Chimeric antigen receptor double-negative T cell medicine for co-expressing CXCR5 and IL-7 and application of chimeric antigen receptor double-negative T cell medicine

Abstract

The invention discloses a chimeric antigen receptor double-negative T cell drug co-expressing CXCR5 and IL-7 and application thereof, the cell drug can co-express CXCR5 and IL-7, and the cell drug comprises EF1-alpha, a CD8 alpha signal peptide, an NKG2D extracellular domain, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a costimulatory factor 4-1BB, a CD3 zeta intracellular signal region, T2A, a chemotactic factor receptor CXCR5, P2A and interleukin IL-7 which are connected in sequence; according to the chimeric antigen receptor double-negative T cell drug co-expressing the CXCR5 and the IL-7 and the application of the chimeric antigen receptor double-negative T cell drug, the CXCR5 and the IL-7 are co-expressed on CAR-DNT cells of targeted NKG2DL, so that the migration and infiltration capacity of the drug to a tumor site is enhanced, the survival and proliferation capacity of the CAR-T cells is improved, the killing effect of the CAR-DNT cells on tumors is improved, and the drug has a good application prospect. The purpose of treating tumors is achieved, and allogenic treatment can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a chimeric antigen receptor double-negative T cell drug co-expressing CXCR5 and IL-7 and uses thereof. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy refers to the modification of T cells in vitro through genetic engineering, and the return of the modified T cells to the patient's body, so that they can directly recognize and bind to specific antigens on the surface of tumor cells in vivo, thereby activating T cells. Activated T cells directly kill tumor cells by releasing cytokines such as perforin and granzyme B. At the same time, they also recruit other endogenous immune cells in the human body to kill tumor cells by releasing cytokines, thereby achieving the purpose of treating tumors. At present, CAR-T cell therapy has achieved good results in the treatment of hematological tumors. CAR-T therapy targeting BCMA and CD19 was approved by the US Food and Drug Administration in 2019 and 2021. However, due to the complex structure and immune microenvironment of solid tumors, CAR-T still faces many challenges in the treatment of solid tumors.

[0003] Double negative T cells (DNTs) are a type of CD3 + CD4 - CD8 - DNT is a subset of T cells that only accounts for about 10% of the total number of T cells in human peripheral blood cells. DNT highly expresses cell surface receptors, such as natural killer group 2 member D (NKG2D), which specifically recognizes and kills tumor cells expressing the corresponding ligand in an MHC-non-restricted manner, but does not cause graft-versus-host disease and host-versus-graft reactions, and can achieve allogeneic therapy. The ligand of NKG2D (NKG2DL) is highly expressed on the surface of various tumor cells, but is usually lowly expressed or not expressed in healthy tissues. At present, a variety of NKG2D-based CAR-T cell therapies have been developed and have achieved significant therapeutic effects. Therefore, DNT-based CAR-T cell therapy is very promising.

[0004] However, another huge challenge facing CAR-T cell therapy is that the complex immune microenvironment of the tumor prevents CAR-T cells from infiltrating into solid tumors, and therefore cannot effectively kill tumor cells. Therefore, it is necessary to find a way to transform CAR-T cells, increase the efficiency of CAR-T proliferation in the body, and enhance its ability to migrate to the tumor site.

[0005] CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family and belongs to the CXC chemokine receptor family. CXCR5 is mainly expressed on the surface of B cells, T cells and dendritic cells, and plays an important role in inflammation, infection and immune response. CXCL13 (chemokine CX-Cmotif ligand 13), also known as B cell chemokine, is a ligand of CXCR5. Studies have shown that CXCL13 is abnormally expressed on the surface of various tumor cells, and the CXCL3 / CXCR5 signaling axis is related to tumor migration and invasion.

[0006] Interleukin-7 (IL-7) is a member of the IL-2 cytokine superfamily. It is a glycoprotein secreted by bone marrow stromal cells, thymic stromal cells, mucosal lymphoid tissues and keratinocytes. IL-7 receptors are mainly present in the bone marrow cell lineage and lymphocytes, and also in NK cells and T cells after stimulation by IL-2 or mitogens. IL-7 must bind to the IL-7 receptor to activate the two signaling pathways of JAK-STAT and PI3K-Akt in the cell, accelerate the cell cycle, and improve the survival and proliferation of cells.

[0007] Therefore, the present invention aims to co-express CXCR5 and IL-7 on the basis of the existing CAR-DNT cell structure targeting NKG2DL, so as to enhance its ability to migrate and infiltrate into the tumor site, and increase the survival and proliferation ability of CAR-T cells, thereby enhancing the killing effect of CAR-DNT cells on tumors and achieving the purpose of treating tumors. Summary of the invention

[0008] The purpose of the present invention is to address the deficiencies of the prior art and provide a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7 and its use, so as to enhance its ability to migrate and infiltrate into the tumor site, and increase the survival and proliferation ability of CAR-T cells, thereby enhancing the killing effect of CAR-DNT cells on tumors and achieving the purpose of treating tumors.

