Engineered T cell based on HLA-DR enhancement as well as preparation method and application of engineered T cell

Abstract

The invention relates to the technical field of immunotherapy, in particular to an HLA-DR enhancement-based engineered T cell and a preparation method and application thereof, and the HLA-DR enhancement-based engineered T cell comprises: a) a chimeric antigen receptor CAR; b) an exogenously expressed HLA-DR molecule, the expression of the HLA-DR enhancing the antitumor activity, and the HLA-DR molecule comprising: (i) an HLA-DR alpha subunit encoded by a nucleotide sequence having at least 90% identity with SEQ ID NO: 1; and / or (ii) an HLA-DRbeta subunit encoded by a nucleotide sequence having at least 90% identity with SEQ ID NO: 2. Therefore, by overexpressing a molecule HLA-DR which is up-regulated in expression on T cells during a cancer-related treatment reaction period of a tumor patient, the molecule HLA-DR is a specific T cell expression change molecule aiming at a responder or a good prognosis person after a clinical tumor patient is treated, so that the function of the CAR-T cells in a tumor killing process is improved, and the reaction durability of the CAR-T cells is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to engineered T cells based on HLA-DR enhancement and a preparation method and application thereof. BACKGROUND

[0002] Chimeric antigen receptor T cell (CAR-T) therapy has made significant clinical breakthroughs in the treatment of hematological malignancies, and multiple approved products have shown excellent efficacy. However, solid tumors account for the vast majority of malignant tumors, and CAR-T therapy for solid tumors is still in the clinical exploration stage, and its efficacy is limited by multiple biological obstacles: including (1) target antigen loss caused by tumor antigen heterogeneity; (2) insufficient T cell persistence and terminal exhaustion phenotype formation; (3) negative regulation of immunosuppressive tumor microenvironment (TME).

[0003] These mechanisms result in insufficient CAR-T cell infiltration, functional attenuation, and low clinical response rate in the treatment of solid tumors.

[0004] Currently, HLA-DR, as an antigen presenting molecule, plays a key role in the process of immune recognition, and as a molecule that is usually up-regulated after T cell activation, it is often associated with T cell activation. Recent studies have found that the higher the proportion of CD8 + HLA + T cell subsets in tumor patients, the better the prognosis of patients, HLA-DR, as an antigen presenting molecule, is usually expressed on the surface of various antigen presenting cells such as B cells, dendritic cells, monocytes / macrophages, etc., but HLA-DR also has the function of interacting with some molecules on the cell surface and affecting cell signal transmission, which also reflects the complex biological function of HLA-DR.

[0005] The problem to be solved by the present application is to overexpress HLA-DR molecules on CAR-T cells to improve the persistence and anti-tumor efficacy of CAR-T cells, and to provide a promising strategy for improving the efficacy of CAR-T cells. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art and provide engineered T cells based on HLA-DR enhancement and a preparation method and application thereof. By overexpressing the molecule HLA-DR that is up-regulated on T cells during the response of tumor patients to cancer-related treatment, it is a molecule that specifically changes the expression of T cells in clinically responsive or well-prognosed tumor patients after treatment, to improve the function of CAR-T cells in the process of tumor killing and improve their response persistence.

[0007] The application is realized by the following technical solutions: on the one hand, an engineered T cell is provided, comprising: a) a chimeric antigen receptor CAR; b) an exogenous HLA-DR molecule, the expression of which enhances anti-tumor activity, and the HLA-DR molecule comprising: (i) an HLA-DR alpha subunit encoded by a nucleotide sequence having at least 90% identity with SEQ ID NO: 1; and / or, (ii) an HLA-DR beta subunit encoded by a nucleotide sequence having at least 90% identity with SEQ ID NO: 2.

[0008] Further, the CAR comprises: an extracellular binding domain targeting a tumor antigen; a hinge region; a transmembrane domain; an intracellular signaling domain comprising a CD3 zeta domain and an optional costimulatory domain.

[0009] Still further, the costimulatory domain is selected from 4-1BB, CD28, OX40, ICOS, CD27 or a combination thereof.

[0010] Further, the extracellular binding domain is a single-chain antibody scFv, and the scFv targets any one or several antigens selected from MSLN, CD276, CD19, HER2, GPC3, CLDN18.2, BCMA, PSMA, MUC1, CD123.

[0011] Still further, the scFv targets MSLN and comprises: (i) an amino acid sequence as shown in SEQ ID NO: 5; or (ii) a variant having at least 90% identity with SEQ ID NO: 5 and retaining the ability to bind MSLN.

