Dual-targeting chimeric antigen receptor, recombinant expression vector, anti-tumor natural killer cell and application

Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to a dual-targeting chimeric antigen receptor, a recombinant expression vector, an anti-tumor natural killer cell and application, and provides a PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3 zeta fusion receptor.The dual-targeting chimeric antigen receptor can target PD-L1 on the surface of a tumor cell, can also recognize NKG2DLs and can be used for preparing anti-tumor natural killer cells. The broad spectrum of the NK cells is greatly improved, and meanwhile, the immune escape of tumor cells is also reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immunotherapy, and includes a dual-targeted chimeric antigen receptor, a recombinant expression vector, an anti-tumor natural killer cell and an application thereof. Background Art

[0002] Tumor immunotherapy can effectively overcome the mechanism of tumor immune escape, thereby reawakening immune cells to eliminate tumor cells. Due to its small side effects and obvious therapeutic effects, it is gradually becoming the development direction of future tumor treatment and is called the fourth tumor treatment technology after surgery, radiotherapy and chemotherapy.

[0003] Chimeric antigen receptor, abbreviated as CAR, with the continuous advancement of CAR technology, tumor immunotherapy is developing at an unprecedented speed, and it also provides new treatment hope for cancer patients. The design and application of CAR structure provides a new approach to cancer treatment. T cells are the first immune cells modified by CAR to specifically target tumor cells, and clinical results show that CAR-T cells have great advantages in the treatment of hematological malignancies. However, with the gradual development of CAR-T technology, it has been gradually discovered that its cell efficacy is limited by the heterogeneity of tumor antigens, and tumor recurrence often occurs. Therefore, how to overcome postoperative recurrence is the direction and idea we have been looking for, so in recent years, we have gradually shifted to CAR-NK cell therapy.

[0004] In recent years, with increasing attention paid to the unique properties and specific cytotoxicity of natural killer cells, abbreviated as NK, there has been growing interest in developing CAR-NK cells for cancer therapy. Compared with CAR-T cells, CAR-NK cells may offer some significant advantages, including: (1) better safety, such as less cytokine release syndrome and neurotoxicity and graft-versus-host disease in an allogeneic setting. (2) multiple mechanisms of activation of cytotoxic activity. (3) high feasibility of “off-the-shelf” manufacturing. CAR-NK cells can be engineered to target multiple antigens, enhance in vivo proliferation and persistence, increase infiltration into solid tumors, overcome resistant tumor microenvironments, and ultimately achieve effective antitumor responses. NK cells appear to be an attractive vehicle for CAR expression because they can be obtained from a variety of sources and can be safely infused regardless of donor-patient matching, which greatly reduces the cost of treatment. CAR-NK cells are known to be effective against hematological malignancies, but an increasing number of preclinical findings suggest that they are also active against non-hematological tumors.

[0005] Although the advantages of CAR-NK cell therapy over CAR-T cell therapy are obvious, the existing antigen receptors may cause tumor recurrence due to antigen escape, so how to prevent antigen escape has become an urgent problem that needs to be solved. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a dual-targeting chimeric antigen receptor, a recombinant expression vector, an anti-tumor natural killer cell and an application.

[0007] A dual-targeting chimeric antigen receptor, which is composed of the PD1-DAP10-CD137 receptor and the NKG2D-IgG4-CD28-CD137-CD3ζ receptor connected in sequence;

[0008] The PD1-DAP10-CD137 receptor is composed of the CD8α-C1 signal peptide, the Myc tag, the PD1 extracellular segment, the DAP10 extracellular transmembrane and intracellular segments, and the CD137-C1 intracellular segment.

[0009] The NKG2D-IgG4-CD28-CD137-CD3ζ receptor is composed of CD8α-C2 signal peptide, FLAG tag, NKG2D ligand binding domain, IgG4 monomer hinge region, CD28 transmembrane and intracellular region, CD137-C2 intracellular segment, and CD3ζ intracellular domain.

[0010] The two can quickly combine in cells to form dual-targeted receptors. Using this chimeric molecular module, on the one hand, NKG2D binds to MICA / MICB on the tumor surface, and PD1 binds to PD-L1 on tumor cells, fully exerting the function of targeting tumor cells; on the other hand, the combined action of NKG2D and DAP10 enhances the activation effect of NK92 cells.

