Multi-functional genetically modified vector, and multi-functional genetically modified immune cells prepared thereby and use thereof

Abstract

Disclosed are a multi-functional genetically modified vector, and multi-functional genetically modified immune cells prepared thereby and the use thereof. The multi-functional genetically modified vector carries an isolated nucleic acid. The isolated nucleic acid comprises: a first nucleic acid molecule for inhibiting the expression of TIGIT; a second nucleic acid molecule encoding a fusion protein comprising IL-15Rα and IL-15; and a third nucleic acid molecule encoding CXCR2, wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are linked. The multi-functional genetically modified vector can inhibit the expression of TIGIT in a host cell and express a fusion protein comprising IL-15Rα and IL-15, and CXCR2. Therefore, the multi-functional genetically modified immune cells prepared by adopting the multi-functional genetically modified vector can improve the survival capacity, the proliferation capacity and the intratumor infiltration capacity thereof, and can also resist immune depletion, thereby further improving the clinical efficacy of immune cells.

Description

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

[0001] The present invention relates to the field of biotechnology, and more specifically, to multifunctional gene-modified vectors, multifunctional gene-modified immune cells prepared therefrom, and their applications. More specifically, the present invention relates to shRNA, isolated nucleic acids, kits, transgenic immune cells, pharmaceutical compositions, methods for enhancing immune cell killing, activation, proliferation, and chemotaxis, and their applications. Background Art

[0002] Gene-modified cell therapy is a novel treatment approach that modifies the genome of a patient's cells to treat disease. This technology utilizes genetic engineering techniques to introduce exogenous genes or regulatory factors into immune cells, endowing them with new functions or enhanced therapeutic potential. Gene-modified cell technology is widely used in immunotherapy with immune cells such as NK cells, T cells, NKT cells, γδT cells, and macrophages.

[0003] Tumors typically possess a physical barrier of fibrous tissue, within which resides a tumor microenvironment characterized by low oxygen, low pH, nutrient deficiency, and high osmotic pressure, and lack a mature vascular supply. Consequently, the tumor microenvironment is highly unfavorable for the localization, infiltration, survival, and proliferation of immune cells within the tumor, and can easily lead to immunosuppression and immune exhaustion.

[0004] Therefore, there is an urgent need to further develop new gene-modified immune cells to improve immune cell survival, cell proliferation, resistance to immune exhaustion and enhance 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 inventors unexpectedly discovered that NK cells have weak in vivo expansion and persistence, poor tumor infiltration, and are susceptible to immune exhaustion or immunosuppression. They constructed a multifunctional gene-modified vector that expresses shRNA to silence the immunosuppressive receptor TIGIT, a cell membrane-expressed IL15 / IL15Rα fusion protein, and the chemokine receptor CXCR2 for use in modifying NK cells. NK cells modified with this multifunctional gene-modified vector can enhance their survival cycle and cell proliferation in tumors, resist immune exhaustion, and improve their ability to infiltrate tumors, thereby further improving their clinical efficacy.

[0008] Based on this, the first aspect of the present invention provides 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 the TIGIT receptor ligand CD155, and thereby enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.

[0009] In its second aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid comprises a first nucleic acid molecule that inhibits the expression of TIGIT; a second nucleic acid molecule that encodes a fusion protein comprising IL-15Rα and IL-15; and a third nucleic acid molecule that encodes CXCR2; wherein the first, second, and third nucleic acid molecules are linked. The nucleic acid isolated according to an embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells and can cause the immune cell membrane to express the IL15 / IL15Rα fusion protein and CXCR2 receptor, thereby improving the long-term survival and proliferation ability of immune cells carrying the isolated nucleic acid in vitro and in vivo, as well as their infiltration ability and anti-tumor activity in tumors.

[0010] In its third aspect, 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 an embodiment of the present invention can be used to produce transgenic immune cells that exhibit high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong tumor infiltration ability.

[0011] In a fourth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes 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 be used to prepare transgenic immune cells that have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong tumor infiltration ability.

[0012] In its fifth aspect, the present invention provides a transgenic immune cell. According to embodiments of the present invention, the transgenic immune cell suppresses TIGIT expression and expresses a fusion protein and CXCR2; the fusion protein comprises IL-15Rα and IL-15. This transgenic immune cell exhibits advantages such as high tumor killing efficiency, strong anti-tumor activity, long-term survival and expansion in vivo, and strong proliferation and chemotaxis.

[0013] In its sixth aspect, 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 an embodiment of the present invention exhibits high tumor killing efficiency, strong anti-tumor activity, and long-term survival and expansion capabilities in vivo and in vitro, as well as strong proliferation and chemotaxis capabilities.

[0014] In its seventh aspect, the present invention provides 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 into which the expression vector has been introduced. This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, and improve the long-term survival and expansion ability, proliferation, and chemotaxis of immune cells in vitro and in vivo. In particular, it can produce immune cells with strong killing, activation, proliferation, and chemotaxis in vitro, which can be used to construct a desired 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 set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by 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 readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0018] FIG1 is a flow cytometry chart showing the silencing efficiency of different TIGIT shRNAs screened in Example 1.

[0019] FIG2 is a flow cytometry chart showing the silencing efficiency of different TIGIT shRNAs screened in Example 1.

