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-modified vectors, multifunctional gene-modified immune cells prepared from them, and their applications Technical Field

[0001] This invention relates to the field of biotechnology, specifically to multifunctional gene-modified vectors and the multifunctional gene-modified immune cells prepared therefrom, and their applications. More specifically, this invention relates to shRNA, isolated nucleic acids, kits, transgenic immune cells, pharmaceutical compositions, a method for enhancing the killing, activation, proliferation, and chemotaxis of immune cells, and their uses. Background Technology

[0002] Gene-modified cell therapy is a novel treatment method that treats diseases by modifying the genome of cells in a patient's body. This technology utilizes genetic engineering to introduce exogenous genes or regulatory factors into immune cells, endowing these cells with new functions or enhanced therapeutic potential. Gene-modified cell technology is widely used for 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 exists a tumor microenvironment characterized by hypoxia, low pH, nutrient deficiency, and high osmotic pressure, and lacks a mature blood supply. Therefore, the tumor microenvironment is highly unfavorable for the localization, infiltration, survival, and proliferation of immune cells within the tumor, and easily leads to immunosuppression and immune exhaustion.

[0004] Therefore, there is an urgent need to further develop new gene-modified immune cells to improve their survival, cell proliferation, resistance to immune exhaustion, and ability to infiltrate tumors. Summary of the Invention

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

[0006] This invention is based on the following discoveries of the inventors:

[0007] The inventors unexpectedly discovered that NK cells exhibit weak in vivo expansion and persistence, poor tumor infiltration ability, and are prone to immune exhaustion or immunosuppression. They constructed a multifunctional gene-modification vector expressing a shRNA that silences the immunosuppressive receptor TIGIT, a cell membrane-expressed IL15 / IL15Rα fusion protein, and the chemokine receptor CXCR2, for modifying NK cells. NK cells modified with this multifunctional gene-modification vector can enhance their survival time 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, in a first aspect, the present invention provides an shRNA. According to embodiments of the present invention, the shRNA comprises the nucleotide sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:11. The shRNA according to embodiments 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, thereby enhancing the infiltration ability and anti-tumor activity of immune cells.

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

[0010] In a third aspect, the present invention provides an expression vector. According to embodiments 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 embodiments of the present invention can be used to prepare transgenic immune cells, which exhibit high tumor-killing efficiency, strong anti-tumor activity, long survival time in vitro and in vivo, strong amplification capacity, and strong tumor infiltration capacity.

[0011] In a fourth aspect, the present invention provides a kit. According to embodiments 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 embodiments of the present invention can prepare transgenic immune cells that exhibit high tumor-killing efficiency, strong anti-tumor activity, long survival time in vitro and in vivo, strong amplification capacity, and strong tumor infiltration capacity.

[0012] In a fifth aspect, the present invention provides a transgenic immune cell. According to embodiments 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. This transgenic immune cell possesses advantages such as high tumor-killing efficiency, strong anti-tumor activity, strong long-term survival and expansion capacity in vivo, and strong proliferation and chemotaxis abilities.

[0013] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments 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 cells described in the fifth aspect of the present invention. The pharmaceutical composition according to embodiments of the present invention exhibits high tumor-killing efficiency, strong anti-tumor activity, long-term survival and expansion capabilities in vivo and in vitro, and strong proliferation and chemotaxis abilities.

[0014] In a 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 includes 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 infused with the expression vector. This method can enhance the killing efficiency of immune cells, enhance the antitumor activity of immune cells, improve the long-term survival and expansion capacity of immune cells in vitro and in vivo, and enhance their proliferation and chemotaxis abilities. In particular, it can prepare immune cells with strong killing, activation, proliferation, and chemotaxis in vitro for constructing desired immune cell models.

[0015] In an eighth aspect of the invention, the invention provides for the use of the isolated nucleic acid described in the second aspect of the invention, the expression vector described in the third aspect of the invention, the transgenic immune cells described in the fifth aspect of the invention, and the pharmaceutical composition described in the sixth aspect of the invention in the preparation of a medicament for the treatment or prevention of tumors.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

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

[0018] Figure 1 is a flow cytometry plot showing the silencing efficiency of different TIGIT shRNAs in Example 1.

[0019] Figure 2 is a flow cytometry plot showing the silencing efficiency of different TIGIT shRNAs in Example 1.

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

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

[0022] Figure 5 shows the results of the multifunctional gene-modified vector promoting the killing activity of NK cells in Example 3.

[0023] Figure 6 shows the results of promoting NK cell survival using the multifunctional gene-modified vector in Example 3.

[0024] Figure 7 shows the results of NK cell proliferation promoted by the multifunctional gene-modified vector in Example 3.

[0025] Figure 8 shows the results of NK cell chemotaxis promoted by the multifunctional gene-modified vector in Example 3.

[0026] Figure 9 shows the results of the in vivo tumor-suppressing effect of the multifunctional gene-modified vector on liver cancer significantly enhanced by Example 4.

