A recombinant CAR19-IL24 gene, a lentivirus vector, a CAR19-IL24-T cell and application

Abstract

The application discloses a recombinant CAR19-IL24 gene, a lentivirus vector, CAR19-IL24-T cells and application, and belongs to the tumor cell immunological technical field. The CAR19-IL24-T cells can be effectively prepared by using the application, the CAR19-IL24-T cells express a chimeric antigen receptor and interleukin 24 which are targeted to tumor antigens, and have good tumor killing capacity. The CAR19-IL24-T cells are safe, specific and effective CART cells, and have important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of tumor cell immunotherapy technology, specifically relating to a recombinant CAR19-IL24 gene, a lentiviral vector, CAR19-IL24-T cells, and their applications. Background Technology

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T cell immunotherapy) is a novel immunotherapy that utilizes chimeric antigen receptor T cells. CAR-T cells are T cells modified to express chimeric antigen receptor (CAR) molecules on their surface. The CAR molecules expressed on the T cell membrane "redirect" the T cells, endowing them with the ability to specifically recognize and kill target cells expressing CAR molecules, thereby specifically eliminating tumor target cells in the patient's body. A chimeric antigen receptor (CAR) generally includes an antigen-binding domain (usually derived from the scFv segment of the antibody-antigen binding region) that recognizes an antibody that recognizes a specific tumor antigen, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction region (usually a co-stimulatory signaling domain and a CD3ζ intracellular region). These components are coupled together in a specific order to form a chimeric antigen receptor (CAR).

[0003] In 1993, Israeli scientist Zelig Eshhar first proposed the concept of CAR-T therapy. In 2011, Professor Carl H. June's team at the University of Pennsylvania achieved a breakthrough in treating a patient with chronic lymphocytic leukemia (CLL) using CAR-T therapy. In 2012, the team successfully used CAR-T cells to treat Emily, a patient with acute lymphoblastic leukemia (ALL), who has remained relapse-free to date. In 2013, *Science* magazine ranked CAR-T cell immunotherapy as the top scientific breakthrough worldwide. By 2017, two CAR-T products had been approved by the US FDA for clinical use, and corresponding CAR-T products in China are currently undergoing clinical approval.

[0004] CAR-T cell immunotherapy has become a breakthrough approach for treating specific human tumors. The therapeutic effect of CAR-T cell immunotherapy is closely related to the design of the recombinant CAR molecule, which determines the type of tumor the CAR-T cells can target and the effectiveness of CAR-T cell therapy. Currently, most reported CARs are second- or third-generation designs, and their efficacy still needs improvement. Further optimization or improvement of CAR design is key to enhancing the efficacy of CAR-T therapy. Studies have reported that combining cytokines IL12, IL15, IL18, and IL33 can enhance the therapeutic effect of CAR-T, suggesting that other interleukins may also enhance the therapeutic effect of CAR-T. Currently, there are no CAR-T cell products that secrete IL-24. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and its application.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0007] A recombinant CAR19-IL24 gene, the sequence of which is shown in SEQ ID NO.1.

[0008] A lentiviral vector carrying the above-mentioned recombinant CAR19-IL24 gene, the lentiviral vector sequence of which is shown in SEQ ID NO.2.

[0009] A T cell expressing the recombinant CAR19-IL24 gene, wherein the T cell expressing the recombinant CAR19-IL24 gene is prepared by transducing the above-mentioned lentiviral vector into T cells.

[0010] A type of T cell that co-expresses CAR and IL24.

[0011] Preferably, the T cells are prepared by linking the expression genes of CAR and IL24 in the same expression cassette of the same vector, and then transducing the vector into T cells.

[0012] Preferably, the T cells are prepared by constructing the CAR expression gene and IL24 in different expression frames of the same vector, and then transducing the vector into T cells.

