TWI837168B - An isolated T cell receptor, a modified cell thereof, an encoding nucleic acid, a recombinant expression vector, a method for preparing TCR modified cells, a pharmaceutical composition and its use - Google Patents

Abstract

The present invention provides an isolated T cell receptor, a modified cell thereof, an encoding nucleic acid and an application thereof. The isolated T cell receptor (TCR) comprises at least one of an α chain and a β chain, both of which comprise a variable region and a constant region, and is characterized in that the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of the variable region of the α chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the β chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 2. The TCR can specifically recognize tumor antigens and avoid possible off-target toxic side effects. Immune cells modified with this TCR have significant anti-tumor effects.

Description

An isolated T cell receptor, a modified cell thereof, an encoding nucleic acid, a recombinant expression vector, a method for preparing TCR-modified cells, a pharmaceutical composition and its use

The present invention belongs to the field of biotechnology, and specifically relates to isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions and applications.

Her2/neu (ERBB2) is a transmembrane protein belonging to the EGFR family. It forms dimers with other family proteins and regulates cell proliferation, differentiation, and carcinogenesis through a series of intracellular signal transduction pathways (see references “Growth Factors, 2008; 26: 263”, “Oncol Biol. Phys, 2004; 58: 903”). Her2/neu protein is overexpressed in a variety of epithelial cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see the literature "Trends in Molecular Med, 2013; 19: 677"), and its expression in primary and metastatic cancer cells is relatively uniform (see the literature "J Clin Oncol, 1998; 8: 103"). Therefore, Her2/neu becomes an appropriate target for targeted therapy.

Herceptin, a humanized monoclonal antibody targeting Her2/neu, can significantly prolong the survival of Her2/neu-positive breast cancer patients (see the literature "N Engl J Med, 2001, 344: 783"). However, the clinical response rate of Herceptin alone in the treatment of Her2-positive metastatic breast cancer is only 11% to 26% (see the literature "J Clin Oncol, 2002; 20: 7169"), indicating that Herceptin alone is not effective for most metastatic breast cancers with high Her2 expression. Although Herceptin combined with chemotherapy can improve the clinical response rate, most breast cancer patients with overexpression of Her2/neu will develop resistance to Herceptin after one year (see the literature "J Clin Oncol, 2001; 19: 2587").

Patients with Her2/neu positive tumors produce endogenous antibodies and T cell responses against Her2/neu antigens (see the reference "Cancer Res, 2005; 65: 650"). Therefore, specific immunotherapy targeting Her2/neu antigens has become a promising treatment approach. T cells that specifically recognize Her2/neu epitope peptide 369-377 can be successfully isolated from ascites of ovarian cancer with high expression of Her2/neu (see the reference "J. Exp. Med. 1995; 181: 2109-2117"). A tumor vaccine targeting the Her2/neu 369-377 polypeptide antigen has entered clinical trials. Although the Phase 1/2 clinical trial showed that this vaccine can induce specific T killer cells targeting the Her2/neu 369-377 polypeptide antigen (see the reference “Breast Care, 2016; 11: 116”), the Phase III clinical trial did not achieve the intended goal of prolonging patient survival (http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast-cancer-halted-after-futility-review). After the development of tumor-specific T cell therapy based on chimeric antigen receptors (CARs) cultured in vitro, it entered clinical trials as the first CAR-T cell therapy targeting Her2/neu antigen for solid tumors, but was terminated due to the occurrence of severe cytokine release syndrome (CRS) leading to patient death (see the literature "Nature Med, 2016; 22: 26"). Severe cytokine storm and neurotoxicity are common toxic reactions in CAR-T therapy (see the reference "Blood, 2016; 127: 3321"), which may be partly related to the unrestricted cell activation of CAR, a non-natural T cell receptor (see the reference "Nat Ned, 2015; 21: 581"), or to the autocrine secretion of cytokines that do not require antigen stimulation (see the reference "Cancer Immunol Res, 2015; 3: 356").

TCR-T therapy, which involves the transfer of T cells modified with specific T cell receptor (TCR) genes, is considered to be the most promising immune cell gene therapy for solid tumors (see the literature “Adv Immunol. 2016; 130: 279-94”). Among them, clinical studies of TCR-T therapy targeting NY-ESO-1 antigen have shown encouraging clinical therapeutic effects (see the literature “Nat Med. 2015 Aug; 21(8): 914-921”). However, the number of specific TCRs that are currently known to target tumor antigens and effectively recognize tumor cells is very limited, thus limiting the widespread application of TCR-T therapy. In addition, although TCR-T therapy does not have the severe cytokine storm toxicity shown in CAR-T therapy, if the target antigen is derived from self-protein, targeting the target antigen with low expression in normal tissue cells may lead to severe autoimmune reaction, namely, on target off tumor toxicity reaction (see the reference "Blood 2009; 114: 535-546"). In addition, in order to obtain high-affinity TCRs that effectively recognize tumor cells, the general strategy is to perform gene point mutations on the complementarity determining regions (CDRs) on the TCR in vitro, or to induce T cells from a humanized mouse T cell library that has not been screened by the central tolerance mechanism (see the reference "Front Immunol. 2013; 4: 363"). However, this high-affinity TCR may cross-react with normal self-proteins and cause damage to normal tissue cells, i.e., severe or even fatal off-target toxicity (see the literature "Curr Opin Immunol 2015; 33: 16-22", see the literature "Sci Transl Med. 2013; 5(197): 197ra103"). Therefore, obtaining a new TCR gene that specifically targets tumor antigens and effectively recognizes tumor cells while avoiding possible off-target toxic side effects is the main challenge for the successful development of TCR-T immune cell gene therapy.

In order to solve the problems existing in the above-mentioned prior art, the present invention provides isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, drug compositions and applications.

Specifically, the present invention provides:

(1) An isolated T cell receptor, comprising at least one of an α chain and a β chain, wherein the α chain and the β chain both comprise a variable region and a constant region, wherein the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of the variable region of the α chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the β chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 2.

(2) The T cell receptor according to (1), wherein the T cell receptor is capable of specifically recognizing the antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule; preferably, the antigen epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3.

(3) The T cell receptor according to (1), wherein the constant region of the α chain and/or the constant region of the β chain are derived from humans; preferably, the constant region of the α chain is replaced in whole or in part by a homologous sequence derived from another species, and/or the constant region of the β chain is replaced in whole or in part by a homologous sequence derived from another species; more preferably, the other species is a mouse.

(4) The T cell receptor according to (1), wherein the constant region of the α chain is modified with one or more disulfide bonds, and/or the constant region of the β chain is modified with one or more disulfide bonds.

(5) The T cell receptor according to (1), wherein the amino acid sequence of the α chain is as shown in SEQ ID NOs: 4, 5 or 6, and the amino acid sequence of the β chain is as shown in SEQ ID NOs: 7, 8 or 9.

(6) An isolated nucleic acid encoding a T cell receptor, comprising a coding sequence of at least one of the α chain and the β chain of the T cell receptor, wherein the α chain coding sequence and the β chain coding sequence both comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and the amino acid sequence encoded by the α chain variable region coding sequence has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence encoded by the β chain variable region coding sequence has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 2.

(7) The nucleic acid according to (6), wherein the nucleic acid is DNA or RNA.

(8) The nucleic acid according to (6), wherein the α-chain variable region encoding sequence is shown as SEQ ID NO: 10, and the β-chain variable region encoding sequence is shown as SEQ ID NO: 11.

(9) The nucleic acid according to (6), wherein the T cell receptor encoded by the nucleic acid is capable of specifically recognizing the antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule; preferably, the antigen epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3.

(10) The nucleic acid according to (6), wherein the α chain constant region coding sequence and/or the β chain constant region coding sequence are derived from humans; preferably, the α chain constant region coding sequence is completely or partially replaced by a homologous sequence derived from other species, and/or the β chain constant region coding sequence is completely or partially replaced by a homologous sequence derived from other species; more preferably, the other species is mouse.

(11) The nucleic acid according to (6), wherein the α chain constant region coding sequence comprises one or more disulfide bond coding sequences, and/or the β chain constant region coding sequence comprises one or more disulfide bond coding sequences.

(12) The nucleic acid according to (6), wherein the α-chain coding sequence is as shown in SEQ ID NOs: 12, 13 or 14, and the β-chain coding sequence is as shown in SEQ ID NOs: 15, 16 or 17.

(13) The nucleic acid according to any one of (6) to (11), wherein the α-chain coding sequence and the β-chain coding sequence are connected by a coding sequence for a cleavable linking polypeptide.

(14) The nucleic acid according to (13), whose sequence is shown in SEQ ID NOs: 18, 19, or 20.

(15) A recombinant expression vector comprising a nucleic acid according to any one of (6) to (14) and/or a complementary sequence thereof, which is operably linked to a promoter. The promoter may be a eukaryotic cell promoter, including a continuous expression promoter and an inducible expression promoter, including (for example): PGK1 promoter, EF-1α promoter, CMV promoter, SV40 promoter, Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, human beta actin promoter.

(16) The recombinant expression vector according to (15), wherein the recombinant expression vector contains a suicide gene coding sequence; wherein the suicide gene is selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or a combination thereof.

(17) The recombinant expression vector according to (16), wherein the suicide gene coding sequence is under the control of a promoter, and the promoter used to control the suicide gene coding sequence is the same as or different from the promoter to which the nucleic acid described in any one of (6) to (14) is linked, and the promoters are independent of each other.

(18) The recombinant expression vector according to (16), wherein the suicide gene coding sequence and the nucleic acid described in any one of (6)-(14) are under the control of the same promoter, and the suicide gene coding sequence is linked to the nucleic acid described in any one of (6)-(14) via a coding sequence of a cleavable linker polypeptide or an internal ribosome entry site (IRES) sequence.

(19) A T cell receptor-modified cell, the surface of which is modified by the T cell receptor described in any one of (1) to (5), wherein the cell includes a primitive T cell or a precursor cell thereof, a NKT cell, or a T cell strain.

(20) The T cell receptor-modified cell according to (19), wherein the cell expresses a suicide gene protein on its cell surface or inside the cell; wherein the suicide gene is selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or a combination thereof.

(21) A method for preparing a cell modified with a T cell receptor according to (19) or (20), comprising the following steps: 1) providing a cell; 2) providing a nucleic acid encoding the T cell receptor according to any one of (1) to (5); 3) transfecting the nucleic acid into the cell.

(22) The method according to (21), wherein the cells in step 1) are autologous or allogeneic.

(23) The method according to (21), wherein the transfection method includes: a viral vector transfection method, preferably, the viral vector includes a γ-retroviral vector or a lentiviral vector; a chemical method, preferably, the chemical method includes a liposome transfection method; a physical method, preferably, the physical method includes an electrotransfection method.

(24) The method according to (21), wherein the nucleic acid described in step 2) is the nucleic acid described in any one of (6) to (14).

(25) Use of the T cell receptor modified cells according to (19) or (20) in the preparation of drugs for treating or preventing tumors and/or cancers.

(26) The use according to (25), wherein the tumor and/or cancer is positive for the antigen Her2/neu and is positive for HLA-A2.

(27) Use of the T cell receptor-modified cells according to (19) or (20) in the preparation of a drug for detecting tumors and/or cancers in a host.

(28) A pharmaceutical composition, wherein the pharmaceutical composition comprises as an active ingredient the T cell receptor modified cells according to (19) or (20), and a pharmaceutically acceptable excipient.

(29) The pharmaceutical composition according to (28), wherein the pharmaceutical composition comprises the T cell receptor modified cells in a total dose range of 1×10 ³ -1×10 ⁹ cells/Kg body weight per patient per treatment course.

(30) The drug composition according to (28), wherein the drug composition is suitable for administration via an artery, vein, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular or intraperitoneal route.

(31) A method for treating tumors and/or cancer, comprising administering cells modified with the T cell receptor according to (19) or (20) to a patient with the tumor and/or cancer.

(32) The method according to (31), wherein the administration dosage of the T cell receptor modified cells is a total dosage range of 1×10 ³ -1×10 ⁹ cells/kg body weight per patient per treatment course.

(33) The method according to (31), wherein the T cell receptor modified cells are administered via artery, vein, subcutaneous, intradermal, intratumoral, intralymphatic, intralymph node, subarachnoid space, intramarrow, intramuscular or intraperitoneal administration.

(34) The method according to (31), wherein the tumor and/or cancer is positive for the antigen Her2/neu and is positive for HLA-A2.

(35) The method according to (31) further comprises administering other drugs for treating tumors and/or drugs for regulating the patient's immune system to the tumor and/or cancer patient. Compared with the prior art, the present invention has the following advantages and positive effects: the present invention successfully induces T cell clones that are specific to Her2/neu antigen epitope polypeptides (such as Her2/neu 369-377 polypeptides) presented by HLA-A2 from the peripheral blood of healthy donors who are HLA-A2 positive, and screens out T cell clones carrying natural TCRs that specifically recognize Her2/neu antigen epitope polypeptides (such as Her2/neu 369-377 polypeptides), thereby obtaining the full sequence of the TCR. This TCR is non-CD8 dependent and has a moderate to high affinity for Her2/neu antigen epitope peptides (such as Her2/neu 369-377 peptides). It can specifically recognize HLA-A2 positive tumor cells that express Her2/neu antigens. In addition, T cell clones carrying this TCR enter the peripheral T cell library after screening by the central immune tolerance mechanism. Killer T cells carrying this TCR once existed in the peripheral blood of normal people and did not cross-react with normal tissue cells that express trace amounts of Her2/neu protein. In addition, in order to avoid the TCR from producing off-target cross-reactions with normal proteins and causing autoimmune toxicity, the information of the key amino acid sites on the Her2/neu 369-377 polypeptide related to the TCR recognition function is first obtained. Based on this, all human normal proteins containing the key amino acid sites recognized by the TCR are searched from the human normal protein database, and antigen epitope peptides that may bind to HLA-A2 molecules are further screened. Experiments show that the TCR does not recognize these epitope peptides from normal proteins with potential cross-reactions. Therefore, the present invention obtains a new TCR that can specifically recognize tumor antigens and avoid possible off-target toxic side effects.

In a further invention, the constant region of TCR is also modified (for example, disulfide bond modification or murinization) so that when this TCR is expressed on immune cells, it can further reduce or avoid the occurrence of mismatch with endogenous TCR.

Immune cells modified with this TCR (e.g., primitive T cells, their precursor cells, NKT cells, T cell lines) can specifically recognize a variety of HLA-A2 ⁺ and Her2/neu ⁺ tumor cell lines and have significant anti-tumor effects. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.