[0027] Figure 10 shows the in vivo tumor-suppressing effect of the multifunctional gene-modified vector on colorectal cancer in Example 4.

[0028] Figure 11 shows 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 PLC / PRF / 5 liver cancer cells in a comparative study.

[0029] Figure 12 shows the transwell experiment results of multifunctional NK cells expressing CXCR2 in the comparative experiment, which showed that their chemotactic ability was higher than that of multifunctional NK cells expressing other chemokine receptors (CXCR1, CXCR3, CXCR4).

[0030] Figure 13 shows the results in the comparative example of the multifunctional transgenic immune cells of the present invention exhibiting strong killing activity against tumor cells of various different tumor types. Detailed Implementation

[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 construed 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 construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated, nor are they in any particular order. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0034] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0036] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0037] In this document, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0038] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0039] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The chimeric antigen receptor, nucleic acid molecule, expression vector, or transgenic immune cell or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0040] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of drugs or transgenic immune cells to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administration of drugs containing cells with chimeric antigen receptors as described herein to an individual in need.

[0041] This invention proposes an shRNA molecule, isolated nucleic acid, expression vector, kit, transgenic immune cells, pharmaceutical composition, a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells, and their uses, which will be described in detail below.

[0042] shRNA molecules

[0043] This invention proposes an shRNA. According to embodiments of the invention, the shRNA comprises the nucleotide sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:11. The shRNA according to embodiments of the 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, thereby enhancing the infiltration ability and anti-tumor activity of immune cells.

[0044] According to embodiments 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. Therefore, using this shRNA can effectively silence TIGIT molecules on immune cells, especially when the resulting immune cells are brought into contact with tumor cells, thereby improving the killing efficiency against tumors with high expression of the TIGIT receptor ligand CD155 and 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. Therefore, using this shRNA can effectively silence TIGIT molecules on immune cells, especially when the resulting immune cells are brought into contact with tumor cells, thereby improving the killing efficiency against tumors with high expression of the TIGIT receptor ligand CD155 and enhancing the anti-tumor activity of immune cells.

[0047] isolated nucleic acids

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

[0049] According to embodiments 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 an amino acid sequence as 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 linker peptide 1.

[0058] According to an embodiment of the present invention, the N-terminus of IL-15Rα is connected to the C-terminus of the linker peptide 1, and the N-terminus of the linker peptide 1 is connected to the C-terminus of IL-15; or the C-terminus of IL-15Rα is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of IL-15.

[0059] According to an embodiment of the present invention, the linker 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 linker peptide 1 has the nucleotide sequence 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 linked together, and the first nucleic acid molecule is located at one end of the second nucleic acid molecule and the third nucleic acid molecule.

[0065] In this paper, the term "the first nucleic acid molecule is located at one end of the second and third nucleic acid molecules" means that the connected second and third nucleic acid molecules are a whole nucleic acid molecule A, and the first nucleic acid molecule is located at the 3' or 5' end of 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, the fourth nucleic acid molecule encoding a linker peptide 2, the linker peptide 2 being cleavable in the immune cells.

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

[0072] According to an embodiment of the present invention, the linker 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 linker peptide 2 comprises P2A or a fragment thereof.

[0074] According to an embodiment of the present invention, the linker 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 operatively linked to the first nucleic acid molecule; and / or a second promoter operatively 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 a first promoter operably connected to the 5' end of the first nucleic acid molecule; and the 3' end of a second promoter operably connected 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 promoters.

[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 enhancement element.

[0080] According to an embodiment of the present invention, the gene transduction efficiency enhancement 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, which is operatively linked to the third nucleic acid molecule, and the sixth nucleic acid molecule encodes a posttranscriptional regulatory element.

[0083] According to an embodiment of the present invention, the posttranscriptional regulatory element is a 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 isolated nucleic acids are, from the 5' end to the 3' end, 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.

[0086] According to an embodiment of the present invention, the isolated nucleic acids are, from the 5' end to the 3' end, sequentially 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.

[0087] According to an embodiment of the present invention, the isolated 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 isolated 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 carrier

[0090] In a third aspect, the present invention provides an expression vector. According to embodiments 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 embodiments of the present invention can be used to prepare transgenic immune cells, which exhibit high tumor-killing efficiency, strong anti-tumor activity, long survival time in vitro and in vivo, strong amplification capacity, and strong tumor infiltration capacity.

[0091] According to embodiments of the present invention, the 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 retroviral vectors, lentiviral vectors, adenovirus-associated virus vectors, and adenovirus-associated virus vectors.

[0094] Reagent test kit

[0095] In a fourth aspect, the present invention provides a kit. According to embodiments 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 embodiments of the present invention can prepare transgenic immune cells that exhibit high tumor-killing efficiency, strong anti-tumor activity, long survival time in vitro and in vivo, strong amplification capacity, and strong tumor infiltration capacity.

