ShRNA for inhibiting TIM-3 gene expression, immune cell and application thereof

Abstract

The invention relates to the technical field of biology, in particular to shRNA for inhibiting TIM-3 gene expression, an immune cell and application of the shRNA. According to the shRNA molecule and the application thereof, the shRNA molecule targets TIM-3 expression, and an efficient, specific and stable silencing effect can be kept; the TIM-3 gene is silenced, so that the killing capability of immune cells on tumor cells is effectively improved; the multiplication capacity and the viability of immune cells in a low-oxygen stress or low-oxygen microenvironment are effectively improved; the multiplication capacity and the viability of immune cells in a complex immunosuppression tumor microenvironment are effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to shRNAs for inhibiting TIM-3 gene expression, immune cells and their applications. Background Art

[0002] Gene-modified cell therapy is a novel treatment method that uses genetic engineering techniques to introduce foreign genes or regulatory factors into immune cells, endowing these immune cells with new functions or stronger therapeutic potential. Gene-modified cell technology is widely applied to immune cells such as NK cells, T cells, NKT cells, γδT cells and macrophages for immunotherapy.

[0003] Currently, immunotherapy with immune cells such as chimeric antigen receptor (CAR)-modified T cells and NK cells is a new targeted immune cell therapy developed in recent years. CAR-T and CAR-NK cells have achieved remarkable results in malignant hematological tumors, but their efficacy in solid tumors is still not ideal. One of the main reasons is that the immunosuppressive microenvironment of solid tumors and the immune escape mechanism of tumor cells themselves lead to dysfunction of CAR-T or CAR-NK cells and their inability to exert anti-tumor immune responses.

[0004] Tumor cells usually express ligands of inhibitory receptors such as TIM-3 (such as Galectin-9, CEACAM1), which inhibit the activation and effector functions of immune cells such as NK cells by binding to inhibitory receptors such as TIM-3 on the surface of immune cells, and even lead to exhaustion of immune cell functions. Tumors usually secrete immunosuppressive factors and metabolites such as TGF-β, and have a tumor microenvironment with hypoxia, low pH, nutrient deficiency, high osmotic pressure, etc., and lack mature blood vessel supply, all of which affect the activity and function of immune cells present in the microenvironment. Therefore, the microenvironment within tumors is very unfavorable for the localization, infiltration, survival, proliferation and function of immune cells, especially prone to inhibiting the functions of immune cells and even causing immune exhaustion.

[0005] Therefore, there is an urgent need to develop new immunotherapy strategies to improve the activity, killing ability and resistance to exhaustion of immune cells in the complex tumor microenvironment and stimulate the potential of immune cell therapy for solid tumors. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide an shRNA molecule and its application, the shRNA molecule targets TIM-3 expression and can maintain an efficient, specific and stable silencing effect; silencing the TIM-3 gene effectively improves the killing ability of immune cells against tumor cells; effectively improves the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment; effectively improves the proliferation ability and survival ability of immune cells in a complex immunosuppressive tumor microenvironment.

[0007] To this end, the first aspect of the present invention provides an shRNA molecule. According to an embodiment of the present invention, the shRNA molecule includes a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is as shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31.

[0008] The present invention proposes an shRNA that specifically targets the TIM-3 molecule, and the shRNA sequence with the best silencing effect is obtained by screening. The shRNA according to the embodiment of the present invention can inhibit the expression of the TIM-3 molecule on immune cells, enhance the killing ability of immune cells against tumors with high expression of the TIM-3 receptor ligand Galectin-9 and CEACAM1, and further improve the anti-tumor activity of immune cells.

[0009] The shRNA targeting TIM-3 expression screened by the present invention can maintain an efficient, specific and stable silencing effect; silencing the TIM-3 gene effectively improves the killing ability of immune cells against tumors; effectively improves the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment; effectively improves the proliferation ability and survival ability of immune cells in a complex immunosuppressive tumor microenvironment.

[0010] According to an embodiment of the present invention, the shRNA molecule further includes a stem-loop structure, the stem-loop structure connects the sense strand fragment and the antisense strand fragment, and the stem-loop structure includes 4-11 nucleotides.

[0011] According to an embodiment of the present invention, the DNA sequence encoding the shRNA molecule is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12.

[0012] The second aspect of the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector includes a DNA sequence expressing the shRNA molecule described in the first aspect.

[0013] According to an embodiment of the present invention, the expression vector is selected from any one of lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

[0014] The third aspect of the present invention provides a microorganism. According to an embodiment of the present invention, the microorganism includes the expression vector described in the second aspect.

[0015] The fourth aspect of the present invention provides a host cell. According to an embodiment of the present invention, the host cell includes the expression vector described in the second aspect.

[0016] The fifth aspect of the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes at least one of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect.

[0017] The sixth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, the microorganism described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug is used for the prevention and / or treatment of tumors.

[0018] According to an embodiment of the present invention, the tumor is a TIM-3 ligand-positive tumor, including solid tumors and hematological tumors.

[0019] According to an embodiment of the present invention, the solid tumors include liver cancer, colon cancer, breast cancer, and cervical cancer.

