening T cells containing a target antigen-specific TCR based on the marker combination. It has been verified that more than 90% of the T cells screened by this method are target antigen-specific T cells, and the screening accuracy is significantly improved compared with the use of CD137 marker alone;

[0040] (2) The results of in vitro and in vivo experiments have verified that the antigen-specific TCRs identified by the marker combination of the present invention exhibit specific killing responses to target cells in both in vitro and in vivo experiments, indicating that screening antigen-specific TCRs based on the marker combination of the present invention has good application prospects in personalized TCR-T therapy. Compared with traditional methods, this method can more effectively screen antigen-specific TCRs, thereby providing the possibility of personalized transformation of T cells to achieve more accurate and efficient virus / tumor attacks;

[0041] (3) The method provided by the present invention uses single-cell TCR+ transcriptome sequencing to obtain the antigen-specific TCR identified by CD137+ / CD200+ markers and its ability to specifically bind to pMHC in vitro. Compared with the RT-PCR method, it can obtain the characteristics of the antigen-specific T cells, including their cell subsets and activation status, while obtaining the T cell TCR sequence.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0044] FIG1 is a flow chart of EBV-LMP1-TCR-T screening according to an embodiment of the present invention;

[0045] FIG2 is a flow cytometry analysis diagram of EBV-LMP1-TCR-T according to an embodiment of the present invention;

[0046] FIG3 is a diagram of the EBV-LMP1-TCR sequence sequenced from a single cell according to an embodiment of the present invention;

[0047] FIG4 is a graph showing transcriptome characteristics of TCR-clonetypes analyzed by single-cell sequencing according to an embodiment of the present invention;

[0048] Figure 5 is a schematic diagram of the EBV-LMP1-TCR-T vector according to an embodiment of the present invention;

[0049] FIG6 is a diagram showing the results of in vitro binding verification of EBV-LMP1-TCR-Jurkat T according to an embodiment of the present invention.

[0050] FIG7 is a diagram showing the results of in vitro binding verification of EBV-LMP1-TCR-primary CD8+T according to an embodiment of the present invention.

[0051] FIG8 is a diagram showing the results of screening TCR sequences specifically targeting TP53-R248Q based on CD137 / CD200 markers and in vitro identification according to an embodiment of the present invention.

[0052] FIG9 is a diagram showing the results of verifying the expression of CD137 / CD200 markers in antigen-specific T cells based on CyTof technology according to an embodiment of the present invention. DETAILED DESCRIPTION

[0053] The present inventors have developed a new marker combination for screening T cells containing target antigen-specific TCRs. The proportion of target antigen-specific T cells in the T cells obtained by screening with this marker combination is more than 90% (compared to CD137 alone). + maker, correspondingly only 40% or even lower), the screening accuracy is significantly improved.

[0054] Furthermore, the inventors have also developed corresponding methods for screening target antigen-specific T cells and methods for obtaining target antigen-specific T cells in large quantities. Compared with traditional methods, these methods can more effectively screen TCRs specific to virus / tumor-specific antigens, thereby providing the possibility for personalized modification of T cells to achieve more accurate and efficient virus / tumor attacks.

[0055] Marker combination

[0056] The present invention relates to a marker combination for screening T cells containing target antigen-specific TCR (also referred to as "target antigen-specific T cells" in the present invention), including CD137 + and CD200 + .

[0057] In the present invention, the term "CD137 + " and "CD137 positive" can be used interchangeably and refer to high expression of CD137 on the cell surface, also known as 4-1BB, which is a T cell co-stimulatory immune checkpoint molecule that is mainly expressed on activated T cells and on many non-T cells such as natural killer cells (NK cells), dendritic cells (DC cells), and macrophages.

[0058] In the present invention, the term "CD200 + " and "CD200 positive" can be used interchangeably to refer to cells that express high levels of CD200 on their surface. + "Subpopulation cells" refer to cell populations that highly express the CD200 antigen. CD200 (OX-2) is a member of the IG superfamily and is expressed on the surface of a large number of cells, including B cells and activated T cells.

[0059] Method for screening target antigen-specific T cells

[0060] The present invention also relates to a method for screening target antigen-specific T cells, comprising steps S1 and S2. Specifically, the method comprises:

[0061] S1, obtain antigen-presenting cells loaded with target antigen and make them interact with CD8 + T cell co-stimulation culture, thereby inducing the expansion of antigen-specific T cells.

[0062] S2, CD8 obtained from the co-culture system in step S1 + T cells were sorted by CD137 + and CD200 + CD8 + T cells, through single-cell TCR sequencing, to obtain the TCR sequence of target antigen-specific T cells.

