CN113493525B

CN113493525B - Synergistic and depletion resistant chimeric antigen receptor T cell and application thereof in preparation of tumor treatment drugs

Info

Publication number: CN113493525B
Application number: CN202010263052.3A
Authority: CN
Inventors: 王恩秀; 汪晨; 陈颖
Original assignee: Nanjing Cart Medical Technologies Ltd
Current assignee: Nanjing Cart Medical Technologies Ltd
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2023-07-11
Anticipated expiration: 2040-04-07
Also published as: CN113493525A

Abstract

The invention discloses a chimeric antigen receptor T cell which has synergy and resists depletion and application thereof in preparing medicines for treating tumors, which comprises the following steps: an antigen binding domain (scFv) and a signaling domain; wherein the signaling domain comprises a first conductive domain and a second conductive domain, the antigen binding domain being in tandem between the first conductive domain and the second conductive domain; the first conducting domain comprises a receptor signaling domain and/or a co-stimulatory domain; the second conductive domain comprises a transmembrane domain DAP12, the transmembrane domain DAP12 being connected or not connected to a co-stimulatory domain and/or a primary signaling domain. The T cells expressing the chimeric antigen receptor can make the chimeric antigen receptor more efficiently and stably expressed in the T cells so as to better play the role of killing target cells, improve the durability of CAR-T, have lower expression of the depletion marker and have richer memory T cells.

Description

Synergistic and depletion resistant chimeric antigen receptor T cell and application thereof in preparation of tumor treatment drugs

Technical Field

The invention belongs to the field of biology, and particularly relates to a chimeric antigen receptor T cell which is synergistic and resistant to exhaustion and application thereof in preparing a tumor treatment drug.

Background

In recent years, although conventional tumor treatment methods such as radiotherapy, chemotherapy and operation treatment can achieve a certain treatment effect, the problems of metastasis, recurrence and low survival rate of patients of tumors are not solved properly. With the development of tumor immunology theory and technology, the role of immune cells in tumor therapy is increasingly emphasized. Among them, the chimeric antigen receptor T cell technology is a cell therapy technology which has been developed very rapidly in recent years.

Chimeric Antigen Receptors (CARs) are the core component of CAR-T, which, by virtue of their ligand binding domain properties, are able to redirect their specificity and reactivity towards selected immune cells, thus conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a wider range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a Tumor Associated Antigen (TAA) binding region (typically an scFv fragment derived from a monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signaling region.

There are still problems with the current use of conventional CAR-T for hematological tumors, limiting its clinical application. The current FDA approved CD19 CAR-T cell drugs respectively take CD28 or 4-1BB as a co-stimulatory domain (Forsberg MH, et al potential of CAR T therapy for relapsed or refractory pediatric and young adult B-cell ALL. Ther Clin Risk Manag,2018,14:1573-1584.Hombach AA,et al.The weal and woe of costimulation in the adoptive therapy of cancer with Chimeric Antigen Receptor (CAR) -redirected T cells. Curr Mol Med,2013,13 (7): 1079-1088), and the two CAR-T cells with the co-stimulatory domain of CD28 have advantages and disadvantages, namely, the CAR-T cells proliferate faster in the early stage of the reaction, can mediate stronger tumor killing capacity, but have stronger side effects and poorer persistence in the later stage of the immune reaction; while 4-1 BB-based CAR-T cells have a poorer ability to proliferate at the early stage of the reaction than CD28 CAR, they have a better persistence, and can maintain a higher cell number at the late stage of the immune reaction (Salter AI, et al phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal,2018,11 (544): eaat6753.Maloney DG. Anti-CD19 CAR T cell therapy for lymphoma-off to the same as in the first place of the reaction, i.e., nat Rev Clin Oncol,2019,16 (5): 279-280.Kawalekar OU,et al.Distinct signaling by Chimeric Antigen Receptors (CARs) contact CD28 signaling domain versus 4-1BB in primary human T cells.Blood,2013,122 (21): 2902.). In addition, it is noted that CAR-T cells also have some side effects during treatment, such as cytokine release syndrome (Cytokine release syndrome, CRS), neurotoxicity, B cell dysfunction, and also have problems with tumor recurrence (Bonifant CL, et al, toxicity and management in CAR T-cell therapy. Mol ter-Oncolytics, 2016,3:16011.Porter DL,et al.Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.Sci Transl Med,2015,7 (303): 303ra139.Maude SL,et al.Managing cytokine release syndrome associated with novel T cell-gating therapies. Cancer J,2014,20 (2): 119-122.Sadelain M.Chimeric antigen receptors:a paradigm shift in immunotherapy.Annu Rev Canc Biol,2017,1:447-466.).

Disclosure of Invention

One of the purposes of the invention is to provide a novel synergistic and exhaustion-resistant Chimeric Antigen Receptor (CAR), and T cells expressing the chimeric antigen receptor can enable the chimeric antigen receptor to be expressed more efficiently and stably in the T cells so as to better play a role in killing target cells, improve the durability of the CAR-T, enable the exhaustion marker to express lower and have richer memory T cells.

The second purpose of the invention is to provide the application of the chimeric antigen receptor T cell in preparing a tumor treatment medicine.