When using the TCR-modified immune cells of the present invention to treat tumors, the cytokine storm and immune rejection reactions caused by CAR-T therapy can be effectively avoided.

The TCR-modified immune cells of the present invention provide a new option for treating HLA-A2 ⁺ and Her2/neu ⁺ tumor patients and have good industrial application prospects.

6A5-TCR-T: Human T cells expressing Her2 TCR-6A5-mC

6A5-EGFR-TCR-T: Human T cells expressing Her2 TCR-6A5-mC and tEGFR

Colo205, HCT116: colorectal cancer cells

Cy: Cyclophosphamide

E75: Her2/neu 369-377 polypeptide

Furin-F2A: coding sequence of cleavable linker polypeptide

Her2-E75: Her2/neu 369-377 polypeptide

Her2/neu:ERBB2

HIV: Human immunodeficiency virus type 1

MDA-MB-231, MCF-7: breast cancer cells

MOCK-T: control T cells

NCI-H446: Lung cancer cells

PANC-1: pancreatic cancer cells

PBMC: peripheral blood mononuclear cells

TCR: specific T cell receptor

U87MG:Neuroglioma cells

Figure 1 shows the phenotype and functional test results of Her2/neu 369-377 polypeptide (Her2-E75)-specific killer T cells induced from HLA-A2 ⁺ normal donor PBMC (specifically #1 PBMC) in Example 1 of the present invention. Figure 1A shows the results of flow cytometry analysis of PBMC cells after two rounds of in vitro stimulation with Her2-E75 antigen polypeptide, stained with CD8-APC antibody and Her2-E75 pentamer-PE, and the right picture shows cells stimulated with polypeptide, and FACS sorting of CD8 ⁺ pentamer ⁺ killer T cell population is performed to obtain T cell clones. The left picture shows control tissue cells without polypeptide stimulation. The horizontal axis represents the fluorescence intensity of CD8 molecule expression, and the vertical axis represents the fluorescence intensity of bound Her2-E75 pentamer. Figure 1B is the phenotypic analysis of CD8 ⁺ E75-tetramer ⁺ killer T cell clones after CD8-APC and Her2-E75 tetramer-PE staining by flow cytometry. The right figure shows that CD8 ⁺ Her2 tetramer ⁺ T cell clone Her2 CTL 6A5 is a purified Her2-E75 polypeptide-specific CTL cell clone. The left figure is a control group CTL cell without polypeptide stimulation. The horizontal axis represents the fluorescence intensity of CD8 molecule expression, and the vertical axis represents the fluorescence intensity of bound Her2-E75 tetramer. Figure 1C shows the main functional fragments of the constructed lentiviral vector carrying the Her2TCR-6A5-mC gene (i.e., "pCDH-EF1α-Her2 TCR vector"). The fragments shown express the TCR gene driven by the EF-1α promoter, and the invariant region fragments of the β chain and α chain of each TCR are mouse invariant region fragments, and the β chain and α chain of the TCR are linked by the coding sequence of the cleavable linking polypeptide (furin-F2A).

Figures 2A to 2C show the phenotypic and functional assay results of peripheral blood mononuclear cells (PBMCs) transfected with the Her2 TCR-6A5-mC TCR gene. Figure 2A shows the results of flow cytometry analysis of PBMCs from two different donors transfected with a lentiviral vector encoding Her2 TCR-6A5-mC, after staining with Her2-E75 tetramer-PE and anti-CD8-APC antibodies. First, lymphocyte populations were separated according to cell morphology and size, and the Her2-E75 tetramer ⁺ cell population was a cell expressing the Her2 TCR-6A5-mC TCR. The horizontal axis represents the fluorescence intensity of CD8 molecule expression, and the vertical axis represents the fluorescence intensity of the bound Her2-E75 tetramer. The percentages shown are the ratios of each positive cell population to the number of lymphocytes isolated. The left panel refers to peripheral blood mononuclear cells provided by one donor (#1 PBMC), and the right panel refers to PBMC provided by a different donor (#2 PBMC). CD8 ⁺ Her2-E75 tetramer ⁺ cells are killer T cells expressing Her2 TCR-6A5-mC. CD8 ^- Her2-E75 tetramer ⁺ cells may be CD4 ⁺ helper T cells expressing Her2 TCR-6A5-mC. Figure 2B shows that T cells expressing Her2 TCR-6A5-mC can recognize the Her2-E75 polypeptide presented by T2 cells. PBMCs from two different donors transfected with lentiviral vectors encoding Her2 TCR-6A5-mC were co-cultured with T2 cells presenting different concentration gradients of Her2-E75 peptide for 16 hours, and the cell supernatants were taken for IFN-γ ELISA analysis. The target cells in the control group were T2 cells presenting EBV virus antigen peptide LMP2 426-434 that can bind to HLA-A2 molecules (not shown in the figure). In the figure, "0.1μg/ml" indicates a T2 cell group presenting 0.1μg/ml of Her2-E75 polypeptide, "0.01μg/ml" indicates a T2 cell group presenting 0.01μg/ml of Her2-E75 polypeptide, "0.001μg/ml" indicates a T2 cell group presenting 0.001μg/ml of Her2-E75 polypeptide, and "0.0001μg/ml" indicates a T2 cell group presenting 0.0001μg/ml of Her2-E75 polypeptide. The vertical axis indicates the concentration of IFN-γ secreted by T cells. Figure 2C shows the results of the CD8 antibody blocking test of T cell function. Among them, when #2 PBMCs transfected with a lentiviral vector encoding Her2 TCR-6A5-mC were co-cultured with Her2-E75 antigen peptide presented by T2 cells, anti-human CD8 antibodies were added to detect whether the function of T cells to secrete IFN-γ was inhibited. In the figure, "T2+Her2-E75" represents the T2 cell group that presented 0.1μg/ml Her2-E75 peptide without the addition of anti-human CD8 antibodies, and "T2+Her2-E75+anti-CD8" represents the T2 cell group that presented 0.1μg/ml Her2-E75 peptide with the addition of anti-human CD8 antibodies. The horizontal axis represents different experimental groups, and the vertical axis represents the concentration of IFN-γ secreted by T cells. "ns" indicates that there is no significant difference between the two experimental groups. In Figures 2B and 2C, each experimental group and control group were replicates, and the results are shown as mean ± SEM.

Figures 3A to 3K show the functional detection results of peripheral blood mononuclear cells (PBMCs) transfected with the Her2 TCR-6A5-mC TCR gene to identify tumor cell lines. Figure 3A shows the expression of HLA-A2 and Her2/neu in different tumor cell lines. The horizontal axis represents different human tumor cell lines. "Colo205" and "HCT116" are colorectal cancer cells; "MDA-MB-231" and "MCF-7" are breast cancer cells; "PANC-1" is pancreatic cancer cells; "U87MG" is neuroglioma cells; "NCI-H446" is lung cancer cells. The vertical coordinate "MFI" represents the mean fluorescence intensity of cells stained with anti-HLA-A2 fluorescent antibody or anti-Her2/neu fluorescent antibody. The white bar represents the expression of Her2/neu on the cell surface, and the black bar represents the expression of HLA-A2 on the cell surface. Figure 3B shows the results of ELISA analysis of IFN-γ in the supernatant of #2 PBMCs transfected with the lentiviral vector encoding the Her2 TCR-6A5-mC TCR gene and co-cultured with cells of different tumor cell lines for 24 hours. Each test group and control group has three wells, and the results are shown as mean ± SME. The horizontal axis represents different target cells, and the vertical axis represents the concentration of IFN-γ secreted by T cells. The effector-target ratio E:T is 5:1. The white bars indicate that the effector cells are control peripheral blood mononuclear cells that are not transfected with the Her2 TCR-6A5-mC TCR gene, and the black bars indicate that the effector cells are peripheral blood mononuclear cells that are transfected with the Her2 TCR-6A5-mC TCR gene. Figures 3C, D, E, F, G, H, I, J, and K show the killing activity of #2 PBMC against different tumor cell lines after transfection with a lentiviral vector encoding the Her2 TCR-6A5-mC TCR gene. The killing activity of Figures 3C-3G was obtained by counting live cells, and the killing activity of Figures 3H-3K was determined by the MTT method, and the reaction time was 24 hours. Among them, Figures 3C and 3H show the results for the tumor cell line MCF-7, Figure 3D shows the results for the tumor cell line HCT116, Figure 3E shows the results for the tumor cell line U87MG, Figure 3F shows the results for the tumor cell line NCI-H446, Figure 3G shows the results for the tumor cell line SKOV3, Figure 3I shows the results for the tumor cell line PANC-1, Figure 3J shows the results for the tumor cell line HEPG2, and Figure 3K shows the results for the tumor cell line HT-29. Each test group and control group has three wells, and the results are shown as mean ± SME. The horizontal axis shows different effect-target ratios E: T. The vertical coordinate shows the percentage of T cell killing rate against target cells. The dot graph shows that the effector cells are control peripheral blood mononuclear cells that are not transfected with the Her2 TCR-6A5-mC TCR gene, and the upper triangle graph shows that the effector cells are peripheral blood mononuclear cells that are transfected with the Her2 TCR-6A5-mC TCR gene. In the MTT killing experiment, another group was added with 10μM paclitaxel as a positive control (shown as a separate lower triangle point in Figures 3H-3K).

Figure 4 shows the key amino acid sites on the Her2-E75 polypeptide recognized by the Her2 TCR-6A5-mC TCR, and the results of the recognition function test for epitope polypeptides from normal human proteins with potential cross-reaction. Figure 4A shows the results of ELISA analysis of IFN-γ detection of cell supernatants after 9 new epitope polypeptides formed in Example 5 were mixed and cultured with T2 cells and #2 PBMCs transfected with lentiviral vectors encoding the Her2 TCR-6A5-mC TCR gene for 24 hours. Each test group and control group were duplicate wells, and the results were shown as mean ± SME. The horizontal axis shows different epitope peptides (T2+ peptides) presented by T2 cells. "E75" is Her2/neu 369-377 peptide, "E75-K1A", "E75-I2A", "E75-F3A", "E75-G4A", "E75-S5A", "E75-L6A", "E75-F8A", "E75-L9A" are Her2/neu 369-377 peptides whose corresponding amino acids are replaced by alanine, and "E75-A7G" is Her2/neu 369-377 peptides whose seventh alanine is replaced by glycine. The vertical axis shows the concentration of IFN-γ secreted by T cells. The effector-target ratio E:T is 5:1. Figure 4B shows the recognition function of Her2 TCR-6A5-mC TCR for epitope peptides from normal human proteins with potential cross-reaction. "E75" is Her2/neu 369-377 polypeptide, polypeptide "B" is NSMA3 93-101 polypeptide, "C" is O11A1 103-111 polypeptide, and "D" is SV2C 687-695 polypeptide. #2 PBMCs transfected with lentiviral vectors encoding the Her2 TCR-6A5-mC TCR gene were co-cultured with T2 cells presenting different concentration gradients of the polypeptide for 24 hours, and the cell supernatant was taken for IFN-γ ELISA analysis. Each test group and control group had three wells, and the results were shown as mean ± SME. The horizontal axis shows that T2 cells present different concentrations of epitope peptides. The vertical axis represents the concentration of IFN-γ secreted by T cells.

Figure 5 shows the main functional fragments of the Her2 TCR-6A5-mC-PGKp-tEGFR lentiviral vector carrying the tEGFR gene and the Her2 TCR-6A5-mC gene constructed in Example 6. The fragments shown include: 1) expressing the TCR gene driven by the EF-1α promoter, the invariant region fragments of the β chain and α chain of each TCR are mouse invariant region fragments, and the β chain and α chain of the TCR are linked by the coding sequence of the cleavable linking polypeptide (furin-F2A); 2) expressing the truncated human EGFR gene fragment driven by the PGK promoter.

Figure 6 shows the killing activity of human T cells expressing Her2 TCR-6A5-mC TCR and human T cells expressing Her2 TCR-6A5-mC and tEGFR against different human tumor cell lines in the experiment of Example 7, wherein Figure 6A shows the experimental results for the tumor cell line C33A, Figure 6B shows the experimental results for the tumor cell line CFPAC-1, and Figure 6C shows the experimental results for the tumor cell line Saos-2. Each test group and control group has three wells, and the results are shown as mean ± SME. The horizontal coordinates of Figures 6A-C show different effect-target ratios E: T. The vertical coordinates show the percentage value of cell killing rate. The dot graph indicates that the effector cells are control human T cells without any TCR transfection, the square graph indicates that the effector cells are human T cells expressing Her2 TCR-6A5-mC TCR, and the upper triangle graph indicates that the effector cells are human T cells expressing Her2 TCR-6A5-mC and tEGFR at the same time.

FIG. 7 shows the curves of tumor volume changes in each group of animals after drug treatment in the experiment of Example 8, with the horizontal axis representing time after drug administration (days) and the vertical axis representing tumor volume (mm ³ ).

The present invention is further explained below through the description of specific implementation methods and with reference to the attached drawings, but this is not a limitation of the present invention. Technical personnel in this field can make various modifications or improvements based on the basic idea of the present invention, but as long as they do not deviate from the basic idea of the present invention, they are all within the scope of the present invention.

In the present invention, the terms "tumor", "cancer", "tumor cell", "cancer cell", "T cell", "T cell receptor", "T cell receptor modification", "TCR variable region", "TCR constant region", "antigen", "antigenic epitope polypeptide", "homologous sequence", "encoding", "antigen presentation", "recombinant DNA expression vector", "promoter", "complementary sequence", "transfection", "autologous", "heterologous", "specific identification", "TCR-T therapy" cover the meanings generally recognized in the field.

