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WO2025108480A1 - Vecteur génétiquement modifié multifonctionnel, cellules immunitaires génétiquement modifiées multifonctionnelles ainsi préparées et leur utilisation - Google Patents

Vecteur génétiquement modifié multifonctionnel, cellules immunitaires génétiquement modifiées multifonctionnelles ainsi préparées et leur utilisation Download PDF

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WO2025108480A1
WO2025108480A1 PCT/CN2024/134157 CN2024134157W WO2025108480A1 WO 2025108480 A1 WO2025108480 A1 WO 2025108480A1 CN 2024134157 W CN2024134157 W CN 2024134157W WO 2025108480 A1 WO2025108480 A1 WO 2025108480A1
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nucleic acid
acid molecule
cells
optionally
seq
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Chinese (zh)
Inventor
王烃
周英利
胡渊
张彩
陈敏华
谢思奇
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Shanghai NK Cell Tech Co Ltd
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Shanghai NK Cell Tech Co Ltd
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C12N15/09—Recombinant DNA-technology
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    • C12N5/0602—Vertebrate cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of biotechnology, and in particular, to a multifunctional gene modification vector and a multifunctional gene modification immune cell prepared therefrom and their applications. More particularly, the present invention relates to a shRNA, an isolated nucleic acid, a kit, a transgenic immune cell, a pharmaceutical composition, a method for enhancing immune cell killing, activation, proliferation and chemotaxis and their applications.
  • Gene-modified cell therapy is a new type of treatment that treats diseases by modifying the genome of cells in the patient's body.
  • This technology uses genetic engineering technology to introduce exogenous genes or regulatory factors into immune cells, giving these immune cells new functions or stronger therapeutic potential.
  • Gene-modified cell technology is widely used in immune cells used for immunotherapy, such as NK cells, T cells, NKT cells, ⁇ T cells and macrophages.
  • Tumors usually have a physical barrier of fibrous tissue, and within the barrier there is a tumor microenvironment with hypoxia, low pH, nutrient deficiency, and high osmotic pressure, and lacks mature vascular supply. Therefore, the microenvironment within the tumor is very unfavorable for the localization, infiltration, survival, and proliferation of immune cells in the tumor, and is prone to immunosuppression and immune exhaustion.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • NK cells have weak in vivo expansion and persistence, poor tumor infiltration ability, and are prone to immune exhaustion or immunosuppression.
  • the inventor constructed a multifunctional gene modification vector that expresses shRNA of the silencing immunosuppressive receptor TIGIT, IL15/IL15R ⁇ fusion protein expressed on the cell membrane, and chemokine receptor CXCR2 for modifying NK cells.
  • NK cells modified by the multifunctional gene modification vector of the present invention can enhance their survival cycle and cell proliferation ability in tumors, resist immune exhaustion, and improve the ability to infiltrate tumors, thereby further improving their efficacy in clinical practice.
  • the present invention proposes a shRNA.
  • the shRNA includes a nucleotide sequence shown in any one of SEQ ID NO:7 to SEQ ID NO:11.
  • the shRNA according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, enhance the killing efficiency of immune cells against tumors with high expression of TIGIT receptor ligand CD155, and thus enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.
  • the present invention proposes an isolated nucleic acid.
  • the isolated nucleic acid includes a first nucleic acid molecule, which inhibits the expression of TIGIT; a second nucleic acid molecule, which encodes a fusion protein, and the fusion protein includes IL-15R ⁇ and IL-15; and a third nucleic acid molecule, which encodes CXCR2; wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected.
  • the nucleic acid isolated according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, and can make the immune cell membrane express IL15/IL15R ⁇ fusion protein and CXCR2 receptor, and can improve the long-term survival and proliferation ability of immune cells carrying the above-mentioned isolated nucleic acid in vivo and in vitro, and its infiltration ability and anti-tumor activity in tumors.
  • the present invention provides an expression vector.
  • the expression vector carries the isolated nucleic acid described in the second aspect of the present invention.
  • the expression vector according to the embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.
  • the present invention provides a kit.
  • the kit comprises the isolated nucleic acid described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention.
  • the kit according to an embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.
  • the present invention proposes a transgenic immune cell.
  • the transgenic immune cell inhibits the expression of TIGIT, and expresses a fusion protein and CXCR2; wherein the fusion protein includes IL-15R ⁇ and IL-15.
