Nucleic acid construct, vector, transgenic immune cell and their application

Abstract

The present invention provides a nucleic acid construct encoding a chimeric antigen receptor and FcRγ, an expression vector, a lentiviral vector, a transgenic immune cell, a pharmaceutical composition and its application, wherein the nucleic acid construct 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 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 immune cell carrying the nucleic acid construct of the present invention has high expression abundance of the chimeric antigen receptor, high tumor cell killing activity, good therapeutic effect and high clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid construct 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 the CAR molecule usually uses the scFv sequence from the antibody, which is usually from species such as mice, rabbits, and alpacas and has strong immunogenicity. Even if the sequence is humanized, it still has a certain immunogenicity, producing anti-drug antibodies (ADA) and weakening the persistence of CAR-T cells in the body. In recent years, many new technologies have been applied to solve this problem, such as using natural receptor protein sequences as receptors to reduce immunogenicity.

[0003] NKp30 receptor is a natural NK cell activating receptor, which is highly expressed on NK cells and can recognize B7H6 molecules, thereby mediating cytotoxicity. B7H6 is a member of the B7 family. B7H6 molecules are usually highly expressed on a variety of primary tumor cells, including leukemia, lymphoma, colorectal cancer, gastric cancer and pancreatic cancer, but are almost not expressed on normal tissue cells. At present, the function of CAR-T cells constructed based on the extracellular segment of NKp30 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 number US14342060, US16882800). In this document or patent, T cells and other cells expressing chimeric antigen receptors were constructed using NKp30 amino acid positions 1-139 as the extracellular segment, and it was observed that the killing activity of immune cells against B7H6-positive tumor cells was enhanced.

[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. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a nucleic acid construct encoding a chimeric antigen receptor and FcRγ, an expression vector, a lentiviral vector, a transgenic immune cell, a pharmaceutical composition and its application. The nucleic acid construct of the present invention encodes a chimeric antigen receptor and an FcRγ molecule, and the immune cell carrying the nucleic acid construct of the present invention can simultaneously express the chimeric antigen receptor and the FcRγ molecule, and the chimeric antigen receptor has a high expression abundance, high tumor cell killing activity, good therapeutic effect, and high clinical application value.

[0006] It should be noted that the present invention is completed based on the following work of the inventors:

[0007] Natural killer (NK) cells are one of the main members of anti-tumor immunity, and they can participate in anti-tumor immune responses through various pathways. NK cell surface highly expresses NK cytotoxic receptors (NCRs) such as NKG2D, NKp30, NKp44, and NKp46, and their related ligands are often highly expressed on malignantly transformed or senescent cells. When NK cells recognize related ligands, they can mediate potent cytotoxic effects and eliminate malignantly transformed or senescent cells. Recent studies have found that there are multiple variants of NKp30, namely NKp30a, NKp30b, NKp30c, NKp30d, NKp30e, and NKp30f. There are significant differences in the functions of different variants. After activating NKp30a, it can mediate the strongest killing activity and IFN-γ secretion ability of NK cells, followed by NKp30b. After activating NKp30c, the killing activity and IFN-γ secretion ability of NK cells are not significantly improved, but the ability to secrete the immunosuppressive factor IL-10 is promoted. Thus, the inventors retained the arginine at position 143 in the NKp30a sequence based on the natural NKp30a sequence, and screened out a NKp30a amino acid sequence (1-147) through a large number of experiments. Further test results show that the chimeric antigen receptor constructed based on the NKp30a amino acid sequence significantly enhances the activation effect of NK cells. The transgenic immune cells thus obtained can effectively kill B7H6-positive tumor cells and are highly safe.

[0008] Furthermore, the inventors creatively co-expressed the above-mentioned chimeric antigen receptor and FcRγ molecule in immune cells. Further test 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.

[0009] Furthermore, the inventors co-expressed different chimeric antigen receptors with FcRγ molecules in immune cells and observed the same results.

[0010] Therefore, in the first aspect of the present invention, the present invention provides a nucleic acid construct. The nucleic acid construct 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,

[0011] 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.

[0012] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0013] According to the embodiments of the present invention, the immune cells carrying the above nucleic acid construct can simultaneously express the chimeric antigen receptor and the FcRγ molecule, and the chimeric antigen receptor is expressed in high abundance, the immune cell killing activity is high, the therapeutic effect is good, and the clinical application value is high.

[0014] In this article, the term "immune cell expressing chimeric antigen receptor", equivalent to "CAR-immune cell", is a transgenic immune cell that expresses a specific chimeric antigen receptor, which is further used for the prevention and / or treatment of a disease. In some cases, combined with the context, it is equivalent to "transgenic immune cell".