[0012] Further, the CAR and the HLA-DR molecule are linked by a self-cleaving polypeptide encoding sequence for co-expression, and the self-cleaving polypeptide is selected from P2A, T2A, E2A or F2A.

[0013] In addition, a recombinant vector is provided, comprising a nucleotide sequence encoding the above-mentioned CAR; a nucleotide sequence encoding the above-mentioned HLA-DR molecule; and a 2A self-cleaving peptide encoding region connecting the CAR and HLA-DR encoding sequences.

[0014] Further, the recombinant vector is a lentiviral vector.

[0015] Also provided is an application based on the above-mentioned engineered T cells, wherein the tumor comprises a hematological tumor or a solid tumor.

[0016] Further, the solid tumor comprises, but is not limited to, one or more of liver cancer, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.

[0017] Beneficial effects The present application improves the function of CAR-T cells in tumor killing and improves the reaction persistence by overexpressing a molecule HLA-DR that is up-regulated on T cells during the response of a tumor patient to cancer-related treatment, which is a T cell expression change specific to the tumor patient who responds to treatment or has a good prognosis.

[0018] The present application improves cell expansion and differentiation of memory cells during CAR-T cell culture by genetic engineering, and improves the effector function, activation level, enhances anti-tumor activity and persistence during the anti-tumor response, and is not prone to exhaustion, and the overexpressed molecule is a molecule that is up-regulated during T cell activation, which is an endogenous molecule and does not cause rejection, and the molecule has the ability of antigen presentation, and has the potential to expand the effect of tumor immune cell response. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The structural schematic diagrams of different transgenic CAR-T cells constructed in the embodiments of the present application are shown. Specifically, they include the vector construction schematic diagrams of MSLN CAR-T cells (expressing only a chimeric antigen receptor targeting MSLN), MSLN-HLA-DRα CAR-T cells (co-expressing MSLN CAR and HLA-DRα), MSLN-HLA-DRβ CAR-T cells (co-expressing MSLN CAR and HLA-DRβ), and MSLN-HLA-DRαβ CAR-T cells (co-expressing MSLN CAR, HLA-DRα, and HLA-DRβ).

[0020] Figure 2 The results of detecting the expression efficiency of CAR molecules by flow cytometry are shown in the graphs. The graphs show the CAR positive rates of MSLN CAR-T cells and their control groups overexpressing HLA-DRα, HLA-DRβ, or HLA-DRαβ, which proves that each CAR-T cell is successfully constructed and has comparable expression efficiency.

[0021] Figure 3Figure 4 is a graph showing the expansion ability of CAR-T cells during in vitro culture. The bar graph shows the cell expansion fold of MSLN CAR-T cells and CAR-T cells overexpressing HLA-DRa, HLA-DRb or HLA-DRab after continuous culture for 8 days, indicating that HLA-DR overexpression can significantly improve the proliferation ability of CAR-T cells.

[0022] Figure 4 Figure 5 is a graph showing the memory differentiation phenotype analysis results of CAR-T cells. The proportion distribution of Naive cells, Central Memory cells (CM), Effector Memory cells and Effector cells in different CAR-T cells is shown by detecting the expression of CCR7 and CD45RO by flow cytometry, indicating that HLA-DR overexpression (especially HLA-DRa) can promote the differentiation of CAR-T cells to Central Memory phenotype.

[0023] Figure 5 Figure 6 is a graph showing the in vitro killing activity detection results of CAR-T cells on tumor cells (OVCAR-3). The killing efficiency of MSLN CAR-T cells and CAR-T cells overexpressing HLA-DR is compared at different effector-target ratios by luciferase reporter system, showing that HLA-DR overexpression can significantly enhance the tumor killing ability of CAR-T cells.

[0024] Figures 6-7 Figure 7 is a graph showing the detection results of cytokine and toxic molecule expression of CAR-T cells after co-incubation with tumor cells. The expression levels of IL-2, perforin and IFN-g are detected by flow cytometry (a) and statistical graph (b) to show that CAR-T cells overexpressing HLA-DR produce higher levels of effector molecules during killing. Figure 6 Figure 8 is a representative flow cytometry graph, Figure 7 Figure 9 is a statistical graph, indicating that CAR-T cells overexpressing HLA-DR have higher activation state and cytotoxic potential.

[0025] Figure 8 Figure 10 is a graph showing the detection results of activation and toxicity related molecule expression of CAR-T cells after co-incubation with tumor cells. The expression of Granzyme B, CD25, CD69 and TNF-a is detected by flow cytometry, showing that CAR-T cells overexpressing HLA-DR have higher activation state and cytotoxic potential.