[0020] FIG3 is a schematic diagram of the construction elements of the multifunctional gene modification vector in Example 2.

[0021] FIG4 is a flow cytometry result diagram of expression verification of each element of the multifunctional gene modification vector in Example 2.

[0022] FIG5 is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting the killing activity of NK cells.

[0023] FIG6 is a graph showing the results of the multifunctional gene-modified vector promoting NK cell survival in Example 3.

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

[0025] FIG8 is a graph showing the results of the multifunctional gene-modified vector promoting NK cell chemotaxis in Example 3.

[0026] FIG9 is a graph showing the results of Example 4 showing that the multifunctional gene-modified vector significantly enhances the in vivo tumor inhibition effect of NK cells on liver cancer.

[0027] FIG10 is a graph showing the results of Example 4 showing that the multifunctional gene-modified vector significantly enhances the in vivo tumor inhibition effect of NK cells on colorectal cancer.

[0028] Figure 11 is a graph showing the results of the killing activity of multifunctional transgenic NK cells with silenced TIGIT and multifunctional transgenic NK cells with silenced other immunosuppressive receptors (TIM3, NKG2A, LAG3) against liver cancer cells PLC / PRF / 5.

[0029] Figure 12 is a graph showing the transwell assay results in which the chemotactic ability of multifunctional NK cells expressing CXCR2 in the comparative example is higher than that of multifunctional NK cells expressing other chemokine receptors (CXCR1, CXCR3, and CXCR4).

[0030] FIG13 is a graph showing the results of the comparative example showing that the multifunctional transgenic immune cells of the present invention have strong killing activity against tumor cells of various tumor types. DETAILED DESCRIPTION

[0031] 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.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of technical features indicated, nor should they be understood to indicate a sequential order. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0033] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0034] In order to make the present invention more easily understood, certain 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.

[0035] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0036] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0037] As used herein, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of being inserted into a suitable host and replicating itself, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the above functions. The vector may 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.

[0038] 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 bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0039] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable route. The chimeric antigen receptors, nucleic acid molecules, expression vectors, genetically modified immune cells, or pharmaceutical compositions of the present invention can be administered via any common route, provided that they reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0040] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or 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 arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the 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, reduce or inhibit the 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.

[0041] The present invention provides an 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 are described in detail below.

[0042] shRNA molecules

[0043] The present invention provides a shRNA. According to an embodiment of the present invention, the shRNA includes the 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 the TIGIT receptor ligand CD155, and thereby enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.

[0044] According to an embodiment of the present invention, the above-mentioned shRNA molecule may further include at least one of the following additional technical features:

[0045] 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, and in particular, the obtained immune cells can be brought into contact with tumor cells to improve the killing efficiency of tumors with high expression of the TIGIT receptor ligand CD155, thereby enhancing the anti-tumor activity of immune cells.

[0046] 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, and in particular, the obtained immune cells can be brought into contact with tumor cells to improve the killing efficiency of tumors with high expression of the TIGIT receptor ligand CD155, thereby enhancing the anti-tumor activity of immune cells.

[0047] Isolated nucleic acids

[0048] In its second aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid comprises a first nucleic acid molecule that inhibits the expression of TIGIT; a second nucleic acid molecule that encodes a fusion protein comprising IL-15Rα and IL-15; and a third nucleic acid molecule that encodes CXCR2; wherein the first, second, and third nucleic acid molecules are linked. The nucleic acid isolated according to an embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells and can cause the immune cell membrane to express the IL15 / IL15Rα fusion protein and CXCR2 receptor, thereby improving the long-term survival and proliferation ability of immune cells carrying the isolated nucleic acid in vitro and in vivo, as well as their infiltration ability and anti-tumor activity in tumors.

[0049] According to an embodiment of the present invention, the isolated nucleic acid may further include at least one of the following additional technical features:

[0050] 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.

[0051] According to an embodiment of the present invention, the TIGIT has the nucleotide sequence shown in SEQ ID NO:21.

[0052] 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.

[0053] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:3.

[0054] According to an embodiment of the present invention, the IL-15 has the amino acid sequence shown in SEQ ID NO: 4.

[0055] According to an embodiment of the present invention, the IL-15Rα has a nucleotide sequence as shown in SEQ ID NO:13.

[0056] According to an embodiment of the present invention, the IL-15 has a nucleotide sequence as shown in SEQ ID NO:14.

[0057] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide 1.

[0058] 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.

[0059] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.

[0060] According to an embodiment of the present invention, the nucleotide sequence encoding the connecting peptide 1 is as shown in SEQ ID NO:12.

[0061] According to an embodiment of the present invention, the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:15.

[0062] According to an embodiment of the present invention, the CXCR2 has the amino acid sequence shown in SEQ ID NO:5.

[0063] According to an embodiment of the present invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:17.

[0064] According to an embodiment of the present invention, the second nucleic acid molecule and the third nucleic acid molecule are connected, and the first nucleic acid molecule is located at one end of the second nucleic acid molecule and the third nucleic acid molecule.

[0065] 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 a whole 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] According to an embodiment of the present invention, the isolated nucleic acid further comprises: 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, and the connecting peptide 2 can be cleaved in the immune cell.