Her2/neu antigen is a tumor-associated antigen. Most high-affinity T cells that recognize Her2/neu antigen are eliminated by the central tolerance mechanism to avoid possible autoimmune reactions (see the reference "Immunol Rev. 2016; 271(1): 127-40"). Therefore, it is very difficult to induce T cell clones with specific recognition of Her2/neu antigen expressed by tumor cells from the peripheral blood T cell pool. Dendritic cells are used to present the Her2/neu 369-377 peptide antigen, and the high-affinity TCR induced from the peripheral blood of patients immunized with the Her2/neu 369-377 peptide vaccine can recognize extremely low amounts of exogenously loaded Her2/neu 369-377 peptide, but cannot recognize the antigen peptide endogenously presented by tumor cells (see the reference "Cancer Res. 1998; 58: 4902-4908"). This may be due to the difference in conformation between the exogenously loaded polypeptide/HLA complex and the HLA/polypeptide complex naturally presented in the cell, or because the Her2/neu 369-377 polypeptide is located in the highly glycosylated region of the Her2 protein, the Her2/neu 369-377 polypeptide naturally presented in the cell may be glycosylated, resulting in differences in TCR recognition conformation (see the literature "Proc. Natl. Acad. Sci. USA 2003; 100: 15029-15034"). In the process of inducing T cells in vitro with Her2/neu 369-377 antigen peptide, high-affinity T cell clones that can only recognize exogenously loaded antigen peptides often gain dominant expansion, while the growth of T cell clones that can specifically recognize endogenous Her2/neu antigen peptides presented by cells is inhibited (see the literature "J Exp Med. 2016 Nov 14; 213(12): 2811-2829"), thus increasing the difficulty of obtaining functional TCRs that can recognize tumor cells. A research group induced allogeneic T cells (Allo-T cells) from HLA-A2 negative peripheral blood, which can specifically recognize the HLA-A2 restricted Her2/neu 369-377 antigen peptide. After transfecting the T cells with the obtained TCR gene, they can not only recognize the Her2/neu 369-377 antigen peptide presented by tumor cells, but also cross-recognize the Her3 and Her4 antigen epitopes of the same family (see the reference "Journal of Immunology, 2008, 180: 8135-8145"). However, TCR-T therapy based on allogeneic allo-TCR has the risk of generating allo-reactions against other normal self-protein antigen epitopes (see references “Int. J. Cancer 2009; 125, 649-655”, “Nat Immunol 2007; 8: 388-97”). Another research group induced Her2/neu 369-377 peptide-specific T cells from the peripheral blood of tumor patients immunized with Her2/neu 369-377 peptide vaccine, and paired the alpha and beta chains from different T cells and screened a high-affinity TCR. T cells transfected with this high-affinity TCR can recognize a variety of HLA-A2 ⁺ Her2/neu ⁺ tumor cells (see the literature "HUMAN GENE THERAPY 2014; 25: 730-739"). This TCR was not obtained from monoclonal T cells, so it is not certain whether this TCR is a natural TCR that exists in the T cell library that has been screened for central tolerance. In order to improve the affinity of TCR for epitope peptides presented by HLA class I molecules, point mutations can be made in the functional region of TCR recognizing epitope peptides, and high-affinity TCRs can be screened. Since Her2/neu protein is also expressed in trace amounts in important organs such as myocardium, lungs, esophagus, kidneys, and bladder (see the literature "Oncogene. 1990 Jul; 5(7): 953-62"), the non-natural high-affinity Her2/neu antigen-specific TCR obtained by the above method has the risk of producing off-target toxic reactions to normal tissues.

Tumor cells highly express Her2/neu protein, so the amount of antigenic peptides presented by HLA on the cell surface will also increase accordingly. The difference in the amount of HLA/antigen peptide complexes on tumor cells and normal cells can become a window for specific T cells to distinguish normal and tumor tissues. The present invention proposes to obtain the sequence of natural TCR from the autologous T cell repertoire, and then express TCR on T cells in vitro, so that the obtained TCR-expressing T cells can recognize the Her2/neu antigens that are increased in tumor cells, which is the key to the successful development of effective and low-toxic TCR-T therapy.

In order to obtain a TCR that can specifically recognize tumor antigens while avoiding possible off-target toxic side effects, the present invention induces T cell clones that are specific to the Her2/neu 369-377 polypeptide presented by HLA-A2 from the peripheral blood of healthy donors who are HLA-A2 positive, and screens out T cell clones that carry natural TCRs with moderate affinity for the Her2/neu 369-377 polypeptide. This is different from the strategy of other research groups to induce Her2/neu 369-377 peptide-specific T cells from the peripheral blood of tumor patients immunized with Her2/neu 369-377 peptide vaccine (see the document "HUMAN GENE THERAPY 2014, 25: 730-739"). The present invention believes that after immunization with Her2/neu 369-377 antigen peptide, specific T cell clones targeting Her2/neu 369-377 peptide will proliferate preferentially, and therefore cannot represent the specific T cell population of Her2/neu 369-377 peptide antigen presented by target cells that naturally exist in the T cell repertoire in the body. The present invention also does not adopt the method of inducing peptide-specific T cells from HLA-A2 negative peripheral blood by other research groups (see the document "The Journal of Immunology, 2010, 184: 1617-1629"). Although it is easier to obtain allo-T cells that recognize Her2/neu 369-377 peptide antigens with high affinity from allogeneic PBMCs, this also increases the allogeneic reaction caused by T cells cross-recognizing other peptides presented by HLA-A2 molecules.

Based on the above concept, the present invention provides an isolated T cell receptor, including at least one of an α chain and a β chain, wherein the α chain and the β chain both contain a variable region and a constant region, and the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of the variable region of the α chain has at least 98%, preferably at least 98.5%, and more preferably at least 99% consistency with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the β chain has at least 98%, preferably at least 98.5%, and more preferably at least 99% consistency with the amino acid sequence shown in SEQ ID NO: 2, as long as it does not significantly affect the effect of the present invention. It is also preferred that the amino acid sequence of the variable region of the α chain is as shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the β chain is as shown in SEQ ID NO: 2.

The variable regions of the TCR α and β chains are used to bind to antigenic peptides/major histocompatibility complex (MHC I), and include three hypervariable regions or complementarity determining regions (CDRs), namely, CDR1, CDR2, and CDR3. The CDR3 region is crucial for the specific recognition of antigenic peptides presented by MHC molecules. The TCR α chain is composed of different V and J gene segments, while the β chain is composed of different V, D, and J gene segments. The corresponding CDR3 region formed by the recombination and combination of specific gene fragments, as well as the palindromic and random nucleotide additions in the binding region, form the specificity of TCR for antigen polypeptide recognition (see the document "Immunobiology: The immune system in health and disease. ^5th edition, Chapter 4, The generation of Lymphocyte antigen receptors"). The MHC class I molecule includes human HLA. The HLA includes: HLA-A, B, C.

More specifically, the T cell receptor can specifically recognize the antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule. The amino acid sequence of the antigen Her2/neu is shown in SEQ ID NO: 21. Preferably, the antigen epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3. The HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A*0201, 0202, 0203, 0204, 0205, 0206 and 0207. Preferably, the HLA-A2 molecule is preferably HLA-A*0201.

In one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is the Her2/neu 369-377 polypeptide (SEQ ID NO: 3). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is an antigenic epitope polypeptide having 4-9 consecutive identical amino acids (e.g., 4, 5, 6, 7, 8 or 9 consecutive identical amino acids) with the Her2/neu 369-377 polypeptide, and the length of these polypeptides is 8-11 amino acids. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 22).

Preferably, the maximum half-reaction polypeptide concentration of the T cell receptor recognizing the Her2/neu 369-377 polypeptide is between 1.0-10 ng/ml. In one embodiment of the present invention, the maximum half-reaction polypeptide concentration is about 1.6 ng/ml-2.9 ng/ml. The term "maximum half-reaction polypeptide concentration" refers to the concentration of the polypeptide required to induce a T cell response to reach 50% of the maximum value. It is reported that the maximum half-reaction peptide concentration of specific T cells against cytomegalovirus (CMV) antigen CMV pp65 (495-503) peptide is between 0.1-1 ng/ml, and this TCR is considered to have high affinity for CMV antigen peptide (see the document "Journal of Immunogical Methds 2007; 320: 119-131"). In the present invention, the T cell receptor has a moderate to high affinity for Her2/neu antigen, thereby avoiding the off-target toxicity that may be caused by high affinity (maximum half-reaction peptide concentration less than 0.1 ng/ml). In addition, the recognition of Her2/neu 369-377 peptide by this T cell receptor is independent of the auxiliary effect of CD8 molecules. CD8 negative CD4 positive T cells expressing this T cell receptor can also specifically recognize the Her2/neu 369-377 peptide presented by HLA-A2 and secrete cytokines, thereby enhancing the function of the killer T cells expressing this T cell receptor.

The exogenous TCR α and β chains expressed by T cells may mismatch with the α and β chains of the TCR of the T cells themselves, which will not only dilute the expression of the correctly matched exogenous TCR, but also make the antigen specificity of the mismatched TCR unclear, thus posing a potential risk of recognizing self-antigens. Therefore, it is better to modify the constant regions of the TCR α and β chains to reduce or avoid mismatching.

In one embodiment, the constant region of the α chain and/or the constant region of the β chain are derived from humans; preferably, the present invention finds that the constant region of the α chain can be replaced in whole or in part by homologous sequences derived from other species, and/or the constant region of the β chain can be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is mouse. The replacement can increase the expression of TCR in cells, and can further improve the specificity of cells modified by the TCR to Her2/neu antigen.

The constant region of the α chain may be modified with one or more disulfide bonds, and/or the constant region of the β chain may be modified with one or more disulfide bonds, such as 1 or 2.

In a specific implementation, two different modified TCRs were prepared. One method is to add a disulfide bond to the constant region of the TCR by point mutation. The method is described in the document "Cancer Res. 2007 Apr 15; 67(8): 3898-903.", which is incorporated herein by reference in its entirety. Her2 TCR-1B5-mC is to replace the corresponding human TCR constant region sequence with the mouse TCR constant region sequence. The method is described in the document "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated herein by reference in its entirety.

In a specific embodiment, the amino acid sequence of the α chain is shown in SEQ ID NOs: 4, 5 or 6, and the amino acid sequence of the β chain is shown in SEQ ID NOs: 7, 8 or 9.

Among them, for the α chain with an amino acid sequence as shown in SEQ ID NO: 4, its sequence is the original human sequence; for the α chain with an amino acid sequence as shown in SEQ ID NO: 5, its constant region is modified with a disulfide bond; for the α chain with an amino acid sequence as shown in SEQ ID NO: 6, its constant region is replaced with a mouse constant region.

Among them, for the β chain with an amino acid sequence as shown in SEQ ID NO: 7, its sequence is the original human sequence; for the β chain with an amino acid sequence as shown in SEQ ID NO: 8, its constant region is modified with 1 disulfide bond; for the β chain with an amino acid sequence as shown in SEQ ID NO: 9, its constant region is replaced with a mouse constant region.

In one specific embodiment, the amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 4, and the amino acid sequence of the β chain is shown in SEQ ID NO: 7. In another specific embodiment, the amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 5, and the amino acid sequence of the β chain is shown in SEQ ID NO: 8. In another specific embodiment, the amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 6, and the amino acid sequence of the β chain is shown in SEQ ID NO: 9.

In other specific embodiments of the present invention, the α chain of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NOs: 4, 5 or 6; for example, the α chain has at least 90%, preferably at least 95%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 4, 5 or 6.

In other specific embodiments of the present invention, the β chain of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9; for example, the β chain has at least 90%, preferably at least 95%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9.

The α chain and/or β chain of the TCR of the present invention can also bind to other functional sequences at the end (e.g., C-terminus), such as the functional region sequences of the co-stimulatory signals CD28, 4-1BB and/or CD3zeta.

The present invention also provides an isolated nucleic acid encoding a T cell receptor, comprising a coding sequence of at least one of the α chain and the β chain of the T cell receptor, wherein the α chain coding sequence and the β chain coding sequence both comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and the amino acid sequence encoded by the α chain variable region coding sequence has at least 98%, preferably at least 98.5%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence encoded by the β chain variable region coding sequence has at least 98%, preferably at least 98.5%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown in NO:2 has at least 98%, preferably at least 98.5%, and more preferably at least 99% identity, as long as it does not significantly affect the effect of the present invention. It is also preferred that the α chain variable region encoding sequence encodes the amino acid sequence shown in SEQ ID NO:1, and the β chain variable region encoding sequence encodes the amino acid sequence shown in SEQ ID NO:2.

The nucleic acid may be DNA or RNA.

Preferably, the α chain variable region coding sequence is as shown in SEQ ID NO: 10, and the β chain variable region coding sequence is as shown in SEQ ID NO: 11.

More specifically, the T cell receptor encoded by the nucleic acid can specifically recognize the antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule.

In one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is the Her2/neu 369-377 polypeptide (SEQ ID NO: 3). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is an antigenic epitope polypeptide having 4-9 consecutive identical amino acids (e.g., 4, 5, 6, 7, 8 or 9 consecutive identical amino acids) with the Her2/neu 369-377 polypeptide, and the length of these polypeptides is 8-11 amino acids. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 22).

Preferably, the maximum half-reaction polypeptide concentration of the T cell receptor encoded by the nucleic acid to recognize the Her2/neu 369-377 polypeptide is between 1.0-10ng/ml (for example, between 3.0-8.0ng/ml, 5.0-7.0ng/ml). In one embodiment of the present invention, the maximum half-reaction polypeptide concentration is about 1.6-2.9ng/ml. In this case, the T cell receptor has a medium-high affinity for the Her2/neu antigen, which can avoid the off-target toxicity that may be caused by high affinity (maximum half-reaction polypeptide concentration is less than 0.1ng/ml).

In one embodiment, the constant region of the α chain and/or the constant region of the β chain are derived from humans; preferably, the constant region coding sequence of the α chain is replaced in whole or in part by a homologous sequence derived from other species, and/or the constant region coding sequence of the β chain is replaced in whole or in part by a homologous sequence derived from other species. More preferably, the other species is a mouse. The replacement can increase the expression of TCR in cells, and can further improve the specificity of cells modified by the TCR to Her2/neu antigen.

The α chain constant region coding sequence may contain one or more disulfide bond coding sequences, and/or the β chain constant region coding sequence may contain one or more disulfide bond coding sequences.

In a specific embodiment, the α chain coding sequence is shown in SEQ ID NOs: 12, 13 or 14, and the β chain coding sequence is shown in SEQ ID NOs: 15, 16 or 17.

Among them, for the α chain shown in the coding sequence of SEQ ID NO: 12, its sequence is the original human sequence; for the α chain shown in the coding sequence of SEQ ID NO: 13, its constant region is modified with a disulfide bond; for the α chain shown in the coding sequence of SEQ ID NO: 14, its constant region is replaced with a mouse constant region.

Among them, for the β chain of the coding sequence shown in SEQ ID NO: 15, its sequence is the original human sequence; for the β chain of the coding sequence shown in SEQ ID NO: 16, its constant region is modified with a disulfide bond; for the β chain of the coding sequence shown in SEQ ID NO: 17, its constant region is replaced with a mouse constant region.

In a specific embodiment, the coding sequence of the α chain of the TCR is shown as SEQ ID NO: 12, and the coding sequence of the β chain is shown as SEQ ID NO: 15. In another specific embodiment, the coding sequence of the α chain of the TCR is shown as SEQ ID NO: 13, and the coding sequence of the β chain is shown as SEQ ID NO: 16. In another specific embodiment, the coding sequence of the α chain of the TCR is shown as SEQ ID NO: 14, and the coding sequence of the β chain is shown as SEQ ID NO: 17.