[0096] Transgenic immune cells

[0097] In a fifth aspect, the present invention provides a transgenic immune cell. According to embodiments 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. This transgenic immune cell possesses advantages such as high tumor-killing efficiency, strong anti-tumor activity, strong long-term survival and expansion capacity in vivo, and strong proliferation and chemotaxis abilities.

[0098] Furthermore, the inventors studied various immunosuppressive receptors, screening and comparing the effects of silencing different immunosuppressive receptors on reversing immunosuppressive signals and resisting immune exhaustion. They also obtained various transgenic immune cells by combining the shRNAs of different immunosuppressive receptors with the aforementioned fusion protein and CXCR2. The results showed that although other immunosuppressive receptors have similar effects to TIGIT, compared to other immunosuppressive receptors, the transgenic immune cells obtained by combining TIGIT-shRNA with the aforementioned fusion protein and CXCR2 (i.e., transgenic immune cells that silence TIGIT and express IL-15Rα, IL-15, and CXCR2) exhibited significantly higher tumor-killing efficiency, stronger anti-tumor activity, stronger long-term survival and proliferation capacity in vivo, and stronger proliferation and chemotaxis. In contrast, transgenic immune cells that silenced other immunosuppressive receptors had disadvantages such as lower tumor-killing efficiency, weaker anti-tumor activity, weaker long-term survival and proliferation capacity in vivo, and weaker proliferation and chemotaxis.

[0099] According to embodiments of the present invention, the above-mentioned transgenic 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 linker peptide 1.

[0105] According to an embodiment of the present invention, the N-terminus of IL-15Rα is connected to the C-terminus of the linker peptide 1, and the N-terminus of the linker peptide 1 is connected to the C-terminus of IL-15; or the C-terminus of IL-15Rα is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of IL-15.

[0106] According to an embodiment of the present invention, the linker 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 alternative 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. These transgenic immune cells exhibit higher tumor-killing efficiency, stronger anti-tumor activity, greater long-term survival and expansion capacity in vivo, and stronger proliferation and chemotaxis capabilities.

[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 a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments 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 cells described in the fifth aspect of the present invention. This pharmaceutical composition exhibits high tumor-killing efficiency, strong anti-tumor activity, long-term survival and expansion capabilities in vivo and in vitro, and strong proliferation and chemotaxis abilities.

[0117] A method to enhance the killing, activation, proliferation and chemotaxis of immune cells

[0118] In a 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 includes 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 infused with the expression vector. This method can enhance the killing efficiency of immune cells, enhance the antitumor activity of immune cells, improve the long-term survival and expansion capacity of immune cells in vitro and in vivo, and enhance their proliferation and chemotaxis abilities. In particular, it can prepare immune cells with strong killing, activation, proliferation, and chemotaxis in vitro for constructing desired immune cell models.

[0119] According to embodiments 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 expression vector is introduced into immune cells by electroporation, transfection, or infection.

[0121] According to an embodiment of the present invention, the immune cell is at least one of T cells, NKT cells, NK cells, and macrophages.

[0122] According to an embodiment of the present invention, the immune cell is an NK cell.

[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 an eighth aspect of the invention, the invention provides for the use of the isolated nucleic acid described in the second aspect of the invention, the expression vector described in the third aspect of the invention, the transgenic immune cells described in the fifth aspect of the invention, and the pharmaceutical composition described in the sixth aspect of the invention in the preparation of a medicament for the treatment or prevention of tumors.

[0127] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0128] According to embodiments of the present invention, the tumor includes solid tumors and hematomas.

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

[0130] According to embodiments 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 carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin.

[0131] According to embodiments of the present invention, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic 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 neoplasm.

[0132] method

[0133] In a ninth aspect, the present invention provides a method for treating or preventing tumors. According to an embodiment of the invention, the method includes administering to a subject a pharmaceutically acceptable dose of the transgenic immune cells described in the fifth aspect of the invention and the pharmaceutical composition described in the sixth aspect of the invention. As is known, transgenic immune cells and pharmaceutical compositions possess advantages such as high tumor-killing efficiency, strong anti-tumor activity, long-term survival and expansion capabilities in vivo and in vitro, and strong proliferation and chemotaxis abilities. Therefore, the use of the aforementioned transgenic immune cells and pharmaceutical compositions can effectively kill tumor cells, thereby effectively preventing and treating tumors.

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

[0135] According to embodiments of the present invention, the tumor includes solid tumors and hematomas.

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

[0137] According to embodiments 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 carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin.

[0138] According to embodiments of the present invention, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic 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 neoplasm.

[0139] The sequence descriptions involved in this invention are detailed in Table 1.

[0140] Table 1: Amino Acid / Nucleotide Sequence Description Table

[0141] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0142] Experiment Example 1: shRNA sequence screening for silencing TIGIT

[0143] 1. RNAi target sequence design

[0144] The gene sequence number of 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 shown below. Five RNAi target sequences were designed and screened from the TIGIT mRNA coding sequence (nucleotide sequence shown as SEQ ID NO:22).