[0011] The amino acid sequence of the CD8α-C1 signal peptide is shown in SEQ ID NO.1;

[0012] The amino acid sequence of the Myc tag is shown in SEQ ID NO.2;

[0013] The amino acid sequence of the extracellular segment of PD1 is shown in SEQ ID NO.3;

[0014] The amino acid sequence of the extracellular transmembrane and intracellular segments of DAP10 is shown in SEQ ID NO.4;

[0015] The amino acid sequence of the intracellular segment of CD137-C1 is shown in SEQ ID NO.5;

[0016] The amino acid sequence of the CD8α-C2 signal peptide is shown in SEQ ID NO.1;

[0017] The amino acid sequence of the FLAG tag is shown in SEQ ID NO.6;

[0018] The amino acid sequence of the NKG2D ligand binding domain is shown in SEQ ID NO.7;

[0019] The amino acid sequence of the hinge region of the IgG4 monomer is shown in SEQ ID NO.8;

[0020] The amino acid sequence of the transmembrane and intracellular regions of CD28 is shown in SEQ ID NO.9;

[0021] The amino acid sequence of the intracellular segment of CD137-C2 is shown in SEQ ID NO.5;

[0022] The amino acid sequence of the CD3ζ intracellular domain is shown in SEQ ID NO.10.

[0023] Preferably, the amino acid sequence of the dual-targeting chimeric antigen receptor is as shown in SEQ ID NO.12.

[0024] Preferably, the nucleotide sequence of the gene encoding the dual-targeted chimeric antigen receptor is shown in SEQ ID NO. 11, the gene encoding the dual-targeted chimeric antigen receptor is driven by a CMV promoter, the first receptor and the second receptor are connected by a P2A peptide, avoiding the phenomenon of uneven target protein expression in the dual promoter. The nucleotide sequence conforms to the codon preference in the human body and can be better expressed in the human body.

[0025] A recombinant expression vector comprises a gene encoding the dual-targeting chimeric antigen receptor.

[0026] A method for preparing a recombinant expression vector comprises the following steps:

[0027] synthesizing the dual-targeting chimeric antigen receptor encoding gene;

[0028] The dual-targeting chimeric antigen receptor encoding gene is connected to a lentiviral expression vector to obtain the recombinant expression vector.

[0029] The lentiviral expression vector is pCDH-CMV-MCS-P2A-copGFP-T2A-Puro, which is obtained by optimizing pCDH-CMV-MCS-EF1-copGFP-T2A-Puro.

[0030] An anti-tumor natural killer cell is obtained by infecting a natural killer cell to be transformed with the recombinant expression vector.

[0031] The NK cells of the present invention have extremely strong cytotoxicity. Compared with the targeting effect of CAR-NK on a certain antigen, PN-CAR-NK92 cells recognize tumor cells through the NKG2D / NKG2DL and PD1 / PD-L1 pathways. Since most tumors express MICA, MICB, and PD-L1, the targeting effect of PN-CAR-NK92 on tumor cells is more broad-spectrum, rather than being limited to one or several specific antigen targets.

[0032] A method for preparing anti-tumor natural killer cells comprises the following steps:

[0033] The recombinant lentiviral expression vector is packaged using auxiliary plasmids pSPAX2 and PMD2G to express a virus carrying the target plasmid;

[0034] The virus carrying the target plasmid is used to infect NK92 cells to obtain the anti-tumor natural killer cells.

[0035] The use of the dual-targeting chimeric antigen receptor, the recombinant expression vector or the anti-tumor natural killer cell in the preparation of anti-tumor drugs.

[0036] Preferably, the anti-tumor drug has the dual-targeting chimeric antigen receptor, the recombinant expression vector or the anti-tumor natural killer cell as the only active ingredient.

[0037] Preferably, the tumor is any one of breast cancer, gastric cancer, and osteosarcoma.

[0038] Immune checkpoints refer to a class of immunosuppressive molecules that regulate immune cell activity through a series of pathways of co-inhibition or co-stimulation signals, prevent excessive immune activation and ensure self-tolerance, thereby avoiding normal tissue damage and destruction. PD1 is one of the main inhibitory molecules. The immune negative regulatory mechanism mediated by PD1 / PD-L1 can inhibit the activation of the MHC-TCR signaling pathway, control the proliferation of T cells and NK cells, cytokine secretion, or response to similar antigens. PD1 is mainly expressed on the surface of activated immune cells, and its ligand PD-L1 is limited in normal tissues, but its expression in most solid tumor cells and immunosuppressive cells is significantly increased. Tumors use the PD1 / PD-L1 negative regulatory mechanism to escape anti-tumor immune responses. Therefore, blocking this immunosuppressive signaling pathway has become an important path for immunotherapy of solid tumors. NKG2DLs are diverse and exist on a variety of tumor cells. High expression of MICA / B can often be detected in liver cancer, ovarian cancer, pancreatic cancer, colon cancer, breast cancer, etc., while NKG2DLs are rarely expressed in healthy cells of the human body. Therefore, in the process of cell carcinogenesis, NK cells that perform immune surveillance in the immune system can well recognize and bind to these ligands through their own NKG2D receptors, thereby triggering the activation effect of NK cells and effectively eliminating a variety of tumor cells.

