CAR-immune cells with knockdown of NKG2A gene and uses thereof

Abstract

The present invention proposes a CAR-immune cell. The NKG2A gene of the CAR-immune cell is silenced, and the NKG2A gene is silenced by knocking out the NKG2A gene of the CAR-immune cell, and the knockout is achieved by a CRISPR / Cas9 system containing at least one sgRNA in the nucleotide sequence shown in SEQ ID NO: 8 to 10. The CAR-immune cell can block or cancel the binding of NKG2A to the ligand HLA-E, prevent the CAR-immune cells entering the tumor microenvironment from being inhibited by the inhibitory microenvironment, and the CAR-immune cell has stronger tumor killing activity and IFN-γ secretion ability, can effectively inhibit tumor cell growth and significantly prolong the survival of mice, and can be used to eliminate or reduce the immune escape mechanism of the tumor or for the prevention and / or treatment of tumors or cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a CAR-immune cell for knocking down the NKG2A gene and a use thereof. More specifically, the present invention relates to a CAR-immune cell, a pharmaceutical composition and a use thereof. Background Art

[0002] In recent years, chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. However, CAR-T cells are prone to produce adverse reactions such as cytokine storm, neurotoxicity, and GVHD in clinical applications, and the therapeutic effect of CAR-T cells on solid tumors is not ideal, making the clinical application of CAR-T cells still face challenges.

[0003] CAR-NK cells have the advantage of good safety over CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; NK cells do not require antigen presentation and are not restricted by MHC, and can directly kill tumor cells; CAR-NK cells can identify and kill tumors with a variety of recognition mechanisms such as CAR dependence and NKR dependence, and have a wide anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development. However, both CAR-NK cells and CAR-T cells face the problem of being easily affected by the tumor immunosuppressive microenvironment when treating solid tumors.

[0004] Therefore, the development of a CAR-immune cell that is less susceptible to the tumor immunosuppressive microenvironment continues. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a CAR-immune cell that knocks down the NKG2A gene, which has the advantages of resisting the negative regulation effect of the tumor immune microenvironment and resisting cell exhaustion.

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] At present, the method of reversing the exhaustion of immune cells caused by the tumor microenvironment usually adopts immune checkpoint blockade therapy, that is, by using immune checkpoint inhibitors, such as monoclonal antibodies of immune checkpoint molecules, to block the binding of inhibitory receptors on the surface of T cells or NK cells with the corresponding ligands on the surface of tumor cells or regulatory T cells, myeloid cells or stromal cells in the tumor microenvironment, thereby weakening the inhibition of immune effector cells such as T cells or NK cells and awakening their ability to effectively kill tumors. However, in clinical practice, the response rate of immune checkpoint blockade therapy is low, only 20%-30%, and it is easy to develop drug resistance.

[0008] As an important immune checkpoint, NKG2A is mainly expressed on the surface of NK cells, and is also expressed on NKT cells and certain CD8+T cell subsets. NKG2A and CD94 are expressed on the cell surface in the form of heterodimers, mainly recognizing the non-classical MHC-I class molecule HLA-E, transmitting inhibitory signals to NK cells, and inhibiting the effector function of NK cells. That is, NKG2A highly expressed in the tumor microenvironment will weaken the activation and anti-tumor function of NK cells and other immune cells.

[0009] Based on this, the inventors found through experiments that compared with immune checkpoint inhibitors, CAR-immune cells with knockdown of the NKG2A gene do not require the preparation and development of monoclonal antibodies. Knocking down the expression of NKG2A can directly block or cancel its binding with the ligand HLA-E, thereby blocking the induction of inhibitory signals from the tumor microenvironment, preventing CAR-immune cells entering the tumor microenvironment from being inhibited by the inhibitory microenvironment, thereby effectively resisting exhaustion and enabling CAR-immune cells to fully exert their anti-tumor effects.

[0010] Therefore, in one aspect of the present invention, the present invention proposes a CAR-immune cell. According to an embodiment of the present invention, the NKG2A gene of the CAR-immune cell is silenced; wherein the immune cells of the CAR-immune cell include at least one of NK cells, T cells, NKT cells and γδT cells. According to an embodiment of the present invention, the CAR-immune cells (especially CAR-NK cells) can block or cancel the binding of NKG2A to the ligand HLA-E, prevent the CAR-immune cells entering the tumor microenvironment from being inhibited by the inhibitory microenvironment, and the CAR-immune cells have stronger tumor killing activity and IFN-γ secretion ability, which can effectively inhibit tumor cell growth and prolong the survival of mice, and can be used to eliminate or reduce the immune escape mechanism of tumors or for the prevention and / or treatment of tumors or cancer.