[0063] As used herein, the term "T cell" refers to T lymphocytes, such as CD4 + T cells or CD8 + T cells, or CD4 + T cells and CD8 + A mixture of T cells. Here, CD4 +T cell groups include T helper cells, such as T helper 1 (Th-1) cells, T helper 2 (Th-2) cells, T helper 17 (Th-17) cells, CD4 + CD25 + Regulatory T cells (Treg), Tr1 cells and T helper 3 (Th-3) cells. CD8 + T cell populations include CD4 - CD8 + Cytotoxic T cells and CD4 + CD8 + Phenotypic T cells (CD4 + CD8 dim ,CD4 dim CD8 bright or CD4 hi CD8 hi ).

[0064] As used herein, the term "antigen" is particularly understood to mean a protein, polypeptide, or peptide. Antigens are in particular polypeptide sequences that are taken up and processed by APCs, and fragments thereof (so-called epitopes) are presented to T cells on MHC molecules. Antigens are also particularly peptides that are presented to T cells together with MHC. In addition, antigens are understood to mean expression plasmids, DNA, or RNA encoding polypeptides. In some embodiments of the present invention, an "antigen" is a polypeptide. As used herein, the term "polypeptide" refers to a polymer of amino acids of any length. The term "polypeptide" also includes the following terms: target epitope, epitope, peptide, oligopeptide, protein, multimeric protein, and aggregates of polypeptides. In addition, the expression "polypeptide" also encompasses polypeptides that exhibit post-translational modifications such as glycosylation, acetylation, phosphorylation, aminoacetylation, and similar modifications. In addition, the expression "polypeptide" is understood to also refer to polypeptides that exhibit one or more amino acid analogs, such as non-natural amino acids; polypeptides with alternative linkages and other modifications known in the art, whether naturally occurring or from non-natural sources.

[0065] As used herein, the term "target antigen" refers to the target for diagnosis / treatment of TCR-T therapy. The target antigen may be a viral antigen (usually an oncogenic viral antigen or an oncogenic virus-derived antigen), a tumor-specific antigen (TSA) or a tumor-associated antigen (TAAs), etc. The term "tumor-specific antigen" refers to a protein molecule / peptide derived from a tumor cell gene mutation or distortion, which is specifically expressed in tumor cells but not present in normal tissues. The term "tumor-associated antigen" refers to an antigen molecule present on tumor cells or normal cells. Tumor-associated antigens are not unique to tumor cells. They can be synthesized in trace amounts in normal cells and are highly expressed / overexpressed when tumor cells proliferate. The term "Oncogenic Viral Antigens" refers to viral proteins expressed by virus-driven tumors, which are commonly used to make conventional preventive vaccines. In one embodiment of the present invention, the target antigen is the EBV-LMP1 target peptide.

[0066] As used herein, the term "antigen-presenting cells (APC)" refers to a class of immune cells that can ingest, process, and present the processed antigens to T cells. The term "antigen-presenting cells" particularly includes so-called professional antigen-presenting cells, such as dendritic cells, monocytes, macrophages, non-professional APCs, such as B cells, as well as vascular endothelial cells, fibroblasts of the skin, epithelial cells of the thymus or thyroid gland, glial cells of the brain, beta cells of the pancreas, and vascular endothelial cells. Non-professional APCs express the class I and class II MHC molecules necessary for interacting with T cells only after being activated by cytokines such as IFN-γ. In addition, T cells can also act as APCs. These APCT cells are produced by intracellular transfer of MHC class I and class II molecules and co-stimulatory molecules such as CD80, CD40 ligand (CD40L), OX40 ligand (OX40L) and 4-1BB ligand (4-1BBL, TNFSF9) due to contact with APCs, especially dendritic cells (DCs). In some embodiments, APCs include monocytes-phagocytes, dendritic cells, B cells, Langerhans cells, and virally infected target cells of tumor cells. In one embodiment of the present invention, the antigen-presenting cells are dendritic cells, which have the strongest antigen-presenting ability. The term "antigen-presenting cells carrying target antigens" refers to the formation of pMHC (peptide-major histocompatibility complex) complexes expressed on the surface of antigen-presenting cells that can be recognized and bound by T cells after the immunogenic antigen peptides in the target antigen are phagocytosed by antigen-presenting cells. The cells are called antigen-presenting cells carrying target antigens.