The aim of the invention can be achieved by the following technical scheme:

the present invention provides a chimeric antigen receptor comprising: an antigen binding domain (scFv) and a signaling domain; wherein the signaling domain comprises a first conductive domain and a second conductive domain, the antigen binding domain being in tandem between the first conductive domain and the second conductive domain; the first conducting domain comprises a receptor signaling domain and/or a co-stimulatory domain; the second conductive domain comprises a transmembrane domain with or without a co-stimulatory domain and/or a primary signaling domain attached thereto.

In some embodiments, the second conductive domain of the invention is linked to an antigen binding domain (scFv) by a tandem signaling domain.

The chimeric antigen receptor disclosed by the invention is changed into a multi-chain form capable of transmitting an activation signal after the antigen binding domain specifically binds to an antigen, and the activation signal is transmitted to immune cells expressing the chimeric antigen receptor, so that the effect of immunotherapy is realized.

In order to improve the safety and curative effect of the chimeric antigen receptor, the chimeric antigen receptor can mediate stronger tumor killing capability, has better persistence and lower expression of a depletion marker, can differentiate more memory T cells, and also provides a plurality of preferable conduction domains.

In some preferred embodiments, the first conductive domain of the invention is free of co-stimulatory domains; i.e. the preferred first conducting domain comprises only the receptor signaling domain.

In some preferred embodiments, the first conductive domain of the invention comprises only a receptor signaling domain; the second conducting domain comprises a transmembrane domain linked to a costimulatory domain; or the second conducting domain comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain, connected in sequence.

In some embodiments, the transmembrane domain of the invention is selected from any one of DAP12, DAP10, cd3ζ, fcεrγ; DAP12 is preferred.

In some embodiments, the receptor signaling domain of the invention is selected from one of KIRS2, KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR DL5B, KIR DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, KIR3DP1, NKp46, NKp30, NKp44, SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, KLRD1/KLRC2, pb1, PILRB, CLEC5A, TREM1, TREM2, CD300 37300E, SIGLEC-14, SIGLEC-15, SIGLEC-16, or NKG 2D; preferably NKp44 or TREM1 or KIRS2; more preferred is KIRS2.

In some embodiments, antigens bound by the antigen binding domains of the invention are associated with malignancy and may be routinely selected in the art according to different tumor targets, e.g., the tumor antigens may be selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CEA, CTLA4, BCMA, CS1, c-Met, EPCAM, EGFR/EGFRvIII, gp100, GPC3, GD-2, IGF1R, IGF-I receptor, MAGE A3, mesothelin, MUC1, NY-ESO-1, HER2, PD1, PSMA, ROR1, WT1, glycolipid F77, or any other tumor antigen or other modification type and any combination thereof.

In some embodiments, the costimulatory domain according to the invention is selected from any of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3; preferably 4-1BB.

In some embodiments, the primary signal domain of the present invention is cd3ζ.

In some embodiments, the tandem signaling domain of the present invention is one or more of F2A, E2A, T2A, P2A, preferably T2A.

The invention also provides a recombinant expression vector with the chimeric antigen receptor.

In one embodiment, the recombinant expression vector of the invention comprises a second conducting domain, a tandem signaling domain, an antigen binding domain, a first conducting domain, connected in sequence.

The invention also provides an immune cell with the chimeric antigen receptor.

Chimeric antigen receptor immune cells of the invention; in some embodiments, the cells are selected from T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells, or embryonic stem cell culture differentiated immune cells.

The invention also provides application of the chimeric antigen receptor, the recombinant expression vector or the immune cells in preparing medicines for treating tumors.

The tumor according to the present invention refers to a new tumor formed by local tissue cell proliferation of an organism under the action of various tumorigenic factors, including but not limited to brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-hodgkin lymphoma, uterine cancer, etc. as well as other unlisted tumors or any combination thereof.

The above-mentioned structural domain is the prior known structural domain, and the nucleotide sequence and the amino acid sequence can be obtained by inquiring the prior art. For example:

the transmembrane domain DAP12 is widely applied to the surfaces of natural killer cells, granulocytes and mononuclear/macrophage and is used for transmitting activation signals, the nucleotide sequence of the transmembrane domain DAP12 is shown as SEQ ID NO.1, and the amino acid sequence of the transmembrane domain DAP12 is shown as SEQ ID NO. 2. Or a polypeptide having 85% -99% identity to its amino acid sequence.

The nucleotide sequence of the tandem signal transduction structural domain T2A is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4. Or a polypeptide having 85% -99% identity to its amino acid sequence.

The costimulatory domain 4-1BB is an active costimulatory molecule, and is expressed on various cells of an immune system, the nucleotide sequence of the costimulatory domain is shown as SEQ ID NO.5, and the amino acid sequence of the costimulatory domain is shown as SEQ ID NO. 6. Or a polypeptide having 85% -99% identity to its amino acid sequence.

The full-length nucleotide sequence of the receptor signal domain NKp44 gene is NCBI, genBank is AJ225109.1, the amino acid sequence is NCBI, and GenBank is CAB39168.1; the full-length nucleotide sequence of the TREM1 gene is shown in NCBI, access: NM_004829.6, amino acid sequence see NCBI, access: np_004820; the full-length nucleotide sequence of the KIRS2 gene is shown in NCBI, access: NM_147130.2, amino acid sequence see NCBI, access: np_667341.1.