[0011] According to an embodiment of the present invention, the immune cells of the CAR-immune cells are NK cells.

[0012] It should be noted that in the present invention, the source of NK cells of CAR-NK cells is not specifically limited, including but not limited to NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSC-derived NK cells, NK-92 and other NK cells from different sources, and is also applicable to CAR-NK cells, CAR-T cells, CAR-NKT cells and CAR-γδT cells.

[0013] According to an embodiment of the present invention, the silencing of the NKG2A gene is achieved by knocking out the NKG2A gene of the CAR-immune cell.

[0014] According to an embodiment of the present invention, the knockout is achieved through the CRISPR / Cas9 system.

[0015] According to an embodiment of the present invention, the sgRNA of the CRISPR / Cas9 system has at least one of the nucleotide sequences shown in SEQ ID NOs: 8 to 10. Thus, the above sgRNA can be used to target and knock down the NKG2A gene in CAR-immune cells (especially CAR-NK cells), and obtain CAR-immune cells with silenced NKG2A genes. The application of the sgRNA can further reduce the expression of other inhibitory receptors such as PD-1 and Tim-3 in CAR-immune cells, thereby improving the tumor killing activity and IFN-γ secretion ability of CAR-immune cells, effectively inhibiting tumor cell growth and prolonging the survival of mice.

[0016] According to an embodiment of the present invention, the knockout is performed in the following manner: an expression vector carrying the sgRNA and a nucleic acid encoding the Cas9 molecule is introduced into the CAR-immune cell to be modified for culture treatment.

[0017] According to an embodiment of the present invention, the culturing treatment time is 24 to 72 hours.

[0018] According to an embodiment of the present invention, the expression vector is a eukaryotic cell expression vector.

[0019] According to an embodiment of the present invention, the CAR of the CAR-immune cell includes: an extracellular region, the extracellular region includes a single-chain antibody and a hinge region, the C-terminus of the single-chain antibody is connected to the N-terminus of the hinge region, and the single-chain antibody specifically recognizes the tumor antigen MSLN; a transmembrane region, the N-terminus of the transmembrane region is connected to the C-terminus of the hinge region of the extracellular region; and an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0020] According to an embodiment of the present invention, the antigen includes at least one selected from mesothelin (MSLN), HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, and BCMA; preferably, the antigen is MSLN.

[0021] According to an embodiment of the present invention, the single-chain antibody includes at least one selected from anti-mesothelin (MSLN) single-chain antibody, anti-HER2 single-chain antibody, anti-EGFR single-chain antibody, anti-GPC3 single-chain antibody, anti-MUC1 single-chain antibody, anti-CEA single-chain antibody, anti-CLDN18.2 single-chain antibody, anti-EpCAM single-chain antibody, anti-GD2 single-chain antibody, anti-PSCA single-chain antibody, anti-CD133 single-chain antibody, anti-CD19 single-chain antibody, anti-CD20 single-chain antibody, anti-CD22 single-chain antibody, anti-CD30 single-chain antibody, anti-CD33 single-chain antibody and anti-BCMA single-chain antibody; preferably, it is an anti-MSLN single-chain antibody.

[0022] According to an embodiment of the present invention, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO: 11. Thus, the single-chain antibody of the CAR-immune cell can target and bind to mesothelin, thereby targeting and killing mesothelin-positive tumor cells (such as pancreatic cancer, ovarian cancer, mesothelioma, etc.).

[0023] According to an embodiment of the present invention, the hinge region is selected from the hinge region of the CD8 molecule.

[0024] According to an embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO:12.

[0025] According to an embodiment of the present invention, the transmembrane region is selected from the transmembrane segment of the CD8 molecule.

[0026] According to an embodiment of the present invention, the transmembrane region has an amino acid sequence as shown in SEQ ID NO:13.

[0027] According to an embodiment of the present invention, the intracellular region includes a co-stimulatory domain and an intracellular signaling domain.

[0028] According to an embodiment of the present invention, the co-stimulatory domain is selected from the intracellular segment of the 41BB molecule.

[0029] According to an embodiment of the present invention, the costimulatory domain has an amino acid sequence as shown in SEQ ID NO:14.

[0030] According to an embodiment of the present invention, the intracellular signaling domain is selected from the intracellular segment of the CD3ζ molecule.

[0031] According to an embodiment of the present invention, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO:15.

[0032] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned CAR-immune cells. The pharmaceutical composition according to an embodiment of the present invention can be used to eliminate or reduce the immune escape mechanism of the tumor or to prevent and / or treat tumors or cancer.

