Multifunctional gene modification vector and multifunctional gene modified immune cells prepared therefrom and their applications

Abstract

The present invention discloses a multifunctional gene modification vector and multifunctional gene modification immune cells prepared therefrom and their applications. The multifunctional gene modification vector carries an isolated nucleic acid, and the isolated nucleic acid includes: a first nucleic acid molecule that inhibits the expression of TIGIT; a second nucleic acid molecule that encodes a fusion protein, the fusion protein including IL-15Rα and IL-15; and a third nucleic acid molecule that encodes CXCR2; wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected. The multifunctional gene modification vector can inhibit the expression of TIGIT in host cells, and express a fusion protein including IL-15Rα and IL-15 and CXCR2, thereby, the multifunctional gene modification immune cells prepared by the multifunctional gene modification vector can improve their survival ability, proliferation ability and tumor infiltration ability, and can also resist immune exhaustion, thereby further improving the clinical efficacy of immune cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a multifunctional gene modification vector and a multifunctional gene modification immune cell prepared therefrom and their applications. More particularly, the present invention relates to a shRNA, an isolated nucleic acid, a kit, a transgenic immune cell, a pharmaceutical composition, a method for enhancing immune cell killing, activation, proliferation and chemotaxis and their applications. Background Art

[0002] Gene-modified cell therapy is a new type of treatment that treats diseases by modifying the genome of cells in the patient's body. This technology uses genetic engineering technology to introduce exogenous genes or regulatory factors into immune cells, giving these immune cells new functions or stronger therapeutic potential. Gene-modified cell technology is widely used in immune cells used for immunotherapy, such as NK cells, T cells, NKT cells, γδT cells and macrophages.

[0003] Tumors usually have a physical barrier of fibrous tissue, and within the barrier there is a tumor microenvironment with hypoxia, low pH, nutrient deficiency, and high osmotic pressure, and lacks mature vascular supply. Therefore, the microenvironment within the tumor is very unfavorable for the localization, infiltration, survival, and proliferation of immune cells in the tumor, and is prone to immunosuppression and immune exhaustion.

[0004] Therefore, there is an urgent need to further develop new genetically modified immune cells to improve immune cell survival, cell proliferation, resistance to immune exhaustion, and increased tumor infiltration ability in tumors. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

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

[0007] The inventor unexpectedly discovered that NK cells have weak in vivo expansion and persistence, poor tumor infiltration ability, and are prone to immune exhaustion or immunosuppression. The inventor constructed a multifunctional gene modification vector that expresses shRNA of the silencing immunosuppressive receptor TIGIT, IL15 / IL15Rα fusion protein expressed on the cell membrane, and chemokine receptor CXCR2 for modifying NK cells. NK cells modified by the multifunctional gene modification vector of the present invention can enhance their survival cycle and cell proliferation ability in tumors, resist immune exhaustion, and improve the ability to infiltrate tumors, thereby further improving their efficacy in clinical practice.

[0008] Based on this, in the first aspect of the present invention, the present invention proposes a shRNA. According to an embodiment of the present invention, the shRNA includes a nucleotide sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:11. The shRNA according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, enhance the killing efficiency of immune cells against tumors with high expression of TIGIT receptor ligand CD155, and thereby enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.

[0009] In the second aspect of the present invention, the present invention proposes an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid includes a first nucleic acid molecule, which inhibits the expression of TIGIT; a second nucleic acid molecule, which encodes a fusion protein, and the fusion protein includes IL-15Rα and IL-15; and a third nucleic acid molecule, which encodes CXCR2; wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected. The nucleic acid isolated according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, and can make the immune cell membrane express IL15 / IL15Rα fusion protein and CXCR2 receptor, and can improve the long-term survival and proliferation ability of immune cells carrying the above-mentioned isolated nucleic acid in vivo and in vitro, and its infiltration ability and anti-tumor activity in tumors.

[0010] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the isolated nucleic acid described in the second aspect of the present invention. The expression vector according to the embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.

[0011] In a fourth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the isolated nucleic acid described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention. The kit according to an embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.

[0012] In the fifth aspect of the present invention, the present invention proposes a transgenic immune cell. According to an embodiment of the present invention, the transgenic immune cell inhibits the expression of TIGIT, and expresses a fusion protein and CXCR2; wherein the fusion protein includes IL-15Rα and IL-15. The transgenic immune cell has the advantages of high tumor killing efficiency, strong anti-tumor activity, strong long-term survival and expansion ability in the body, and strong proliferation and chemotaxis ability.

[0013] In the sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the transgenic immune cell described in the fifth aspect of the present invention. The pharmaceutical composition according to the embodiment of the present invention has high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability.

[0014] In the seventh aspect of the present invention, the present invention proposes a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells. According to an embodiment of the present invention, the method comprises introducing the expression vector described in the third aspect of the present invention or the expression vector in the kit described in the fourth aspect of the present invention into immune cells; and culturing the immune cells introduced with the expression vector. This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, improve the long-term survival and amplification ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially can prepare immune cells with strong killing, activation, proliferation and chemotaxis in vitro for constructing the required immune cell model.

[0015] In the eighth aspect of the present invention, the present invention proposes the use of the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the transgenic immune cell described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a drug for treating or preventing tumors.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0018] Figure 1 This is a flow cytometry graph of screening different TIGIT shRNA silencing efficiencies in Example 1.

[0019] Figure 2 This is a flow cytometry chart of the screening of different TIGIT shRNA silencing efficiencies in Example 1.

[0020] Figure 3 It is a schematic diagram of the construction elements of the multifunctional gene modification vector in Example 2.

[0021] Figure 4 This is a flow cytometry result diagram of the expression verification of each element of the multifunctional gene modification vector in Example 2.

[0022] Figure 5 This is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting the killing activity of NK cells.

[0023] Figure 6 This is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting the survival of NK cells.