[0009] The present invention is achieved by a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7, wherein the cell drug can co-express CXCR5 and IL-7, and the cell drug comprises: EF1-α, CD8α signal peptide, NKG2D extracellular segment, CD8α hinge region, CD8α transmembrane region, co-stimulatory molecule 4-1BB, CD3ζ intracellular signal region, T2A, chemokine receptor CXCR5, P2A, and interleukin IL-7 connected in sequence;

[0010] The nucleotide sequence of the CD8α signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the NKG2D signal peptide is shown in SEQ ID NO.3, the nucleotide sequence of the CD8α hinge region is shown in SEQ ID NO.5, the nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.7, the nucleotide sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.9, the nucleotide sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.11, the nucleotide sequence of T2A is shown in SEQ ID NO.13, the nucleotide sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.15, the nucleotide sequence of P2A is shown in SEQ ID NO.17, and the nucleotide sequence of interleukin IL-7 is shown in SEQ ID NO.19.

[0011] The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.2, the amino acid sequence of the NKG2D signal peptide is shown in SEQ ID NO.4, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.6, the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.8, the amino acid sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.10, the amino acid sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.12, the amino acid sequence of T2A is shown in SEQ ID NO.14, the amino acid sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.16, the amino acid sequence of P2A is shown in SEQ ID NO.18, and the amino acid sequence of interleukin IL-7 is shown in SEQ ID NO.20.

[0012] Wherein, the nucleotide sequence of the CD8α signal peptide is shown as SEQ ID NO.1.

[0013] SEQ ID NO.1:

[0014] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0015] The nucleotide sequence of the NKG2D signal peptide is shown in SEQ ID NO.3.

[0016] SEQ ID NO.3:

[0017] ATGTTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTG GTGAAGTCATATCATTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG

[0018] The nucleotide sequence of the CD8α hinge region is shown in SEQ ID NO.5.

[0019] SEQ ID NO.5:

[0020] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCACAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0021] The nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.7.

[0022] SEQ ID NO.7:

[0023] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0024] The nucleotide sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.9.

[0025] SEQ ID NO.9:

[0026] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0027] The nucleotide sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.11.

[0028] SEQ ID NO.11:

[0029] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0030] The nucleotide sequence of T2A is shown in SEQ ID NO.13.

[0031] SEQ ID NO.13:

[0032] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0033] The nucleotide sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.15.

[0034] SEQ ID NO.15:

[0035]

[0036] The nucleotide sequence of P2A is shown in SEQ ID NO.17.

[0037] SEQ ID NO.17:

[0038] GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGTGACGTCGAGGAGAATCCTGGTCCC

[0039] The nucleotide sequence of interleukin IL-7 is shown in SEQ ID NO.19.

[0040] SEQ ID NO.19:

[0041] ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGC ATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGA GGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCA ACCAACAAAGAGTTTGGAAGAAAATAAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC

[0042] In this example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.2.

[0043] SEQ ID NO.2:

[0044] MALPVTALLLPLALLLHAARP

[0045] The amino acid sequence of the signal peptide is shown in SEQ ID NO.4.

[0046] SEQ ID NO.4:

[0047] MLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0048] The amino acid sequence of the hinge region is shown in SEQ ID NO.6.

[0049] SEQ ID NO.6:

[0050] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0051] The amino acid sequence of the transmembrane region is shown in SEQ ID NO.8.

[0052] SEQ ID NO.8:

[0053] IYIWAPLAGTCGVLLLSLVITLYC

[0054] The amino acid sequence of the co-stimulatory molecule is shown in SEQ ID NO.10.

[0055] SEQ ID NO.10:

[0056] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0057] The amino acid sequence of the intracellular signal region is shown in SEQ ID NO.12.

[0058] SEQ ID NO.12:

[0059] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0060] The amino acid sequence is shown in SEQ ID NO.14.

[0061] SEQ ID NO.14:

[0062] EGRGSLLTCGDVEENPGP

[0063] The amino acid sequence of the chemokine receptor is shown in SEQ ID NO.16.

[0064] SEQ ID NO.16:

[0065] MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEIL FAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSLSESENATSLTTF

[0066] The amino acid sequence is shown in SEQ ID NO.18.

[0067] SEQ ID NO.18:

[0068] ATNFSLLKQAGDVEENPGP

[0069] The amino acid sequence of the interleukin is shown in SEQ ID NO.20.

[0070] SEQ ID NO.20:

[0071] MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

[0072] On the other hand, the present invention also provides a method for preparing a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7, the method for preparing a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7 comprising:

[0073] S10, construction of pLL3.7-NKG2D-CAR-CXCR5-IL-7 plasmid, obtaining the gene sequence of the target gene Homo sapiens CXC motif chemokine receptor5 (CXCR5) and Homo sapiens interleukin7 (IL-7), designing primers containing homology arms based on the CDS sequences of CXCR5 and IL-7, using T cell cDNA as a template, using PCR to obtain the target gene fragment and recovering it on gel, using restriction endonuclease EcoRI to perform single enzyme digestion on the pLL3.7-NKG2D-CAR vector, recovering the linear vector after enzyme digestion on gel, using homologous recombinase to construct plasmid pLL3.7-NKG2D-CAR-CXCR5-IL-7, and sequencing to verify the successful construction of the plasmid;

[0074] S20, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging;

[0075] S30, CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation.