[0033] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0034] In another aspect of the present invention, the present invention proposes a use of the aforementioned CAR-immune cell or the aforementioned pharmaceutical composition in the preparation of a drug, wherein the drug is used to eliminate or reduce the immune escape mechanism of the tumor.

[0035] In another aspect of the present invention, the present invention proposes a use of the aforementioned CAR-immune cell or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors or cancer.

[0036] In this article, the term "cancer" or "tumor" can be any unregulated cell growth. Exemplarily, it can be non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, etc.

[0037] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor, including but not limited to non-small cell lung cancer, papillary thyroid cancer, multiforme glioblastoma, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, leukemia, lymphoma.

[0038] In another aspect of the present invention, the present invention proposes a method for eliminating or reducing the immune escape mechanism of the tumor or preventing and / or treating a tumor or cancer. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the aforementioned CAR-immune cells to the subject. As previously described, according to the aforementioned CAR-immune cells (such as CAR-NK cells), the binding of NKG2A and ligand HLA-E of immune cells (such as NK cells) can be directly blocked or cancelled, eliminating or reducing the immune escape mechanism of the tumor, and effectively inhibiting the growth of tumor cells or killing tumor cells, thereby preventing and / or treating tumors or cancer. In addition, the pharmaceutical composition comprising the above-mentioned CAR-immune cells can effectively eliminate or reduce the immune escape mechanism of the tumor and inhibit the growth of tumor cells or kill tumor cells. Thus, the method can eliminate or reduce the immune escape mechanism of the tumor or be used to prevent and / or treat tumors or cancer.

[0039] The effective amount of the CAR-immune cells and pharmaceutical compositions of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment condition, several separate doses may be given per day, or the dose may be reduced proportionally.

[0040] The CAR-immune cells and pharmaceutical compositions of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions.

[0041] In another aspect of the present invention, the present invention proposes a method for reducing the expression of PD-1 and / or Tim-3 of CAR-immune cells. According to an embodiment of the present invention, the method comprises: introducing an sgRNA molecule as shown in any one of the nucleotide sequences of SEQ ID NO: 8 to 10 into a cell. Introducing sgRNA into cells according to the method of an embodiment of the present invention can not only reduce the expression of NKG2A in CAR-immune cells (especially CAR-NK cells), but also further reduce the expression of PD-1 and / or Tim-3 in CAR-immune cells. For example, in scientific research, it is used to reduce the expression of multiple inhibitory receptors such as NKG2A, PD-1 and / or Tim-3 in cells, and obtain cells that meet the target for subsequent research.

[0042] According to an embodiment of the present invention, the immune cells of the CAR-immune cells include at least one of NK cells, T cells, NKT cells and γδT cells. Exemplarily, the immune cells are NK cells or T cells.

[0043] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0045] Figure 1 This is the CRISPR NKG2A KO plasmid map in Example 1 of the present invention;

[0046] Figure 2 is the editing efficiency of different sgRNA sequences in Example 1 of the present invention;

[0047] Figure 3 NK-92 and MSLN-CAR-NKG2A in Example 2 of the present invention low The expression level of NKG2A in NK-92 cells;

[0048] Figure 4 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 2 of the present invention low Expression levels of PD-1 and Tim-3 in NK-92 cells;

[0049] Figure 5 The expression of mesothelin (MSLN) on the cell surface of A1847, Hey, HO8910 and A2780 in Example 2 of the present invention;

[0050] Figure 6 The expression of HLA-E on the surface of A1847, HO8910 and A2780 cells in Example 2 of the present invention;

[0051] Figure 7 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 2 of the present invention low The killing efficiency of NK-92 cells on different target cells;

[0052] Figure 8 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 2 of the present invention lowThe secretion levels of granzyme B and perforin in NK-92 cells;

[0053] Fig. 9 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 2 of the present invention low IFN-γ secretion capacity of NK-92 cells;

[0054] Fig.10 This is a flow chart of subcutaneous tumor bearing of ovarian cancer A1847 cells in Example 3 of the present invention;

[0055] Fig.11 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 3 of the present invention low Tumor volume of mice in the NK-92 treatment group;

[0056] Fig.12 NK-92, α-MSLN-CAR-NK-92 and α-MSLN-CAR-NKG2A in Example 3 of the present invention low Survival of mice in the NK-92 treatment group. DETAILED DESCRIPTION

[0057] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0058] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0059] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0060] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0061] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0062] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition that interacts in a harmful manner, their use is also within the scope of the present invention.