[0145] Based on the designed RNAi target sequence, shRNA sequences for constructing shRNA lentiviral vectors were designed. The coding sequences of the designed TIGIT shRNAs (shTG1 to shTG5, nucleotide sequences as shown in SEQ ID NO: 7–11) and the nonsense control sequence (shSCR, nucleotide sequence as shown in SEQ ID NO: 23) have positive, circular, antisense, and termination sequences. Based on the restriction enzyme sites of the pLKO.1-EGFP lentiviral vector, AgeI and EcoRI restriction sites were selected to insert the shRNA sequences, and the resulting plasmids were named pLKO.1-shTG1-EGFP to pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP, respectively.

[0146] 2. Packaging of Lentivirals

[0147] Take 5 × 10⁵ 293T cells in the logarithmic growth phase 6 One cell line was seeded into a 10 cm cell culture dish, and 10 mL of DMEM medium was added. The cells were incubated overnight at 37°C with 5% CO2. When the cell density reached 80-90%, 10 mL of fresh DMEM medium was added, and the cell culture dish was kept in the incubator for later use. Lentiviral packaging systems were prepared by adding 6 μg of psPAX2 and 3 μg of pMD2.G lentiviral helper plasmids, and 6 μg of lentiviral vector plasmids (pLKO.1-shTG1-EGFP~pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP) to 250 μL of serum-free DMEM medium to prepare plasmid mixtures. 15 μL of... Add to 235 μL of serum-free DMEM medium and mix well. The mixture was added to the plasmid mixture separately, mixed well, and incubated at room temperature for 15 min. The mixture was then added to 293T cell culture dishes. After 24 h, the medium was changed, and the culture dishes were returned to a 37℃, 5% CO2 incubator. After 48 h, the cell supernatant was collected, centrifuged at 400×g for 5 min to remove cell debris, and the supernatant was filtered through a 0.45 μm filter into 50 mL centrifuge tubes to obtain viral solutions of different TIGIT shRNA (shTG1~shTG5) and nonsense control sequences (shSCR). The viral solutions were concentrated with 5×PEG8000 solution, and the centrifuge tubes were mixed thoroughly by inverting and incubated overnight at 4℃. After centrifugation at 4℃, 4000×g for 20 min, the supernatant was discarded, and the viral pellet was resuspended in an appropriate amount of serum-free DMEM. The pellet was then transferred to EP tubes and stored at -80℃.

[0148] 3. Detection of silencing efficiency of different TIGIT shRNAs

[0149] NK cells were infected with different TIGIT shRNAs (shTG1–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 resuspended in an appropriate amount of α-MEM medium to adjust the NK cell density to 5×10⁻⁶ cells / mL. 5 Cells / mL. 5 × 10⁵ cells / mL were added to each well of a 24-well plate. 5 One NK cell, 0.2 mL of virus concentrate prepared in step 2 of Example 1, 0.8 mL of α-MEM medium, and protamine (final concentration 8 μg / mL) were mixed thoroughly. The mixed 24-well plate was placed in a 37°C, 5% CO2 incubator. After 24 h, 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 min, and resuspended in a small amount of fresh α-MEM medium. The NK cells were then transferred to a cell culture flask, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added for continued culture. The cell medium was changed every 2 days, and IL-2 (final concentration 200 IU / mL) was added.

[0150] On day 6 after infection with different TIGIT shRNAs (shTG1–shTG5) and nonsense control sequences (shSCR) lentiviruses, TIGIT expression in infected NK cells was detected by flow cytometry. Infected NK cells were transferred to flow cytometry tubes, 3 mL of 1×PBS was added, and the cells were centrifuged at 100×g for 5 min. The supernatant was discarded, and the infected NK cell pellet was resuspended and washed once again with 1×PBS. After resuspending the infected NK cell pellet, APC-labeled anti-human TIGIT antibody (Biolegend) was added for staining, and the cells were incubated at room temperature for 30 min. After washing twice with 1×PBS, 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 GFP tags, GFP-positive cells represent infected cells. In the same tube, the positivity rate of TIGIT on GFP-positive cells (A) and the positivity rate of TIGIT on GFP-negative cells (B) were analyzed separately. The calculation formula was: TIGIT silencing efficiency % = (1-A / B) × 100%.

[0152] The results showed that, compared with uninfected NK cells or the nonsense control group (shSCR), the expression of TIGIT in positive cells infected with different TIGIT shRNAs (shTG1-shTG5) was significantly reduced, as shown in Figure 1. Among them, the silencing efficiency of shTG1 and shTG3 sequences was the highest, reaching over 70%, as shown in Figure 2.

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

[0154] 1. Construction of multifunctional gene modification vectors

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

[0156] 2. Multifunctional gene-modified vector packaging of lentiviruses

[0157] 6 μg of psPAX2 and 3 μg of pMD2.G, and 6 μg of 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 medium to prepare plasmid mixtures and mixed thoroughly. Lentiviral viruses were packaged using the method described in 1.2 of Example 1, thereby obtaining lentiviruses lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5 packaged with different multifunctional gene-modified vectors.