[0039] Based on the above content, the present invention uses the whole gene synthesis technology to synthesize the PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fusion receptor, that is, only the extracellular domain of the PD1 molecule is retained as the first recognition target, and the extracellular transmembrane and intracellular segments of DAP10 and the intracellular segments of the co-stimulatory molecule 41BB are used to replace the intracellular inhibitory signal domain of the PD1 receptor. This chimeric molecular module is used to play the role of signal conversion "switch", thereby reversing the PD1 / PD-L1 immune negative regulation mechanism. At the same time, the extracellular domain of the NKG2D molecule is selected as the second recognition target, and the CD28 transmembrane and intracellular segments, the 41BB intracellular segments and CD3ζ are used as co-stimulatory molecules. When the PD1 extracellular signal domain recognizes the PD-L1 molecule on the tumor cell, the negative regulation signal is transmitted downward, which triggers the activation of the DAP10 activating receptor pathway and becomes a positive activation signal. At the same time, when the extracellular domain of the NKG2D molecule recognizes the NKG2DLs molecules on tumor cells, it can also activate NK cells, and under the synergistic action of the co-stimulatory molecules 41BB and CD3ζ, it greatly enhances the anti-tumor therapeutic effect of NK cells.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a dual-targeted chimeric antigen receptor that can target PD-L1 on the surface of tumor cells and recognize NKG2DLs, greatly improving the broad spectrum of NK cells and reducing the immune escape of tumor cells.

[0042] The anti-tumor NK cells of the present invention can be allogeneically infused with little damage to normal cells, will not cause an excessive immune response in the body, and can achieve accurate recognition and specific killing of solid tumor cells, with stronger cytotoxicity and better therapeutic effect on tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the structure of the recombinant lentiviral vector CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH containing a gene encoding a dual-targeted chimeric antigen receptor of the present invention;

[0044] Figure 2 This is a schematic diagram of the mRNA level expression of PD1 and NKG2D in the molecular module PN-CAR-NK92 cells after lentiviral transfection of NK92 in Example 3 of the present invention;

[0045] Figure 3 This is a schematic diagram of flow cytometry detection of PD1 and NKG2D in the molecular module PN-CAR-NK92 cells after lentiviral transfection of NK92 in Example 4 of the present invention;

[0046] Figure 4 This is a schematic diagram of flow cytometry detection of PD-L1 and MICA / MICB in EMT6-PD-L1 and MGC-803 tumor cells in Example 5 of the present invention;

[0047] Figure 5 This is a graph showing the killing efficiency of NK92 and single-module PD1-CAR-NK92, NKG2D-CAR-NK92 and dual-module PN-CAR-NK92 cells against EMT6-PD-L1 and MGC-803 tumor cells in Example 6 of the present invention.

[0048] Figure 6 This is a schematic diagram of flow cytometry detection of PD-L1 and MICA / MICB of KATO-III and U2OS tumor cells in Example 5 of the present invention;

[0049] Figure 7 This is a graph showing the killing efficiency of NK92 and PN-CAR-NK92 cells against KATO-III and U2OS tumor cells in Example 6 of the present invention.

[0050] Figure 8Schematic diagram of the rational design of PN-CAR-NK92 and the anti-tumor effect of modified NK cells in the present invention. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0052] Example 1

[0053] A dual-targeting chimeric antigen receptor consists of PD1-DAP10-CD137 receptor and NKG2D-IgG4-CD28-CD137-CD3ζ receptor.

[0054] The PD1-DAP10-CD137 receptor is composed of CD8α-C1 signal peptide, Myc tag, PD1 extracellular segment, DAP10 extracellular transmembrane and intracellular segment, and CD137-C1 intracellular segment.

[0055] The amino acid sequence of the CD8α-C1 signal peptide is: MALPVTALLLPLALLLHAARP, recorded as SEQ ID NO.1.

[0056] The amino acid sequence of the Myc tag is: EQKLISEEDL, recorded as SEQ ID NO.2.

[0057] The amino acid sequence of the extracellular segment of PD1 is: FLDSPDRPWNPPTFSPALLVVTEGDNATFT CSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRD FHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPS PSPRPAGQFQTLV, recorded as SEQ ID NO.3.

[0058] The amino acid sequence of the extracellular transmembrane and intracellular segments of DAP10 is: QTTPGERSSLPAFYPGTSGSC SGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGR G, recorded as SEQ ID NO.4.

[0059] The amino acid sequence of the intracellular segment of CD137-C1 is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, recorded as SEQ ID NO.5.

[0060] The NKG2D-IgG4-CD28-CD137-CD3ζ receptor is composed of CD8α-C2 signal peptide, FLAG tag, NKG2D ligand binding domain, IgG4 monomer hinge region, CD28 transmembrane and intracellular region, CD137-C2 intracellular segment, and CD3ζ intracellular domain.