[0024] Figure 7 This is a graph showing the result of the multifunctional gene-modified vector promoting NK cell proliferation in Example 3.

[0025] Figure 8 This is a graph showing the result of the multifunctional gene-modified vector in Example 3 promoting NK cell chemotaxis.

[0026] Fig. 9 This is a result diagram showing that the multifunctional gene-modified vector in Example 4 significantly enhances the in vivo tumor inhibition effect of NK-92 cells on liver cancer.

[0027] Fig.10 This is a result diagram showing that the multifunctional gene-modified vector in Example 4 significantly enhances the in vivo tumor inhibition effect of NK-92 cells on colorectal cancer. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated, nor is there any order of precedence. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0030] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0031] In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0032] In the present invention, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0033] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or situation can but does not necessarily occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0034] In this article, the term "vector" or "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes vectors with multiple of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0035] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0036] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The chimeric antigen receptor, nucleic acid molecule, expression vector or transgenic immune cell or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection.

[0037] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

[0038] The present invention provides a shRNA molecule, an isolated nucleic acid, an expression vector, a kit, a transgenic immune cell, a pharmaceutical composition, a method for enhancing immune cell killing, activation, proliferation and chemotaxis and uses thereof, which will be described in detail below.

[0039] shRNA molecules

[0040] The present invention proposes a shRNA. According to an embodiment of the present invention, the shRNA includes a nucleotide sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:11. The shRNA according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, enhance the killing efficiency of immune cells against tumors with high expression of TIGIT receptor ligand CD155, and thus enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.

[0041] According to an embodiment of the present invention, the above shRNA molecule may further include at least one of the following additional technical features:

[0042] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 7. Thus, the shRNA can be used to effectively silence TIGIT molecules on immune cells, especially by contacting the obtained immune cells with tumor cells, which can improve the killing efficiency of tumors with high expression of TIGIT receptor ligand CD155 and enhance the anti-tumor activity of immune cells.

[0043] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 9. Thus, the shRNA can be used to effectively silence TIGIT molecules on immune cells, especially by contacting the obtained immune cells with tumor cells, which can improve the killing efficiency of tumors with high expression of TIGIT receptor ligand CD155 and enhance the anti-tumor activity of immune cells.

[0044] Isolated nucleic acids

[0045] In the second aspect of the present invention, the present invention proposes an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid includes a first nucleic acid molecule, which inhibits the expression of TIGIT; a second nucleic acid molecule, which encodes a fusion protein, and the fusion protein includes IL-15Rα and IL-15; and a third nucleic acid molecule, which encodes CXCR2; wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected. The nucleic acid isolated according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, and can make the immune cell membrane express IL15 / IL15Rα fusion protein and CXCR2 receptor, and can improve the long-term survival and proliferation ability of immune cells carrying the above-mentioned isolated nucleic acid in vivo and in vitro, and its infiltration ability and anti-tumor activity in tumors.

[0046] According to an embodiment of the present invention, the isolated nucleic acid may further include at least one of the following additional technical features:

[0047] According to an embodiment of the present invention, the first nucleic acid molecule carries the shRNA described in the first aspect of the present invention.

[0048] According to an embodiment of the present invention, the TIGIT has the nucleotide sequence shown in SEQ ID NO:21.

[0049] According to an embodiment of the present invention, the N-terminus of the IL-15Rα is connected to the C-terminus of the IL-15, or the C-terminus of the IL-15Rα is connected to the N-terminus of the IL-15.

[0050] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:3.

[0051] According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO:4.

[0052] According to an embodiment of the present invention, the IL-15Rα has a nucleotide sequence as shown in SEQ ID NO:13.

[0053] According to an embodiment of the present invention, the IL-15 has a nucleotide sequence as shown in SEQ ID NO:14.

[0054] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide 1.

[0055] According to an embodiment of the present invention, the N-terminus of the IL-15Rα is connected to the C-terminus of the connecting peptide 1, and the N-terminus of the connecting peptide 1 is connected to the C-terminus of the IL-15; or the C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the IL-15.

[0056] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.

[0057] According to an embodiment of the present invention, the nucleotide sequence encoding the connecting peptide 1 is as shown in SEQ ID NO:12.

[0058] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:15.

[0059] According to an embodiment of the present invention, the CXCR2 has the amino acid sequence shown in SEQ ID NO:5.

[0060] According to an embodiment of the present invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:17.

[0061] According to an embodiment of the present invention, the second nucleic acid molecule is connected to the third nucleic acid molecule, and the first nucleic acid molecule is located at one end of the second nucleic acid molecule and the third nucleic acid molecule.

[0062] Herein, the term "the first nucleic acid molecule is located at one end of the second nucleic acid molecule and the third nucleic acid molecule" means that the connected second nucleic acid molecule and the third nucleic acid molecule are an integral nucleic acid molecule A, and the first nucleic acid molecule is located at the 3' end or the 5' end of the nucleic acid molecule A.

[0063] According to an embodiment of the present invention, the 3' end of the first nucleic acid molecule is connected to the 5' end of the second nucleic acid molecule, and the 3' end of the second nucleic acid molecule is connected to the 5' end of the third nucleic acid molecule.

[0064] According to an embodiment of the present invention, the 3' end of the first nucleic acid molecule is connected to the 5' end of the third nucleic acid molecule, and the 3' end of the third nucleic acid molecule is connected to the 5' end of the second nucleic acid molecule.

[0065] According to an embodiment of the present invention, the 3' end of the second nucleic acid molecule is connected to the 5' end of the third nucleic acid molecule, and the 3' end of the third nucleic acid molecule is connected to the 5' end of the first nucleic acid molecule.

[0066] According to an embodiment of the present invention, the 3' end of the third nucleic acid molecule is connected to the 5' end of the second nucleic acid molecule, and the 3' end of the second nucleic acid molecule is connected to the 5' end of the first nucleic acid molecule.