[0067] As used herein, the term "co-stimulation" refers to the reaction required to fully activate T cells. "Co-stimulation culture" refers to the process in which cultured cells undergo a co-stimulatory response, which is accompanied by the complete activation of T cells. The complete activation of T cells requires the synergistic action of a first signal and a second signal, wherein the first signal preliminarily activates the T cells and the second signal is a co-stimulatory signal that fully activates the T cells. The first signal is generated by TCR recognizing the MHC / antigen peptide complex presented by APC, and the second signal is generated by the interaction of co-stimulatory molecules on the surface of APC with corresponding co-stimulatory molecules on the surface of T cells. In an embodiment of the present invention, unactivated CD8 + T cells are co-stimulated with antigen-presenting cells containing target antigens to make CD8 + The corresponding TCR on the T cell surface recognizes the MHC / antigen peptide complex presented by the APC, and the co-stimulatory molecules on the APC surface interact with the CD8 + The corresponding co-stimulatory molecules on the surface of T cells interact with each other, causing CD8 + T cells are fully activated. In an embodiment of the invention, the "co-stimulation culture" system includes IL-21, IL-2, IL-7 and IL-15. In an embodiment of the invention, the "co-stimulation culture" is cultured in an incubator at 37°C and 5% CO2 concentration. In an embodiment of the invention, the "co-stimulation culture" time is not less than 10 days, more preferably not less than 20 days, more preferably not less than 24 days, for example 25 days, 30 days.

[0068] T cells are fully activated by costimulatory culture to obtain activated T cells. As used herein, the term "activated T cells" refers to T cells that are stimulated by the first antigen contact through the T cell receptor on the basis of the unactivated state before the first antigen attack. The T cell receptor recognizes the peptide presented on the MHC molecule, resulting in so-called T cell receptor crosslinking. Another prerequisite for T cell activation is the second signal, which is mediated by the interaction between the costimulatory molecules and their ligands on the APC and the T cell. This activation leads to a signal cascade inside the T cell, ultimately leading to proliferation and the occurrence of various effector functions. In addition, activated T cells are characterized by transient expression of characteristic surface molecules such as CD40 ligands, 4-1BB, CD69 and / or CD25. The activation of unactivated T cells to activated T cells after the initial contact with the antigen on the surface of professional antigen-presenting cells is also called "initialization" (priming). The term "activated T cells" is synonymous with the term "effector T cells" and also includes memory T cells that are specifically reactivated in vivo by re-contacting with their antigens. As used herein, the term "unactivated T cell" refers to a T cell that exhibits a certain antigen specificity but has not yet made its first contact with its antigen. Naive T cells, such as naive CD4+ T cells, exhibit a marker such as CD45RA on their surface.

[0069] As used in the present invention, the term "T cell recognition" refers to dual recognition, meaning that T cells can only recognize antigenic peptides obtained by APC-processed target antigens, and simultaneously recognize MHC molecules associated with the antigenic peptides. For example, CD4+ T cells recognize antigenic peptides and MHC class II molecules; CD8+ T cells recognize antigenic peptides and MHC class I molecules. The T cell recognition process involves two TCR recognition proteins, TCRα and TCRβ, in which case the TCR is an αβ receptor. In embodiments of the present invention, the T cell recognition process involves both TCRα and TCRβ. The term "αβT cell" refers to a T cell that produces αβ receptors.

[0070] As used herein, the term "sorting" refers to separation and selection, and "sorting" of cells refers to the technique of separating a cell from a variety of cell samples. Cell sorting methods include density gradient centrifugation, immunodensity centrifugation, immunomagnetic bead method, flow cytometry, etc. In one embodiment of the present invention, CD137 + and CD200 + CD8 + CD8 T cells obtained from costimulatory cultures +In one embodiment of the present invention, the CD8 T cells cultured in step S1 are isolated by flow cytometry using CD200 and CD137 labeled with different fluorescent dyes. + T cells are sorted, preferably, the fluorescent dye is Cy5, FITC, APC, PI, 7-AAD, PE, PerCP or a composite dye formed by a combination thereof, such as Percp / Cy5-5 or CD137-PE-Cy7.

[0071] In some embodiments of the present invention, the isolated CD137 + and CD200 + CD8 + Among T cells, target antigen-specific T cells account for more than 85%, more preferably higher than 90%, and more preferably higher than 91%.

[0072] Methods for constructing lentiviral vectors

[0073] The present invention also relates to a method for constructing a lentiviral vector, comprising steps SA and SB. Specifically, the method comprises:

[0074] SA, using the "method for screening target antigen-specific T cells" of the present invention to screen target antigen-specific T cells, analyze the TCR sequence on their surface to obtain a sequence encoding the target antigen-specific T cell TCR, which is called the target gene sequence.

[0075] SB, constructing a lentiviral vector containing an exogenous gene sequence, wherein the exogenous gene sequence includes the target gene sequence obtained in step SA.