[0020] The seventh aspect of the present invention provides a method for reducing the expression of the TIM-3 gene in a target cell. According to an embodiment of the present invention, the method includes: introducing the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the target cell,

[0021] or infecting the target cell with the microorganism described in the third aspect.

[0022] The target cells include, but are not limited to, immune cells for immunotherapy such as NK cells, T cells, NKT cells, Treg cells, γδT cells, and macrophages.

[0023] The eighth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect in the preparation of a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit is used to reduce the expression of TIM-3 and / or NKG2A and / or TIGIT in a target cell, and / or increase the expression of at least one of NKG2D, NKp30, and CD16 in the target cell.

[0024] The ninth aspect of the present invention provides a method for enhancing the killing ability of immune cells against tumor cells. According to an embodiment of the present invention, the method includes:

[0025] Introducing the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infecting the immune cells with the microorganism described in the third aspect, so that the TIM-3 gene is lowly expressed in the immune cells.

[0026] The tenth aspect of the present invention provides a reagent or a kit for enhancing the killing ability of immune cells against tumor cells. According to an embodiment of the present invention, the reagent or the kit includes at least one of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect.

[0027] The eleventh aspect of the present invention provides a method for enhancing the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment. According to an embodiment of the present invention, the method includes:

[0028] Introducing the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infecting the immune cells with the microorganism described in the third aspect, so that the TIM-3 gene is lowly expressed in the immune cells.

[0029] The twelfth aspect of the present invention provides a method for enhancing the proliferation ability and survival ability of immune cells in a tumor microenvironment. According to an embodiment of the present invention, the method includes:

[0030] Introducing the shRNA molecule described in the first aspect and / or the expression vector described in the second aspect into the immune cells, or infecting the immune cells with the microorganism described in the third aspect, so that the TIM-3 gene is lowly expressed in the immune cells.

[0031] The thirteenth aspect of the present invention provides the use of the shRNA molecule described in the first aspect, the expression vector described in the second aspect, and the microorganism described in the third aspect in the following:

[0032] Enhancing the killing ability of immune cells against tumor cells;

[0033] To improve the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment;

[0034] To improve the proliferation ability and survival ability of immune cells in an immunosuppressive tumor microenvironment,

[0035] Optionally, the tumor is a TIM-3 ligand-positive tumor, including solid tumors and hematological tumors.

[0036] Current studies have shown that during the in vitro activation and expansion of immune cells or in the tumor microenvironment, the TIM-3 immune checkpoint molecule is often highly expressed. Therefore, when immune cells enter a complex tumor microenvironment containing a large amount of TIM-3 ligand Galectin-9 protein derived from tumor cells, etc., their immune killing ability will be greatly inhibited. Based on this, the inventor constructed an shRNA expressing a silenced immunosuppressive receptor TIM-3 for modifying immune cells.

[0037] The immune cells modified by silencing TIM-3 of the present invention significantly enhanced the in vitro killing ability against tumor cells highly expressing the TIM-3 ligand. The inventor also unexpectedly found that silencing TIM-3 could improve the proliferation ability and survival ability of immune cells in a hypoxic and tumor microenvironment. Moreover, the inventor also unexpectedly found through experiments that the application of this shRNA molecule could further reduce the expression of other inhibitory receptors (such as NKG2A, TIGIT, etc.) of immune cells and increase the expression of activation receptors (such as NKG2D, NKp30, CD16) of immune cells, thereby further eliminating or resisting the influence of various immunosuppressive signals in the tumor microenvironment and avoiding immune exhaustion. Therefore, the targeted TIM-3 shRNA designed by the present invention not only improves its antitumor activity through the blocking effect of its own inhibitory signaling pathway, but also can further enhance its ability to adapt to and resist the tumor microenvironment, enabling it to maintain vitality, proliferation ability and killing activity even in a complex hypoxic and tumor immunosuppressive microenvironment, thereby increasing its potential for treating solid tumors and having clinical application value for preventing and / or treating tumors or cancers.

[0038] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0040] Figure 1 Shows the detection of the expression level of TIM3 protein in NK cells expanded from peripheral blood in Example 1 by flow cytometry;

[0041] Figure 2 It shows the expression levels of TIM-3 mRNA in NK cells transfected with different TIM-3 shRNAs in Example 1 detected by qRT-PCR;

[0042] Figure 3 It shows the expression levels of TIM-3 protein in NK cells transfected with different TIM-3 shRNAs in Example 1 detected by flow cytometry. Among them, from bottom to top in the left figure are the expression levels of TIM-3 protein in NK cells transfected with NTC, TIM3-sh01 to TIM3-sh16, and the right figure is the column statistical result chart;

[0043] Figures 4A-4B It shows the levels of TIM-3 ligand expression in different tumor cells detected by flow cytometry in Example 2;

[0044] Figure 5 It shows the killing efficiencies of NK cells transfected with TIM-3 shRNA and NTC respectively in Example 2 against tumor cells with high expression of TIM-3 ligand;