[0067] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a fourth nucleic acid molecule, which is arranged between the second nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a connecting peptide 2, and the connecting peptide 2 can be cut in the immune cell.

[0068] According to an embodiment of the present invention, the connecting peptide 2 includes a 2A peptide or a fragment thereof.

[0069] According to an embodiment of the present invention, the connecting peptide 2 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

[0070] According to an embodiment of the present invention, the connecting peptide 2 includes P2A or a fragment thereof.

[0071] According to an embodiment of the present invention, the connecting peptide 2 has an amino acid sequence shown in SEQ ID NO:16.

[0072] According to an embodiment of the present invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:18.

[0073] According to an embodiment of the present invention, the isolated nucleic acid further includes: a first promoter, which is operably linked to the first nucleic acid molecule; and / or a second promoter, which is operably linked to the second nucleic acid molecule.

[0074] According to an embodiment of the present invention, the isolated nucleic acid further comprises: the 3' end of the first promoter is operably connected to the 5' end of the first nucleic acid molecule; the 3' end of the second promoter is operably connected to the 5' end of the second nucleic acid molecule.

[0075] According to an embodiment of the present invention, the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter.

[0076] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a fifth nucleic acid molecule, which is arranged between the first nucleic acid molecule and the second promoter, and the fifth nucleic acid molecule encodes a gene transduction efficiency enhancing element.

[0077] According to an embodiment of the present invention, the gene transduction efficiency enhancing element is cPPT / CTS.

[0078] According to an embodiment of the present invention, the fifth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:19.

[0079] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a sixth nucleic acid molecule, wherein the sixth nucleic acid molecule is operably linked to the third nucleic acid molecule, and the sixth nucleic acid molecule encodes a post-transcriptional regulatory element.

[0080] According to an embodiment of the present invention, the post-transcriptional regulatory element is WPRE.

[0081] According to an embodiment of the present invention, the sixth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:20.

[0082] According to an embodiment of the present invention, the separated nucleic acids are, from the 5' end to the 3' end, the first nucleic acid molecule, the fifth nucleic acid molecule, the second nucleic acid molecule, the fourth nucleic acid molecule, the third nucleic acid molecule, and the sixth nucleic acid molecule.

[0083] According to an embodiment of the present invention, the separated nucleic acids are, from the 5' end to the 3' end, the first nucleic acid molecule, the fifth nucleic acid molecule, the third nucleic acid molecule, the fourth nucleic acid molecule, the second nucleic acid molecule, and the sixth nucleic acid molecule.

[0084] According to an embodiment of the present invention, the separated nucleic acids are, from the 5' end to the 3' end, the fifth nucleic acid molecule, the third nucleic acid molecule, the fourth nucleic acid molecule, the second nucleic acid molecule, the sixth nucleic acid molecule, and the first nucleic acid molecule.

[0085] According to an embodiment of the present invention, the separated nucleic acids are, from the 5' end to the 3' end, the fifth nucleic acid molecule, the second nucleic acid molecule, the fourth nucleic acid molecule, the third nucleic acid molecule, the sixth nucleic acid molecule, and the first nucleic acid molecule.

[0086] Expression vector

[0087] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the isolated nucleic acid described in the second aspect of the present invention. The expression vector according to the embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.

[0088] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0089] According to an embodiment of the present invention, the vector of the expression vector is a non-pathogenic viral vector.

[0090] According to an embodiment of the present invention, the viral vector includes at least one selected from a retroviral vector, a lentiviral vector and an adenovirus-associated viral vector.

[0091] Reagent test kit

[0092] In a fourth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the isolated nucleic acid described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention. The kit according to an embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.

[0093] Genetically modified immune cells

[0094] In the fifth aspect of the present invention, the present invention proposes a transgenic immune cell. According to an embodiment of the present invention, the transgenic immune cell inhibits the expression of TIGIT, and expresses a fusion protein and CXCR2; wherein the fusion protein includes IL-15Rα and IL-15. The transgenic immune cell has the advantages of high tumor killing efficiency, strong anti-tumor activity, strong long-term survival and expansion ability in the body, and strong proliferation and chemotaxis ability.

[0095] Furthermore, the inventors studied a variety of immunosuppressive receptors, screened and compared the effects of silencing different immunosuppressive receptors on reversing immunosuppressive signals and resisting immune exhaustion, and obtained a variety of transgenic immune cells by combining shRNA of different immunosuppressive receptors with the above-mentioned fusion protein and CXCR2. The results showed that although other immunosuppressive receptors have similar effects to TIGIT, compared with other immunosuppressive receptors, the transgenic immune cells obtained by combining TIGIT-shRNA with the above-mentioned fusion protein and CXCR2 (i.e., silencing TIGIT, expressing IL-15Rα, IL-15 and CXCR2 in the transgenic immune cells) have more obvious high tumor killing efficiency, strong anti-tumor activity, strong long-term survival and expansion ability in the body, and strong proliferation and chemotaxis, while the transgenic immune cells silencing other immunosuppressive receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak long-term survival and expansion ability in the body, and weak proliferation and chemotaxis.

[0096] According to an embodiment of the present invention, the genetically modified immune cells may further include at least one of the following additional technical features:

[0097] According to an embodiment of the present invention, the TIGIT has the amino acid sequence shown in SEQ ID NO:1.

[0098] According to an embodiment of the present invention, the N-terminus of the IL-15Rα is connected to the C-terminus of the IL-15, or the C-terminus of the IL-15Rα is connected to the N-terminus of the IL-15.

[0099] According to an embodiment of the present invention, the IL-15Rα has the amino acid sequence shown in SEQ ID NO:3.

[0100] According to an embodiment of the present invention, the IL-15 has the amino acid sequence shown in SEQ ID NO:4.

[0101] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide 1.

