Nucleic acid, vector, and transgenic immune cell and use thereof

Abstract

Provided are a nucleic acid encoding a chimeric antigen receptor and FcRγ, an expression vector, a lentiviral vector, a transgenic immune cell, and a pharmaceutical composition and a use thereof. The nucleic acid comprises a first nucleic acid molecule and a second nucleic acid molecule. The first nucleic acid molecule encodes the chimeric antigen receptor, and the second nucleic acid molecule encodes FcRγ, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region, and an intracellular region, the N-terminal of the transmembrane region is linked to the C-terminal of the extracellular region, and the N-terminal of the intracellular region is linked to the C-terminal of the transmembrane region. According to an immune cell carrying the nucleic acid, the expression abundance of a chimeric antigen receptor is high, the tumor cell killing activity is high, the efficacy is good, and the clinical application value is high.

Description

Nucleic acid, vector, transgenic immune cell and their application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid encoding a chimeric antigen receptor and FcRγ, an expression vector, a lentiviral vector, a transgenic immune cell, a pharmaceutical composition and applications thereof. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. The antigen recognition region of CAR molecules typically utilizes antibody-derived fragment (scFv) sequences, typically from species such as mice, rabbits, and alpacas, and possesses strong immunogenicity. Even with humanized sequences, some immunogenicity persists, generating anti-drug antibodies (ADAs) that impair the persistence of CAR-T cells in vivo. In recent years, numerous new technologies have been applied to address this issue, such as using native receptor protein sequences as receptors to reduce immunogenicity.

[0003] The NKp30 receptor is a natural NK cell activating receptor that is highly expressed on NK cells and can recognize the B7H6 molecule, thereby mediating cytotoxicity. B7H6 is a member of the B7 family. The B7H6 molecule is usually highly expressed on a variety of primary tumor cells, including leukemia, lymphoma, colorectal cancer, gastric cancer, and pancreatic cancer, but is almost not expressed on normal tissue cells. Currently, the function of CAR-T cells constructed based on the NKp30 extracellular segment as a receptor for B7H6 recognition has been verified in research (An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo. J Immunol. 2012 Sep 1; 189(5): 2290-9. doi: 10.4049 / jimmunol.1103495. Patent application numbers US14342060, US16882800). In this document or patent, NKp30 amino acid positions 1-139 were used as the extracellular segment to construct T cells and other cells expressing chimeric antigen receptors, and it was observed that the immune cells had an enhanced killing activity against B7H6-positive tumor cells.

[0004] Based on the above research and development status, further research is needed to investigate immune cells expressing chimeric antigen receptors with higher killing activity.

[0005] Summary of the Invention

[0006] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides nucleic acids encoding chimeric antigen receptors and FcRγ, expression vectors, lentiviral vectors, transgenic immune cells, pharmaceutical compositions, and uses thereof. The nucleic acids of the present invention encode chimeric antigen receptors and FcRγ molecules. Immune cells carrying the nucleic acids of the present invention can simultaneously express the chimeric antigen receptors and FcRγ molecules. The chimeric antigen receptors are expressed at high abundance, exhibit high tumor cell killing activity, exhibit good therapeutic efficacy, and have high clinical application value.

[0007] It should be noted that the present invention is completed based on the following work of the inventors:

[0008] Natural killer (NK) cells are a key component of anti-tumor immunity, participating in anti-tumor immune responses through multiple pathways. NK cell surface expression of NK cytotoxicity receptors (NCRs) such as NKG2D, NKp30, NKp44, and NKp46 is high, and their corresponding ligands are often highly expressed on malignantly transformed or senescent cells. Upon recognition of these ligands, NK cells can mediate potent cytotoxicity, eliminating these malignantly transformed or senescent cells. Recent studies have revealed multiple variants of NKp30, namely NKp30a, NKp30b, NKp30c, NKp30d, NKp30e, and NKp30f. These variants exhibit significant functional differences. Activation of NKp30a mediates the strongest NK cell cytotoxicity and IFN-γ secretion, followed by NKp30b. Activation of NKp30c, however, does not significantly enhance NK cell cytotoxicity or IFN-γ secretion, but instead enhances the secretion of the immunosuppressive factor IL-10. Thus, the inventors retained the arginine at position 143 in the natural NKp30a sequence and, through extensive experiments, identified an NKp30a amino acid sequence (1-147). Further experimental results demonstrated that a chimeric antigen receptor constructed based on this NKp30a amino acid sequence significantly enhanced NK cell activation. The resulting genetically modified immune cells were able to effectively kill B7H6-positive tumor cells and were highly safe.