  • the transgenic immune cell has the advantages of high tumor killing efficiency, strong anti-tumor activity, strong long-term survival and expansion ability in the body, and strong proliferation and chemotaxis ability.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the transgenic immune cell described in the fifth aspect of the present invention.
  • the pharmaceutical composition according to the embodiment of the present invention has high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability.
  • the present invention proposes a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells.
  • the method comprises introducing the expression vector described in the third aspect of the present invention or the expression vector in the kit described in the fourth aspect of the present invention into immune cells; and culturing the immune cells introduced with the expression vector.
  • This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, improve the long-term survival and amplification ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially can prepare immune cells with strong killing, activation, proliferation and chemotaxis in vitro for constructing the required immune cell model.
  • 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.
  • Figure 1 is a flow cytometer chart showing the silencing efficiency of different TIGIT shRNAs screened in Example 1.
  • Figure 2 is a flow cytometry chart of the silencing efficiency of different TIGIT shRNAs screened in Example 1.
  • FIG. 3 is a schematic diagram of the construction elements of the multifunctional gene modification vector in Example 2.
  • FIG. 4 is a flow cytometry result diagram of expression verification of each element of the multifunctional gene modification vector in Example 2.
  • FIG. 5 is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting the killing activity of NK cells.
  • FIG. 6 is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting the survival of NK cells.
  • FIG. 7 is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting NK cell proliferation.
  • FIG8 is a graph showing the results of the multifunctional gene-modified vector in Example 3 promoting NK cell chemotaxis.
  • FIG. 9 is a graph showing the results of Example 4 showing that the multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of NK cells on liver cancer.
  • FIG. 10 is a graph showing the results of Example 4 showing that the multifunctional gene-modified vector significantly enhances the in vivo tumor-suppressing effect of NK cells on colorectal cancer.
  • Figure 11 is a graph showing the results of the killing activity of multifunctional transgenic NK cells with TIGIT silenced and multifunctional transgenic NK cells with other immunosuppressive receptors (TIM3, NKG2A, LAG3) against liver cancer cells PLC/PRF/5 in the comparative example.
  • FIG. 12 is a graph showing the results of a transwell experiment 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, CXCR4).
  • FIG. 13 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.
  • first and second are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated, nor is there any order of precedence. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, “plurality” means two or more.
  • the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or situation can but does not necessarily occur, and the description includes cases where the event or situation occurs and cases where it does not occur.
  • the term "vector” or "expression vector” generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and/or translation of DNA or RNA.
  • the vector also includes vectors with multiple of the above functions.
  • the vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce a desired expression product by culturing a suitable host cell containing the vector.
  • the term "pharmaceutical composition” generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.
  • the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means.
  • the chimeric antigen receptor, nucleic acid molecule, expression vector or transgenic immune cell or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration.
  • the composition of the present invention is administered by intravenous injection.
  • treatment refers to the use of drugs to obtain the desired pharmacological and/or physiological effects.
  • the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease.
  • Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
  • Treatment covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.
  • the present invention provides a shRNA molecule, an isolated nucleic acid, an expression vector, a kit, a transgenic immune cell, a pharmaceutical composition, a method for enhancing immune cell killing, activation, proliferation and chemotaxis and uses thereof, which will be described in detail below.
  • the present invention proposes a shRNA.
  • the shRNA includes a nucleotide sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 11.
  • the shRNA according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, enhance the killing efficiency of immune cells against tumors with high expression of TIGIT receptor ligand CD155, and thus enhance the infiltration ability of immune cells and the anti-tumor activity of immune cells.
  • the above shRNA molecule may further include at least one of the following additional technical features:
  • the shRNA has a nucleotide sequence as shown in SEQ ID NO: 7.
  • the shRNA can be used to effectively silence TIGIT molecules on immune cells, especially by contacting the obtained immune cells with tumor cells, which can improve the killing efficiency of tumors with high expression of TIGIT receptor ligand CD155 and enhance the anti-tumor activity of immune cells.
  • the shRNA has a nucleotide sequence as shown in SEQ ID NO: 9.
  • the shRNA can be used to effectively silence TIGIT molecules on immune cells, especially by contacting the obtained immune cells with tumor cells, which can improve the killing efficiency of tumors with high expression of TIGIT receptor ligand CD155 and enhance the anti-tumor activity of immune cells.
  • the present invention proposes an isolated nucleic acid.