[0071] According to an embodiment of the present invention, the connecting peptide 2 includes a 2A peptide or a fragment thereof.

[0072] 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.

[0073] According to an embodiment of the present invention, the connecting peptide 2 includes P2A or a fragment thereof.

[0074] According to an embodiment of the present invention, the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO:16.

[0075] According to an embodiment of the present invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:18.

[0076] According to an embodiment of the present invention, the isolated nucleic acid further comprises: 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.

[0077] According to an embodiment of the present invention, the isolated nucleic acid further comprises: the 3' end of the first promoter is operably linked to the 5' end of the first nucleic acid molecule; the 3' end of the second promoter is operably linked to the 5' end of the second nucleic acid molecule.

[0078] 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.

[0079] According to an embodiment of the present invention, the isolated nucleic acid further comprises: a fifth nucleic acid molecule disposed between the first nucleic acid molecule and the second promoter, wherein the fifth nucleic acid molecule encodes a gene transduction efficiency enhancing element.

[0080] According to an embodiment of the present invention, the gene transduction efficiency enhancing element is cPPT / CTS.

[0081] According to an embodiment of the present invention, the fifth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:19.

[0082] 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.

[0083] According to an embodiment of the present invention, the post-transcriptional regulatory element is WPRE.

[0084] According to an embodiment of the present invention, the sixth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:20.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] expression vector

[0090] In its third aspect, 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 an embodiment of the present invention can be used to produce transgenic immune cells that exhibit high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong tumor infiltration ability.

[0091] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0092] According to an embodiment of the present invention, the vector of the expression vector is a non-pathogenic viral vector.

[0093] According to an embodiment of the present invention, the viral vector includes at least one selected from a retroviral vector, a lentiviral vector, an adeno-associated viral vector, and an adeno-associated viral vector.

[0094] Reagent test kit

[0095] In a fourth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes 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 be used to prepare transgenic immune cells that have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong tumor infiltration ability.

[0096] Genetically modified immune cells

[0097] In its fifth aspect, the present invention provides a transgenic immune cell. According to embodiments of the present invention, the transgenic immune cell suppresses TIGIT expression and expresses a fusion protein and CXCR2; the fusion protein comprises IL-15Rα and IL-15. This transgenic immune cell exhibits advantages such as high tumor killing efficiency, strong anti-tumor activity, long-term survival and expansion in vivo, and strong proliferation and chemotaxis.

[0098] 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 and expressing IL-15Rα, IL-15 and CXCR2 in the transgenic immune cells) have more obvious tumor killing efficiency, strong anti-tumor activity, long-term survival and expansion ability in the body, as well as strong proliferation and chemotaxis. Transgenic immune cells that silence 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.

[0099] 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:

[0100] According to an embodiment of the present invention, the TIGIT has the amino acid sequence shown in SEQ ID NO:1.

[0101] 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.

[0102] According to an embodiment of the present invention, the IL-15Rα has the amino acid sequence shown in SEQ ID NO:3.

[0103] According to an embodiment of the present invention, the IL-15 has the amino acid sequence shown in SEQ ID NO:4.

[0104] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide 1.

[0105] 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.

[0106] According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.

[0107] According to an embodiment of the present invention, the fusion protein has the amino acid sequence shown in SEQ ID NO: 2.

[0108] According to an embodiment of the present invention, the CXCR2 has the amino acid sequence shown in SEQ ID NO:5.

[0109] According to an embodiment of the present invention, the transgenic immune cells are obtained by introducing the aforementioned expression vector into immune cells.

[0110] In some optional embodiments of the present invention, the immune cells include at least one of T cells, NKT cells, NK cells and macrophages.

[0111] 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.

[0112] 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.

[0113] 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 cell lines.

[0114] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, Treg cells and γδT cells.

[0115] Pharmaceutical composition

[0116] In its sixth aspect, 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. This pharmaceutical composition exhibits high tumor killing efficiency, strong anti-tumor activity, and long-term survival and expansion capabilities in vivo and in vitro, as well as strong proliferation and chemotaxis capabilities.

[0117] A method for enhancing immune cell killing, activation, proliferation and chemotaxis

[0118] In its seventh aspect, the present invention provides 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 into which the expression vector has been introduced. This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, and improve the long-term survival and expansion ability, proliferation, and chemotaxis of immune cells in vitro and in vivo. In particular, it can produce immune cells with strong killing, activation, proliferation, and chemotaxis in vitro, which can be used to construct a desired immune cell model.

[0119] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0120] 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.

[0121] 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.

[0122] According to an embodiment of the present invention, the immune cells are NK cells.

[0123] 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 cell lines.

[0124] According to an embodiment of the present invention, the T cells include CD4 + T cells, CD8 + T cells, Treg cells and γδT cells.

[0125] use

[0126] 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.

[0127] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0128] According to an embodiment of the present invention, the tumor includes solid tumors and hematological tumors.

[0129] According to an embodiment of the present invention, the solid tumor is a tangible tumor occurring in an organ.

[0130] 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, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and skin squamous cell carcinoma.

[0131] According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms.