[0009] Furthermore, the inventors creatively co-expressed the above-mentioned chimeric antigen receptor and FcRγ molecule in immune cells. Further experimental results showed that the expression abundance of the aforementioned NKp30 receptor was further increased, and the tumor cell killing activity of immune cells was significantly improved.

[0010] Furthermore, the inventors co-expressed different chimeric antigen receptors with FcRγ molecules in immune cells and observed the same results.

[0011] Therefore, in the first aspect of the present invention, the present invention provides a nucleic acid. The nucleic acid comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a chimeric antigen receptor and the second nucleic acid molecule encodes FcRγ; wherein,

[0012] The chimeric antigen receptor includes an extracellular region, a transmembrane region and an intracellular region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0013] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0014] According to the embodiments of the present invention, the immune cells carrying the above nucleic acid can simultaneously express chimeric antigen receptors and FcRγ molecules, and the chimeric antigen receptors are expressed in high abundance, the immune cell killing activity is high, the therapeutic effect is good, and the clinical application value is high.

[0015] As used herein, the term "chimeric antigen receptor-expressing immune cell" is equivalent to "CAR-immune cell," which is a genetically modified immune cell that expresses a specific chimeric antigen receptor and is further used for the prevention and / or treatment of a disease. In some cases, depending on the context, it is equivalent to "transgenic immune cell."

[0016] According to an embodiment of the present invention, the extracellular region has B7H6 binding activity. According to an embodiment of the present invention, the nucleic acid encodes a chimeric antigen receptor and an FcRγ molecule that can recognize B7H6 molecules, thereby achieving high expression of the chimeric antigen receptor on the cell surface.

[0017] According to an embodiment of the present invention, the extracellular region includes an extracellular segment of NKp30 or a fragment thereof. According to an embodiment of the present invention, the nucleic acid encodes an NKp30 chimeric antigen receptor and an FcRγ molecule, thereby achieving high expression of the NKp30 chimeric antigen receptor on the cell surface, thereby obtaining immune cells with high abundance of NKp30 chimeric antigen expression and high tumor cell killing activity.

[0018] According to an embodiment of the present invention, the extracellular region has an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3 or an amino acid sequence of a conservatively modified form thereof.

[0019] According to an embodiment of the present invention, the extracellular region has an amino acid sequence as shown in SEQ ID NO: 1. Thus, the tumor cell killing activity of the NKp30 chimeric antigen receptor encoded by the nucleic acid is further improved.

[0020] According to an embodiment of the present invention, the intracellular region includes a costimulatory domain and an intracellular signaling domain.

[0021] According to an embodiment of the present invention, the C-terminus of the costimulatory factor domain is connected to the N-terminus of the intracellular signaling domain.

[0022] According to an embodiment of the present invention, the costimulatory domain is selected from at least one of CD3ζ, CD28, 4-1BB, 2B4, DAP10, DAP12, CD27, CD40, OX40, ICOS intracellular segment or a fragment thereof.

[0023] According to an embodiment of the present invention, the costimulatory domain is the CD28 intracellular segment or a fragment thereof.

[0024] According to an embodiment of the present invention, the intracellular segment of CD28 has an amino acid sequence as shown in SEQ ID NO:4.

[0025] According to an embodiment of the present invention, the intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule or a fragment thereof.

[0026] According to an embodiment of the present invention, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:5.

[0027] According to an embodiment of the present invention, the transmembrane region is selected from one of the transmembrane region of the NKp30 molecule, the transmembrane region of the CD3ζ molecule, and the transmembrane region of the CD28 molecule.

[0028] According to an embodiment of the present invention, the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 14, 15 or 20.

[0029] According to an embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 6 or 8.

[0030] In this way, transgenic immune cells with high expression abundance of NKp30 chimeric antigen receptor, further enhanced tumor cell killing activity and higher safety can be obtained.

[0031] According to an embodiment of the present invention, the chimeric antigen receptor has a nucleotide sequence as shown in SEQ ID NO: 7 or 9. Thus, the expression abundance of the NKp30 chimeric antigen receptor on the cell surface is further increased.

[0032] According to an embodiment of the present invention, the FcRγ has an amino acid sequence as shown in SEQ ID NO: 10. Thus, the abundance of NKp30 chimeric antigen receptor expression on the cell surface can be further increased, and the tumor cell killing activity of immune cells can be further enhanced.