[0102] According to an embodiment of the present invention, the N-terminus of the IL-15Rα is connected to the C-terminus of the connecting peptide 1, and the N-terminus of the connecting peptide 1 is connected to the C-terminus of the IL-15; or the C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the IL-15.

[0103] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.

[0104] According to an embodiment of the present invention, the fusion protein has the amino acid sequence shown in SEQ ID NO:2.

[0105] According to an embodiment of the present invention, the CXCR2 has the amino acid sequence shown in SEQ ID NO:5.

[0106] According to an embodiment of the present invention, the transgenic immune cells are obtained by introducing the aforementioned expression vector into immune cells.

[0107] In some optional embodiments of the present invention, the immune cells include at least one of T cells, NKT cells, NK cells and macrophages.

[0108] According to an embodiment of the present invention, the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells and macrophages.

[0109] According to an embodiment of the present invention, the transgenic immune cells are derived from NK cells. The transgenic immune cells have higher tumor killing efficiency, stronger anti-tumor activity, stronger long-term survival and expansion ability in the body, and stronger proliferation and chemotaxis ability.

[0110] According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

[0111] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

[0112] Pharmaceutical composition

[0113] In the sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the transgenic immune cell described in the fifth aspect of the present invention. The pharmaceutical composition has high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability.

[0114] A method for enhancing immune cell killing, activation, proliferation and chemotaxis

[0115] In the seventh aspect of the present invention, the present invention proposes a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells. According to an embodiment of the present invention, the method comprises introducing the expression vector described in the third aspect of the present invention or the expression vector in the kit described in the fourth aspect of the present invention into immune cells; and culturing the immune cells introduced with the expression vector. This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, improve the long-term survival and amplification ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially can prepare immune cells with strong killing, activation, proliferation and chemotaxis in vitro for constructing the required immune cell model.

[0116] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0117] According to an embodiment of the present invention, the introduction of the expression vector into the immune cells is performed by electroporation, transfection or infection.

[0118] According to an embodiment of the present invention, the immune cell is at least one of a T cell, a NKT cell, a NK cell and a macrophage.

[0119] According to an embodiment of the present invention, the immune cells are NK cells.

[0120] According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

[0121] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

[0122] use

[0123] In the eighth aspect of the present invention, the present invention proposes the use of the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the transgenic immune cell described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of a drug for treating or preventing tumors.

[0124] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0125] According to an embodiment of the present invention, the tumor includes solid tumors and hematological tumors.

[0126] According to an embodiment of the present invention, the solid tumor is a tangible tumor occurring in an organ.

[0127] According to an embodiment of the present invention, the solid tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer and melanoma.

[0128] According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma.

[0129] method

[0130] In the ninth aspect of the present invention, the present invention proposes a method for treating or preventing tumors. According to an embodiment of the present invention, the method comprises: administering to a subject a pharmaceutically acceptable dose of the transgenic immune cells described in the fifth aspect of the present invention and the pharmaceutical composition described in the sixth aspect of the present invention. As can be seen from the foregoing, transgenic immune cells and pharmaceutical compositions have the advantages of high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability. Therefore, the use of the above-mentioned transgenic immune cells and pharmaceutical compositions can effectively kill tumor cells, thereby effectively preventing and treating tumors.

[0131] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0132] According to an embodiment of the present invention, the tumor includes solid tumors and hematological tumors.

[0133] According to an embodiment of the present invention, the solid tumor is a tangible tumor occurring in an organ.

[0134] According to an embodiment of the present invention, the solid tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer and melanoma.

[0135] According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma.

[0136] The sequence descriptions involved in the present invention are shown in Table 1.

[0137] Table 1: Amino acid / nucleotide sequence description

[0138]

[0139]

[0140]

[0141]

[0142] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0143] Experimental Example 1: Screening of shRNA sequences that silence TIGIT

[0144] 1. RNAi target sequence design

[0145] The gene number of Homo sapiens T cell immunoreceptor with Ig and ITIMdomains (TIGIT) was found in the NCBI website as NM_173799.4, and the coding sequence (CDS) of TIGIT mRNA is as follows. In the TIGIT mRNA coding sequence (nucleotide sequence as shown in SEQ ID NO: 22), 5 RNAi target sequences were designed for screening.

[0146] According to the designed RNAi target sequence, the shRNA sequence for constructing the shRNA lentiviral vector was designed. The coding sequence of the designed TIGIT shRNA (shTG1 to shTG5, the nucleotide sequence is shown in SEQ ID NO: 7 to 11) and the nonsense control sequence (shSCR, the nucleotide sequence is shown in SEQ ID NO: 23) have the structure of sense, loop, antisense, and termination sequences. According to the restriction site of the pLKO.1-EGFP lentiviral vector, the AgeI and EcoRI restriction sites were selected to insert the shRNA sequence respectively, and the obtained plasmids were named pLKO.1-shTG1-EGFP to pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP respectively.

[0147] 2. Lentivirus Packaging

[0148] Take 5×10 293T cells in the logarithmic growth phase 6 Inoculate cells in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37°C, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G, 6μg lentiviral vector plasmids (pLKO.1-shTG1-EGFP~pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP) to 250μL serum-free DMEM medium, prepare the plasmid mixtures, and mix well. Add 15μL Add to 235 μL serum-free DMEM medium and mix well. The mixed solution was added to the above plasmid mixture at one time, mixed, and incubated at room temperature for 15 minutes. The above mixed solution was added to the 293T cell culture dish. After 24 hours, the liquid was changed, and the culture dish was returned to the 37°C, 5% CO2 incubator. After 48 hours, the cell supernatant was collected, centrifuged at 400×g for 5 minutes, and the cell debris was removed. The supernatant was filtered into a 50mL centrifuge tube with a 0.45μm filter head to obtain different TIGIT shRNA (shTG1~shTG5) and nonsense control sequence (shSCR) lentivirus virus liquids, and 5×PEG8000 solution was added to concentrate the virus liquid. The centrifuge tube was turned upside down to mix evenly, and placed in a 4°C refrigerator overnight. Centrifuge at 4°C, 4000×g for 20 minutes, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0149] 3. Detection of silencing efficiency of different TIGIT shRNAs

