Engineered CAR-NK cell as well as preparation method and application thereof

Abstract

The invention relates to an engineered CAR-NK cell as well as a preparation method and application thereof. The engineered CAR-NK cell expresses a CAR molecule and a membrane-bound IL-15, the membrane-bound IL-15 comprises a signal peptide and an IL-15 which are connected in sequence, and the signal peptide comprises an IL-15 LSP signal peptide. A novel engineered CAR-NK cell is designed, CAR molecules and membrane combined IL-15 are simultaneously expressed in the NK cell, and a specific IL-15 signal peptide is designed and used, so that the cell amplification in the preparation process can be promoted, the viability of the CAR-NK cell and the tumor cell killing ability can be improved, and the CAR-NK cell has a wide application prospect in the fields of tumor treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical bioengineering, and relates to an engineered CAR-NK cell and its preparation method and application, in particular to a CD56-positive CD3-negative CAR-NK cell expressing membrane-bound IL-15 and its preparation method and application. Background Art

[0002] Natural Killer (NK) cells are phenotypically characterized by the expression of CD56 and the non-expression of CD3. NK cells account for about 5% - 15% of the peripheral blood circulating lymphocytes and belong to non-specific immune cells. NK cells have multiple biological functions, and these functions are regulated by the activation and inhibition of precise and complex activating and inhibitory receptors. NK cells can directly recognize abnormal cells without prior sensitization or HLA typing, and then play a killing role through pathways such as inducing apoptosis of target cells by releasing cytotoxic granules such as perforin and granzyme, with a rapid onset and showing strong activity in lysing abnormal cells. When the receptors of NK cells bind to the corresponding ligands, NK cells are activated, and the activated NK cells can synthesize and secrete various important cytokines such as IFN-γ and TNF-α, playing an immunomodulatory role on a variety of immune cells, thus becoming the core cells of innate immunity. NK cells are the first line of defense against tumors and pathogen infections in the human body. Currently, NK cell immunotherapy is developing into a new tumor treatment method with broad prospects. In order to enhance the anti-tumor activity and specificity of NK cells, chimeric antigen receptor (CAR) is applied to genetically engineer NK cells, and the development of CAR-NK cells is a newly emerging tumor immunotherapy method in recent years.

[0003] At present, CAR-NK cell therapy is just starting out, with only a few officially announced clinical trials. Anti-CD19 CAR-NK cells are mainly used to treat CD19-positive lymphoma and blood cancer because CD19 is a well-studied and comprehensively tested target with relatively good safety and efficacy. However, in current research, it has been found that CAR-NK cells have a relatively short survival period in the body, the persistence of the tumor-killing effect of CAR-NK is not as good as that of CAR-T, and at the same time, the proliferation of CAR-NK after contacting cancer target cells is also not as good as that of CAR-T. Due to these limitations, special functional genes need to be added during the preparation of CAR-NK cells to enable CAR-NK cells to simultaneously express cytokines that can maintain the in-vivo survival and proliferation of NK cells. For example, CN116410336A discloses the construction and application of CAR-NK cells that can highly express chimeric antigen receptors and secrete the functional stimulatory factor IL-15, providing a CAR molecule that can highly express and an autocrine functional stimulatory molecule IL-15-based CAR-CD19-IPS-iNK cells differentiated from induced pluripotent stem cells. However, IL-15 expressed in a secreted form will be diluted in the human body, resulting in a decrease in the effective concentration; on the other hand, as an inflammatory factor, IL-15 may have side effects on normal tissues, etc., and the secreted IL-15 may still stimulate CD8-positive T cells around NK cells. It can be seen that how to achieve attenuation of toxicity and enhancement of efficacy in this regard is a problem that needs to be solved in the development of CAR-NK cells.

[0004] In summary, designing CAR-NK cells that can kill tumors with long-term and high efficiency and have high safety is of great significance for the field of tumor treatment. Summary of the Invention

[0005] In view of the deficiencies of the prior art and the actual needs, the present invention provides an engineered CAR-NK cell, its preparation method and application, and designs an engineered CAR-NK cell with high survival ability and tumor cell killing ability, providing new ideas for tumor treatment.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides an engineered CAR-NK cell, which expresses a CAR molecule and membrane-bound IL-15 (interleukin 15), and the membrane-bound IL-15 includes a signal peptide and IL-15 connected in sequence, and the signal peptide includes the IL-15LSP signal peptide.

[0008] In the present invention, novel engineered CAR-NK cells are designed to simultaneously express a CAR molecule and a membrane-bound mature IL-15 protein in the CAR-NK cells, and a specific signal peptide is used in the membrane-bound IL-15 to further enhance the biological function of the CAR-NK cells modified with the chimeric antigen receptor CAR, significantly improving their survival ability and tumor cell killing ability. In addition, they have high proliferation ability, which is conducive to large-scale preparation.

[0009] Preferably, the membrane-bound IL-15 further includes a transmembrane domain.

[0010] Preferably, the membrane-bound IL-15 includes an IL-15LSP signal peptide, IL-15, and a transmembrane domain connected in sequence.

[0011] Preferably, the transmembrane domain includes the hinge transmembrane region of CD8.

[0012] Preferably, the nucleic acid sequence of the membrane-bound IL-15 includes the sequence shown in SEQ ID NO.1.

[0013] Preferably, the amino acid sequence of the membrane-bound IL-15 includes the sequence shown in SEQ ID NO.2.

[0014] Preferably, the raw material cells of the engineered CAR-NK cells are derived from at least one of peripheral blood, cord blood, placental tissue, or induced pluripotent stem cells.

[0015] Preferably, the CAR molecule includes a chimeric antigen receptor and / or a chimeric switch receptor.

[0016] In the present invention, the simultaneous expression of a CAR molecule and a membrane-bound IL-15 in CAR-NK cells realizes the enhancement of the biological function of CAR-NK cells. It can be understood that in the art, CAR molecules are theoretically applicable to the present invention without special limitations. For example, NKG2D CAR, CD19CAR, and BCMACAR, etc. The amino acid sequence of the NKG2D CAR includes the sequence shown in SEQ ID NO.3, the amino acid sequence of the CD19CAR includes the sequence shown in SEQ ID NO.4, and the amino acid sequence of the BCMACAR includes the sequence shown in SEQ ID NO.5.