[0061] The amino acid sequence of the CD8α-C2 signal peptide is identical to that of the CD8α-C1 signal peptide.

[0062] The amino acid sequence of the FLAG tag is: DYKDDDDK, recorded as SEQ ID NO.6.

[0063] The amino acid sequence of the NKG2D ligand binding domain is: LFNQEVQIPLTESYCGPCPKN WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSY HWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCS TPNTYICMQRTV, recorded as SEQ ID NO.7.

[0064] The amino acid sequence of the hinge region of the IgG4 monomer is: ESKYGPPAPPAP, recorded as SEQ ID NO.8.

[0065] The amino acid sequence of the transmembrane and intracellular regions of CD28 is: FWVLVVVGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS, recorded as SEQ ID NO.9.

[0066] The amino acid sequence of the intracellular segment of CD137-C2 and the amino acid sequence of the intracellular segment of CD137-C1.

[0067] The amino acid sequence of the CD3ζ intracellular domain is: RVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, recorded as SEQ ID NO.10.

[0068] The PD1-DAP10-CD137 receptor was prepared according to the following steps:

[0069] The first receptor consisting of a nucleotide fragment containing the extracellular segment of PD1 (1-147aa), the extracellular transmembrane and intracellular segment of DAP10 (19-93aa), and the intracellular (214-255aa) nucleotide fragment of CD137 (also known as 4-1BB) was synthesized by the method of whole gene synthesis.

[0070] The NKG2D-IgG4-CD28-CD137-CD3ζ receptor was prepared according to the following steps:

[0071] A second receptor consisting of the NKG2D extracellular domain sequence, which was sequentially connected to the IgG4 hinge region sequence, the CD28 transmembrane region sequence (153-220aa), the CD137 intracellular domain and the CD3ζ intracellular domain, was synthesized by the whole gene synthesis method.

[0072] The dual-targeting chimeric antigen receptor was prepared by the following steps:

[0073] Through the method of whole gene synthesis, the nucleotide fragment of the first receptor and the nucleotide fragment of the second receptor were connected in series, and the two were connected by the self-cleaving peptide P2A to obtain a dual-targeted chimeric antigen receptor, namely PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment.

[0074]

[0075] The amino acid sequence of the dual-targeting chimeric antigen receptor is: MALPVTALLLPLALLLHAARPDY KDDDDKLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVESKYGPPAPPAPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, denoted as SEQ IDNO.12.

[0076] The nucleotide sequence of CD8α-C1 signal peptide is: ATGGCGCTTCCGGTGACTGCTCTACT TCTACCGTTGGCTCTGCTGCTCCACGCCGCTCGCCCT, recorded as SEQ ID NO.13.

[0077] The nucleotide sequence of the Myc tag is: GAACAGAAGCTCATCTCGGAGGAAGACCT G, recorded as SEQ ID NO.14.

[0078] The extracellular nucleotide sequence of PD1: TTCCTGGACAGCCCCGACCGCCCGTGGAATCCA CCCACCTTTTCACCGGCCCTGCTGGTGGTTACAGAGGGCGACAACGCCACGTTCACCTGCTCCTTCTCCAACACGTCGGAGAGTTTCGTCCTCAACTGGTACAGGATGTCTCCCAGCAACCAGACCGACAAACTTGCTGCGTTCCCCGAGGATCGCAGCCAGCCGGGCCAGGATTGCCGATTCAGAGTCACCCAGCTGCCCAACGGTCGGGACTTTCATATGTCCGTGGTCCGCGCCCGCCGCAACGACTCTGGCACCTACCTGTGCGGGGCCATCTCTTTGGCTCCCAAGGCCCAAATCAAGGAGAGCCTGCGCGCCGAGCTCCGCGTAACCGAGCGGCGTGCAGAGGTGCCAACCGCTCACCCCTCTCCTAGCCCTAGGCCTGCCGGACAGTTCCAGACCCTGGTG, denoted as SEQ ID NO.15.

[0079] The nucleotide sequence of the extracellular transmembrane and intracellular segments of DAP10 is: CAGACTACTCCCGGTGAGC GCAGTTCCCTGCCTGCGTTTTATCCGGGGACCAGCGGCTCCTGTTCTGGATGTGGTTCGCTGTCCTTGCCCCTGCTGGCCGGCTTGGTGGCGGCGGACGCCGTGGCTTCCCTGCTGATTGTCGGTGCCGTGTTCCTGTGCGCCCGGCCTCGCCGTTCCCCAGCGCAGGAGGACGGCAAAGTGTACATTAATATGCCCGGCCGCGGC, denoted as SEQ ID NO.16.

[0080] The nucleotide sequence of the intracellular segment of CD137-C1 is: AAGCGCGGGCGAAAGAAGCTGCT GTACATCTTCAAGCAGCCATTTATGCGTCCTGTGCAGACGACCCAGGAGG AAGATGGCTGTTCATGCCGCTTCCCGGAGGAGGAGGAGGGCGGCTGCGA GTTG, denoted as SEQ ID NO.17.