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

[0159] CD3-positive cells from peripheral blood mononuclear cells (PBMCs) were negatively selected using CD3 magnetic beads to enrich NK cells. These sorted cells were then seeded into pre-coated culture flasks and cultured using cytokines such as IL-2, following the method described in CN 202310035787.4. On day 7 of culture, the cultured NK cells were infected with lentiviruses (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5) prepared in step 2 of Example 2. The medium was changed on day 9, and culture continued. Flow cytometry was performed on day 11 to detect the expression of various elements on the NK cells. The flow cytometry method was as follows: 1×10⁻⁶ cells were injected with lentiviruses prepared in step 2 of Example 2.6 Cells were added to flow cytometry tubes for staining. Different staining protocols were followed by antibody administration, and the cells were incubated at room temperature for 30 minutes. After washing twice with 1×PBS, the cell pellet was resuspended, divided into two aliquots, and analyzed by flow cytometry.

[0160] The first batch included PerCP / Cyanine 5.5-labeled anti-human CD3 antibody (Biolegend) and Brilliant Violet 785. TM The first sample contained anti-human CD56 antibody (Biolegend), APC-labeled anti-human TIGIT antibody (Biolegend), and PE-labeled anti-human CXCR2 antibody (Biolegend); the second sample contained PerCP / Cyanine 5.5-labeled anti-human CD3 antibody (Biolegend) and Brilliant Violet 785. TM Anti-human CD56 antibody (Biolegend), APC-labeled anti-human IL-15Rα antibody (Biolegend), and PE-labeled anti-human IL-15 antibody (Invitrogen).

[0161] This embodiment exemplifies the results of NK cell infection with lentivirus packaged in the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results showed that uninfected NK cells highly expressed the TIGIT receptor but did not express CXCR2 and mbIL15RF molecules (see Figure 4A). After infection with the lentivirus packaged in the multifunctional gene-modified 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 comprising IL-15Rα and IL-15) was significantly increased. These results indicate that all three functional elements (the first, second, and third nucleic acid molecules) in the multifunctional gene-modified vector can be efficiently expressed.

[0162] Example 3: In vitro functional assay of NK cells prepared using a multifunctional gene-modified vector

[0163] 1. Construction of multifunctional NK cells

[0164] Collect NK cells in the logarithmic growth phase, centrifuge at 100×g for 5 min to harvest the cells, resuspend the cells in an appropriate amount of α-MEM medium, and adjust the NK cell density to 5×10⁶ cells / year. 5 Cells / mL. 5 × 10⁵ cells / mL were added to each well of a 24-well plate. 51 mL of concentrated lentivirus solution (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5) was mixed with protamine (final concentration 8 μg / mL) and incubated at 37℃ in a 5% CO2 incubator. After 24 h, the NK cell status was observed, the medium was changed, and the infected NK cells were transferred into EP tubes, centrifuged at 100×g for 5 min, and resuspended in a small amount of fresh α-MEM medium. The infected NK cells were then transferred into cell culture flasks, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added respectively, and the cells were cultured for another 48 h. The culture was then expanded, and the post-infection NK cell status was adjusted for further expansion. After infection, CXCR2-positive cells were sorted by flow cytometry to obtain multifunctional NK cells infected with different lentivirus concentrates (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5), which were then used for subsequent in vitro functional experiments.

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

[0166] The inventors tested the cytotoxic activity of NK cells prepared from a multifunctional gene-modified vector. The specific method is as follows: 1×10 4 Human HepG2 liver cancer cells or Hey ovarian cancer cells were seeded into 96-well E plates and cultured overnight using the iCELLigence system for impedance-based real-time cell analysis (RTCA). The next day, 3 × 10⁶ cells were seeded... 4 One NK cell or pluripotent NK cell was inserted into the corresponding well, and then cell impedance was monitored for 2-3 hours using an RTCA system. The cell killing efficiency was calculated as (target cell impedance without effector cells - target cell impedance with effector cells) × 100 ÷ target cell impedance without effector cells.

[0167] This embodiment exemplifies the results of preparing multifunctional NK cells using the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that the multifunctional NK cells prepared using the multifunctional gene-modified vector of this invention have significantly higher killing efficiency against HepG2 liver cancer cells or Hey ovarian cancer cells than unmodified NK cells, as detailed in Figures 5A and 5B.

[0168] 3. Multifunctional gene-modification vectors promote NK cell survival.

[0169] The inventors further verified the effect of the multifunctional gene-modified vector on promoting NK cell survival. The specific method is as follows: NK cells of the same number and the multifunctional NK cells prepared in step 1 of Example 3 were respectively inoculated into 24-well plates, and different IL-2 concentrations (200 IU / mL and 0 IU / mL) were set. Flow cytometry was performed every 24 hours to detect apoptosis rate. The flow cytometry detection of apoptosis rate was performed according to the kit instructions (Linke Biotechnology, catalog number AP101), briefly as follows: cells were collected in EP tubes, washed once by centrifugation with 1×PBS solution, and resuspended. 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube. After gentle vortexing, the cells were incubated at room temperature in the dark for 5 min, and then resuspended for flow cytometry detection. Cell viability was the proportion of cells that were negative for both Annexin V-FITC and PI staining.