[0013] The present invention will be further described below:

[0014] The recombinant CAR19-IL24 gene of this invention combines interleukin-24 (IL-24) and a chimeric antigen receptor (CAR), comprising a nucleic acid sequence encoding the following portions: an extracellular region containing an antigen-binding portion that specifically recognizes tumor antigens; a transmembrane portion; and intracellular regions containing CD28 cytoplasmic functional regions, 41BB cytoplasmic functional regions, and CD3zeta cytoplasmic functional regions, which can be linked in any order, and autocrine IL-24. Transfection of T cells using the recombinant CAR19-IL24 gene and a vector containing this gene is highly efficient, and the transfected positive T cells exhibit good proliferative capacity. Furthermore, taking CAR19.IL-24, which specifically recognizes CD19 molecules, as an example, the recombinant CAR19.IL24 gene of this invention has high specificity for the B-cell tumor antigen CD19, prompting CAR19.IL24-T cells to specifically kill CD19+ B-cell tumors.

[0015] Preliminary experimental results and analysis of this invention indicate that IL-24 expression enhances the therapeutic potential of CAR-T therapy. On one hand, IL-24 can enhance T cell proliferation and activation. T cell proliferation and activation potential is one of the key factors affecting the therapeutic effect of CAR-T products. T cells with stronger proliferation and activation potential are more likely to proliferate in large quantities after in vitro culture and reinfusion into patients, and are more conducive to maintaining long-term anti-tumor activity in vivo. On the other hand, IL-24 exhibits anti-tumor effects against various tumor cells, such as inducing tumor-specific apoptosis, inhibiting tumor angiogenesis, and suppressing tumor cell invasion and metastasis. Furthermore, the IL-24 molecule is relatively small, making it easier to add an IL-24 expression cassette to the CAR sequence. Based on the above experimental and analytical results, this invention proposes for the first time an enhanced CAR19 structure design incorporating IL-24 (CAR19-IL24), and constructs a lentiviral vector LV-CAR19-IL24 carrying this sequence. Subsequently, CAR-T cells expressing the recombinant CAR19-IL24 gene were prepared. The aim is to demonstrate the effectiveness and safety of this CAR19-IL24 design, laying the foundation for the clinical application of CAR19-IL24-T cells.

[0016] Taking CAR19.IL-24, which specifically recognizes the CD19 molecule, as an example:

[0017] 1) Based on existing reports and bioinformatics methods, this invention first identifies the CAR19, T2A, and IL24 CDS sequences that specifically recognize CD19 molecules, and then synthesizes the CAR sequence and CAR19-IL24 sequence through gene synthesis.

[0018] 2) The CAR19 and CAR19-IL24 sequences were ligated into the lentiviral vector Addgene: #12252 using molecular cloning technology. The two sequences were linked via T2A (the DNA sequences mentioned above are not yet included in the appendix).

[0019] 3) Peripheral blood from normal volunteers was collected by density gradient centrifugation to separate mononuclear cells (PBMCs), and then negatively sorted to obtain total T cells (PanT cells). These cells were then stimulated with CD3 / 28 magnetic beads and supplemented with IL-2 for in vitro expansion.

[0020] 4) Package lentivirus carrying the recombinant CAR19-IL24 gene, transduce PanT cells stimulated with CD3 / 28 magnetic beads for 24 hours, and prepare enhanced CARART cells expressing the recombinant CAR19-IL24 gene.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) Sequencing confirmed that none of the constructed lentiviral vector sequence elements had mutations.

[0023] 2) IL24-enhanced CAR19.IL-24-T cells prepared using lentiviral vectors exhibited higher antitumor activity compared to CAR19-T cells. After lentiviral transduction, T cells were identified by PCR to confirm successful integration of the CAR sequence, and the successful expression of the CAR molecule was confirmed by flow cytometry, Western blot, and Q-PCR.

[0024] 3) Cell proliferation capacity assays confirmed that CAR19.IL-24-T cells have a stronger proliferative capacity.

[0025] 4) In vitro co-culture cytotoxicity assays confirmed that CAR19.IL-24-T cells exhibited a stronger tumor-killing effect. Furthermore, based on the principles of this invention, different combinations of the extracellular-intracellular domains of CAR with IL-24 can be selected to adapt to different tumor cell types. Different linker elements can be selected to construct IL-24 and CAR molecules in different sequences within the same expression cassette. IL-24 and CAR can be expressed separately in different expression cassettes on T cells.