In another embodiment, the α chain coding sequence and the β chain coding sequence are connected by a coding sequence of a cleavable linking polypeptide, which can increase the expression of TCR in cells. The term "cleavable linking polypeptide" means that the polypeptide plays a connecting role and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding the polypeptide is translated by ribosome skipping, thereby separating the polypeptides connected thereto from each other. Examples of cleavable linking polypeptides are known in the art, such as F2A polypeptides, and F2A polypeptide sequences include but are not limited to F2A polypeptides from picornaviruses and similar 2A sequences from other viruses. For example, the cleavable/ribosome skipping 2A linker sequence can be derived from different viral genomes, including F2A (foot-and-mouth disease virus 2A), T2A (thosea asigna virus 2A), P2A (porcine teschovirus-1 2A) and E2A (equine rhinitis A virus 2A). In addition, the cleavable linker polypeptide also includes a canonical four amino acid motif that can be cleaved by the furin enzyme, i.e., the R-X-[KR]-R amino acid sequence. The TCR encoded by this embodiment is a single-chain chimeric T cell receptor. After the single-chain chimeric T cell receptor is expressed, the cleavable linking polypeptide connecting the α chain and the β chain will be cleaved by a specific enzyme in the cell, thereby forming equal amounts of free α chain and β chain.

The α and β chains constituting the single-chain chimeric TCR can also be replaced in whole or in part by homologous sequences from other species as described above, and/or modified with (encoding) one or more disulfide bonds.

In a specific embodiment, the sequence of the nucleic acid is shown in SEQ ID NOs: 18, 19, or 20.

Preferably, the nucleotide sequence of the nucleic acid is codon optimized to increase gene expression, protein translation efficiency and protein expression, thereby enhancing the ability of TCR to recognize antigens. Codon optimization includes but is not limited to modification of the translation activation region, alteration of mRNA structural fragments, and use of different codons encoding the same amino acid.

In other embodiments, the sequence of the TCR encoding nucleic acid may be mutated, including removal, insertion and/or replacement of one or more amino acid codons, so that the function of the expressed TCR to recognize the Her2/neu antigen remains unchanged or is enhanced. For example, in one embodiment, conservative amino acid substitution is performed, including replacing an amino acid in the variable region of the above-mentioned TCR α chain and/or β chain with another amino acid with similar structural and/or chemical properties. The term "similar amino acid" refers to amino acid residues with similar polarity, charge, solubility, hydrophobicity, hydrophilicity and other properties. The mutated TCR still has the biological activity of recognizing the above-mentioned Her2/neu antigen polypeptide presented by the target cell. In another embodiment, TCR maturation modification is performed, that is, the amino acids in the complementary determining region 2 (CDR2) and/or CDR3 regions in the variable regions of the above-mentioned TCR α chain and/or β chain are removed, inserted and/or replaced, thereby changing the affinity of TCR binding to Her2/neu antigen.

The present invention also provides an isolated mRNA transcribed from the DNA according to the present invention.

The present invention also provides a recombinant expression vector, which contains the nucleic acid (e.g., DNA) according to the present invention, and/or its complementary sequence, which is effectively linked to a promoter.

Preferably, in the recombinant expression vector, the DNA of the present invention is suitably and effectively linked to a promoter, enhancer, terminator and/or polyA signal sequence.

The combination of the above-mentioned functional elements of the recombinant expression vector of the present invention can promote the transcription and translation of DNA and enhance the stability of mRNA.

The basic framework of the recombinant expression vector can be any known expression vector, including plasmids or viruses. Viral vectors include but are not limited to (for example) retroviral vectors (the virus prototype is Moloney murine leukemia virus (MMLV)) and lentiviral vectors (the virus prototype is human immunodeficiency virus type I (HIV)). The recombinant vector expressing the TCR of the present invention can be obtained by conventional recombinant DNA technology in the art.

In one embodiment, the expression of the α chain and β chain genes on the recombinant expression vector can be driven by two different promoters, including various known types, such as strong expression, weak expression, continuous expression, inducible, tissue-specific, and differentiation-specific promoters. The promoter can be viral or non-viral (e.g., eukaryotic cell promoter), such as CMV promoter, promoter on LTR of MSCV, EF1-α promoter, and PGK-1 promoter, SV40 promoter, Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, and human β-actin promoter. The driving directions of the two starters can be the same or opposite.

In another embodiment, the expression of the α chain and β chain genes on the recombinant expression vector can be driven by the same promoter, for example, in the case of encoding a single-chain chimeric T cell receptor, the nucleotide sequence of the α chain and the nucleotide sequence of the β chain are connected by the Furin-F2A polypeptide coding sequence.

In other embodiments, the recombinant expression vector may contain coding sequences of other functional molecules in addition to the α-chain and β-chain genes. One embodiment includes expressing a self-fluorescent protein (such as GFP or other fluorescent proteins) for in vivo tracking imaging. Another embodiment includes expressing an inducible suicide gene system, such as inducing expression of herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing expression of Caspase 9 (iCasp9) protein. The expression of these "safety-switches" can increase the safety of the TCR gene-modified cells of the present invention when used in vivo (see the reference "Front. Pharmacol., 2014; 5: 1-8). Therefore, the recombinant expression vector may contain a suicide gene coding sequence, and the suicide gene may be selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or Alternatively, the suicide gene coding sequence is under the control of a promoter, and the promoter used to control the suicide gene coding sequence and the promoter to which the nucleic acid described in the present invention is connected may be the same or different and are independent of each other. Alternatively, the suicide gene coding sequence and the nucleic acid described in the present invention are under the control of the same promoter, and the suicide gene coding sequence can be cleavably linked to the coding sequence of the polypeptide or the internal ribosome entry site (IRES (internal ribosome entry site) sequence, which is linked to the nucleic acid described in the present invention. The coding sequence of the cleavable linker polypeptide can be the cleavable/ribosome skipping 2A linker sequence described above, which can come from different viral genomes, including F2A, T2A, P2A and E2A. Another embodiment includes expressing human tropism factor receptor genes, such as CCR2, which can bind to the corresponding tropism factor ligands highly expressed in tumor tissues, thereby increasing the homing of cells modified by the TCR gene of the present invention in tumor tissues.

The present invention also provides a T cell receptor-modified cell, the surface of which is modified by the T cell receptor described in the present invention, wherein the cell includes a primitive T cell or a precursor cell thereof, a NKT cell, or a T cell strain.

The "modification" in "T cell receptor modification" means that the cells express the T cell receptor described in the present invention through gene transfection, that is, the T cell receptor is anchored on the cell membrane of the modified cell through the transmembrane region and has the function of recognizing the antigen polypeptide/MHC complex.

In some embodiments, the T cell receptor-modified cell expresses a suicide gene protein on its cell surface or in the cell, and the suicide gene can be selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or a combination thereof. This can increase the safety of the T cell receptor-modified cell for use in vivo.

The present invention also provides a method for preparing cells modified with the T cell receptor according to the present invention, comprising the following steps: 1) providing cells; 2) providing nucleic acid encoding the T cell receptor of the present invention; 3) transfecting the nucleic acid into the cells.

The cells described in step 1) may be derived from mammals, including humans, dogs, mice, rats, and transgenic animals thereof. The cells may be autologous or allogeneic. Allogeneic cells include cells from identical twins, allogeneic stem cells, and genetically modified allogeneic T cells.

The cells described in step 1) include primitive T cells or their precursor cells, NKT cells, or T cell strains. The term "naive T cells" refers to mature T cells in peripheral blood that have not yet been activated by corresponding antigens. These cells can be isolated by methods known in the art. For example, T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, umbilical cord blood, and tumor tissue. In one embodiment, T cells can be derived from hematopoietic stem cells (HSCs), including those from bone marrow, peripheral blood or umbilical cord blood, and are isolated and obtained by stem cell marker molecules such as CD34. In one embodiment, T cells can be derived from induced pluripotent stem cells (iPS cells), including introducing specific genes or specific gene products into somatic cells, so that the somatic cells are transformed into stem cells, and then induced to differentiate into T cells or their precursor cells in vitro. T cells can be obtained by common methods such as density gradient centrifugation, and examples of density gradient centrifugation include Ficoll or Percoll density centrifugation. One embodiment is to obtain a product of enriched T cells from peripheral blood using plasma separation apheresis or leukapheresis. One embodiment is to label specific cell populations with antibodies and then obtain enriched CD8 ⁺ or CD4 ⁺ T cells by magnetic bead separation (such as the CliniMACS® system (Miltenyi Biotec)) or flow cytometry separation.

Preferably, the T cell precursor cell is a hematopoietic stem cell. The coding gene of the TCR of the present invention can be directly introduced into the hematopoietic stem cell, and then transferred into the body to further differentiate into mature T cells; the coding gene can also be introduced into T cells that are differentiated and matured from hematopoietic stem cells under specific conditions in vitro.

The cells can be resuspended in a freezing solution and stored in liquid nitrogen. Common freezing solutions include but are not limited to PBS solutions containing 20% DMSO and 80% human serum albumin. The cells are frozen at -80°C at a temperature drop of 1°C per minute and then stored in the gas phase of a liquid nitrogen tank. Other freezing methods are to freeze the cells in the freezing solution directly at -80°C or in liquid nitrogen.

The nucleic acid described in step 2) is the nucleic acid described in the present invention, including the DNA and RNA.

The transfection includes physical, biological and chemical methods. The physical method is to introduce the TCR gene into the cell in the form of DNA or RNA through calcium phosphate precipitation, liposomes, microinjection, electroporation, gene guns and other methods. Currently, there are commercial instruments, including electrotransfer instruments, such as Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), Multiporator (Eppendort, Germany). The biological method is to introduce the TCR gene into the cell through DNA or RNA vectors. The above-mentioned retrovirus vectors (such as γ retrovirus vectors) are commonly used tools for transfection and insertion of foreign gene fragments into animal cells (including human cells). Other viral vectors come from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adenovirus-related viruses. The chemical method is to introduce polynucleotides into cells, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, micelles and liposomes. Regardless of how the TCR gene is introduced into cells, various detection methods are used to analyze whether the target gene is introduced into the target cells. The detection methods include common molecular biology methods (such as Southern blotting and Northern blotting, RT-PCR and PCR, etc.), or common biochemical methods (such as ELISA and Western blotting), as well as the methods mentioned in the present invention.

Preferably, the transfection is performed by a retroviral vector or a lentiviral vector.

After transfection, the cells can be cultured according to their respective conventional methods and conditions according to the actual application. For example, T cells can be expanded in vitro after being activated by the TCR/CD3 complex on the surface and auxiliary stimulatory molecules (such as CD28). Stimulants that activate TCR, CD3 and CD28 (such as anti-TCR, CD3 or CD28 antibodies) can be adsorbed on the surface of the culture container, or on the surface of the co-culture (such as magnetic beads), or can be directly added to the cell culture medium for co-culture. Another embodiment is to co-culture T cells with trophoblasts, which express auxiliary stimulatory molecules or corresponding ligands, including but not limited to HLA-A2, β2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.

According to the usual mammalian cell in vitro culture method, T cells are cultured and expanded under appropriate culture conditions. For example, cells can be passaged when they reach a confluence of more than 70%, and fresh culture medium is generally replaced every 2 to 3 days. When the cells reach a certain number, they can be used directly or frozen as described above. The in vitro culture time can be within 24 hours or as long as 14 days or longer. The frozen cells can be thawed and used in the next step.

In one embodiment, cells can be cultured in vitro for a few hours to 14 days, or any number of hours in between. T cell culture conditions include the use of basal culture media, including but not limited to RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo. Other conditions required for cell survival and proliferation include but are not limited to the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF-β and TNF-α, other culture supplements (including amino acids, sodium pyruvate, vitamin C, 2-hydroxyethanol, growth hormone, growth factor). The cells can be placed under appropriate culture conditions, for example, the temperature can be at 37°C, 32°C, 30°C or room temperature, and the air condition can be, for example, air containing 5% CO ₂ .

The present invention also provides the use of the T cell receptor modified cells according to the present invention in the preparation of drugs for treating or preventing tumors and/or cancers.

The tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, stomach cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer and brain cancer.

The present invention also provides the use of cells modified with T cell receptors according to the present invention in the preparation of drugs for detecting tumors and/or cancers in a host.

In one embodiment of the present invention, a sample of tumor and/or cancer cells taken from a host can be contacted with the T cell receptor-modified cells of the present invention at a certain concentration, and the degree of reaction between the two can be used to determine whether the tumor and/or cancer is HLA-A2 positive or HLA-A2 negative, and whether it expresses the antigen Her2/neu.

The present invention also provides a drug composition, wherein the drug composition comprises the T cell receptor modified cells according to the present invention as an active ingredient, and a pharmaceutically acceptable excipient.

The pharmaceutical composition preferably contains the T cell receptor modified cells in a total dosage range of 1×10 ³ -1×10 ⁹ cells/Kg body weight per patient per treatment course, including any number of cells between the two endpoints. Preferably, each treatment course is 1-3 days, and is administered 1-3 times per day. One or more treatment courses can be given to the patient according to actual conditions and needs.

The pharmaceutically acceptable excipients include pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives, including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, preservatives, etc.

The drug composition can be administered through a suitable route of administration, which is suitable for administration through an artery, vein, subcutaneous, intradermal, intratumoral, intralymphatic, intralymph node, subarachnoid space, intramarrow, intramuscular or intraperitoneal.

The present invention also provides a method for treating tumors and/or cancers, comprising administering cells modified with the T cell receptor according to the present invention to patients with tumors and/or cancers.

The tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, stomach cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer and brain cancer.

The dosage of the T cell receptor modified cells is preferably 1×10 ³ -1×10 ⁹ cells/kg body weight per patient per course of treatment. Preferably, each course of treatment is 1-3 days, and the cells are administered 1-3 times per day. One or more courses of treatment may be given to the patient according to actual conditions and needs.

The T cell receptor modified cells can be administered through a suitable administration route, which is suitable for administration through an artery, vein, subcutaneous, intradermal, intratumoral, intralymphatic, intralymph node, subarachnoid space, intramarrow, intramuscular or intraperitoneal.

After entering the body of the treated subject, the T cell receptor-modified cells can eliminate tumor cells expressing Her2/neu antigen and/or change the microenvironment of tumor tissue to induce other anti-tumor immune responses.