[0026] Figure 9 Figure 11 is a graph showing the detection results of exhaustion related molecule expression of CAR-T cells after co-incubation with tumor cells. The double positive proportion of PD-1 and TIM-3 is detected by flow cytometry, indicating that CAR-T cells overexpressing HLA-DRa or HLA-DRb do not significantly up-regulate exhaustion markers, while the HLA-DRab co-expression group shows certain exhaustion phenotype under certain conditions. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0028] The extracellular antigen binding domain in the present application can also be referred to as a "single chain antibody" (scFv), which refers to an antibody fragment with the ability to bind to an antigen, which is formed by a hinge connection between the amino acid sequences of the antibody light chain variable region (VL region) and the heavy chain variable region (VH region). In some embodiments, the single chain antibody (scFv) is from a screened or existing antibody. The antibody of interest can be a human antibody, including a human-mouse chimeric antibody and a humanized antibody.

[0029] The nucleotide fragment encoding the protein in the present application is defined in the present application as a part of the nucleic acid sequence that directly determines the amino acid sequence of the protein product (such as a single chain antibody, a hinge region, a transmembrane region and an intracellular costimulatory domain).

[0030] The chimeric antigen receptor polypeptide (CAR) in the present application is an artificially modified receptor polypeptide, which can anchor a specific molecule (such as an antibody) that recognizes a tumor cell surface antigen on an immune cell (such as a T cell), so that the immune cell recognizes a tumor antigen or a viral antigen and kills a tumor cell or a virus-infected cell. The CAR usually comprises, in order, an optional signal peptide, a polypeptide such as a single chain antibody that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region and an intracellular signaling region.

[0031] The CAR-T cell of the present application can express a chimeric antigen receptor polypeptide and overexpress HLA-DRa or HLA-DRb. The chimeric antigen receptor polypeptide expressed by the CAR-T cell comprises a signal peptide, a humanized MSLN single chain antibody, a hinge region, a CD8a transmembrane domain, a cytoplasmic signaling domain 41BB and CD3zeta.

[0032] The coding DNA fragment (nucleotide sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2) of the HLA-DRa or HLA-DRb is obtained from the NCBI website. HLA-DR is a heterodimeric protein, and the expression of HLA-DRa or HLA-DRb is inconsistent in T cell sequencing data, so the present application overexpresses HLA-DRa or HLA-DRb respectively or simultaneously.