[0076] The pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging method comprises:

[0077] S201, pLL3.7-NKG2D-CAR-CXCR5-IL-7 transfection;

[0078] S202, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus collection and concentration;

[0079] S203, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus titer determination.

[0080] The CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation method comprises:

[0081] S301, human PBMCs were sorted using CD4 and CD8 magnetic beads to remove CD4 + 、CD8 +T cells, CD3 and CD28 antibodies were added to the cell suspension after sorting to activate DNT cells. After 48 hours, the DNT cells were centrifuged and the medium was changed to remove the antibodies. After 6 days of continuous culture, DNT cells were obtained and the proportion of DNT cells was detected by flow cytometry.

[0082] S302, DNTs were seeded in 24-well plates, 5 × 10 5 cells, with a total of 4 wells, namely: negative control group without virus infection, pLL3.7-Mock negative control group, pLL3.7-NKG2D-CAR group and pLL3.7-NKG2D-CAR-CXCR5-IL-7 group, add the corresponding volume of virus solution at MOI=10:1, collect the cells after 24 hours, centrifuge at 400g for 5 minutes, discard the culture medium, and add fresh culture medium;

[0083] S303, cells were collected 48 hours after lentiviral infection, and the NKG2D expression efficiency, i.e., the virus infection positive rate, was detected by flow cytometry. At the same time, the expression level of CXCR5 of CAR-DNT after lentiviral infection was detected by flow cytometry. DNT cells infected with pLL3.7-NKG2D-CAR-CXCR5-IL-7 successfully overexpressed CXCR5.

[0084] S304, the three groups of CAR-DNT cells were collected 48 hours after infection, the cells were lysed with Trizol, total RNA was extracted, and reverse transcription was performed. RT-PCR was used to analyze the expression of IL-7 at the transcriptional level in CAR-DNT cells infected with lentivirus.

[0085] The present invention also provides the use of a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 in the preparation of an anti-tumor drug, thereby enhancing the killing effect of CAR-DNT cells on tumors, achieving the purpose of treating tumors, and realizing allogeneic treatment.

[0086] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0087] The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 provided by the present invention and its use aims to enhance the migration and infiltration ability to the tumor site and increase the survival and proliferation ability of CAR-T cells by co-expressing CXCR5 and IL-7 on CAR-DNT cells targeting NKG2DL, thereby enhancing the killing effect of CAR-DNT cells on tumors, achieving the purpose of treating tumors, and realizing allogeneic treatment.

[0088] The present invention constructs a NKG2D-CAR-DNT cell that can target tumor cells with high expression of NKG2DL and kill them; it is proved that after co-expression of CXCR5 and IL-7, the migration ability of NKG2D-CAR-DNT cells to tumors is enhanced; at the same time, after co-expression of CXCR5 and IL-7, the anti-tumor activity of NKG2D-CAR-DNT cells is significantly enhanced, and it is verified by in vitro and in vivo experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 A schematic diagram of PCR amplification results is shown.

[0090] Figure 2 A schematic diagram of the single enzyme digestion results of the pLL3.7-NKG2D-CAR plasmid is shown.

[0091] Figure 3 A schematic diagram showing the scale of flow cytometry detection of DNT cells is shown.

[0092] Figure 4 A schematic diagram of the structure of a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 is shown.

[0093] Figure 5 The figure shows the result of flow cytometry detection of NKG2D positive rate.

[0094] Figure 6 The figure shows the result of flow cytometry detection of CXCR5 expression in CAR-DNT after lentivirus infection.

[0095] Figure 7 The figure shows the test results of RT-PCR detection of IL-7 expression level.

[0096] Figure 8 A schematic diagram of the CAR-DNT cell killing rate detection results is shown.

[0097] Fig. 9 The figure shows the test diagram of the in vitro migration effect of CAR-DNT.

[0098] Fig.10 A schematic diagram of the in vivo imaging detection results of mice is shown.

[0099] Fig.11 The proportion of CAR-T cells in tumors was detected by flow cytometry. DETAILED DESCRIPTION

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0101] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0102] The DNA sequences involved in the present invention are all synthesized by conventional methods, and the experimental conditions and operations not mentioned in the examples of the present invention are all carried out according to conventional methods in the art or conditions recommended by the manufacturer.

[0103] Example 1

[0104] The present invention provides a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7. Figure 4 A schematic structural diagram of a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 is shown. The cell drug can co-express CXCR5 and IL-7, and the cell drug includes: EF1-α, CD8α signal peptide, NKG2D extracellular segment, CD8α hinge region, CD8α transmembrane region, co-stimulatory molecule 4-1BB, CD3ζ intracellular signal region, T2A, chemokine receptor CXCR5, P2A, and interleukin IL-7 connected in sequence.