[0033] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 11. Thus, the expression abundance of the NKp30 chimeric antigen receptor on the cell surface is further increased.

[0034] The method further comprises a third nucleic acid molecule, wherein the third nucleic acid molecule encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the third nucleic acid molecule.

[0035] According to an embodiment of the present invention, the first linker is selected from at least one of P2A, T2A, E2A, and F2A. Thus, immune cells with relatively uniform expression of chimeric antigen receptors and FcRγ on the cell membrane can be obtained, further increasing the expression abundance of the NKp30 chimeric antigen receptor on the cell surface.

[0036] According to an embodiment of the present invention, the first linker is P2A. Thus, immune cells can be obtained that uniformly express chimeric antigen receptors and FcRγ on the cell membrane, further increasing the expression abundance of NKp30 chimeric antigen receptors on the cell surface.

[0037] According to an embodiment of the present invention, the first linker has an amino acid sequence as shown in SEQ ID NO: 12. Thus, immune cells are obtained that uniformly express chimeric antigen receptor and FcRγ on their cell membranes.

[0038] According to an embodiment of the present invention, the first linker has a nucleotide sequence as shown in SEQ ID NO: 13. Thus, the expression abundance of the NKp30 chimeric antigen receptor on the cell surface is further increased.

[0039] According to an embodiment of the present invention, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 16 or 17. Thus, immune cells with significantly increased expression abundance of NKp30 chimeric antigen receptor on the cell surface and further enhanced tumor cell killing activity are obtained.

[0040] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0041] In a second aspect of the present invention, an expression vector is provided. The constructed expression vector can effectively express the chimeric antigen receptor and the FcRγ.

[0042] When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control elements. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0043] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred 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 expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0044] According to an embodiment of the present invention, the nucleic acid can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0045] As used herein, the term "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, and the like. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable immune cells, they can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby achieving substantial expression of the protein encoded by the nucleic acid molecules on the cell membrane, thereby obtaining transgenic immune cells.

[0046] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0047] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0048] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of retrovirus, lentivirus and adeno-associated virus.

[0049] According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus.

[0050] In a third aspect of the present invention, the present invention provides a transgenic immune cell. The transgenic immune cell comprises:

[0051] Expressing the aforementioned chimeric antigen receptor; or carrying the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned lentiviral vector.

[0052] The immune cells according to the embodiments of the present invention are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned chimeric antigen receptor and the aforementioned FcRγ under appropriate conditions.

[0053] According to an embodiment of the present invention, the transgenic immune cells are optionally selected from T cells, NK cells, NKT cells, γδT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells, iPSC-derived or embryonic stem cell cultured and differentiated immune cells. The aforementioned chimeric antigen receptor and the aforementioned FcRγ of the present invention can be transduced into immune cells such as T, NK, NKT, γδT, macrophages, iPSCs through expression vectors (such as lentiviral vectors), thereby being expressed on the surface of these immune cells.

[0054] According to an embodiment of the present invention, the immune cells of the transgenic immune cells are NK cells.

[0055] The present invention does not impose strict restrictions on the source of NK cells, and NK cells can be derived from peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, or NK-92 cells.

[0056] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises:

[0057] The aforementioned nucleic acid, the aforementioned expression vector, the aforementioned lentiviral vector or the aforementioned transgenic immune cell.

[0058] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0059] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0060] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the chimeric antigen receptor, nucleic acid, expression vector, or transgenic immune cell of the present invention, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0061] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, lentiviral vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0062] In the fifth aspect of the present invention, the present invention provides a use of the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned lentiviral vector, and the aforementioned transgenic immune cell in the preparation of a drug, wherein the drug is used to prevent and / or treat related diseases mediated by the B7H6 target.

[0063] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0064] In some specific embodiments of the present invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0065] In a specific embodiment of the present invention, the cancer is colorectal cancer.

[0066] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, lentiviral vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0067] The present invention also provides a method for preventing and / or treating diseases mediated by the B7H6 target. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the above-mentioned transgenic immune cells or the above-mentioned pharmaceutical composition to a subject.

[0068] According to an embodiment of the present invention, the B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

[0069] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0070] In some specific embodiments of the present invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0071] In a specific embodiment of the present invention, the cancer is colorectal cancer.

[0072] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, lentiviral vectors, and transgenic immune cells are also applicable to the treatment methods for this disease and will not be elaborated here.