[0033] The amino acid sequence of HLA-DRa is shown in SEQ ID NO: 6; The amino acid sequence corresponding to HLA-DRa (the amino acid sequence corresponding to the nucleic acid sequence of SEQ ID NO: 1): SEQ ID NO: 6 MAISGVPVLGFFIIAVLMSAQESWAIKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTENVVCALGLTVGLVGIIIGTIFIIKGLRKSNAAERRGPL; SEQ ID NO: 1 ATGGCCATAAGTGGAGTCCCTGTGCTAGGATTTTTCATCATAGCTGTGCTGATGAGCGCTCAGGAATCATGGGCTATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAATCCTGACCAATCAGGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAGGAGACGGTCTGGCGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATAGCTGTGGACAAAGCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGATCACCAATGTACCTCCAGAGGTAACTGTGCTCACGAACAGCCCTGTGGAACTGAGAGAGCCCAACGTCCTCATCTGTTTCATCGACAAGTTCACCCCACCAGTGGTCAATGTCACGTGGCTTCGAAATGGAAAACCTGTCACCACAGGAGTGTCAGAGACAGTCTTCCTGCCCAGGGAAGACCACCTTTTCCGCAAGTTCCACTATCTCCCCTTCCTGCCCTCAACTGAGGACGTTTACGACTGCAGGGTGGAGCACTGGGGCTTGGATGAGCCTCTTCTCAAGCACTGGGAGTTTGATGCTCCAAGCCCTCTCCCAGAGACTACAGAGAACGTGGTGTGTGCCCTGGGCCTGACTGTGGGTCTGGTGGGCATCATTATTGGGACCATCTTCATCATCAAGGGATTGCGCAAAAGCAATGCAGCAGAACGCAGGGGGCCTCTG The amino acid sequence of HLA-DRP is shown in SEQ ID NO: 7. The amino acid sequence corresponding to HLA-DRP (the amino acid sequence corresponding to the nucleic acid sequence of SEQ ID NO: 2): SEQ ID NO: 7 MVCLRLPGGSCMAVLTVTLMVLSSPLALAGDTRPRFLEYSTSECHFFNGTERVRYLDRYFHNQEENVRFDSDVGEFRAVTELGRPDAEYWNSQKDLLEQKRGRVDNYCRHNYGVVESFTVQRRVHPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKTGVVSTGLIHNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKMLSGVGGFVLGLLFLGAGLFIYFRNQKGHSGLQPRGFLS; SEQ ID NO: 2 ATGGTGTGTCTGAGGCTCCCTGGAGGCTCCTGCATGGCAGTTCTGACAGTGACACTGATGGTGCTGAGCTCCCCACTGGCTTTGGCTGGGGACACCAGACCACGTTTCTTGGAGTACTCTACGTCTGAGTGTCATTTCTTCAATGGGACGGAGCGGGTGCGGTACCTGGACAGATACTTCCATAACCAGGAGGAGAACGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGTGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAAGCGGGGCCGGGTGGACAACTACTGCAGACACAACTACGGGGTTGTGGAGAGCTTCACAGTGCAGCGGCGAGTCCATCCTAAGGTGACTGTGTATCCTTCAAAGACCCAGCCCCTGCAGCACCATAACCTCCTGGTCTGTTCTGTGAGTGGTTTCTATCCAGGCAGCATTGAAGTCAGGTGGTTCCGGAATGGCCAGGAAGAGAAGACTGGGGTGGTGTCCACAGGCCTGATCCACAATGGAGACTGGACCTTCCAGACCCTGGTGATGCTGGAAACAGTTCCTCGGAGTGGAGAGGTTTACACCTGCCAAGTGGAGCACCCAAGCGTGACAAGCCCTCTCACAGTGGAATGGAGAGCACGGTCTGAATCTGCACAGAGCAAGATGCTGAGTGGAGTCGGGGGCTTTGTGCTGGGCCTGCTCTTCCTTGGGGCCGGGCTGTTCATCTACTTCAGGAATCAGAAAGGACACTCTGGACTTCAGCCAAGAGGATTCCTGAGC; Taking the CAR molecule vector as an example, the pCDH-EF1a-MSLN plasmid is subjected to SaII enzyme cutting, and then the above obtained coding DNA fragment is recombined into the pCDH-EF1a-MSLN plasmid by using a homologous recombination enzyme. The CAR nucleotide sequence of the pCDH-EF1a-MSLN plasmid targeting MSLN is shown in SEQ ID NO: 3.

[0034] SEQ ID NO: 3 In the practice of the present application, any CAR can be selected; the CAR includes but is not limited to one or more of CD19-CAR, CD20-CAR, CD123-CAR, HER2-CAR, GPC3-CAR, BCMA-CAR, MUC1-CAR, PSMA-CAR, NKG2D-CAR, CD276-CAR, CLDN18.2-CAR.

[0035] In the practice of the present application, any CAR can be selected; the CAR includes but is not limited to one or more of CD19-CAR, CD20-CAR, CD123-CAR, HER2-CAR, GPC3-CAR, BCMA-CAR, MUC1-CAR, PSMA-CAR, NKG2D-CAR, CD276-CAR, CLDN18.2-CAR.

[0036] SEQ ID NO: 4 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSG; A recombinant lentivirus vector, comprising: (a) a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising a single-chain antibody (scFv) targeting MSLN, a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain, wherein the nucleotide sequence of the CAR is set forth in SEQ ID NO: 3; (b) a nucleotide sequence encoding an HLA-DR molecule, the sequence selected from: (i) an HLA-DRa-encoding sequence set forth in SEQ ID NO: 1; and / or (ii) an HLA-DR beta coding sequence as set forth in SEQ ID NO: 2; (c) a 2A self-cleavage peptide coding region connecting the coding sequences of (a) and (b) to achieve co-expression, the 2A peptide being selected from P2A, T2A, E2A or F2A; (d) an EF1 alpha promoter driving the expression of the sequences of (a)-(c); (e) a lentiviral backbone element comprising a 5' LTR, a 3' LTR and a packaging signal ψ.

[0037] A method for preparing an engineered T cell, comprising: 1) T cell activation: human peripheral blood-derived CD3 + T cells are co-incubated with CD3 / CD28 antibody magnetic beads and activated at 37°C, 5% CO2 for 24-72 hours; 2) Virus transduction: the lentiviral vector of claims 7-8 is added to the activated T cells at a multiplicity of infection MOI = 5-20; polybrene is added at a final concentration of 6 μg / mL; centrifugation is performed at 32°C, 1000 x g for 1.5 hours; 3) Cell expansion: the transduced cells are placed in RPMI1640 medium containing 10% FBS and 50-200 IU / mL IL-2; incubation is performed at 37°C, 5% CO2, and regular half-volume medium replacement or expansion culture is performed.