[0150] Different TIGIT shRNAs (shTG1 to shTG5) and nonsense control sequences (shSCR) were used to infect NK-92 cells. NK-92 cells in the logarithmic growth phase were harvested by centrifugation at 100×g for 5 min, and appropriate amounts of α-MEM medium were added to resuspend the NK-92 cells. The NK-92 cell density was adjusted to 5×10 5 5×10 5 NK-92 cells, 0.2mL of virus concentrate prepared in step 2 of this embodiment 1, 0.8mL of α-MEM medium and protamine (final concentration 8μg / mL), and mix well. The 24-well plate after mixing was placed in a 37°C, 5% CO2 incubator for culture. After 24h, the state of the NK-92 cells was observed, the liquid was changed, the infected NK-92 cells were transferred to an EP tube, centrifuged at 100×g for 5min, a small amount of fresh α-MEM medium was added to resuspend the NK-92 cells, and the NK-92 cells were transferred to a cell culture flask, and 10mL of fresh α-MEM medium and IL-2 (final concentration of 200IU / mL) were added to continue culture. The cell liquid was changed every 2 days, and IL-2 (final concentration of 200IU / mL) was added.

[0151] On the 6th day after infection with different TIGIT shRNA (shTG1-shTG5) and nonsense control sequence (shSCR) lentiviruses, the expression of TIGIT in infected NK-92 cells was detected by flow cytometry. The infected NK-92 cells were transferred into a flow tube, 3 mL of 1×PBS solution was added, centrifuged at 100×g for 5 min, the supernatant was discarded, the infected NK-92 cell pellet was flicked, and washed again with 1×PBS solution. After resuspending the infected NK-92 cell pellet, APC-labeled anti-human TIGIT antibody (Biolegend) was added for staining and incubated at room temperature for 30 min. After washing twice with 1×PBS solution, the infected NK-92 cell pellet was resuspended and then subjected to flow cytometry detection.

[0152] Since the TIGIT shRNA (shTG1-shTG5) and nonsense control sequence (shSCR) lentiviral vectors contain GFP tags, GFP-positive cells represent positively infected cells. In the same tube, the positive rate of TIGIT on GFP-positive cells (A) and the positive rate of TIGIT on GFP-negative cells (B) were analyzed separately. Calculation formula: TIGIT silencing efficiency % = (1-A / B) × 100%.

[0153] The results showed that compared with uninfected NK-92 cells or nonsense control sequence group (shSCR), the expression of TIGIT in cells infected with different TIGITshRNA (shTG1~shTG5) lentiviruses was significantly decreased. For specific results, see Figure 1 Among them, the silencing efficiency of shTG1 and shTG3 sequences is the highest, reaching more than 70%. For specific results, see Figure 2 .

[0154] Example 2: Construction of a multifunctional gene modification vector and verification of expression of each element

[0155] 1. Construction of multifunctional gene modification vector

[0156] The base sequence of cPPT / CTS-EF1a-mbIL15RF-P2A-CXCR2-WPRE was synthesized by whole gene synthesis and cloned into the lentiviral vector (pLKO.1-shTG1-EGFP to pLKO.1-shTG5-EGFP) prepared in the above Example 1 through restriction sites EcoRI and KpnI. After sequencing verification, pLKO.1-multi-functional-vector-shTG1 to pLKO.1-multi-functional-vector-shTG5 plasmids were obtained, i.e., the multifunctional gene modification vector plasmid involved in the present invention. This example exemplarily shows the construction of pLKO.1-multi-functional-vector-shTG3 multifunctional gene modification vector and the schematic diagram of the elements, see Figure 3 .

[0157] 2. Multifunctional gene modification vector packaging lentivirus

[0158] 6 μg psPAX2 and 3 μg pMD2.G of lentiviral packaging auxiliary plasmids, and 6 μg multifunctional gene-modified lentiviral vector plasmids (pLKO.1-multi-functional-vector-shTG1~pLKO.1-multi-functional-vector-shTG5) were added to 250 μL of serum-free DMEM culture medium to prepare a plasmid mixture and mixed evenly. The lentivirus was packaged by the method described in 1.2 of Example 1 to obtain lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5 packaged with different multifunctional gene-modified vectors.

[0159] 3. Verification of expression of each element of the multifunctional gene modification vector

[0160] CD3 positive cells in peripheral blood mononuclear cells (PBMC) are removed by negative selection with CD3 magnetic beads, NK cells in PBMC are enriched, and the sorted cells are inoculated in pre-coated culture bottles for culture. Referring to the method described in CN 202310035787.4, IL-2 and other cytokines are used for induction culture. On the 7th day of culture, the cultured NK cells were infected with the lentivirus (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5) prepared in step 2 of Example 2. The culture was continued after the medium was changed on the 9th day. Flow cytometry was performed on the 11th day to detect the expression of various elements on NK cells. The method of flow cytometry is as follows: 1×10 6 Each cell was added to the flow cytometry tube for staining, and the antibodies were added for staining according to different staining schemes, and incubated at room temperature for 30 minutes. After washing twice with 1× PBS solution, the cell pellet was resuspended and divided into two parts, and then flow cytometry was performed on the machine.

[0161] The first portion was added with PerCP / Cyanine5.5 labeled anti-human CD3 antibody (Biolegend), Brilliant Violet785 TM The second batch was added with anti-human CD56 antibody (Biolegend), APC-labeled anti-human TIGIT antibody (Biolegend) and PE-labeled anti-human CXCR2 antibody (Biolegend); the second batch was added with PerCP / Cyanine5.5-labeled anti-human CD3 antibody (Biolegend), Brilliant Violet 785 TM Labeled anti-human CD56 antibody (Biolegend), APC-labeled anti-human IL-15Rα antibody (Biolegend) and PE-labeled anti-human IL-15 antibody (Invitrogen).