[0105] CAR-T cell immunotherapy, as a new type of T cell adoptive transfer therapy, has significant efficacy in the treatment of blood tumors, but there are still many problems and challenges in the treatment of solid tumors. The main reason is that the proliferation ability of CAR-T cells in the body will be affected by the immune microenvironment, and T cells are difficult to migrate to the tumor site, thus affecting their efficiency in killing tumor cells. In addition, graft-versus-host disease is also one of the obstacles that need to be overcome in CAR-T treatment. Studies have confirmed that DNT can be safely and effectively used in allogeneic immunotherapy without causing graft-versus-host disease, cytokine release syndrome and neurotoxicity.

[0106] The present invention modifies DNT cells with NKG2D-CAR, aiming to overexpress NKG2D so as to better target cancer cells expressing NKG2DL. Compared with T cells, loading CAR structure on DNT is safer.

[0107] Wherein, the nucleotide sequence of the CD8α signal peptide is shown as SEQ ID NO.1.

[0108] SEQ ID NO.1:

[0109] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0110] The nucleotide sequence of the NKG2D signal peptide is shown in SEQ ID NO.3.

[0111] SEQ ID NO.3:

[0112] ATGTTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTG GTGAAGTCATATCATTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG

[0113] The nucleotide sequence of the CD8α hinge region is shown in SEQ ID NO.5.

[0114] SEQ ID NO.5:

[0115] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCACAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0116] The nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.7.

[0117] SEQ ID NO.7:

[0118] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0119] The nucleotide sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.9.

[0120] SEQ ID NO.9:

[0121] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0122] The nucleotide sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.11.

[0123] SEQ ID NO.11:

[0124] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0125] The nucleotide sequence of T2A is shown in SEQ ID NO.13.

[0126] SEQ ID NO.13:

[0127] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0128] The nucleotide sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.15.

[0129] SEQ ID NO.15:

[0130]

[0131] The nucleotide sequence of P2A is shown in SEQ ID NO.17.

[0132] SEQ ID NO.17:

[0133] GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGTGACGTCGAGGAGAATCCTGGTCCC

[0134] The nucleotide sequence of interleukin IL-7 is shown in SEQ ID NO.19.

[0135] SEQ ID NO.19:

[0136] ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGC ATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGA GGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCA ACCAACAAAGAGTTTGGAAGAAAATAAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC

[0137] In this example, the amino acid sequence of the signal peptide is shown in SEQ ID NO.2.

[0138] SEQ ID NO.2:

[0139] MALPVTALLLPLALLLHAARP

[0140] The amino acid sequence of the signal peptide is shown in SEQ ID NO.4.

[0141] SEQ ID NO.4:

[0142] MLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0143] The amino acid sequence of the hinge region is shown in SEQ ID NO.6.

[0144] SEQ ID NO.6:

[0145] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0146] The amino acid sequence of the transmembrane region is shown in SEQ ID NO.8.

[0147] SEQ ID NO.8:

[0148] IYIWAPLAGTCGVLLLSLVITLYC

[0149] The amino acid sequence of the co-stimulatory molecule is shown in SEQ ID NO.10.

[0150] SEQ ID NO.10:

[0151] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0152] The amino acid sequence of the intracellular signal region is shown in SEQ ID NO.12.

[0153] SEQ ID NO.12:

[0154] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0155] The amino acid sequence is shown in SEQ ID NO.14.

[0156] SEQ ID NO.14:

[0157] EGRGSLLTCGDVEENPGP

[0158] The amino acid sequence of the chemokine receptor is shown in SEQ ID NO.16.

[0159] SEQ ID NO.16:

[0160] MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEIL FAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSLSESENATSLTTF

[0161] The amino acid sequence is shown in SEQ ID NO.18.

[0162] SEQ ID NO.18:

[0163] ATNFSLLKQAGDVEENPGP

[0164] The amino acid sequence of the interleukin is shown in SEQ ID NO.20.

[0165] SEQ ID NO.20:

[0166] MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

[0167] The present invention co-expresses CXCR5 and IL-7 on CAR-DNT cells targeting NKG2DL for the first time, with the aim of enhancing their ability to migrate and infiltrate into tumor sites, and increasing the survival and proliferation ability of CAR-T cells, thereby enhancing the tumor-killing effect of CAR-DNT cells, achieving the purpose of treating tumors, and realizing allogeneic treatment.

[0168] Example 2

[0169] The embodiment of the present invention also provides a method for preparing a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7. The method for preparing a chimeric antigen receptor double negative T cell drug that co-expresses CXCR5 and IL-7 specifically comprises:

[0170] S10, construction of pLL3.7-NKG2D-CAR-CXCR5-IL-7 plasmid, obtaining the gene sequence of the target gene Homo sapiens CXC motif chemokine receptor5 (CXCR5) and Homo sapiens interleukin7 (IL-7), designing primers containing homology arms based on the CDS sequences of CXCR5 and IL-7, using T cell cDNA as a template, using PCR to obtain the target gene fragment and recovering it on gel, using restriction endonuclease EcoRI to perform single enzyme digestion on the pLL3.7-NKG2D-CAR vector, recovering the linear vector after enzyme digestion on gel, using homologous recombinase to construct plasmid pLL3.7-NKG2D-CAR-CXCR5-IL-7, and sequencing to verify the successful construction of the plasmid;