[0017] SEQ ID NO.3:

[0018] MLLLVTSLLLCELPHPAFLLIPGAHAFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVASNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0019] SEQ ID NO.4:

[0020] MLLLVTSLLLCELPHPAFLLIPGAHADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0021] SEQ ID NO.5:

[0022] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSASNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0023] In a second aspect, the present invention provides a method for preparing the engineered CAR-NK cells described in the first aspect, the preparation method comprising:

[0024] Introducing nucleic acid molecules encoding the CAR molecule and membrane-bound IL-15 into NK cells and culturing them.

[0025] Preferably, after genetic integration modification, the NK cells co-express the CAR molecule and membrane-bound IL-15.

[0026] Preferably, the introducing method includes viral or non-viral gene transfection.

[0027] Preferably, the culturing includes co-culturing with engineered K562 artificial antigen-presenting cells.

[0028] Preferably, after genetic integration modification, the engineered K562 artificial antigen-presenting cells co-express CD137L, IL-21, IL-15 and the antigen corresponding to the CAR molecule.

[0029] In the present invention, CAR-NK cells have enhanced cell expansion ability during in vitro preparation and can be massively expanded after co-culture with engineered K562 artificial antigen-presenting cells in vitro.

[0030] In a third aspect, the present invention provides the use of the engineered CAR-NK cells described in the first aspect in the preparation of a product for eliminating cells expressing the corresponding CAR target.

[0031] Preferably, the cells expressing the corresponding CAR target include tumor cells.

[0032] Preferably, the tumor cells include at least one of blood cancer cells, ovarian cancer cells, triple-negative breast cancer cells, pancreatic cancer cells, colorectal cancer cells, or bladder cancer cells, etc.

[0033] In a fourth aspect, the present invention provides a pharmaceutical composition, which includes the engineered CAR-NK cells described in the first aspect.

[0034] In a fifth aspect, the present invention provides a method for eliminating cells expressing the CAR target in a subject, the method including administering to the subject a pharmaceutically effective amount of the engineered CAR-NK cells described in the first aspect.

[0035] Preferably, the way of administering the drug to the subject is by intravenous, intra-arterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intralesional, percutaneous, or subcutaneous local injection or perfusion of the engineered CAR-NK cells described in the first aspect.

[0036] In the present invention, methods for eliminating cells expressing the CAR target (such as tumor cells, etc.) can be further developed based on the engineered CAR-NK cells and can be applied to the fields of disease treatment purposes or non-treatment purposes.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The present invention designs novel engineered CAR-NK cells, which simultaneously express the CAR molecule and membrane-bound IL-15 in NK cells, and designs and uses a specific signal peptide to further improve the cell expansion ability, survival ability, and tumor cell killing ability during the preparation process, and has broad application prospects in the fields such as tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the genomic organization and gene expression of interleukin 15.

[0040] Figure 2Schematic diagram of the plasmid for preparing CAR-NK expressing membrane-bound IL-15. GM CSF SP: GM CSF signal peptide; IL-15LSP: IL-15 long signal peptide; mIL-15: IL-15 mature protein; CD8H-TM: CD8 hinge / transmembrane domain for anchoring the IL-15 molecule to the cell membrane.

[0041] Figure 3 Schematic diagram of the process for preparing CAR-NK cells.

[0042] Figure 4A Expression result diagram of the NKG2D ligand of the K562 artificial antigen-presenting cell line for amplifying NKG2D CAR-NK cells.

[0043] Figure 4B Expression result diagram of CD37L, IL-21, and IL-15 of the K562 artificial antigen-presenting cell line for amplifying NKG2D CAR-NK cells.

[0044] Figure 5A Result diagram for characterizing the K562 artificial antigen-presenting cell line for amplifying CD19-specific CAR-NK cells.

[0045] Figure 5B Result diagram for characterizing the K562 artificial antigen-presenting cell line for amplifying BCMA-specific CAR-NK cells.

[0046] Figure 6A Result diagram for flow cytometry detection of three different CAR-NK cells. The three CAR-NK cells use three different CAR plasmids: NKG2D CAR, mIL-15 / NKG2D CAR with membrane-bound IL-15 guided by the conventional GM-CSF signal peptide, and LSPmIL-15 / NKG2D CAR with membrane-bound IL-15 guided by the LSP signal peptide.

[0047] Figure 6B Result diagram for the expression level of NKG2D CAR on the surface of three CAR-NK cells.

[0048] Figure 7A Result diagram for the expression level of NKG2D CAR on LSPmIL-15 / NKG2D CAR-NK cells under the stimulation of the NKG2D K562 artificial antigen-presenting cell line.

[0049] Figure 7B Result diagram for the number of LSPmIL-15 / NKG2D CAR-NK cells under the stimulation of the NKG2D K562 artificial antigen-presenting cell line.

[0050] Figure 8Figure showing the co-expression results of membrane-bound IL-5 and NKGD CAR on the surface of NKG2D CAR-NK cells. Mock NK cells (simulated electroporated NK cells) were used as a control.

[0051] Figure 9 Figure showing the comparison results of the expression of NK cell surface receptors on NKG2D CAR-NK cells. Mock NK cells (simulated electroporated NK cells) were used as a control. The data shown are the mean ± SD from three different donors.

[0052] Figure 10 Figure showing the comparison results of the expression of immunosuppressive factors on NKG2D CAR-NK cells. Mock NK cells (simulated electroporated NK cells) were used as a control. The data shown are the mean ± SD from three different donors.

[0053] Figure 11A Schematic diagram of the preparation process of CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR using human umbilical cord blood cells as raw materials.

[0054] Figure 11B Figure showing the results of the expression level of NKG2D CAR on NK cells stimulated by the NKG2D K562 artificial antigen-presenting cell line.

[0055] Figure 11C Figure showing the results of the number of CAR-NK cells stimulated by the NKG2D K562 artificial antigen-presenting cell line.

[0056] Figure 12A Figure showing the number of early CD34 cells in the preparation of human iPSC cells + cells.

[0057] Figure 12B Figure showing the colony formation results of iPSC cells in the preparation of human iPSC cells.

[0058] Figure 12C Figure showing the expression of pluripotency markers in the preparation of human iPSC cells, immunohistochemistry results.

[0059] Figure 12D Figure showing the expression of pluripotency markers in the preparation of human iPSC cells, flow cytometry detection results.

[0060] Figure 12E Figure showing the expression of pluripotency markers in the preparation of human iPSC cells, RT-PCR results.

[0061] Figure 12F Figure showing the karyotype analysis results of human iPSC cells in the preparation.

[0062] Figure 13ANKG2D CAR-expressing human iPSC cell single-cell clone NKG2D CAR expression result diagram.