[0081] The nucleotide sequence of CD8α-C2 signal peptide is: ATGGCCTTACCAGTGACCGCCTTGCTC CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG, recorded as SEQ ID NO.18.

[0082] The nucleotide sequence of the FLAG tag is: GACTACAAAGACGATGACGACAAG, recorded as SEQ ID NO.19.

[0083] The nucleotide sequence of the NKG2D ligand binding domain is: TTATTCAACCAAGAAGTTCA AATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCAT TGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG, recorded as SEQID NO.20.

[0084] The nucleotide sequence of the hinge region of the IgG4 monomer is: GAATCTAAATATGGGCCTCCAGCA CCACCCGCGCCT, recorded as SEQ ID NO.21.

[0085] The nucleotide sequence of the transmembrane and intracellular region of CD28 is: TTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC, recorded as SEQ ID NO.22.

[0086] The nucleotide sequence of the intracellular segment of CD137-C2 is: AAACGGGGCAGAAAGAAACTCCT GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGA AGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA CTG, recorded as SEQ ID NO.23.

[0087] The nucleotide sequence of the CD3ζ intracellular domain is: AGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAA CTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCTCGA, recorded as SEQID NO.24.

[0088] Example 2

[0089] The method for preparing the recombinant expression vector of the gene encoding the dual-targeting chimeric antigen receptor comprises the following steps:

[0090] The first receptor composed of the nucleotide fragment containing the extracellular segment of PD1 (1-147aa), the extracellular transmembrane and intracellular segment of DAP10 (19-93aa), and the intracellular segment of CD137 (also known as 4-1BB) (214-255aa) was synthesized by the method of total gene synthesis. The NKG2D extracellular domain sequence was connected with the hinge region sequence of IgG4 (monomer mutant type: ESKYGPPAPPAP), the transmembrane region sequence of CD28 (153-220aa), the intracellular segment of CD137 and the intracellular domain of CD3ζ in sequence. The two were connected by the self-cleaving peptide P2A. The nucleotide fragment of the first receptor and the nucleotide fragment of the second receptor were connected in series to obtain the PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment.

[0091] Synthesizing the encoding gene of the dual-targeting chimeric antigen receptor, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11;

[0092] Connecting the dual-targeting chimeric antigen receptor encoding gene to a lentiviral expression vector comprises the following steps:

[0093] The dual-targeting chimeric antigen receptor PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ fragment and the lentiviral vector (pCDH-CMV-MCS-P2A-copGFP-T2A-Puro) were double-digested (XbaI and XhoI), and the dual-targeting chimeric antigen receptor fragment and the lentiviral vector were connected using T4 DNA ligase. Finally, the complete lentiviral plasmid was sequenced, and the sequencing results were consistent with the expected sequences of each fragment, thereby obtaining the recombinant expression vector.

[0094] Among them, the method for obtaining the lentiviral vector can be found in the article with DOI number 10.1016 / j.molimm.2023.01.010, titled Engagement of an optimized lentiviral vector enhances the expression and cytotoxicity of CAR in human NK cells, and authored by Changjiang Guo et al.

[0095] Example 3

[0096] The preparation of highly cytotoxic anti-tumor NK cells in this embodiment includes the following steps:

[0097] (I) Lentivirus packaging

[0098] 1. Extraction of lentiviral packaging plasmid and target plasmid

[0099] 1.1 Plasmid transformation

[0100] Take stbl3 competent cells and E. coli TOP10 competent cells, add 20ng of the target plasmid to stbl3 competent cells, add 1μg of lentiviral packaging auxiliary plasmid pSPAX2 and 1μg of lentiviral packaging auxiliary plasmid PMD2G to TOP10 competent cells, mix them evenly, incubate them on ice for 30min, immediately put them in a 42℃ water bath for heat shock for 90s, and then put them in an ice bath for 2min. Then put them in a 37℃ constant temperature shaking incubator, 220rpm / min, shake and culture for 1h, take 100μL and evenly spread it on LB solid medium, and culture them in a 37℃ constant temperature incubator for 13h.

[0101] Among them, stbl3 competent cells were purchased from TransGenBiotech, catalog number L681212.

[0102] The target plasmid is: CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH.

[0103] The LB solid culture medium contains ampicillin antibiotic (Amp) at a concentration of 50 μg / mL, which is added at a volume ratio of 1:1000.

[0104] 1.2 Plasmid extraction

[0105] 1) Small extraction: Pick the monoclonal colonies from the above culture plates respectively, inoculate them into 5 mL of fresh LB medium, place them in a 37°C shaker, 220 rpm / min, and oscillate at constant temperature for 12-14 hours; wherein, the LB medium contains Amp at a concentration of 50 μg / mL, which is added at a ratio of 1:1000.