[0063] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0064] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0065] Example 1: NKG2A low Preparation of CAR-NK cells

[0066] 1. Construction of CAR expression plasmid and CRISPR NKG2A KO plasmid

[0067] 1.1 Construction of CRISPR NKG2A KO plasmid

[0068] The method for knocking down NKG2A designed by the present invention first uses a gene mutation detection method to screen the sgRNA sequence that effectively silences NKG2A, and then constructs a CRISPR / Cas 9 vector (LentiCRISPR-NKG2A-KO) that silences NKG2A. The CRISPR NKG2A KO plasmid map is shown in Figure 1 .

[0069] Among them, the nucleotide sequence of sgRNA1 is shown in SEQ ID NO: 8 in the sequence listing; the nucleotide sequence of sgRNA2 is shown in SEQ ID NO: 9 in the sequence listing; and the nucleotide sequence of sgRNA3 is shown in SEQ ID NO: 10 in the sequence listing.

[0070] GAAGCTCATTGTTGGGATCC (SEQ ID NO: 8);

[0071] AACAACTATCGTTACCACAG(SEQ ID NO:9);

[0072] TGAACAGGAAATAACCTATG (SEQ ID NO: 10).

[0073] The sgRNA targeting NKG2A was designed through the synthego website, the sgRNA was annealed and phosphorylated, the Lent CRISPR V2 vector was digested with Esp3I restriction endonuclease, and then the phosphorylated NKG2A-sgRNA was connected to the linearized vector. After transformation and plating, single clone colonies were picked for shaking experiment, and the bacterial solution was sent to a sequencing company for sequencing, which proved that the sequence of the inserted gene fragment was correct. The results showed that the LentiCRISPR-NKG2A-KO vector was successfully constructed.

[0074] Screening of NKG2A-sgRNA sequences: In order to screen out the sgRNA sequence with the highest efficiency in silencing NKG2A, the LentiCRISPR-NKG2A-KO plasmid was transfected into 293T cells, the medium was changed after 12 hours, the cells were collected after 48 hours, and the genome was extracted. Conventional PCR technology was used to amplify the specific region containing the sgRNA targeting site. After the PCR product was annealed, a protrusion was generated at the mutation site because the mutated single strand could not be completely complementary to the wild-type strand that did not mutate. This protrusion was then recognized and cut by T7E1, forming two broken short strands. Use Imagine J to perform grayscale analysis on the bands obtained from the agarose gel electrophoresis experiment. The results are shown in Figure 2 The results showed that sgRNA2 had a higher editing efficiency than sgRNA1 and sgRNA3, and the lentiviral vector constructed with the sgRNA2 sequence was used for subsequent experiments.

[0075] 1.2 Construction of anti-MSLN-CAR vector:

[0076] The CAR vector (anti-MSLN-CAR) sequence targeting mesothelin designed by the present invention comprises a CSF2R signal peptide, an extracellular segment targeting and recognizing MSLN (anti-MSLN scFv), a CD8 Hinge region, a CD8a transmembrane segment, a 4-1BB intracellular co-stimulatory signal domain, and an intracellular signal transduction molecule CD3ζ.

[0077] The full-length sequence of the Anti-MSLN-CAR gene is shown in SEQ ID NO: 1 in the sequence listing;

[0078]

[0079] The nucleotide sequence of the CSF2R signal peptide is shown in SEQ ID NO: 2 in the sequence listing;

[0080] ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG (SEQ ID NO: 2).

[0081] The nucleotide sequence of the anti-MSLN single-chain antibody is shown in SEQ ID NO: 3 in the sequence listing;

[0082] ATCCCCGACATCCAGATGGCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGCAGGTATCCTGCAGAGCATCTGGCTATAGTATCAATACTTACTATATGCAGTGGGTGCGGCAGGCCCCTGGAGCAGGCCTTGAGTGGATGGGCGTTATCAACCCCAGTGGTGTCACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACTTTGACCAACGACACGTCCACAAACACAGTCTACATGCAGTTGAACAGTCTGACATCTGCCGACACGGCCGTCTACTACTGTGCGAGATGGGCCTTATGGGGGGACTTCGGTATGGACGTCTGGGGCAAGGGAACCCTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACCATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGT(SEQ ID NO:3).

[0083] The amino acid sequence of the anti-MSLN single-chain antibody encoded by SEQ ID NO: 3 (SEQ ID NO: 11) is as follows:

[0084] IPDIQMAQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKR(SEQ ID NO: 11);

[0085] The nucleotide sequence of the CD8 Hinge region is shown in SEQ ID NO: 4 in the sequence listing;

[0086] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 4).

[0087] The amino acid sequence of the CD8 Hinge region encoded by SEQ ID NO: 4 (SEQ ID NO: 12) is as follows:

[0088] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 12);

[0089] The nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO: 5 in the sequence listing;

[0090] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 5).