[0063] Figure 13B IL-15 expression result diagram of NKG2D CAR-expressing human iPSC cell single-cell clone.

[0064] Figure 14A Three-stage differentiation flow chart of NKG2D CAR-expressing human iPSC cells differentiating into CAR-NK cells.

[0065] Figure 14B CD34 + and CD44 + expression result diagram after the first stage of differentiation of NKG2D CAR-expressing human iPSC cells differentiating into CAR-NK cells.

[0066] Figure 14C Result diagram of membrane-bound IL-15 promoting cell expansion during the preparation of NKG2D CAR-NK using human iPSC cells as raw materials.

[0067] Figure 14D Result diagram of the effect of killing cancer cells in vitro.

[0068] Figure 14E Result diagram of NK cell receptor expression on iPSC cell-derived CAR-NK cells.

[0069] Figure 15A CD56 + CD3 - positive rate result diagram of co-expressing LSPmIL-15 and CD19 CAR-NK cells prepared using human peripheral blood cells as raw materials.

[0070] Figure 15B CD56 + CD3 - positive rate result diagram of co-expressing LSPmIL-15 and BCMACAR-NK cells prepared using human peripheral blood cells as raw materials.

[0071] Figure 16 Result diagram showing that membrane-bound IL-15 guided by the LSP signal peptide promotes the in vitro survival of NKG2D CAR-NK cells more effectively than membrane-bound IL-15 guided by the conventional GM-CSF signal peptide in the absence of IL-2.

[0072] Figure 17To compare with the membrane-bound IL-15 guided by the conventional GM-CSF signal peptide, the result graph of the membrane-bound IL-15 guided by the LSP signal peptide promoting the in vitro survival of NKG2D CAR-NK cells more effectively under the condition of low-concentration IL-2 (10 IU / mL).

[0073] Figure 18 To compare with the membrane-bound IL-15 guided by the conventional GM-CSF signal peptide, the result graph of the membrane-bound IL-15 guided by the LSP signal peptide promoting the in vitro survival of NKG2D CAR-NK cells more effectively under the condition of high-concentration IL-2 (100 IU / mL).

[0074] Figure 19A The result graph of the survival of LSPmIL-15 / NKG2D CAR-NK cells in normal tumor-free NSG mice.

[0075] Figure 19B The result graph of the survival of LSPmIL-15 / NKG2D CAR-NK cells in tumor-bearing NSG mice.

[0076] Figure 20 The result graph of the in vitro cancer cell killing effects of mIL-15 / NKG2D CAR-NK and LSPmIL-15 / NKG2D CAR-NK cells.

[0077] Figure 21 The result graph of the in vivo tumor killing effect of LSPmIL-15 / NKG2D CAR-NK cells. Detailed implementation manners

[0078] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0079] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0080] In the specific embodiments of the present invention, the following experimental materials are adopted:

[0081] Wild-type K562 cells of human chronic myeloid leukemia cell line were purchased from ATCC, colorectal cancer cell line HCT-116, ovarian cancer cell line SKOV-3, and human acute myeloid leukemia cell line KG-1 were from ATCC cell bank, PBMCs were extracted from peripheral blood of solid tumor patients or healthy donors; MNCs from umbilical cord blood were purchased from Maishun Biotechnology Co., Ltd. iPSCs were derived from CD34 + Cell reprogramming. The other reagents or consumables not mentioned are from conventional reagent manufacturers in the field or are prepared by conventional means in the field. The culture medium for SKOV3 cells is MCCOY (Gibco) + 10% FBS (Gibco), the culture medium for HCT-116 cells is 1640 (Gibco) + 10% FBS (Gibco), and the culture medium for KG-1 cells and K562 cells is IMDM (Gibco) + 10% FBS (Gibco). The NK cell culture medium is AIM medium (Gibco) + 5% AB serum (Gemini). The IPSC culture medium is mTeSRPlus (Stemcell).

[0082] Human recombinant IL-2 (hrIL2) was purchased from Beijing Shuanglu Company, and P3Primary Cell 4D-Nucleofector XKit was purchased from Lonza; CD3, CD56, CD137L, CD16, CD94, NKG2A, NKG2D, NKG2C, CD158, CD34, tra-1-60, SSEA4, CD19 antibody, BCMA antibody, OCT4, nanog antibody, Biotin-conjugated anti-human IL-15, IL21 antibody and streptavidin (Streptavidin) were purchased from Biolegend; Strep-tag II antibody was purchased from GenScript; Anti-tra-1-81 antibody was purchased from abcam; hULBP-1, hULBP-2 / 5 / 6, hULBP-3, hULBP-4, and MICA / B antibodies were purchased from R&D system. The flow cytometer was purchased from Agilent, model Novocyte2060R; the real-time killing detector was purchased from ACEA Bio, model xCELLigence RTCA DP; the experimental NCG mice were purchased from the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences.

[0083] The NKG2D-specific K562 cells in the examples were genetically engineered to express membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), and CD37L. Membrane-bound human IL-15 was formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 30-162 of UniprotKB P40933), and the hinge transmembrane region of human CD8 (amino acids 128-213 of UniprotKB P01732); membrane-bound human IL-21 was formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE4), the hinge constant region of human IGHG4 (amino acids 99-327 of UniprotKB P01861), and the transmembrane region of human CD4 (amino acids 397-418 of UniprotKB P01730).

[0084] The K562-CD19 cells in the examples were genetically engineered to express membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), CD37L, and CD19. Membrane-bound human IL-15 was formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 30-162 of UniprotKB P40933), and the hinge transmembrane region of human CD8 (amino acids 128-213 of UniprotKB P01732); membrane-bound human IL-21 was formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE4), the hinge constant region of human IGHG4 (amino acids 99-327 of UniprotKB P01861), and the transmembrane region of human CD4 (amino acids 397-418 of UniprotKB P01730); the sequence of CD19 was UniProt KB P15391.

[0085] The K562-BCMA cells used in the examples are cells inactivated by γ-ray treatment (100 Gy). The K562-BCMA cells are genetically engineered to express membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), CD37L, and BCMA. Membrane-bound human IL-15 is formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 49-162 of UniprotKB P40933), and the hinge transmembrane region of human CD8 (amino acids 128-213 of UniprotKB P01732); membrane-bound human IL-21 is formed by fusing the GM-CSF Rα signal peptide (amino acids 1-22 of UniprotKBP15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE4), the hinge constant region of human IGHG4 (amino acids 99-327 of UniprotKB P01861), and the transmembrane region of human CD4 (amino acids 397-418 of UniprotKB P01730); the sequence of BCMA is the amino acid sequence of positions 1-77 of UniProt KB-Q02223.

