Preparation and application of CAR-NK cells with enhanced infiltration ability into tumor sites

Abstract

The present invention provides a transgenic cell and its application. The transgenic cell expresses a chimeric antigen receptor and a chemokine receptor, and the chemokine receptor includes CXCR2 and CXCR6. The transgenic cell can simultaneously express and secrete the chimeric antigen receptor and the chemokine receptor, enabling the transgenic cell to target tumor antigens, improving the ability of CAR-lymphocytes to infiltrate into solid tumors, enhancing the anti-tumor activity of CAR lymphocytes at the same time, and being applied to the treatment of solid tumors and hematological tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to the preparation of CAR-NK cells and their use in tumor treatment. More specifically, the present invention relates to a construct, expression vector, transgenic cell, pharmaceutical composition and their use for increasing the infiltration ability of CAR-NK cells into tumor sites. Background Art

[0002] In recent years, chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. However, CAR-T cells are prone to adverse reactions such as cytokine storm, neurotoxicity, and graft-versus-host disease (GVHD) in clinical applications. In addition, the current treatment effect of CAR-T cells on solid tumors is still not ideal, making the clinical application of CAR-T cells still face challenges. CAR-NK cells have the advantage of good safety compared with CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; NK cells can play a direct role in killing tumor cells without antigen presentation and are not restricted by MHC; CAR-NK cells can recognize and kill tumors through multiple recognition mechanisms such as CAR dependence and NKR dependence, and have a broad anti-tumor spectrum. Therefore, CAR-NK cells have broad application prospects in anti-tumor treatment and have become a hot spot in the field of cell immunotherapy research and development. However, one of the problems faced by CAR-NK cell therapy for solid tumors is that NK cells are difficult to infiltrate into solid tumors. An important limiting factor affecting the clinical results of NK cell infusion in solid tumor patients is the failure of a sufficient amount of NK cells to migrate to the tumor site to play an anti-tumor role. Effectively transporting NK cells to the tumor site is the key to the success of cancer immunotherapy. Therefore, improving the infiltration ability of NK cells into solid tumors has become an important research and development strategy for genetically modified NK cells.

[0003] Chemokines and chemokine receptors play an important role in the migration of lymphocytes such as T or NK cells to tumors. Lymphocytes can be attracted and recruited by chemokines secreted by tumors through chemokine receptors on the cell surface and are guided into the tumor site. CXCR2 is a receptor for multiple chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, and CXCL8). These chemokines are highly expressed in various solid tumors such as liver cancer, kidney cancer, breast cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, pancreatic cancer, and ovarian cancer, and are related to tumor proliferation, angiogenesis, invasion and metastasis, and the formation of an immunosuppressive microenvironment. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to some extent.

[0005] The inventors found that CXCR2 is usually only expressed in memory B cells and certain myeloid cells (such as neutrophils and basophils), and is not expressed in human T cells and NK cells. Therefore, this is the reason why T cells and NK cells are difficult to infiltrate into the tumor site. Therefore, overexpressing CXCR2 in lymphocytes is expected to effectively promote the aggregation of T cells or NK cells to the tumor site and effectively improve the anti-tumor effect of T cells or NK cells. Moreover, the inventors found that when CXCR2 receptor is co-expressed in CAR-lymphocytes, compared with co-expressing CXCR6 receptor, its chemotactic and targeting ability to tumors is stronger, and its killing ability to tumor cells is stronger.

[0006] Therefore, the present invention proposes a CAR-lymphocyte, the single-chain antibody on its surface can recognize tumor antigens of all solid tumors and hematological tumors and can express CXCR2 receptor intracellularly, enhancing the migration ability of lymphocytes to the tumor site and effectively improving the killing effect of lymphocytes on tumors.

[0007] Therefore, in the first aspect of the present invention, the present invention proposes a construct. According to an embodiment of the present invention, the construct includes a first nucleic acid and a second nucleic acid. Among them, the first nucleic acid encodes a chimeric antigen receptor, and the second nucleic acid encodes a chemokine receptor.

[0008] According to an embodiment of the present invention, the construct can encode a chimeric antigen receptor and a chemokine receptor simultaneously. Introducing the construct of the embodiment of the present invention into lymphocytes, the chimeric antigen receptor and the chemokine receptor are expressed on the surface of the lymphocytes. Among them, the chimeric antigen receptor can enable lymphocytes to target tumor antigens and locate on the cell surface expressing the antigen. In addition, the chemokine receptor can increase the infiltration ability of lymphocytes to the tumor site and effectively improve the tumor killing effect of lymphocytes.