  • the isolated nucleic acid includes a first nucleic acid molecule, which inhibits the expression of TIGIT; a second nucleic acid molecule, which encodes a fusion protein, and the fusion protein includes IL-15R ⁇ and IL-15; and a third nucleic acid molecule, which encodes CXCR2; wherein the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are connected.
  • the nucleic acid isolated according to the embodiment of the present invention can inhibit the expression of TIGIT molecules on immune cells, and can make the immune cell membrane express IL15/IL15R ⁇ fusion protein and CXCR2 receptor, and can improve the long-term survival and proliferation ability of immune cells carrying the above-mentioned isolated nucleic acid in vivo and in vitro, and its infiltration ability and anti-tumor activity in tumors.
  • the isolated nucleic acid may further include at least one of the following additional technical features:
  • the first nucleic acid molecule carries the shRNA described in the first aspect of the present invention.
  • the TIGIT has a nucleotide sequence shown in SEQ ID NO:21.
  • the N-terminus of the IL-15R ⁇ is connected to the C-terminus of the IL-15, or the C-terminus of the IL-15R ⁇ is connected to the N-terminus of the IL-15.
  • the IL-15R ⁇ has an amino acid sequence as shown in SEQ ID NO:3.
  • the IL-15 has an amino acid sequence as shown in SEQ ID NO:4.
  • the IL-15R ⁇ has a nucleotide sequence as shown in SEQ ID NO:13.
  • the IL-15 has a nucleotide sequence as shown in SEQ ID NO:14.
  • the fusion protein further includes a connecting peptide 1.
  • the N-terminus of the IL-15R ⁇ 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-15R ⁇ 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.
  • the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.
  • the connecting peptide 1 is encoded with a nucleotide sequence as shown in SEQ ID NO:12.
  • the second nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:15.
  • the CXCR2 has an amino acid sequence shown in SEQ ID NO:5.
  • the third nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:17.
  • 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.
  • the term "the first nucleic acid molecule is located at one end of the second nucleic acid molecule and the third nucleic acid molecule” means that the connected second nucleic acid molecule and the third nucleic acid molecule are an integral nucleic acid molecule A, and the first nucleic acid molecule is located at the 3' end or the 5' end of the nucleic acid molecule A.
  • 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.
  • 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.
  • 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.
  • 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.
  • the isolated nucleic acid further comprises: a fourth nucleic acid molecule, which is arranged between the second nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a connecting peptide 2, and the connecting peptide 2 can be cut in the immune cell.
  • the connecting peptide 2 includes a 2A peptide or a fragment thereof.
  • the connecting peptide 2 includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.
  • the connecting peptide 2 includes P2A or a fragment thereof.
  • the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO:16.
  • the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:18.
  • the isolated nucleic acid further includes: a first promoter, which is operably linked to the first nucleic acid molecule; and/or a second promoter, which is operably linked to the second nucleic acid molecule.
  • the isolated nucleic acid further includes: the 3’ end of the first promoter is operably connected to the 5’ end of the first nucleic acid molecule; the 3’ end of the second promoter is operably connected to the 5’ end of the second nucleic acid molecule.
  • the first promoter and the second promoter are independently selected from U6, H1, CMV, EF-1, LTR or RSV promoter.
  • the isolated nucleic acid further comprises: a fifth nucleic acid molecule, which is arranged between the first nucleic acid molecule and the second promoter, and the fifth nucleic acid molecule encodes a gene transduction efficiency enhancing element.
  • the gene transduction efficiency enhancing element is cPPT/CTS.
  • the fifth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:19.
  • 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.
  • the post-transcriptional regulatory element is WPRE.
  • the sixth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:20.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides an expression vector.
  • the expression vector carries the isolated nucleic acid described in the second aspect of the present invention.
  • the expression vector according to the embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.
  • the above expression vector may further include at least one of the following additional technical features:
  • the vector of the expression vector is a non-pathogenic viral vector.
  • the viral vector includes at least one selected from a retroviral vector, a lentiviral vector, an adenovirus-associated viral vector, and an adeno-associated viral vector.
  • the present invention provides a kit.
  • the kit comprises the isolated nucleic acid described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention.
  • the kit according to an embodiment of the present invention can prepare transgenic immune cells, which have high tumor killing efficiency, strong anti-tumor activity, long survival time in vivo and in vitro, strong proliferation ability, and strong infiltration ability in tumors.