Other partial domains according to the invention may be as shown in Table 1, or polypeptides having 85% -99% identity to their amino acid sequences.

[ Table 1 ]

The invention has the beneficial effects that:

(1) The CAR structure provided by the invention has stronger killing effect on tumors through in vitro function experiments;

(2) The CAR structure related by the invention can secrete higher cytokine level when being stimulated by antigen, and has good anti-tumor effect;

(3) The CAR structure of the invention has stronger anti-tumor effect through in vivo functional experiments;

(4) The signaling domains described herein, when combined with the 4-1BB domain with the transmembrane receptor DAP12 to form the signaling domain of the CAR, and the receptor signaling domain is not linked to the costimulatory domain, can induce activation of immune cells when the CAR specifically binds to a ligand in the target, resulting in a more stable and stronger immune response.

(5) The CAR structures of the invention enable lower levels of expression of the depletion marker.

(6) The CAR structure provided by the invention has more memory type T cells, such as Tscm, tcm and the like.

Drawings

FIG. 1 is a lentiviral vector containing different signal domain structures;

FIG. 2 is a schematic diagram containing different signal domain structures;

FIG. 3 is a graph showing the positive rate of expression of a CAR structure recognizing the CD19 antigen on the surface of T cells detected by flow cytometry 7 days after infection of KT4 CAR-T cells with lentivirus;

FIG. 4 is a graph of the killing effect of NTD/CAR-T cells on antigen-positive tumor cell lines;

FIG. 5 is secretion of IFN-gamma by NTD/CAR-T cells under antigen positive stimulation;

FIG. 6 is the secretion of IL-2 by NTD/CAR-T cells under antigen positive stimulation;

FIG. 7 is a graph of the anti-tumor effect of CAR-T cells in mice.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedure, in which specific conditions are not noted in the examples, is generally carried out according to conventional conditions, for example, those described in the guidelines for molecular cloning experiments (third edition, J. Sam Brookfield et al) or according to the manufacturer's recommendations. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

EXAMPLE 1 construction of CAR lentiviruses containing 5 elements DAP12-4-1BB-T2A-CD19 (scFv) -KIRS2

To demonstrate that CAR-T cells containing the intracellular signaling domain of DAP12-4-1BB-KIRS2 are more advantageous than previously reported CAR-T cells containing the 4-1BB-CD3 zeta, DAP12-KIRS2, DAP12-4-1BB-CD3 zeta-KIRS 2-4-1BB, DAP12-41BB-KIRS2-4-1BB, DAP12-KIRS2-4-1BB and DAP12-CD3 zeta-KIRS 2-4-1BB stimulatory signals, it is desirable to construct viral vectors containing a combination of different stimulatory signals, respectively. In this example, the structure of extracellular recognition antigen is unified with a single-chain antibody targeting B-cell malignancy (CD 19), and the following 8 chimeric antigen receptors (fig. 1 and 2) are respectively required to be constructed:

CD19(scFv)-CD8α-4-1BB-CD3ζ (KT1)

DAP12-T2A-CD19(scFv)-KIRS2 (KT2)

DAP12-4-1BB-CD3ζ-T2A-CD19(scFv)-KIRS2 (KT3)

DAP12-4-1BB-T2A-CD19(scFv)-KIRS2 (KT4)

DAP12-4-1BB-CD3ζ-T2A-CD19(scFv)-KIRS2-4-1BB (KT5)

DAP12-41BB-T2A-CD19(scFv)-KIRS2-4-1BB (KT6)

DAP12-T2A-CD19(scFv)-KIRS2-4-1BB (KT7)

DAP12-CD3ζ-T2A-CD19(scFv)-KIRS2-4-1BB (KT8)

1. synthesis of Gene sequences of chimeric antigen receptors targeting B cell malignancies containing different intracellular stimulatory signals

The synthesis of single chain antibody scFv (CD 19) against B cell malignancy, which contains natural killer activator receptor (DAP 12), T2A, activator co-stimulatory molecule 4-1BB, and anti-B cell malignancy in turn, the structure of which is shown in figure 1, the nucleotide sequence of DAP12 is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of T2A is shown in SEQ ID NO.3, the amino acid sequence is shown in SEQ ID NO.4, the nucleotide sequence of 4-1BB is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6, the nucleotide sequence of single chain antibody (CD 19) scFv against B cell malignancy is shown in SEQ ID NO.7, the amino acid sequence is shown in SEQ ID NO.8, the nucleotide and amino acid sequence of KIRS2 are shown in the patent (targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, publication No. CN 107580628A), the nucleotide sequence of CD8 alpha-4-1 BB-CD3 is shown in the patent No.3, and the amino acid sequence of zeta 9 is shown in the patent No. 35.