[0009] According to an embodiment of the present invention, the above construct may further include at least one of the following additional technical features:

[0010] According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, the extracellular region includes a single-chain antibody and a CD8 hinge region, the single-chain antibody includes a heavy-chain variable region and a light-chain variable region, the extracellular single-chain antibody of the extracellular region can specifically recognize tumor antigens, and the C-terminus of the single-chain antibody is connected to the N-terminus of the CD8 hinge region.

[0011] A transmembrane region, the transmembrane region includes a CD8 transmembrane region, the N-terminus of the CD8 transmembrane region is connected to the C-terminus of the CD8 hinge region of the extracellular region and is embedded in the cell cell membrane;

[0012] The intracellular region, the N-terminus of which is connected to the C-terminus of the transmembrane region, and the intracellular region includes a 4-1BB co-stimulatory factor domain and a CD3ζ intracellular signaling segment.

[0013] According to an embodiment of the present invention, the tumor antigen is selected from embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including at least one of MSLN (mesothelin), HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, GD2, IL-13RA2, B7-H3, CD133, ROR1, CD19, CD20, CD22, CD30, CD33, BCMA.

[0014] According to an embodiment of the present invention, the chemokine receptor includes at least one selected from CXCR2 and CXCR6.

[0015] According to an embodiment of the present invention, the chemokine is CXCR2. The expression of CXCR2 enables lymphocytes to have a stronger ability to infiltrate into tumors.

[0016] According to an embodiment of the present invention, the single-chain antibody has the amino acid sequence shown in SEQ ID NO.1; the CD8 transmembrane region has the amino acid sequence shown in SEQ ID NO.2; the 4-1BB co-stimulatory factor domain has the amino acid sequence shown in SEQ ID NO.3; the CD3ζ intracellular signaling segment has the amino acid sequence shown in SEQ ID NO.4; the CD8 hinge region has the amino acid sequence shown in SEQ ID NO:5; CXCR2 has the amino acid sequence shown in SEQ ID NO.6; CXCR6 has the amino acid sequence shown in SEQ ID NO.7.

[0017] DIQMAQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKR(SEQ ID NO.1)

[0018] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO.2)

[0019] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO.3)

[0020] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO.4)

[0021] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO.5)

[0022] MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLYLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL (SEQ ID NO.6)

[0023] MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLVLVISIFYHKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSMLILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDKLICGYHDEAISTVVLATQMTLGFFLPLLTMIVCYSVIIKTLLHAGGFQKHRSLKIIFLVMAVFLLTQMPFNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFRKNFWKLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQ(SEQ ID NO.7)

[0024] According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged to express the chimeric antigen receptor and the chemokine receptor in a transgenic cell, and the chimeric antigen receptor and the chemokine receptor are in a non-fused form. According to an embodiment of the present invention, after expression, the chimeric antigen receptor and the chemokine receptor exist independently and can independently perform their respective functions of targeting and chemotaxing tumor cells.

[0025] According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged on the same vector.

[0026] According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged on different vectors.

[0027] According to an embodiment of the present invention, the internal ribosome entry site sequence is arranged between the first nucleic acid molecule and the second nucleic acid molecule, and the internal ribosome entry site has the nucleotide sequence shown in SEQ ID NO: 8.

[0028] TGAGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATG(SEQ IDNO:8)

[0029] According to an embodiment of the present invention, the third nucleic acid molecule is disposed between the first nucleic acid molecule and the second nucleic acid molecule, and the third nucleic acid molecule encodes a self-cleaving peptide P2A, and the self-cleaving peptide P2A can be cleaved in the transgenic cell.

[0030] According to an embodiment of the present invention, the self-cleaving peptide P2A has the amino acid sequence shown in SEQ ID NO: 9.

[0031] ATNFSLLKQAGDVEENPGP(SEQ ID NO:9)

[0032] According to an embodiment of the present invention, the first promoter, the first promoter is operably linked to the first nucleic acid molecule; similarly, a second promoter, the second promoter is operably linked to the second nucleic acid molecule. In this way, the nucleic acid molecule linked to the promoter can initiate the transcription process alone and does not interfere with the expression of other nucleic acid molecules.