[0073] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable means. The transgenic immune cells or pharmaceutical compositions of the present invention may be administered via any common route, provided they reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0074] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

[0075] The effective amount of the transgenic immune cells and pharmaceutical compositions described herein may vary depending on the mode of administration and the severity of the disease to be treated. Preferably, the selection of the effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0076] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0077] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0079] FIG1 is a schematic diagram of the structures of different chimeric antigen receptors according to Example 1 of the present invention;

[0080] FIG2 is a flow cytometry result diagram showing the NKR expression levels on different NKR-NK cells according to Example 2 of the present invention;

[0081] FIG3 is a bar graph showing the mean fluorescence intensity (MFI) of NKR expression on different NKR-NK cells detected by flow cytometry according to Example 2 of the present invention;

[0082] FIG4 is a statistical result diagram showing the killing efficiency of different NKR-NK-92 cells on NCI-H716 cells according to Example 2 of the present invention. DETAILED DESCRIPTION

[0083] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0084] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0085] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0086] Terms and Definitions

[0087] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0088] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0089] As used herein, the term "FcRγ" is equivalent to "Fc receptor γ chain," "Fc receptor-γ," and binds to IgG class antibodies.

[0090] In this article, the term "(G4S) n " is equivalent to "(Gly4Ser) n ", which means 4 glycines and 1 serine are repeated n times. It is a widely used type of linker peptide and can be located at V H C-terminal and V L N terminal, can also be located at V L C-terminal and V H Between the N-termini. Currently, (G4S)3 is commonly used. Glycine, the amino acid with the smallest molecular weight and shortest side chain, increases side chain flexibility, while serine, the most hydrophilic amino acid, increases the hydrophilicity of the linker peptide. Therefore, (G4S)3 exhibits excellent stability and activity.

[0091] In this document, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0092] The sequences involved in the present invention are detailed in Table 1.

[0093] Table 1: Nucleotide / amino acid sequence description

[0094] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0095] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0096] Example 1: Preparation of NK cells expressing different NKR structures

[0097] 1. Construction of different NKR structures

[0098] Different CAR sequences were synthesized by whole gene synthesis and cloned into the lentiviral vector pCDG-EF1-MCS-TA2-copGFP through the restriction sites XbaI and BamHI. After sequencing verification, the pCDH-EF1a-NKR1, pCDH-EF1a-NKR2, pCDH-EF1a-NKR3 and pCDH-EF1a-NKR4 expression plasmid vectors were obtained.

[0099] The schematic diagram of the genetic element structure of the chimeric antigen receptor of this example is shown in Figure 1, and the nucleotide / amino acid sequences are shown in Table 1. The amino acid sequences of NKp30 1-147, NKp30 1-139, and NKp30 1-201 are shown in SEQ ID NOs: 1 to 3, respectively, and the nucleotide sequences of NKR1, NKR2, NKR3, and NKR4 are shown in SEQ ID NOs: 16 to 19.

[0100] 2. Lentivirus packaging

[0101] 5×10 293T cells in the logarithmic growth phase were taken 6 Inoculate in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37℃, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G lentiviral packaging auxiliary plasmids, and 6μg target gene vector plasmids to 250μL serum-free DMEM medium to prepare a plasmid mixture, and mix evenly. Add to 235 μL serum-free DMEM medium and mix well. Add the mixed solution to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixed solution to the 293T cell culture dish. Change the medium after 24 hours, put the culture dish back into the 37°C, 5% CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by inverting the centrifuge tube, and place it in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus pellet, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0102] 3. Lentiviral infection of human NK-92 cells

[0103] NK-92 cells in the logarithmic growth phase were harvested by centrifugation at 100 × g for 5 min, and appropriate amount of α-MEM medium was added to resuspend the cells to adjust the cell density to 5 × 10 5 5×10 cells / mL were seeded into 24-well plates. 5 NK-92 cells, 0.2 mL of virus concentrate, 0.8 mL of α-MEM medium, and protamine (final concentration 8 μg / mL) were mixed thoroughly. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cells were observed, the medium was changed, and the infected cells were transferred to an EP tube. The cells were centrifuged at 100×g for 5 minutes. A small amount of fresh α-MEM medium was added to resuspend the cells. The cells were transferred to a cell culture flask, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added and cultured for another 48 hours. The cells were transferred to a flow cytometer, 3 mL of 1× PBS solution was added, and the cells were centrifuged at 100×g for 5 minutes. The supernatant was discarded, the cell pellet was flicked, and the cells were washed again with 1× PBS. GFP expression was detected using a flow cytometer. The culture was continued to expand, and the infected NK-92 cells were adjusted for amplification. The infected NK-92 cells were sorted for GFP-positive NKR-NK92 cells by flow cytometry for use in later experiments.