[0171] It should be noted that when obtaining the gene sequence of the target gene Homo sapiens CXC motif chemokine receptor5 (CXCR5) and Homo sapiens interleukin7 (IL-7), the gene sequence of Homo sapiens CXC motif chemokine receptor5 (CXCR5) was found on the NCBI website: NM_001716.5. And the gene sequence of Homo sapiens interleukin7 (IL-7) was found: NM_000880.4. At the same time, Figure 1 The schematic diagram of PCR amplification results is shown, thereby characterizing the use of PCR to obtain the target gene fragment and perform gel recovery results. Figure 1 In the figure, M: DNA molecular weight standard; 1: CXCR5; 2: IL-7. Figure 2The schematic diagram of the single enzyme digestion result of pLL3.7-NKG2D-CAR plasmid is shown. Figure 2 In the figure, M: DNA molecular weight standard; 1: pLL3.7-NKG2D-CAR; 2: pLL3.7-NKG2D-CAR / EcoRI.

[0172] S20, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging;

[0173] Wherein, the pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging method comprises:

[0174] S201, pLL3.7-NKG2D-CAR-CXCR5-IL-7 transfection;

[0175] It should be noted that pLL3.7-NKG2D-CAR-CXCR5-IL-7 transfection is a plasmid transfection process, and the specific steps of transfection are:

[0176] S2011, 293T cells were passaged one day in advance, and lentiviral packaging was performed when the cell confluence was about 80%;

[0177] S2012, psPA×2, pMD2.G, target plasmid, PEI, and DMEM medium were placed at room temperature for 5 min;

[0178] S2013, take 500 μL of DMEM into a 1.5 mL EP tube, add 50 μL of PEI, mix well, and let stand at room temperature for 5 min;

[0179] S2014, take 5 μg of vector plasmid, 5 μg of psPA×2, 3 μg of pMD2.G, add 500 μL of DMEM, and let stand at room temperature for 5 min;

[0180] S2015, add the prepared PEI-DMEM solution into the DMEM containing the plasmid and let stand at room temperature for 20 min;

[0181] S2016, slowly drop 1 mL of DNA / PEI mixture into the 293T culture dish prepared the day before, mix gently, and incubate in a 37°C incubator;

[0182] S2017, after 6-8 h, replace the DMEM medium containing 1% serum and place in a 37°C incubator for further incubation.

[0183] S202, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus collection and concentration;

[0184] In this embodiment, the method for collecting and concentrating the pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus specifically includes:

[0185] S2021, 48 h after plasmid transfection, the supernatant was collected, 10 mL of fresh DMEM medium containing 1% serum was added and culture was continued for 72 h, the supernatant was collected again and mixed with the supernatant collected at 48 h;

[0186] S2022, then centrifuged at 4000 g for 10 min at 4 °C, and collected the supernatant;

[0187] S2023, the supernatant obtained by filtration using a 0.22 μm filter;

[0188] S2024, transfer the filtered virus supernatant into an ultracentrifuge tube and centrifuge at 15000g for 2h. Discard the supernatant after centrifugation, add X-vivo medium, and repeatedly pipette and incubate at 4°C overnight;

[0189] S2025, divide the virus solution into appropriate volumes, store it at -80°C, and take a small amount of virus for titer determination.

[0190] S203, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus titer determination.

[0191] It should be noted that the specific steps for determining the lentiviral titer are as follows:

[0192] S2031, digest 293T cells, count after centrifugation, make cell suspension in serum-containing medium, and adjust the cell density to 4×10 5 / mL, add 0.5mL of cell suspension to each well of a 24-well culture plate;

[0193] S2032, 0 μL, 0.5 μL, 1 μL, and 1.5 μL of virus solution were added to each well;

[0194] S2033, flow cytometry detection of target gene expression in infected cells after 48 h;

[0195] S2034, the titer is calculated using the following formula:

[0196]

[0197] After the virus was collected and concentrated, the titer of pLL3.7-NKG2D-CAR-CXCR5-IL-7 was 2108 after titer detection.

[0198] S30, CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation.

[0199] The CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation method comprises:

[0200] S301, human PBMCs were sorted using CD4 and CD8 magnetic beads to remove CD4 + 、CD8 + T cells, CD3 and CD28 antibodies were added to the cell suspension after sorting to activate DNT cells, and the DNT cells were centrifuged and replaced after 48 hours, and the antibodies were removed. After 6 days of continuous culture, DNT cells were obtained, and the proportion of DNT cells was detected by flow cytometry. Figure 3 A schematic diagram showing the proportions of DNT cells detected by flow cytometry is shown;

[0201] S302, DNT was inoculated in a 24-well plate, 5 × 105 cells per well, and 4 wells in total, namely: negative control group without virus infection, pLL3.7-Mock negative control group, pLL3.7-NKG2D-CAR group and pLL3.7-NKG2D-CAR-CXCR5-IL-7 group, and the corresponding volume of virus solution was added at MOI = 10:1. After 24 hours, the cells were collected and centrifuged at 400g for 5 minutes, the culture medium was discarded, and fresh culture medium was added;