[0033] According to an embodiment of the present invention, the first promoter and the second promoter are each independently selected from CMV, EF-1, and RSV promoters.

[0034] It should be noted that the "construct" described in this application can be either the target gene sequence or the vector into which the target gene sequence is introduced.

[0035] According to an embodiment of the present invention, the construct is a non-pathogenic viral vector.

[0036] According to an embodiment of the present invention, the non-pathogenic virus is selected from retroviruses, lentiviruses, adenoviruses, and other related viruses.

[0037] According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus.

[0038] According to an embodiment of the present invention, the first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 10;

[0039]

[0040] According to an embodiment of the present invention, the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 11.

[0041]

[0042] According to an embodiment of the present invention, the expression vector is a lentiviral vector.

[0043] According to an embodiment of the present invention, the expression vector is an adenovirus vector, a non-pathogenic vector or a retroviral vector.

[0044] In a third aspect of the present invention, the present invention provides a lentiviral vector. According to an embodiment of the present invention, the lentiviral vector has the nucleotide sequence shown in SEQ ID NO: 12.

[0045]

[0046] After the lentiviral vector according to the embodiment of the present invention is introduced into the recipient cell, a chimeric antigen receptor and a chemokine receptor can be simultaneously expressed on the membrane surface of the recipient cell.

[0047] In a fourth aspect of the present invention, the present invention provides a transgenic cell. According to an embodiment of the present invention, the transgenic cell carries the construct, expression vector, lentiviral vector described above, or expresses a chimeric antigen receptor and a chemokine receptor, and the chimeric antigen receptor and the chemokine receptor are in a non-fused form.

[0048] According to an embodiment of the present invention, the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.

[0049] In a fifth aspect of the present invention, the present invention provides a CAR-lymphocyte. According to an embodiment of the present invention, the CAR-lymphocyte carries the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, or expresses a chimeric antigen receptor and a chemokine receptor, and the chimeric antigen receptor and the chemokine receptor are in a non-fused form.

[0050] According to an embodiment of the present invention, the chimeric antigen receptor and the chemokine receptor are as defined in the first aspect of the present invention.

[0051] According to an embodiment of the present invention, the CAR-lymphocyte includes at least one selected from the group consisting of NK-92 cells, peripheral blood NK cells, cord blood NK cells, iPSCs, CAR-T cells, CAR-NKT cells, CAR-γδT cells, and CAR-macrophages.

[0052] In a sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, and the CAR-lymphocyte described in the fifth aspect of the present invention.

[0053] According to an embodiment of the present invention, the pharmaceutical composition further includes: a pharmaceutically acceptable excipient.

[0054] In the seventh aspect of the present invention, the present invention provides a use of a pharmaceutical composition in the preparation of a medicament. According to an embodiment of the present invention, the construct described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, the lentiviral vector described in the third aspect of the present invention, the transgenic cell described in the fourth aspect of the present invention, the CAR-lymphocyte described in the fifth aspect of the present invention, and the pharmaceutical composition described in the sixth aspect of the present invention can be used for the immunotherapy of solid tumors or hematological tumors.

[0055] According to an embodiment of the present invention, the solid tumors include at least one selected from the tangible tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioblastoma, kidney cancer, breast cancer, prostate cancer, melanoma.

[0056] According to an embodiment of the present invention, the hematological tumors include at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma in blood cells and the hematopoietic system. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a structural schematic diagram of a CAR targeting MSLN according to Example 1 of the present invention and a structural schematic diagram of a CAR connecting P2A and CXCR2 or CXCR6 receptor. Among them, SP represents a nucleotide sequence encoding a signal peptide, α-MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single-chain antibody, CD8 hinge+TM represents a nucleotide sequence encoding a CD8 hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB co-stimulatory factor domain, CD3ζ represents a nucleotide sequence encoding an intracellular region of CD3Z, P2A represents a nucleotide sequence encoding a P2A self-cleaving peptide, CXCR2 represents a nucleotide sequence encoding the full length of CXCR2, and CXCR6 represents a nucleotide sequence encoding the full length of CXCR6;

[0058] Figure 2 It is a result diagram of quantitative PCR for detecting chemokine expression in pancreatic cancer cells according to Example 2 of the present invention;