[0132] method

[0133] In its ninth aspect, the present invention provides 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 previously mentioned, the transgenic immune cells and pharmaceutical composition exhibit advantages such as high tumor-killing efficiency, strong anti-tumor activity, long-term survival and expansion in vivo and in vitro, and strong proliferation and chemotaxis. Therefore, the transgenic immune cells and pharmaceutical composition described above can effectively kill tumor cells, thereby effectively preventing and treating tumors.

[0134] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0135] According to an embodiment of the present invention, the tumor includes solid tumors and hematological tumors.

[0136] According to an embodiment of the present invention, the solid tumor is a tangible tumor occurring in an organ.

[0137] 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, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and skin squamous cell carcinoma.

[0138] According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms.

[0139] The sequences involved in the present invention are detailed in Table 1.

[0140] Table 1: Amino acid / nucleotide sequence description

[0141] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0142] Experimental Example 1: Screening of shRNA sequences that silence TIGIT

[0143] 1. RNAi target sequence design

[0144] The gene number for Homo sapiens T cell immunoreceptor with Ig and ITIM domains (TIGIT) was found on the NCBI website as NM_173799.4. The coding sequence (CDS) of TIGIT mRNA is as follows. Five RNAi target sequences were designed within the TIGIT mRNA coding sequence (nucleotide sequence shown in SEQ ID NO: 22) for screening.

[0145] Based on the designed RNAi target sequence, shRNA sequences were designed for constructing shRNA lentiviral vectors. The designed TIGIT shRNA coding sequences (shTG1 to shTG5, nucleotide sequences shown in SEQ ID NOs: 7-11) and the nonsense control sequence (shSCR, nucleotide sequence shown in SEQ ID NO: 23) comprise sense, loop, antisense, and terminator sequences. Based on the restriction sites of the pLKO.1-EGFP lentiviral vector, the shRNA sequences were inserted into the AgeI and EcoRI restriction sites, respectively. The resulting plasmids were named pLKO.1-shTG1-EGFP, pLKO.1-shTG5-EGFP, and pLKO.1-shSCR-EGFP, respectively.

[0146] 2. Lentivirus Packaging

[0147] 5×10 293T cells in the logarithmic growth phase were taken 6 The cells were seeded into a 10cm cell culture dish, 10mL of DMEM medium was added, and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density in the cell culture dish reached 80-90%, 10mL of fresh DMEM medium was replaced, and the cell culture dish was placed in the incubator for standby use. The lentiviral packaging system was prepared by adding 6μg psPAX2 and 3μg pMD2.G, 6μg of lentiviral vector plasmids (pLKO.1-shTG1-EGFP~pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP) to 250μL of serum-free DMEM culture medium, and the plasmid mixtures were prepared and mixed evenly. 15μL Add to 235 μL serum-free DMEM medium and mix well. The mixture was added to the plasmid mixture at once, mixed, and incubated at room temperature for 15 minutes. The mixture was added to the 293T cell culture dish. After 24 hours, the medium was changed and the culture dish was returned to the 37°C, 5% CO2 incubator. After 48 hours, the cell supernatant was collected and centrifuged at 400×g for 5 minutes to remove cell debris. The supernatant was filtered with a 0.45μm filter into a 50mL centrifuge tube to obtain the virus solution of different TIGIT shRNA (shTG1~shTG5) and nonsense control sequence (shSCR) lentivirus. 5×PEG8000 solution was added to concentrate the virus solution. The centrifuge tube was mixed upside down and placed in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus pellet, transfer to an EP tube, and store in a -80°C refrigerator.

[0148] 3. Silencing efficiency detection of different TIGIT shRNAs

[0149] NK cells were infected with different TIGIT shRNAs (shTG1 to shTG5) and nonsense control sequences (shSCR) lentiviruses. NK cells in the logarithmic growth phase were harvested by centrifugation at 100 × g for 5 min, and the NK cells were resuspended in an appropriate amount of α-MEM medium to adjust the NK cell density to 5 × 10 5 5×10 5 NK cells, 0.2mL virus concentrate prepared in step 2 of the present embodiment 1, 0.8mL α-MEM culture medium and protamine (final concentration 8μg / mL), and mix well. The 24-well plate after the mixture is placed in a 37°C, 5% CO2 incubator and cultured. After 24h, the NK cell state is observed, the liquid is changed, the infected NK cells are transferred into an EP tube, 100 × g is centrifuged for 5min, a small amount of fresh α-MEM culture medium is added to resuspend the NK cells, the NK cells are transferred into a cell culture flask, 10mL fresh α-MEM culture medium and IL-2 (final concentration is 200IU / mL) are added to continue to culture. Cell liquid is changed every 2 days, and IL-2 (final concentration is 200IU / mL) is added.

[0150] On day 6 after infection with different TIGIT shRNAs (shTG1-shTG5) and a nonsense control sequence (shSCR) lentivirus, TIGIT expression in infected NK cells was assessed by flow cytometry. The infected NK cells were transferred to a flow cytometry tube, 3 mL of 1× PBS solution was added, and the cells were centrifuged at 100×g for 5 minutes. The supernatant was discarded, and the infected NK cell pellet was flicked and washed again with 1× PBS solution. The infected NK cell pellet was resuspended and stained with an APC-conjugated anti-human TIGIT antibody (Biolegend) and incubated at room temperature for 30 minutes. After washing twice with 1× PBS solution, the infected NK cell pellet was resuspended and analyzed by flow cytometry.