[0038] All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making creative efforts fall within the scope of protection of the present application. The experimental methods not specified in the following examples are generally performed according to conventional conditions or according to the conditions recommended by the manufacturers.

[0039] The reagents and raw materials used in the embodiments and comparative examples of the present application can be obtained through commercial channels unless otherwise specified.

[0040] Example 1: Preparation of CAR-T cells Construction of recombinant plasmids: The recombinant plasmids are transformed into Stb13 competent cells, and colony screening is performed on plates containing ampicillin antibiotic. After expansion culture of single colonies, the plasmids are extracted by a plasmid extraction kit, and the recombinant lentiviral expression plasmids pCDH-EF1 alpha-MSLN, pCDH-EF1 alpha-MSLN-P2A-HLA-DR alpha, pCDH-EF1 alpha-MSLN-T2A-HLA-DR beta, pCDH-EF1 alpha-MSLN-P2A-HLA-DR alpha-T2A-HLA-DR beta with correct sequencing results are preserved.

[0041] Lentivirus preparation: 293T cell plating: Take a T75 culture bottle of 80% confluent 293T cells, pour off the culture medium, wash twice with saline, add 1 ml of 0.05% trypsin, digest for 2 min at 37°C, add culture medium to stop digestion, count 10 μl on a counting plate, then plate 1×10 6 6 cells per well in a 6-well plate; 20 hours later, replace the transfection medium with 2.5 ml, and 2 hours later, add the transfection system consisting of the main plasmid (pCDH-EF1α-MSLN) and packaging plasmids psPAX2 and pMD2.G to the well plate using a calcium transfection kit, gently shake, and place in a culture incubator; 6 hours later, replace with 3 ml of complete culture medium; 48 hours later, collect the virus supernatant (pCDH-EF1α-MSLN-P2A-HLA-DRα, pCDH-EF1α-MSLN-T2A-HLA-DRβ, and pCDH-EF1α-MSLN-P2A-HLA-DRα-T2A-HLA-DRβ lentivirus preparation steps are the same).

[0042] Preparation of purified T cells: Dilute the peripheral blood of a healthy person with saline, then mix with lymph separation liquid at a ratio of 2:1, centrifuge at 2500 rpm, speed up 5, speed down 5, centrifuge for 25 min, then aspirate the white membrane layer and count, then centrifuge at 1500 rpm for 5 min, discard the supernatant, add an appropriate amount of buffer and CD3 magnetic beads according to the counting result, incubate at 4°C for 20 min, add buffer to the magnetic sorting column fixed in the magnetic field, add the incubated cell suspension, and constantly add buffer during the process until the T cell suspension is completely washed out, obtaining CD3 + T cells, then count, add 1×10 7 CD3 + T cells to CD3 / CD28 activation beads 100 ul for activation stimulation.

[0043] T cell infection: After 2 days of T cell activation, add polybrene (also known as hexadimethrine bromide) to the collected virus supernatant, with a final concentration of 6 μg / mL, add 2-3 ml of virus supernatant (control MOI of 10) per 1×10 6 T cells, centrifuge at 1000 g and 32°C for 1.5 h, and replace the fresh culture medium after completion.

[0044] Culture and expand CAR-T cells. CAR-T cells after infection were added into 1640 medium (containing 10% heat-inactivated FBS and 100 IU / ml IL-2) and placed in an incubator, and fresh 1640 medium was replaced every 24 h. The gene expression efficiency was detected after 3 days of infection, and CAR-T cells were obtained.

[0045] Example 1: Expression efficiency of CAR molecules The difference between the preparation method of CAR-T cells of the present comparative example 1 and the preparation method of MSLN CAR-T cells is only that the master plasmid is different; The master plasmid in the comparative example 1 is pCDH-EF1α-MSLN-P2A-HLA-DRα, pCDH-EF1α-MSLN-T2A-HLA-DRβ, and pCDH-EF1α-MSLN-P2A-HLA-DRα-T2A-HLA-DRβ plasmid, and the content not mentioned is exactly the same as MSLN CAR-T cells. The CAR-T cells prepared in the comparative example 1 are referred to as MSLN-HLA-DRα CAR-T cells or MSLN-HLA-DRβ CAR-T cells or MSLN-HLA-DRαβ CAR-T cells, and the structure of the transgene of the above-mentioned cells is shown in Figure 1 .