  • the present invention proposes a transgenic immune cell.
  • the transgenic immune cell inhibits the expression of TIGIT, and expresses a fusion protein and CXCR2; wherein the fusion protein includes IL-15R ⁇ and IL-15.
  • the transgenic immune cell has the advantages of high tumor killing efficiency, strong anti-tumor activity, strong long-term survival and expansion ability in the body, and strong proliferation and chemotaxis ability.
  • 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 transgenic immune cells obtained by combining TIGIT-shRNA with the above-mentioned fusion protein and CXCR2 i.e., silencing TIGIT, expressing IL-15R ⁇ , IL-15 and CXCR2 in the transgenic immune cells
  • the transgenic immune cells silencing other immunosuppressive receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak long-term survival and expansion ability in the body, and weak proliferation and chemotaxis.
  • the genetically modified immune cells may further include at least one of the following additional technical features:
  • the TIGIT has the amino acid sequence shown in SEQ ID NO:1.
  • the N-terminus of the IL-15R ⁇ is connected to the C-terminus of the IL-15, or the C-terminus of the IL-15R ⁇ is connected to the N-terminus of the IL-15.
  • the IL-15R ⁇ has the amino acid sequence shown in SEQ ID NO:3.
  • the IL-15 has the amino acid sequence shown in SEQ ID NO:4.
  • the fusion protein further includes a connecting peptide 1.
  • the N-terminus of the IL-15R ⁇ 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-15R ⁇ 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.
  • the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:6.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO:2.
  • the CXCR2 has the amino acid sequence shown in SEQ ID NO:5.
  • the transgenic immune cells are obtained by introducing the aforementioned expression vector into immune cells.
  • the immune cells include at least one of T cells, NKT cells, NK cells and macrophages.
  • the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells and macrophages.
  • 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.
  • 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.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells, Treg cells and ⁇ T cells.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the isolated nucleic acid described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the transgenic immune cell described in the fifth aspect of the present invention.
  • the pharmaceutical composition has high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability.
  • the present invention proposes a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells.
  • the method comprises introducing the expression vector described in the third aspect of the present invention or the expression vector in the kit described in the fourth aspect of the present invention into immune cells; and culturing the immune cells introduced with the expression vector.
  • This method can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, improve the long-term survival and amplification ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially can prepare immune cells with strong killing, activation, proliferation and chemotaxis in vitro for constructing the required immune cell model.
  • the above method may further include at least one of the following additional technical features:
  • the introduction of the expression vector into the immune cells is performed by electroporation, transfection or infection.
  • the immune cell is at least one of a T cell, a NKT cell, a NK cell and a macrophage.
  • the immune cells are NK cells.
  • 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.
  • peripheral blood NK cells umbilical cord blood NK cells
  • iPSC induced pluripotent cell
  • the T cells include CD4 + T cells, CD8 + T cells, Treg cells and ⁇ T cells.
  • 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.
  • the above-mentioned use may further include at least one of the following additional technical features:
  • the tumor includes solid tumors and hematological tumors.
  • the solid tumor is a tangible tumor occurring in an organ.
  • 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.
  • 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 tumors.
  • the present invention proposes a method for treating or preventing tumors.
  • 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.
  • transgenic immune cells and pharmaceutical compositions have the advantages of high tumor killing efficiency, strong anti-tumor activity, long-term survival and amplification ability in vivo and in vitro, and strong proliferation and chemotaxis ability. Therefore, the use of the above-mentioned transgenic immune cells and pharmaceutical compositions can effectively kill tumor cells, thereby effectively preventing and treating tumors.
  • the above method may further include at least one of the following additional technical features:
  • the tumor includes solid tumors and hematological tumors.
  • the solid tumor is a tangible tumor occurring in an organ.
  • 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.
  • 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 tumors.
  • TIGIT Homo sapiens T cell immunoreceptor with Ig and ITIM domains
  • the shRNA sequence for constructing the shRNA lentiviral vector was designed.
  • the designed TIGIT shRNA coding sequence (shTG1 to shTG5, nucleotide sequence as shown in SEQ ID NO: 7 to 11) and nonsense control sequence (shSCR, nucleotide sequence as shown in SEQ ID NO: 23) have the structure of sense, loop, antisense, and termination sequences.