[0086] The amino acid sequence of PiggyBac transposase is from GenBank AAA87375.2. The hinge regions of NKG2D CAR, CD19CAR, and BCMACAR are from amino acids 99-110 of IgG4 (UniProtKB-P01861), the transmembrane regions are from amino acids 153-179 of CD28 (UniProtKB-P10747), the intracellular domains are from amino acids 209-255 of 4-1BB (UniProtKB-Q07011), and amino acids 52-164 of CD3ζ (UniProtKB-P20963). The long signal peptide of IL-15LSP is from amino acids 1-48 of UniprotKB P40933. Membrane-bound IL-15 guided by the LSP signal peptide is formed by fusing amino acids 1-48 of human IL-15 UniprotKB P40933 (IL-15LSP), amino acids 49-162 of human IL-15 UniprotKB P40933 (mature IL-15), and the hinge transmembrane region (CD8H-TM) of human CD8, amino acids 128-213 of UniprotKB P01732. The DNA and amino acid sequences of membrane-bound IL-15 guided by the IL-15LSP signal peptide are shown as SEQ IDNO.1 and SEQ ID NO.2, respectively.

[0087] SEQ ID NO.1:

[0088] atgagaatttcgaaaccacatttgagaagtatttccatccagtgctacttgtgtttacttctaaacagtcattttctaactgaagctggcattcatgtcttcattttgggctgtttcagtgcagggcttcctaaaacagaagccaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctgctagcttcgtgccggtcttcctgccagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaccacaggaactga。

[0089] SEQ ID NO.2:

[0090] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN。

[0091] Schematic diagram of interleukin-15 genomic organization and gene expression is as Figure 1 shown. IL-15 is encoded by a 34-kb region on human chromosome 4q31. Its gene contains 9 exons (1 - 8 and 4A), and 8 introns. Four of the exons (5 - 8) encode the mature protein. There are 2 subtypes of IL-15 mRNA, which produce 2 different precursor proteins with different signal peptide lengths: the signal peptide of IL-15LSP is a long signal peptide containing 48 amino acids, while the signal peptide of IL-15SSP is 21 amino acids in length. The two precursor proteins produce the same mature protein, but they have different intracellular trafficking. LSP IL-15 targets the Golgi apparatus, early endosomes, and the endoplasmic reticulum secretion pathway. SSP IL-15 is not secreted and is confined to the cytoplasm and nucleus.

[0092] Example 1

[0093] This example relates to plasmids for CAR-NK preparation and the preparation process.

[0094] To produce NK cells expressing different CAR genes, chimeric antigen receptor CAR plasmid constructs targeting three different cell surface proteins were constructed. Most of the CAR plasmid constructs simultaneously carried the DNA sequence of membrane-bound IL-15 (schematic diagram is as Figure 2As shown. First, the NKG2D CAR plasmid contains the extracellular domain (Ectodomain) of the NKG2D receptor expressed in tandem with a streptavidin tag (STII Tag) (where the STII Tag is used to detect the expression of NKG2D CAR), a 4-1BB co-stimulatory domain, the CD3zeta cytoplasmic domain as the activation domain, and TIR is the terminal inverted repeat sequence in the piggyBac transposon system, which is used for the recognition and binding of transposase and subsequent gene cleavage and insertion. The other parts of the mIL-15 / NKG2D CAR plasmid are the same as the NKG2D CAR plasmid, but carry membrane-bound IL-15, which is co-expressed with NKG2D CAR through the IRES sequence. The membrane-bound IL-15 contains a mature IL-15 sequence (mIL-15), with the GM-CSF signal peptide (GM-CSF SP) sequence connected at the front end and the hinge and transmembrane (CD8H-TM) domain sequence of CD8α connected at the back end. Preferably, by applying the mature IL-15 sequence with the IL-15LSP signal peptide to replace the mature IL-15 sequence in the mIL-15 / NKG2D CAR plasmid, we also constructed the LSPmIL-15 / NKG2D CAR plasmid. When constructing the CAR plasmids targeting CD19 and BCMA, we replaced the extracellular domain sequence of the NKG2D receptor in the LSPmIL-15 / NKG2D CAR plasmid with the CD19- and BCMA-specific scFv domain sequences, generating the LSPmIL-15 / CD19CAR and LSPmIL-15 / BCMACAR plasmids.

[0095] Example 2

[0096] In this example, CAR-specific K562 artificial antigen-presenting cells were constructed for the expansion of CAR-NK cells.

[0097] To construct NKG2D CAR-specific K562 artificial antigen-presenting cells, K562 cells expressing NKG2D ligands were resuspended in 10 mL of Opti-MEM and centrifuged at 200×g for 5 min. The cell pellet was resuspended in 100 μL of P3 buffer, and CD37L, mIL-21, and mIL-15 plasmids and PiggyBac transposase plasmid were added. After mixing, the suspension was transferred to a Lonza electroporation cuvette. Then, the electroporation cuvette was placed in the Lonza 4D-NucleofectorTM X Unit (in the single electroporation cuvette module) for electroporation. After electroporation, the K562 cell suspension in the electroporation cuvette was slowly transferred to a well of a 6-well plate pre-added with K562 medium (IMDM + 10% FBS) for cell culture. On the 5th day after electroporation, puromycin screening was carried out at a concentration of 200 μg / mL for 1 month, and the medium was changed every 2 days. After 1 month, single cells were sorted into 96-well plates using a cell sorter BD FACSAria (BD Biosciences), and flow cytometry analysis was performed on the expanded single-cell clones to detect the expression of the transgene. The expression of NKG2D ligands in the selected NKG2D CAR-specific K562 single-cell lines was as Figure 4A shown. The positive rates of ULBP-1, ULBP-2 / 5 / 6, ULBP-3, and ULBP-4 were 13.20, 53.43, 0.46, and 1.44%, respectively, and the positive rate of MIC A / B was 71.47%. The expression of other related functional genes in the selected K562 single-cell lines was as Figure 4B shown. The positive rates of CD137L, mIL-21, and mIL-15 were 99.95, 99.26, and 99.35%, respectively.

[0098] To construct CD19CAR- and BCMA-specific K562 artificial antigen-presenting cell lines, the above-engineered K562 cells were resuspended separately, CD19 plasmid and BCMA plasmid were added respectively, and they were co-electroporated with the PiggyBac transposase plasmid, and then single-cell cloning was performed by sorting according to the above method. Figure 5A and 5B respectively show the expression of CD19 and BCMA on the surface of K562 cells.