[0045] Figure 6 It shows the expression of inhibitory receptors NKG2A, TIGIT, LAG3, PD-1 and the expression of activating receptors NKG2D, NKp30, NKp44 and CD16 on the surface of NK cells with silenced TIM-3 detected by flow cytometry in Example 3;

[0046] Figure 7 A - D in it show the proliferation and survival results of NK cells with silenced TIM-3 under normoxia and hypoxia conditions in Example 4. Among them, A and B show the proliferation results of NK cells with silenced TIM-3 under normoxia and hypoxia conditions; C and D show the survival results of NK cells with silenced TIM-3 and control cells under hypoxia conditions;

[0047] Figure 8 A and B in it show the concentrations of Galectin-9 protein in the culture supernatants of different tumor cells (PLC-PRF-5, HepG2, SW620, HCT116, MDA-MB-231 and Hela cells) and non-tumor cells (HEK293 and HMC3) detected by ELISA;

[0048] Figure 9A - D in the figure show the proliferation and survival results of TIM - 3 - silenced NK cells and control cells in Example 5 in normal medium and tumor - conditioned medium; among them, A and B show the proliferation results of TIM - 3 - silenced NK cells and control cells in normal medium and tumor - conditioned medium; C and D show the survival results of TIM - 3 - silenced NK cells and control cells in tumor - conditioned medium. Detailed implementation mode

[0049] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0050] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0051] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0052] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0053] In this document, the term "comprising" or "including" is an open - ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0054] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0055] TIM-3 (T cell immunoglobulin and mucin-domain containing-3, also known as CD366, HAVCR2) is a negatively regulated immune checkpoint protein and a transmembrane protein encoded by the HAVCR2 gene. It was first discovered in CD4+ and CD8+ effector T cells capable of secreting IFN-γ. Now it is found that TIM-3 is widely expressed in a variety of immune cells, including: CD4+ T cell subsets (Th1 / Th17 cells), regulatory T cells (Tregs), CD8+ T cell subsets, and innate immune cells (DCs, NK cells, monocytes, macrophages, etc.). TIM-3 has multiple ligands, such as galectin-9 (Galectin-9, the main ligand), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM-1), high mobility group protein B1 (HMGB1), phosphatidylserine (PtdSer). Multiple studies have shown that TIM-3 binds to its important ligands to transmit negative regulatory signals and inhibit the activation and function of immune cells. Therefore, TIM-3 is considered an important immune checkpoint, which can prevent the over-activation of immune cells, maintain the body's homeostasis, and play a key role in the regulation of transplantation immune tolerance, autoimmunity, allergy, and asthma. However, in the tumor or chronic inflammation microenvironment, immune cells overexpress TIM-3. After TIM-3 binds to its ligand, it inhibits the activities of immune effector cells such as T cells and NK cells, which is the key factor leading to the exhaustion of T cell and NK cell functions in the tumor microenvironment and the main reason for tumor immune escape. Therefore, it is urgent to further design and develop new immunotherapy strategies targeting TIM-3 to improve the killing ability and resistance to exhaustion of immune cells in the complex tumor microenvironment and stimulate the potential of immune cells to treat solid tumors. The present invention is made based on the above content.

[0056] According to a specific embodiment of the present invention, the present invention provides an shRNA molecule, including a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is as shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31. The target nucleotide sequences of these shRNA molecules are as shown in SEQ ID NO:20, which can inhibit the expression of TIM-3 gene.

[0057] According to a specific embodiment of the present invention, the shRNA molecule further comprises a stem-loop structure that connects the sense strand fragment and the antisense strand fragment, and the stem-loop structure comprises 4-11 nucleotides. Sequences of those stem-loop structures commonly used in the art when designing shRNA molecules are applicable to the shRNA molecules of the present invention and are all covered within the protection scope of the present invention.

[0058] According to a specific embodiment of the present invention, the DNA sequence encoding the shRNA molecule is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12.

[0059] According to a specific embodiment of the present invention, the present invention provides an expression vector comprising the DNA sequence expressing the aforementioned shRNA molecule.

[0060] According to a specific embodiment of the present invention, the expression vector further contains a promoter that drives the transcription of RNA polymerase III.

[0061] According to a specific embodiment of the present invention, the expression vector is a lentiviral vector. It should be noted that the expression vector includes but is not limited to the PLKO.1-CMV-PURO lentiviral vector.

[0062] According to a specific embodiment of the present invention, the expression vector is a recombinant vector obtained by inserting the DNA sequence expressing the aforementioned shRNA molecule into a lentiviral vector.

[0063] According to a specific embodiment of the present invention, the present invention provides a microorganism comprising the aforementioned expression vector. The microorganism can be, for example, a recombinant bacterium or a recombinant virus. The recombinant virus can be, for example, a lentivirus or others, and all are covered within the protection scope of the present invention.

[0064] It should be noted that the present invention has designed multiple DNA sequences expressing shRNA molecules for the target gene TIM-3. The shRNA molecules expressed by these DNA sequences have different silencing efficiencies for the gene TIM-3. To further improve the silencing efficiency of the target gene, multiple vectors can also be introduced into the recombinant microorganism simultaneously, and these vectors carry the DNA sequences of different shRNA molecules respectively.