The method for treating tumors and/or cancers also includes administering other drugs for treating tumors and/or drugs for regulating the patient's immune system to patients with tumors and/or cancers to enhance the number and function of the T cell receptor-modified cells in the body. The other drugs for treating tumors include but are not limited to: chemotherapy drugs, such as cyclophosphamide, fludarabine; radiotherapy; immunosuppressants, such as cyclosporine, azathioprine, methotrexate, mycophenolate, FK50; antibodies, such as anti-CD3, IL-2, IL-6, IL-17, TNFα antibodies.

In certain embodiments, the method for treating tumors and/or cancers further comprises administering to the patient other drugs for treating tumors and/or cancers, and/or drugs for regulating the patient's immune system, to eliminate the number and function of the T cell receptor-modified immune cells carrying the suicide gene in the body when the T cell receptor-modified immune cells produce severe toxic side effects. The other drugs for treating tumors and/or cancers include but are not limited to: chemically induced dimerization (CID) drugs, AP1903, phosphorylated ganciclovir, anti-Cd20 antibodies, anti-CMYC antibodies, and anti-EGFR antibodies.

The present invention also provides the use of the isolated T cell receptor for detecting the proliferation or survival of the TCR-T cells in patients treated with the TCR-modified T cells (i.e., TCR-T cells), thereby conducting drug metabolism research and understanding the efficacy and toxicity of the TCR-T cells. Specifically, the TCR sequence can be used as a primer to detect the number of TCR-T cells carrying the TCR in the body by PCR. Compared with the method of analyzing by flow cytometry after staining with fluorescently labeled HLA/peptide complex multimers, this application requires less cells and is more sensitive.

The following examples further explain or illustrate the content of the present invention, but these examples should not be understood as limiting the scope of protection of the present invention.

example

Unless otherwise specified, the experimental methods used in the following examples are all carried out using conventional experimental procedures, operations, materials and conditions in the field of bioengineering.

Unless otherwise specified, the percentage concentration (%) of each reagent refers to the volume percentage concentration (%(v/v)) of the reagent.

Materials and methods

Cell line: The cell line used to prepare lentiviral particles is 293T cells (ATCC CRL-3216). The presenting cell line used to present antigenic peptides is T2 cells (174xCEM.T2, ATCC CRL-1992). The tumor cell lines used for functional testing are human colorectal cancer colo205 cells (ATCC CCL-222), HT-29 cells (HTB-38) and HCT116 cells (ATCC CCL-247), human breast cancer MDA-MB-231 cells (ATCC HTB-26) and MCF7 cells (ATCC CHTB-22), human ovarian cancer SKOV3 cells (ATCC HTB-77), human pancreatic cancer PANC-1 cells (ATCC CRL-1469), human neuroglioma U87MG cells (ATCC HTB-14), human hepatocellular carcinoma HepG2 cells (ATCC HB-8065), human non-small cell lung cancer NCI-H460 cells (ATCC HTB177) and small cell lung cancer NCI-H446 cells (ATCC HTB-171), human cervical cancer cells C33A (ATCC HTB-31), human osteosarcoma cells Saos-2 (ATCC HTB-85), and human pancreatic cancer cells CFPAC-1 (ATCC CRL-1918). The cell lines were maintained and cultured in RPMI-1640 complete medium (Lonza, cat#12-115F), and RPMI-1640 complete medium was added with 10% fetal calf serum FBS (ATCC 30-2020), 2mmol/L L-glutamine, 100μg/ml penicillin and 100μg/ml streptomycin.

Peripheral blood products: Unless otherwise specified, the human peripheral blood products (including peripheral blood mononuclear cells) from healthy donors used in the following experiments were obtained from Pacific Blood Center in San Francisco (#1 PBMC and #2 PBMC were Trima residual cell fractions #R32334 and #R33941 from the Apheresis collection kit, respectively).

Trypan blue staining method: Wash the cells with PBS, digest them with trypsin, suspend the cells in PBS, add trypan blue dye solution with a final concentration of 0.04% (w/v), count under a microscope, dead cells will be stained light blue, and live cells will not be stained. The number of live cells is taken as the final data.

In vitro induction of Her2/neu 369-377 specific killer T cells (CTL): Peripheral blood was centrifuged on Ficoll-Paque Premium (Sigma-Aldrich, cat# GE-17-5442-02) density gradient (×400g) for 30 minutes to obtain single nucleated cells (PBMC). First, the HLA-A2 phenotype of cells was detected by staining with fluorescein FITC-labeled anti-HLA-A2 antibody (Biolegend, cat#343303). After flow cytometry analysis (the flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and the results were analyzed using Flowjo software (Flowjo)), RNA of positive cells was extracted, reverse transcribed into cDNA and cloned into a vector, and then HLA gene sequencing analysis was performed to determine that the cell type was HLA-A*0201. HLA-A2-positive PBMC cells were cultured in the culture wells of a 24-well culture plate, and the culture medium was the above-mentioned RPMI-1640 complete medium. 2×10e6/ml PBMC were added to each well, and Her2/neu 369-377 peptide (Her2-E75, synthesized by Peptide2.0, 10μg/ml dissolved in DMSO) was added to the final concentration of 1μg/ml. After culturing in an incubator at 5% CO ₂ and 37°C for 16-24 hours, the following cytokines were added at the final concentration: human IL-2 (Peprotech, cat# 200-02) 100IU/ml, human IL-7 (Peprotech, cat# 200-07) 5ng/ml, and human IL-15 (Peprotech, cat# 200-15) 5ng/ml. After 10 to 14 days of culture, the cultured T cells were re-stimulated with antigens: 10e6 cultured cells obtained above were added to each well of a 24-well plate, and 2×10e6 HLA-A2 positive PBMC cells treated with 25μg/ml mitomycin C (Santa Cruz Biotechnology, cat#SC-3514) for 2 hours were added as trophoblasts. Her2/neu 369-377 polypeptide was added to each well at a final concentration of 1μg/ml. After overnight culture, IL-2 100IU/ml, IL-7 5ng/ml, and IL-15 5ng/ml (final concentration) were added. After two rounds of the above antigen stimulation and re-stimulation, the expanded T cells were collected for phenotypic analysis and T cell cloning.

Flow cytometric analysis and single cell isolation; The phenotype of T cells expressing the Her2/neu 369-377-specific TCR was analyzed by flow cytometry. The cells to be tested were collected and placed in a 1.5 ml tube (the number of cells was about 10e5), washed _once with 1 ml DPBS solution (2.7 mM KCl, 1.5 _mM KH2PO4, 136.9 _mM NaCl, 8.9 mM _Na2HPO4.7H2O , pH 7.4), and replaced in 100 μl DPBS containing 1% calf serum, _and 5 μl of fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat# 300912) and 10 μl of fluorescein PE-labeled Her2-E75/HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co., cat# T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer, Proimmune, cat# F214-2A-D), incubated on ice for 30 minutes _, washed twice with DPBS solution, and resuspended in 100μl PBS solution ( _{8mM Na2HPO4} , 136mM NaCl, 2mM _KH2PO4 , 2.6mM KCl, pH7.2-7.4) for flow cytometric analysis. The flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and the results were analyzed using Flowjo software (Flowjo). T cell clones were obtained by single cell separation using a flow cytometer (FACS sorter) and then cultured. PBMCs stimulated with Her2/neu369-377 polypeptide antigen were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75/HLA-A2 pentamer, and then separated by flow cytometry (model: Sony cell sorter SH800). After a single CD8 ⁺ Her2-E75/HLA-A2 pentamer ⁺ cell was sorted into a single well of a 96-well culture plate, HLA-A2-positive PBMC cells treated with 25μg/ml mitomycin C for 2 hours were added, 10e5 cells per well, 1μg/ml Her2/neu 369-377 peptide was added and cultured overnight, and then RPMI-1640 complete culture medium containing IL-2 100IU/ml, IL-7 5ng/ml, and IL-15 5ng/ml was added. Fresh culture medium containing the cytokine was replaced every 3-4 days, and T cell clone growth was observed under a microscope. The proliferated T cells are collected and restimulated with antigens as described above to obtain a sufficient number of cells for phenotypic or functional testing, and RNA is extracted for TCR gene cloning.

T cell function test: In order to detect the ability of T cells transfected with TCR genes to recognize antigen epitope peptides, 10e5 T cells transfected with TCR genes and 10e5 T2 cells were added to each well of a 96-well plate and mixed and cultured in 100μl/well RPMI-1640 complete medium. Each test group was duplicated. Then, Her2/neu 369-377 peptides of different final concentrations (1μg/ml, 0.5μg/ml, 0.1μg/ml, 0.05μg/ml, 0.01μg/ml, 0.005μg/ml, 0.001μg/ml and 0.0001μg/ml) were added and cultured overnight in an incubator at 5% CO ₂ and 37°C. In order to determine the key amino acid sites of the epitope antigen recognized by the TCR, 10e5 T cells transfected with the TCR gene and 10e5 T2 cells were added to each well of a 96-well plate, and then the epitope peptide to be tested was added at a final concentration of 0.1 μg/ml and then placed in an incubator at 5% CO ₂ and 37°C for overnight culture. After 24 hours, the supernatant was collected and the IFN-γ in the supernatant was detected using the human IFN-γ ELISA Read-set-Go kit (eBioscience, cat#88-7316) or the human IFN-γ DuoSet ELISA kit (R&D Systems, cat#DY285B) according to the manufacturer's instructions.

In order to detect the ability of T cells transfected with TCR genes to recognize tumor cell lines, a certain amount of PBMC cells transfected with TCR genes and tumor cells were added to each well of a 96-well plate as target cells according to different effector-target ratios. After 24 hours of culture, the supernatant was collected to detect the secreted interferon-γ in the supernatant. Each test group was a duplicate well or triplicate well. In the antibody function blocking test, anti-human CD8 antibody (Biolegend, cat# 300912) with a final concentration of 10μg/ml was added to the cell culture wells at the same time, and the cells were placed in an incubator at 5% CO ₂ and 37°C for overnight culture. After 18-24 hours, the cell supernatant was collected and IFN-γ in the supernatant was detected using human IFN-γ ELISA Read-set-Go kit (eBioscience, cat#88-7316) or human IFN-γ DuoSet ELISA kit (R&D Systems, cat#DY285B) according to the manufacturer's instructions.

In order to detect the ability of T cells transfected with TCR genes to kill tumor cells, 1×10e4 target cells were added to each well of a 24-well culture plate and cultured for 24 hours to allow the target cells to completely adhere to the wall. The suspended cells were removed and a certain number of T cells transfected with TCR genes were added according to the set effector-target ratio. After 24 hours of culture, the suspended cells were removed and the adherent cells were collected by trypsin digestion and trypan blue staining to count the live cells. Cytotoxicity % = (initial live cell number of target cells - live cell number of target cells at the end of culture) / initial live cell number of target cells × 100. Each experimental group was tested in duplicate or triplicate wells, and the significant differences were analyzed using the student's t-test. Alternatively, the killing activity was detected using the MTT method.

MTT method description:

Digest the logarithmic growth phase cells with trypsin, collect them by centrifugation after termination, blow them evenly to prepare single cell suspension; adjust the cell concentration to 0.1~10×10 ⁴ /ml with cell culture medium (adjust the number of inoculated cells according to the growth conditions of different cells), inoculate them into 96-well cell culture plates, the culture system is 100μl/well, and culture them in a 37℃, 5% CO ₂ incubator overnight to allow the cells to fully adhere to the wall, reaching 70~80% on the next day; count with a counting plate, and use a countstar counter to verify the accuracy of the count. Take out the 96-well plate, add 100μl of pre-prepared T cell and TCR-T cell suspension, vortex slightly before adding the sample, add 100μl of the corresponding cell culture serum-free medium to the blank control well; place in a 37℃, 5% CO ₂ incubator and culture for 24 hours; take out the cells after 24 hours, centrifuge at 400g, and after 10 minutes, take out 180μl of the medium and put it into a new 96-well plate, and keep the sample for the subsequent ELISA detection of supernatant IFN-γ level. The detection steps can refer to the detection instructions. Note: The supernatant can be frozen at -80℃ for subsequent detection. Add 100μl of complete medium to each well, add 10μl of MTT solution (5mg/ml, i.e. 0.5% MTT) to each well, and continue to culture for 4-6h; set up an effector cell control group, 4 hours after adding MTT, centrifuge at 300g for 5 minutes, centrifuge the effector cells stained with MTT to the bottom of the plate, discard the supernatant, and then add DMSO for detection. Add 150μl of DMSO to each well, shake at low speed on a rocking bed for 10 minutes to fully dissolve the crystals, and detect its absorbance at 490nm on an enzyme marker.

Obtaining monoclonal TCR genes: Total RNA was purified from T cell clones using the Zymo Quick-RNA Microprep kit (Zymo Research, cat# R1050), and cDNA was obtained using the Smarter RACE 5'/3' kit (Takara Bio, USA, cat# 634858) using this as a template. PCR was performed using the 5'-CDS primer and the TCR β chain 3' primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO: 24) and the α chain 3' primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 25) to amplify the full sequence gene fragments of TCR α and β, and cloned into the pRACE vector (Takara Bio, USA, cat# 634858). Transform competent bacteria Stellar (Takara Bio, USA, cat#636763) and obtain plasmid for sequencing.

Preparation of recombinant TCR lentiviral expression vector: The viral vector used to express TCR is a replication-defective lentiviral vector, including: a lentiviral vector pCDH-EF1α-MCS-(PGK-GFP) expressing GFP, which can be purchased from System Biosciences (Cat# CD811A-1); and a vector pCDH-EF1α-MCS that does not express GFP, which is obtained by removing the PGK promoter and GFP gene on the pCDH-EF1α-MCS-(PGK-GFP) vector using conventional techniques in the art. According to the obtained TCR gene sequence, the complete gene sequence of TCR β chain and α chain as well as the cleavable F2A sequence and Furin enzyme fragment between them were synthesized and linked to the multiple cloning site downstream of the EF-1α promoter of the vector. The transcription order of TCR insertion was TCR β chain (without stop codon), Furin enzyme fragment, F2A fragment, TCR α chain (for methods, see the literature "Gene Ther. 2008 Nov; 15 (21): 1411-1423"). The vector expressing GFP is driven by the reverse PGK promoter. The vector that does not express GFP is the one that has the PGK promoter and GFP fragment removed.