[0106] 2) Large extraction: 200 μL of the bacterial solution treated in step 1) was inoculated into 200 mL of LB medium, shaken at 37° C., 220 rpm / min, and the OD value was measured to be about 1.8; wherein the LB medium contained 200 μL of Amp.

[0107] 3) Plasmids were extracted using the OMEGA Endotoxin-free Plasmid Extraction Kit, and the plasmid concentration and purity were measured using a Nanodrop ultra-micro spectrophotometer, and then stored in a -20°C refrigerator for later use. The OMEGA Endotoxin-free Plasmid Extraction Kit was purchased from OmegaBio-Tek, USA, with the product number D6926-03, and the Nanodrop ultra-micro spectrophotometer was purchased from Thermo Electric (Shanghai) Technology Instrument Co., Ltd., USA, model: NanoDrop2000.

[0108] 2. Lentivirus packaging process

[0109] When the 293T cells reached a density of 80%-90%, fresh DMEM high-glucose medium containing 6% fetal bovine serum was used for incubation in an incubator for 1 hour, and virus packaging was performed after 1 hour.

[0110] The calcium phosphate kit was used for packaging. The packaging steps were as follows: the target plasmid CMV-PD1-DAP10-41BB-P2A-NKG2D-Ig4-CD28-41BB-CD3ζ-pCDH was mixed with two auxiliary plasmids pSPAX2 and PMD2G in a ratio of 4:3:1 to obtain a mixed plasmid, the mixed plasmid was added to the CaCl2 solution provided by the calcium phosphate kit, pipetted to mix, added dropwise to BBS at a uniform speed, pipetted to mix, incubated at room temperature for 20 minutes to obtain a mixed solution, the above mixed solution was added to 293T cells, gently mixed, placed in an incubator for 12 hours, the supernatant was removed, PBS was gently washed once, and 15 mL of fresh DMEM culture medium containing 10% FBS was replaced to collect 48h and 72h viruses respectively.

[0111] Among them, fetal bovine serum was purchased from Hyclone with the catalog number SV30265.01B, DMEM high-glucose medium was purchased from Hyclone with the catalog number WH0021D031, and calcium phosphate kit was purchased from Beyotime with the catalog number C0508.

[0112] 3. Concentration of Lentivirus

[0113] The collected virus supernatant was centrifuged to remove cell debris, PEG-8000 was added, and the mixture was centrifuged at 4°C for more than 12 h to collect virus particles. The concentrated virus particles were resuspended in pre-cooled PBS to obtain concentrated PD1-DAP10-41BB virus solution, which was stored in aliquots at -80°C to avoid repeated freezing and thawing.

[0114] 4. Determination of lentiviral titer

[0115] The qPCR lentiviral titer kit with the catalog number LV900 of abm was used for determination. The lentiviral titer obtained in this example was higher than 1×10 9 .

[0116] (II) Lentiviral transfection of NK92 cells

[0117] Take 3-5×10 5 NK92 cells / well were inoculated in a 24-well plate, and concentrated PD1-DAP10-41BB virus solution was added, MOI = 50-100, and polybrene was added to each well at a final concentration of 8 μg / mL, mixed, and placed in a 37°C incubator for culture. Centrifugation was performed after 12-15 hours, the virus supernatant was removed, and fresh culture medium was replaced and cultured. After culture, lentivirus-infected NK92 cells were obtained, which were recorded as PN-CAR-NK92. The rational design of PN-CAR-NK92 and the anti-tumor effect of modified NK cells are shown in Figure 8 shown.

[0118] Among them, polybrene was purchased from Yishen Biotechnology with the product number 40804ES76.

[0119] (III) QPCR detection of PD1 and NKG2D mRNA expression levels in cells

[0120] PN-CAR-NK92 and NK92 cells were collected 72 hours after lentivirus infection. The mRNA of both was extracted, and the mRNA levels of PD1 and NKG2D in PN-CAR-NK92 and NK92 cells were detected. The detection primers (5'-3') used were:

[0121] PD1-F: GTGTCACACAACTGCCCAAC, recorded as SEQ ID NO. 25;

[0122] PD1-R: CCGCAGGCTCTCTTTGATCT, recorded as SEQ ID NO. 26;

[0123] NKG2D-F: GAGTGATTTTTCAACACGATGGC, recorded as SEQ ID NO. 27;

[0124] NKG2D-R: ACAGTAACTTTCGGTCAAGGGAA, recorded as SEQ ID NO. 28;

[0125] GAPDH-F: GAGGACCTGACCTGCCGTCT, recorded as SEQ ID NO. 29;

[0126] GAPDH-R: GGAGGAGTGGGTGTCGCTGT, recorded as SEQ ID NO. 30.