2. Construction of lentiviral vectors expressing chimeric antigen receptors

pELNS T2A-CD19-KIRS2 is stored by Nanjing Kanji medical science, inc., or is constructed according to the methods disclosed in the literature (Enxiu Wang et al generation of point T-cell Immunotherapy for Cancer Using DAP-Based, multichain, chimeric immunoacceptors.2015, cancer Immunology Research,3 (7): 815). The following procedure for constructing lentiviral vectors using KT4 as an example chimeric antigen receptor was followed, DAP12-41BB gene synthesis was synthesized by the biological engineering (Shanghai) Limited and provided with pUC57-DAP12-41BB plasmids, plasmids pELNS T2A-CD19-KIRS2 and pUC57-DAP12-41BB were digested with SmaI, bamHI-HF by double digestion (purchased from NEB) and the digestion reaction was performed in accordance with the instructions to obtain a DNA fragment of pELNS T2A-CD19-KIRS2 fragment of about 9000bp and DAP12-41BB fragment of about 500bp, then DNA fragment recovery was performed using agarose gel recovery kit (Tiangen Biochemical Co., ltd.) for specific procedures, and the obtained pELNS T2A-CD19-KIRS2 and DAP12-41BB were recovered, and the desired fragment pELNS 12-41 and the vector fragment pES 19-KTkaRS 2 were ligated by means of T4 ligase (purchased from DAP 4) to obtain chimeric antigen receptor pELNS 2A-KIRS 2 (designated as DAP-41). mu.L of lentiviral vector KT4 was transformed into competent cells of E.coli TOP10 (purchased from Bio-technology Co., ltd. In Nanj, anjie, nanj) and cultured at 37℃for 15 hours, then the selected monoclonal was selected, and after culturing at 37℃for 14 hours, the plasmid was extracted by using a plasmid extraction kit (purchased from Takara, inc.), and the specific method was as described in the specification.

Respectively constructing pELNS CD19-CD8 alpha-41 BB-CD3 zeta (KT 1 for short) according to the method; DAP12-T2A-CD19-KIRS2 (KT 2 for short); DAP12-41BB-CD3 ζ -T2A-CD19-KIRS2 (abbreviated as KT 3); DAP12-41BB-T2A-CD19-KIRS2 (KT 4 for short); pELNS DAP12-41BB-CD3 ζ -T2A-CD19-KIRS2-41BB (abbreviated as KT 5); pELNS DAP12-41BB-T2A-CD19-KIRS2-41BB (KT 6 for short); pELNS DAP12-T2A-CD19-KIRS2-41BB (KT 7 for short); pELNS DAP12-CD3 ζ -T2A-CD19-KIRS2-41BB (KT 8 for short) lentiviral vector.

3. Lentivirus package

The implementation package lentivirus adopts a calcium phosphate transfection method, and the kit used by the method is purchased from Shanghai Biyundian biotechnology Co., ltd, and comprises the following specific steps:

(1) Passage of 293T cells on alternate days

Each T150 cell flask was seeded 5X 10 ⁶ Individual cells. After 48 hours, the cell number should reach 20-25 million/bottle.

(2) 293T cell laying bottle

a) Taking 1T 150 cell flask as an example, cells were gently washed twice with about 15ml of 1 XPBS;

b) 3ml of 0.25% pancreatin-2.21 mM EDTA was added;

c) Waiting for cell shedding, 12ml of 10% (wt) FBS (DMEM medium (from Gibico) available from burning) was added to the cells that had been shed;

d) The cells were collected and transferred to a sterile centrifuge tube, 1000rpm, and centrifuged for 10 minutes;

e) The supernatant was aspirated off and the pellet was resuspended in 10ml DMEM medium of 10% (wt) FBS;

f) Cell count, 12×10 based on cell concentration ⁶ The volume required for the individual cells;

g) The cells were combined with 25ml of DMEM medium containing 10% (wt) FBS, placed in T150 cell flasks and gently shaken to allow uniform distribution of the cells to the bottom of the flasks for overnight incubation in a 5% co2 incubator at 37 ℃.

(3) Cell transfection

Cells were observed to reach approximately 80% -90% cell density, at which point transfection could begin.

a) The culture solution is gently sucked off 30-60 minutes before transfection;

b) Mixing plasmid DNA and calcium chloride solution, taking a T150 bottle as an example, 28ug pRSV. Rev (purchased from Invitrogen corporation), 28ug pGAG-Pol (purchased from Invitrogen corporation), 11ug pVSVG (purchased from Invitrogen corporation), 23ug recombinant lentiviral expression plasmid KT1-KT8, were added to 1.5ml calcium chloride solution, and mixed well;

c) Adding 1.5ml of BBS solution into a 15ml sterile centrifuge tube, uniformly mixing the DNA-calcium chloride solution by using a 1ml gun head, dripping the mixture into the BBS solution, rapidly uniformly mixing the mixture, and incubating the mixture for 25 to 30 minutes at room temperature;

d) The DNA-calcium chloride-BBS mixture was added dropwise to the T150 flask with a 5ml pipette. Culturing in a cell incubator containing 5% carbon dioxide at 37 ℃ for 6h, and changing the liquid;

e) After 6h, the liquid is changed. The plate was gently shaken several times to suspend some of the calcium phosphate precipitate thoroughly, the culture solution containing the calcium phosphate precipitate was aspirated, and 20ml of fresh 5% (wt) FBS DMEM medium was added to continue the culture.

(4) Primary collection of viral supernatants

a) Collecting the culture supernatant of 293T cells transfected the previous day into a centrifuge tube, centrifuging at 1000rpm for 5 minutes, marking, and temporarily storing in a refrigerator at 4 ℃;

b) 20ml of 5% (wt) FBS DMEM medium, preheated beforehand, was added to the cell flask, and the cell incubator was continued to culture overnight at 37 ℃.