[0170] This embodiment exemplifies the results of preparing multifunctional NK cells using the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that in the absence of IL-2 (IL-20 IU / mL), the survival rate of the unmodified NK cell group decreased significantly from 24 hours, with most cells undergoing apoptosis by 72 hours. In contrast, the multifunctional NK cell group maintained a high cell viability even under complete IL-2 withdrawal conditions, with very few cells undergoing apoptosis (see Figure 6). These results demonstrate that the multifunctional gene-modified vector can play an important role in promoting NK cell survival.

[0171] 4. Multifunctional gene-modified vectors promote NK cell proliferation.

[0172] The inventors further verified the effect of the multifunctional gene-modified vector on NK cell proliferation. The specific method is as follows: NK cells of the same number and the multifunctional NK cells prepared in step 1 of Example 3 were respectively inoculated into T25 cell culture flasks, and different IL-2 concentrations (200 IU / mL, 20 IU / mL, and 0 IU / mL) were set. Trypan blue staining and cell counting were performed every 48 hours. Cell proliferation curves were plotted after culturing for 8 days.

[0173] This embodiment exemplifies the results of preparing multifunctional NK cells using the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that the multifunctional NK cells exhibited significantly stronger proliferation capacity than unmodified NK cells at the same IL-2 concentration under different IL-2 conditions, as detailed in Figure 7. These experimental results demonstrate that the multifunctional gene-modified vector can significantly promote NK cell proliferation.

[0174] 5. Multifunctional gene-modified vectors promote the chemotaxis of NK cells.

[0175] The inventors further investigated the chemotactic ability of pluripotent NK cells using a transwell assay. 1×10⁻⁶ cells were introduced into the upper chamber of the transwell. 6 100 NK cells or pluripotent NK cells were cultured in a cell culture chamber with 600 μL of serum-free α-MEM medium, and chemokine CXCL8 at concentrations of 1, 10, and 100 ng / mL were added to induce chemoattraction. The cells were then returned to the cell culture incubator, and after 48 h, the cells in the lower chamber were collected for cell counting.

[0176] This embodiment exemplifies the results of preparing multifunctional NK cells using the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that, compared to unmodified NK cells, the number of multifunctional NK cells migrating to the lower chamber was significantly greater, and this migration was dose-dependent on IL-8 concentration. These experimental results demonstrate that the multifunctional gene-modified vector can significantly promote the chemotactic ability of NK cells.

[0177] Example 4: NK cells prepared from multifunctional gene-modified vectors exhibit potent in vivo tumor-suppressing activity.

[0178] 1. Multifunctional gene-modified vectors significantly enhance the in vivo tumor-suppressing effect of NK cells against liver cancer.

[0179] This embodiment establishes a mouse subcutaneous tumor-bearing model using human hepatocellular carcinoma HepG2 cells to observe the therapeutic effect of the multifunctional NK cells prepared in step 1 of embodiment 3 on the hepatocellular carcinoma tumor-bearing model. The specific method is as follows: Eight 6-week-old NCG mice were selected for subcutaneous tumor implantation in the axilla, with a tumor-bearing dose of 5 × 10⁻⁶ cells / mL. 6 Each mouse received 5 HepG2 cells. On day 3 after tumor implantation, mice underwent NK cell infusion therapy and were randomly assigned to three groups based on tumor size: untreated group, NK cell therapy group, and multifunctional NK cell therapy group. In the NK cell or multifunctional NK cell therapy group, NK cells were infused via tail vein at a dose of 5 × 10⁻⁶ cells / mouse. 6 One cell per treatment per week, for a total of 4 treatments, with an intraperitoneal injection of 5×10 cells every 3-4 days. 4 IU of IL-2 maintains NK cell activity in vivo. Tumor volume is measured weekly, and tumor growth curves are plotted.

[0180] This embodiment exemplifies the results of preparing multifunctional NK cells using the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3). The results show that, compared to the unmodified NK cell therapy group, the multifunctional NK cells prepared based on the multifunctional gene-modified vector of this invention significantly enhanced the inhibitory effect on liver cancer cell growth; see Figure 9 for specific results.

[0181] 2. Multifunctional gene-modified vectors significantly enhance the in vivo tumor-suppressing effect of primary NK cells against colorectal cancer.