  • pLKO.1-EGFP lentiviral vector AgeI and EcoRI restriction sites were selected to insert the shRNA sequence respectively, and the obtained plasmids were named pLKO.1-shTG1-EGFP to pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP respectively.
  • lentiviral packaging system prepares the lentiviral packaging system, add 6 ⁇ g psPAX2 and 3 ⁇ g pMD2.G, 6 ⁇ g of lentiviral vector plasmids (pLKO.1-shTG1-EGFP ⁇ pLKO.1-shTG5-EGFP and pLKO.1-shSCR-EGFP) to 250 ⁇ L of serum-free DMEM medium, prepare the plasmid mixtures, and mix them evenly. Add 15 ⁇ L Add to 235 ⁇ L serum-free DMEM medium and mix well. The mixed solution was added to the above plasmid mixture at one time, mixed, and incubated at room temperature for 15 minutes. The above mixed solution was added to the 293T cell culture dish.
  • the liquid was changed, and the culture dish was returned to the 37°C, 5% CO2 incubator. After 48 hours, the cell supernatant was collected, centrifuged at 400 ⁇ g for 5 minutes, and the cell debris was removed. The supernatant was filtered into a 50mL centrifuge tube with a 0.45 ⁇ m filter head to obtain different TIGIT shRNA (shTG1 ⁇ shTG5) and nonsense control sequence (shSCR) lentivirus virus liquids, and 5 ⁇ PEG8000 solution was added to concentrate the virus liquid. The centrifuge tube was mixed upside down and placed in a 4°C refrigerator overnight.
  • NK cells Different TIGIT shRNA (shTG1-shTG5) and nonsense control sequence (shSCR) lentivirus were used to infect NK cells.
  • NK cells Take NK cells in the logarithmic growth phase, harvest NK cells by centrifugation at 100 ⁇ g for 5 min, add an appropriate amount of ⁇ -MEM medium to resuspend NK cells, and adjust the NK cell density to 5 ⁇ 10 5 cells/mL.
  • the mixed 24-well plate was placed in a 37°C, 5% CO 2 incubator for culture. After 24 hours, observe the status of NK cells, change the medium, transfer the infected NK cells into EP tubes, centrifuge at 100 ⁇ g for 5 minutes, add a small amount of fresh ⁇ -MEM medium to resuspend the NK cells, transfer the NK cells into cell culture bottles, add 10mL of fresh ⁇ -MEM medium and IL-2 (final concentration of 200IU/mL) and continue to culture. Change the cell medium every 2 days and add IL-2 (final concentration of 200IU/mL).
  • TIGIT shRNA shTG1-shTG5
  • nonsense control sequence shSCR
  • GFP-positive cells represent positively infected cells.
  • A the positive rate of TIGIT on GFP-positive cells
  • B the positive rate of TIGIT on GFP-negative cells
  • Example 2 Construction of a multifunctional gene modification vector and verification of expression of each element
  • the base sequence of cPPT/CTS-EF1a-mbIL15RF-P2A-CXCR2-WPRE was synthesized by whole gene synthesis and cloned into the lentiviral vector (pLKO.1-shTG1-EGFP ⁇ pLKO.1-shTG5-EGFP) prepared in the above Example 1 through the restriction sites EcoRI and KpnI.
  • pLKO.1-multi-functional-vector-shTG1 ⁇ pLKO.1-multi-functional-vector-shTG5 plasmids were obtained, i.e., the multifunctional gene modification vector plasmid involved in the present invention.
  • This example exemplarily shows the construction of the pLKO.1-multi-functional-vector-shTG3 multifunctional gene modification vector and a schematic diagram of the elements, see Figure 3 for details.
  • CD3 positive cells in peripheral blood mononuclear cells 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 bottles for culture.
  • IL-2 and other cytokines were used for induction 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, and the culture was continued after changing the medium on the 9th day. Flow cytometry was performed on the 11th day to detect the expression of each element on the NK cells.
  • the method of flow cytometry is as follows: 1 ⁇ 10 6 cells were added to the flow tube for staining, and antibodies were added for staining according to different staining schemes, and incubated at room temperature for 30 minutes. After washing twice with 1 ⁇ PBS solution, the cell pellet was resuspended and divided into two parts, and flow cytometry was performed on the machine.
  • the first portion 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 TIGIT antibody (Biolegend) and PE-labeled anti-human CXCR2 antibody (Biolegend); the second portion 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-15R ⁇ antibody (Biolegend) and PE-labeled anti-human IL-15 antibody (Invitrogen).