(5) Viral supernatant was collected a second time (48 h/fourth day).

(6) Filtering the supernatant

The supernatants collected from the two steps were pooled together and filtered through a 0.45 μm filter to remove cell debris.

(7) Virus concentration

Centrifuge at 12000rpm at 4℃overnight.

(8) Virus storage

After centrifugation, the whole supernatant was poured, resuspended in fresh 5% (wt) FBS in DMEM medium, and virus split-packed and rapidly stored in-80℃refrigerator for further use.

(9) Lentivirus titer assay

a) Virus infection of 293T cells

293T cells were plated in 24-well plates before infection, 200. Mu.L of purified concentrated virus was added to 293T cells, and after 24 hours, the cells were collected by centrifugation at 1500rpm for 5 minutes after 72 hours of infection with DMEM medium containing 10% FBS (wt), and the genome was extracted.

b) Extraction of genome

The genome extraction kit was purchased from Takara company and was operated according to the kit instructions.

c) qPCR assay of viral titres

The reaction system is as follows: probe qPCR Mix 12.5. Mu.L (purchased from Takara), upstream primer 0.5. Mu.L (synthesized by biological engineering (Shanghai) Co., ltd.), downstream primer 0.5. Mu.L (synthesized by biological engineering (Shanghai) Co., ltd.), probe 1. Mu.L (synthesized by biological engineering (Shanghai) Co., ltd.), template 2. Mu.L, sterilized water 8.5. Mu.L, reaction system 25. Mu.L, reaction conditions were set according to the instructions, after the reaction was completed, data were analyzed by analytical software, and virus titer was calculated from standard curve. The results of the calculation showed that the virus titer was 1×10 ⁷ TU/ml。

Example 2 viral infection of T cells

1. Isolated activation of T cells and viral infection

(1) Isolation of human peripheral blood mononuclear cells

Peripheral blood was collected with a blood collection tube containing an anticoagulant, centrifuged at 3000rpm for 30min, and upper plasma was collected and centrifuged at 5000rpm for 10min. Adding into lymphocyte separating liquid (purchased from Tianjin city, ocean biologicals science and technology Co., ltd.) according to volume ratio of 1:1, gradient centrifuging at 3000rpm for 30min, and layering from top to bottom: the first layer is a plasma layer; the second layer is lymphocyte white membrane layer; the third layer is a transparent separating liquid layer; and a fourth layer of red blood cells. The lymphocyte buffy coat was aspirated, washed 2 times with normal saline, centrifuged twice at 1500rpm for 10min, the cells resuspended in PBS, and human peripheral blood mononuclear cells were cultured by adding 5% autologous plasma +300IU/ml recombinant human IL-2 +KBBM581 complete medium.

(2) Lentivirus infection of T lymphocytes

Freshly prepared mononuclear cells PBMC were cultured with complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+KBM581, IL-2 was purchased from R & D Systems, KBM581 was purchased from Corning, CD3/CD28 Dynabeads immunomagnetic beads (purchased from invrotogen) were added to activate T cells on day 0, lentiviral infection was performed for the first 3 days, and corresponding lentiviral vectors KT1, KT2, KT3, KT4, KT5, KT6, KT7, KT8 were added respectively at MOI=1, uninfected T lymphocytes were used as blank control, and after 48 hours the medium was replaced with complete medium containing 5% autologous plasma +300IU/ml recombinant human IL-2+KBM581, and the culture was continued for 7-9 days.

2. Detection of CAR Positive Rate in T cells

The virus-infected T cells cultured to day 7 were centrifuged at 1200rpm for 5min, the supernatant was discarded to collect cells, the cells were resuspended in PBS containing 1% FBS by volume fraction, and the cells were adjusted to a density of 1X 10 ⁵ Biotin-labeled CD19 protein (biosway) was added, and then strepavidin-PE (BD Biosciences) was added, incubated at 4℃for 30min, washed 2 times with PBS, and detected by an up-flow cytometer, and the results showed that after 7 days of incubation, CAR-T cell CAR positive rate: the positive rate of KT1 virus infection group is 41%, the positive rate of KT2 virus infection group is 52%, the positive rate of KT3 virus infection group is 59%, the positive rate of KT4 virus infection group is 65% (figure 3), the positive rate of KT5 virus infection group is 49%, the positive rate of KT6 virus infection group is 59%, the positive rate of KT7 virus infection group is 59%, the positive rate of KT8 virus infection group is 59%, the invention is seenThe virus infection is shown to have better expression.