[0104] Example 2: Investigation of biological functions of different NKR-NK cells

[0105] In this example, the inventors investigated the expression levels of NKR molecules in different NKR-NK cells obtained in Example 1 and their killing activity against NKp30 ligand B7H6-positive tumors at the cellular level.

[0106] 1. Investigation of the expression levels of different NKR receptors on NK cells

[0107] In this invention, the extracellular recognition domain of the NKR molecule utilizes the extracellular segment of the NKp30 receptor, which is specifically recognized by the NKp30 antibody. Therefore, NKp30 antibodies can be used to detect NKR molecule expression. Since NK cells endogenously express NKp30 molecules, NK cells not infected with the lentivirus serve as a negative control group for NKR molecule expression.

[0108] First, flow cytometry was used to detect the expression levels of NKR molecules on NKR1-NK-92, NKR2-NK-92, NKR3-NK-92, and NKR4-NK-92 cells.

[0109] The results are shown in Figures 2 and 3.

[0110] The results of the NKR receptor expression level investigation showed that: (1) the expression level of NKp30 on NKR1-NK-92, NKR2-NK-92, NKR3-NK-92, and NKR4-NK-92 cells was significantly higher than that on NK-92 cells not infected with lentivirus; (2) the expression level of NKp30 on NKR1-NK-92 and NKR2-NK-92 cells was significantly higher than that on NKR3-NK-92 and NKR4-NK-92 cells. Among them, the expression level of NKR1 molecule was the highest.

[0111] The above results show that the chimeric antigen receptor with an extracellular region of NKp30:1-139 (NKp30-CAR, refer to the sequence disclosed in US14342060) has a high expression level on immune cells. When the immune cells simultaneously express FcRγ molecules, the expression level of the chimeric antigen receptor with an extracellular region of NKp30:1-147 is further improved.

[0112] It should be noted that when immune cells simultaneously express FcRγ molecules and a chimeric antigen receptor with an extracellular domain of NKp30:1-139 (such as NKp30-CAR), the inventors also observed increased expression of the chimeric antigen receptor. The inventors also co-expressed different chimeric antigen receptors with FcRγ molecules in immune cells and observed the same results.

[0113] 2. In vitro cytotoxicity of NK-92 cells after modification of different NKR receptor genes

[0114] The inventors selected colorectal cancer cell line NCI-H716 cells that highly express the NKp30 ligand B7-H6 as target cells and performed in vitro cytotoxicity tests on different NKR-NK cells.

[0115] The specific method is as follows: NCI-H716 cells were fluorescently labeled with CFSE and the cells were stained with 2x10 4 NK-92 cells or different NKR-NK-92 cells were plated / well in a 96-well plate and incubated with NCI-H716 cells for 4 hours. Propidium Iodide (PI) was then added to distinguish live and dead cells, and the killing efficiency was measured by flow cytometry. The effector cell to target cell ratio was 2:1.

[0116] The test results are shown in Figure 4.

[0117] The results of in vitro killing activity investigation showed that the killing efficiency of NK-92 cells modified with the NKR1 gene of the present invention against B7-H6-positive NCI-H716 cells was significantly higher than that of NK-92 cells and NKp30-CAR-NK-92 cells.

[0118] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0119] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A nucleic acid, characterized in that comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a chimeric antigen receptor, and the second nucleic acid molecule encodes FcRγ; wherein, The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; The extracellular region has B7H6 binding activity and has an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3 or an amino acid sequence of a conservatively modified form thereof.

2. The nucleic acid according to claim 1, characterized in that The extracellular region has an amino acid sequence as shown in SEQ ID NO:

1.

3. The nucleic acid according to claim 1, characterized in that The intracellular region includes a costimulatory domain and an intracellular signaling domain; the C-terminus of the costimulatory domain is connected to the N-terminus of the intracellular signaling domain.

4. The nucleic acid according to claim 3, characterized in that The co-stimulatory domain is selected from at least one of CD3ζ, CD28, 4-1BB, 2B4, DAP10, DAP12, CD27, CD40, OX40, ICOS intracellular segment or a fragment thereof.

5. The nucleic acid according to claim 3, characterized in that The co-stimulatory domain is the CD28 intracellular segment or a fragment thereof.