[0162] This example shows the results of NK cell infection by lentivirus packaged with a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3). The results showed that the NK cells in the uninfected group highly expressed TIGIT receptors, but did not express CXCR2 and mbIL15RF molecules. Figure 4A. After infection with lentivirus packaged with a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3), the expression of TIGIT receptor on NK cells was significantly reduced, and the expression of CXCR2 and mbIL15RF molecules (encoding fusion proteins, which include IL-15Rα and IL-15) was significantly increased. These results show that the three functional elements (first nucleic acid molecule, second nucleic acid molecule, and third nucleic acid molecule) in the multifunctional gene modification vector can be efficiently expressed.

[0163] Example 3: In vitro functional detection of NK cells prepared by multifunctional gene-modified vectors

[0164] 1. Construction of multifunctional NK-92 cells

[0165] NK-92 cells in the logarithmic growth phase were aspirated and centrifuged at 100 × g for 5 min to harvest the cells. An appropriate amount of α-MEM medium was added to resuspend the cells and the NK-92 cell density was adjusted to 5 × 10 5 5×10 5 NK-92 cells, 1mL of concentrated lentivirus solution (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5) were mixed with protamine (final concentration 8μg / mL) and cultured in a 37℃, 5% CO2 incubator. After 24h, observe the status of NK-92 cells, change the medium, transfer the infected NK-92 cells into EP tubes, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the infected NK-92 cells into cell culture bottles, add 10mL of fresh α-MEM medium and IL-2 (final concentration of 200IU / mL) and continue to culture for 48h. Continue to expand the culture and adjust the status of the infected NK-92 cells for amplification. The infected NK-92 cells were sorted by flow cytometry for CXCR2-positive cells as infection-positive cells, that is, multifunctional NK-92 cells infected with different lentivirus concentrates (lentivirus-multi-functional-shTG1 to lentivirus-multi-functional-shTG5) were constructed and used for subsequent in vitro functional experiments.

[0166] 2. Multifunctional gene-modified vector promotes the killing activity of NK cells

[0167] The inventors tested the killing activity of NK cells prepared by the multifunctional gene-modified vector. The specific method is as follows: 1×10 4Adherent target HepG2 and Hey cells were seeded into 96-well E-plates and monitored overnight in culture using the impedance-based real-time cell analysis (RTCA) iCELLigence system. 4 NK-92 or multifunctional NK-92 cells were respectively inoculated into the corresponding wells, and then the cell impedance was monitored for 2-3 hours using the RTCA system. The formula for calculating the 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.

[0168] This example shows the results of multifunctional NK-92 cells prepared by the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that the killing efficiency of multifunctional NK-92 cells prepared by the multifunctional gene-modified vector of the present invention on liver cancer HepG2 cells or ovarian cancer Hey cells is significantly higher than that of NK-92 cells. Figure 5 A and Figure 5 B.

[0169] 3. Multifunctional gene modification vector promotes the survival of NK cells

[0170] The inventors further verified the effect of the multifunctional gene-modified vector on NK cell survival. The specific method is as follows: the same number of NK-92 cells and the multifunctional NK-92 cells prepared in step 1 of Example 3 were plated in a 24-well plate, and different IL-2 concentrations (200 IU / mL and 0 IU / mL) were set, and the apoptosis rate was detected by flow cytometry every 24 hours. The apoptosis rate was detected by flow cytometry according to the steps of the kit instructions (Lianke Bio, catalog number AP101), which is briefly as follows: collect cells in EP tubes, add 1×PBS solution, centrifuge and wash once, and resuspend the cells. Add 5 μL Annexin V-FITC and 10 μL PI to each tube. After gentle vortex mixing, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow detection. The cell viability is the ratio of double negatives of Annexin V-FITC and PI staining.

[0171] This example shows the results of multifunctional NK-92 cells prepared by a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3). The results showed that in the absence of IL-2 (IL-20 IU / mL), the cell survival rate of the NK-92 cell group decreased significantly from 24 hours, and most cells had undergone apoptosis by 72 hours; while the multifunctional NK-92 cell group cells could still maintain a high cell survival rate even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis. For details, see Figure 6These results indicate that multifunctional gene modification vectors can play an important role in promoting NK cell survival.

[0172] 4. Multifunctional gene modification vector promotes the proliferation of NK cells

[0173] The inventors further verified the effect of the multifunctional gene-modified vector on NK cell proliferation. The specific method is as follows: the same number of NK-92 cells and the multifunctional NK-92 cells prepared in step 1 of Example 3 were plated in T25 cell culture flasks, and different IL-2 concentrations (200 IU / mL, 20 IU / mL and 0 IU / mL) were set, and trypan blue staining cell counts were performed every 48 hours. Observe and culture until the 8th day, and draw a cell proliferation curve

[0174] This example shows the results of multifunctional NK-92 cells prepared by multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3). The results show that the proliferation ability of multifunctional NK-92 cells under different IL-2 concentrations is significantly stronger than that of NK-92 cells under the same IL-2 concentration. Figure 7 The above test results show that the multifunctional gene modification vector can significantly promote the proliferation of NK cells.

[0175] 5. Multifunctional gene-modified vector promotes chemotaxis of NK cells

[0176] The inventors further investigated the chemotactic ability of multifunctional NK-92 cells by transwell experiment. 1×10 6 NK-92 or multifunctional NK-92 cells were cultured in the lower chamber, and 600 μL of serum-free α-MEM medium was added to the lower chamber, and chemokine CXCL8 at concentrations of 1, 10, and 100 ng / mL was added for chemotaxis. The cells were returned to the cell culture incubator, and the cells in the lower chamber were collected after 48 hours for cell counting.