Example 3 evaluation of in vitro killing effect of virally infected CAR-T cells

(1) Culturing target cells MCF7-CD19 cells (CD 19 positive cell line), 293T (CD 19 negative cell line) and effector cells KT1, KT2, KT3, KT4, KT5, KT6, KT7 and KT8 respectively to obtain 8 groups of CAR-T cells;

(2) Collecting target cells and effector cells, centrifuging at 1500rpm for 5min, and discarding the supernatant;

(3) Resuspension of target cells and effector cells with 10% fbs+1640 complete medium;

(4) Using a real time cell analysis System (RTCA), 50. Mu.L 1640 medium was added to the wells of E-Plate16

(5) Determining that the selected well contacts normally using RTCA assay baseline;

(6) Setting the effective target ratio to be 0:1, 1:1, 5:1 and 10:1;

(7) E-Plate16 was removed, and 50. Mu.L of the well-mixed target cell suspension was added to each well in accordance with the effective target ratio so that the number of cells per well was 10 ⁴ cells/50μL；

(8) E-Plate16 was placed in an incubator at 37℃with 5% CO ₂ Culturing overnight under the condition;

(9) The next day, E-Plate16 was removed, 50. Mu.L of the corresponding effector cells were added, and the killing rate after 8h of addition of effector cells was calculated;

(10)

the killing effect of the CAR-T of the KT3 and KT4 groups on CD19 antigen positive tumor cells is superior to that of the KT1 and KT2 groups, the killing effect of the CAR-T of the KT1 and KT2 groups on CD19 antigen positive tumor cells is superior to that of the KT5 and KT6 groups, the killing effect of the CAR-T of the KT5 and KT6 groups on CD19 antigen positive tumor cells is superior to that of the KT7 and KT8 groups, and the killing effect of the CAR-T of the KT3 and KT4 groups on CD19 antigen positive tumor cells is particularly obviously higher than that of the KT7 and KT8 groups. The results of FIG. 4 show that the in vitro killing experiment result shows that the structural combination of KT3 and KT4 groups has strong anti-tumor activity on the basis of improving the safety of CAR, and the design of the CAR signal region is beneficial to clinical application.

Example 4 detection of cytokine secretion by virally infected CAR-T cells

(1) Cytokine detection was performed using the R & D Elisa kit.

(2) Dilution of standard: preparing 7 centrifuge tubes with 1ml, numbering the centrifuge tubes in sequence, firstly adding 500 mu L of standard substance diluent into each centrifuge tube, then adding 500 mu L of standard substance with original concentration into 1 centrifuge tube with the number being coded, fully and uniformly mixing, and then adding 500 mu L of standard substance diluent into a second centrifuge tube in the centrifuge tube, fully and uniformly mixing; adding 500 mu L of the mixture into a third centrifuge tube, and fully and uniformly mixing; adding 500 mu L of the mixture into a fourth centrifuge tube, and fully and uniformly mixing; adding 500 mu L of the mixture into a fifth centrifuge tube, and fully and uniformly mixing; adding 500 mu L of the mixture into a sixth centrifuge tube, and fully and uniformly mixing; then, 500. Mu.L of the mixture was added to the seventh centrifuge tube, and the mixture was thoroughly mixed.

(3) Standard substance holes are arranged on the enzyme-labeled coated plate, 100 mu L of standard substances with different concentrations are sequentially added, and each concentration is 2-3 parallel holes.

(4) Sample adding: blank holes (blank control Kong Yongshui is used for replacing, enzyme-labeled reagent and biotin-labeled antibody are used as usual) and sample holes to be measured are respectively arranged, 100 mu L of sample is firstly added into the sample holes to be measured on the enzyme-labeled coating plate, the sample is added to the bottom of the holes of the enzyme-labeled plate, the hole walls are not touched as much as possible, and the sample is gently shaken and uniformly mixed.

(5) Incubation: incubate for 2h at room temperature.

(6) Washing: the liquid was discarded, the liquid in the dry well was sucked with a water-absorbent paper, 200. Mu.L of the washing liquid was added to each well, and after standing for 30 seconds, the liquid in the dry well was sucked with a water-absorbent paper, and this was repeated 3 times.

(7) Adding an antibody: 100. Mu.L of detection antibody was added to the enzyme-labeled coated plate.

(8) Incubation: and (5) the same as the operation.

(9) Washing: and (6) the same as the operation.

(10) And (3) marking: 100. Mu.L of horseradish peroxidase labeled streptavidin was added to each well.

(11) Incubation: incubate for 20min at room temperature in the dark.

(12) Washing: and (6) the same as the operation.

(13) Color development: adding 100 mu L of color development liquid into each hole, gently shaking and mixing, and standing at room temperature for 20min in dark place.

(14) And (3) terminating: the reaction was terminated by adding 50. Mu.L of a stop solution to each well.

(15) And (3) measuring: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank value zeroed, and the measurement was performed within 15 minutes after the addition of the stop solution.

Target cells expressing antigen were selected for co-culture with KT4 CAR-T, and the level of IL-2 and IFN-gamma secretion in response to antigen stimulation was detected for KT4 CAR-T, and Raji (CD 19 positive cell line) was selected for target cells, 293T (CD 19 negative cell line) to show that KT4 CAR-T specifically secreted IL-2 and IFN-gamma when stimulated with CD19 antigen, and that KT4 CAR-T significantly secreted IFN-gamma and IL-2 when co-cultured with CD19 positive target cells Raji (FIGS. 5 and 6), indicating that KT4 CAR has a response to antigen positive tumor cells.

On the other hand, when KT1, KT2, KT3, KT4, KT5, KT6, KT7, KT8 CAR-T were compared to the positive target cells Raji, IL-2 and IFN-gamma cytokine levels were secreted from each group. As shown in fig. 5, the secretion levels of KT3 and KT4 CAR-T are significantly improved compared with those of the CAR-T of each other group, and the amount of secreted cytokines is extremely low because of weak anti-tumor effects of KT7 and KT8 CAR-T.