[0099] In the present invention, the ligand molecules, cytokines, etc. expressed by the engineered cells and required for NK cell activation can be secreted or membrane-bound. The ligand molecules required for NK cell activation may include CD137L and / or OX40L, and the cytokines required for NK activation may include any one or a combination of at least two of human IL-12, human IL-15, human IL-18 or human IL-21. The cell-based artificial antigen-presenting cells need to be pretreated with γ-rays (50-150 Gy), x-rays (50-150 Gy) or mitomycin C (20 μg / mL) for inactivation before use.

[0100] Example 3

[0101] In this example, membrane-bound IL-15 guided by the LSP signal peptide promotes cell expansion during the preparation of NKG2D CAR-NK using human peripheral blood cells as raw materials.

[0102] To examine whether membrane-bound IL-15 guided by the LSP signal peptide can promote cell expansion during the preparation of NKG2D CAR-NK using human peripheral blood cells as raw materials, according to Figure 3 the procedure, three different NKG2D CAR plasmids were used to prepare three kinds of NKG2D CAR-NK cells: NKG2D CAR-NK, mIL-15 / NKG2D CAR-NK and LSPmIL-15 / NKG2D CAR-NK cells. Figure 6A As shown by the representative flow cytometry detection, the positive rates of NK cells expressing CD56 + CD3 - in the three kinds of NKG2D CAR-NK cells were 99.60%, 92.90% and 99.98% respectively, with no significant difference. The positive rates of NKG2D CAR detected by streptavidin tag STII Tag were 61.70%, 90.10% and 98.40% respectively. The CAR plasmid with mIL-15 significantly increased the expression of NKG2D CAR, but there was no significant difference between mIL-15 and LSP mIL-15. Figure 6BData presenting the positive rates of NKG2DCAR in multiple samples, where the number of samples of NKG2D CAR-NK cells was 5, the number of samples of mIL-15 / NKG2D CAR-NK cells was 8, and the number of samples of LSPmIL-15 / NKG2D CAR-NK cells preferably developed in the present invention was as high as 16. The data from multiple samples confirmed that the positive rates of NKG2D CAR in the two types of CAR-NK cells with mIL-15 were significantly higher (p<0.0001) statistically than those in the CAR-NK cells without mIL-15. The positive rate of NKG2D CAR in the CAR-NK cells with mIL-15 was 92.51±7.57% (n = 24), and the positive rate of NKG2D CAR in the CAR-NK cells without mIL-15 was 57.96±10.05% (n = 5). However, there was no significant statistical difference in the positive rates of NKG2D CAR between mIL-15 and LSP mIL-15. The positive rate of NKG2D CAR in the former was 91.00±8.11% (n = 8), and the positive rate of NKG2D CAR in the latter was 93.27±7.44% (n = 16).

[0103] In Figure 7A it, the inventor presented data on the continuous increase in the positive rate of NKG2D CAR during the preparation of LSPmIL-15 / NKG2D CAR-NK cells under the stimulation of NKG2D CAR-specific K562 artificial antigen-presenting cells. After plasmid electroporation and the first round (Cycle) of K562 cell stimulation, the positive rate of NKG2D CAR was 19.65±1.61% (n = 8). After the second round of K562 cell stimulation, the positive rate of NKG2D CAR increased to 57.45±13.25% (n = 8). After the third round of K562 cell stimulation, the positive rate of NKG2D CAR increased to 82.83±10.18% (n = 8). When the preparation of CAR-NK cells was completed after the fourth round of K562 cell stimulation, the positive rate of NKG2D CAR was 93.57±5.13% (n = 8), that is, more than 90% of NK cells expressed NKG2DCAR. These data demonstrated the importance of NKG2D CAR-specific K562 artificial antigen-presenting cells in the preparation of high-purity NKG2D CAR-NK cells. In Figure 7BIn addition, data on the production process of LSPmIL-15 / NKG2D CAR-NK cells are further presented. During the stimulation with NKG2D CAR-specific K562 artificial antigen-presenting cells, the number of NKG2D CAR-positive NK cells continuously increases. After four rounds of K562 cell stimulation, the total amplification multiple can reach 590,000 times. Therefore, combining the data in Figures 6 and 7, the present invention reveals that the membrane-bound IL-15 guided by the LSP signal peptide combined with NKG2D CAR-specific K562 artificial antigen-presenting cells can effectively enrich and amplify NKG2D CAR-NK cells, and the CAR expression in the final product can reach more than 90%, and the amplification multiple can reach hundreds of thousands of times.

[0104] From Figures 8 to 10 , data on the characterization of LSPmIL-15 / NKG2D CAR-NK cells are provided. The first content of cell characterization is the expression of membrane-bound IL-15. When performing flow cytometry detection, antibodies against IL-15 and antibodies against the streptavidin tag STII Tag are applied simultaneously. Figure 8 It shows that mIL-15 / NKG2D CAR-NK and LSPmIL-15 / NKG2D CAR-NK cells co-express IL-15 and NKG2D CAR, while mock electroporated blank control NK cells (mock NK) do not express IL-15 and NKG2D CAR. Since the conventional flow cytometry method can only detect the expression of cell surface proteins, Figure 8 the results confirm that the expressed IL-15 is membrane-bound IL-15. In Figure 9 , the similarities and differences in the expression levels of surface proteins, especially some functional receptors, of three types of NK cells, namely mock NK cells, mIL-15 / NKG2D CAR-NK cells, and LSPmIL-15 / NKG2D CAR-NK cells, are compared. Except that CAR-NK cells highly express NKG2D CAR and NKp44 receptors, there are no differences in the expression of other surface proteins among the three types of cells. NKp44 is a member of the NK cell cytotoxic receptor (NCR) family, and the mechanism and significance of the upregulation of membrane-bound IL-15 on NKp44 are not yet clear. Since the preparation of CAR-NK using Figure 3 the method requires about 28 days and 4 rounds of K562 cell stimulation, and NK cells will show a state of functional exhaustion under excessive stimulation. The exhaustion state of the prepared NK cell product is confirmed by examining the expression of NK cell-related immune checkpoint inhibitory receptors. Figure 10The expression of three immune checkpoint inhibitory receptors, TIM-3, LAG-3, and PD-1, on the surface of three types of cells was still at a low expression level. The expression of TIGIT on mock NK cells was higher than 60% (66.18 ± 4.66%, n = 4). However, after the application of membrane-bound IL-15, the expression of TIGIT was downregulated, especially on the surface of LSPmIL-15 / NKG2D CAR-NK cells, where the expression of TIGIT was downregulated to 33.87 ± 9.66% (n = 3). Compared with mock NK cells, the difference was statistically significant (p < 0.05). This result indicates that the expression of membrane-bound IL-15 can prevent NK cell exhaustion.