[0076] As used herein, the term “TCR sequence” refers to the amino acid sequence of a T cell antigen receptor (TCR). When the T cell is an αβT cell, the TCR of the activated αβT cell has a TCRα protein and a TCRβ protein, and the amino acid sequence of the TCR includes both the amino acid sequence of the TCRα protein and the amino acid sequence of the TCRβ protein. The method for obtaining the amino acid sequence of the protein is well known to those skilled in the art. In an embodiment of the invention, the amino acid sequence of the TCRα protein and the TCRβ protein can be obtained by single-cell sequencing. In a preferred embodiment of the present invention, after obtaining the aforementioned amino acid sequence, the corresponding sequences of the TCRα protein and the TCRβ protein with the highest frequency of occurrence are sequence optimized, and their constant regions are modified to avoid mismatching between the exogenous TCR and the endogenous TCR of the T cell. In a preferred embodiment of the present invention, after obtaining the aforementioned amino acid sequence, the two amino acid sequences are spliced ​​with a linker sequence to obtain a fusion protein consisting of the TCRα protein-linker-TCRβ protein. Such a linker can be selected from at least one of P2A, T2A, E2A, F2A and Furin-cleavage, and the amino acid sequences of these linkers should be well known in the art.

[0077] As used herein, the term "target gene" refers to a gene sequence encoding a target antigen-specific T cell TCR. In an embodiment of the present invention, the amino acid sequences of the TCRα protein and the TCRβ protein are analyzed to obtain a gene sequence encoding the TCRα protein (first sequence) and a gene sequence encoding the TCRβ protein (second sequence). The "target gene" includes both a gene sequence encoding the TCRα protein and a gene sequence encoding the TCRβ protein. In a preferred embodiment of the present invention, after obtaining the corresponding target gene sequence, codon optimization is performed on it to increase the expression of the encoded protein.

[0078] As used herein, the term "exogenous gene" refers to the gene fragment inserted into a lentiviral vector. Exogenous genes include target genes, as well as tag genes that promote expression and genes encoding linkers for connecting multiple target gene fragments. In a preferred embodiment of the present invention, the gene sequence encoding the TCRα protein and the gene sequence encoding the TCRβ protein are connected via a sequence encoding a linker to form the exogenous gene, allowing the TCRα and TCRβ proteins to be expressed simultaneously in a single expression vector.

[0079] As used herein, the term "lentiviral vector" has the meaning conventionally understood by those skilled in the art. It refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1). Lentiviral vectors carrying exogenous genes, with the assistance of lentiviral packaging plasmids and cell lines, are packaged into infectious viral particles, which then infect cells or living tissues to achieve expression of the exogenous gene in them. Lentiviral vectors can effectively integrate exogenous genes or exogenous shRNAs into host chromosomes, thereby achieving the effect of persistent expression of the target sequence. In terms of infectivity, they can effectively infect a variety of cell types, including neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, and stem cells, thereby achieving excellent gene therapy effects. For some difficult-to-transfect cells, such as primary cells, stem cells, and undifferentiated cells, the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or target shRNA, and greatly increase the probability of the target gene or target shRNA integrating into the host cell genome, making it relatively convenient and rapid to achieve long-term, stable expression of the target gene or target shRNA. In a specific embodiment, the lentiviral vector described herein is a first to third generation HIV-1 lentiviral vector system. Those skilled in the art are familiar with the composition of the first to third generation HIV-1 lentiviral vector system, and the first to third generation HIV-1 lentiviral vector system can also be purchased commercially, for example, from Invitrogen, Takara, etc. In an embodiment of the present invention, a lentiviral expression vector containing the gene of interest is constructed by inserting a gene sequence encoding the TCRα protein into the lentiviral vector system. In an embodiment of the present invention, a lentiviral expression vector containing the gene of interest is constructed by inserting a gene sequence encoding the TCRβ protein into the lentiviral vector system. In a preferred embodiment of the present invention, a lentiviral expression vector containing the gene of interest is constructed by inserting a gene sequence encoding a fusion protein consisting of the TCRα protein-linker-TCRβ protein into the lentiviral vector system.

[0080] Methods for constructing target antigen-specific TCR-T

[0081] The present invention also relates to a method for constructing target antigen-specific TCR-T cells, comprising steps Sa, Sb and Sc.

[0082] Sa, using packaging cells to package the lentiviral vector obtained by the method for constructing a lentiviral vector to obtain packaging cells for producing lentiviral particles.

[0083] Sb, culturing the packaging cells obtained in step Sa to express the lentiviral particles.

[0084] Sc, use the lentiviral particles obtained in step Sb to infect CD8 +T cells, thereby obtaining target antigen-specific TCR-T cells.