[0127] Test results such as Figure 2As shown in the figure, it can be seen that the mRNA expression level of PD1 / NKG2D in PN-CAR-NK92 cells is greatly improved compared with that in NK92 cells.

[0128] Example 4

[0129] Flow cytometry was used to detect the expression of target genes PD1 and NKG2D

[0130] PN-CAR-NK92 and NK92 cells were obtained after 72 hours of lentivirus infection. Centrifuged at 300 g for 5 min and counted 1×10 cells. 6 , wash 1-2 times with PBS containing 1% FBS, hereinafter referred to as washbuffer, add APC-labeled anti-human PD1 flow cytometry antibody and BV421-labeled anti-human NKG2D flow cytometry antibody in 100 μL system, incubate on ice in dark for 30 minutes, wash 2-3 times with washbuffer. Finally, add 400 μL PBS solution to resuspend the cells, and perform detection and analysis by flow cytometer.

[0131] Among them, PD1 flow cytometry antibody was purchased from BD Company with catalog number 320822, and the flow cytometer model was BD FACSCantoII.

[0132] PD1 test results Figure 3 As shown, it can be seen that the expression rate of PD1 in PN-CAR-NK92 is about 94.3%, and the expression rate of NKG2D is about 98.1%, which is greatly improved compared with NK92 cells.

[0133] Example 5

[0134] Flow cytometry was used to detect the expression of PD-L1 in mouse breast cancer cells EMT6-PD-L1, human gastric cancer cells MGC-803, human gastric cancer cells KATO-III and human osteosarcoma cells U2OS, and the expression of MICA and MICB in NKG2DL

[0135] PD-L1 is expressed, but MICA / MICB are not expressed in mouse breast cancer cells EMT6-PD-L1; PD-L1 is not expressed, but MICA / MICB are expressed in human gastric cancer cells MGC-803; PD-L1 is not expressed, but MICA / MICB are not expressed in human gastric cancer cells KATO-III; PD-L1 is expressed, but MICA / MICB are expressed in human osteosarcoma cells U2OS.

[0136] Mouse breast cancer cells EMT6-PD-L1, human gastric cancer cells MGC-803, human gastric cancer cells KATO-III, and human osteosarcoma cells U2OS in the logarithmic growth phase were centrifuged at 300 g for 5 min and the cells were counted to 1×106 , wash 1-2 times with PBS containing 1% FBS, hereinafter referred to as washbuffer, add APC-labeled anti-human PD-L1 flow cytometry antibody and PE-labeled anti-human MICA / B flow cytometry antibody in 100 μL system, incubate on ice in the dark for 30 minutes, wash 2-3 times with washbuffer. Finally, add 400 μL PBS solution to resuspend the cells and perform detection and analysis by flow cytometer.

[0137] Among them, PD-L1 flow cytometry antibody was purchased from BD with the catalog number 2460257, MICA / B flow cytometry antibody was purchased from BD with the catalog number 320906, and the flow cytometer model was BD FACSCantoII.

[0138] PD-L1 test results Figure 4 As shown, in EMT6-PD-L1 cells, PD-L1 is expressed at 98.1%, while MICA / MICB is basically not expressed; in MGC-803 cells, PD-L1 is basically not expressed, while MICA / MICB is expressed at 98.6%; in KATO-III cells, PD-L1 and MICA / MICB are basically not expressed; in U2OS cells, PD-L1 is expressed at about 84.5%, and MICA / MICB is expressed at about 97.6%. Figure 6 .

[0139] Example 6

[0140] PN-CAR-NK92 in vitro cytotoxicity assay

[0141] 1. Cell Preparation

[0142] EMT6-PD-L1, MGC-803, KATO-III and U2OS cells in the logarithmic growth phase after the transfer of luciferase were taken and inoculated into 96-well plates to ensure that 1×10 4 Each cell type was cultured in 3 replicate wells in a 5% CO2, 37°C incubator overnight.

[0143] 2. Experimental design

[0144] The experimental groups were set up into 2 groups, each group was based on different effector cells, namely PD1-CAR-NK92, NKG2D-CAR-NK92, PN-CAR-NK92 cells and NK92, with an effector-target ratio of 5:1. At the same time, 3 groups of controls were set up, including target cell control wells, sample maximum enzyme activity control wells, and co-incubation wells, with a total volume of 100 μL per well. Among them, the sample maximum enzyme activity control wells were target cell wells that were not treated with effector cells for subsequent lysis.

[0145] 3. Luciferase Assay

[0146] This experiment uses ONE-GloTM LuciferaseAssay System Promega for detection. Effector cells and target cells are placed in a 5% CO2, 37°C incubator and co-incubated for 5 hours. One hour before the scheduled detection point, take out the 96-well plate from the cell incubator and add Lysis Solution (10×) to the "sample maximum enzyme activity control well" in an amount of 10% of the original culture solution volume. After adding Lysis Solution, repeatedly blow and mix, and then continue to culture in the incubator. After the co-incubation time is reached, add the detection solution and detect on the microplate reader.