Example 5 anti-tumor Effect of CAR-T cells in mice

Nalm6 cells in 5% CO ₂ Culturing in an incubator at 37 ℃ and the culture medium is DMEM high-sugar culture medium containing 10% FBS. NGG mice were purchased from Jiangsu Jiuzhikang biotechnology limited, 4 week old, body weight (17±2) g, female. 2.5X10 ⁶ A/mL Nalm6 cell suspension was inoculated subcutaneously to the right of the mice in an amount of 0.2 mL/mouse, and the mice were cultured for 20d to form tumors. 2.5X10 ⁷ The CAR-T cell suspension/mL was administered by intravenous injection in an amount of 0.2 mL/mouse, and the tumor volume of the mice was measured at intervals of 3-5 d.

Intravenous injection of PBS (control group) and 8 groups of CAR-T including KT1, KT2, KT3, KT4, KT5, KT6, KT7 and KT8, observation of tumor volume change in mice shows that: mice in the control group died at 16d, whereas the experimental groups KT3 and KT4 had tumor disappearance at 16d, with KT3 and KT4 groups mice having earliest tumor disappearance times in vivo compared to the remaining groups, followed by KT1 and KT2, and again KT5 and KT6 (fig. 7). In mice, 8 groups of CAR-T of different structure had varying degrees of anti-tumor effect on tumors of positive antigens.

Example 6 CAR-T cell surface depletion marker expression level

To evaluate the effect of KT1-KT8 on the depletion marker by antigen stimulation, CD19 antigen positive target cells 293T-CD19 were selected for co-culture with CAR-T at a 1:1 target ratio after 12 days of CAR-T culture, and CAR-T cell surface depletion markers PD-1, lag-3, tim-3 expression were detected after 1 day of co-culture (table 1). In vitro demonstrated minimal expression of PD-1 in KT4, suggesting that KT4 may have a lower inhibition of PD-L1 mediation. Likewise, LAG3 and Tim3 were minimally expressed in KT4, and the results indicate that the levels of depletion markers generated on the surface of CAR-T cells in 8 sets of different structures vary from structure to structure.

TABLE 1 CAR-T cell surface depletion marker expression profiles

	KT1	KT2	KT3	KT4	KT5	KT6	KT7	KT8
									PD1(％)	28.1	26.7	16.3	8.5	33.7	36.4	48.9	44.6
LAG3(％)	30.2	30.8	22.9	10.6	25.3	29.7	33.3	21.4
									Tim3(％)	25.3	14.6	20.8	4.7	21.4	25.3	35.4	34.9

Example 7 CAR-T cell differentiation subtype case

To evaluate the T cell differentiation subtype at day 10 of KT1-KT8 production, the proportion of each cell population was examined Tn, tscm, tcm, tem, temRA by CD3-APC-cy7, CD4-APC, CCR7-FITC, CD45RA-PerCP-Cy5.5, CD45 RO-Amylan (Table 2). The differentiation subtypes of CAR-T cells of different structures in group 8 are more diverse. Among the subtypes representing early memory types are Tscm, tn and Tcm. The results show that KT3 and KT4 memory cells account for a relatively large number, wherein KT3 is mainly CD8+ cells, and KT4 is mainly CD4+ cells.

TABLE 2T cell differentiation subtype cases

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing agricultural university

<120> a chimeric antigen receptor T cell which is synergistic and resistant to depletion and its use in the preparation of a medicament for treating tumors

