Engineered NK cell as well as preparation method and application thereof

Abstract

The invention provides an engineered NK cell as well as a preparation method and application thereof. The engineered NK cell comprises one or more overexpressed proteins selected from the group consisting of: SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1. The invention also relates to a method for preparing the engineered NK cell. According to the application, screening of a human overexpression transcription factor library is carried out on NK cells obtained by inducing healthy human peripheral blood mononuclear cells, six potential transcription factor targets capable of improving the killing function of the NK cells are obtained through a biosignal analysis method on the NK cells, and the NK cells of overexpression transcription factors are constructed. Compared with a non-overexpressed NK cell treatment strategy, several target spots with a good effect on solid tumors can significantly improve the anti-tumor effect of the NK cells by about 20%-50%, and the survival time of tumor-bearing NSG mice is significantly prolonged.

Description

Technical Field

[0001] The present invention belongs to the fields of medical immunotherapy and biopharmaceuticals, and more particularly, relates to engineered NK cells and their preparation methods and applications. Background Art

[0002] Natural Killer cells (NK cells) are an important part of the human immune system and belong to a type of innate immune cells. NK cells account for 10%-15% of human peripheral blood lymphocytes. Like T cells, NK cells also have killing ability, and the regulation of their cell effector functions is achieved through the dynamic balance between inhibitory killer cell immunoglobulin-like receptors (KIR) and activating cell surface receptors. NK cells achieve functional activation through activating receptors NKG2D, natural cytotoxic receptors NKp30, NKp44 (CD336, NCR2), and NKp46 (CD335, NCR1). On the other hand, inhibitory receptors, such as the CD94 / NKG2A heterodimer, can recognize various forms of MHC-I molecules, thereby inhibiting NK cell function and avoiding the destruction of healthy autologous cells. At the same time, NK cells also express receptors for a variety of activating cytokines (such as IL-12, IL-15, etc.) and receptors for immunosuppressive factors (such as TGFβ). NK cells have the following several killing mechanisms against target cells. By releasing cytoplasmic granules containing perforin and granzyme, and expressing members of the tumor necrosis factor (TNF) family, they induce apoptosis of tumor cells by interacting with their corresponding receptors, thereby directly killing tumor cells. In addition, antibody-dependent cell cytotoxicity (ADCC) mediated by the Fc receptor CD16 can trigger the killing of target cells by NK cells. However, for solid tumors, which account for the vast majority of tumors, due to their complex tumor microenvironment (TME), NK cells face many limitations in the treatment of solid tumors. For example, hypoxia, high concentrations of adenosine, reactive oxygen species (ROS), and immunosuppressive cells and factors in the microenvironment can all inhibit the anti-tumor activity of NK cells. At the same time, NK cells also have problems such as low virus transduction efficiency, which further affects the application of NK cells in immunotherapy.

[0003] In molecular biology, transcription factors (TFs) alter gene expression and determine cell states by binding to specific sequences in the genome. The human genome contains over 1,800 transcription factor loci, encoding over 3,500 isoforms, forming a vast network of possible regulatory outcomes. Overexpression of a single transcription factor can drive profound changes in cell fate. For example, a single transcription factor has been shown to direct the differentiation of pluripotent stem cells into many different types of cells, including muscle cells and neurons. Overexpression of a combination of transcription factors can produce even greater changes in gene regulatory networks. For instance, overexpression of four "Yamanaka factors" (Oct4, Sox2, Klf4, and c-Myc) can reprogram fibroblasts into stem cells. It has been reported that they can regulate the migration and homing of natural killer (NK) cells to the tumor microenvironment. These findings highlight the powerful ability of transcription factors to drive changes in cell states and underscore the utility of using transcription factor overexpression to understand the gene expression programs that control cell fate.

[0004] Therefore, an increasing number of studies are dedicated to how to use gene editing technologies to enhance and restore the killing of tumors, especially malignant solid tumor cells, by NK cells, thereby improving the efficacy of NK cell-based immunotherapy.

[0005] However, as mentioned above, for solid tumors, which account for the vast majority of tumors, due to their complex tumor microenvironment (TME), the killing effect of NK cells is limited during the treatment process, greatly restricting its therapeutic effect. Therefore, the design of NK cells for solid tumors urgently needs to be optimized. In addition, there is little existing research on NK gene editing, and this gene editing strategy severely relies on the understanding of existing immunological knowledge, with only a few known targets available for selection. There is still a huge space for exploration regarding whether there are unknown potential targets specific to chimeric antigen receptor (CAR) cells that specifically recognize tumor surface antigens of various specificities. Summary of the Invention

[0006] The present invention uses a screening system for a high-throughput overexpression transcription factor library to discover key transcription factors that can significantly enhance the anti-tumor activity of NK cells, clarify the mechanism of action of regulating the anti-tumor ability of NK cells, and further apply it to the construction of CAR-NK, providing new targets and strategies for improving the clinical efficacy of NK immunotherapy for solid tumors.