[0202] S303, cells were collected 48 hours after lentiviral infection, and the NKG2D expression efficiency, i.e., the virus infection positive rate, was detected by flow cytometry. At the same time, the expression of CXCR5 in CAR-DNT after lentiviral infection was detected by flow cytometry. DNT cells infected with pLL3.7-NKG2D-CAR-CXCR5-IL-7 successfully overexpressed CXCR5, while Figure 5 The flow cytometry results of NKG2D positive rate are shown. The positive rate of pLL3.7-NKG2D-CAR-CXCR5-IL-7 infected DNT cells is about 99.6%. Figure 6 The results of flow cytometry detection of CXCR5 expression in CAR-DNT after lentivirus infection are shown. Figure 6 It can be concluded that DNT cells infected with pLL3.7-NKG2D-CAR-CXCR5-IL-7 successfully overexpressed CXCR5.

[0203] S304, the three groups of CAR-DNT cells were collected 48 hours after infection, the cells were lysed with Trizol, total RNA was extracted, and reverse transcription was performed. RT-PCR was used to analyze the expression of IL-7 at the transcriptional level in CAR-DNT cells infected with lentivirus.

[0204] It should be noted that Figure 7 The results of the RT-PCR test for IL-7 expression level are shown. After lentiviral infection, the IL-7 gene was significantly upregulated at the transcriptional level. Figure 7 In the figure, M: DNA molecular weight standard; 1: β-actin (Mock-CAR-DNT); 2: β-actin (NKG2D-CAR-DNT); 3: β-actin (CXCR5 / IL-7-NKG2D-CAR-DNT); 4: IL-7 (Mock-CAR-DNT); 5: IL-7 (NKG2D-CAR-DNT); 6: IL-7 (CXCR5 / IL-7-NKG2D-CAR-DNT).

[0205] Cytokines can affect the proliferation, differentiation and survival of cytotoxic T cells, and therefore have great application potential in cellular immunotherapy. Cytokine IL-7 has been shown to promote the proliferation and persistence of T cells, and can promote the differentiation of T cells into memory T cells. Co-expression of IL-7 on CAR-DNT cells can enhance the proliferation ability of CAR-DNT cells, thereby enhancing their tumor-killing efficiency. Since pancreatic cancer expresses a large amount of chemokine CXCL13, co-expression of its receptor CXCR5 on CAR-DNT cells can enhance the migration ability of CAR-DNT cells to the tumor site and increase tumor-infiltrating T cells, thereby achieving better anti-tumor effects.

[0206] The embodiment of the present invention co-expresses CXCR5 and IL-7 on NKG2D-CAR-DNT cells targeting NKG2DL, and proves through in vitro and in vivo experiments that after co-expressing CXCR5 and IL-7, CAR-DNT cells have better persistence, stronger migration ability to tumor tissue, and better anti-tumor activity.

[0207] Example 3

[0208] Overexpression of CXCR5 and IL-7 can promote the tumor killing efficiency of NKG2D-CAR-DNT.

[0209] 1. CXCR5 / IL-7-NKG2D-CAR-DNT in vitro killing verification experiment

[0210] 1) Co-culture of PANC-1 and CAR-DNT cells

[0211] The cultured PANC-1-luc cells with high expression of NKG2DL were cultured at 3×10 5 The number of per well was inoculated into a 96-well plate treated with low adsorption;

[0212] Mock-CAR-DNT, NKG2D-CAR-DNT and CXCR5 / IL-7-NKG2D-CAR-DNT were inoculated into 96-well plates seeded with tumor cells at effector-target ratios of 0.25:1, 0.5:1, and 1:1, respectively. Each group had three replicates, and each well was replenished to 200 μL.

[0213] The culture plate after cell mixing was placed in a 37°C incubator for 16 h;

[0214] After 16 h, collect the cells in each well into a 1.5 mL EP tube, centrifuge at 4500 rpm for 5 min, discard the supernatant, add 100 μL of cell lysis buffer, transfer to a 96-well white ELISA plate, and use a chemiluminescence instrument to detect the bioluminescence intensity.

[0215] 2) Analysis of CAR-DNT killing efficiency

[0216] The measured bioluminescence intensity is used to calculate the target cell apoptosis rate according to the formula, thereby obtaining the killing efficiency of CAR-DNT cells. The calculation formula is as follows:

[0217]

[0218] like Figure 8 As shown, a schematic diagram of the CAR-DNT cell killing rate detection results is shown. Compared with the control group, CXCR5 / IL-7-NKG2D-CAR-DNT cells have a higher killing efficiency against PANC-1, and the killing effect is more significant as the effect-target ratio increases. When the effect-target ratio is 4:1, the killing rate of CXCR5 / IL-7-NKG2D-CAR-DNT is 67.42%, which is significantly different from Mock-CAR-DNT (46.41%) and NKG2D-CAR-DNT (36.81%). The above results fully prove that overexpression of CXCR5 and IL-7 can significantly enhance the killing ability of NKG2D-CAR-DNT against tumor cells expressing NKG2DL.