Preparation of recombinant TCR lentiviral particles: TCR lentiviral particles were obtained by transfecting 293T/293FT cells with Lipofectaine 2000 transfection reagent (Invitrogen, #11668019). 293T/293FT cells and transfection procedures were prepared according to the manufacturer's instructions. Transfection was performed in a 6-well culture plate. First, Opti-MEM 1 culture medium (Thermo Fisher, cat#51985091) was used to prepare a liposome mixed solution of the transfection plasmid. According to the manufacturer's instructions, 6 μl of lipofectaine 2000 reagent, 0.8 μg of TCR lentiviral vector plasmid, and 1.8 μg of viral packaging plasmid of the pCDH system (SBI, cat#LV500A-1) were added to 250 μl of the culture medium. After incubation for 25 minutes, the mixture was added to the 293T/293FT cell culture wells. After culturing for 16 hours at 5% CO ₂ and 37°C, the culture medium was changed to DMEM medium without FBS (Thermo Fisher, cat#11965092). After culturing for 24 hours and 48 hours, the cell supernatant was collected and centrifuged at 2000 g for 10 minutes. The virus supernatant obtained after filtering with a 0.4 μm filter membrane was concentrated using lentivirus concentrate (GeneCopoeiaTM#LPR-LCS-01) according to the manufacturer's instructions and used to infect cells.

Recombinant TCR lentiviral transfection of human T cells: Frozen primary PBMC cells were thawed and cultured in RPMI-1640 complete medium for 24 hours. Dead cells were removed by Ficoll-Paque Premium density gradient centrifugation (×400g) for 30 minutes, and placed in 24-well plate culture wells treated with 2μg/ml anti-human CD3 antibody (Biolegend, OKT3 clone cat#317303) and 2μg/ml anti-human CD28 antibody (Biolegend, cat#302914) (100μl DPBS solution containing the above CD3 antibody and CD28 antibody was added to each well) for 24 hours. The cell concentration was 2×10e6/ml. Alternatively, Dynabead human T-CD3/CD8 magnetic beads (Thermo Fisher, cat#11131D), stimulate and activate PBMC cells according to the manufacturer's instructions. Collect cells after 24 hours of culture, add 100μl concentrated TCR lentiviral particles (3×10e8Tu/ml) to the wells of a 24-well plate, and continue to culture with RPMI-1640 complete culture medium or X-VIVO15 (Lonza#04-418Q) containing IL-2100IU/ml, IL-7 5ng/ml, and IL-15 5ng/ml, and replace the culture medium containing the above cytokines every 3 days. You can also use RestroNectin pre-treated culture plates (Takara, cat# T110A) and infect activated PBMC cells with viruses according to the manufacturer's instructions. Phenotypic and functional tests can generally be performed after 72 hours. Transfection of T cell lines is also carried out according to the above steps. If the viral vector is marked with GFP, GFP-positive cells can generally be observed under a fluorescence microscope 48 hours after transfection.

Example 1: Inducing Her2/neu 369-377 polypeptide (Her2-E75 epitope polypeptide) to specifically kill T cells from the peripheral blood of HLA-A2 positive normal donors

In this example, a low concentration of 1 μg/ml Her2/neu 369-377 polypeptide was used to induce polypeptide-specific killer T cells from HLA-A2-positive normal PBMC (#2) after two rounds of in vitro stimulation, and flow cytometry analysis and single cell separation were performed. The specific method is as described above. The results are as follows: The right figure of Figure 1A shows that 0.024% of the lymphocytes are CD8-positive killer T cells that can bind to Her2/neu 369-377/HLA-A2 pentamer (i.e., Her2-E75 pentamer), and the control cells in the left figure that were not stimulated with Her2 polypeptide did not show CD8-positive pentamer-positive cells. The results show that in the natural T cell pool, the number of specific T cells that recognize the Her2/neu 369-377 antigen peptide is very small. Despite the small number, this group of T cells that can recognize the Her2/neu 369-377 peptide can still be clearly distinguished. In addition, according to the fluorescence intensity of binding to the Her2-E75 pentamer, the positive cells include high-affinity T cells and low-affinity T cells. 300 CD8-positive pentamer-positive cells were separated by flow cytometry and then cultured for monoclonal culture. After two rounds of antigen peptide restimulation and cytokine expansion, a proliferating T cell clone Her2 CTL clone 6A5 (referred to as Her2 CTL 6A5) was obtained from these 300 separated single T cells. The right picture of Figure 1B shows that 97.9% of CD8 ⁺ CTL cells can bind to Her2/neu 369-377/HLA-A2 tetramers (i.e., Her2-E75 tetramers), indicating that this purified T cell clone is not mixed with other irrelevant cells. The left picture shows control T cells that cannot bind to Her2-E75 tetramers.

Example 2: Obtaining the complete sequence of Her2/neu 369-377 polypeptide-specific TCR

In this example, total RNA was directly purified from a certain amount of Her2 CTL 6A5 cells obtained in Example 1, and paired TCR α chain and β chain gene sequences (i.e., the two chains can together form a functional TCR that recognizes antigenic polypeptides) were obtained by 5'-RACE RT-PCR. The TCR encoded by the pair is called "Her2 TCR-6A5". The amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 4, and the encoding sequence is shown in SEQ ID NO: 12, and the amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 7, and the encoding sequence is shown in SEQ ID NO: 15. This TCR exists in the peripheral T cell pool of HLA-A2 positive normal people, and will not cross-react with normal cells that express Her2/neu protein in trace amounts to cause autoimmune reactions. In order to detect the antigenic specificity and function of the obtained TCR, the TCR α and β chain sequences were cloned into a replication-deficient lentiviral expression vector. Figure 1C shows a schematic diagram of the constructed TCR lentiviral vector structure fragment. The constant region of the TCR α and β chains was replaced by a mouse sequence from a human sequence and connected by a cleavable linker polypeptide. The expression of the 6A5 TCR α and β chains is driven by the EF-1α promoter. This promoter is a highly expressed promoter in eukaryotic cells and will not be affected by factors such as methylation, resulting in loss of function. It is suitable for long-term expression of exogenous genes in vivo. The TCR α chain and β chain are connected by the F2A polypeptide sequence. The TCR α chain and β chain genes can be transcribed at the same time and translated through ribosome skipping, so that the TCR α chain and β chain polypeptides are separated from each other. This ensures the consistency of the expression of the TCR α chain and β chain, thereby forming TCR dimers more efficiently. There is also a furin enzyme cleavage site between the TCR α chain and β chain, which is used to remove the redundant peptide segment at the carboxyl end of the β chain.

The nucleotide sequence of TCR β chain and α chain linked by a cleavable linking polypeptide, in which the constant region is replaced by the human sequence and the mouse sequence (SEQ ID NO: 20) (the corresponding TCR is Her2 TCR-6A5-mC, the amino acid sequence is shown in SEQ ID NO: 23) is connected to the above-mentioned vector to obtain the Her2 TCR-6A5-mC recombinant lentiviral vector. The Her2 TCR-6A5-mC gene fragments were amplified by PCR and cloned into the downstream of the EF1-promoter of the above-mentioned lentiviral vector (i.e., pCDH-EF1α-MCS): the β fragment of Her2 TCR-6A5-mC carrying the mouse constant region sequence was amplified by the 5′ primer 5′-AGAGCTAGCGAATTCAACATGGGCTGCAGGCTGCTC-3′ (SEQ ID NO: 26) and the 3′ primer 5′-GGATCGCTTGGCACGTGAATTCTTTCTTTTGACCATAGCCAT-3′ (SEQ ID NO: 27); the α gene of Her2 TCR-6A5-mC carrying the mouse constant region sequence was amplified by the 5′ primer 5′-TCCAACCCTGGGCCCATGCTCCTGTTGCTCATACCAGTG-3′ (SEQ ID NO: NO: 28) and 3' primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID NO: 29). PCR was performed using the Q5 High Fidelity PCR Kit (NEB, cat#M0543S) with the following reaction conditions: 98°C for 30 seconds, followed by 25 cycles of 98°C for 10 seconds, 65°C for 10 seconds, and 72°C for 3 minutes. The obtained TCR fragment was cloned into the MCS region downstream of the EF1α promoter in the pCDH-EF1α-MCS vector.

The constructed recombinant TCR lentiviral expression vector was prepared according to the aforementioned method to obtain the respective recombinant TCR lentiviral particles.

Example 3: Normal peripheral blood T cells were transfected with Her2 TCR-6A5-mC recombinant lentivirus to express a specific TCR that can recognize the Her2/neu 369-377 polypeptide.

In order to further verify whether the TCR obtained by the present invention can be expressed in primary T cells and has the function of recognizing Her2/neu antigen polypeptide, peripheral blood T cells from two different normal donors activated by CD3/CD28 antibodies were transfected with recombinant lentiviral particles carrying the Her2 TCR-6A5-mC gene (Her2 TCR-6A5-mC recombinant lentiviral vector), and the cells were collected 14 days later for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows: Figure 2A shows that lymphocytes in the peripheral blood mononuclear cells of both donors (#1 PBMC and #2 PBMC, respectively) can bind to Her2-E75 tetramers, indicating that the Her2 TCR-6A5-mC expressed by these cells can specifically recognize the Her2/neu antigen peptide presented by HLA-A2. The results also show that among the Her2-E75 tetramer-positive cells (i.e., expressing Her2 TCR-6A5-mC), the positive rate of CD8 ⁺ T killer cells and CD8 ^- lymphocytes is similar. ^CD8- lymphocytes are likely to be CD4 ⁺ T helper cells. If the transfection efficiency of lentivirus-infected CD8 ⁺ and CD4 ⁺ T cells is the same, it means that the exogenous Her2/neu 369-377-specific TCR on CD4 ⁺ cells can effectively bind to the Her2-E75 tetramer. This also further shows that the transfected Her2 TCR-6A5-mC can effectively bind to the Her2/HLA-A2 complex without the auxiliary function of CD8 molecules, that is, Her2 TCR-6A5-mC recognizes the Her2/neu 369-377 epitope peptide presented by HLA-A2 and is CD8-independent. CD4 cells expressing Her2 TCR-6A5-mC TCR secrete cytokines after recognizing Her2 antigen, which can not only assist the function of killer T cells and their survival time in the body, but also induce specific T cells targeting endogenous tumor antigens by regulating the tumor microenvironment, thereby enhancing anti-tumor immunity.

10e5 TCR-transfected PBMC cells were added to each well of a 96-well plate and co-cultured with different concentrations of Her2/neu 369-377 antigen peptide presented by T2 cells (1×10e5 per well) (Her2/neu 369-377 antigen peptide was diluted 10-fold starting from 0.1μg/ml to obtain different groups with final concentrations of 0.1μg/ml, 0.01μg/ml, 0.001μg/ml and 0.0001μg/ml). The IFN-γ secreted by T cells in the supernatant was detected to determine the function of the TCR-expressing PBMC cells to specifically recognize the Her2/neu 369-377 peptide. Figure 2B shows that PBMC expressing Her2 TCR-6A5-mC can be activated by Her2/neu 369-377 antigen peptide presented by T2 cells to secrete IFN-γ, indicating that primary T cells expressing exogenous Her2 TCR-6A5-mC can specifically recognize Her2/neu 369-377 peptide presented by HLA-A2 molecules. The ability to recognize antigen peptides is related to the expression level of exogenous TCR on T cells. The half-maximum reaction (EC50) peptide concentrations of two different donors' PBMCs transfected with Her2 TCR-6A5-mC for recognizing antigen peptides were estimated by curve fitting to be approximately 1.6 ng/ml and 2.9 ng/ml, respectively (IC50 Tool program, http://www.ic50.tk/). Although this reaction sensitivity is lower than the EC50 of high-affinity TCRs that recognize foreign antigens such as viral antigens (EC50 is about 10e-10M) (see the literature "CANCER RESEARCH 1998, 58.4902-4908" and "HUMAN GENE THERAPY 2014, 25: 730-739"), it is still within the medium-to-high TCR affinity range that can recognize common tumor-related antigens (as described in the literature "Eur J Immunol (2012) 42: 3174-9").

Figure 2C shows that when T cells were co-cultured with antigenic peptides presented by T2 cells (T2+Her2-E75, i.e., Her2/neu 369-377 peptide), the function of T cells to secrete IFN-γ was not significantly inhibited after adding anti-human CD8 antibodies. This indicates that the function of exogenous TCR to recognize Her2/neu 369-377 antigenic peptides does not require the auxiliary effect of CD8 molecules, and also shows that the recognition function of the Her2 TCR-6A5-mC TCR described in the present invention is non-CD8 function-dependent.

Example 4: Her2/neu 369-377 peptide-specific TCR expressed by normal peripheral blood T cells after transfection with Her2 TCR-6A5-mC recombinant lentivirus can recognize HLA-A2 ⁺ Her2/neu ⁺ tumor cells

First, the expression of HLA-A2 and Her2/neu in selected tumor cell lines was tested. The tumor cell lines included colorectal cancer Colo205 and HCT116, breast cancer MDA-MB-231 and MCF-7, pancreatic cancer PANC-1, neuroglioma U87MG, and small cell lung cancer NCI-H446. Tumor cells were stained with anti-HLA-A2 antibody (BD Bioscences, cat#561341) and anti-human CD340 (erbB2) antibody (Biolegend, cat#324406) and then analyzed by flow cytometry. The results in Figure 3A show that Colo205, MDA-MB-231, MCF-7, HCT116, and PANC-1 are all HLA-A2 ⁺ Her/neu ⁺ ; U87MG is HLA-A2 ⁺ , Her2/neu ^- ; and NCI-H446 is negative for both HLA-A2 and Her2/neu . These tumor cell lines not only originate from different tissues, but also express different HLA-A2 and Her2/neu . Among them, U87MG and NCI-H446 cells can be used as negative controls for Her2 TCR-6A5-mC T cell function detection.

After adding 1×10e4 tumor cells to each well of a 96-well plate, a certain amount of PBMC cells transfected with Her2 TCR-6A5-mC TCR or PBMC cells not transfected with Her2 TCR-6A5-mC TCR were added to each well of the 96-well plate as a control group according to the effector-target ratio (5:1). The effector-target ratio was 5:1. T cells were co-cultured with different tumor cell lines, and then the secreted IFN-γ in the supernatant was detected. The specific method is as described above. The results are as follows: Figure 3B shows that T cells expressing Her2 TCR-6A5-mC can be activated by HLA-A2 ⁺ Her2/neu ⁺ tumor cell lines and secrete IFN-γ, including colorectal cancer Colo205 and HCT116, breast cancer MDA-MB-231 and MCF-7, and pancreatic cancer PANC-1. However, the control group HLA-A2 ⁺ Her2/neu ^- neuroglioma U87MG and HLA-A2 ^- Her2/neu ^- lung cancer NCI-H446 could not activate T cells transfected with Her2 TCR-6A5-mC, indicating that Her2 TCR-6A5-mC TCR can specifically recognize Her2/neu antigens presented by HLA-A2 on the surface of tumor cells. Control T cells derived from PBMC of the same donor, cultured in parallel but not transfected with Her2 TCR-6A5-mC, could not be activated by the listed tumor cell lines, indicating that the response to tumor cells is not non-specific. The results also showed that the ability of Her2 TCR-6A5-mC T cells to recognize Her2/neu antigen presented by HLA-A2 is not closely related to the expression of HLA-A2 and Her2/neu molecules on the surface of tumor cells. Different tumor cells may have different inhibitory effects on T cells. On the other hand, the expression level on the cell surface does not necessarily reflect the total expression level of Her2/neu. The Her2/neu expressed by some tumor cells is mainly present in the cell cytoplasm, and these antigens are more easily presented by HLA-A2 (see the reference "J Immunol 2006; 177: 5088-5097").