[0151] Because the TIGIT shRNA (shTG1-shTG5) and nonsense control sequence (shSCR) lentiviral vectors contain a GFP tag, GFP-positive cells represent positively infected cells. In the same tube, the TIGIT positivity rate in GFP-positive cells (A) and the TIGIT positivity rate in GFP-negative cells (B) were analyzed separately. Calculation formula: TIGIT silencing efficiency (%) = (1-A / B) × 100%.

[0152] The results showed that TIGIT expression was significantly decreased in cells infected with different TIGIT shRNA (shTG1-shTG5) lentiviruses compared to uninfected NK cells or a nonsense control sequence (shSCR). See Figure 1 for specific results. Among them, the shTG1 and shTG3 sequences had the highest silencing efficiency, exceeding 70%. See Figure 2 for specific results.

[0153] Example 2: Construction of a multifunctional gene modification vector and verification of expression of each element

[0154] 1. Construction of a multifunctional gene modification vector

[0155] The base sequence of cPPT / CTS-EF1a-mbIL15RF-P2A-CXCR2-WPRE was synthesized by whole gene synthesis and cloned into the lentiviral vectors (pLKO.1-shTG1-EGFP to pLKO.1-shTG5-EGFP) prepared in Example 1 above through the restriction sites EcoRI and KpnI. After sequencing verification, the pLKO.1-multi-functional-vector-shTG1 to pLKO.1-multi-functional-vector-shTG5 plasmids were obtained, which are the multifunctional gene modification vector plasmids involved in the present invention. This example exemplifies the construction of the pLKO.1-multi-functional-vector-shTG3 multifunctional gene modification vector and a schematic diagram of its elements, see Figure 3 for details.

[0156] 2. Multifunctional gene modification vector packaging lentivirus

[0157] 6 μg psPAX2 and 3 μg pMD2.G of the lentiviral packaging helper plasmids, and 6 μg of the multifunctional gene-modified lentiviral vector plasmids (pLKO.1-multi-functional-vector-shTG1 to 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 lentiviruses lentivirus-multi-functional-shTG1 to lentivirus-multi-functional-shTG5 packaged with different multifunctional gene-modified vectors.

[0158] 3. Verification of the expression of each element of the multifunctional gene modification vector

[0159] CD3 positive cells in peripheral blood mononuclear cells (PBMC) were removed by negative selection with CD3 magnetic beads, NK cells in PBMC were enriched, and the sorted cells were inoculated in pre-coated culture flasks for culture. Referring to the method described in CN 202310035787.4, IL-2 and other cytokines were 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. After changing the medium on the 9th day, the culture was continued. On the 11th day, flow cytometry was performed to detect the expression of various elements on the NK cells. The flow cytometry method is as follows: 1×106 Each cell was added to a flow cytometry tube for staining. Antibodies were added according to different staining protocols and incubated at room temperature for 30 minutes. After washing twice with 1× PBS solution, the cell pellet was resuspended and divided into two aliquots for flow cytometry analysis.

[0160] The first part was added with PerCP / Cyanine5.5 labeled anti-human CD3 antibody (Biolegend), Brilliant Violet 785 TM The second part 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 part 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).

[0161] This example exemplifies the results of NK cell infection with a lentivirus packaged with a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3). The results showed that the TIGIT receptor was highly expressed on the NK cells of the uninfected group, and CXCR2 and mbIL15RF molecules were not expressed, as shown in Figure 4A. After infection with a lentivirus packaged with a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3), the expression of the TIGIT receptor on NK cells was significantly reduced, while the expression of CXCR2 and mbIL15RF molecules (encoding a fusion protein including IL-15Rα and IL-15) was significantly increased. These results indicate that the three functional elements (first nucleic acid molecule, second nucleic acid molecule, third nucleic acid molecule) in the multifunctional gene modification vector can be efficiently expressed.

[0162] Example 3: In vitro functional assay of NK cells prepared with multifunctional gene-modified vectors

[0163] 1. Construction of multifunctional NK cells

[0164] NK cells in the logarithmic growth phase were aspirated and centrifuged at 100 × g for 5 min to harvest the cells. Appropriate amount of α-MEM medium was added to resuspend the cells and the NK cell density was adjusted to 5 × 10 5 5×10 5For each NK cell, 1 mL of concentrated lentivirus solution (lentivirus-multi-functional-shTG1 to lentivirus-multi-functional-shTG5) was added to protamine (final concentration 8 μg / mL) and mixed evenly. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the NK cell status was observed, the medium was changed, and the infected NK cells were transferred to EP tubes. Centrifuged at 100×g for 5 minutes, and a small amount of fresh α-MEM medium was added to resuspend the cells. The infected NK cells were transferred to cell culture flasks, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added to each flask and cultured for another 48 hours. The culture was then expanded, and the status of the infected NK cells was adjusted for amplification. The infected NK cells were sorted by flow cytometry, and the CXCR2-positive cells were used as infection-positive cells. That is, multifunctional NK 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.