[0059] Figure 3 It is a result diagram of ELISA for detecting the secretion level of chemokine CXCL8 in pancreatic cancer tumor cells according to Example 2 of the present invention;

[0060] Figure 4 It is a result diagram of ELISA for detecting the secretion level of chemokine CXCL16 in pancreatic cancer tumor cells according to Example 2 of the present invention;

[0061] Figure 5It is a graph showing the detection results of the expression levels of CXCR2 in NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention;

[0062] Figure 6 It is a graph showing the detection results of the expression levels of CXCR6 in NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention;

[0063] Figure 7 It is a graph showing the detection results of the chemotactic abilities of NK-92, anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR2-NK-92, and anti-MSLN CAR-CXCR6-NK-92 cells according to Example 2 of the present invention;

[0064] Figure 8 It is a graph showing the detection results of the in vitro killing abilities of NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 2 of the present invention;

[0065] Figure 9 It is a graph showing the detection results of the anti-tumor abilities of NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR2-NK-92 cells according to Example 3 of the present invention;

[0066] Figure 10 It is a graph showing the detection results of the infiltration of NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR2-NK-92 cells into tumors according to Example 3 of the present invention; Detailed implementation manners

[0067] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the drawings. Wherein, the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention, but should not be construed as a limitation to the present invention.

[0068] During the description of the present invention, the relevant terms in this article are explained and described. These explanations and descriptions are only for the convenience of understanding the solution and should not be regarded as a limitation to the protection solution of the present invention.

[0069] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0070] In this text, the terms "optionally", "optional", or "option" generally mean that the subsequently described event or condition may but does not necessarily occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0071] "Operably linked" in this text means that an exogenous gene is linked to a vector such that control elements within the vector, such as transcriptional control sequences, translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of a regulatory system, the expression of the aforementioned isolated nucleic acid can be effectively achieved, and thus a large amount of the protein encoded by the isolated nucleic acid can be obtained in vitro.

[0072] In this application, a transgenic cell that simultaneously expresses a chimeric antigen receptor and an immune stimulatory molecule is constructed. Among them, the antigens targeted by the chimeric antigen receptor include embryonic proteins, glycoprotein antigens, squamous cell antigens, etc., including at least one of MSLN, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, EpCAM, PSCA, GD2, IL-13RA2, B7-H3, CD133, ROR1, CD19, CD20, CD22, CD30, CD33, BCMA, which are located on the cell surface expressing the antigen. The construct can express at least one of the CXCR2 and CXCR6 receptors, improve the ability of CAR-lymphocytes to infiltrate into solid tumors, and at the same time enhance the anti-tumor activity of CAR-lymphocytes, and is applied to the treatment of solid tumors and hematological tumors.

[0073] Embodiments of the present invention will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0074] It should be noted that in the following embodiments, the "plasmid" and the "vector" have the same meaning and can be used interchangeably.

[0075] Example 1: Preparation of Anti-MSLN-CAR-CXCR2-NK cells

[0076] 1. Construction of the CAR expression plasmid

[0077] The sequence of the CAR vector targeting mesothelin (anti-MSLN-CAR) designed by the present invention includes an extracellular segment (anti-MSLN scFv) targeting and recognizing MSLN, a CD8 hinge (Hinge) region and transmembrane segment, an intracellular co-stimulatory factor domain of 4-1BB and an intracellular signal transduction molecule CD3ζ, and a CXCR2 gene fragment linked by a P2A self-cleaving peptide. For the structural schematic diagram of the gene elements, see Figure 1 。

[0078] Construction of the anti-MSLN-CAR-CXCR2 vector: Using the pSBbi-MSLN CAR-GP plasmid as a template to amplify the anti-MSLN-CAR gene fragment, amplifying the CXCR2 or CXCR6 gene fragment from the pENTER-CXCR2 or pENTER-CXCR6 vector (Vigenebio), ligating the obtained CXCR2 or CXCR6 gene fragment after enzyme digestion to the pSBbi-MSLN CAR-GP plasmid to form an anti-MSLN-CAR-CXCR2 or anti-MSLN-CAR-CXCR6 fragment. After transformation, identification, and correct sequencing, amplifying the anti-MSLN-CAR-CXCR2 or anti-MSLN-CAR-CXCR6 fragment and inserting it between the AsiSI and MIuI restriction enzyme cleavage sites of the lentiviral vector pLent-EF1a-P2A-GFP to construct the pLent-EF1a-anti-MSLN-CAR-P2A-CXCR2-GFP or pLent-EF1a-anti-MSLN-CAR-P2A-CXCR6-GFP vector, and verifying the sequence by sequencing.