[0219] 2. CXCR5 / IL-7-NKG2D-CAR-DNT in vitro migration assay

[0220] 1) Construction of in vitro cell migration model

[0221] The migration experiment of CAR-DNT was carried out in a 24-well plate using a 3 m Transwell chamber.

[0222] Add 5 × 10 5Mock-CAR-DNT, NKG2D-CAR-DNT or CXCR5 / IL-7-NKG2D-CAR-DNT cells were added to the lower chamber, and the cells were cultured in a cell culture incubator for 4 hours.

[0223] 2) Evaluation of CAR-DNT migration ability in vitro

[0224] After 4 hours, the chamber was taken out, and the CAR-DNT cells in the lower chamber were counted. The cell migration index was calculated according to the formula as follows:

[0225]

[0226] Fig. 9 The in vitro migration effect test diagram of CAR-DNT is shown. Fig. 9 As shown in the figure, under the influence of tumor supernatant, CAR-DNT will show a certain migration ability. Compared with the control group, the migration ability of CXCR5 / IL-7-NKG2D-CAR-DNT was significantly improved. The above results show that NKG2D-CAR-T overexpressing CXCR5 and IL-7 has better migration ability in vitro.

[0227] 3. Verification of the anti-tumor effect of CXCR5 / IL-7-NKG2D-CAR-DNT in vivo

[0228] 1) Construction of CAR-DNT treatment model in NSG mice

[0229] Prepare 6-8 week old female NSG mice and mark each mouse with an ear tag;

[0230] The PANC-1-luc cells were adjusted to a cell density of 2 × 10 7 / mL, add an equal volume of Matrigel and mix well;

[0231] The hair on one side of the mouse's back was cleaned with a razor, and 100uL of cell suspension was drawn into the mouse with a 1mL syringe for subcutaneous tumor loading;

[0232] Observe the tumor formation on the back of the mouse, measure the size of the tumor with a vernier caliper, and calculate the tumor volume. The calculation formula is as follows:

[0233]

[0234] When the average tumor volume reaches 100 mm 3 The mice were divided into three groups: PBS group, Mock-CAR-DNT group, NKG2D-CAR-DNT group and CXCR5 / IL-7-NKG2D-CAR-DNT group;

[0235] Prepare CAR-DNT cells and resuspend the cells in PBS to adjust the density to 5×10 7 Pieces / mL;

[0236] Each mouse was injected with 100 μL of cell suspension through the tail vein, and the PBS group was injected with 100 μL of PBS as the control group.

[0237] 2) Evaluation of the therapeutic effect of CAR-DNT in vivo

[0238] The tumor volume of mice was measured every three days, and the mice were imaged every seven days. When the tumor volume reached 2000mm 3 The mice were euthanized at 4 °C. Fig.10 Figure 2 shows a schematic diagram of the results of in vivo imaging of mice. Fig.10 As shown, the bioluminescence intensity of the PBS group and the Mock-CAR-DNT group was the strongest, indicating that their tumors grew fastest. Both the NKG2D-CAR-DNT group and the CXCR5 / IL-7-NKG2D-CAR-DNT group showed certain anti-tumor effects, and the treatment effects of the two groups were similar in the first seven days of treatment. As the treatment time increased, the CXCR5 / IL-7-NKG2D-CAR-DNT group showed better tumor treatment effects, which were significantly better than the treatment effects of the other three groups. The above results show that co-expression of CXCR5 and IL-7 can enhance the inhibitory effect of NKG2D-CAR-DNT on tumors in vivo.

[0239] On the 21st day of treatment, mouse tumor tissue was obtained and ground, filtered through a 0.22 μm filter membrane, and a single cell suspension was obtained. 0.5 μl Anti-Human-CD3 (BV421) antibody was added to label CD3 + T cells were incubated at 4°C for 30 minutes, washed once with 1×PBS, resuspended in 200 μL of 1×PBS, transferred to a flow cytometer, and tested on the flow cytometer. Fig.11 As shown in the figure, after CAR-DNT treatment, CAR-DNT cells can be detected in the tumor tissues of mice, and more T cells can be detected in the tumors of mice in the CXCR5 / IL-7-NKG2D-CAR-DNT group compared with the Mock-CAR-DNT group and the NKG2D-CAR-DNT group. The above results show that compared with the control group, CXCR5 / IL-7-NKG2D-CAR-DNT has a stronger ability to migrate to tumor tissues.

[0240] In summary, the present invention provides a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 and its use. The chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 and its use provided by the present invention co-expresses CXCR5 and IL-7 on CAR-DNT cells targeting NKG2DL, with the aim of enhancing their migration and infiltration capabilities to the tumor site, and increasing the survival and proliferation capabilities of CAR-T cells, thereby enhancing the tumor-killing effect of CAR-DNT cells, achieving the purpose of treating tumors, and can realize allogeneic treatment.

[0241] The present invention constructs a NKG2D-CAR-DNT cell that can target tumor cells with high expression of NKG2DL and kill them; it is proved that after co-expression of CXCR5 and IL-7, the migration ability of NKG2D-CAR-DNT cells to tumors is enhanced; at the same time, after co-expression of CXCR5 and IL-7, the anti-tumor activity of NKG2D-CAR-DNT cells is significantly enhanced, and it is verified by in vitro and in vivo experiments.