[0105] Example 4

[0106] In this example, human umbilical cord blood cells were used as raw materials to prepare CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR.

[0107] In addition to using human peripheral blood cells as raw materials to prepare NKG2D CAR-NK as described above, the present invention also tested whether it is possible to use human umbilical cord blood cells as raw materials to prepare CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR. Umbilical cord blood cells have the advantages of low immunogenicity, low requirement for matching, and strong proliferation ability, and have unique clinical application value compared with peripheral blood cells. Figure 11A The shown preparation process is similar Figure 3 , but cord blood mononuclear cells CBMC were used. It was found that as long as the NKG2D CAR-specific K562 artificial antigen-presenting cells were stimulated for 3 rounds, the expression of NKG2D CAR in some samples could be close to 90% ( Figure 11B ). Similar to the situation in Example 3, under the stimulation of the NKG2D CAR-specific K562 cell line, the expression level of NKG2D CAR on NK cells increased continuously in each round. For example, the positive rate was 16.00 ± 6.55% (n = 3) after the first round of amplification, 49.07 ± 10.76% (n = 3) after the second round of amplification, and could reach 75.73 ± 10.76% (n = 3) after the third round of amplification ( Figure 11C ).

[0108] Example 5

[0109] In this example, human iPSC cells were used as raw materials to prepare CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR.

[0110] Whether using human peripheral blood or human umbilical cord blood as raw materials to prepare CAR-NK cells, the raw materials will be used up after a certain period of time. Induced pluripotent stem cells (iPSCs) are pluripotent stem cell lines prepared by reprogramming, which can be infinitely amplified in vitro and have the potential to differentiate into any cell type in the body. Therefore, using iPSCs as raw materials, large-scale library construction can be achieved through in vitro mass amplification, realizing the consistency of batch production quality, stable products, and determined quality, and having broad clinical application prospects when preparing CAR-NK cells. In view of this, the present invention provides a method for preparing CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR using human iPS cells as raw materials. First, cytokines are used to enrich CD34 + cells ( Figure 12A ), and then a free vector with an oriP / EBNA-1 (EB nuclear antigen-1) backbone expressing reprogramming factors Oct4, Sox2, Lin28, Klf4, and L-Myc is used to reprogram CD34 + cells into human iPSC cells in a virus-free and transgene-free manner. Complete iPSC cell colonies can be formed within 3 months ( Figure 12B ). The generated human iPSCs express pluripotency markers Nanog, Oct4, Tra-1-81, Tra-1-60, SSEA4, and oriP / EBNA-1 ( Figures 12C - 12E ), and an iPSC cell line with karyotype identified as non-aberrant is selected to establish an iPSC cell bank ( Figure 12F ). Before preparing CAR-NK cells co-expressing LSPmIL-15 and NKG2D CAR, an iPSC cell line co-expressing LSPmIL-15 and NKG2D CAR was first established. The specific method is as follows: collect iPSCs and resuspend them in 10 mL of Opti-MEM, centrifuge at 300×g for 10 min, resuspend the cell pellet in 100 μL of P3 buffer, add the LSP mIL15 / NKG2D CAR plasmid (with the structure as Figure 2 ) and the PiggyBac transposase plasmid, mix well and transfer to a Lonza electroporation cuvette. Then place the electroporation cuvette in a Lonza 4D-NucleofectorTM X Unit (in the single electroporation cuvette module) for electroporation. After the electroporation is completed, slowly transfer the cell suspension in the electroporation cuvette to a T75 flask pre-added with iPSC cell medium for cell culture. After 2 weeks, use a cell sorter to sort single cells into a 96-well plate, and perform flow cytometry analysis on the amplified single cell clones to detect the expression of the transgene. The expression of NKG2D CAR in the iPSC single cell lines used for screening is as Figure 13AAs shown, the expression of IL-15 in the screened iPSC single-cell lines is as Figure 13B shown. The iPSC cell line co-expressing LSPmIL-15 and NKG2D CAR forms LSP mIL15 / NKG2D CAR-NK cells derived from iPSC cells through three stages: hematopoietic stem cell differentiation, lymphocyte differentiation, and NK cell expansion ( Figure 14A ). Figure 14B It shows that during the first-stage hematopoietic stem cell differentiation, although the number of hematopoietic stem cells generated from CAR-iPSC cells is relatively small compared with wild-type iPSC cells, there is no difference in the number of CD34-positive cells, while the number of CD34 / CD44 double-positive cells is slightly more than that of the double-positive cells derived from wild-type iPSC cells. During the third-stage NK cell expansion, γ-ray irradiated inactivated NKG2D CAR-specific K562 cells were used again to stimulate NK cell expansion. After 6 rounds of stimulation, the NK cell expansion reached more than 1 million times, and the expansion multiple of NKG2D CAR-NK with LSP mIL15 was significantly higher than that of NK cells from wild-type iPSC cells ( Figure 14C ). When detecting the expression of NK cell receptors by flow cytometry, LSPmIL15 / NKG2D CAR-NK cells derived from iPSC cells highly expressed NKG2D CAR, CD56, NKG2A, NKG2C, NKp30, CD94, and CD117, but did not express CD3 and NKp80 ( Figure 14D ). Finally, during the killing function test, compared with NK cells from wild-type iPSC cells, LSPmIL15 / NKG2D CAR-NK cells derived from iPSC cells killed target cells faster and had a stronger killing effect ( Figure 14E ).

[0111] Example 6

[0112] In this example, CAR-NK cells co-expressing LSPmIL-15 and CD19 or BCMA were prepared using human peripheral blood cells as raw materials.