[0091] The amino acid sequence of the CD8 transmembrane region encoded by SEQ ID NO: 5 (SEQ ID NO: 13) is as follows:

[0092] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO: 13);

[0093] The nucleotide sequence of the 4-1BB costimulatory region is shown in SEQ ID NO: 6 in the sequence listing;

[0094] AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 6).

[0095] The amino acid sequence of the 4-1BB co-stimulatory region encoded by SEQ ID NO: 6 (SEQ ID NO: 14) is as follows:

[0096] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14);

[0097] The nucleotide sequence of the intracellular region of CD3ζ is shown in SEQ ID NO: 7 in the sequence listing;

[0098] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO: 7).

[0099] The amino acid sequence of the CD3ζ intracellular region encoded by SEQ ID NO: 7 (SEQ ID NO: 15) is as follows:

[0100] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGSVQPPRTPTTPFTCRPCPL (SEQ ID NO: 15);

[0101] The inventors used the pSBbi-MSLN CAR-GP plasmid as a template to amplify the anti-MSLN-CAR gene fragment, and inserted the anti-MSLN-CAR fragment into the EcoRI and AsiSI restriction sites of the lentiviral vector pCDH-CMV-MCS-EF1a-RFP to construct the pCDH-CMV-anti-MSLN-CAR-EF1a-RFP vector.

[0102] 2. Lentivirus packaging and virus concentration

[0103] 2.1 Preparation of LentiCRISPR-NKG2A-KO lentivirus and concentrated viral fluid: 5×10 293T cells in logarithmic growth phase were taken. 6 Inoculate in a 10 cm culture dish, add 10 mL of DMEM medium, and culture overnight in an incubator at 37°C and 5% CO2. When the 293T cell density reaches 80%, replace with 10 mL of fresh DMEM medium and continue to culture in an incubator at 37°C and 5% CO2. Prepare lentiviral packaging system: Add 6 μg of psPAX2 plasmid, 3 μg of pMD2.G plasmid and 6 μg of LentiCRISPR-NKG2A-KO plasmid to 250 μL of serum-free DMEM medium, mix well, and prepare DNA mixture; add 15 μL Add to 235 μL of serum-free DMEM medium and mix well. Add the mixture to the DNA mixture at one time, let it stand to mix, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. Change the solution after 24 hours. Put the culture dish back into the incubator at 37°C and 5% CO2. Collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove the cell debris, and filter the supernatant with a 0.45μm filter into a new 50ml centrifuge tube. Add 5×PEG8000 solution, mix evenly by turning the centrifuge tube upside down, and place it in a 4°C refrigerator overnight. Then centrifuge at 4°C and 4000×g for 20 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, divide it into EP tubes, obtain the virus concentrate, and store it in a -80°C refrigerator.

[0104] 2.2 Preparation of lentivirus for packaging pCDH-CMV-anti-MSLN-CAR-EF1a-RFP and its concentrated virus solution: 5×10 293T cells in the logarithmic growth phase were taken. 6 Inoculate in a 10 cm culture dish, add 10 mL of DMEM medium, and culture overnight in an incubator at 37°C and 5% CO2. When the 293T cell density reaches 80%, replace with 10 mL of fresh DMEM medium and continue to culture in an incubator at 37°C and 5% CO2. Prepare lentiviral packaging system: Add 6 μg of psPAX2 plasmid, 3 μg of pMD2.G plasmid and 6 μg of pCDH-CMV-anti-MSLN-CAR-EF1a-RFP plasmid to 250 μL of serum-free DMEM medium, mix well, and prepare a DNA mixture; add 15 μL Add to 235 μL of serum-free DMEM medium and mix well. Add the mixture to the DNA mixture at one time, let it stand to mix, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. Change the liquid after 24 hours. Put the culture dish back into the incubator at 37°C and 5% CO2. Collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove the cell debris, and filter the supernatant with a 0.45μm filter into a new 50ml centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by turning the centrifuge tube upside down, and place it in a 4°C refrigerator overnight. Then centrifuge at 4°C and 4000×g for 20 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, divide it into EP tubes, obtain the virus concentrate, and store it in a -80°C refrigerator.

[0105] 3. Lentiviral titer detection

[0106] Take 293T cells in the logarithmic growth phase and adjust the concentration of 293T cells to 1×10 5 / mL. Take a 24-well plate and add 1mL of cell suspension (1×10 5 / well), set up 3 cell gradients. Place in an incubator at 37°C and 5% CO2 for overnight culture. First dilute the concentrated virus solution obtained in step 2: take a 1.5mL EP tube, draw 60μL of virus concentrate into the EP tube, dilute with 540μL DMEM culture medium, and mix well. Replace the medium with fresh culture medium, draw 5μL and 50μL of virus solution into the corresponding wells, mark them, and then put the culture plate back into the incubator at 37°C and 5% CO2. After 72h, harvest the cells, detect the RFP expression rate of 293T cells by flow cytometry, and convert the virus titer according to the formula.