[0085] As used herein, the terms "packaging cell" and "packaging cell line" are used interchangeably, and any known packaging cell line for producing viral vectors can be used in the present invention. In some embodiments of the present invention, the packaging cell line used is selected from HEK293, HEK293 T, HEK 293E, HEK293FT, 293 Vec, TE671, HT1080, or HeLa cell lines. In a preferred embodiment, the packaging cell line is HEK293T.

[0086] As used in the present invention, the term "packaging" refers to the process of transfecting a packaging cell line with a lentiviral vector carrying an exogenous gene together with a packaging vector. The method of packaging a lentiviral vector using a packaging cell line is conventional in the art, and those skilled in the art can package the lentiviral vector with reference to the instructions of commercially available lentiviral vectors. In one embodiment of the present invention, the packaging cell line is cultured, and the lentiviral vector carrying the exogenous target gene, the packaging vector, and the transfer vector are co-transfected into a packaging cell line (HEK293T) in an exponential growth phase, and the PEG8000 concentration method / sucrose density gradient ultracentrifugation method is used to concentrate and purify the resulting lentiviral particles. Optionally, GFP fluorescence counting, absolute quantitative qPCR, and RT-qPCR are used to measure the purified lentiviral particles.

[0087] As used in the present invention, the term "virus-infected T cells" refers to the process by which viruses activate T cells. In one embodiment of the present invention, lentiviral particles and CD8 + T cells were co-cultured to obtain a large number of virus-infected CD8 + T cells.

[0088] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified. The cytokines used in the following examples are all purchased from Peprotech.

[0089] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0090] Experimental methods

[0091] Preparation of dendritic cells by differentiation of human peripheral monocytes

[0092] (1) CD14 was isolated from the peripheral blood of healthy donors using magnetic beads (Miltenyi Biotec, Cat. No. 130-050-201). + Monocytes were cultured in AIM-V culture medium (Thermo) containing 1% human serum, human GM-CSF (Peprotech, 100 ng / mL), and IL-4 (50 ng / mL) for 3 days. After changing the medium, the culture was continued for 2 days to induce the differentiation of monocytes into MoDCs.

[0093] (2) After 5 days of differentiation, cytokines such as TNF-α (10 ng / mL), IL-1β (10 ng / mL), IL-6 (10 ng / mL), and Prostaglandin E2 (Peprotech, 1 μg / mL) were added to induce DC maturation.

[0094] (3) After 24 hours, mature DC cells were harvested and the expression of mature DC markers was detected by flow cytometry. Phenotype of mature DCs: CD14 - ,CD11c + ,CD40 + ,CD80 + ,CD83 + ,CD86 + ,HLA-ABC + ,HLA-DR + The obtained mature DCs cells were collected and used for subsequent loading with EBV-LMP1 antigen polypeptide.

[0095] Target antigen-loaded dendritic cells and CD8 + Costimulatory culture of T lymphocytes

[0096] (1) Add 5 μg / ml of the EBV-LMP1 target peptide to the mature DCs. Seal with Parafilm and place in a MACSmix rotator. Rotate and bind for 4 hours in a cell culture incubator. After incubation of mDCs with EBV-LMP1-peptide, irradiate the EBV-LMP1-peptide-loaded DCs with a dose of 40 Gy using an irradiator. After irradiation, collect the EBV-LMP1-peptide-loaded DCs by centrifugation into a 15 ml centrifuge tube.

[0097] (2) Lymphoprep density gradient centrifugation was used to obtain PBMCs from healthy donor blood and EasySep TM Isolation of CD8 from PBMCs using the Human CD8 Positive Selection Kit + T cells, EBV-LMP1-peptide-loaded DCs were mixed with CD8 + T cells were added to a 12-well plate and cultured, and IL-21 with a final concentration of 30 ng / ml and EBV-LMP1 peptide at 5 μg / ml were added to the culture system.

[0098] (3) Place the 12-well plate in a 37°C, 5% CO2 cell culture incubator and incubate overnight. On Day 1, add 10 ng / ml of IL-2, IL-7, and IL-15 to each well. The plate is then placed in a cell culture incubator at 37°C, 5% CO2 for 10 days. Change the medium halfway every 3 days during the culture process.

[0099] (4) On Day 14, Irradiated EBV-LMP1-peptide-loaded DCs were prepared and subjected to a second round of in vitro stimulation with cells in each culture system, and the stimulation was continued for 10 days.

[0100] (5) On Day 25, cells were aspirated from each well of the plate, washed twice with FACS buffer, and then CD200-Percep / Cy5-5, CD137-PE-Cy7, CD8a-APC, and PE-HLA-A0201-restricted-LMP1-Tetramer (MBL company) were added. After mixing, the cells were incubated at 4°C in the dark for 30 min. After the incubation, the cells were washed three times with FACS buffer, and the cells were resuspended in 2 ml of FACS buffer containing 1× DAPI working solution and CD8 was detected. + / Dextramer + (%) ratio detection.