[0113] In addition to preparing CAR-NK cells co-expressing LSPmIL-15 and NKG2D, LSPmIL-15 / CD19CAR plasmid and LSPmIL-15 / BCMACAR plasmid in Figure 2 were also used to prepare CAR-NK cells co-expressing LSPmIL-15 and CD19, and CAR-NK cells co-expressing LSPmIL-15 and BCMA, respectively. The preparation method was similar to Figure 3 , PBMC was extracted from peripheral blood, and after CD3-positive cell depletion and counting, γ-ray irradiated inactivated CD19 or BCMA CAR-specific K562 single-cell lines were addedFigure 5A and Figure 5B ), cultured with AIMV + 5% human AB serum, and 50 IU / mL IL-2 was added after 2 - 3 days. Two days later, the cells were resuspended in 10 mL of Opti-MEM, centrifuged at 300×g for 10 min, the cell pellet was resuspended in 100 μL of P3 buffer, LSPmIL-15 / CD19 or BCMACAR plasmid and PiggyBac transposase plasmid were added, and after mixing, it was transferred to a Lonza electroporation cuvette. Then the electroporation cuvette was placed in the Lonza 4D-NucleofectorTM X Unit (in the single electroporation cuvette module) for electroporation. After the electroporation, the cell suspension in the electroporation cuvette was slowly transferred to a T75 flask pre-added with medium (IMV + 5% human AB serum) for cell culture. After 7 - 10 days of co-culture in one round, the cells were counted, amplified and analyzed, and the transgenic expression was examined by flow cytometry. At the same time, the cells were continued to be co-cultured with the γ-ray irradiated inactivated CD19 or BCMA CAR-specific K562 single cell line for the second round of co-culture, and so on for four rounds of co-culture. During the culture process, the CAR positive rate in LSPmIL-15 / CD19CAR-NK cells ( Figure 15A ) and LSPmIL-15 / BCMACAR-NK cells ( Figure 15B ) continuously increased. At the end of four rounds of co-culture, the CD19CAR positive rate of the former was 78.14% while the CD56 + CD3 - positive rate was 93.37% ( Figure 15A ), and the BCMA CAR positive rate of the latter was 91.05% while the CD56 + CD3 - positive rate was 99.32% ( Figure 15B ).

[0114] Example 7

[0115] This example tests the ability of membrane-bound IL-15 guided by the LSP signal peptide to promote the in vitro survival of CAR-NK cells.

[0116] To compare the differences in the ability of membrane-bound IL-15 prepared using a conventional GM-CSF signal peptide and membrane-bound IL-15 prepared using the IL-15LSP signal peptide in supporting the in vitro proliferation of CAR-NK cells and maintaining the in vitro survival of CAR-NK cells, the present invention discloses that the IL-15LSP signal peptide exhibits stronger functions in supporting the in vitro proliferation and survival of CAR-NK cells. Specifically, three different types of NK cells were cultured under three different conditions: LSPmIL-15 / NKG2D CAR-NK cells, mIL-15 / NKG2D CAR-NK cells, and mock electroporated NK cells (mock NK cells). Among them, LSPmIL-15 / NKG2D CAR-NK cells were produced by combining PiggyBac transposase, NKG2D CAR, membrane-bound IL-15 carrying the LSP signal peptide with NKG2D-specific K562. mIL-15 / NKG2D CAR-NK cells were produced by combining piggyBac transposase, NKG2D CAR, conventional membrane-bound IL-15 without the LSP signal peptide with NKG2D-specific K562. Mock electroporated NK cells (mock NK cells) were only subjected to the electroporation process and combined with NKG2D-specific K562 for production. The three different culture conditions were: without adding IL-2 (no IL-2, Figure 16 ), adding low-concentration IL-2 (IL-2: 10 IU / mL, Figure 17 ), and adding high-concentration IL-2 (IL-2: 100 IU / mL, Figure 18 ).

[0117] As Figure 16 shown, under the condition of not adding IL-2, mock NK cells all died after surviving for about 7 days, while the two types of NKG2D CAR-NK cells with membrane-bound IL-15 not only did not die but also could proliferate during the initial 3 weeks. Compared with mIL-15 / NKG2D CAR-NK cells without using the IL-15LSP signal peptide, LSPmIL-15 / NKG2D CAR-NK cells using the IL-15LSP signal peptide exhibited stronger cell proliferation ability, and the difference was statistically significant ( Figure 16) Therefore, LSPmIL-15 significantly plays a stronger role in supporting the proliferation of NK cells. Without adding IL-2, the mIL-15 / NKG2D CAR-NK cells without the IL-15LSP signal peptide can survive for about 56 days, while the LSPmIL-15 / NKG2D CAR-NK cells with the IL-15LSP signal peptide can survive for about 63 days. Therefore, LSPmIL-15 can also play a better role in supporting the survival of NK cells.

[0118] In the condition of providing low concentration ( Figure 17 ) and high concentration ( Figure 18 ) of IL-2, it was found that although IL-2 alone can support the survival of mock NK cells and is positively correlated with the IL-2 concentration, IL-2 alone does not support the proliferation of NK cells. Even when using high-concentration IL-2, no increase in the number of NK cells higher than the initial seeding number was observed. Combining Figure 16 's findings, it shows that membrane-bound IL-15 has a stronger ability to support cell proliferation than adding IL-2, and further reveals that when membrane-bound IL-15 is applied together with added IL-2, it shows a synergistic effect in supporting the proliferation and survival of NK cells, with more cell proliferation and longer cell survival observed, and it is positively correlated with the IL-2 concentration ( Figures 16 - 18 ). LSPmIL-15 plays a more significant role in supporting the proliferation and survival of NK cells.

[0119] Example 8

[0120] This example tests the in vivo survival ability of LSPmIL-15 / NKG2D CAR-NK cells.

[0121] In this example, the survival time of LSPmIL-15 / NKG2D CAR-NK cells in tumor-free and tumor-bearing mice was examined. NSG immunodeficient mice were used in the experiment. In the first experiment, 1×10^7 LSPmIL-15 / NKG2D CAR-NK cells were intravenously injected into 5 tumor-free NSG mice, and blood samples were collected on days 3, 7, 14, 21, and 28 after injection for analysis. On days 3 and 7 after injection, the percentage of CAR-NK cells in the mice remained at a relatively high level, accounting for about 4% of all peripheral blood PBMC cells, and began to gradually decline from day 14 and was hardly detectable by day 28 ( Figure 19A)。The results of this animal experiment were consistent with those of the in vitro experiment, showing that even with the support of LSPmIL-15, NK cells disappeared after surviving for a certain period of time. Since an important application of CAR-NK cells is cancer treatment, to further examine the survival time of LSPmIL-15 / NKG2D CAR-NK cells in vivo under tumor-bearing conditions, first, 1×10^6 HCT-116-Luc colorectal cancer cells were inoculated into NSG mice, and then 1×10^7 LSPmIL-15 / NKG2D CAR-NK cells were injected via intraperitoneal injection 7 days later. Blood samples were collected on days 3, 7, 14, 21, 28, 35, and 42 for analysis. The inventors observed that between days 21 and 28, the percentage of LSPmIL-15 / NKG2D CAR-NK cells in peripheral blood increased, which was higher than the percentage detected between days 3 and 7( Figure 19B ). Since HCT-116-Luc colorectal cancer cells express NKG2D ligands, this increase should be due to the proliferation of CAR-NK cells stimulated by cancer cells. The survival time of LSPmIL-15 / NKG2D CAR-NK cells in tumor-bearing mice was also significantly longer than that in tumor-free mice. The cell percentage detected on day 42 after injection (2.014%) was 66% of the cell percentage on day 3 after injection (3.046%), that is, more than half of the injected LSPmIL-15 / NKG2D CAR-NK cells were still alive.