[0007] Therefore, on the one hand, the present invention provides an engineered NK cell comprising one or more overexpressed proteins selected from the group consisting of SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1.

[0008] SOX1, whose full name is "SRY-box Transcription Factor 1", has the amino acid sequence shown in MYSMMMET DLHSPGGAQAPTNLSGPAGAGGGGGGGGGGGGGGGAKANQDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKVMSEAEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLLKKDKYSLAGGLLAAGAGGGGAAVAMGVGVGVGAAAVGQRLESPGGAAGGGYAHVNGWANGAYPGSVAAAAAAAAMMQEAQLAYGQHPGAGGAHPHAHPAHPHPHHPHAHPHNPQPMHRYDMGALQYSPISNSQGYMSASPSGYGGLPYGAAAAAAAAAGGAHQNSAVAAAAAAAAASSGALGALGSLVKSEPSGSPPAPAHSRAPCPGDLREMISMYLPAGEGGDPAAAAAAAAQSRLHSLPQHYQGAGAGVNGTVPLTHI (SEQ ID NO:1).

[0009] ZNF205, whose full name is "Zinc Finger Protein 205", has the amino acid sequence shown in MSADGGGIQDTQD KETPPEVPDRGHPHQEMPSKLGEAVPSGDTQESLHIKMEPEEPHSEGASQEDGAQGAWGWAPLSHGSKEKALFLPGGALPSPRIPVLSREGRTRDRQMAAALLTAWSQMPVTFEDVALYLSREEWGRLDHTQQNFYRDVLQKKNGLSLGFPFSRPFWAPQAHGKGEASGSSRQAGDEKEWRGACTGAVEVGQRVQTSSVAALGNVKPFRTRAGRVQWGVPQCAQEAACGRSSGPAKDSGQPAEPDRTPDAAPPDPSPTEPQEYRVPEKPNEEEKGAPESGEEGLAPDSEVGRKSYRCEQCGKGFSWHSHLVTHRRTHTGEKPYACTDCGKRFGRSSHLIQHQIIHTGEKPYTCPACRKSFSHHSTLIQHQRIHTGEKPYVCDRCAKRFTRRSDLVTHQGTHTGAKPHKCPICAKCFTQSSALVTHQRTHTGVKPYPCPECGKCFSQRSNLIAHNRTHTGEKPYHCLDCGKSFSHSSHLTAHQRTHRGVRPYACPLCGKSFSRRSNLHRHEKIHTTGPKALAMLMLGAAAAGALATPPPAPT (SEQ ID NO:2).

[0010] TCF7L1, whose full name is "Transcription Factor 7-Like 1", has the amino acid sequence shown in (SEQ ID NO:3): MPQLGGGGGG GGGGSGGGGGSSAGAAGGGDDLGANDELIPFQDEGGEEQEPSSDSASAQRDLDEVKSSLVNESENQSSSSDSEAERRPQPVRDTFQKPRDYFAEVRRPQDSAFFKGPPYPGYPFLMIPDLSSPYLSNGPLSPGGARTYLQMKWPLLDVPSSATVKDTRSPSPAHLSNKVPVVQHPHHMHPLTPLITYSNDHFSPGSPPTHLSPEIDPKTGIPRPPHPSELSPYYPLSPGAVGQIPHPLGWLVPQQGQPMYSLPPGGFRHPYPALAMNASMSSLVSSRFSPHMVAPAHPGLPTSGIPHPAIVSPIVKQEPAPPSLSPAVSVKSPVTVKKEEEKKPHVKKPLNAFMLYMKEMRAKVVAECTLKESAAINQILGRKWHNLSREEQAKYYELARKERQLHSQLYPTWSARDNYGKKKKRKREKQLSQTQSQQQVQEAEGALASKSKKPCVQYLPPEKPCDSPASSHGSMLDSPATPSAALASPAAPAATHSEQAQPLSLTTKPETRAQLALHSAAFLSAKAAASSSGQMGSQPPLLSRPLPLGSMPTALLASPPSFPATLHAHQALPVLQAQPLSLVTKSAH