[0065] According to a specific embodiment of the present invention, the present invention provides a host cell comprising the aforementioned expression vector. The host cell can be, for example, various immune cells, such as NK cells, T cells, B cells, NKT cells, γδT cells, Tregs, macrophages, and various cells derived from iPSCs, etc.

[0066] According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising at least one of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism.

[0067] According to a specific embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0068] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, the aforementioned microorganism, the aforementioned host cell, and the aforementioned pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors. The drug can also be used to eliminate or mitigate the immune escape mechanism of tumors.

[0069] According to a specific embodiment of the present invention, the tumor is a TIM-3 ligand-positive tumor, including solid tumors and hematological tumors;

[0070] According to a specific embodiment of the present invention, the solid tumors include, but are not limited to, liver cancer, colon cancer, breast cancer, and cervical cancer.

[0071] According to a specific embodiment of the present invention, the present invention provides a method for reducing the expression of TIM-3 gene in target cells, comprising: introducing the aforementioned shRNA molecule and / or the aforementioned expression vector into the target cells,

[0072] or infecting the target cells with the aforementioned microorganism.

[0073] It should be noted that there are no particular restrictions on the type of the target cells. For example, they can be NK cells, T cells, B cells, NKT cells, γδT cells, Tregs, macrophages, and various cells derived from iPSCs, etc.

[0074] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism in the preparation of a reagent or kit for reducing the expression of TIM-3 and / or NKG2A and / or TIGIT in target cells, and / or increasing the expression of at least one of NKG2D, NKp30, and CD16 in target cells.

[0075] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the killing ability of immune cells against tumor cells, and the method includes:

[0076] Introducing the aforementioned shRNA molecule and / or the aforementioned expression vector into the immune cells, or infecting the immune cells with the aforementioned microorganism, such that the TIM-3 gene is lowly expressed in the immune cells.

[0077] According to a specific embodiment of the present invention, the present invention provides a reagent or kit for enhancing the ability of immune cells to kill tumor cells, and the reagent or kit includes at least one of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism.

[0078] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the proliferation ability and survival ability of NK cells under hypoxic stress or in a hypoxic microenvironment, including:

[0079] Introducing the aforementioned shRNA molecule and / or the aforementioned expression vector into the immune cells, or infecting the immune cells with the aforementioned microorganism, such that the TIM-3 gene is lowly expressed in the immune cells.

[0080] According to a specific embodiment of the present invention, the present invention provides a method for enhancing the proliferation ability and survival ability of immune cells in a tumor microenvironment, and the method includes:

[0081] Introducing the aforementioned shRNA molecule and / or the aforementioned expression vector into the immune cells, or infecting the immune K cells with the aforementioned microorganism, such that the TIM-3 gene is lowly expressed in the immune cells.

[0082] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned shRNA molecule, the aforementioned expression vector, and the aforementioned microorganism in the following:

[0083] Enhancing the killing ability of immune cells against tumor cells;

[0084] Enhancing the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment;

[0085] Enhancing the proliferation ability and survival ability of immune cells in a tumor immunosuppressive microenvironment.

[0086] According to a specific embodiment of the present invention, the tumor includes solid tumors and hematological tumors.

[0087] The shRNA molecule provided by the present invention, the most direct function of silencing the TIM-3 gene is to block the inhibitory signal mediated by TIM-3, weaken the inhibition of the tumor microenvironment on immune cells, resist exhaustion, and improve the killing ability against tumor cells. However, there is no relevant report on the regulation of TIM-3 on cell proliferation and the hypoxic microenvironment. This study unexpectedly found that silencing the TIM-3 gene can not only improve the killing function of immune cells, but also improve the survival and proliferation ability of immune cells in the hypoxic and immunosuppressive microenvironments.

[0088] For the sequence description involved in the present invention, please refer to Table 1 for details.

[0089] Table 1: Table of amino acid / nucleotide sequence description

[0090]

[0091]

[0092]

[0093] The present invention provides an shRNA for inhibiting the expression of the TIM-3 gene and its application. The target nucleotide sequence of the shRNA for inhibiting the expression of the TIM-3 gene is as shown in SEQ ID NO: 20. The above shRNA can continuously, stably, highly efficiently and specifically inhibit the expression of the TIM-3 gene. Inhibiting the expression of TIM-3 in immune cells can significantly improve the killing ability of immune cells, and improve the survival ability and proliferation ability of immune cells in complex tumor microenvironments and hypoxic microenvironments, thereby improving the therapeutic effect of immune cells in tumor immunotherapy. The present invention provides a new idea and new strategy for improving the ability of immune cells to resist the inhibition of the tumor microenvironment and immune exhaustion and enhancing the anti-tumor immune efficacy of immune cells, especially playing a promoting role in improving the efficacy of immune cell treatment for solid tumors.