[0242] It should be noted that, for the above-mentioned embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the described order of actions, because according to the present invention, some steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0243] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7, characterized in that: The cell medicine can co-express CXCR5 and IL-7, and the cell medicine includes: EF1-α, CD8α signal peptide, NKG2D extracellular segment, CD8α hinge region, CD8α transmembrane region, co-stimulatory molecule 4-1BB, CD3ζ intracellular signal region, T2A, chemokine receptor CXCR5, P2A, and interleukin IL-7 connected in sequence; The nucleotide sequence of the CD8α signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the NKG2D signal peptide is shown in SEQ ID NO.3, the nucleotide sequence of the CD8α hinge region is shown in SEQ ID NO.5, the nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.7, the nucleotide sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.9, the nucleotide sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.11, the nucleotide sequence of T2A is shown in SEQ ID NO.13, the nucleotide sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.15, the nucleotide sequence of P2A is shown in SEQ ID NO.17, and the nucleotide sequence of interleukin IL-7 is shown in SEQ ID NO.

19.

2. The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 according to claim 1, characterized in that: The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.2, the amino acid sequence of the NKG2D signal peptide is shown in SEQ ID NO.4, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.6, the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.8, the amino acid sequence of the co-stimulatory molecule 4-1BB is shown in SEQ ID NO.10, the amino acid sequence of the CD3ζ intracellular signal region is shown in SEQ ID NO.12, the amino acid sequence of T2A is shown in SEQ ID NO.14, the amino acid sequence of the chemokine receptor CXCR5 is shown in SEQ ID NO.16, the amino acid sequence of P2A is shown in SEQ ID NO.18, and the amino acid sequence of interleukin IL-7 is shown in SEQ ID NO.

20.

3. The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 according to claim 1, characterized in that: The method for preparing a chimeric antigen receptor double-negative T cell drug that co-expresses CXCR5 and IL-7 comprises: S10, construction of pLL3.7-NKG2D-CAR-CXCR5-IL-7 plasmid, obtaining the gene sequence of the target gene Homo sapiens CX-Cmotif chemokine receptor5 (CXCR5) and Homo sapiens interleukin7 (IL-7), designing primers containing homology arms based on the CDS sequences of CXCR5 and IL-7, using T cell cDNA as a template, using PCR to obtain the target gene fragment and recovering it on gel, using restriction endonuclease EcoRI to perform single enzyme digestion on the pLL3.7-NKG2D-CAR vector, recovering the linear vector after enzyme digestion on gel, using homologous recombinase to construct plasmid pLL3.7-NKG2D-CAR-CXCR5-IL-7, and sequencing to verify the successful construction of the plasmid; S20, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging; S30, CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation.

4. The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 according to claim 3, characterized in that: The pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentiviral packaging method comprises: S201, pLL3.7-NKG2D-CAR-CXCR5-IL-7 transfection; S202, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus collection and concentration; S203, pLL3.7-NKG2D-CAR-CXCR5-IL-7 lentivirus titer determination.

5. The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 according to claim 3, characterized in that: The CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation method comprises: S301, human PBMCs were sorted using CD4 and CD8 magnetic beads to remove CD4 + 、CD8 + T cells, CD3 and CD28 antibodies were added to the cell suspension after sorting to activate DNT cells. After 48 hours, the DNT cells were centrifuged and the medium was changed to remove the antibodies. After 6 days of continuous culture, DNT cells were obtained and the proportion of DNT cells was detected by flow cytometry. S302, DNTs were seeded in 24-well plates, 5 × 10 5 cells, with a total of 4 wells, namely: negative control group without virus infection, pLL3.7-Mock negative control group, pLL3.7-NKG2D-CAR group and pLL3.7-NKG2D-CAR-CXCR5-IL-7 group, add the corresponding volume of virus solution at MOI=10:1, collect the cells after 24 hours, centrifuge at 400g for 5 minutes, discard the culture medium, and add fresh culture medium; S303, cells were collected 48 hours after lentiviral infection, and the NKG2D expression efficiency, i.e., the virus infection positive rate, was detected by flow cytometry. At the same time, the expression level of CXCR5 of CAR-DNT after lentiviral infection was detected by flow cytometry. DNT cells infected with pLL3.7-NKG2D-CAR-CXCR5-IL-7 successfully overexpressed CXCR5.

6. The chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 according to claim 5, characterized in that: The CXCR5 / IL-7-NKG2D-CAR-DNT cell preparation method further comprises: S304, the three groups of CAR-DNT cells were collected 48 hours after infection, the cells were lysed with Trizol, total RNA was extracted, and reverse transcription was performed. RT-PCR was used to analyze the expression of IL-7 at the transcriptional level in CAR-DNT cells infected with lentivirus.

7. Use of the chimeric antigen receptor double negative T cell drug co-expressing CXCR5 and IL-7 as claimed in any one of claims 1 to 6 in the preparation of anti-tumor drugs.

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