[0107] Titer (TU / ml) = 100,000 (target cells) × (% of GFP-positive cells / 100) / volume of supernatant (in mL).

[0108] 4. Lentivirus infection of human NK cells

[0109] 4.1 Package pCDH-CMV-anti-MSLN-CAR-EF1a-RFP lentivirus to infect human NK-92 cells: Take NK-92 cells in the logarithmic growth phase, add 2 mL of α-MEM medium to resuspend the cells, and adjust the cell density of NK-92 cells to 5×10 5 5×10 5 NK-92 cells, 1 μL protamine (final concentration 8 μg / mL) and 1 mL of the virus concentrate of the packaged pCDH-CMV-anti-MSLN-CAR-EF1a-RFP lentivirus obtained in step 2.2. Place in an incubator at 37°C and 5% CO2 for culture. After 24 hours of culture, observe the cell state, change the medium, and obtain infected cells. Transfer the infected cells to an EP tube, centrifuge at 100×g for 5 minutes, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the infected cells to a cell culture flask, add 10 mL of fresh α-MEM medium and IL-2 (final concentration of 200 IU / mL) to continue culture. After the infected cells are amplified, transfer the infected cells to a flow tube, add 3 mL of 1×PBS to resuspend the cells, centrifuge at 100×g for 5 minutes, discard the supernatant, flick the cell pellet, and repeat once. Detect the expression rate of NKG2A by flow cytometry. Continue to expand the culture and adjust the state of NK-92 cells after infection. RFP-positive NK-92 cells were sorted using a sorting flow cytometer to obtain anti-MSLN-CAR-NK-92 cells.

[0110] 4.2 LentiCRISPR-NKG2A-KO packaged lentivirus infected anti-MSLN-CAR-NK-92 cells: Take anti-MSLN-CAR-NK-92 cells in the logarithmic growth phase, add 2 mL of α-MEM medium to resuspend the cells, and adjust the cell density of NK-92 cells to 5×10 5 5×10 5anti-MSLN-CAR-NK-92 cells, 1 μL protamine (final concentration 8 μg / mL) and 1 mL of the virus concentrate of the packaged LentiCRISPR-NKG2A-KO lentivirus obtained in step 2.1. Place in an incubator at 37°C and 5% CO2 for 24 hours, observe the cell state, change the medium, and obtain infected cells. Transfer the infected cells to an EP tube, centrifuge at 100×g for 5 minutes, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the infected cells to a cell culture flask, add 10 mL of fresh α-MEM medium and IL-2 (final concentration of 200 IU / mL) to continue culturing. After the infected cells are amplified, transfer the infected cells to a flow tube, add 3 mL of 1×PBS to resuspend the cells, centrifuge at 100×g for 5 minutes, discard the supernatant, flick the cell pellet, and repeat once. The expression rate of NKG2A is detected by flow cytometry. Continue to expand the culture and adjust the state of anti-MSLN-CAR-NK-92 cells after infection. After flow cytometry sorting, the purity of anti-MSLN-CAR-NK-92 cells after infection was close to 99%, and NKG2A low anti-MALN CAR-NK-92 cells, referred to as CAR-NK cells.

[0111] Example 2: Expression level and biological function identification of NKG2A in CAR-NK cells

[0112] 1. Expression level of NKG2A and other inhibitory receptors in CAR-NK-92 cells

[0113] Flow cytometry was used to compare the expression levels of NKG2A in anti-MSLN CAR-NK-92 cells before and after NKG2A knockdown. It was found that NK-92 cells highly expressed NKG2A, with a positive rate close to 100%. After gene knockout using CRISPR / Cas9 technology, 80.4% of CAR-NK cells became CAR-NK cells with low NKG2A expression. For details, see Figure 3 .

[0114] At the same time, we observed whether silencing NKG2A would also affect the expression of other exhaustion-related molecules. We isolated NK-92 cells, CAR-NK-92 cells, and NKG2A low Anti-MALN CAR-NK-92 cells were co-incubated with ovarian cancer cell A1847 at a ratio of 1:1 for 72 hours, and NK cells were collected. The expression of inhibitory receptors such as PD-1, Tim-3, and TIGIT on the surface of CAR-NK-92 cells was detected by flow cytometry. The results showed that compared with CAR-NK-92 cells and NK-92 cells, NKG2A lowThe expression of PD-1 and Tim-3 on the surface of anti-MALN CAR-NK-92 cells was significantly reduced, and there was no significant difference in the expression of TIGIT. For details, see Figure 4 Therefore, the results further indicate that silencing the expression of NKG2A on the surface of CAR-NK cells also weakens the expression of other inhibitory receptors such as PD-1 and Tim-3, promoting the balance between CAR-NK cell activation receptors and inhibitory receptors to tilt toward activation analysis, thereby improving activation and function, and enhancing the resistance of CAR-NK cells to tumor microenvironment-induced exhaustion.