[0094] Next, the solutions of the present disclosure will be explained in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0095] Example 1 Design and screening of shRNA for silencing the TIM-3 gene

[0096] 1. Design of RNAi target sequence

[0097] The gene ID of Homo sapiens HAVCR2 hepatitis A virus cellular receptor 2 (TIM-3) found on the NCBI website is NM_032782.5, and the coding sequence (CDS) of the TIM-3 gene is shown as SEQ ID NO:19. Sixteen RNAi target sequences were designed and screened in the TIM-3 gene coding sequence (the nucleotide sequence is shown as SEQ ID NO:20).

[0098] According to the designed RNAi target sequences, shRNA sequences for constructing shRNA lentiviral vectors were designed. The coding sequences of the designed TIM-3 shRNA (TIM-3-sh01 to TIM-3-sh16, the nucleotide sequences are shown as SEQ ID NO:2 to 17) and the non-sense control sequence (Non-targeting control, NTC, the nucleotide sequence is shown as SEQ ID NO:18) have the structure of sense, loop, antisense, and termination sequences. According to the restriction enzyme sites of the PLKO.1-CMV-PURO lentiviral vector, the AgeI and EcoRI restriction enzyme sites were selected to insert the shRNA sequences respectively, and the obtained plasmids were named pLKO.1-TIM-3-sh01 to pLKO.1-TIM-3-sh16 and PLKO.1-NTC respectively.

[0099] 2. Packaging of Lentivirus and Concentration of Virus Liquid

[0100] Take 5×10 6 cells in the logarithmic growth phase and inoculate them into a 10-cm cell culture dish, add 10 mL of DMEM medium, and culture them overnight in a 37°C, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80% - 90%, replace it with 10 mL of fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby. Prepare the lentivirus packaging system and add the lentivirus packaging mixture to the 293T cell culture dish. After 24 hours, change the medium, put the culture dish back into the 37°C, 5% CO2 incubator. After 48 hours, collect the cell supernatants respectively, centrifuge at 400×g for 5 minutes to remove cell debris, filter the supernatant through a 0.45-μm syringe filter into a 50-mL centrifuge tube to obtain the virus liquids of different TIM-3 shRNA (TIM-3-sh01 to TIM-3-sh16) and the non-sense control sequence (NTC) lentivirus respectively, and concentrate the virus liquids. The concentrated virus is used to infect cells or stored in an -80°C refrigerator.

[0101] 3. Detection of TIM3 Protein Expression Level in Peripheral Blood-expanded NK Cells by Flow Cytometry

[0102] NK cells were isolated from the donor's peripheral blood using an NK cell sorting and purification kit, and the sorted and purified NK cells were appropriately amplified in vitro. The expression level of TIM3 protein in NK cells was detected by flow cytometry. The NK cells were transferred into a flow tube, 3 mL of 1×PBS solution was added, and centrifuged at 100×g for 5 min. The supernatant was discarded, the NK cell pellet was flicked up, and washed again with 1×PBS solution. After resuspending the NK cell pellet, APC-labeled anti-human TIM-3 antibody (Biolegend) was added for staining and incubated at room temperature for 30 min. After washing twice with 1×PBS solution, the NK cells were resuspended and analyzed by flow cytometry. The results are as Figure 1 shown, indicating that NK cells expanded from peripheral blood express a relatively high level of TIM3 protein.

[0103] 4. Detection of the silencing efficiency of different TIM-3 shRNAs

[0104] NK cells were infected with different TIM-3 shRNAs (TIM-3-sh01 to TIM-3-sh16) and a nonsense control sequence (NTC) lentivirus. The expanded NK cells were counted, and the required number of NK cells was taken, centrifuged at 100×g for 5 min to harvest the NK cells, resuspended in an appropriate amount of NK cell medium, and the NK cell density was adjusted to 5×10 5 cells / mL. 5×10 5 NK cells, 0.2 mL of the virus concentrate prepared in "2. Lentivirus packaging and virus liquid concentration" of Example 1 of this embodiment, 0.8 mL of NK cell medium, and a pro-infection reagent were added to a 24-well plate and mixed evenly. The 24-well plate with the evenly mixed solution was placed in an incubator at 37°C and 5% CO2 for culture. After 24 h, the status of the NK cells was observed, and a fresh NK cell medium with double the volume was supplemented. The NK cells were transferred to a cell culture flask and continued to be cultured. Fresh medium was supplemented every 2 days, and pro-NK cell growth factors such as IL2 were added.

[0105] After infection of NK cells with different TIM-3 shRNAs (TIM-3-sh01 to TIM-3-sh16) and the nonsense control sequence (NTC) lentivirus, puromycin drug screening was performed. On the 7th day after infection, the expression of TIM-3 in the infected NK cells was detected by quantitative real-time PCR (qPCR) and flow cytometry, respectively.

[0106] qRT-PCR: Transfer the infected NK cells into a 15 mL centrifuge tube, centrifuge at 300×g for 5 min, discard the supernatant, resuspend and wash the cell pellet with PBS, centrifuge at 300×g for 5 min, discard the supernatant, add 1 mL of Trizol reagent to lyse the cell pellet, and transfer it into a 1.5 mL RNase Free EP tube. Extract the total cellular RNA, reverse transcribe it into cDNA, and then perform real-time fluorescence quantitative PCR using TIM-3 specific primers to detect the mRNA expression level of the TIM-3 gene.