[0101] Flow cytometry and single-cell sequencing to obtain CD137 + / CD200 + CD8 + TCR sequence of T

[0102] (1) CD8 in flow cytometry culture system + / CD137 + / CD200 + T cells, 10×Genomics single-cell sequencing technology to obtain CD137 + / CD200+ TCRα and TCRβ chain sequences of T cells.

[0103] (2) After obtaining the TCR sequence through single-cell sequencing, the most frequently occurring TCRα / βchain sequence is optimized and its constant region is modified to avoid mismatch between the exogenous TCR and the endogenous TCR of the T cell; the expression sequence is codon optimized to increase protein expression; P2A and Furin-cleavage are used to enable TCRα and TCRβ to be expressed simultaneously in one expression vector.

[0104] (3) Constructing a lentiviral expression vector, inserting the above-mentioned TCRα and TCRβ sequences into the same lentiviral expression vector, and performing virus packaging through 293T cells to produce EBV-LMP1 antigen-specific TCR-specific virus particles.

[0105] Infect Jurkat T cells with EBV-LMP1-TCR lentivirus to construct EBV-LMP1-TCR-Jurkat T

[0106] (1) Jurkat T-CD8 cells (from ACRO Biosystems) were revived and passaged for 2 generations until stable. Then, 1e6 cells were plated into 6-well plates at a density of 1e6 / well and cultured in 3 ml of 1640 complete medium per well.

[0107] (2) Jurkat T-CD8 cells were infected with EBV-LMP1-TCR lentiviral particles and activated. 10 μg / ml polybrene was added and centrifuged at 500 × g for 90 min at 30°C. After centrifugation, the cells were placed in a cell culture incubator at 37°C with 5% CO2 for 3 days, followed by a second round of infection with EBV-LMP1-TCR lentivirus.

[0108] (3) 24 h after the second round of EBV-LMP1-TCR lentivirus infection, the supernatant was discarded by centrifugation and replaced with fresh 1640 complete medium for continued culture.

[0109] HLA-A*0201 restricted EBV-LMP1-TCR-T in vitro binding assay

[0110] (1) Three days after the second round of EBV-LMP1-TCR lentivirus infection, EBV-LMP1-TCR-Jurkat T cells were aspirated, washed twice with FACS buffer, and PE-HLA-A0201-restricted-LMP1-Tetramer and CD8a-APC antibody were added for staining. CD8 was detected by FACS. + / Tetramer +(%) ratio to determine whether EBV-LMP1-TCR-T can specifically bind to PE-HLA-A0201-restricted-LMP1-Tetramer.

[0111] Infect activated T cells with EBV-LMP1-TCR / TP53-R248Q-TCR lentivirus to construct TCR-T

[0112] (1) CD8+ T cells were isolated from PBMCs using the EasySepTM Human CD8 Positive Selection Kit at a ratio of 1:2.5 CD8+ T cells:CD3 / 28 Dynabeads. CD8+ T cells were activated in vitro using CD3 / 28 Dynabeads at 37°C for 48 h.

[0113] (2) EBV-LMP1-TCR / TP53-R248Q-TCR lentiviral particles were used to infect activated CD8+ T cells. 10 μg / ml polybrene was added and centrifuged at 800 g for 90 min at 30°C. After centrifugation, the cells were placed in a cell culture incubator at 37°C with 5% CO2 overnight, followed by a second round of infection with the EBV-LMP1-TCR / TP53-R248Q-TCR lentivirus.

[0114] (3) 48 h after the second round of EBV-LMP1-TCR / TP53-R248Q-TCR lentivirus infection, some TCR-T cells were collected, washed with FACS buffer, and stained with corresponding Tetramer-PE and CD8a-APC antibodies. The CD8+ / Tetramer+ (%) ratio was detected by FACS.

[0115] CyTof (mass cytometry) verifies the expression of CD markers in T cells

[0116] (1) After two rounds of stimulation with autologous mDCs loaded with CMV-pp65 peptides, CMV-pp65-tetramer antibodies were used to flow cytometry isolate TCR-T cells specific for the CMV-pp65 antigen;

[0117] (2) The sorted Tetramer-positive and Tetramer-negative cells were subjected to single-cell sequencing using CyTof to analyze the expression of extracellular markers of antigen-specific and antigen-nonspecific T cells;

[0118] (3) Use CyTof software to analyze and visualize the generated multidimensional data results, and analyze and statistically analyze the expression of CD137 and CD200 molecules in Tetramer-positive and Tetramer-negative cells