[0115] 2. Expression of mesothelin and NKG2A ligand HLA-E in tumor cells

[0116] The expression of mesothelin (MSLN) on the cell surface of human ovarian cancer cell lines A1847, HO8910 and A2780 was detected by flow cytometry. The results are shown in Figure 5 The results showed that A1847 cells expressed high levels of mesothelin (positive rate was 97.2%), Hey cells expressed mesothelin at a moderate level (positive rate was 32.8%), while HO8910 and A2780 cells expressed mesothelin at very low levels, with positive rates of 1.83% and 0.56%, respectively.

[0117] Studies have shown that NKG2A ligand HLA-E is highly expressed on the surface of ovarian cancer tissues and various ovarian cancer cells. The inventors detected the expression of HLA-E on the surface of human ovarian cancer cell lines A1847, HO8910, and A2780 by flow cytometry. The results are shown in Figure 6 The results showed that HO8910 cells expressed high levels of HLA-E (positive rate 95.5%), A1847 cells expressed at a medium level (positive rate 36.0%), and A2780 cells expressed a low level of HLA-E (positive rate 17.4%). Therefore, the inventors selected HO8910 and A1847 cells as HLA-E positive target cells and A2780 cells as HLA-E negative target cells.

[0118] 3. In vitro killing of NK-92 cells after NKG2A knockdown

[0119] NK-92, anti-MSLN CAR-NK-92, NKG2A low Anti-NSLN CAR-NK-92 was used as effector cells, and ovarian cancer cell lines A1847, HO8910, and A2780 were used as target cells. The effector-target ratio was set to 10:1, 5:1, and 2.5:1. The effector cells and target cells were co-incubated for 5 h. The LDH release method was used to detect the killing efficiency of effector cells on target cells. The results are shown in Figure 7The results showed that when the effector-target ratio was 10:1, NKG2A low The killing efficiency of anti-NSLN CAR-NK-92 cells on A1847 cells that moderately expressed the NKG2A ligand HLA-E was 63.77±4.39%, which was significantly higher than that of anti-MSLN CAR-NK-92 group (49.77±2.78%) and NK-92 group (37.60±1.50%). low The killing efficiency of anti-NSLN CAR-NK-92 on HO8910 cells with high expression of HLA-E was 52.10±4.52%, which was significantly higher than that of anti-MSLN CAR-NK-92 group (32.70±2.0%) and NK-92 group (30.20±0.95%); while there was no significant difference in the killing efficiency of the three groups of effector cells on A2780 cells with low expression of HLA-E. Therefore, the above results indicate that knocking down NKG2A can significantly improve the killing ability of CAR-NK cells against HLA-E positive tumor cells.

[0120] In addition, the inventors also tested the degranulation of NK cells with or without NKG2A knockdown. Various effector cells were co-incubated with ovarian cancer cells at an effector-target ratio of 10:1 for 5 h, and the cell culture supernatant was collected. The secretion levels of granzyme B and perforin in the supernatant were detected by ELISA. The results are shown in Figure 8 The results showed that after co-incubation with A1847 cells with moderate HLA-E expression and HO8910 cells with high HLA-E expression, NKG2A low The levels of granzyme B and perforin secreted by anti-NSLN CAR-NK-92 cells were significantly higher than those in the CAR-NK-92 group and the NK-92 group. However, after co-incubation with A2780 cells that lowly express HLA-E, there was no significant difference in the levels of granzyme B and perforin secreted by effector cells in each group. It was further verified that knocking down NKG2A can significantly increase the degranulation level and killing function of CAR-NK cells against HLA-E positive tumor cells.