[0011] KLF11, also known as "Krüppel-like Factor 7", has the amino acid sequence shown in MHTPDFAGPDDARAVD IMDICESILERKRHDSERSTCSILEQTDMEAVEALVCMSSWGQRSQKGDLLRIRPLTPVSDSGDVTTTVHMDAATPELPKDFHSLSTLCITPPQSPDLVEPSTRTPVSPQVTDSKACTATDVLQSSAVVARALSGGAERGLLGLEPVPSSPCRAKGTSVIRHTGESPAACFPTIQTPDCRLSDSREGEEQLLGHFETLQDTHLTDSLLSTNLVSCQPCLHKSGGLLLTDKGQQAGWPGAVQTCSPKNYENDLPRKTTPLISVSVPAPPVLCQMIPVTGQSSMLPAFLKPPPQLSVGTVRPILAQAAPAPQPVFVGPAVPQGAVMLVLPQGALPPPAPCAANVMAAGNTKLLPLAPAPVFITSSQNCVPQVDFSRRRNYVCSFPGCRKTYFKSSHLKAHLRTHTGEKPFNCSWDGCDKKFARSDELSRHRRTHTGEKKFVCPVCDRRFMRSDHLTKHARRHMTTKKIPGWQAEVGKLNRIASAESPGSPLVSMPASA (SEQ ID NO:4).

[0012] ZNF844, whose full name is "Zinc Finger Protein 844", has the amino acid sequence shown in MDLVAFEDVAVNF TQEEWSLLDPSQKNLYREVMQETLRNLASIGEKWKDQNIEDQYKNPRNNLRSLLGERVDENTEENHCGETSSQIPDDTLNKKTSPGVKSCESSVCGEVFVGHSSLNRHIRADTAHKPSEYQEYGQEPYKCQQRKKAFRCHPSFQMQEKAHTGEKLYDCKECGKTFISHSSIQRHMIMHNGDGTYKCKFCGKACPCLSIYLIHERVHTGEKPYKCKQCGKAFSYSTSLQIHERTHTGEKPYECKECGKAFGSPNSLYEHRRTHTGEKPYECKQCGKAFRWFHSFQIHERTHSEEKAYECTKCGKAFKCPSYLCRHEVTHSGKKPCECKQCGKALSYLNFQRHMKMHTRMRPYKCKTVEKPLILPVRFEDMKELTLERNLMNASTVVKPSIVPVPFTIMKGLTLERNPMNVSSVVKPSFLPLPFDIMKGLTLERNRMSVSNVGKPSDLPHTFKCMEGLTLKRNPMNVSSVVKPSFFPLPFDIMKGLTLERNPMSVSNVGKPSHLPHTFKCMKGLTLESNCMNLNNVKKPLDLSETFKFMKRHTLERNPIRNMEKHSTISLPFKYMQQCTEDRMPMNVKSVTKHSYLPRSFEYMQEHTLERNPMNVRNAEKRSIIFLLCVYTKGCTLERNHINVRIVGKHSVCLVPFVDIKGLTLE (SEQ ID NO:5).

[0013] PSIP1, whose full name is "PC4 and SFRS1 interacting protein 1", has the amino acid sequence shown in MTRDF KPGDLIFAKMKGYPHWPARVDEVPDGAVKPPTNKLPIFFFGTHETAFLGPKDIFPYSENKEKYGKPNKRKGFNEGLWEIDNNPKVKFSSQQAATKQSNASSDVEVEEKETSVSKEDTDHEEKASNEDVTKAVDITTPKAARRGRKRKAEKQVETEEAGVVTTATASVNLKVSPKRGRPAATEVKIPKPRGRPKMVKQPCPSESDIITEEDKSKKKGQEEKQPKKQPKKDEEGQKEEDKPRKEPDKKEGKKEVESKRKNLAKTGVTSTSDSEEEGDDQEGEKKRKGGRNFQTAHRRNMLKGQHEKEAADRKRKQEEQMETEHFAL (SEQ ID NO:6).

[0014] In some embodiments, the engineered NK cells comprise overexpressed SOX1. In some other embodiments, the engineered NK cells comprise overexpressed ZNF205. In some other embodiments, the engineered NK cells comprise overexpressed TCF7L1. In some other embodiments, the engineered NK cells comprise overexpressed KLF11. In some other embodiments, the engineered NK cells comprise overexpressed ZNF844. In some other embodiments, the engineered NK cells comprise overexpressed PSIP1.

[0015] In some embodiments, the engineered NK cells further comprise overexpressed chimeric antigen receptor (CAR).