[0015] 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 construct encodes a chimeric antigen receptor and an FcRγ molecule capable of recognizing a B7H6 molecule, thereby achieving high expression of the chimeric antigen receptor on the cell surface.

[0016] According to an embodiment of the present invention, the extracellular region includes an extracellular segment of NKp30 or a fragment thereof. The nucleic acid construct according to an embodiment of the present invention encodes a NKp30 chimeric antigen receptor and an FcRγ molecule, thereby achieving high expression of the NKp30 chimeric antigen receptor on the cell surface, and obtaining immune cells with high abundance of NKp30 chimeric antigen expression and high tumor cell killing activity.

[0017] 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.

[0018] 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 construct is further improved.

[0019] According to an embodiment of the present invention, the intracellular region includes a co-stimulatory domain and an intracellular signaling domain.

[0020] According to an embodiment of the present invention, the C-terminus of the co-stimulatory factor domain is connected to the N-terminus of the intracellular signaling domain.

[0021] According to an embodiment of the present invention, 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.

[0022] According to an embodiment of the present invention, the co-stimulatory domain is the CD28 intracellular segment or a fragment thereof.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 is further increased, and the tumor cell killing activity of immune cells is further improved.

[0032] 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.

[0033] 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.

[0034] 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 expressing chimeric antigen receptors and FcRγ more uniformly on the cell membrane can be obtained, further increasing the expression abundance of NKp30 chimeric antigen receptors on the cell surface.

[0035] According to an embodiment of the present invention, the first linker is P2A. Thus, immune cells uniformly expressing chimeric antigen receptors and FcRγ on the cell membrane can be obtained, further increasing the expression abundance of NKp30 chimeric antigen receptors on the cell surface.

[0036] 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 receptors and FcRγ on their cell membranes.

[0037] 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.

[0038] According to an embodiment of the present invention, the nucleic acid construct 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 improved tumor cell killing activity are obtained.

[0039] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0040] In the second aspect of the present invention, the present invention provides an expression vector. The expression vector carries the aforementioned nucleic acid construct. Thus, the constructed vector can effectively express the aforementioned chimeric antigen receptor and the aforementioned FcRγ.

[0041] When the above-mentioned nucleic acid molecule is connected to an expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0042] 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 the expression of 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 that contains the vector.

[0043] 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 a 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.

[0044] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable immune cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of the regulatory system, thereby achieving a large amount of expression of the protein encoded by the nucleic acid molecule on the cell membrane, thereby obtaining transgenic immune cells.

[0045] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0046] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0047] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of a retrovirus, a lentivirus and an adeno-associated virus.

[0048] According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus.

[0049] In a third aspect of the present invention, the present invention provides a transgenic immune cell. The transgenic immune cell comprises:

[0050] Expressing the aforementioned chimeric antigen receptor; or carrying the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned lentiviral vector.

[0051] 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.

[0052] According to an embodiment of the present invention, 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, umbilical cord blood NK cells, NK-92 cells, iPSC-derived or embryonic stem cells cultured and differentiated from any of the above immune cells. The aforementioned chimeric antigen receptor and the aforementioned FcRγ of the present invention can be transduced to immune cells such as T, NK, NKT, γδT, macrophages, iPSC, etc. through expression vectors (such as lentiviral vectors), thereby expressing on the surface of these immune cells.

[0053] According to an embodiment of the present invention, the immune cells of the transgenic immune cells are NK cells.

[0054] The present invention does not impose strict restrictions on the source of NK cells, and the NK cells may be peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, NK-92 cells, and the like.

[0055] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises:

[0056] The aforementioned nucleic acid construct, the aforementioned expression vector, the aforementioned lentiviral vector or the aforementioned transgenic immune cell.

[0057] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0058] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0059] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the chimeric antigen receptor, nucleic acid, expression vector or transgenic immune cell of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0060] 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 described in detail here.

[0061] In the fifth aspect of the present invention, the present invention provides a use of the aforementioned nucleic acid construct, 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.

[0062] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0063] 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.

[0064] In a specific embodiment of the present invention, the cancer is colorectal cancer.

[0065] 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 described in detail here.

[0066] 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 transgenic immune cell or the above pharmaceutical composition to a subject.

[0067] According to an embodiment of the present invention, the B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

[0068] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0069] 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.

[0070] In a specific embodiment of the present invention, the cancer is colorectal cancer.

[0071] 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 methods of treating the disease and will not be described in detail here.

[0072] In this article, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The transgenic immune cells or pharmaceutical compositions of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection.

[0073] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

[0074] The effective amount of the transgenic immune cells and pharmaceutical compositions of the present invention 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 a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

[0075] 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.