[0026] In summary, this invention combines interleukin-24 and chimeric antigen receptors to achieve better therapeutic effects in tumor immunotherapy. This invention can effectively prepare CAR19-IL24-T cells, which express chimeric antigen receptors targeting tumor antigens and interleukin-24. Compared with CD19-CART cells, CAR19-IL24-T cells have stronger tumor-killing function and better proliferation capacity. Furthermore, it has been demonstrated that CAR19-IL24-T cells are a safe, specific, and effective CART cell line with significant clinical application value. Attached Figure Description

[0027] Figure 1 Schematic diagram of CAR19 and CAR19-IL24;

[0028] Figure 2 A map showing the intracellular protein expression levels of CAR19 and CAR19-IL24 using Western Blot;

[0029] Figure 3 To observe GFP expression in CAR19-IL24-T cells using an inverted fluorescence microscope;

[0030] Figure 4 To detect CAR19 molecules on the surfaces of CAR19-T and CAR19-IL24-T using flow cytometry;

[0031] Figure 5 The figure shows the functional assay results of CAR19.IL-24-T cells; in the figure, E: utility cells, namely CAR19-IL24-T cells transduced by LV-CAR19-IL24, CAR19-T cells transduced by LV-CAR19, and T cells transduced by LV-GFP from the same batch; T: target cells, which are CD19-positive Raji cells; the apoptosis of CD19-positive cells (i.e., Raji cells) was detected using Annexin-V APC+, and the vertical axis represents the apoptosis rate of target cells;

[0032] Figure 6 Figure showing the amplification of IL-24 from an existing plasmid containing the IL-24 sequence using PCR;

[0033] Figure 7 This is a diagram showing the conditions for bacterial culture PCR identification. Detailed Implementation

[0034] 1. Construction of CAR19 expression cassette and lentiviral vector

[0035] (1) Based on existing reports and bioinformatics methods, the FMC63scFv sequence that specifically recognizes CD19 molecules was found. The transmembrane region and intracellular signal transduction region sequences were designed and the gene was synthesized (CAR19). Bbs1 restriction sites were designed at both ends of the CAR19 sequence. This sequence was loaded into the pUC57 plasmid (pUC57-CAR19).

[0036] (2) pUC57-CAR19 was digested with BbsI to generate BamH1 and AgeI sticky ends at the 5' and 3' ends of the CAR19 sequence, respectively. Simultaneously, the second-generation lentiviral vector plasmid pHQ-009 was digested with both BamH1 and AgeI. (Enzyme digestion system attached)

[0037] (3) The pHQ-009 backbone (7379 bp) and CAR19 sequence (1534 bp) were recovered from the gel. Plasmid pHQ-010 was constructed using T4 DNA Ligase ligation. Ligation time was no more than 10 min at room temperature (25℃) or overnight in a water bath at 16℃. (Ligation system attached)

[0038] (4) Take 5 μl of the ligation product and convert it using Stbl3. (Stbl3 conversion system attached)

[0039] (5) After 14 hours, select single clones for bacterial culture PCR identification. (See attached bacterial culture PCR system and conditions)

[0040] (6) Collect PCR-positive clones from bacterial culture for Sanger sequencing identification.

[0041] (7) Take the clone whose Sanger sequencing sequence is consistent with the theoretical sequence and perform plasmid extraction pHQ-010, which is a second-generation lentiviral vector plasmid containing the CAR19 sequence.

[0042] 2. Construction of the CAR19.IL-24 expression cassette and lentiviral vector

[0043] (1) IL-24 was amplified from an existing plasmid containing the IL-24 sequence using PCR, with XmaI and HindIII restriction sites introduced using upstream and downstream primers, respectively. (See Table 1 for the plasmid PCR system and conditions; see [link to table 1] for details on amplifying IL-24 from an existing plasmid containing the IL-24 sequence using PCR.) Figure 6 )

[0044] Table 1

[0045]

[0046] (2) The PCR product was purified by gel extraction (723 bp). It was ligated into the pGEM-T-easy vector using T4 DNA Ligase. The amount added to the ligation system was estimated based on the OD value, backbone, and fragment length, with the insert-to-backbone molar ratio between 3:1 and 10:1 (see Table 2).