[0016] In further embodiments, the chimeric antigen receptor comprises:

[0017] (a) A signal peptide, for example, the signal peptide is derived from human CD8α, for example, the amino acid sequence of the signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID NO:7);

[0018] (b) Extracellular antigen-binding domain. For example, the extracellular antigen-binding domain comprises a GD2-binding protein or a mutant thereof. For example, the amino acid sequence of the extracellular antigen-binding domain is: GD2-binding protein: "EVQL LQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRA" (SEQ ID NO:8);

[0019] (c) Hinge region. For example, the hinge region is derived from human CD8α. For example, the amino acid sequence of the hinge region is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD (SEQ ID NO:9);

[0020] (d) Transmembrane domain. For example, the transmembrane domain is derived from human CD8α. For example, the amino acid sequence of the transmembrane domain is IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:10);

[0021] (e) Intracellular co-stimulatory signaling domain. For example, the intracellular co-stimulatory signaling domain is derived from human 4-1BB. For example, the amino acid sequence of the intracellular co-stimulatory signaling domain is KRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:11);

[0022] (f) Intracellular stimulatory signaling domain. For example, the intracellular stimulatory signaling domain is derived from human CD3ζ. For example, the amino acid sequence of the intracellular stimulatory signaling domain is RVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SE Q ID NO:12).

[0023] In some embodiments, the chimeric antigen receptor may be GD2-41BB-CAR, and its amino acid sequence is MALPVTALLLPLALLLHAARPEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:13).

[0024] In some embodiments, the engineered NK cells are obtained by genetically engineering NK cells to overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1.

[0025] In a further embodiment, the engineered NK cells are obtained by genetically engineering NK cells to simultaneously (1) overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, and (2) overexpress a chimeric antigen receptor (CAR).

[0026] In some embodiments, the genetic engineering means may be: infection by viral vectors (such as lentivirus infection, adenovirus, adeno-associated virus, and retroviral vectors, etc.), transposon-mediated, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat, such as CRISPR activation, etc.), and non-coding RNAs (such as Long-noncoding RNA, microRNA, etc.).

[0027] In some embodiments, the NK cells may be peripheral blood-derived NK cells (PB-NK), umbilical cord blood-derived NK cells (Umbilical Cord Blood, UCB-NK), induced pluripotent stem cell-derived NK cells (Induced Pluripotent Stem Cells, Ipsc-NK), and immortalized tumor-derived NK cells (such as NK-92), and so on.

[0028] In some embodiments, the engineered NK cells are obtained by a method comprising the following steps: co-culturing NK cells derived from healthy donors with feeder cells in vitro, adding a lentivirus suspension loaded with the coding sequences of one or more transcription factors selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 and an optional CAR, and then performing centrifugal infection with a cationic agent. The number of days of in vitro culture can be, for example, 10 days, but is not limited thereto.

[0029] In a specific embodiment, the engineered NK cells are obtained by a method comprising the following steps: co-culturing NK cells derived from healthy donors with feeder cells in vitro at a cell number ratio of 1:1. After 10 days of co-culture, the NK cells are transferred to a 24-well plate pre-coated with Retronectin at a density of 0.5 Million / well. Then, a lentivirus suspension loaded with the coding sequences of one or more transcription factors selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 and an optional CAR and a cationic agent are added, followed by centrifugal infection. After 24 hours, the cells are replenished with medium. The positive infection rate can be detected 72 hours after centrifugal infection, and subsequent functional experiments can be performed.

[0030] In the present invention, the "feeder cells" refer to the parental K562 erythroleukemia cell line modified by viral / non-viral transduction methods to overexpress NK cell-stimulating proteins (such as 4-1BB ligand, membrane-bound interleukin 21, etc.), which are used to provide growth support for NK cells in in vitro culture. In some embodiments, the feeder cells can be commercially available products, such as ZY-NKZ-0104 from Zhongying Biology in Hangzhou.

[0031] On the other hand, the present invention provides a method for preparing engineered NK cells, which includes the step of genetically engineering NK cells to overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1.

[0032] In a further embodiment, the method for preparing engineered NK cells includes the steps of genetically engineering NK cells to simultaneously (1) overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, and (2) overexpress a chimeric antigen receptor (CAR).

[0033] In some embodiments, the genetic engineering means can be: infection by viral vectors (such as lentivirus infection, adenovirus, adeno-associated virus, and retroviral vectors, etc.), transposon-mediated, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat, such as CRISPR activation, etc.), and non-coding RNAs (such as Long-noncoding RNA, microRNA, etc.).

[0034] In some embodiments, the NK cells can be peripheral blood-derived NK cells (PB-NK), umbilical cord blood-derived NK cells (UCB-NK), induced pluripotent stem cell-derived NK cells (Induced Pluripotent Stem Cells, Ipsc-NK), and immortalized tumor-derived NK cells (such as NK-92), etc. In a further embodiment, the NK cells can be obtained by ex vivo expansion. The ex vivo expansion can be achieved by ex vivo expansion methods based on cytokines (such as IL-2, IL-15, IL-21, etc.), expansion methods using feeder cells, magnetic bead enrichment methods, etc.