[0079] 1.2 Packaging of lentivirus and concentration of virus solution

[0080] Take 5×10 6 293T cells in the logarithmic growth phase and inoculate them into a 10-cm culture dish, add 10 mL of DMEM medium, and culture overnight in an incubator at 37°C and 5% CO2. When the cell density reaches 80%, replace it with 10 mL of fresh DMEM medium and continue to culture in the incubator. Prepare the lentivirus packaging system: Add 6 μg of the psPAX2 plasmid, 3 μg of the pMD2.G plasmid, and 6 μg of the target gene vector plasmid to 250 μL of serum-free DMEM medium, mix well to prepare a DNA mixture; add 15 mL to 235 μL of serum-free DMEM medium and mix well. The mixed solution was added to the DNA mixed solution at one time, left standing and mixed evenly, and incubated at room temperature for 15 min. The mixed solution was added to a 293T cell culture dish. After 24 h, the medium was changed, and the fluorescence positive rate was observed at the same time. The culture dish was placed back into the 37 °C, 5% CO2 incubator. After 48 h, the cell supernatant was collected, centrifuged at 400×g for 5 min to remove cell debris, and the supernatant was filtered through a 0.45 mm filter tip into a new 50 ml centrifuge tube. 5×PEG8000 solution was added, and the centrifuge tube was inverted up and down to mix evenly, and then placed in a 4 °C refrigerator overnight. The centrifuge tube was placed at 4 °C and centrifuged in a 4000×g centrifuge for 20 min. The supernatant was discarded, and the virus precipitate was resuspended with an appropriate amount of serum-free DMEM medium, aliquoted into EP tubes, and stored in an -80 °C refrigerator.

[0081] 1.3 Lentivirus titer detection

[0082] Take 293T cells in the logarithmic growth phase and adjust the concentration to 1×10 5 / mL. Take a 24-well plate and add 1 mL of cell suspension (1×10 5 / well) to each well, and set 3 gradients for the volume of virus added. Incubate overnight in a 37 °C, 5% CO2 incubator. First, dilute the concentrated virus solution 10-fold: Take a 1.5 mL EP tube, pipette 60 μL of the virus concentrate into the EP tube, and dilute it with 540 μL of DMEM medium, and mix evenly. Change the medium of 293T cells with fresh DMEM medium, and pipette 5 μL, 50 μL, and 500 μL of the diluted virus solution into the corresponding wells respectively, make marks, and then place the culture plate back into the 37 °C, 5% CO2 incubator. After 24 h, aspirate the virus solution in the well plate, and then add 1 mL of fresh DMEM medium. After 72 h, harvest the cells by trypsin digestion, and use a flow cytometer to detect the GFP expression rate of 293T cells. Calculate the virus titer according to the following formula:

[0083] Titer (TU / mL) = (C × N × D × 1000) / V

[0084] Where: C = GFP positive rate detected by flow cytometry

[0085] N = Number of cells at the time of infection (about 1×105)

[0086] D = Dilution factor of the virus vector

[0087] V = Volume of the diluted virus added

[0088] 1.4 Lentivirus infection of human NK cells

[0089] Take NK-92 cells (purchased from ATCC) in the logarithmic growth phase, harvest the cells by centrifugation at 100×g for 5 min, add an appropriate amount of α-MEM medium to resuspend the cells, and adjust the cell density to 5×105 cells / mL. In a 24-well plate, 5×10 5 NK-92 cells were seeded respectively. 1 mL of the virus concentrate was mixed evenly with protamine (purchased from Solarbio, final concentration 8 μg / mL), and placed in an incubator at 37°C and 5% CO2 for culture. After 24 hours, the cell status was observed, the medium was changed, the infected cells were transferred into an EP tube, centrifuged at 100×g for 5 minutes, resuspended with a small amount of fresh α-MEM medium, transferred into 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 into a flow tube, 3 mL of 1×PBS solution was added, centrifuged at 100×g for 5 minutes, the supernatant was discarded, the cell pellet was flicked up, and washed again with 1×PBS solution. The expression rate of GFP was detected using a flow cytometer. The culture was continued to expand, and the status of the infected NK-92 cells was adjusted for amplification. The infected NK-92 cells were sorted by a flow cytometer to obtain GFP-positive CAR-NK-92 cells for later experiments.