1×10e4 target cells were added to each well of the culture plate, and a certain number of PBMC cells transfected with TCR genes were added according to the set effector-target ratio (1:1, 5:1, 10:1, 20:1, 40:1). After 24 hours, the killing activity of T cells against tumor cells was measured. Figure 3C-K shows that compared with control T cells without TCR transfection, T cells expressing Her2 TCR-6A5-mC TCR can specifically recognize and kill HLA-A2 ⁺ Her2/neu ⁺ tumor cell lines MCF-7, HCT116, PANC-1 and HEPG-2. The killing ability is dose-dependent with the number of Her2 TCR-6A5-mC T cells. However, the control group HLA-A2 ⁺ Her2/neu ^- neuroglioma U87MG, HLA-A2-Her2/neu + SKOV3 and HT-29, and HLA-A2 ^- Her2/neu ^- lung cancer NCI-H446 could not be specifically killed by Her2 TCR-6A5-mC T cells. The results also showed that when Her2 TCR-6A5-mC T cells increased to a certain number, they showed significant specific recognition and killing functions for HLA-A2 ⁺ Her2/neu ⁺ tumor cells. When the effector-target ratio was lower than 10:1, the specific killing function was not obvious, which may be related to the number of Her2/neu epitope peptides presented by HLA-A2 on the surface of tumor cells. To further enhance the sensitivity of Her2 TCR-6A5-mC T cells to recognize and kill tumor cells, one strategy is to increase the amount of tumor target cells expressing HLA-A2 and Her2/neu.

Example 5: The Her2/neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells after transfection with Her2 TCR-6A5-mC recombinant lentivirus does not recognize epitope polypeptides from normal human proteins with potential cross-reactions that can bind to HLA-A2 molecules.

The Her2 TCR-6A5-mC TCR is derived from T cells in the peripheral blood of healthy donors. Since the TCR exists in the normal T cells (T cell repertoire) in the peripheral blood, it usually does not recognize the self-proteins of normal tissues and produce off-target toxic reactions. In order to further improve the safety of T cells expressing the TCR in clinical use, this embodiment first determines the key amino acid sites (motifs) related to the recognition function of the Her2 TCR-6A5-mC TCR by comparing and screening the antigen epitope peptides (alanine scanning). Each amino acid on the Her2-E75 polypeptide KIFGSLAFL is replaced with alanine to perform single mutation. Since the seventh amino acid of the Her2-E75 polypeptide is alanine itself, it is replaced with glycine during single mutation. The resulting neo-epitope peptides were synthesized and tested for their ability to activate T cells expressing Her2 TCR-6A5-mC TCR. Since alanine maintains the basic framework of the secondary structure of the polypeptide chain and has a relatively small residue side chain, the role of the specific residue replaced by it on the biological activity of the polypeptide can be determined. For antigen epitope peptides, the key amino acid sites associated with the recognition function of Her2 TCR-6A5-mC TCR can be determined. The 9 neo-epitope peptides (final concentration of 0.1 μg/ml) were mixed and cultured with T2 cells and #2 PBMCs transfected with a lentiviral vector encoding the Her2 TCR-6A5-mC TCR gene for 24 hours, and the cell supernatant was taken for ELISA analysis of IFN-γ detection. The effector-target ratio E:T is 5:1. The results in Figure 4A show that after the amino acid residues at positions 1, 2, 3, 4, 5, 6, 8, and 9 of the Her2-E75 peptide were replaced by alanine or the alanine at position 7 was replaced by glycine, the newly formed epitope peptides had different abilities to activate Her2 TCR-6A5-mC TCR to secrete interferons. Compared with Her2-E75, the ability of the antigen epitope peptide to activate Her2 TCR-6A5-mC TCR was enhanced when the lysine residue at position 1 was replaced. When the alanine at position 7 was replaced by glycine, the phenylalanine at position 8 and the leucine at position 9 were replaced by alanine, the ability of the epitope peptide to activate Her2 TCR-6A5-mC TCR was reduced. However, when the isoleucine at position 2, the phenylalanine at position 3, the glycine at position 4, the serine at position 5 and the leucine at position 6 were replaced by alanine, the ability of the antigen epitope peptide to activate Her2 TCR-6A5-mC TCR was significantly reduced. The results show that the amino acid residues at positions 2, 3, 4, 5, and 6 are crucial for the recognition function of Her2 TCR-6A5-mC TCR. The amino acid side chains at these positions may form anchoring sites for epitope peptides to bind to HLA-A2 molecules or binding sites for TCR specific recognition. Changing the amino acid residues at these positions will cause the peptide to lose the antigen specificity recognized by Her2 TCR-6A5-mC TCR, while the amino acid residues at other positions contribute relatively little to the recognition function of Her2 TCR-6A5-mC TCR. Therefore, normal human proteins containing isoleucine at position 2, phenylalanine at position 3, glycine at position 4, serine at position 5, and leucine at position 6 are likely to be recognized by Her2 TCR-6A5-mC TCR and produce cross-reactions. In order to obtain all normal human proteins containing the above key amino acid residue sites, the human normal protein database (https://prosite.expasy.org/cgi-bin/prosite/PSScan.cgi) was searched with the sequence "X-I-F-G-S-L-X-X-X", where "X" can be any of the 21 common amino acids. A total of 13 different normal human protein sequences contain the -2I-3F-4G-5S-6L- sequence. Table 1 shows the protein name, the epitope position and epitope sequence containing the -2I-3F-4G-5S-6L- sequence. In order for these peptides to become epitope peptides recognized by Her2 TCR-6A5-mC TCR, they must first be able to bind to HLA-A2. The binding ability of the peptide to HLA-A2 can be predicted by the HLA/peptide binding prediction software (http://www.cbs.dtu.dk/services/NetMHC/). Table 1 also shows the predicted affinity of the peptide to HLA-A2, and the ranking of the affinity of the peptide to HLA-A2 in the affinity of the known natural epitope peptides that bind to HLA-A2. "Affinity (nM)" refers to the predicted affinity of the epitope peptide to HLA-A2. "%rank" refers to the affinity ranking of the epitope polypeptide binding to HLA-A2 in the affinity ranking of known natural epitope polypeptides binding to HLA-A2. The smaller the number, the higher the affinity. "Binding level" is the ability of the epitope polypeptide to bind to HLA-A2. "SB" (strong binding) means that the polypeptide has a high affinity to HLA-A2. Usually, %rank <0.5 is set as strong affinity, 0.5 <%rank <2 is set as weak affinity, and %rank >2 is set as no binding. Usually, affinity <50nM and %rank <0.5 are considered to be high affinity for the polypeptide to bind to HLA-A2. The results showed that, like the Her2/neu 369-377 peptide, the NSMA3 93-101 peptide, the O11A1 103-111 peptide, and the SV2C 687-695 peptide all contained the -2I-3F-4G-5S-6L-sequence and were likely to be the predicted epitope peptides that bind to HLA-A2 with high affinity. In order to detect whether the above potential epitope peptides derived from normal human protein, containing the -2I-3F-4G-5S-6L-sequence and binding to HLA-A2 molecules with high affinity can be recognized by the Her2 TCR-1B5-mC TCR, it was detected whether T cells expressing Her2 TCR-1B5-mC can be activated by the epitope peptide presented by T2 cells and secrete interferon-γ. #2 PBMCs transfected with a lentiviral vector encoding the Her2 TCR-6A5-mC TCR gene were co-cultured with T2 cells presenting different concentrations of the peptide for 24 hours, and the cell supernatant was taken for ELISA analysis of IFN-γ. Figure 4B shows the results of the supernatant of peripheral blood mononuclear cells (PBMCs) transfected with the Her2 TCR-6A5-mC TCR gene and co-cultured with different concentrations of epitope peptides presented by T2 cells to examine the secretion of IFN-γ. The results showed that, except for the Her2/neu 369-377 peptide, the other three predicted antigen epitope peptides could not activate Her2 TCR-1B5-mC T cells, indicating that the predicted epitope peptides derived from normal human proteins could not be recognized by Her2 TCR-1B5-mC TCR, thereby reducing the risk of Her2 TCR-6A5-mC TCR recognizing normal proteins and causing off-target side effects.

Example 6: Preparation of Her2 TCR-6A5-mC-PGKp-tEGFR lentiviral vector plasmid carrying tEGFR gene and Her2 TCR-6A5-mC gene

First, a truncated human EGFR (tEGFR) gene fragment was synthesized (by Integrated DNA Technologies, Inc., USA). tEGFR is composed of a nucleotide sequence encoding a human GM-CSF receptor signal peptide fragment (a nucleic acid fragment encoding a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor leader peptide) fused with a nucleotide sequence encoding human EGFR functional fragment (EGFR domains) III, IV and transmembrane spanning components (SEQ ID NO: 30) (i.e., a fragment of 2233-3306 bp in the nucleotide sequence of GenBank No. KX055828, see the reference "Cancer Immunol Res. 2016 Jun; 4(6): 509-19."). The extracellular domain III has a binding site for cetuximab (Erbitux®, the first IgG1 monoclonal antibody against EGF receptor approved by the US FDA) (see the literature "Blood. 2011 Aug 4; 118(5): 1255-1263."). The tEGFR fragment was obtained by PCR amplification using the synthetic tEGFR gene fragment as a template with the 5' primer 5'-GACGAGAGCGGCCTGACCATGCTTC-3' (SEQ ID NO: 31) and the 3' primer 5'-GCACAGTCGCTCGAGTCACATGAAGAG-3' (SEQ ID NO: 32). PCR was performed using the Q5 high-fidelity PCR kit (NEB, cat#M0543S), and the reaction conditions were: 98°C for 30 seconds, followed by 25 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 1 minute. The tEGFR fragment obtained by PCR amplification was cloned downstream of the PGK promoter on the Her2 TCR-6A5-mC recombinant lentiviral vector. Downstream of tEGFR is the addition of the BGH poly A signal (bovine growth hormone polyadenylation signal). The Her2 TCR-6A5-mC-PGKp-tEGFR recombinant lentiviral vector carrying the tEGFR gene was sequenced in its entirety to confirm that the inserted gene sequences were correct. Figure 5 shows a schematic diagram of the constructed TCR lentiviral vector structure fragment.

PBMC cells transfected with Her2 TCR-6A5-mC-PGKp-tEGFR lentivirus were stained with anti-EGFR antibody (Anti-EGFR antibody [EGFR1] (PE/Cy7 ®) (ab239309) obtained from Abcam) and analyzed by flow cytometry to confirm the expression of tEGFR.

Human T cells transfected with Her2 TCR-6A5-mC-PGKp-tEGFR lentivirus can simultaneously express Her2 TCR-6A5-mC TCR and the "safe conversion molecule" tEGFR. After tEGFR expression, the safety of the TCR gene-modified cells of the present invention in vivo can be increased. When necessary, anti-EGFR antibodies (such as cetuximab) can be used to eliminate the TCR gene-modified cells of the present invention in the patient's body.

Example 7: Killing effects of human T cells expressing Her2 TCR-6A5-mC and human T cells expressing Her2 TCR-6A5-mC and tEGFR on different human tumor cells

First, the expression of HLA-A2 and Her2/neu in the selected tumor cell lines was detected. Human tumor cell lines include cervical cancer cell C33A, osteosarcoma cell Saos-2, and pancreatic cancer cell CFPAC-1. Tumor cells were stained with anti-HLA-A2 antibody (BD Bioscences, cat#561341) and anti-human CD340 (erbB2) antibody (Biolegend, cat#324406) and then flow cytometry analysis was performed. The test results showed that cervical cancer cell C33A, osteosarcoma cell Saos-2, and pancreatic cancer cell CFPAC-1 were all HLA-A2 ⁺ Her/neu ⁺ .

Human T cells expressing Her2 TCR-6A5-mC and human T cells expressing Her2 TCR-6A5-mC and tEGFR were prepared according to the aforementioned method (i.e., the method of "recombinant TCR lentivirus transfected human T cells"), wherein the human T cells expressing Her2 TCR-6A5-mC were normal peripheral blood T cells transfected with Her2 TCR-6A5-mC recombinant lentivirus, and the human T cells expressing Her2 TCR-6A5-mC and tEGFR were normal peripheral blood T cells transfected with Her2 TCR-6A5-mC-PGKp-tEGFR recombinant lentivirus, and the peripheral blood mononuclear cells used in the preparation were obtained from allcells, USA (Cat. No. PB005F, specification 100 million, frozen).

2×10e4 tumor cells were added to each well of a 96-well culture plate as target cells. Control T cells ("MOCK-T"), human T cells expressing Her2 TCR-6A5-mC ("6A5-TCR-T"), and human T cells expressing Her2 TCR-6A5-mC and tEGFR ("6A5-EGFR-TCR-T") were added to the corresponding wells at an effector-target ratio (E:T) of 1:1, 5:1, 10:1, and 20:1, respectively. Three replicate wells were set up for each group. After incubation at 37°C in a 5% carbon dioxide incubator for 24 hours, an MTT assay was performed to obtain the corresponding killing results.

Figure 6A-C shows that compared with control human T cells without any TCR transfection, human T cells expressing Her2 TCR-6A5-mC TCR and human T cells expressing Her2 TCR-6A5-mC and tEGFR can specifically recognize and kill HLA-A2 ⁺ Her2/neu ⁺ tumor cell lines C33A, CFPAC-1, and Saos-2. The killing ability is dose-dependent with human T cells expressing Her2 TCR-6A5-mC TCR or human T cells expressing Her2 TCR-6A5-mC and tEGFR.

It can be seen that the Her2/neu 369-377 peptide-specific TCR and the TCR carrying the EGFR suicide gene expressed by normal peripheral blood T cells after transfection with Her2 TCR-6A5-mC recombinant lentivirus and Her2 TCR-6A5-EGFR-mC can recognize and then kill cervical cancer cells C33A, osteosarcoma cells Saos-2, and pancreatic cancer cells CFPAC-1.