[0165] 2. Multifunctional gene-modified vectors promote the killing activity of NK cells

[0166] The inventors tested the killing activity of NK cells prepared by multifunctional gene modified vectors. The specific method is as follows: 1×10 4 Target cells, human liver cancer cells HepG2 or human ovarian cancer cells Hey, were seeded into 96-well E-plates and monitored overnight in culture using the impedance-based real-time cell analysis (RTCA) iCELLigence system. 4 Individual NK cells or multifunctional NK cells were plated into the corresponding wells, and the cell impedance was monitored for 2-3 hours using the RTCA system. The cell killing efficiency was calculated as (impedance of target cells without effector cells - impedance of target cells with effector cells) × 100 ÷ impedance of target cells without effector cells.

[0167] This example demonstrates the results of multifunctional NK cells prepared using a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that multifunctional NK cells modified with the multifunctional gene-modified vector of the present invention were significantly more effective at killing HepG2 liver cancer cells or Hey ovarian cancer cells than unmodified NK cells, as shown in Figures 5A and 5B.

[0168] 3. Multifunctional gene modification vector promotes NK cell survival

[0169] The inventors further verified the effect of the multifunctional gene-modified vector on the survival of NK cells. The specific method is as follows: the same number of NK cells and the multifunctional NK cells prepared in step 1 of Example 3 were plated in 24-well plates, 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 flow cytometry detection of apoptosis rate was performed according to the steps of the kit instructions (Lianke Bio, product number AP101), which is briefly as follows: collect the cells in an EP tube, 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 gently vortexing to mix, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow cytometry. The cell viability is the ratio of double negative Annexin V-FITC and PI staining.

[0170] This example exemplifies the results of multifunctional NK 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-20IU / mL), the cell survival rate of the non-genetically modified NK cell group decreased significantly from 24h, and most cells had undergone apoptosis by 72h; while the multifunctional NK cell group cells were able to maintain a high cell viability even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis, as shown in Figure 6. These results show that the multifunctional gene modification vector can play an important role in promoting the survival of NK cells.

[0171] 4. Multifunctional gene modification vector promotes the proliferation of NK cells

[0172] 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 cells and the multifunctional NK 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. Trypan blue staining was performed every 48 hours to count the cells. The cells were observed and cultured until the 8th day, and the cell proliferation curve was drawn.

[0173] This example demonstrates the results of multifunctional NK cells prepared using a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that the proliferation capacity of multifunctional NK cells under different IL-2 concentrations is significantly stronger than that of unmodified NK cells under the same IL-2 concentration, as shown in Figure 7. These experimental results show that the multifunctional gene-modified vector can significantly promote the proliferation of NK cells.

[0174] 5. Multifunctional gene-modified vectors promote NK cell chemotaxis

[0175] The inventors further investigated the chemotactic ability of multifunctional NK cells by transwell experiment. 6 For each NK cell or multifunctional NK cell, add 600 μL of serum-free α-MEM medium to the lower chamber and add chemokine CXCL8 at concentrations of 1, 10, and 100 ng / mL for chemotaxis. Return the cells to the cell culture incubator and collect the cells in the lower chamber after 48 hours for cell counting.

[0176] This example demonstrates the results of multifunctional NK cells prepared using a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that compared to unmodified NK cells, significantly more multifunctional NK cells migrated to the lower chamber, and the relationship was dose-dependent with IL-8 concentration. These experimental results demonstrate that the multifunctional gene-modified vector can significantly enhance the chemotactic ability of NK cells.

[0177] Example 4: NK cells prepared by multifunctional gene-modified vectors have strong tumor-suppressing activity in vivo

[0178] 1. The multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of NK cells on liver cancer

[0179] In this example, a subcutaneous tumor-bearing model of mice was established using human liver cancer HepG2 cells to observe the therapeutic effect of the multifunctional NK cells prepared in step 1 of Example 3 on the liver cancer tumor-bearing model. 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 5×10 6 HepG2 cells / mouse. On the third day after tumor loading, mice were treated with NK cell transfusion and randomly divided into untreated group, NK cell treatment group and multifunctional NK cell treatment group according to tumor size. In the NK cell or multifunctional NK cell treatment group, NK cells were transfused through the tail vein at a therapeutic dose of 5×10 6 cells / time / week, for a total of 4 treatments, and 5×10 4 IU of IL-2 was used to maintain NK cell activity in vivo. Tumor volume was measured once a week, and tumor growth curves were drawn.

[0180] This example demonstrates the results of multifunctional NK cells prepared using a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that compared to the unmodified NK cell treatment group, multifunctional NK cells prepared using the multifunctional gene-modified vector of the present invention significantly enhanced the ability to inhibit the growth of liver cancer cells. See Figure 9 for specific results.

[0181] 2. Multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of primary NK cells on colorectal cancer

[0182] In this example, a human colorectal cancer mouse heterotopic transplant tumor model was established using the human colorectal cancer cell line NCI-H716 cells. Human peripheral blood 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 NK cells from different peripheral blood sources, and the therapeutic effect of multifunctional NK cells on the human colorectal cancer model was observed.