[0076] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0078] Figure 1 Schematic diagram of the structure of different chimeric antigen receptors in Example 1 of the present invention;

[0079] Figure 2 This is a flow cytometry result diagram of NKR expression levels on different NKR-NK cells in Example 2 of the present invention;

[0080] Figure 3 This is a bar graph of the mean fluorescence intensity (MFI) of NKR expression on different NKR-NK cells detected by flow cytometry in Example 2 of the present invention;

[0081] Figure 4 This is a statistical result diagram of the killing efficiency of different NKR-NK-92 cells on NCI-H716 cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0082] 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.

[0083] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. 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, the meaning of "plurality" is two or more.

[0084] The endpoints and any values ​​of the ranges disclosed in this article 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 endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[0085] Terms and Definitions

[0086] 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.

[0087] 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.

[0088] As used herein, the term "FcRγ" is equivalent to "Fc receptor γ chain", "Fc receptor-γ", and binds to IgG class antibodies.

[0089] 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, in which glycine is the amino acid with the smallest molecular weight and the shortest side chain, which can increase the flexibility of the side chain, and serine is the most hydrophilic amino acid, which can increase the hydrophilicity of the connecting peptide. Therefore, (G4S)3 has good stability and activity.

[0090] In this document, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0091] The sequence descriptions involved in the present invention are shown in Table 1.

[0092] Table 1: Nucleotide / amino acid sequence description

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0099] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0100] Example 1: Preparation of NK cells expressing different NKR structures

[0101] 1. Construction of different NKR structures

[0102] 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.

[0103] The schematic diagram of the gene element structure of the chimeric antigen receptor in this example is shown in Figure 1 The nucleotide / amino acid sequences are shown in Table 1. Among them, 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.

[0104] 2. Lentivirus packaging

[0105] Take 5×10 293T cells in the logarithmic growth phase 6 Inoculate in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37°C, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare a lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G, a lentiviral packaging auxiliary plasmid, and 6μg target gene vector plasmid to 250μL serum-free DMEM medium to prepare a plasmid mixture, and mix well. Add 15μL Add to 235 μL serum-free DMEM medium and mix well. Add the mixture to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. Change the liquid 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 the 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 turning the centrifuge tube upside down, 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 precipitate, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0106] 3. Lentivirus infection of human NK-92 cells

[0107] NK-92 cells in the logarithmic growth phase were aspirated and centrifuged at 100 × g for 5 min to harvest the cells. An appropriate amount of α-MEM medium was added to resuspend the cells and the cell density was adjusted to 5 × 10 5 5×10 cells / mL were seeded in 24-well plates. 5 NK-92 cells, 0.2mL virus concentrate, 0.8mL α-MEM medium and protamine (final concentration 8μg / mL), mix well. Place in a 37℃, 5% CO2 incubator for culture. After 24h, observe the cell state, change the medium, transfer the infected cells to an EP tube, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells to a cell culture bottle, add 10mL fresh α-MEM medium and IL-2 (final concentration of 200IU / mL) and continue to culture for 48h. Transfer the cells to a flow tube, add 3mL 1×PBS solution, centrifuge at 100×g for 5min, discard the supernatant, flick the cell pellet, and wash again with 1×PBS solution. Use a flow cytometer to detect the expression rate of GFP. Continue to expand the culture and adjust the state of the infected NK-92 cells for amplification. The infected NK-92 cells are sorted by flow cytometry for GFP-positive NKR-NK92 cells for later experiments.

[0108] Example 2: Investigation of biological functions of different NKR-NK cells

[0109] 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.

[0110] 1. Investigation of the expression levels of different NKR receptors on NK cells

[0111] In the present invention, the extracellular recognition domain of the NKR molecule uses the extracellular segment of the NKp30 receptor, which can be specifically recognized by the NKp30 antibody, so the NKp30 antibody can be used to detect the expression of the NKR molecule. Since NK cells endogenously express NKp30 molecules, NK cells that are not infected with lentivirus are used as the negative control group for NKR molecule expression during the detection.

[0112] 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.

[0113] The results are as follows Figure 2 , Figure 3 shown.

[0114] The results of the investigation of NKR receptor expression levels 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.

[0115] 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.

[0116] It should be noted that when immune cells simultaneously express FcRγ molecules and chimeric antigen receptors with an extracellular region 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.

[0117] 2. Investigation of in vitro cytotoxic activity of NK-92 cells after modification of different NKR receptor genes

[0118] The inventors selected colorectal cancer cell line NCI-H716 cells with high expression of NKp30 ligand B7-H6 as target cells and performed in vitro killing activity detection on different NKR-NK cells.

[0119] The specific method is as follows: NCI-H716 cells were fluorescently labeled with CFSE at 2x10 4 / well were inoculated into 96-well plates, and NK-92 or different NKR-NK-92 cells were inoculated and incubated with NCI-H716 cells for 4 hours. PI staining was then added to distinguish between live and dead cells, and the killing efficiency was detected by flow cytometry. The ratio of effector cells to target cells was 2:1.