[0046] Detection process: The expression efficiency of CAR molecules was detected. After 3 days of culture of cells after lentivirus infection, APC Protein L-labeled CAR-T cells were used to detect the expression of gene editing on the surface of CAR-T cells by flow cytometry.

[0047] The results are shown in Figure 2 : The expression efficiency of CAR molecules is about 80%, and MSLN CAR-T cells and comparative example 1 CAR-T cells are effectively prepared for subsequent experimental verification.

[0048] Example 2: Expansion ability of CAR-T cells Changes in the expansion of CAR-T cells during in vitro culture after preparation of CAR-T cells; 1×10 6 CAR-T cells were added to each well of a 12-well plate, and continuously cultured for 8 days, during which time half-volume replacement and transfer to more cell culture wells were performed at appropriate times. After 8 days, cell counting was performed, and the results showed that the cell expansion multiples of MSLN-HLA-DRα CAR-T cells or MSLN-HLA-DRβ CAR-T cells or MSLN-HLA-DRαβ CAR-T cells were significantly increased compared with MSLN CAR-T cells, indicating that overexpression of HLA-DR molecules on CAR-T cells can improve the expansion ability of CAR-T cells. The results are shown in Figure 3 .

[0049] Effect Example 3: Memory differentiation ability of CAR-T cells After the preparation of CAR-T cells, the changes in memory differentiation during the in vitro culture of the cells were detected. After the CAR-T cells were cultured in vitro for 12 days, the cultured CAR-T cells were collected, centrifuged at 500g for 5 min, and the supernatant was discarded. 1 mL of Flow buffer was added, vortexed, centrifuged at 500g for 5 min, and the supernatant was discarded. Antibody suspension was prepared by adding 100 μl of PBS, 1 μl of APC anti-human CCR7, and 1 μl of PE anti-human CD45RO per tube. After mixing, the cells were added, vortexed, and incubated at 4°C in the dark for 30 min. 1 mL of Flow buffer was added, vortexed, centrifuged at 500g for 5 min, and 200 μl of Flow buffer was added. The relevant indicators were detected by flow cytometry, and the proportion of each cell subpopulation in the MSLN CAR-T, MSLN-HLA-DRα CAR-T cell or MSLN-HLA-DRβ CAR-T cell or MSLN-HLA-DRαβ CAR-T cell group was calculated. CCR7 + CD45RO - Subgroup is Naïve cell, CCR7 + CD45RO + is Central Memory cell (CM), CCR7 - CD45RO + is Effector Memory cell, CCR7 - CD45RO - is Effector cell. The results are shown in Figure 4 .

[0050] The results show that HLA-DR overexpression can increase the central memory differentiation of CAR-T cells, and HLA-DRα overexpression significantly increases the central memory differentiation of CAR-T cells, and reduces the effector differentiation of CAR-T cells, indicating that HLA-DR overexpression can significantly increase the proportion of memory cells in CAR-T preparations, reduce the terminal differentiation, and retain the anti-tumor potential.

[0051] Effect Example 4: Killing ability, activation and effector function of CAR-T cells The killing ability of CAR-T cells after 24h in vitro co-incubation with tumor cells was detected. The ovarian cancer cell line OVCAR-3 was selected as the target cell (overexpressing luciferase), and after counting, 2x10 4Cells, set three wells, after overnight, according to the effective target ratio 2:1, 1:1, 1:2, add MSLN CAR-T, MSLN-HLA-DRα CAR-T cells or MSLN-HLA-DRβ CAR-T cells or MSLN-HLA-DRαβ CAR-T cells or NSLN CAR-T cells as control, 24h, add 1ul D-fluorescein potassium salt with a concentration of 100uM to each well of the 96-well plate. After 10 minutes, use small animal live imaging instrument to detect the total light particle value of each well of the 96-well plate. Analysis found that HLA-DR overexpression can significantly improve tumor killing ability. The calculation of statistical results is shown in Figure 5 .