Example 8: Effects of human T cells expressing Her2 TCR-6A5-mC on the growth of subcutaneous tumors in mice

This experiment used the NOD-SCID immune-deficient Colo205 colorectal cancer mouse model after cyclophosphamide (Cy) injection to simulate the human immune environment, and detected the inhibitory effect of human T cells expressing Her2 TCR-6A5-mC (prepared according to the aforementioned "recombinant TCR lentivirus transfected human T cells" method, in which the peripheral blood mononuclear cells used were obtained from allcells, USA (catalog number PB005F, specification 100 million, frozen)) on tumor growth, proving its basic drug efficacy.

The NOD-SCID immunodeficient mice used in this experiment were 6-week-old female mice (Vitamin Liva). Each mouse was subcutaneously inoculated with Colo205 colorectal cancer cells on the ventral side. The inoculation dose was 3×10 ⁶ cells. Seven days after inoculation, 18 tumor-bearing mice with an average tumor size of about 100 mm ³ were selected and randomly divided into 3 groups, with 6 mice in each group. The day of grouping was set as day 0. The first group was the blank control group (Cy+IL-2+PBS (it)), in which cyclophosphamide (Baxter, 8D231A) 200 mg/Kg per mouse was intraperitoneally injected on day 0, and the dosage was 100 μl. On the first day, each mouse was injected intratumorally (abbreviated as "it") with 100μl PBS solution (obtained from Cellmax, catalog number CBS101.05); at the same time, IL-2 (obtained from Jiangsu Jinsi Li Company, trade name Intercon-180350101) was injected subcutaneously in the neck, 100,000 IU per mouse. On the second day, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. On the third day, each mouse was injected intratumorally with 100μl PBS solution; at the same time, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. On the fourth day, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. On the fifth day, each mouse was injected intratumorally with 100μl PBS solution; at the same time, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. On the sixth day, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. On the 7th day, IL-2 was injected subcutaneously in the neck, 100,000 IU per mouse. The second group was the control T cell group (Cy+IL-2+T(it)), and the dosing regimen was basically the same as the first group, except that on the 1st, 3rd and 5th days, instead of PBS solution, 100 μl of control T cell suspension without any TCR transfection was injected into the tumor (the suspension medium was PBS), and the number of cells was 2×10 ⁷ per mouse. The third group was the Her2 TCR-6A5-mC T cell group of the present invention (Cy+IL-2+TCR-6A5T(it)). The dosing regimen was basically the same as that of the first group, except that on days 1, 3, and 5, instead of PBS solution, 100 μl of human T cell suspension expressing Her2 TCR-6A5-mC was injected intratumorally, with a cell number of 2×10 ⁷ per mouse. In the third group, when 100 μl of Her2 TCR-6A5-mC T cells were given to each mouse, the cells were freshly prepared according to the aforementioned preparation method, and the culture time was about 10 days from the time of transfection of PBMC with Her2 TCR-6A5-mC recombinant lentivirus according to the expected different dosing times, and the Her2 TCR-6A5-mC TCR positivity rate of the cells was about 40%. Starting from day 0, the tumor volume was measured every 2-4 days. The changes in the average tumor volume of animals in each group are shown in Figure 7.

As shown in Figure 7, in the NOD-SCID immunodeficient Colo205 colorectal cancer mouse model after cyclophosphamide injection, intratumoral injection of human T cell suspension expressing Her2 TCR-6A5-mC can inhibit tumor growth. There is a significant difference of p value less than 0.001 between the second and third groups (indicated as "***" in the figure). It can be seen that human T cells expressing Her2 TCR-6A5-mC have a significant therapeutic effect compared with control T cells without any TCR transfection.

Discuss

The difference in sensitivity of different tumor cell lines to specific T cells may be related to the different levels of Her2/neu antigen peptide/HLA-A2 complex expressed by tumor cells, or may be related to the different inhibitory effects of tumor cells themselves on T cell function. Although high-affinity TCRs that specifically recognize Her2/neu 369-377 peptides can be obtained by induction of Her2/neu 369-377 peptides in vitro, these high-affinity TCRs often cannot recognize Her2/neu antigens presented by tumor cells (see the literature "Cancer Res. 1998; 58: 4902-4908", "Cancer Immunol. Immunother. 2008; 57: 271-280"). One reason may be that the conformation of the exogenous Her2/neu 369-377 peptide bound to the HLA-A2 molecule is different from the conformation of the peptide/HLA complex presented in the cell (see the reference "Journal of Immunology, 2008, 180: 8135-8145"). Another possible reason is that the Her2/neu 369-377 polypeptide acts as a mimotope antigen, and the induced specific TCR can recognize both the Her2/neu 369-377 polypeptide and similar polypeptides presented by tumor cells, such as the Her2/neu 373-382 polypeptide (see the reference "J Immunol. 2013 Jan 1; 190(1): 479-488"). However, although the high-affinity TCR has a high affinity for the Her2/neu 369-377 polypeptide presented by HLA-A2, it cannot effectively recognize the corresponding mimotope polypeptide presented by tumor cells and kill tumor cells. The TCR that specifically recognizes the Her2/neu 369-377 polypeptide described in the present invention can target the Her2/neu 369-377 polypeptide presented by tumor cells to specifically recognize and kill tumor cells.

Since most high-affinity T cells that recognize self-antigens are eliminated by the central tolerance mechanism, the TCRs that can recognize Her2/neu antigens naturally existing in the peripheral T cell pool are mostly of medium and low affinity. Another high-affinity TCR that can recognize tumor cells and is independent of CD8 function is selected through functional testing after multiple α chains and β chains are paired from the Her2/neu 373-382 peptide-specific T cell population (see the literature "HUMAN GENE THERAPY 2024, 25: 730-739"; WO/2016/133779). Since it is not directly obtained from specific monoclonal T cells, it is not certain whether this TCR exists in the peripheral natural T cell pool. It is generally believed that the therapeutic effect of high-affinity T cell transfer therapy is better than that of low-affinity T cells targeting the same antigen (see the literature "Clin Exp Immunol (2015) 180: 255-70"). However, high-affinity TCR itself is prone to produce autoimmune reactions that recognize self-antigens (see the literature "Blood (2009) 114: 535-46"). TCRs that have not been screened by the central tolerance mechanism will also recognize normal tissues with low antigen expression, or produce cross-reactive off-target toxicity against other similar self-antigen epitopes (see the literature "Sci Transl Med (2013) 5: 197ra103", "Blood (2013) 122: 863-71"). Another reason for selecting high affinity TCRs is that the functions of these TCRs are independent of the auxiliary function of CD8, and thus they can obtain auxiliary effects on the function of CD8 ⁺ killer T cells by transfecting CD4 ⁺ T cells. The TCR of the present invention recognizes the Her2/neu 369-377 polypeptide with medium to high affinity, and the function of the TCR is independent of the auxiliary function of CD8, and thus is suitable for modification of T cells in relay transfer therapy. The TCR of the present invention cannot recognize all potential epitope peptides derived from normal proteins obtained by comparison screening methods and computer-assisted prediction software, thereby further avoiding the potential risk of cross-reactions against normal proteins.

In summary, the present invention provides a complete sequence of Her2/neu 369-377 polypeptide-specific TCR α and β chains induced from an HLA-A2 ⁺ autologous peripheral T cell pool. Primary killer T cells expressing this TCR and TCR with modified constant regions after transfection can recognize a variety of HLA-A2 ⁺ Her2/neu ⁺ tumor cells. This provides a new method and approach for the development and clinical application of T cells modified by specific TCR to treat tumors.

<110> Hou Yafei, Hangzhou Kangwanda Pharmaceutical Technology Co., Ltd.

<120> An isolated T cell receptor, a modified cell thereof, an encoding nucleic acid, a recombinant expression vector, a method for preparing TCR-modified cells, a pharmaceutical composition and its use

<130> FI-193972-59: 50TW

<150> CN201810972150.7

<151> 2018-08-24

<160> 32

<170> PatentIn version 3.5

<210> 1

<211> 132

<212> PRT

<213> Homo sapiens

<400> 1

Claims (39)

A separated T cell receptor comprises at least one of an α chain and a β chain, wherein both the α chain and the β chain comprise a variable region and a constant region, wherein the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of the variable region of the α chain is as shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the β chain is as shown in SEQ ID NO: 2. The T cell receptor as described in item 1 of the patent application, wherein the T cell receptor is capable of specifically recognizing the antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule. The T cell receptor as described in item 2 of the patent application, wherein the antigen epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3. The T cell receptor as described in Item 1 of the patent application, wherein the constant region of the α chain and/or the constant region of the β chain are derived from humans. The T cell receptor as described in item 4 of the patent application, wherein the constant region of the α chain is replaced in whole or in part by a homologous sequence derived from another species, and/or the constant region of the β chain is replaced in whole or in part by a homologous sequence derived from the other species. The T cell receptor as described in Item 5 of the patent application, wherein the other species is a mouse. The T cell receptor as described in item 1 of the patent application, wherein the constant region of the α chain is modified with one or more disulfide bonds, and/or the constant region of the β chain is modified with one or more disulfide bonds. The T cell receptor as described in item 1 of the patent application, wherein the amino acid sequence of the α chain is shown as SEQ ID NOs: 4, 5 or 6, and the amino acid sequence of the β chain is shown as SEQ ID NOs: 7, 8 or 9. A separated nucleic acid encoding a T cell receptor, comprising a coding sequence of at least one of the α chain and the β chain of the T cell receptor, wherein the α chain coding sequence and the β chain coding sequence both comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence encoded by the α chain variable region coding sequence is as shown in SEQ ID NO: 1, and the amino acid sequence encoded by the β chain variable region coding sequence is as shown in SEQ ID NO: 2. The nucleic acid as described in item 9 of the patent application, wherein the nucleic acid is DNA or RNA. The nucleic acid as described in item 9 of the patent application, wherein the α chain variable region coding sequence is shown as SEQ ID NO: 10, and the β chain variable region coding sequence is shown as SEQ ID NO: 11. The nucleic acid as described in item 9 of the patent application scope, wherein the T cell receptor encoded by the nucleic acid can specifically recognize an antigen epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule. The nucleic acid as described in item 12 of the patent application, wherein the antigen epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3. The nucleic acid as described in Item 9 of the patent application, wherein the α chain constant region coding sequence and/or the β chain constant region coding sequence are derived from humans. The nucleic acid as described in item 14 of the patent application, wherein the α chain constant region coding sequence is completely or partially replaced by a homologous sequence derived from another species, and/or the β chain constant region coding sequence is completely or partially replaced by a homologous sequence derived from the other species. The nucleic acid as described in Item 15 of the patent application, wherein the other species is a mouse. A nucleic acid as described in item 9 of the patent application, wherein the α chain constant region coding sequence comprises one or more disulfide bond coding sequences, and/or the β chain constant region coding sequence comprises one or more disulfide bond coding sequences. The nucleic acid as described in item 9 of the patent application, wherein the α chain coding sequence is shown in SEQ ID NOs: 12, 13 or 14, and the β chain coding sequence is shown in SEQ ID NOs: 15, 16 or 17. A nucleic acid as described in any one of items 9 to 17 of the patent application, wherein the α chain coding sequence and the β chain coding sequence are connected by a coding sequence for a cleavable linking polypeptide. The nucleic acid as described in item 19 of the patent application scope, whose sequence is shown in SEQ ID NOs: 18, 19, or 20. A recombinant expression vector, which contains a nucleic acid described in any one of items 9 to 20 of the patent application scope, and/or its complementary sequence, which is effectively linked to a promoter. A recombinant expression vector as described in item 21 of the patent application, wherein the recombinant expression vector contains a suicide gene coding sequence; wherein the suicide gene is selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or a combination thereof. A recombinant expression vector as described in Item 22 of the patent application, wherein the suicide gene coding sequence is under the control of a promoter, and the promoter used to control the suicide gene coding sequence is the same as or different from the promoter to which the nucleic acid is linked, and is independent of each other. The recombinant expression vector as described in item 22 of the patent application, wherein the suicide gene coding sequence and the nucleic acid are under the control of the same promoter, and the suicide gene coding sequence is linked to the nucleic acid through a cleavable linker polypeptide coding sequence or an internal ribosome entry site sequence. A T cell receptor-modified cell, the surface of which is modified by the T cell receptor described in any one of items 1 to 8 of the patent application, wherein the cell includes a primitive T cell or a precursor cell thereof, a NKT cell, or a T cell strain. The T cell receptor-modified cell as described in Item 25 of the patent application, wherein the cell expresses a suicide gene protein on its cell surface or inside the cell; wherein the suicide gene is selected from: iCasp9, HSV-TK, mTMPK, truncated EGFR, truncated CD19, truncated CD20 or a combination thereof. A method for preparing a cell modified with a T cell receptor as described in item 25 or 26 of the patent application, comprising the following steps: 1) providing a cell; 2) providing a nucleic acid encoding a T cell receptor as described in any one of items 1 to 8 of the patent application; 3) transfecting the nucleic acid into the cell. As described in item 27 of the patent application, the cells in step 1) are from autologous or allogeneic sources. As described in item 27 of the patent application, the transfection method includes: using a viral vector transfection method, a chemical method and a physical method. As described in item 29 of the patent application, the viral vector comprises a γ-retroviral vector or a lentiviral vector. As described in item 29 of the patent application, the chemical method includes liposome transfection. As described in item 29 of the patent application, the physical method includes electrotransfection. As described in item 27 of the patent application, the nucleic acid in step 2) is a nucleic acid as described in any one of items 9 to 20 of the patent application. A use of a T cell receptor modified cell as described in claim 25 or 26 in the preparation of a drug for treating or preventing a tumor and/or cancer, wherein the tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive. The use as described in item 34 of the patent application, wherein the drug for treating or preventing tumors and/or cancers also includes other drugs for treating tumors and/or drugs for regulating the patient's immune system. A use of a T cell receptor-modified cell as described in claim 25 or 26 in the preparation of a drug for detecting whether a host's tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive. A drug composition, wherein the drug composition comprises as an active ingredient a T cell receptor-modified cell as described in item 25 or 26 of the patent application, and a pharmaceutically acceptable excipient. The pharmaceutical composition as described in claim 37, wherein the pharmaceutical composition comprises the T cell receptor modified cells in a total dosage range of 1×10 ³ -1×10 ⁹ cells/Kg body weight per patient per course of treatment. The drug composition as described in claim 37, wherein the drug composition is suitable for administration via an artery, vein, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular or intraperitoneal route.