[0035] In some embodiments, the method for preparing the engineered NK cells comprises the following steps: co-culturing NK cells derived from a healthy donor with feeder cells in vitro, adding a lentiviral suspension loaded with the coding sequences of one or more transcription factors selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 and an optional CAR, and a cationic agent, followed by centrifugal infection. The number of days of in vitro culture can be, for example, 10 days, but is not limited thereto.

[0036] In a specific embodiment, the method for preparing the engineered NK cells comprises the following steps: co-culturing NK cells derived from a healthy donor with feeder cells in vitro at a cell number ratio of 1:1. After 10 days of co-culture, transfer the NK cells to a 24-well plate pre-incubated with Retronectin at a quantity of 0.5 Million / well, add an appropriate amount of a lentiviral suspension loaded with the coding sequences of one or more transcription factors selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 and an optional CAR, and a cationic agent, followed by centrifugal infection. After 24 hours, replenish the cells with liquid. The positive infection rate can be detected 72 hours after centrifugal infection, and subsequent functional experiments can be carried out.

[0037] In the preparation method of the present invention, the feeder cells are as described above.

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising the aforementioned engineered NK cells and a pharmaceutically acceptable carrier.

[0039] On another aspect, the present invention provides the use of the aforementioned engineered NK cells or the aforementioned pharmaceutical composition in the preparation of an anti-tumor drug.

[0040] In some embodiments, the tumor is selected from glioblastoma (GBM).

[0041] On yet another aspect, the present invention provides a method for treating a tumor in a subject, comprising administering a therapeutically effective amount of the aforementioned engineered NK cells to a subject in need thereof.

[0042] In some embodiments, the tumor is selected from glioblastoma (GBM).

[0043] This application screened a human overexpressed transcription factor library for NK cells induced from healthy human peripheral blood mononuclear cells (PBMC). Six potential transcription factor targets that could enhance the killing function of NK cells were obtained through bioinformatics analysis of NK cells. NK cells overexpressing transcription factors were constructed, and the killing effects of the NK cells overexpressing transcription factors on several glioblastoma stem cells (GSC) were further verified at the cellular and animal levels. Compared with the NK cell treatment strategy without overexpression, several targets (such as ZNF205 and TCF7L1) that had good effects on solid tumors could significantly improve the anti-tumor effect of NK cells by about 20%-50% and significantly extend the survival time of tumor-bearing NSG mice. Brief Description of the Drawings

[0044] Figure 1 : Flow chart of high-throughput overexpression transcription factor library screening on PB-NK.

[0045] Figure 2 : In vitro screening results of human transcription factor overexpression library on PB-NK from two healthy donors.

[0046] Figure 3 : Detection of the killing effects of NK cells overexpressing transcription factor targets on different glioblastoma stem cells at different effector-to-target ratios and co-culture times of 24 hours (A, tumor cells were added once at 0 hour and detected after 24 hours) and 48 hours (B, tumor cells were added at 0 and 24 hours, and then detected after another 24 hours (a total of 48 hours)) using an enzyme-linked immunosorbent assay reader.

[0047] Figure 4 : Flow chart of NK treatment for orthotopic tumor-bearing mice.

[0048] Figure 5 : Graph showing the effects of NK cell treatment overexpressing ZNF205 on the survival time (A) and tumor signal generation (B) of orthotopic tumor-bearing mice.

[0049] Figure 6 : Graph showing the effects of NK cell treatment overexpressing TCF7L1 on the survival time (A) and tumor signal generation (B) of orthotopic tumor-bearing mice.

[0050] Figure 7: The killing of different glioblastoma stem cells by NK cells overexpressing GD2-41BB-CAR, NK cells overexpressing ZNF205, and NK cells overexpressing both GD2-41BB-CAR and ZNF205 was determined using an enzyme-linked immunosorbent assay (ELISA) at different effector-to-target ratios and co-culture times of 24 hours (A) and 48 hours (B).

[0051] In the figure, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Detailed implementation manners

[0052] In the following, the present invention will be described in detail by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0054] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0055] Example 1: Screening for key transcription factors that enhance NK cell therapy in vitro using a human transcription factor overexpression library.