  • This example exemplifies the results of NK cell infection by lentivirus packaged with a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3).
  • the results showed that the 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.
  • the expression of the TIGIT receptor on NK cells was significantly reduced, and the expression of CXCR2 and mbIL15RF molecules (encoding fusion proteins, which include IL-15R ⁇ and IL-15) was significantly increased.
  • Example 3 In vitro functional detection of NK cells prepared by multifunctional gene-modified vectors
  • NK cells Absorb NK cells in the logarithmic growth phase, harvest the cells by centrifugation at 100 ⁇ g for 5min, add an appropriate amount of ⁇ -MEM medium to resuspend the cells, and adjust the NK cell density to 5 ⁇ 10 5 /mL. Inoculate 5 ⁇ 10 5 NK cells in 24-well plates, add 1mL of concentrated lentivirus solution (lentivirus-multi-functional-shTG1 ⁇ lentivirus-multi-functional-shTG5) and protamine (final concentration 8 ⁇ g/mL) respectively, and mix evenly. Place in a 37°C, 5% CO 2 incubator for culture.
  • concentrated lentivirus solution lentivirus-multi-functional-shTG1 ⁇ lentivirus-multi-functional-shTG5
  • protamine final concentration 8 ⁇ g/mL
  • NK cells After 24h, observe the state of NK cells, change the medium, transfer the infected NK cells to EP tubes, centrifuge at 100 ⁇ g for 5min, add a small amount of fresh ⁇ -MEM medium to resuspend the cells, transfer the infected NK cells to cell culture bottles, add 10mL of fresh ⁇ -MEM medium and IL-2 (final concentration of 200IU/mL) respectively, and continue to culture for 48h. Continue to expand the culture, adjust the state of the infected NK cells for amplification.
  • the infected NK cells were sorted by flow cytometry to obtain CXCR2-positive cells 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 in subsequent in vitro functional experiments.
  • Multifunctional gene-modified vector promotes the killing activity of NK cells
  • the specific method is as follows: 1 ⁇ 10 4 target cells, human liver cancer cells HepG2 or human ovarian cancer Hey cells, were inoculated into a 96-well E plate, and monitored overnight in culture using the impedance-based real-time cell analysis (RTCA) iCELLigence system.
  • RTCA real-time cell analysis
  • 3 ⁇ 10 4 NK cells or multifunctional NK cells were respectively inoculated into the corresponding wells, and then the cell impedance was monitored again for 2-3 hours using the RTCA system.
  • the formula for calculating the cell killing efficiency is (impedance of target cells without effector cells - impedance of target cells with effector cells) ⁇ 100 ⁇ impedance of target cells without effector cells.
  • This example shows the results of multifunctional NK cells prepared by the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3).
  • the results show that the killing efficiency of multifunctional NK cells of the multifunctional gene-modified vector of the present invention on liver cancer HepG2 cells or ovarian cancer Hey cells is significantly higher than that of NK cells without gene modification, see Figure 5A and Figure 5B for details.
  • Multifunctional gene modification vector promotes the survival of NK cells
  • the inventors further verified the effect of the multifunctional gene-modified vector on NK cell survival.
  • the specific method is as follows: NK cells with the same number of cells and the multifunctional NK cells prepared in step 1 of Example 3 are plated in 24-well plates, and different IL-2 concentrations (200 IU/mL and 0 IU/mL) are set, and flow cytometry is performed every 24 hours to detect the apoptosis rate.
  • the flow cytometry detection of apoptosis rate was performed according to the steps in the kit instructions (Lianke Bio, catalog number AP101), which is briefly as follows: collect cells in EP tubes, add 1 ⁇ PBS solution, centrifuge and wash once, and resuspend the cells.
  • This example shows the results of multifunctional NK cells prepared by a multifunctional gene modification vector (pLKO.1-multi-functional-vector-shTG3).
  • the results showed that the cell survival rate of the non-genetically modified NK cell group decreased significantly from 24h in the absence of IL-2 (IL-20IU/mL), 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.
  • Multifunctional gene modification vector promotes the proliferation of NK cells
  • 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, and trypan blue staining cell counts were performed every 48 hours. Observe and culture until the 8th day, and draw a cell proliferation curve
  • This example shows the results of multifunctional NK cells prepared by a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3).