[0090] Example 2: Detection of the expression and chemotactic ability of CXCR2 or CXCR6 in Anti-MSLN CAR-NK cells and determination of the killing function of Anti-MSLN CAR-CXCR2-NK cells

[0091] 2.1 Detection of highly expressed chemokines in pancreatic cancer tumor cells

[0092] Total RNA was extracted from the pancreatic cancer tumor cell line Capan-2 cells (purchased from ATCC), and cDNA was synthesized. The expression of chemokines CXCL8 (IL-8), CXCL10, CXCL12, CXCL16 and CCL18 was detected by quantitative PCR. The results showed that Capan-2 cells highly expressed CXCL8 and CXCL16 ( Figure 2 ). Further, the contents of CXCL8 and CXCL16 secreted by the pancreatic cancer tumor cell lines AsPC-1 cells (purchased from ATCC) and Capan-2 cells were detected by ELISA. The results showed that both AsPC-1 and Capan-2 cells secreted CXCL8 and CXCL16, and the contents of CXCL8 and CXCL16 secreted by Capan-2 cells were significantly higher than those of AsPC-1 cells ( Figure 3 and Figure 4 ). Therefore, the chemokine receptors CXCR2 and CXCR6 corresponding to CXCL8 and CXCL16 were selected as targets to enhance the chemotactic ability of NK cells to tumors such as pancreatic cancer that highly express CXCL8 and CXCL16 chemokines.

[0093] 2.2 Expression of CXCR2 or CXCR6 in CAR-NK-92 cells

[0094] Flow cytometry was used to detect the expression of CXCR2 or CXCR6 in anti-MSLN CAR-CXCR2-NK-92 and anti-MSLN CAR-CXCR6-NK-92 cells respectively. The results showed that the expression rate of CXCR2 on anti-MSLN CAR-CXCR2-NK-92 cells was 99.0%( Figure 5 ), and the expression rate of CXCR6 on anti-MSLN CAR-CXCR6NK-92 cells was 90.5%( Figure 6 ), while unmodified NK-92 cells (cells not expressing CXCR2, CXCR6, and CAR) and anti-MSLN CAR-NK-92 cells had basically no CXCR2 and CXCR6 expression( Figure 5 and Figure 6 ). This indicates that CXCR2 or CXCR6 was successfully expressed on the surface of NK cells.

[0095] 2.3 Comparison of chemotactic ability between Anti-MSLN CAR-CXCR2 NK-92 and Anti-MSLN CAR-CXCR6 NK-92 cells

[0096] The chemotactic ability of NK cells was detected by transwell assay. 3×10 5 NK-92 cells were seeded in the upper chamber of the transwell chamber, and 600 μL of pancreatic cancer Capan-2 cell culture supernatant was added to the lower chamber. After culturing at 37 °C for 4 h, the cells in the lower chamber were collected and counted. The results showed that the migration rate of anti-MSLN CAR-CXCR2-NK-92 cells was 23.38% ± 2.30%, which was significantly higher than that of anti-MSLN CAR-NK-92, anti-MSLN CAR-CXCR6-NK-92, and NK-92 cells( Figure 7 ).

[0097] 2.4 In vitro killing function of Anti-MSLN CAR-CXCR2 NK-92 cells

[0098] Using NK-92, anti-MSLN CAR-NK-92, and anti-MSLN CAR-CXCR2-NK-92 cells as effector cells and the pancreatic cancer cell line Capan-2 as target cells, effector-to-target ratios of 5:1, 2.5:1, and 1.25:1 were set. The effector cells and target cells were co-incubated for 5 h, and the killing efficiency of the effector cells against the target cells was detected by the LDH release method. The results showed that the killing efficiencies of anti-MSLN CAR-CXCR2-NK-92 cells and anti-MSLN CAR-NK-92 cells against pancreatic cancer cell Capan-2 were significantly higher than those of unmodified NK-92 cells( Figure 8 ). This indicates that the expression of CXCR2 does not affect the killing effect of CAR-NK92 cells.