[0177] This example shows the results of multifunctional NK-92 cells prepared by a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3). The results show that compared with NK-92 cells, the number of multifunctional NK-92 cells migrating to the lower chamber is significantly greater, and is dose-dependent with the concentration of IL-8. The above test results show that the multifunctional gene modification vector can significantly promote the chemotaxis of NK cells.

[0178] Example 4: NK cells prepared by multifunctional gene-modified vectors have strong in vivo tumor-suppressing activity

[0179] 1. The multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of NK-92 cells on liver cancer

[0180] In this example, a mouse subcutaneous tumor-bearing model was established using human liver cancer HepG2 cells to observe the therapeutic effect of the multifunctional NK-92 cells prepared in step 1 of Example 3 on the liver cancer tumor-bearing model. The specific method is as follows: 6-week-old NCG mice (8 mice) were selected for subcutaneous tumor bearing in the armpits, and the tumor bearing dose was 5×10 6 HepG2 cells / mouse. On the third day after tumor loading, mice were treated with NK cell reinfusion and randomly divided into untreated group, NK-92 cell treatment group and multifunctional NK-92 cell treatment group according to tumor size. In the NK-92 or multifunctional NK-92 cell treatment group, NK cells were reinfused through the tail vein for treatment at a dose of 5×10 6 cells / time / week, for a total of 4 treatments, and 5×10 4 IU of IL-2 maintained the activity of NK cells in vivo. The tumor volume was measured once a week and the tumor growth curve was drawn.

[0181] This example shows the results of multifunctional NK-92 cells prepared by the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that compared with the NK-92 cell treatment group, the multifunctional NK-92 cells prepared based on the multifunctional gene-modified vector of the present invention have a significantly enhanced effect of inhibiting the growth of liver cancer cells. For specific results, see Fig. 9 .

[0182] 2. The multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of primary NK cells on colorectal cancer

[0183] In this example, a human colorectal cancer mouse heterotopic transplant tumor model was established using human colorectal cancer cell line NCI-H716 cells, and human peripheral blood primary NK cells were infected with the lentivirus prepared in step 2 of Example 2 (lentivirus-multi-functional-shTG1 to lentivirus-multi-functional-shTG5) to prepare multifunctional primary NK cells from different peripheral blood sources, and the therapeutic effect of multifunctional primary NK cells on the human colorectal cancer model was observed.

[0184] The specific method is as follows: 8 6-week-old NCG mice were selected for subcutaneous tumor bearing in the axilla, with a tumor bearing dose of 1×10 7 NCI-H716 cells / mouse. On day 9 after tumor loading, the screening tumor volume was 50mm 3About 100 mice were used in the experiment and randomly divided into an untreated group, a non-genetically modified primary NK cell treatment group, and a multifunctional primary NK cell treatment group according to the size of the tumor. The mice in the non-genetically modified primary NK cell treatment group were treated once every 2 days for a total of 3 times, with a tail vein infusion dose of 8×10 6 CD56 + NK cells, a total of 2.4×10 7 CD56 + 5×10 NK cells were injected intraperitoneally every 2 days 4 IU of IL-2 maintained the activity of NK cells in vivo; the mice in the multifunctional primary NK cell treatment group were reinfused into the tail vein once, with a dose of 4×10 6 Multifunctional primary NK cells / cell, no IL-2 injection adjuvant therapy. The tumor volume was observed 1-2 times a week, and the tumor growth curve was drawn.

[0185] This example shows the results of multifunctional primary NK cells prepared by lentivirus (lentivirus-multi-functional-shTG3). The results show that compared with the non-genetically modified primary NK cell treatment group, at a lower cell treatment dose, the multifunctional primary NK cell treatment group still showed a stronger tumor inhibition effect, with a tumor inhibition rate of up to 64.5%. For specific results, see Fig.10 .

[0186] The above test results indicate that NK cells prepared based on the multifunctional gene-modified vector of the present invention have significantly enhanced anti-tumor activity against tumors such as ovarian cancer, liver cancer, and colorectal cancer, and can be used at lower dosages, which is expected to break through the bottleneck of poor therapeutic effect of immune cell therapy on solid tumors.

[0187] Comparative Example

[0188] 1. By the method in step 1 of Example 2, shRNAs that silence different immunosuppressive receptors are combined with the above-mentioned expression fusion protein and CXCR2 to prepare multifunctional gene modification vectors that silence different immunosuppressive receptors, and obtain multiple transgenic immune cells that silence different immunosuppressive receptors. The method in Example 3 is used to detect the killing activity, proliferation promotion, survival and chemotaxis of multiple transgenic immune cells that silence different immunosuppressive receptors.

[0189] The results showed that although other immunosuppressive receptors have similar effects to TIGIT, compared with silencing other immunosuppressive receptors, the transgenic immune cells obtained by combining silencing TIGIT with the above-mentioned expression of fusion protein and CXCR2 (i.e., silencing TIGIT and expressing IL-15Rα, IL-15 and CXCR2 in the transgenic immune cells) have more obvious tumor killing activity, strong anti-tumor activity, strong long-term survival and expansion ability, and strong proliferation and chemotaxis ability, while the transgenic immune cells with silencing other immunosuppressive receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak long-term survival and expansion ability in the body, and weak proliferation and chemotaxis ability.

[0190] 2. By the method in step 1 of Example 2, the shRNA that silences TIGIT is combined with the expression of different chemokine receptors to prepare transgenic immune cells that silence TIGIT and express different chemokines, and the killing activity and chemotaxis ability are compared; further, the shRNA that silences TIGIT is combined with the above-mentioned expression of fusion protein and different chemokine receptors to obtain a variety of transgenic immune cells expressing different chemokine receptors. The method in Example 3 is used to detect the chemotaxis ability, killing activity, proliferation and survival ability of a variety of transgenic immune cells expressing different chemokine receptors.