[0120] The test results are as follows Figure 4 shown.

[0121] The results of in vitro killing activity investigation showed that the killing efficiency of NK-92 cells modified by the NKR1 gene of the present invention on B7-H6-positive NCI-H716 cells was significantly higher than that of NK-92 cells and NKp30-CAR-NK-92 cells.

[0122] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0123] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nucleic acid construct, 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 amino acid sequence of the extracellular region is the amino acid sequence shown in SEQ ID NO: 1; The intracellular region includes a co-stimulatory domain and an intracellular signal transduction domain; the co-stimulatory domain is the intracellular segment of CD28; the amino acid sequence of the intracellular segment of CD28 is the amino acid sequence shown in SEQ ID NO: 4; the intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule; the amino acid sequence of the intracellular segment of the CD3ζ molecule is the amino acid sequence shown in SEQ ID NO: 5; The transmembrane region is selected from one of the transmembrane region of the CD3ζ molecule and the transmembrane region of the CD28 molecule; the amino acid sequence of the transmembrane region is the amino acid sequence shown in SEQ ID NO: 14 or 15.

2. The nucleic acid construct according to claim 1, characterized in that The amino acid sequence of the chimeric antigen receptor is the amino acid sequence shown in SEQ ID NO: 6 or 8.

3. The nucleic acid construct according to claim 1, characterized in that The nucleotide sequence of the chimeric antigen receptor is the nucleotide sequence shown in SEQ ID NO: 7 or 9.

4. The nucleic acid construct according to claim 1, characterized in that The amino acid sequence of FcRγ is the amino acid sequence shown in SEQ ID NO:

10.

5. The nucleic acid construct according to claim 1, characterized in that The nucleotide sequence of the second nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:

11.

6. The nucleic acid construct according to claim 4, 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.

7. The nucleic acid construct according to claim 6, characterized in that The first linker is selected from at least one of P2A, T2A, E2A and F2A.

8. The nucleic acid construct according to claim 6, characterized in that The first linker is P2A.

9. The nucleic acid construct according to claim 6, characterized in that The amino acid sequence of the first linker is the amino acid sequence shown in SEQ ID NO:

12.

10. The nucleic acid construct according to claim 6, characterized in that The nucleotide sequence of the first linker is the nucleotide sequence shown in SEQ ID NO:

13.

11. The nucleic acid construct according to claim 1, characterized in that The nucleotide sequence of the nucleic acid construct is the nucleotide sequence shown in SEQ ID NO: 16 or 17.

12. An expression vector, characterized in that: Carrying the nucleic acid construct according to any one of claims 1 to 11.

13. The expression vector according to claim 12, characterized in that The expression vector is a non-pathogenic viral vector.

14. The expression vector according to claim 13, characterized in that The non-pathogenic virus is selected from one of a retrovirus and an adeno-associated virus.

15. The expression vector according to claim 13, characterized in that The non-pathogenic virus is a lentivirus.

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

17.

17. A transgenic immune cell, characterized in that: The transgenic immune cells include: Carrying the nucleic acid molecule according to any one of claims 1 to 11, the expression vector according to any one of claims 12 to 15, or the lentiviral vector according to claim 16.

18. The genetically modified immune cell according to claim 17, characterized in that: The transgenic immune cells are selected from at least one of T cells, NK cells and macrophages.

19. The genetically modified immune cell according to claim 18, characterized in that: The T cells are selected from at least one of NKT cells and γδT cells, and the NK cells are selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells, and NK-92 cells.

20. The transgenic immune cell according to any one of claims 18 or 19, characterized in that: The T cells, NK cells or macrophages are derived from iPSCs or differentiated from embryonic stem cells.

21. The genetically modified immune cell according to claim 17, characterized in that: The transgenic immune cells are selected from NK cells.

22. A pharmaceutical composition, characterized in that include: The nucleic acid construct of any one of claims 1 to 11, the expression vector of any one of claims 12 to 15, the lentiviral vector of claim 16 or the transgenic immune cell of any one of claims 17 to 21.

23. The pharmaceutical composition according to claim 22, characterized in that Further including: Pharmaceutically acceptable excipients.

24. Use of the nucleic acid construct of any one of claims 1 to 11, the expression vector of any one of claims 12 to 15, the lentiviral vector of claim 16, the transgenic immune cell of any one of claims 17 to 21, or the pharmaceutical composition of any one of claims 22 to 23 in the preparation of a drug for preventing and / or treating a B7H6 target-mediated related disease; the B7H6 target-mediated related disease is colorectal cancer.

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