[0119] Example 1, EBV-LMP1-TCR-T screening process

[0120] The whole experiment (Figure 1) was carried out by CD8 + After two rounds of stimulation with autologous mDCs loaded with EBV-LMP1 peptides, the specificity of EBV-LMP1 antigen-specific TCR-T cells was analyzed using CD200-Percep / Cy5-5, CD137-PE-Cy7, CD8a-APC and HLA-A0201-restricted-LMP1-Tetramer-PE. The flow cytometry results (Figure 2, A) showed that after two rounds of mDCs-EBV-LMP1 peptide stimulation, the CD8a + / Tetramer + The proportion of T cells increased from 0.002% to 0.79%, indicating that EBV-LMP1-specific TCR-T cells were specifically expanded in vitro. + / CD137 + T cells were analyzed and CD8a + / CD137 + 37.4% of T cells were EBV-LMP1-Tetramer + cells (Figure 2, B). In addition, analysis revealed that CD8a + / CD200 + 41.8% of T cells were EBV-LMP1-Tetramer + cells, and their proportions were similar to those of CD8a + / CD137 + Similar (Figure 2, C). We then analyzed CD8a + / CD137 + / CD200 + Tetramer in cells + The results showed that up to 92.2% of CD137 + / CD200 + Tetramer + This indicates that the combination of CD200 and CD137 markers can screen viral / tumor neoantigens with high sensitivity. Compared with traditional methods, this method can more effectively screen antigen-specific TCRs (Figure 2, D).

[0121] Example 2: Single-cell sequencing to obtain EBV-LMP1-TCR sequences

[0122] After two rounds of stimulation with autologous mDCs loaded with EBV-LMP1 peptides, EBV-LMP1 antigen-specific TCR-T cells were flow cytometry sorted using CD137 and CD200 antibodies. The sorted EBV-LMP1-TCR-T cells were subjected to single-cell sequencing using the 10× Genomics method to obtain the α and β chain sequences of the EBV-LMP1-TCR-T cells. After single-cell sequencing, the top 10 frequencies obtained by sequencing were ranked. The single-cell sequencing results (Figure 3) showed that after sorting, the 1#TCR clone accounted for 70.97% and the 2#TCR clone accounted for 23.16%, demonstrating the good specificity of this antigen-specific T cell priming experiment.

[0123] Example 3: Single-cell sequencing analysis of TCR-clonetype transcriptome characteristics

[0124] After single-cell sequencing, transcriptome analysis of activation-related genes was performed on clones 1#, 2#, and other TCR-T cells. Single-cell transcriptome sequencing (Figure 4) showed that the expression levels of TNFRSF9 (CD137), GZMB, IFNγ, and IL2RA (CD25) in clones 1# and 2# were significantly higher than those in other TCRs, demonstrating the high specificity of this in vitro antigen-specific T cell priming experiment. The specific TCR-T cells were successfully activated and expanded in the in vitro costimulation experiment.

[0125] Example 4. Construction and sequence optimization of EBV-LMP1-TCR-T vector

[0126] After sequencing the EBV-LMP1-TCR (clonotype ID: 1), the sequence was optimized, including: (1) modification of the constant region to prevent mispairing with the endogenous TCR chain; (2) optimization of the expression sequence for humanized codons to increase TCR protein expression; and (3) simultaneous expression of the alpha and beta chains on a single plasmid using P2A and furin-cleavage. Subsequently, the optimized EBV-LMP1-TCR sequence (Figure 5) was cloned into a lentiviral expression vector.

[0127] Example 5, EBV-LMP1-TCR-T in vitro binding experiment

[0128] EBV-LMP1-TCR lentivirus was packaged in 293T cells. Jurkat T-CD8 cell line (ACRO Biosystems) was infected with EBV-LMP1-1# / 2#-TCR lentivirus twice and then EBV-LMP1-HLA*A:0201-Tetramer / mTCRβC was stained. Flow cytometry results showed that Tetramer + / mTCRβC + Jurkat T-CD8-LMP1-1# / 2#-TCR cell clustering was obvious (Figure 6). Subsequently, primary CD8+ T cells were infected with EBV-LMP1-1# / 2#-TCR lentivirus twice and then flow cytometry was performed. The results showed that the EBV-LMP1-TCR-T screened and identified in this project can be normally expressed in Jurkat T-CD8 cells and primary CD8+ T cells, and can specifically bind to EBV-LMP1-HLA*A:0201-Tetramer (Figure 7). This result shows that the method of screening antigen-specific TCRs based on CD200 and CD137 markers is feasible. This method has good application prospects in personalized TCR-T therapy.