[0191] The results showed that although other chemokine receptors have similar effects to CXCR2, compared with other chemokine receptors, the transgenic immune cells obtained by combining silencing TIGIT with the above-mentioned expression of fusion protein and CXCR2 (i.e., TIGIT is silenced in the transgenic immune cells, and IL-15Rα, IL-15 and CXCR2 are expressed) have high tumor killing activity, strong anti-tumor activity, strong survival and proliferation ability, and strong chemotaxis, while the transgenic immune cells expressing other chemokine receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak survival and proliferation ability, and weak chemotaxis.

[0192] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0193] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A shRNA, characterized in that The nucleotide sequence of the shRNA is shown in any one of SEQ ID NO:7 to SEQ ID NO:

11.

2. The shRNA according to claim 1, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO:

7.

3. The shRNA according to claim 1, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO:

9.

4. An isolated nucleic acid, characterized in that include: A first nucleic acid molecule, wherein the first nucleic acid molecule inhibits the expression of TIGIT, and the first nucleic acid molecule is the shRNA according to any one of claims 1 to 3; A second nucleic acid molecule, wherein the second nucleic acid molecule encodes a fusion protein, wherein the fusion protein includes IL-15Rα, IL-15, and connecting peptide 1, wherein the N-terminus of the IL-15Rα is connected to the C-terminus of the connecting peptide 1, and the N-terminus of the connecting peptide 1 is connected to the C-terminus of the IL-15; or The C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the IL-15; a third nucleic acid molecule encoding CXCR2; a fourth nucleic acid molecule, disposed between the second nucleic acid molecule and the third nucleic acid molecule, wherein the fourth nucleic acid molecule encodes a connecting peptide 2, wherein the connecting peptide 2 can be cleaved in immune cells, and wherein the connecting peptide 2 includes P2A or a fragment thereof; a first promoter, the first promoter being operably linked to the first nucleic acid molecule; and / or a second promoter, the second promoter being operably linked to the second nucleic acid molecule; The first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter; A fifth nucleic acid molecule is disposed between the first nucleic acid molecule and the second promoter, wherein the fifth nucleic acid molecule encodes a gene transduction efficiency enhancing element, and the gene transduction efficiency enhancing element is cPPT / CTS; as well as a sixth nucleic acid molecule, wherein the sixth nucleic acid molecule is operably linked to the third nucleic acid molecule, and the sixth nucleic acid molecule encodes a post-transcriptional regulatory element, wherein the post-transcriptional regulatory element is WPRE; wherein the separated nucleic acids are, from the 5' end to the 3' end, the first nucleic acid molecule, the fifth nucleic acid molecule, the second nucleic acid molecule, the fourth nucleic acid molecule, the third nucleic acid molecule, and the sixth nucleic acid molecule; Wherein, the amino acid sequence of IL-15Rα is shown in SEQ ID NO: 3; The amino acid sequence of IL-15 is shown in SEQ ID NO:4; The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 6; The amino acid sequence of CXCR2 is shown in SEQ ID NO:5; The amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO:

16.

5. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of TIGIT is shown in SEQ ID NO:

21.

6. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the IL-15Rα is shown in SEQ ID NO:

13.

7. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of IL-15 is shown in SEQ ID NO:

14.

8. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence encoding the connecting peptide 1 is shown in SEQ ID NO:

12.

9. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO:

15.

10. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the third nucleic acid molecule is shown in SEQ ID NO:

17.

11. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the fourth nucleic acid molecule is shown in SEQ ID NO:

18.

12. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the fifth nucleic acid molecule is shown in SEQ ID NO:

19.

13. The isolated nucleic acid according to claim 4, characterized in that The nucleotide sequence of the sixth nucleic acid molecule is shown in SEQ ID NO:

20.

14. An expression vector carrying the isolated nucleic acid according to any one of claims 4 to 13.

15. The expression vector according to claim 14, characterized in that The vector of the expression vector is a non-pathogenic viral vector.

16. The expression vector according to claim 15, characterized in that The viral vector comprises at least one selected from a retroviral vector, a lentiviral vector and an adenovirus-associated viral vector.

17. A kit, characterized in that: include: The isolated nucleic acid according to any one of claims 4 to 13 or the expression vector according to any one of claims 14 to 16.

18. A transgenic immune cell, characterized in that: The transgenic immune cell is obtained by introducing the expression vector according to any one of claims 14 to 16 into an immune cell.

19. The genetically modified immune cell according to claim 18, characterized in that: The transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells and macrophages.

20. The genetically modified immune cell according to claim 19, characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

21. The genetically modified immune cell according to claim 19, characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

22. A pharmaceutical composition, characterized in that include: The isolated nucleic acid according to any one of claims 4 to 13, the expression vector according to any one of claims 14 to 16, and the transgenic immune cell according to any one of claims 18 to 21.

23. A method for enhancing immune cell killing, activation, proliferation and chemotaxis in vitro, characterized in that: include: Introducing the expression vector according to any one of claims 14 to 16 or the expression vector in the kit according to claim 17 into immune cells; The immune cells into which the expression vector has been introduced are cultured.

24. The method according to claim 23, characterized in that The introduction of the expression vector into immune cells is carried out by electroporation, transfection or infection.

25. The method according to claim 23, characterized in that The immune cell is at least one of a T cell, a NKT cell, a NK cell and a macrophage.

26. The method according to claim 23, characterized in that The immune cells are NK cells.

27. The method according to claim 25, characterized in that The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells and NK-92 cells.

28. The method according to claim 25, characterized in that The T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

29. Use of the transgenic immune cell according to any one of claims 18 to 21 or the pharmaceutical composition according to claim 22 in the preparation of a drug for treating or preventing a tumor, wherein the tumor includes a solid tumor and a blood tumor, wherein the solid tumor is a tangible tumor occurring in an organ, wherein the solid tumor includes at least one selected from liver cancer and colorectal cancer, and wherein the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma.

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