[0047] Table 2

[0048]

[0049] (3) Take 5 μl of the ligation product, convert it with DH5α, and perform "blue-white" screening.

[0050] Dissolve DH5α competent cells in an ice bath for 5 minutes.

[0051] Add 5 μl of ligation product / 100 μl of competent cells to it.

[0052] Let it stand in ice water for 30 minutes.

[0053] Water bath at 42℃ for 90 seconds. Do not shake.

[0054] Quickly transfer to ice water to cool for 3 minutes.

[0055] Add 900 μL of culture medium without Amp resistance and place in a shaker at 37°C at 150 rpm for 45 min.

[0056] Prepare the plates for blue-white screening (ampicillin resistance plates containing IPTG / X-Gal): Spread 10 μl of 100 mM IPTG and 40 μl of 20 mg / ml X-gal onto the surface of the ampicillin resistance plate, and incubate at room temperature in the dark for 30 minutes to allow absorption. Take an appropriate amount of the bacterial suspension from f) and spread it onto the plate from g), place it in a 37°C incubator, and invert it for 14 hours.

[0057] (4) Select white clones for Sanger sequencing verification. Select clones whose Sanger sequencing sequences are consistent with the theoretical sequences for plasmid uptake using pHQ-040.

[0058] (5) pHQ-040 was digested with Xma1 and HindIII; pUC57-CAR19 was digested with Age1 and HindIII. Prepare the pHQ-040 reaction system according to the table below (see Table 3):

[0059] Table 3

[0060]

[0061] Prepare the pUC57-CAR19 reaction system according to the table below (Table 4):

[0062] Table 4

[0063]

[0064] (6) The IL-24 sequence (723 bp) and pUC57-CAR19 backbone (4290 bp) were recovered from pHQ-040 using a gel extraction method. Plasmid pHQ-036 was constructed using T4 DNA Ligase. Ligation time was no more than 10 min at room temperature (25°C) or overnight in a water bath at 16°C. (Ligation system attached)

[0065] The amount added to the linker system was estimated based on the OD value, backbone, and fragment length, and the system was prepared with a molar ratio of insert fragment to backbone between 3:1 and 10:1 (Table 5).

[0066] Table 5

[0067]

[0068] (7) Take 5 μl of the ligation product and convert it using DH5α. (DH5α conversion system attached)

[0069] a) Dissolve DH5α competent cells in an ice bath for 5 minutes.

[0070] b) Add 5 μl of ligation product / 100 μl of competent cells to it.

[0071] c) Let stand in ice water for 30 minutes.

[0072] d) Water bath at 42℃ for 90 seconds. Do not shake.

[0073] e) Quickly transfer to ice water to cool for 3 minutes.

[0074] f) Add 900 μL of culture medium without Amp resistance and place in a shaker at 37°C at 150 rpm for 45 min.

[0075] g) Take an appropriate amount of the bacterial suspension from f) and spread it onto an Amp-resistant plate. Place it in a 37°C incubator and invert it for 14 hours.

[0076] (8) After 14 hours, select single clones for bacterial culture PCR identification. (See attached bacterial culture PCR system and conditions; PCR conditions diagram is attached.) Figure 7 )

[0077] Single clones were picked up with a sterile tip and transferred to 300 μl LB medium, then placed in a shaker at 37°C and 150 rpm for 45 min before being used for the following PCR (Table 6):

[0078] Table 6

[0079]

[0080] (9) Collect PCR-positive clones from bacterial culture for Sanger sequencing identification.

[0081] (10) Take clones whose sequences are consistent with the theoretical sequences identified by Sanger sequencing and perform plasmid extraction pHQ-036.

[0082] (11) pHQ-036 was digested with Bbs1 and the CAR19.IL-24 sequence of 2215 bp was recovered by gel digestion; at the same time, the second-generation lentiviral vector plasmid pHQ-009 was digested with BamH1 and Age1 and the pHQ-009 backbone of 7379 bp was recovered by gel digestion; plasmid pHQ-035 was constructed by ligation with T4 DNA Ligase, which is a second-generation lentiviral sequence containing the CAR19.IL-24 sequence.