[0122] Example 9

[0123] This example tested the cancer cell killing ability of LSPmIL-15 / NKG2D CAR-NK cells.

[0124] The efficacy of CAR-NK cell tumor therapy depends on their cancer cell killing ability. In this example, the anti-cancer effects of LSPmIL-15 / NKG2D CAR-NK cells were examined in vitro and in vivo. Using three cancer cell lines, namely, intestinal cancer cell line HCT-116, ovarian cancer cell line SKOV-3, and acute myeloid leukemia cell line KG-1, and a short-term (3 h) europium release cytotoxicity assay killing experiment method, the anti-cancer effects of the three effector cells in vitro were compared. The three effector cells were LSPmIL-15 / NKG2D CAR-NK cells, mIL-15 / NKG2D CAR-NK cells, and NK cells treated with mock electroporation (Mock NK). During detection, the ratio of effector cells to target cells (E:T) was increased from 2.5:1 to 20:1. The results showed that compared with mIL-15 / NKG2D CAR-NK cells and Mock NK cells, LSPmIL-15 / NKG2D CAR-NK cells showed better killing effects. At the highest E:T ratio of 20:1, they could kill almost 100% of the three different cancer cells in vitro ( Figure 20 ). Combining the fact that there was no obvious difference in the expression of NKG2D CAR on mIL-15 / NKG2D CAR-NK cells and LSPmIL-15 / NKG2D CAR-NK cells at the end of CAR-NK cell preparation in Example 3, and that LSPmIL-15 / NKG2D CAR-NK cells showed stronger cell proliferation ability than mIL-15 / NKG2D CAR-NK cells in Example 7, the better killing effect of LSPmIL-15 / NKG2D CAR-NK cells observed in this example should be related to the rapid proliferation of these CAR-NK cells during the contact with cancer cells.

[0125] In addition, the anti-cancer effect of mIL-15 / NKG2D CAR-NK cells in vivo was further examined in a mouse tumor model. The mouse tumor model was a mouse xenograft model established by injecting 3×10^6 SKOV-3-Luc human ovarian cancer cells into the abdominal cavity of mice. On the 7th day after tumor inoculation, the mice were randomly divided into three groups of 6 mice each, and were intraperitoneally injected with PBS solvent, 1×10^7 Mock NK cells, or 1×10^7 LSPmIL-15 / NKG2D CAR-NK cells, respectively. After 7 days, the tumor progression was observed by Xenogen in vivo imaging software v2.5, and fluorescence images were obtained and analyzed. Figure 21It was shown that before treatment, the tumor signals of the three groups of mice were at similar levels. After treatment, the PBS and Mock NK cell groups showed similar tumor growth trends, while significantly weakened tumor signals were observed in the mice treated with LSPmIL-15 / NKG2D CAR-NK cells, and the tumor signal intensity in all 6 mice was lower than the pre-treatment level.

[0126] In summary, the present invention designs a novel engineered CAR-NK cell that simultaneously expresses a CAR molecule and membrane-bound IL-15 in NK cells, and designs the use of a specific signal peptide to further improve the cell expansion ability, survival ability, and tumor cell killing ability during the preparation process, showing broad application prospects in the field of tumor treatment and other fields.

[0127] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived within the technical scope disclosed by the present invention by those skilled in the art all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An engineered CAR-NK cell, characterized in that: The engineered CAR-NK cells express CAR molecules and membrane-bound IL-15; The membrane-bound IL-15 comprises a signal peptide and IL-15 connected in sequence; The signal peptide includes an IL-15LSP signal peptide.

2. The engineered CAR-NK cell according to claim 1, characterized in that The membrane-bound IL-15 also includes a transmembrane domain; Preferably, the membrane-bound IL-15 comprises an IL-15LSP signal peptide, IL-15 and a transmembrane domain which are sequentially connected.

3. The engineered CAR-NK cell according to claim 2, characterized in that: The transmembrane domain includes the hinge transmembrane region of CD8.

4. The engineered CAR-NK cell according to any one of claims 1 to 3, characterized in that The nucleic acid sequence of the membrane-bound IL-15 includes the sequence shown in SEQ ID NO.1; Preferably, the amino acid sequence of the membrane-bound IL-15 comprises the sequence shown in SEQ ID NO.

2.

5. The engineered CAR-NK cell according to any one of claims 1 to 4, characterized in that: The raw material cells of the engineered CAR-NK cells are derived from at least one of peripheral blood, umbilical cord blood, placental tissue or induced pluripotent stem cells.

6. The engineered CAR-NK cell according to any one of claims 1 to 5, characterized in that: The CAR molecule comprises a chimeric antigen receptor and / or a chimeric switch receptor.

7. The method for preparing an engineered CAR-NK cell according to any one of claims 1 to 6, characterized in that: The preparation method comprises: The nucleic acid molecules encoding the CAR molecules and membrane-bound IL-15 are introduced into NK cells and cultured.

8. The method for preparing engineered CAR-NK cells according to claim 7, characterized in that: The NK cells are modified by gene integration to co-express the CAR molecule and membrane-bound IL-15; Preferably, the introduction method includes viral or non-viral gene transfection; Preferably, the culturing comprises co-culturing with engineered K562 artificial antigen presenting cells; Preferably, the engineered K562 artificial antigen-presenting cells are modified by gene integration to co-express CD137L, IL-21, IL-15 and the antigens corresponding to the CAR molecules.

9. Use of the engineered CAR-NK cells according to any one of claims 1 to 6 in the preparation of a product for eliminating cells expressing corresponding CAR targets; Preferably, the cells expressing the corresponding CAR target include tumor cells; Preferably, the tumor cells include at least one of blood cancer cells, ovarian cancer cells, triple-negative breast cancer cells, pancreatic cancer cells, colorectal cancer cells or bladder cancer cells.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the engineered CAR-NK cells according to any one of claims 1-6.

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