[0183] The specific method is as follows: 8 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, with a tumor-bearing dose of 1×10 7 NCI-H716 cells / mouse. On day 9 after tumor implantation, the screening tumor volume was 50 mm 3 About 100 mice were used in the experiment and randomly divided into an untreated group, a non-genetically modified NK cell treatment group, and a multifunctional NK cell treatment group according to the size of the tumor. The mice in the non-genetically modified 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 + NK cells were injected intraperitoneally every 2 days at a dose of 5 × 10 4 IU of IL-2 maintained the activity of NK cells in vivo; mice in the multifunctional NK cell treatment group were reinfused into the tail vein once, with a dose of 4×10 6 Multifunctional NK cells / mouse, no IL-2 injection adjuvant therapy. Tumor volume was measured 1-2 times a week, and tumor growth curves were drawn.

[0184] This example demonstrates the results of multifunctional NK cells prepared with a lentivirus (lentivirus-multi-functional-shTG3). The results show that, compared with the unmodified NK cell treatment group, the multifunctional NK cell treatment group still exhibited a stronger tumor suppression effect at a lower cell treatment dose, with a tumor inhibition rate of up to 64.5%. See Figure 10 for specific results.

[0185] The above test results show 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 the dosage is lower when used, which is expected to break through the bottleneck of poor treatment effect of immune cell therapy on solid tumors.

[0186] Comparative Example

[0187] 1. Using the method described in step 1 of Example 2, shRNAs that silence different immunosuppressive receptors were combined with the above-described expression fusion protein and CXCR2 to prepare multifunctional gene-modified vectors that silence different immunosuppressive receptors, thereby obtaining a variety of transgenic immune cells that silence different immunosuppressive receptors. The methods described in Example 3 were used to test the cytotoxicity, proliferation-promoting, survival, and chemotactic abilities of the various transgenic immune cells that silenced different immunosuppressive receptors.

[0188] The inventors compared the killing activity of multifunctional transgenic NK cells that silenced TIGIT with multifunctional transgenic NK cells that silenced other immunosuppressive receptors (TIM3, NKG2A, LAG3) against liver cancer cells PLC / PRF / 5. The results are shown in Figure 11. The killing efficiency of multifunctional NK cells that silenced TIGIT (sh-TIGIT+CXCR2) on liver cancer PLC / PRF / 5 cells was significantly higher than that of NK cells that were not genetically modified and NK cells that silenced other different immunosuppressive receptors (TIM3, NKG2A, LAG3) (sh-TIM3+CXCR2, sh-NKG2A+CXCR2, sh-LAG3+CXCR2).

[0189] Although other immunoinhibitory receptors have similar effects to TIGIT, compared with silencing other immunoinhibitory 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 high 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 that silence other immunoinhibitory 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. Using the method in step 1 of Example 2, combine TIGIT-silencing shRNA with cells expressing different chemokine receptors to prepare transgenic immune cells that silence TIGIT and express different chemokines, and compare their cytotoxicity and chemotactic ability. Furthermore, combine TIGIT-silencing shRNA with the aforementioned fusion protein and different chemokine receptors to obtain multiple transgenic immune cells expressing different chemokine receptors. Use the method in Example 3 to detect the chemotactic ability, cytotoxicity, proliferation, and survival of multiple transgenic immune cells expressing different chemokine receptors.

[0191] The inventors detected and compared the chemotactic ability of multifunctional NK cells expressing different chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4) through transwell experiments. As shown in Figure 12, in the presence of different chemokines (CXCL1, CXCL8, CXCL8+CXCL10+CXCL12), the chemotactic ability of multifunctional NK cells expressing CXCR2 was significantly higher than that of multifunctional NK cells expressing other chemokine receptors (CXCR1, CXCR3, CXCR4). The above experimental results show that multifunctional transgenic immune cells expressing CXCR2 have a stronger ability to infiltrate and chemotactically move into tumor tissue than immune cells expressing other chemokine receptors.

[0192] 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 and IL-15Rα, IL-15 and CXCR2 are expressed in the transgenic immune cells) have high tumor killing activity, strong anti-tumor activity, strong survival and proliferation ability, and strong chemotactic ability, 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 chemotactic ability.

[0193] The inventors also demonstrated that the invented multifunctional transgenic immune cells (NK cells) exhibited strong cytotoxic activity against a variety of tumor types. As shown in Figure 13, the multifunctional NK cells of the present invention exhibited stronger cytotoxic activity against colorectal adenocarcinoma SW620 cells, colon cancer HCT116 cells, breast cancer MDA-MB-231 cells, cervical cancer HeLa cells, and chronic myeloid leukemia K562 cells than unmodified NK cells.

[0194] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0195] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A shRNA, characterized in that It includes the nucleotide sequence 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 shRNA has a nucleotide sequence as shown in SEQ ID NO:7; Optionally, the shRNA has a nucleotide sequence as shown in SEQ ID NO:

9.

3. An isolated nucleic acid, characterized in that include: a first nucleic acid molecule that inhibits expression of TIGIT; a second nucleic acid molecule encoding a fusion protein comprising IL-15Rα and IL-15; and a third nucleic acid molecule encoding CXCR2; Wherein, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected.