[0147] Cytotoxicity (%) = (RLU min-RLU sample) / (RLU min-RLU max)×100.

[0148] The calculated results showed that for EMT6-PD-L1 cells, the killing effects of single-module PD1-CAR-NK92 and dual-module were significantly higher than those of the control NK92 group, and NKG2D-CAR-NK92 cells were basically not killed; for MGC-803 cells, the killing effects of single-module NKG2D-CAR-NK92 and dual-module were significantly higher than those of the control NK92 group, and PD1-CAR-NK92 cells were basically not killed; for U2OS cells, at a ratio of 5:1, the cytotoxicity of PN-CAR-NK92 was even stronger than that of NK92, and the toxicity increased by about 6 times. For KATO-III cells that do not express PD-L1 and MICA / MICB, the cytotoxicity did not change. Figure 5 and Figure 7 .

[0149] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0150] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0151] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A dual-targeting chimeric antigen receptor, characterized in that: It is composed of the PD1-DAP10-CD137 receptor and the NKG2D-IgG4-CD28-CD137-CD3ζ receptor in sequence; The PD1-DAP10-CD137 receptor is composed of the CD8α-C1 signal peptide, the Myc tag, the PD1 extracellular segment, the DAP10 extracellular transmembrane and intracellular segments, and the CD137-C1 intracellular segment. The NKG2D-IgG4-CD28-CD137-CD3ζ receptor is composed of CD8α-C2 signal peptide, FLAG tag, NKG2D ligand binding domain, IgG4 monomer hinge region, CD28 transmembrane and intracellular region, CD137-C2 intracellular segment, and CD3ζ intracellular domain in sequence; The amino acid sequence of the CD8α-C1 signal peptide is shown in SEQ ID NO.1; The amino acid sequence of the Myc tag is shown in SEQ ID NO.2; The amino acid sequence of the extracellular segment of PD1 is shown in SEQ ID NO.3; The amino acid sequence of the extracellular transmembrane and intracellular segments of DAP10 is shown in SEQ ID NO.4; The amino acid sequence of the intracellular segment of CD137-C1 is shown in SEQ ID NO.5; The amino acid sequence of the CD8α-C2 signal peptide is shown in SEQ ID NO.1; The amino acid sequence of the FLAG tag is shown in SEQ ID NO.6; The amino acid sequence of the NKG2D ligand binding domain is shown in SEQ ID NO.7; The amino acid sequence of the hinge region of the IgG4 monomer is shown in SEQ ID NO.8; The amino acid sequence of the transmembrane and intracellular regions of CD28 is shown in SEQ ID NO.9; The amino acid sequence of the intracellular segment of CD137-C2 is shown in SEQ ID NO.5; The amino acid sequence of the CD3ζ intracellular domain is shown in SEQ ID NO.

10.

2. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that The amino acid sequence of the dual-targeting chimeric antigen receptor is shown in SEQ ID NO.

12.

3. The dual-targeting chimeric antigen receptor according to claim 1, characterized in that The nucleotide sequence of the gene encoding the dual-targeting chimeric antigen receptor is shown in SEQ ID NO.

11.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene described in claim 2.

5. A method for preparing the recombinant expression vector according to claim 4, characterized in that: The steps include: synthesizing the encoding gene of the dual-targeting chimeric antigen receptor; The coding gene is connected to a lentiviral expression vector to obtain the recombinant expression vector. The lentiviral expression vector is pCDH-CMV-MCS-P2A-copGFP-T2A-Puro, which is obtained by optimizing pCDH-CMV-MCS-EF1-copGFP-T2A-Puro.

6. An anti-tumor natural killer cell, characterized in that: The anti-tumor natural killer cells are obtained by infecting the natural killer cells to be transformed with the recombinant expression vector described in claim 4.

7. A method for preparing the anti-tumor natural killer cells according to claim 6, characterized in that: The steps include: Using auxiliary plasmids pSPAX2 and PMD2G to package the recombinant expression vector to express a virus carrying the encoding gene; The virus carrying the coding gene is used to infect NK92 cells to obtain the anti-tumor natural killer cells.

8. Use of the dual-targeting chimeric antigen receptor according to claim 1, the recombinant expression vector according to claim 4, or the anti-tumor natural killer cell according to claim 6 in the preparation of an anti-tumor drug.

9. The use according to claim 8, characterized in that: The anti-tumor drug has the dual-targeting chimeric antigen receptor, the recombinant expression vector or the anti-tumor natural killer cell as the only active ingredient.

10. The use according to claim 9, characterized in that: The tumor is any one of breast cancer, gastric cancer, and osteosarcoma.

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