[0083] 3. Preparation of CAR 19 and CAR19.IL-24 lentiviruses (see Table 7):

[0084] Table 7

[0085]

[0086] 4. Preparation of CAR 19-T cells and CAR19.IL-24-T cells

[0087] (1) Peripheral blood density gradient centrifugation was used to separate mononuclear cells (PBMCs) from normal volunteers, and then negative sorting was used to obtain total T cells (PanT cells). The cells were then stimulated with CD3 / 28 magnetic beads and IL-2 was added for in vitro expansion.

[0088] (2) CAR19 and CAR19.IL-24 lentiviruses were transduced into PanT cells stimulated with CD3 / 28 magnetic beads for 24 hours to prepare CAR19-T cells and enhanced CARART cells expressing recombinant CAR19.IL-24 gene.

[0089] 5. Detection of CAR19.IL-24-T cells

[0090] CAR expression level detection

[0091] See Figure 2 The arrows indicate the location of the CAR19 and IL24 proteins, respectively. The binding protein marker 26616 (ThermoFisher) indicates that the intracellular expression of CAR19 and CAR19-IL24 matches the expected sizes of the CAR19 and IL24 proteins.

[0092] See Figure 3After T cells were transduced using the lentiviral vector LV-CAR19-IL24, the GFP carried by it was expressed within the cells. The left image is a bright-field image, and the right image is a fluorescence image from the same field of view. This indicates that the T cells transduced with the lentiviral vector LV-CAR19-IL24 expressed GFP, indicating successful viral transduction and the expression of CAR19-IL24.

[0093] See Figure 4 After transducing T cells using the lentiviral vectors LV-CAR19 and LV-CAR19-IL24, the CAR19 molecules carried by these vectors are expressed on the surface of the T cells. Using APC-conjugated antibodies to detect CAR19 molecules, the M1 region in the figure shows a clear positive signal, indicating a high expression level of CAR19 molecules on the surface of T cells. The left figure shows that 55.96% of T cells expressed CAR19 molecules after transduction with the lentiviral vector LV-CAR19, and the right figure shows that 63.8% of T cells expressed CAR19 molecules after transduction with LV-CAR19-IL24.