[0107] The results are as Figure 2 shown, indicating that compared with uninfected NK cells or the nonsense control sequence group (NTC), the expression levels of TIM-3 mRNA in cells infected with different TIM-3 shRNAs (TIM-3-sh01 to TIM-3-sh16) lentiviruses are different. Some shRNAs show significant silencing effects, while some shRNAs have insignificant silencing effects or even lead to a slight increase in TIM-3 expression. Among them, the sequences of TIM-3-sh01, sh02, sh04, sh05, sh06, sh08, and sh09 have relatively high silencing efficiencies, up to about 80%, and can maintain a stable silencing effect.

[0108] Flow cytometry: Transfer the infected NK cells into a flow cytometry tube, add 3 mL of 1×PBS solution, centrifuge at 100×g for 5 min, discard the supernatant, resuspend the infected NK cell pellet, and wash it once again with 1×PBS solution. After resuspending the infected NK cell pellet, add APC-labeled anti-human TIM-3 antibody (Biolegend) for staining and incubate at room temperature for 30 min. Then add 1×PBS solution to wash twice, resuspend the infected NK cell pellet, and perform flow cytometry analysis.

[0109] Respectively, count the mean fluorescence intensity (MFI) of TIM-3 in the NK cell control group (NTC) cells and the NK cells with silenced TIM-3 (TIM-3-sh01 to TIM-3-sh16), and calculate the TIM-3 silencing efficiency.

[0110] The results show that compared with uninfected NK cells or the nonsense control sequence group (NTC), the expression levels of TIM-3 protein in cells infected with different TIM-3 shRNAs (TIM-3-sh01 to TIM-3-sh16) lentiviruses are different. Some shRNAs show significant silencing effects, while some shRNAs have insignificant silencing effects or even no silencing effect. For the specific results, please refer to Figure 3Among them, TIM-3-sh01, sh02, sh04, sh05, sh06, sh08, sh09, and sh11 sequences have high silencing efficiency, about 70% to 80%, and can maintain a stable knockdown effect. For specific results, see Figure 3 .

[0111] Example 2 Silencing TIM-3 improves NK cell killing ability

[0112] The inventors detected the killing activity of NK cells with silenced TIM-3. It is known that Galectin-9 and CEACAM1 are important ligands of TIM-3. They bind to the receptor TIM-3 on the surface of NK cells and inhibit the killing activity of NK cells. The specific method for detecting the killing activity of NK cells is as follows: Detect the expression of TIM-3 ligands Galectin-9 and CEACAM1 in liver cancer cells (PLC-PRF-5, HepG2), colon cancer cells (SW620, HCT116), breast cancer cells MDA-MB-231 and cervical cancer Hela cells by flow cytometry, analyze the results, and determine that HepG2 and SW620 highly express two TIM-3 ligands (Galectin-9 and CEACAM1), PLC-PRF-5, HCT116, MDA-MB-231 and Hela cells highly express one TIM-3 ligand (Galectin-9), see for details. Figure 4A and 4B .

[0113] 5×10 4 Target cells PLC-PRF-5, HepG2, SW620, HCT116, MDA-MB-231 and Hela cells were seeded into 96-well E plates and monitored in real time using the impedance-based real-time cell analysis (RTCA) iCELLigence system. 4 NK cells or NK cells with silenced TIM-3 (TIM3-sh08 sequence) were respectively inoculated into the corresponding wells, and then the cell impedance was monitored for 4 to 6 hours using the RTCA system. The calculation formula for cell killing efficiency is (impedance of target cells without effector cells - impedance of target cells with effector cells) × 100 ÷ impedance of target cells without effector cells.

[0114] This example shows the killing results of NK cells with TIM-3 silenced. The results show that the killing efficiency of NK cells with TIM-3 silenced by the present invention on tumors with high expression of TIM-3 ligands is significantly higher than that of NK cells. For details, see Figure 5 Other shRNA sequences with high silencing efficiency also had the same improvement in efficacy.

[0115] Example 3 Detection of the Expression of Other Inhibitory Receptors and Activating Receptors of NK Cells with TIM-3 Silenced

[0116] The inventors used flow cytometry to detect and compare the expression levels of TIM-3 in NK cells before and after TIM-3 knockdown. The results are shown in Figure 3 . It was found that after gene knockdown by shRNA technology, the level of TIM-3 expression in NK cells was significantly reduced. At the same time, it was observed whether silencing TIM-3 would also affect the expression of other exhaustion-related molecules. After co-incubating NK cells with colon cancer cells SW620 at a ratio of 1:1 for 72 hours, NK cells were collected, and flow cytometry was used to detect the expression of inhibitory receptors such as NKG2A, TIGIT, LAG3, PD-1 and activating receptors NKG2D, NKp30, NKp44 and CD16 on the surface of NK cells. The results are shown in Figure 6 . The results showed that compared with NK cells, the expression of NKG2A and TIGIT on the surface of NK cells with TIM-3 silenced (TIM3-sh08 sequence) was significantly reduced, and there was no significant change in LAG3 and PD-1; the expression of activating receptors NKG2D, NKp30 and CD16 was significantly increased, while the expression of NKp44 had no obvious change. Therefore, the results further illustrate that silencing the expression of TIM-3 on the surface of NK cells by the method of the present invention will weaken the expression of other inhibitory receptors such as NKG2A and TIGIT, while enhancing the expression of activating receptors such as NKG2D, NKp30 and CD16, promoting the balance of activating receptors and inhibitory receptors of NK cells to tilt towards the activation direction, thereby improving activation and function, and enhancing the resistance of NK cells to tumor microenvironment-induced exhaustion. Specifically, see Figure 6 . Other shRNA sequences with high silencing efficiency also have the same effect.