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 339

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaa 339

<210> 2

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys

<210> 3

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccct 57

<210> 4

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 5

<211> 42

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 6

<211> 126

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 7

<211> 726

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Ala Cys

1 5 10 15

Ala Gly Ala Cys Thr Ala Cys Ala Thr Cys Cys Thr Cys Cys Cys Thr

20 25 30

Gly Thr Cys Thr Gly Cys Cys Thr Cys Thr Cys Thr Gly Gly Gly Ala

35 40 45

Gly Ala Cys Ala Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala

50 55 60

Gly Thr Thr Gly Cys Ala Gly Gly Gly Cys Ala Ala Gly Thr Cys Ala

65 70 75 80

Gly Gly Ala Cys Ala Thr Thr Ala Gly Thr Ala Ala Ala Thr Ala Thr

85 90 95

Thr Thr Ala Ala Ala Thr Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys

100 105 110

Ala Gly Ala Ala Ala Cys Cys Ala Gly Ala Thr Gly Gly Ala Ala Cys

115 120 125

Thr Gly Thr Thr Ala Ala Ala Cys Thr Cys Cys Thr Gly Ala Thr Cys

130 135 140

Thr Ala Cys Cys Ala Thr Ala Cys Ala Thr Cys Ala Ala Gly Ala Thr

145 150 155 160

Thr Ala Cys Ala Cys Thr Cys Ala Gly Gly Ala Gly Thr Cys Cys Cys

165 170 175

Ala Thr Cys Ala Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Cys

180 185 190

Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Ala Ala Cys Ala Gly

195 200 205

Ala Thr Thr Ala Thr Thr Cys Thr Cys Thr Cys Ala Cys Cys Ala Thr

210 215 220

Thr Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Ala Ala

225 230 235 240

Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Cys Ala Cys Thr Thr

245 250 255

Ala Cys Thr Thr Thr Thr Gly Cys Cys Ala Ala Cys Ala Gly Gly Gly

260 265 270

Thr Ala Ala Thr Ala Cys Gly Cys Thr Thr Cys Cys Gly Thr Ala Cys

275 280 285

Ala Cys Gly Thr Thr Cys Gly Gly Ala Gly Gly Gly Gly Gly Gly Ala

290 295 300

Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys Ala Cys

305 310 315 320

Ala Gly Gly Thr Gly Gly Cys Gly Gly Thr Gly Gly Cys Thr Cys Gly

325 330 335

Gly Gly Cys Gly Gly Thr Gly Gly Thr Gly Gly Gly Thr Cys Gly Gly

340 345 350

Gly Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala Thr Cys Thr Gly Ala

355 360 365

Gly Gly Thr Gly Ala Ala Ala Cys Thr Gly Cys Ala Gly Gly Ala Gly

370 375 380

Thr Cys Ala Gly Gly Ala Cys Cys Thr Gly Gly Cys Cys Thr Gly Gly

385 390 395 400

Thr Gly Gly Cys Gly Cys Cys Cys Thr Cys Ala Cys Ala Gly Ala Gly

405 410 415

Cys Cys Thr Gly Thr Cys Cys Gly Thr Cys Ala Cys Ala Thr Gly Cys

420 425 430

Ala Cys Thr Gly Thr Cys Thr Cys Ala Gly Gly Gly Gly Thr Cys Thr

435 440 445

Cys Ala Thr Thr Ala Cys Cys Cys Gly Ala Cys Thr Ala Thr Gly Gly

450 455 460

Thr Gly Thr Ala Ala Gly Cys Thr Gly Gly Ala Thr Thr Cys Gly Cys

465 470 475 480

Cys Ala Gly Cys Cys Thr Cys Cys Ala Cys Gly Ala Ala Ala Gly Gly

485 490 495

Gly Thr Cys Thr Gly Gly Ala Gly Thr Gly Gly Cys Thr Gly Gly Gly

500 505 510

Ala Gly Thr Ala Ala Thr Ala Thr Gly Gly Gly Gly Thr Ala Gly Thr

515 520 525

Gly Ala Ala Ala Cys Cys Ala Cys Ala Thr Ala Cys Thr Ala Thr Ala

530 535 540

Ala Thr Thr Cys Ala Gly Cys Thr Cys Thr Cys Ala Ala Ala Thr Cys

545 550 555 560

Cys Ala Gly Ala Cys Thr Gly Ala Cys Cys Ala Thr Cys Ala Thr Cys

565 570 575

Ala Ala Gly Gly Ala Cys Ala Ala Cys Thr Cys Cys Ala Ala Gly Ala

580 585 590

Gly Cys Cys Ala Ala Gly Thr Thr Thr Thr Cys Thr Thr Ala Ala Ala

595 600 605

Ala Ala Thr Gly Ala Ala Cys Ala Gly Thr Cys Thr Gly Cys Ala Ala

610 615 620

Ala Cys Thr Gly Ala Thr Gly Ala Cys Ala Cys Ala Gly Cys Cys Ala

625 630 635 640

Thr Thr Thr Ala Cys Thr Ala Cys Thr Gly Thr Gly Cys Cys Ala Ala

645 650 655

Ala Cys Ala Thr Thr Ala Thr Thr Ala Cys Thr Ala Cys Gly Gly Thr

660 665 670

Gly Gly Thr Ala Gly Cys Thr Ala Thr Gly Cys Thr Ala Thr Gly Gly

675 680 685

Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala Gly Gly

690 695 700

Ala Ala Cys Cys Thr Cys Ala Gly Thr Cys Ala Cys Cys Gly Thr Cys

705 710 715 720

Thr Cys Cys Thr Cys Ala

725

<210> 8

<211> 242

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 9

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

Claims

1. A chimeric antigen receptor, characterized in that: is shown as KT3 or KT4 below:

KT3：DAP12-4-1BB-CD3ζ-T2A- CD19 scFv-KIRS2

KT4：DAP12-4-1BB-T2A- CD19 scFv-KIRS2

wherein: the nucleotide sequence of DAP12 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the nucleotide sequence of T2A is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4; the nucleotide sequence of 4-1BB is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6; the nucleotide sequence of the anti-CD19 scFv is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO. 8; the amino acid sequence of KIRS2 is: HRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA; the CD3 zeta amino acid sequence is shown as SEQ ID NO.9.

2. A recombinant expression vector comprising the chimeric antigen receptor of claim 1.

3. An immune cell comprising the chimeric antigen receptor of claim 1.

4. The immune cell of claim 3, wherein the cell is selected from the group consisting of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells.

5. Use of the chimeric antigen receptor of claim 1, the recombinant expression vector of claim 2 or the immune cell of claim 3 in the preparation of a medicament for treating a tumor; the tumor includes breast cancer, lymphoma or leukemia.

6. The use according to claim 5, wherein the lymphoma comprises non-hodgkin's lymphoma or hodgkin's lymphoma.