[0094] 6. Functional detection of CAR19-IL-24-T cells

[0095] Cytotoxicity detection

[0096] Figure 5 The results showed that when the E:T cell ratio was 5-8, both CAR19-IL24-T cells and CAR19-T cells could promote apoptosis of target cells. More importantly, the apoptosis-promoting effect of CAR19-IL24-T cells was significantly better than that of CAR19-T cells. sequence list <110> central south university <120> A recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cells and their applications <130> MP22013838F <150> CN201811517078.5 <151> 2018-12-12 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 2947 <212> DNA <213> Artificial sequence <220> <223> Recombinant CAR19-IL24 gene <400> 1 gatcgccacc atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacaccagc 60 attcctcctg atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct 120 gggagacaga gtcaccatca gttgcagggc aagtcaggac attagtaat atttaaattg 180 gtatcagcag aaaccagatg gaactgttaa actcctgatc taccatacat cagattaca 240 ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat 300 tagcaacctg gagcaagaag atattgccac ttacttttgc caacaggta atacgcttcc 360 gtacacgttc ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa 420 gcccggatct ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg 480 cctggtggcg ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc 540 cgactatggt gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt 600 aatatggggt agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat 660 caaggacaac tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac 720 agccatttac tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg 780 gggtcaagga acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc 840 900 ttgtccaagt cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg 960 gggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag 1020 gagtaagagg agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg 1080 gcccacccgc aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc 1140 1200 1260 ccgggaccct gagatggggg gaaagccgag aaagaac cctcaggaag gcctgtacaa 1320 tgaactgcag aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg 1380 ccggaggggc aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac 1440 ctacgacgcc cttcacatgc aggccctgcc ccctcgctat gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaccgggt atgaatttc aacagaggct gcaaagcctg tggactttag ccagcagacc cttctgccct cctttgctgg cgacagcctc 1620 tcaaatgcag atggttgtgc tcccttgcct gggttttacc ctgcttctct ggagccaggt 1680. atcaggggcc cagggccaag aattccactt tgggccctgc caagtgaagg gggttgttcc 1740. ccagaaactg tgggaagcct tctgggctgt gaaagacact atgcaagctc aggataacat cacgagtgcc cggctgctgc agcaggaggt tctgcagaac gtctcggatg ctgagagctg ttaccttgtc cacaccctgc tggagttcta cttgaaaact gttttcaaaa actaccacaa 1980. 1980. 1980. 1980. 1980. 1980. 1980. 1980. 1980 catcgtgtca caactgcaac ccagtcaaga aaatgagatg ttttccatca gagacagtgc acacaggcgg tttctgctat tccggagagc attcaaacag ttggacgtag aagcagctct gaccaaagcc cttggggaag tggacattct tctgacctgg atgcagaaat tctacaagct cgagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt 2220 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 2280 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 2340 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 2400 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 2460 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 2520 catcttcttc areacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 2580 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggagg gcaacatcct 2640 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 2700 gaaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 2760 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 2820 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 2880 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 2940 caagtaa 2947 <210> 2 <211> 9594 <212> DNA <213> Artificial sequence <220> <223> Lentiviral vector sequence <400> 2 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag 60 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg 120 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact 180 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct 240 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 300 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 360 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 420 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 480 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 540 ttttgactag cggaggctag aaggagag atgggtgcga gagcgtcagt attaagcggg 600 ggagaattag atcgcgatgg gaaaaaatttc ggttaaggcc aggggaag aaaaatata 660 aattaaaaca tatagtatgg gcaagcagggg agctagaacg attcgcagtt aatcctggcc 720 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 780 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 840 aaaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaaaaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg has 900 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatg 960 agggacaatt gagaagtga attaataa tataaagtag taaaaattga accattagga 1020 gtagcaccca ccaaggcaaa gagagagtg gtgcagagag aaaaaagagc agtggaata 1080 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatggggcgc agcctcaatg 1140 acgctgacgg tacaggccag acaattattg tctgtatag tgcagcagca gaacaatttg 1200 ctgagggcta tgaggcgca acaccatctg ttgcactca cagtctgggg catcaagcag 1260 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1320 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1380 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1440 aacaattaca caagcttaat acactcctta attgaagaat cgcaaacca gcaagaaaag 1500 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1560 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1620 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1680 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1740 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1800 cggttaactt ttaaaaagaaa aggggggatt ggggggtaca gtgcaggggga aagaatagta 1860 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 1920 aattttatcg atcacgagac tagcctcgag aagcttgata tcgaattcca cggggttggg 1980 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 2040 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag 2100 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 2160 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 2220 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 2280 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga 2340 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg 2400 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg 2460 aatcaccgac ctctctcccc agggggatcg ccaccatgct tctcctggtg acaagccttc 2520 tgctctgtga gttaccacac ccagcattcc tcctgatccc agacatccag atgacacaga 2580 ctacatcctc cctgtctgcc tctctgggag acagagtcac catcagttgc agggcaagtc 2640 aggacattag taaatattta aattggtatc agcagaaacc agatggaact gttaaactcc 2700 tgatctacca tacatcaaga ttacactcag gagtcccatc aaggttcagt ggcagtgggt 2760 ctggaacaga ttattctctc accattagca acctggagca agaagatatt gccacttact 2820 tttgccaaca gggtaatacg cttccgtaca cgttcggagg ggggactaag ttggaaataa 2880 caggctccac ctctggatcc ggcaagcccg gatctggcga gggatccacc aagggcgagg 2940 tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg tccgtcacat 3000 gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc cagcctccac 3060 gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac tataattcag 3120 ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt ttcttaaaaa 3180 tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat tattactacg 3240 gtggtagcta tgctatggac tactggggtc aaggaacctc agtcaccgtc tcctcagcgg 3300 ccgcaattga agttatgtat cctcctcctt acctagacaa tgagaagagc aatggaacca 3360 ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga ccttctaagc 3420 ccttttgggt gctggtggtg gttggggg tcctggctg ctatagcttg ctagtacag 3480 tggcctttat tattctctg gtgaggagta agaggagcag gctcctgcac agtgactaca 3540 tgaacatgac tccccgccgc cccggccca cccgcaagca ttaccagccc tatgccccac 3600 cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca gacgcccccg 3660 cgtaccagca gggccagaac cagctctata acgagctca tctaggacga aggaggagt 3720 acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag ccgagaagga 3780 agaaccctca ggaggcctg tacaatgaac tgcagaaaga gagatggcg gaggcctaca 3840 gtgagattgg gatgaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg 3900 gtctcagtac agccaccaag gataccg acgccctca catgcaggcc ctgccccctc 3960 gctatgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac 4020 cgggtatgaa tttcacag aggctgcaaa gcctgtggac tttagccagc agaccctct 4080 gccctccttt gctggcgaca gccctcaa tgcagatggt tgtgctccct tgcctggtt 4140 ttaccctgct tctctggagc caggtatcag gggcccaggg ccaagaattc cactttgggc 4200 cctgccaagt gaagggggtt gttccccaga aactgtggga agccttctgg gctgtgaaag 4260 acactatgca agctcaggat aacatcacga gtgcccggct gctgcagcag gaggttctgc 4320 agaacgtctc ggatgctgag agctgttacc ttgtccacac cctgctggag ttctacttga 4380 aaactgtttt caaaaactac cacaatagaa cagttgaagt caggactctg aagtcattct 4440 ctactctggc caacaacttt gttctcatcg tgtcacaact gcaacccagt caagaaaatg 4500 agatgttttc catcagagac agtgcacaca ggcggtttct gctattccgg agagcattca 4560 aacagttgga cgtagaagca gctctgacca aagcccttgg ggaagtggac attcttctga 4620 cctggatgca gaaattctac aagctcgagg gcagaggaag tctgctaaca tgcggtgacg 4680 tcgaggagaa tcctggccca ccggtcgcca ccatggtgag caagggcgag gagctgttca 4740 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 4800 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 4860 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 4920 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 4980 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 5040 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 5100 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 5160 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 5220 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 5280 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 5340 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 5400 tcactctcgg catggacgag ctgtacaagt aaagcggccg cgtcgacaat caacctctgg 5460 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 5520 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 5580 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 5640 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 5700 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 5760 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 5820 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 5880 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 5940 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 6000 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattcgagct cggtaccttt 6060 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 6120 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc 6180 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 6240 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 6300 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag 6360 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 6420 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 6480 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 6540 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccag ttccgcccat 6600 tctccgcccc atggctgact aattttttt atttatgcag aggccgaggc cgcctcggcc 6660 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcgtcgag 6720 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac 6780 aacgtcgtga ctgggaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 6840 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 6900 gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg 6960 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 7020 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 7080 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 7140 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 7200 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 7260 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 7320 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 7380 cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 7440 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 7500 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 7560 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 7620 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 7680 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 7740 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 7800 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 7860 agtaagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 7920 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 7980 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 8040 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 8100 acttactcta gcttccccggc aacaattaat agactggatg gaggcggata aagttgcagg 8160 accacttctg cgctcggccc ttccggctgg ctggtttat gctgataaat ctggagccgg 8220 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 8280 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaata gacagatcgc 8340 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 8400 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatccttt 8460 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 8520 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 8580 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 8640 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 8700 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 8760 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 8820 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 8880 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 8940 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 9000 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 9060 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 9120 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 9180 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 9240 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 9300 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 9360 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 9420 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 9480 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 9540 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgca 9594

Claims

1. A recombinant CAR19-IL24 gene combining interleukin-24 (IL-24) and chimeric antigen receptor (CAR) for the preparation of a drug for the specific killing of CD19. + The use of the recombinant CAR19-IL24 gene in a drug for B-cell tumors, wherein the sequence of the recombinant CAR19-IL24 gene is shown in SEQ ID NO.1.

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