[0052] Collect CAR-T cells in 1.5ml EP tubes, centrifuge at 500g for 5min, discard the supernatant, add 1mL Flowbuffer, vortex, centrifuge at 500g for 5min, discard the supernatant, add 100ul PBS, 0.5ul eBioscience™Fixable Viability Dye eFluor™ 506, 1ul APC-A700 anti-human CD45, 1ul PE anti-human IL-2, 1ul APC anti-human perforin, 1ul PC7 anti-human IFN-γ antibody suspension, mix well, add cells, vortex, 4℃ avoid light incubation for 30min, add 1mL Flow buffer, vortex, centrifuge at 500g for 5min, add 200ul Flow buffer, flow cytometry detection related indicators, statistics MSLN CAR-T, MSLN-HLA-DRα CAR-T cells or MSLN-HLA-DRβ CAR-T cells or MSLN-HLA-DRαβ CAR-T cells group of positive cell population ratio or MFI value.

[0053] The results are shown in Figures 6-7 , after analysis found that after 24h co-incubation with tumor cells, MSLN-HLA-DRα CAR-T cells or MSLN-HLA-DRβ CAR-T cells or MSLN-HLA-DRαβ CAR-T cells compared with MSLN CAR-T cells, IL-2, perforin and IFN-γ expression significantly increased.

[0054] Detect the expression of cytotoxic molecules and cytokines of CAR-T cells after 24h in vitro killing. Select ovarian cancer cell line OVCAR-3 as target cells (overexpress luciferase), count and plate into 12-well plates, 2x10 4The three wells were set up, and the cells were adhered for 12 h. MSLN CAR-T, MSLN-HLA-DRa CAR-T cells, or MSLN-HLA-DRb CAR-T cells, or MSLN-HLA-DRa b CAR-T cells were added at an effector target ratio of 2:1, 1:1, and 1:2, respectively, with NSLN CAR-T cells as a control. After 24 h, the CAR-T cells in the wells were collected with 1.5 ml EP tubes, centrifuged at 500 g for 5 min, and the supernatant was discarded. Then, 1 mL Flow buffer was added, vortexed, centrifuged at 500 g for 5 min, and the supernatant was discarded. CAR-T toxicity molecule detection GranzymeB expression, cell activation molecule CD25 and CD69 expression, and cytokine detection TNF-a expression: 100 μl PBS, 0.5 μl eBioscience™ Fixable Viability Dye eFluor™ 506 dead and alive fuel, 1 μl APC-A700 anti-human CD45, 1 μl APC anti-human GranzymeB, 1 μl PE anti-human CD25, 1 μl BV421 anti-human CD69, and 1 μl BV785 anti-human TNF-a were added to each tube to prepare an antibody suspension. After mixing, the cells were added, vortexed, and incubated at 4°C in the dark for 30 min. Then, 1 mL Flow buffer was added, vortexed, centrifuged at 500 g for 5 min, and 200 μl Flow buffer was added. The relevant indicators were detected by flow cytometry, and the proportion of positive cell groups or the MFI values of the MSLN CAR-T, MSLN-HLA-DRa CAR-T cells, or MSLN-HLA-DRb CAR-T cells, or MSLN-HLA-DRa b CAR-T cells were calculated. The results are shown in Figures 6-8 As shown in the results, after 24 h of co-incubation with tumor cells, the GranzymeB, CD25, and TNF-a expressions of MSLN-HLA-DRa CAR-T cells or MSLN-HLA-DRb CAR-T cells or MSLN-HLA-DRa b CAR-T cells were significantly higher than those of MSLN CAR-T cells. The CD69 expression of MSLN-HLA-DRa CAR-T cells or MSLN-HLA-DRa b CAR-T cells was significantly higher than that of MSLN CAR-T cells.

[0055] Based on the above experimental results, it can be found that CAR-T cells overexpressing HLA-DR can enhance the killing ability of CAR-T cells on tumor cells, improve the expression of cytokines and toxic molecules of CAR-T cells, and have a higher level of cell activation after tumor killing.

[0056] Example 5: Exhausted differentiation of CAR-T cells The expression of exhaustion-related molecules of CAR-T cells was detected after 24h in vitro killing. The ovarian cancer cell line OVCAR-3 was selected as the target cell (overexpressing luciferase), and after counting, 2x10 4 cells were plated in a 12-well plate, three replicates were set up, and the cells were attached for 12h. The effector-to-target ratio was set at 2:1, 1:1, and 1:2, and MSLN CAR-T, MSLN-HLA-DRa CAR-T cells, or MSLN-HLA-DRb CAR-T cells, or MSLN-HLA-DRa b CAR-T cells were added, with NSLN CAR-T cells as a control. After 24h, the CAR-T cells in the supernatant were collected with a 1.5ml EP tube, centrifuged at 500g for 5min, and the supernatant was discarded. 1mL Flow buffer was added, vortexed, centrifuged at 500g for 5min, and the supernatant was discarded. CAR-T exhaustion molecules PD-1 and TIM3 expression: 100ul PBS, 0.5ul eBioscience™ Fixable Viability Dye eFluor™ 506, 1ul BV650 anti-human TIM3, and 1ul PE anti-human PD-1 were added to prepare an antibody suspension. After mixing, the cells were added, vortexed, and incubated at 4°C in the dark for 30min. 1mL Flow buffer was added, vortexed, centrifuged at 500g for 5min, and 200ul Flow buffer was added. The relevant indicators were detected by flow cytometry, and the double-positive proportion of MSLN CAR-T, MSLN-HLA-DRa CAR-T cells, or MSLN-HLA-DRb CAR-T cells, or MSLN-HLA-DRa b CAR-T cells was calculated.