4. The isolated nucleic acid according to claim 3, characterized in that The first nucleic acid molecule carries the shRNA according to any one of claims 1 to 2; Optionally, the TIGIT has the nucleotide sequence shown in SEQ ID NO: 21; Optionally, 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; Optionally, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 3; Optionally, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 4; Optionally, the IL-15Rα has a nucleotide sequence as shown in SEQ ID NO: 13; Optionally, the IL-15 has a nucleotide sequence as shown in SEQ ID NO: 14; Optionally, the fusion protein further comprises a connecting peptide 1; Optionally, 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; Optionally, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO: 6; Optionally, the nucleotide sequence encoding the connecting peptide 1 is shown in SEQ ID NO: 12; Optionally, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 15; Optionally, the CXCR2 has an amino acid sequence as shown in SEQ ID NO: 5; Optionally, the third nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

17.

5. The isolated nucleic acid according to claim 4, characterized in that 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; Optionally, the isolated nucleic acid further comprises: 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, and the connecting peptide 2 can be cleaved in immune cells; Optionally, the connecting peptide 2 comprises a 2A peptide or a fragment thereof; Optionally, the connecting peptide 2 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof; Optionally, the connecting peptide 2 comprises P2A or a fragment thereof; Optionally, the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO: 16; Optionally, the fourth nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 18; Optionally, the isolated nucleic acid further comprises: 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; Optionally, the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter; Optionally, the isolated nucleic acid further comprises: a fifth nucleic acid molecule, disposed between the first nucleic acid molecule and the second promoter, wherein the fifth nucleic acid molecule encodes a gene transduction efficiency enhancing element; Optionally, the gene transduction efficiency enhancing element is cPPT / CTS; Optionally, the fifth nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 19; Optionally, the isolated nucleic acid further comprises: a sixth nucleic acid molecule, the sixth nucleic acid molecule being operably linked to the third nucleic acid molecule, the sixth nucleic acid molecule encoding a post-transcriptional regulatory element; Optionally, the post-transcriptional regulatory element is WPRE; Optionally, the sixth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:

20.

6. The isolated nucleic acid according to claim 5, characterized in that The separated nucleic acid is sequentially a first nucleic acid molecule, a fifth nucleic acid molecule, a second nucleic acid molecule, a fourth nucleic acid molecule, a third nucleic acid molecule, and a sixth nucleic acid molecule from the 5' end to the 3' end; or The separated nucleic acid is, from the 5' end to the 3' end, a first nucleic acid molecule, a fifth nucleic acid molecule, a third nucleic acid molecule, a fourth nucleic acid molecule, a second nucleic acid molecule, and a sixth nucleic acid molecule; or The separated nucleic acid is, 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; or 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.

7. An expression vector carrying the isolated nucleic acid according to any one of claims 3 to 6; Optionally, the vector of the expression vector is a non-pathogenic viral vector; Optionally, the viral vector comprises at least one selected from a retroviral vector, a lentiviral vector, an adeno-associated viral vector and an adeno-associated viral vector.

8. A kit, characterized in that: include: The isolated nucleic acid according to any one of claims 3 to 6 or the expression vector according to claim 7.

9. A transgenic immune cell, characterized in that: The transgenic immune cells inhibit the expression of TIGIT, and express the fusion protein and CXCR2; Wherein, the fusion protein includes IL-15Rα and IL-15.

10. The genetically modified immune cell according to claim 9, characterized in that The TIGIT has an amino acid sequence as shown in SEQ ID NO: 1; Optionally, 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; Optionally, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 3; Optionally, the IL-15 has an amino acid sequence as shown in SEQ ID NO: 4; Optionally, the fusion protein further comprises a connecting peptide 1; Optionally, 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; Optionally, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO: 6; Optionally, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 2; Optionally, the CXCR2 has an amino acid sequence as shown in SEQ ID NO: 5; Optionally, the transgenic immune cell is obtained by introducing the expression vector of claim 7 into an immune cell; Optionally, the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells and macrophages, preferably NK cells; Optionally, 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 cell lines; Optionally, the T cells include CD4 + T cells, CD8 + T cells, Treg cells and γδT cells.

11. A pharmaceutical composition, characterized in that include: The isolated nucleic acid according to any one of claims 2 to 6, the expression vector according to claim 7, and the transgenic immune cell according to any one of claims 9 to 10.

12. A method for enhancing immune cell killing, activation, proliferation and chemotaxis, characterized in that: include: Introducing the expression vector according to claim 7 or the expression vector in the kit according to claim 8 into immune cells; Cultivating immune cells into which the expression vector has been introduced; Optionally, the introduction of the expression vector into the immune cell is performed by electroporation, transfection or infection; Optionally, the immune cell is at least one of a T cell, a NKT cell, a NK cell and a macrophage; Optionally, the immune cells are NK cells; Optionally, 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 cell lines; Optionally, the T cells include CD4 + T cells, CD8 + T cells and γδ T cells.

13. Use of the isolated nucleic acid according to any one of claims 3 to 6, the expression vector according to claim 7, the transgenic immune cell according to any one of claims 9 to 10, or the pharmaceutical composition according to claim 11 in the preparation of a drug for treating or preventing a tumor; Optionally, the tumor includes solid tumors and hematological tumors; Optionally, the solid tumor is a tangible tumor occurring in an organ; Optionally, the solid tumor comprises 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, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and skin squamous cell carcinoma; Optionally, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms.

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