[0117] Example 4 Silencing TIM-3 Promotes the Proliferation and Survival of NK Cells in a Hypoxic Environment

[0118] The inventors detected the survival and proliferation abilities of NK cells with TIM-3 silenced in a hypoxic tumor microenvironment. The specific method is as follows: Hypoxia is one of the characteristics of the malignant tumor microenvironment. Cobalt chloride (COCl2) is a chemical hypoxia mimetic that can induce cells to be in a hypoxic stress microenvironment in vitro. The same number (2×10 5 ) of NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates, and 60 μM COCl2 was added to the medium to induce hypoxic stress conditions, and the proliferation ability and survival ability of the cells in the hypoxic microenvironment were detected.

[0119] Cell proliferation experiment: Under normoxic conditions, the same number (2×10 4) The NK cells and the NK cells with TIM-3 silenced were seeded in 12-well plates, maintained under normoxic conditions, and the cells were counted every 48 h and the culture medium was replenished to plot the cell proliferation curve under normoxia. Under hypoxic conditions, the same number (2×10 5 ) of NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates, and 60 μM COCl2 was added to the culture medium to induce hypoxic stress conditions. The cells were counted every 48 h and the culture medium was replenished to plot the cell proliferation curve under hypoxia.

[0120] Cell viability assay: Under hypoxic conditions, the same number (2×10 5 ) of NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates, and 60 μM COCl2 was added to the culture medium to induce hypoxic stress conditions. After maintaining the hypoxic conditions for 3 days, the Annexin V-APC / 7-AAD Apoptosis Kit was used to detect cell survival / apoptosis.

[0121] This example exemplarily shows the proliferation and survival results of NK cells with TIM-3 silenced under normoxic (Normoxia) and hypoxic (Hypoxia) stress conditions. The results show that the NK cells with TIM-3 silenced by the present invention do not affect the proliferation ability under normoxia ( Figure 7 A and B in), but improve their proliferation ability and survival ability in the hypoxic microenvironment ( Figure 7 C and D in). Other shRNA sequences with high silencing efficiency also have the same effect of improvement.

[0122] Example 5 Silencing TIM-3 Promotes the Proliferation and Survival of NK Cells in the Complex Tumor Microenvironment

[0123] The inventors detected the survival ability and proliferation ability of NK cells with TIM-3 silenced (TIM3-sh08 sequence) in the complex tumor microenvironment. The specific method is as follows: The collected tumor-conditioned culture medium (containing various inhibitory factors and metabolites secreted by tumor cells, etc., which can simulate the tumor microenvironment) was detected by ELISA to show that the conditioned culture media of PLC-PRF-5, HepG2, SW620, HCT116, MDA-MB-231 and Hela cells contained different concentrations of Galectin-9, indicating that the tumor-conditioned culture medium contained components that inhibited the immune activity of NK cells (the culture supernatants of normal non-tumor cells human embryonic kidney cells HEK-293T and human microglial cells HMC3 did not contain Galectin-9), see specifically Figure 8 respectively. The same number (2×10 5)NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates, and the collected tumor-conditioned medium was added to the wells at a ratio of 1:1 to detect the proliferation ability and survival ability of the cells in the complex tumor microenvironment.

[0124] Collection of tumor-conditioned culture medium: Tumor cells in the logarithmic growth phase were selected and cultured to a confluence of about 80%. The old medium was removed, and the cells were gently washed 2-3 times with PBS to remove residual serum and poorly conditioned cells. Then, 10-20 mL of serum-free medium was added and the cells were cultured for another 24-48 hours (during this period, tumor cells secrete various factors into the medium); the supernatant of the medium was collected into a 50 mL centrifuge tube, centrifuged at 1000 g for 10 minutes to remove cell debris and impurities, and filtered through a 0.22 μm filter. The obtained tumor-conditioned culture medium was used for cell culture experiments.

[0125] Cell proliferation experiment: Under normal culture conditions, the same number (2×10 4 ) of NK cells and NK cells with TIM-3 silenced were seeded in 12-well plates. The cells were counted every 48 hours and the medium was replenished, and the cell proliferation curve under normoxia was plotted. Under the simulated tumor microenvironment culture conditions, the same number (2×10 4 ) of NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates. The collected tumor-conditioned culture medium was added at a ratio of 1:1 to simulate the tumor microenvironment culture conditions. The cells were counted every 48 hours and the medium was replenished, and the cell proliferation curve in the tumor microenvironment was plotted.