[0129] Example 6: Screening of TCR sequences specifically targeting TP53-R248Q based on CD137 / CD200 markers and in vitro identification

[0130] The discovery and in vitro binding assay of the TCR sequence targeting EBV-LMP1 demonstrated that the combination of CD200 and CD137 markers can be used to screen for viral antigen-specific TCR sequences with high sensitivity and accuracy. Compared with traditional methods, this approach can more effectively screen for antigen-specific TCRs. Subsequently, we used this marker combination (CD137+ / CD200+) to screen for TP53-R248Q tumor neoantigen-specific TCRs in vitro. The specific process is shown in Figure 8A. Subsequently, primary CD8+ T cells were infected with the TP53-R248Q-TCR lentivirus twice and then analyzed by flow cytometry. The results showed that the TP53-R248Q-TCR identified and screened in this project was normally expressed in primary CD8+ T cells and specifically bound to the TP53-R248Q-HLA*A:1101-tetramer (Figure 8B). This result demonstrates that the method of screening for antigen-specific TCRs based on CD200 and CD137 markers is also feasible for screening for tumor neoantigen-specific TCRs. Taken together, these results demonstrate that combining CD200 and CD137 markers can more efficiently screen viral / tumor neoantigen-specific TCRs.

[0131] Example 7: Verification of the expression of CD137 / CD200 markers in antigen-specific T cells based on CyTof technology

[0132] After two rounds of stimulation with autologous mDCs loaded with CMV-pp65peptides, CMV-pp65-Tetramer antibodies were used to flow cytometry isolate CMV-pp65 antigen-specific TCR-T cells. The sorted CMV-pp65-TCR-T cells and Tetramer-negative cells were subjected to CyTof single-cell sequencing to analyze the expression of extracellular markers of antigen-specific and antigen-nonspecific T cells (Figure 9A). After CyTof single-cell sequencing, the sequencing results (Figures 9B-C) showed that CMV-pp65 antigen-specific TCR-T cells significantly upregulated CD137 and CD200 markers, while CD137 and CD200 markers were lowly expressed in antigen-nonspecific T cells. This experimental result once again demonstrated that the combination of CD200 and CD137 markers can more efficiently screen for viral / tumor neoantigen-specific TCRs.

[0133] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A use of a marker combination, characterized in that: Used to screen target antigen-specific T cells, the marker combination includes CD137 + and CD200 + .

2. A method for screening target antigen-specific T cells, characterized in that: The method comprises the steps of: S1, obtaining antigen-presenting cells loaded with target antigens, and co-stimulating the antigen-presenting cells with T cells for culture; S2, sort CD137 from T cells cultured in step S1 + and CD200 + T cells, thereby obtaining the target antigen-specific T cells; preferably, the T cells are CD8+ T cells.

3. The method according to claim 2, characterized in that The target antigens are viral antigens, tumor-specific antigens (TSAs) and / or tumor-associated antigens (TAAs).

4. The method according to claim 2, characterized in that: The viral antigen is selected from at least one of CMV, HPV and EBV; And / or, the tumor-specific antigen is selected from: at least one of point-mutated TP53 and KRAS; And / or, the tumor-associated antigen is selected from at least one of NY-ESO-1 and WT1.

5. The method according to claim 2, characterized in that: The target antigen is a peptide segment derived from EBV-LMP1.

6. The method according to claim 2, characterized in that The antigen presenting cells are selected from at least one of the virus-infected target cells of monocytes-phagocytes, dendritic cells, B cells, Langerhans cells and tumor cells.

7. A method for constructing a lentiviral vector, characterized in that: The method comprises the steps of: SA, using the method described in any one of claims 2-6 to screen and obtain target antigen-specific T cells, analyzing their TCR sequences to obtain the target gene sequence encoding the TCR sequence; and SB, constructing a lentiviral vector containing an exogenous gene sequence, wherein the exogenous gene sequence includes the target gene sequence.

8. The method according to claim 7, characterized in that The target gene sequence includes a first sequence and a second sequence, wherein the first sequence encodes TCRα protein and the second sequence encodes TCRβ protein.

9. The method according to claim 7, characterized in that: The exogenous gene sequence includes a first sequence, a sequence encoding a linker, and a second sequence connected in sequence; wherein the connecting element is selected from at least one of P2A and T2A.

10. A method for constructing a target antigen-specific TCR-T cell, characterized in that: The method comprises the steps of: Sa, using packaging cells to package the lentiviral vector obtained by the method of any one of claims 7 to 9 to obtain packaging cells for producing lentiviral particles; Sb, culturing the packaging cells to express the lentiviral particles; and Sc, use the lentiviral particles obtained in step Sb to infect CD8 + T cells, thereby obtaining the target antigen-specific TCR-T cells.

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