  • the results show that the proliferation ability of multifunctional NK cells under different IL-2 concentrations is significantly stronger than that of non-genetically modified NK cells under the same IL-2 concentration, as shown in Figure 7.
  • the above test results show that the multifunctional gene-modified vector can significantly promote the proliferation of NK cells.
  • Multifunctional gene-modified vector promotes chemotaxis of NK cells
  • the inventors further investigated the chemotactic ability of multifunctional NK cells through transwell experiments.
  • 1 ⁇ 10 6 NK cells or multifunctional NK cells were inoculated into the upper chamber of the transwell chamber, 600 ⁇ L of serum-free ⁇ -MEM medium was added to the lower chamber, and chemokine CXCL8 was added at concentrations of 1, 10, and 100 ng/mL for chemotaxis.
  • the cells were returned to the cell culture incubator, and the cells in the lower chamber were collected for cell counting after 48 hours.
  • This example shows the results of multifunctional NK cells prepared by a multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3).
  • the results show that compared with non-genetically modified NK cells, the number of multifunctional NK cells migrating to the lower chamber is significantly greater, and is dose-dependent with the concentration of IL-8.
  • the above test results show that the multifunctional gene-modified vector can significantly promote the chemotaxis of NK cells.
  • Example 4 NK cells prepared by multifunctional gene-modified vectors have strong in vivo tumor-suppressing activity
  • a subcutaneous tumor-bearing model of mice was established with 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: 6-week-old NCG mice (8 mice) were selected for subcutaneous tumor-bearing in the armpits, and the tumor-bearing dose was 5 ⁇ 10 6 HepG2 cells/mouse.
  • the mice were treated with NK cell reinfusion and randomly divided into an untreated group, an NK cell treatment group, and a multifunctional NK cell treatment group according to the size of the tumor.
  • NK cells were reinfused through the tail vein for treatment, with a treatment dose of 5 ⁇ 10 6 cells/time/week, for a total of 4 treatments, and 5 ⁇ 10 4 IU of IL-2 was injected intraperitoneally every 3 to 4 days to maintain the in vivo activity of NK cells.
  • the tumor volume was measured once a week, and the tumor growth curve was drawn.
  • This example shows the results of multifunctional NK cells prepared by the multifunctional gene-modified vector (pLKO.1-multi-functional-vector-shTG3).
  • the results show that compared with the unmodified NK cell treatment group, the multifunctional NK cells prepared based on the multifunctional gene-modified vector of the present invention have a significantly enhanced effect of inhibiting the growth of liver cancer cells.
  • a human colorectal cancer mouse heterotopic transplant tumor model was established using human colorectal cancer cell line NCI-H716 cells, and 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.
  • mice 6-week-old NCG mice (8 mice) were selected for subcutaneous tumor bearing in the armpits, and the tumor bearing dose was 1 ⁇ 10 7 NCI-H716 cells/mouse.
  • mice with a tumor volume of about 50 mm 3 were selected for 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 tumor volume.
  • mice in the non-genetically modified NK cell treatment group were treated once every 2 days for a total of 3 times, and each tail vein reinfusion dose was 8 ⁇ 10 6 CD56 + NK cells, and a total of 2.4 ⁇ 10 7 CD56 + NK cells were reinfused, and 5 ⁇ 10 4 IU of IL-2 was injected intraperitoneally every 2 days to maintain the in vivo activity of NK cells; mice in the multifunctional NK cell treatment group were treated once by tail vein reinfusion, and the dose was 4 ⁇ 10 6 multifunctional NK cells/mouse, respectively, and no IL-2 injection adjuvant therapy was performed. The tumor volume was observed 1-2 times a week, and the tumor growth curve was drawn.
  • This example shows the results of multifunctional NK cells prepared by lentivirus (lentivirus-multi-functional-shTG3).
  • the results show that compared with the unmodified NK cell treatment group, at a lower cell treatment dose, the multifunctional NK cell treatment group still showed a stronger tumor inhibition effect, with a tumor inhibition rate of up to 64.5%.
  • Figure 10 For specific results, see Figure 10.
  • NK cells prepared based on the multifunctional gene-modified vector of the present invention have significantly enhanced anti-tumor activity against tumors such as ovarian cancer, liver cancer, and colorectal cancer, and can be used at lower dosages, which is expected to break through the bottleneck of poor therapeutic effect of immune cell therapy on solid tumors.