[0056] Step 1: NK cells (from blood banks) induced from healthy human peripheral blood mononuclear cells (PBMCs) were used, and feeder cells (Hangzhou Zhongying Biotechnology, ZY-NKZ-0104) that were irradiated and overexpressed the NK-stimulating molecule 4-1BBL and membrane-bound IL-21 (mbIL-21) were used for in vitro stimulation and expansion culture of NK cells. Meanwhile, a lentivirus loaded with a barcode library (Transcription Factor Library, see Joung, J. et al. (2023). A transcription factor atlas of directed differentiation. Cell 186, 209–229.) including different splicing forms of all human transcription factors was successfully produced using a baboon envelope pseudotyped lentiviral vector (BaEV-LV, see Girard-Gagnepain, A., Amirache, F., Costa, C., Le′vy, C., Frecha, C., Fusil, F., Ne`gre, D., Lavillette, D., Cosset, F.L., and Verhoeyen, E. (2014). Baboon retrovirus envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early cytokine-stimulated and resting HSCs. Blood 124, 1221–1231.). Step 2: After 10 days of in vitro expansion of NK cells, the NK cells were transferred to a 24-well plate pre-incubated with Retronectin at a density of 0.5 million cells per well. After adding an appropriate amount of the above lentiviral suspension and a cationic agent (protamine sulfate), the NK cells were centrifuged for infection and re-supplemented with culture medium 24 hours later. Step 3: 72 hours after centrifugation infection, the NK cells were challenged in vitro with the glioblastoma cell line MGG6 (the tumor cell line was a gift from Professor Hiroaki Wakimoto) for multiple rounds. The co-culture time for each round of challenge was 24 hours, and a total of 2 rounds of challenge were performed. Step 4: NKp46-positive, NKp46-negative NK cells, and unsorted NK cells were collected as a control group by flow cytometry sorting. Step 5: Cell genomic DNA was extracted for library construction and sequencing. Step 6: Transcription factors specifically enriched in NKp46-positive NK cells compared to NKp46-negative NK cells and control NK cells were identified by bioinformatics analysis. The flow chart is shown inFigure 1 。

[0057] The result analysis diagram can be seen in Figure 2 。 Figure 2 The data shows that by taking the intersection of the NK screening results from two healthy donors, selecting the log2 value of the enrichment fold of NKp46-positive NK cells greater than or equal to 1 compared to NKp46-negative cells, and restricting the size of the overexpressed genes to less than 2000 bp, a total of 6 potential targets were obtained for further functional verification.

[0058] Table 1: Six potential human transcription factor targets generated from in vitro screening results

[0059]

[0060] Example 2: Construction of NK cells overexpressing the above 6 transcription factor targets of SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 respectively

[0061] The amino acid sequences of the six transcription factors, SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, were cloned into the pCDH-CMV-eGFP (CD511B-1, systembio) plasmid, and the uncloned plasmid (empty plasmid, vector) was used as the control group. Taking a 10 cm dish as an example, the plasmid transfection ratio was as follows: target plasmid: psPAX2 (12260, Addgene): BaEV (Girard-Gagnepain, A., Amirache, F., Costa, C., Le′vy, C., Frecha, C., Fusil, F., Ne`gre, D., Lavillette, D., Cosset, F.L., and Verhoeyen, E. (2014). Baboon retrovirus envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early cytokine-stimulated and resting HSCs. Blood 124, 1221–1231.) = 8.6 μg: 8.6 μg: 7 μg, and virus packaging was carried out in HEK293T cells. After 48 hours, the cell supernatant containing the virus was collected and concentrated by overnight centrifugation at 3000 rcf. At the same time, NK cells derived from healthy donors were co-cultured in vitro with feeder cells (ZY-NKZ-0104, Hangzhou Zhongying Biotechnology) at a cell number ratio of 1:1. After 10 days of co-culture, the NK cells were transferred to a 24-well plate pre-incubated with Retronectin at a quantity of 0.5 Million / well, and appropriate lentivirus suspensions and cationic agents loaded with SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1 respectively were added for centrifugal infection. After 24 hours, the cells were replenished with fluid. The positive infection rate could be detected 72 hours after centrifugal infection, and subsequent functional experiments were carried out. The NK cells obtained using the empty plasmid were empty plasmid NK cells (NK + Vector).

[0062] Example 3: Killing effect of NK cells overexpressing 6 transcription factor targets on glioblastoma stem cells

[0063] Glioblastoma cells MGG6 (target cells) were placed in a 96-well plate at a density of 10,000 cells / well. The NK cells (effector cells) constructed in Example 2 were co-cultured with the target cells at effector-to-target ratios of 1:1 / 1:2 / 1:4, and three replicate wells were set up for each sample. After 24 and 48 hours of co-culture, flow cytometry was used to detect and calculate the percentage of effector cells killing target cells, and a killing curve was plotted. At the same time, an untreated control group (Control) and a group of empty vector NK cells (NK+Vector) using empty vector NK cells as effector cells were set up.