[0126] Cell survival experiment: Under the simulated tumor microenvironment culture conditions, the same number (2×10 5 ) of NK cells or NK cells with TIM-3 silenced were seeded in 12-well plates. The collected tumor-conditioned culture medium was added at a ratio of 1:1 to simulate the tumor microenvironment culture conditions. After 3 days, the Annexin V-APC / 7-AAD Apoptosis Kit was used to detect the cell survival / apoptosis situation.

[0127] This example exemplarily shows the proliferation ( Figure 9 A and B in) and survival results ( Figure 9 C and D in) of NK cells with TIM-3 silenced under normal culture conditions and complex tumor microenvironment stress conditions. The results show that under normal culture conditions, the control group of NK cells and NK cells with TIM-3 silenced have the same proliferation ability. However, under the complex tumor microenvironment culture conditions, the proliferation and survival abilities of the control group of NK cells are significantly inhibited. Compared with the control group, the NK cells with TIM-3 silenced have improved proliferation and survival abilities. For details, see Figure 9Other shRNA sequences with high silencing efficiency also have the same improvement in efficacy.

[0128] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation manners" or "some examples", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0129] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A shRNA molecule, characterized in that, It includes a sense strand fragment and an antisense strand fragment, the sequences of the sense strand fragment and the antisense strand fragment are complementary, and the sequence of the sense strand fragment is as shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:

31.

2. The shRNA molecule according to claim 1, wherein The shRNA molecule further includes a stem-loop structure, the stem-loop structure connects the sense strand fragment and the antisense strand fragment, and the stem-loop structure includes 4-11 nucleotides.

3. The shRNA molecule according to claim 1, wherein The DNA sequence encoding the shRNA molecule is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:

12.

4. An expression vector, characterized in that, It includes a DNA sequence expressing the shRNA molecule according to any one of claims 1-3.

5. The expression vector according to claim 4, wherein The expression vector is selected from any one of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

6. A microorganism, characterized in that, It includes the expression vector according to claim 4 or 5.

7. A host cell, characterized in that, It includes the expression vector according to claim 4 or 5.

8. A pharmaceutical composition, characterized in that, It includes at least one of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, and the microorganism according to claim 6.

9. Use of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, the microorganism according to claim 6, the host cell according to claim 7, and the pharmaceutical composition according to claim 8 in the preparation of a drug, characterized in that, The drug is used for preventing and / or treating tumors.

10. The use according to claim 9, characterized in that, The tumor is a TIM-3 ligand-positive tumor, including solid tumors and hematological tumors; Optionally, the solid tumor includes liver cancer, colon cancer, breast cancer, and cervical cancer.

11. A method for reducing the expression of TIM-3 gene in target cells, characterized in that, It includes: Introducing the shRNA molecule according to any one of claims 1-3 and / or the expression vector according to claim 4 or 5 into a target cell, or infecting the target cell with the microorganism according to claim 6.

12. Use of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, and the microorganism according to claim 6 in the preparation of a reagent or a kit, characterized in that, The reagent or kit is used for reducing the expression of TIM-3 and / or NKG2A and / or TIGIT in a target cell, and / or increasing the expression of at least one of NKG2D, NKp30, and CD16 in the target cell.

13. A method for enhancing the killing ability of immune cells against tumor cells, characterized in that, The method includes: Introducing the shRNA molecule according to any one of claims 1-3 and / or the expression vector according to claim 4 or 5 into the immune cell, or infecting the immune cell with the microorganism according to claim 6, so that the TIM-3 gene is lowly expressed in the immune cell.

14. A reagent or kit for enhancing the ability of immune cells to kill tumor cells, characterized in that, The reagent or kit includes at least one of the shRNA molecule according to any one of claims 1-3, the expression vector according to claim 4 or 5, and the microorganism according to claim 6.

15. A method for enhancing the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment, characterized in that, The method includes: Introducing the shRNA molecule according to any one of claims 1-3 and / or the expression vector according to claim 4 or 5 into the immune cell, or infecting the immune cell with the microorganism according to claim 6, so that the TIM-3 gene is lowly expressed in the immune cell.

16. A method for enhancing the proliferation ability and survival ability of immune cells in the tumor microenvironment, characterized in that, The method includes: Introduce the shRNA molecule described in any one of claims 1-3 and / or the expression vector described in claim 4 or 5 into the immune cells, or infect the immune cells with the microorganism described in claim 6, so that the TIM-3 gene is lowly expressed in the immune cells.

17. Use of the shRNA molecule described in any one of claims 1-3, the expression vector described in claim 4 or 5, and the microorganism described in claim 6 in the following: Enhance the killing ability of immune cells against tumor cells; Improve the proliferation ability and survival ability of immune cells under hypoxic stress or in a hypoxic microenvironment; Improve the proliferation ability and survival ability of immune cells in an immunosuppressive tumor microenvironment, Optionally, the tumor is a TIM-3 ligand-positive tumor, including solid tumors and hematological tumors.

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