  • step 1 of Example 2 shRNAs that silence different immunosuppressive receptors are combined with the above-mentioned expression fusion protein and CXCR2 to prepare multifunctional gene modification vectors that silence different immunosuppressive receptors, and obtain multiple transgenic immune cells that silence different immunosuppressive receptors.
  • the method in Example 3 is used to detect the killing activity, proliferation promotion, survival and chemotaxis of multiple transgenic immune cells that silence different immunosuppressive receptors.
  • the inventors compared the killing activity of multifunctional transgenic NK cells with TIGIT silencing and multifunctional transgenic NK cells with 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 with TIGIT silencing (sh-TIGIT+CXCR2) against liver cancer PLC/PRF/5 cells is significantly higher than that of NK cells that have not been genetically modified and NK cells with other different immunosuppressive receptors (TIM3, NKG2A, LAG3) silencing (sh-TIM3+CXCR2, sh-NKG2A+CXCR2, sh-LAG3+CXCR2).
  • 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-15R ⁇ , IL-15 and CXCR2 in the transgenic immune cells
  • the transgenic immune cells with silencing other immunosuppressive receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak long-term survival and expansion ability in the body, and weak proliferation and chemotaxis ability.
  • the shRNA that silences TIGIT is combined with the expression of different chemokine receptors to prepare transgenic immune cells that silence TIGIT and express different chemokines, and the killing activity and chemotaxis ability are compared; further, the shRNA that silences TIGIT is combined with the above-mentioned expression of fusion protein and different chemokine receptors to obtain a variety of transgenic immune cells expressing different chemokine receptors.
  • the method in Example 3 is used to detect the chemotaxis ability, killing activity, proliferation and survival ability of a variety of transgenic immune cells expressing different chemokine receptors.
  • the inventors detected and compared the chemotactic ability of multifunctional NK cells expressing different chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4) through transwell experiments.
  • CXCR1, CXCR2, CXCR3, CXCR4 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 tissues than immune cells expressing 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-15R ⁇ , IL-15 and CXCR2 are expressed in the transgenic immune cells
  • TIGIT is silenced and IL-15R ⁇ , IL-15 and CXCR2 are expressed in the transgenic immune cells
  • the transgenic immune cells expressing other chemokine receptors have the disadvantages of low tumor killing efficiency, weak anti-tumor activity, weak survival and proliferation ability, and weak chemotaxis.
  • NK cells multifunctional transgenic immune cells
  • the invented multifunctional transgenic immune cells have strong killing activity against tumor cells of various tumor types.
  • the multifunctional NK cells of the present invention have stronger killing activity against colorectal adenocarcinoma SW620 cells, colon cancer HCT116 cells, breast cancer MDA-MB-231 cells, cervical cancer Hela cells and chronic myeloid leukemia cells K562 than NK cells that have not been genetically modified.

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Abstract

L'invention concerne un vecteur génétiquement modifié multifonctionnel, et des cellules immunitaires génétiquement modifiées multifonctionnelles ainsi préparées et leur utilisation. Le vecteur génétiquement modifié multifonctionnel porte un acide nucléique isolé. L'acide nucléique isolé comporte : une première molécule d'acide nucléique pour inhiber l'expression de TIGIT ; une deuxième molécule d'acide nucléique codant pour une protéine de fusion contenant IL-15Rα et IL-15 ; et une troisième molécule d'acide nucléique codant pour CXCR2, la première molécule d'acide nucléique, la deuxième molécule d'acide nucléique et la troisième molécule d'acide nucléique étant liées. Le vecteur génétiquement modifié multifonctionnel peut inhiber l'expression de TIGIT dans une cellule hôte et exprimer une protéine de fusion comportant IL-15Rα et IL-15, et CXCR2. Par conséquent, les cellules immunitaires génétiquement modifiées multifonctionnelles préparées en adoptant le vecteur génétiquement modifié multifonctionnel peuvent améliorer leur capacité de survie, leur capacité de prolifération et leur capacité d'infiltration intratumorale, et peuvent également résister à l'épuisement immunitaire, ce qui améliore encore l'efficacité clinique des cellules immunitaires.
PCT/CN2024/134157 2023-11-24 2024-11-25 Vecteur génétiquement modifié multifonctionnel, cellules immunitaires génétiquement modifiées multifonctionnelles ainsi préparées et leur utilisation Pending WO2025108480A1 (fr)

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