6. The nucleic acid according to claim 5, characterized in that The intracellular segment of CD28 has an amino acid sequence as shown in SEQ ID NO:

4.

7. The nucleic acid according to claim 3, characterized in that The intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule or a fragment thereof.

8. The nucleic acid according to claim 7, characterized in that The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:

5.

9. The nucleic acid according to claim 1, characterized in that The transmembrane region is selected from one of the transmembrane region of the NKp30 molecule, the transmembrane region of the CD3ζ molecule, and the transmembrane region of the CD28 molecule.

10. The nucleic acid according to claim 9, characterized in that The transmembrane region has an amino acid sequence as shown in SEQ ID NO: 14, 15 or 20.

11. The nucleic acid according to claim 1, characterized in that The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 6 or 8.

12. The nucleic acid according to claim 1, characterized in that The chimeric antigen receptor has a nucleotide sequence as shown in SEQ ID NO: 7 or 9.

13. The nucleic acid according to claim 1, characterized in that The FcRγ has an amino acid sequence as shown in SEQ ID NO:

10.

14. The nucleic acid according to claim 1, characterized in that The second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

11.

15. The nucleic acid according to claim 13 or 14, characterized in that The method further comprises a third nucleic acid molecule, wherein the third nucleic acid molecule encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the third nucleic acid molecule.

16. The nucleic acid according to claim 15, characterized in that The first linker is selected from at least one of P2A, T2A, E2A and F2A.

17. The nucleic acid according to claim 16, characterized in that The first linker is P2A.

18. The nucleic acid according to claim 15, characterized in that The first linker has an amino acid sequence as shown in SEQ ID NO:

12.

19. The nucleic acid according to claim 15, characterized in that The first linker has a nucleotide sequence as shown in SEQ ID NO:

13.

20. The nucleic acid according to claim 1, characterized in that The nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 16 or 17.

21. An expression vector, characterized in that Carrying the nucleic acid according to any one of claims 1 to 20.

22. The expression vector according to claim 21, characterized in that The expression vector is a non-pathogenic viral vector.

23. The expression vector according to claim 22, characterized in that The non-pathogenic virus is selected from one of a retrovirus, a lentivirus and an adeno-associated virus.

24. The expression vector according to claim 22, characterized in that The non-pathogenic virus is a lentivirus.

25. A lentiviral vector, characterized in that The lentiviral expression vector carries a nucleotide sequence as shown in SEQ ID NO: 16 or SEQ ID NO:

17.

26. A transgenic immune cell, characterized in that: The transgenic immune cells include: Carrying the nucleic acid according to any one of claims 1 to 20, the expression vector according to any one of claims 21 to 24, or the lentiviral vector according to claim 25.

27. The transgenic immune cell according to claim 26, characterized in that The transgenic immune cells are selected from T cells, NK cells, NKT cells, γδT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, peripheral blood NK cells, cord blood NK cells, NK-92 cells, iPSC-derived or embryonic stem cell-cultured and differentiated immune cells.

28. The transgenic immune cell according to claim 26, characterized in that The transgenic immune cells are optionally selected from NK cells.

29. A pharmaceutical composition, characterized in that include: The nucleic acid of any one of claims 1 to 20, the expression vector of any one of claims 21 to 24, the lentiviral vector of claim 25, or the transgenic immune cell of any one of claims 26 to 28.

30. The pharmaceutical composition according to claim 29, characterized in that Further including: Pharmaceutically acceptable excipients.

31. Use of the nucleic acid according to any one of claims 1 to 20, the expression vector according to any one of claims 21 to 24, the lentiviral vector according to claim 25, the transgenic immune cell according to any one of claims 26 to 28, or the pharmaceutical composition according to claim 29 or 30 in the preparation of a drug for preventing and / or treating related diseases mediated by the B7H6 target.

32. The use according to claim 31, characterized in that The B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

33. The use according to claim 31, characterized in that The tumor is a solid tumor or a blood tumor.

34. The use according to claim 31, characterized in that The tumor or cancer is at least one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

35. A method for treating and / or preventing B7H6 target-mediated related diseases, characterized in that: include: A pharmaceutically acceptable amount of the transgenic immune cell according to any one of claims 26 to 28 or the pharmaceutical composition according to claim 29 or 30 is administered to a subject.

36. The method according to claim 35, characterized in that The B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

37. The use according to claim 36, characterized in that The tumor is a solid tumor or a blood tumor.

38. The use according to claim 36, characterized in that The tumor or cancer is at least one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

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