[0182] In this embodiment, a human colorectal cancer mouse ectopic xenograft model was established using human colorectal cancer cell line NCI-H716. Human peripheral blood NK cells were infected with lentiviruses (lentivirus-multi-functional-shTG1~lentivirus-multi-functional-shTG5) prepared in step 2 of Example 2 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: Eight 6-week-old NCG mice were selected for subcutaneous tumor implantation in the axilla, with a tumor-bearing dose of 1×10⁻⁶. 7 NCI-H716 cells / device. On day 9 post-tumor implantation, tumors with a volume of 50 mm were selected. 3 Approximately 100 mice were used in the experiment and randomly divided into three groups based on tumor size: an untreated group, a non-genetically modified NK cell therapy group, and a multifunctional NK cell therapy group. Mice in the non-genetically modified NK cell therapy group were treated every two days for a total of three treatments, with each treatment involving a tail vein infusion dose of 8 × 10⁻⁶. 6 CD56 + NK cells, a total of 2.4 × 10⁻⁶ were infused. 7 CD56 + NK cells, and intraperitoneal injection of 5×10 every 2 days. 4 IU of IL-2 maintains NK cell activity in vivo; mice in the multifunctional NK cell therapy group received one tail vein infusion, with a dose of 4×10⁻⁶. 6 One multifunctional NK cell per animal, without adjuvant IL-2 injection. Tumor volume is monitored 1-2 times per week, and tumor growth curves are plotted.

[0184] This embodiment exemplifies the results of multifunctional NK cells prepared using lentivirus (lentivirus-multifunctional-shTG3). The results show that, compared to the unmodified NK cell therapy group, the multifunctional NK cell therapy group exhibited a stronger tumor-suppressing effect at a lower cell therapy dose, with a tumor inhibition rate as high as 64.5%. See Figure 10 for specific results.

[0185] The above experimental 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 applied, which is expected to overcome the bottleneck of poor efficacy of immunotherapy for solid tumors.

[0186] Comparative Example

[0187] 1. Using the method described in step 1 of Example 2, shRNAs silencing different immunosuppressive receptors were combined with the aforementioned fusion protein and CXCR2 to prepare multifunctional gene-modified vectors silencing different immunosuppressive receptors, resulting in various transgenic immune cells with silenced immunosuppressive receptors. The killing activity, proliferation, survival, and chemotaxis of these transgenic immune cells with silenced immunosuppressive receptors were then tested using the method described in Example 3.

[0188] The inventors compared the killing activity of TIGIT-silenced pluripotent transgenic NK cells with other immunosuppressive receptors (TIM3, NKG2A, LAG3) against PLC / PRF / 5 liver cancer cells. The results, shown in Figure 11, indicate that the killing efficiency of TIGIT-silenced pluripotent NK cells (sh-TIGIT+CXCR2) against PLC / PRF / 5 liver cancer cells was significantly higher than that of unmodified NK cells and NK cells with silenced receptors (TIM3, NKG2A, LAG3) (sh-TIM3+CXCR2, sh-NKG2A+CXCR2, sh-LAG3+CXCR2).

[0189] Although other immunosuppressive receptors have similar effects to TIGIT, compared with silencing other immunosuppressive receptors, transgenic immune cells obtained by combining TIGIT with the above-mentioned fusion protein and CXCR2 expression (i.e., transgenic immune cells that silence TIGIT and express IL-15Rα, IL-15 and CXCR2) have more significant effects in killing tumors, strong anti-tumor activity, long-term survival and expansion capacity, and strong proliferation and chemotaxis. In contrast, transgenic immune cells that silence other immunosuppressive receptors have disadvantages such as low tumor killing efficiency, weak anti-tumor activity, weak long-term survival and expansion capacity in vivo, and weak proliferation and chemotaxis.

[0190] 2. Using the method described in step 1 of Example 2, TIGIT-silenced shRNA was combined with receptors expressing different chemokines to prepare transgenic immune cells that silenced TIGIT and expressed different chemokines. The cytotoxic activity and chemotactic ability were compared. Further, the TIGIT-silenced shRNA was combined with the aforementioned fusion protein and different chemokine receptors to obtain various transgenic immune cells expressing different chemokine receptors. The chemotactic ability, cytotoxic activity, proliferation, and survival ability of these various transgenic immune cells expressing different chemokine receptors were detected using the method described in Example 3.

[0191] The inventors used transwell assays to detect and compare the chemotactic capacity of pluripotent NK cells expressing different chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4). As shown in Figure 12, in the presence of different chemokines (CXCL1, CXCL8, CXCL8+CXCL10+CXCL12), the chemotactic capacity of pluripotent NK cells expressing CXCR2 was significantly higher than that of pluripotent NK cells expressing other chemokine receptors (CXCR1, CXCR3, CXCR4). These experimental results indicate that pluripotent transgenic immune cells expressing CXCR2 have a stronger ability to infiltrate and chemotactically attract tumor tissue than immune cells expressing other chemokine receptors.

[0192] Although other chemokine receptors have similar effects to CXCR2, compared with other chemokine receptors, transgenic immune cells obtained by combining silencing TIGIT with the above-mentioned fusion protein and CXCR2 (i.e., transgenic immune cells that silence TIGIT and express IL-15Rα, IL-15 and CXCR2) have high tumor-killing activity, strong anti-tumor activity, strong survival and proliferation ability, and strong chemotactic ability. In contrast, transgenic immune cells expressing other chemokine receptors have disadvantages such as low tumor-killing efficiency, weak anti-tumor activity, weak survival and proliferation ability, and weak chemotactic ability.