[0099] Example 3: Anti-tumor ability and ability to infiltrate into tumors of Anti-MSLN CAR-CXCR2 NK-92 cells

[0100] A pancreatic cancer xenograft model was established by subcutaneous tumor inoculation with pancreatic cancer Capan-2 cells to observe the therapeutic effect of anti-MSLN CAR-CXCR2 NK92 cells on pancreatic cancer. Six-week-old female nude mice were selected and inoculated subcutaneously in the axilla with a tumor inoculation dose of 5×10 6 Capan-2 cells per mouse. Two weeks later, when the tumor volume reached about 100 mm 3 , treatment began. First, the mice were randomly divided into a control group, an NK-92 cell treatment group, an anti-MSLN CAR-NK-92 cell treatment group, and an anti-MSLN CAR-CXCR2-NK-92 cell treatment group. The mice in the treatment groups were injected with 5×10 6 effector cells per 100 μL per mouse via the tail vein. The control group was injected with an equal volume of 1×PBS solution. Injections were performed once every week, and IL-2 (5×10 4 IU / mouse) was injected intraperitoneally every 3 days. The tumor volume of the mice was measured every three days. Treatment was observed for a total of 56 days. Tumor tissues were taken for photography and the tumor volume was measured( Figure 9 ). The results showed that the tumor volume in the anti-MSLN CAR-CXCR2-NK-92 cell treatment group was significantly smaller than that in the other three groups, indicating that CAR-NK cells expressing CXCR2 have stronger anti-tumor ability in vivo.

[0101] To further clarify the infiltration of NK cells, we detected the infiltration of NK cells in tumor tissues by immunofluorescence. As Figure 10As shown, the results showed that there were almost no NK cells in the tumor tissues of the untreated group, a small number of NK cells in the tumor tissues of the NK-92 treatment group, an increased number of infiltrating NK cells in the tumor tissues of the anti-MSLN CAR-NK-92 cell treatment group compared to the untreated and NK92 treatment groups, and the number of infiltrating NK cells in the tumor tissues of the anti-MSLN CAR-CXCR2-NK-92 cell treatment group was significantly higher than that of the anti-MSLN CAR-NK-92 cell treatment group. This indicates that CAR-NK cells expressing CXCR2 have a significantly enhanced ability to infiltrate into solid tumors.

[0102] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0103] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0104] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A construct, characterized in that, Comprising: A first nucleic acid encoding a chimeric antigen receptor; And A second nucleic acid encoding a self-cleaving peptide and a chemokine receptor; Wherein The first nucleic acid has the nucleotide sequence shown in SEQ ID NO: 10; The second nucleic acid has the nucleotide sequence shown in SEQ ID NO:

11.

2. An expression vector, characterized in that, Comprising the construct according to claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector is a lentiviral vector.

4. The expression vector according to claim 2, characterized in that, The expression vector is an adenoviral vector, a non-pathogenic vector or a retroviral vector.

5. A lentiviral vector, characterized in that, Having the nucleotide sequence shown in SEQ ID NO:

12.

6. A transgenic cell, characterized in that, Carrying the construct according to claim 1, the expression vector according to any one of claims 2-4 or the lentiviral vector according to claim 5.

7. A CAR-lymphocyte, characterized in that, The lymphocyte carries the construct according to claim 1, the expression vector according to any one of claims 2-4 or the lentiviral vector according to claim 5.

8. The CAR-lymphocyte according to claim 7, characterized in that, The lymphocyte comprises at least one selected from the group consisting of NK-92 cells, peripheral blood NK cells, and cord blood NK cells.

9. The CAR-lymphocyte according to claim 7, characterized in that, The CAR-lymphocytes comprise at least one selected from the group consisting of CAR-T cells, CAR-NKT cells, and CAR-γδT cells.

10. A pharmaceutical composition, characterized in that, Comprising: The CAR-lymphocytes according to any one of claims 7-9.

11. The pharmaceutical composition according to claim 10, characterized in that, Further comprising: A pharmaceutically acceptable excipient.

12. Use of the construct according to claim 1, the expression vector according to any one of claims 2-4, the lentiviral vector according to claim 5, the transgenic cell according to claim 6, the CAR-lymphocyte according to any one of claims 7-9, or the pharmaceutical composition according to any one of claims 10-11 in the preparation of a drug for the immunotherapy of pancreatic cancer.

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