[0064] The results analysis is shown in Figure 3 . Figure 3 The data shows that the NK cells overexpressing ZNF205 and TCF7L1 constructed in this application exhibited superior anti-tumor killing ability compared to empty vector NK cells at different effector-to-target ratios and during 24-hour and 48-hour co-culture.

[0065] Example 4: Therapeutic effect of NK cells overexpressing ZNF205 on tumor-bearing mice

[0066] Patient-derived glioblastoma cells MGG6 (20,000 cells / 3 μl PBS) were injected into the intracranial cavity of NOD / SCID / IL2Rg- / -(NSG: NOD SCID Gamma; 005557, JAX Lab) mice using a stereotactic injector, with the coordinates of Bregma x = +2.0 mm, y = -2.0 mm, and z = -3.5 mm. Seven days after tumor implantation, in-situ NK cell injection therapy (2,000,000 cells / 36 μl PBS) was performed, and an untreated control group (denoted as Control or No treatment) and a group of empty vector NK cells (Vector+NK) using empty vector NK cells as effector cells were set up. The flow chart is shown in Figure 4 . After tumor injection, the tumor development was recorded, and the time points when the mice showed neurological symptoms were recorded, and a survival curve of tumor-bearing mice was plotted.

[0067] The results analysis is shown in Figure 5 . Figure 5 The data shows that the survival time of mice treated with NK cells overexpressing ZNF205 exceeded 100 days and no detectable tumor signals were generated, while although the survival time of tumor-bearing mice treated with empty vector NK cells was prolonged, tumor occurrence occurred to varying degrees. This result confirms that compared to the empty vector NK cells of the prior art, the NK cells overexpressing ZNF205 constructed in this application showed significantly excellent effects in tumor treatment.

[0068] Example 5: Therapeutic effect of NK cells overexpressing TCF7L1 on tumor-bearing mice

[0069] Patient-derived glioblastoma cells MGG6 (20,000 cells / 3 μl PBS) were injected into the intracranial cavity of NOD / SCID / IL2Rg- / - (NSG: NOD SCID Gamma; 005557, JAX Lab) mice using a stereotactic injector at the coordinates of Bregma x = +2.0 mm, y = -2.0 mm, and z = -3.5 mm. Seven days after tumor implantation, in-situ NK cell injection therapy (2,000,000 cells / 36 μl PBS) was performed. Meanwhile, a control group without NK treatment (denoted as Control or No treatment) and a vector + NK group using empty vector NK cells as effector cells were set up. The flow chart is shown in Figure 4 . After tumor injection, the tumor development was recorded, and the time points when the mice showed neurological symptoms were recorded, and the survival curve of the tumor-bearing mice was plotted.

[0070] The result analysis is shown in Figure 6 . Figure 6 The data showed that the survival time of the mice treated with NK cells overexpressing TCF7L1 exceeded 100 days and no detectable tumor signals were generated. Although the survival time of the tumor-bearing mice was prolonged in the mice treated with empty vector NK cells, tumor occurrence occurred to varying degrees. This result confirmed that compared with the empty vector NK cells of the prior art, the NK cells overexpressing TCF7L1 constructed in this application showed significantly excellent effects in tumor treatment.

[0071] Example 7: Construction of NK cells overexpressing GD2-41BB-CAR and ZNF205 separately or simultaneously The amino acid sequences of GD2-41BB-CAR and ZNF205 were separately or simultaneously linked by P2A (ATNFSLLKQAGDVEENPGP, SEQ ID NO:14) and cloned into the pCDH-CMV-eGFP plasmid. The uncloned plasmid (empty plasmid, vector) was used as a control group. Taking a 10 cm dish as an example, the plasmid transfection ratio was: target plasmid: psPAX2: BaEV = 8.6 μg: 8.6 μg: 7 μg, and virus packaging was carried out in HEK293T cells. After 48 hours, the cell supernatant containing the virus was collected and the virus was concentrated by overnight centrifugation at 3000 rcf. At the same time, NK cells derived from healthy donors and feeder cells were co-cultured in vitro at a cell number ratio of 1:1. After 10 days of co-culture, the NK cells were transferred to a 24-well plate pre-incubated with Retronectin at a quantity of 0.5 Million / well, and an appropriate amount of lentivirus suspension loaded with ZNF205 and a cationic agent were added and then centrifuged for infection. After 24 hours, the cells were replenished with liquid. The positive infection rate could be detected 72 hours after centrifugal infection, and subsequent functional experiments were carried out.

[0072] The amino acid sequence of the chimeric antigen receptor (GD2-41BB-CAR) is MALPVTALLLPLALLLHAARPEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:13).