[0193] Furthermore, the inventors demonstrated that the invented multifunctional transgenic immune cells (NK cells) exhibit strong killing activity against tumor cells of various tumor types. As shown in Figure 13, the multifunctional NK cells of this invention showed stronger killing activity against SW620 colorectal adenocarcinoma cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, HeLa cervical cancer cells, and K562 chronic myeloid leukemia cells compared to unmodified NK cells.

[0194] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0195] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An shRNA, characterized in that, 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: The first nucleic acid molecule inhibits TIGIT expression; A second nucleic acid molecule, the second nucleic acid molecule encoding a fusion protein, the fusion protein comprising IL-15Rα and IL-15; and A third nucleic acid molecule, wherein the third nucleic acid molecule encodes CXCR2; The first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are linked together.

4. The isolated nucleic acid according to claim 3, characterized in that, The first nucleic acid molecule carries the shRNA as described in 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 the amino acid sequence 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 includes linker peptide 1; Optionally, the N-terminus of IL-15Rα is connected to the C-terminus of the linker peptide 1, and the N-terminus of the linker peptide 1 is connected to the C-terminus of IL-15. or The C-terminus of IL-15Rα is connected to the N-terminus of linker peptide 1, and the C-terminus of linker peptide 1 is connected to the N-terminus of IL-15. Optionally, the linker peptide 1 has the amino acid sequence shown in SEQ ID NO:6; Optionally, the nucleotide sequence encoding the linker 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 and the third nucleic acid molecule are linked together, 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 is disposed between the second and third nucleic acid molecules, the fourth nucleic acid molecule encoding a linker peptide 2, which can be cleaved in immune cells; Optionally, the linker peptide 2 comprises a 2A peptide or a fragment thereof; Optionally, the linker peptide 2 comprises at least one of P2A, T2A, E2A, and F2A, or a fragment thereof; Optionally, the linker peptide 2 comprises P2A or a fragment thereof; Optionally, the linker 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, operatively linked to the first nucleic acid molecule; and / or A second promoter is operatively linked to the second nucleic acid molecule; Optionally, the first promoter and the second promoter are each independently selected from U6, H1, CMV, EF-1, LTR or RSV promoters; Optionally, the isolated nucleic acid further comprises: A fifth nucleic acid molecule is disposed between the first nucleic acid molecule and the second promoter, and the fifth nucleic acid molecule encodes a gene transduction efficiency enhancement element; Optionally, the gene transduction efficiency enhancement 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, which is operatively linked to the third nucleic acid molecule, and which encodes a posttranscriptional regulatory element; Optionally, the post-transcriptional regulatory element is a WPRE; Optionally, the sixth nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

20.

6. The isolated nucleic acid according to claim 5, characterized in that, The isolated nucleic acids are, from the 5' end to the 3' end, sequentially designated as 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; or The isolated nucleic acids are, from 5' to 3', sequentially designated as the first nucleic acid molecule, fifth nucleic acid molecule, third nucleic acid molecule, fourth nucleic acid molecule, second nucleic acid molecule, and sixth nucleic acid molecule; or The isolated nucleic acids are, from 5' to 3', sequentially named as the fifth, third, fourth, second, sixth, and first nucleic acid molecules; or The isolated 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 for the expression vector is a non-pathogenic viral vector; Optionally, the viral vector includes at least one selected from retroviral vectors, lentiviral vectors, adenovirus-associated virus vectors, and adenovirus-associated virus vectors.

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

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

10. The transgenic immune cells according to claim 9, characterized in that, The TIGIT has the amino acid sequence 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 the amino acid sequence 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 includes linker peptide 1; Optionally, the N-terminus of IL-15Rα is connected to the C-terminus of the linker peptide 1, and the N-terminus of the linker peptide 1 is connected to the C-terminus of IL-15. or The C-terminus of IL-15Rα is connected to the N-terminus of linker peptide 1, and the C-terminus of linker peptide 1 is connected to the N-terminus of IL-15. Optionally, the linker peptide 1 has the amino acid sequence shown in SEQ ID NO:6; Optionally, the fusion protein has the amino acid sequence 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 cells are obtained by introducing the expression vector of claim 7 into immune cells; 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 the killing, activation, proliferation, and chemotaxis of immune cells, characterized in that, include: The expression vector of claim 7 or the expression vector in the kit of claim 8 is introduced into immune cells; The immune cells into which the expression vector was introduced were cultured; Optionally, the introduction of the expression vector into immune cells is performed by electroporation, transfection, or infection; Optionally, the immune cells are at least one of T cells, NKT cells, NK cells, and macrophages; 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 γδT 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 medicament for the treatment or prevention of tumors; Optionally, the tumor includes solid tumors and hematomas; Optionally, the solid tumor is a tangible tumor occurring in an organ; Optionally, 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 carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin. Optionally, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasm.

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