[0057] The results are shown in Figure 9 After 24h of co-incubation with tumor cells, the PD-1 + TIM3 + expression of MSLN-HLA-DRa CAR-T cells or MSLN-HLA-DRb CAR-T cells did not increase significantly compared to MSLN CAR-T cells. The PD-1 + TIM3 + expression of MSLN-HLA-DRa b CAR-T cells increased significantly compared to MSLN CAR-T cells.

[0058] From the above experimental results, it can be found that overexpression of HLA-DRa or HLA-DRb in CAR-T cells does not increase PD-1 +TIM3 + The proportion of double positive cells indicates that the exhaustion-related molecules thereof will not be significantly up-regulated, and the overexpression of HLA-DR molecules after co-incubation with tumor cells will not increase the exhaustion differentiation of CAR-T cells in the case of enhancing the activation and function of CAR-T cells.

[0059] In the implementation of the present application, the scFV sequence can target tumor antigens, and according to the type of cancer, the scFv sequence can be adjusted according to the expression of the specific target. Taking ovarian cancer as an example, the amino acid sequence of the MSLN-scFv sequence is shown in SEQ ID NO: 5.

[0060] SEQ ID NO: 5 QVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIK.

[0061] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing detailed description of the present application is made with reference to the foregoing embodiments, for the skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement, within the spirit and principles of the present application, any modification, equivalent replacement, improvement, etc., should be included in the scope of protection of the present application.

Claims

1. An engineered T cell, characterized in that, Comprise: a) a chimeric antigen receptor (CAR); b) an exogenously expressed HLA-DR molecule, which enhances anti-tumor activity, and which comprises: (i) an HLA-DR alpha subunit encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1; and / or, (ii) an HLA-DR beta subunit encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO:

2.

2. The engineered T cell of claim 1, wherein, The CAR comprises: an extracellular binding domain targeting a tumor antigen; a hinge region; a transmembrane domain; an intracellular signaling domain comprising a CD3 zeta domain and optionally a costimulatory domain.

3. The engineered T cell of claim 2, wherein, The costimulatory domain is selected from 4-1BB, CD28, OX40, ICOS, CD27, or a combination thereof.

4. The engineered T cell of claim 2, wherein, The extracellular binding domain is a single chain antibody (scFv) or a nanobody (VHH) targeting molecule, and the targeting molecule targets any one or several of MSLN, CD276, CD19, HER2, GPC3, CLDN18.2, BCMA, PSMA, MUC1, CD123.

5. The engineered T cell of claim 4, wherein, The scFv targets MSLN and comprises: (i) an amino acid sequence as set forth in SEQ ID NO: 5; or (ii) a variant that is at least 90% identical to SEQ ID NO: 5 and retains the ability to bind MSLN.

6. The engineered T cell of claim 1, wherein, The CAR is linked to the HLA-DR molecule by a self-cleaving polypeptide encoding sequence for co-expression of the two, and the 2A self-cleaving peptide is selected from P2A, T2A, E2A, or F2A.

7. A recombinant vector, characterized in that, A nucleotide sequence encoding the CAR of claims 1-6; a nucleotide sequence encoding the HLA-DR molecule of claims 1-6; a 2A self-cleaving peptide encoding region linking the CAR and HLA-DR encoding sequences.

8. The recombinant vector of claim 7, wherein, The recombinant vector is a lentiviral vector.

9. Use of the engineered T cell according to any one of claims 1 to 6, characterized in that The tumor comprises a hematological tumor or a solid tumor.

10. Use according to claim 9, characterized in that, The solid tumor comprises, but is not limited to, one or more of liver cancer, pancreatic cancer, ovarian cancer, cholangiocarcinoma, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, and breast cancer.

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