[0073] Example 8: Killing effect of NK cells overexpressing GD2-41BB-CAR and ZNF205 separately or simultaneously constructed in Example 7 on glioblastoma stem cells

[0074] Glioblastoma cells MGG6 (target cells) were placed in a 96-well plate at a density of 10,000 cells / well. NK cells (effector cells) were co-cultured with target cells at effector-to-target ratios of 1:1 / 1:2 / 1:4, and each sample had 3 replicate wells. After 24 and 48 hours of co-culture, the percentage of effector cells killing target cells was detected by flow cytometry and calculated, and a killing curve was plotted. At the same time, an untreated control group (Control) and a vector group (Vector) using empty vector NK cells as effector cells were set up.

[0075] The results analysis is shown in Figure 7 。 Figure 7 The data show that the NK cells overexpressing GD2-41BB-CAR and ZNF205 simultaneously constructed in this application exhibited superior anti-tumor killing ability compared to the other three groups of NK cells at different effector-to-target ratios and co-culture times of 24 hours and 48 hours.

Claims

1. An engineered NK cell that comprises one or more overexpressed proteins selected from the group consisting of SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1.

2. The engineered NK cell according to claim 1, wherein: the engineered NK cell comprises overexpressed SOX1; the engineered NK cell comprises overexpressed ZNF205; the engineered NK cell comprises overexpressed TCF7L1; the engineered NK cell comprises overexpressed KLF11; the engineered NK cell comprises overexpressed ZNF844; or the engineered NK cell comprises overexpressed PSIP1.

3. The engineered NK cells according to claim 1 or 2, wherein, The engineered NK cell further comprises an overexpressed chimeric antigen receptor, preferably, the chimeric antigen receptor comprises: (a) a signal peptide, for example, the signal peptide is derived from human CD8α, for example, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:7; (b) an extracellular antigen-binding domain, for example, the extracellular antigen-binding domain comprises a GD2-binding protein or a mutant thereof, for example, the amino acid sequence of the extracellular antigen-binding domain is as shown in SEQ ID NO:8; (c) a hinge region, for example, the hinge region is derived from human CD8α, for example, the amino acid sequence of the hinge region is as shown in SEQ ID NO:9; (d) a transmembrane domain, for example, the transmembrane domain is derived from human CD8α, for example, the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO:10; (e) an intracellular co-stimulatory signaling domain, for example, the intracellular co-stimulatory signaling domain is derived from human 4-1BB, for example, the amino acid sequence of the intracellular co-stimulatory signaling domain is as shown in SEQ ID NO:11; (f) an intracellular stimulatory signaling domain, for example, the intracellular stimulatory signaling domain is derived from human CD3ζ, for example, the amino acid sequence of the intracellular stimulatory signaling domain is as shown in SEQ ID NO:12, more preferably, the chimeric antigen receptor is GD2-41BB-CAR, and its amino acid sequence is as shown in SEQ ID NO:

13.

4. The engineered NK cells according to any one of claims 1-3, wherein, The engineered NK cell is obtained by genetically engineering an NK cell to overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, preferably, the engineered NK cell is obtained by genetically engineering an NK cell to simultaneously (1) overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, and (2) overexpress a chimeric antigen receptor.

5. The engineered NK cells according to any one of claims 1-4, wherein, The NK cell is a peripheral blood-derived NK cell, a cord blood-derived NK cell, an induced pluripotent stem cell-derived NK cell, or an immortalized tumor-derived NK cell (such as NK-92).

6. A method for preparing engineered NK cells, which comprises the step of genetically engineering NK cells to overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1.

7. The preparation method according to claim 6, wherein, The method for preparing the engineered NK cells comprises the steps of genetically engineering NK cells to simultaneously (1) overexpress one or more proteins selected from SOX1, ZNF205, TCF7L1, KLF11, ZNF844, and PSIP1, and (2) overexpress a chimeric antigen receptor.

8. The preparation method according to claim 6, wherein, The NK cells are NK cells derived from peripheral blood, cord blood, induced pluripotent stem cells, or immortalized tumor-derived NK cells. Preferably, the NK cells can be obtained by ex vivo expansion. For example, the ex vivo expansion is by a cytokine-based expansion method, an expansion method using feeder cells, or an ex vivo expansion by magnetic bead enrichment.

9. A pharmaceutical composition comprising the engineered NK cells according to any one of claims 1-5 and a pharmaceutically acceptable carrier.

10. Use of the engineered NK cells according to any one of claims 1-5 or the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug. Preferably, the tumor is selected from glioblastoma multiforme (GBM).

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