[0121] 4. IFN-γ secretion level of NK-92 cells after NKG2A knockdown

[0122] The ELISA technique was used to detect the changes in the IFN-γ secretion capacity of NK-92 cells after NKG2A knockdown. lowAnti-NSLN CAR-NK-92) were co-incubated with ovarian cancer cells for 5 h, with an effector-target ratio of 10:1. The cell culture supernatant was collected and the level of IFN-γ in the supernatant was detected by ELISA. The results are shown in Fig. 9 The results showed that after co-incubation with HO8910 cells that highly expressed HLA-E, NKG2A low The level of IFN-γ expressed by anti-NSLN CAR-NK-92 cells was significantly higher than that of anti-MSLN CAR-NK92 and NK-92 groups; after co-incubation with A1847 cells with moderate expression of HLA-E, NKG2A low The level of IFN-γ expressed by anti-NSLN CAR-NK-92 cells was significantly higher than that of the NK-92 group. However, after co-incubation with A2780 cells that lowly express HLA-E, the level of IFN-γ secretion in each group did not change significantly. Therefore, the above experiments show that knocking down NKG2A can significantly improve the IFN-γ secretion ability of CAR-NK cells when they come into contact with HLA-E positive tumor cells.

[0123] Example 3: NKG2A low Anti-tumor ability of CAR-NK cells in vivo and survival of tumor-bearing mice

[0124] Ovarian cancer A1847 cells, which are both mesothelin and HLA-E positive, were subcutaneously implanted to establish an ovarian cancer xenograft model to observe the therapeutic effect of NKG2A knockdown CAR-NK cells on ovarian cancer. Four-week-old female nude mice were selected and subcutaneously implanted with tumors in the axilla. The tumor-bearing dose was 2×10 per mouse. 6 A1847 cells, one week later, when the tumor volume reached 100mm 3 Treatment begins around 2 hours after the procedure. Fig.10 First, the mice were randomly divided into a control group, a NK-92 cell treatment group, an anti-MSLN CAR-NK-92 cell treatment group, and a NKG2A low The anti-MSLN CAR-NK-92 cell treatment group. The mice in the treatment group were injected with 1×10 effector cells via tail vein. 7 The control group was injected with an equal volume of 1× PBS solution once every week, and IL-2 (5×10 4 IU / mouse). The tumor volume of mice was measured every three days, and the tumor growth curve and mouse survival curve were drawn. Fig.11 and Fig.12 .

[0125] The results showed that compared with the tumor-bearing control group, all treatment groups significantly inhibited tumor growth, and NKG2A lowThe anti-MSLN CAR-NK-92 cell treatment group had the best therapeutic effect, and the tumor volume was significantly smaller than that of the CAR-NK-92 group and the NK-92 group ( Fig.11 ). Moreover, NKG2A low Compared with the CAR-NK-92 group and the NK-92 group, the anti-MSLN CAR-NK-92 cell treatment group significantly prolonged the survival period of mice and improved the survival rate of mice ( Fig.12 ). Therefore, the above experiments show that CAR-NK cells with NKG2A knockdown have tumoricidal effects in vivo, can resist the inhibitory effects of the tumor microenvironment, resist functional exhaustion, and exert a strong anti-tumor effect.

[0126] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0127] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A CAR-immune cell for treating ovarian cancer with moderate or high expression of HLA-E, characterized in that: The NKG2A gene of the CAR-immune cell is silenced, and the NKG2A gene silencing is achieved by knocking out the NKG2A gene of the CAR-immune cell; The knockout is achieved by the CRISPR / Cas9 system; The sgRNA of the CRISPR / Cas9 system has a nucleotide sequence as shown in SEQ ID NO: 9; The CAR of the CAR-immune cell includes: An extracellular region, the extracellular region comprising a single-chain antibody and a hinge region, the C-terminus of the single-chain antibody is connected to the N-terminus of the hinge region, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO: 11, and the hinge region has an amino acid sequence as shown in SEQ ID NO: 12; a transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the hinge region of the extracellular region, and the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 13; an intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, the intracellular region comprises a costimulatory domain and an intracellular signaling domain, the costimulatory domain has an amino acid sequence as shown in SEQ ID NO: 14, and the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO: 15; Wherein, the immune cells of the CAR-immune cells are NK cells.

2. The CAR-immune cell according to claim 1, characterized in that The knockout is performed in the following manner: The expression vector carrying the sgRNA and the nucleic acid encoding the Cas9 molecule is introduced into the CAR-immune cells to be modified for culture treatment.

3. The CAR-immune cell according to claim 2, characterized in that: The culture treatment time is 24 to 72 hours.

4. The CAR-immune cell according to claim 2, characterized in that: The expression vector is a eukaryotic cell expression vector.

5. A pharmaceutical composition, characterized in that include: The CAR-immune cell according to any one of claims 1 to 4; as well as Optionally, a pharmaceutically acceptable carrier or excipient.

6. Use of the CAR-immune cell according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a drug, The medicine is used for preventing and / or treating ovarian cancer, wherein the ovarian cancer is ovarian cancer with moderate HLA-E expression and ovarian cancer with high HLA-E expression.

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