CN107903326B

CN107903326B - Chimeric antigen receptor comprising C3aR intracellular domain, lentiviral vector, expression cell and drug

Info

Publication number: CN107903326B
Application number: CN201810001909.7A
Authority: CN
Inventors: 杜欣; 翁建宇; 赖沛龙; 陈晓梅
Original assignee: Guangdong General Hospital Guangdong Academy of Medical Sciences
Current assignee: Guangdong General Hospital Guangdong Academy of Medical Sciences
Priority date: 2018-01-02
Filing date: 2018-01-02
Publication date: 2020-06-30
Anticipated expiration: 2038-01-02
Also published as: CN107903326A; WO2019134213A1

Abstract

The invention provides a chimeric antigen receptor comprising a C3aR endodomain, a lentiviral vector, an expression cell and a drug, wherein the chimeric antigen receptor comprises a C3aR endodomain comprising an ectodomain capable of binding an antigen, a transmembrane domain and at least one endodomain selected from a C3aR endodomain or an endodomain of a signaling region in tandem with a C3aR endodomain. The chimeric antigen receptor can improve T17 cell subset, eliminate the effect of immunosuppression of regulatory T cells, remarkably improve the in vitro killing efficiency of tumor target cells, remarkably improve the killing effect of second generation CAR T cells on tumors, and provide a new idea and selection for the CAR-T cell treatment field.

Description

Chimeric antigen receptor comprising C3aR intracellular domain, lentiviral vector, expression cell and drug

Technical Field

The invention belongs to the technical field of cell immunotherapy of tumors, and particularly relates to a chimeric antigen receptor containing a C3aR intracellular domain, and also relates to nucleic acid and application of the chimeric antigen receptor containing a coding C3aR intracellular domain.

Background

Chimeric Antigen Receptor (CAR) T cells are a milestone event expected to cure tumors, and CAR-T technology is a gene engineering technology that transfects a segment of single chain antibody (scFv) that recognizes tumor antigens and intracellular activation motif recombinant genes onto T lymphocytes to achieve better tumor recognition and killing effects. CAR-T molecules typically include an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Wherein the extracellular hinge region is formed by connecting the heavy chain variable region and the light chain variable region of the single-chain antibody through a peptide segment; the transmembrane region is mainly derived from a molecular transmembrane region such as CD8, CD28 or 4-1 BB; the intracellular signal region is mainly from intracellular segment chimeras of T cell signaling molecules such as CD28, 4-1BB, CD3zeta, CD27 or OX-40. The high avidity, high specificity scFv determines that CAR-T targets a certain antigen, and once bound to the antigen, the intracellular segment of the CAR transmits activation and costimulatory signals to T cells, activating the T cells and thus effectively targeting and killing tumor cells. Two activation signals are required for the activation of T cells, namely a first signal for the binding of TCR-CD3 on the surface of the T cells and MHC-I molecules to be activated, and the killing activity of the T cells on tumor cells is determined; the binding of costimulatory molecules on the surface of T cells to the corresponding ligands is the second signal for activation, determining T cell proliferation. Briefly, CAR-T cells recognize specific molecules on the surface of tumor cells through an antigen-antibody recognition pattern, and then activate, proliferate and exert cell killing functions through their intracellular signaling.

The primary CAR molecule has a structure comprising scFv for recognizing tumor cell surface antigen, a transmembrane domain and an intracellular domain of TCR complex CD3zeta for activating T cells, the primary CAR-T cells have short survival time in vivo, have weak killing power on tumor cells and are not ideal in curative effect, the primary CAR molecule has an enhanced ability to activate T cells in order to fully simulate a dual-signaling model of T lymphocyte activation in human bodies, the secondary CAR molecule starts to generate the secondary CAR, the secondary CAR molecule mainly has 1 costimulatory signaling domain added in an intracellular segment domain, the secondary CAR reported co-stimulatory ligands are mainly concentrated on antigen presenting cells of associated co-stimulatory molecules on specific binding T cells, including CD7, B7-1(CD80), B7-2(CD86), PD-L1, PD-L137, 4-1, CAR-40, CAR OS-L296, CAR, CD 2011-7, CD-8-7-T lymphocytes are more excellent than the primary lymphocyte stimulating T-cell proliferation, the primary lymphocyte stimulating T-cell stimulating effect, the primary lymphocyte stimulating T-cell stimulating effect, the primary lymphocyte stimulating T-cell stimulating effect of CD-T cell in vitro, the primary lymphocyte stimulating T cell, the primary lymphocyte stimulating T-7 cell stimulating effect, the primary lymphocyte stimulating T-T cell stimulating effect of CD-cell, the primary lymphocyte stimulating T cell stimulating T-cell stimulating effect, the primary lymphocyte stimulating T cell stimulating T-7 cell, the primary lymphocyte stimulating T-cell stimulating effect of the primary lymphocyte stimulating T-T cell stimulating T-7 cell stimulating T cell, the stimulating T cell stimulating T-cell stimulating effect of the stimulating T cell expressing the stimulating effect, the stimulating T cell stimulating effect, the stimulating cell stimulating effect of the stimulating T-7 cell stimulating T cell expressing the stimulating effect of the stimulating T cell expressing the stimulating cell in vivo, stimulating effect of the stimulating T cell expressing the stimulating effect of the stimulating cell expressing the stimulating effect of the stimulating effect of leukemia cell expressing the stimulating effect of human body, stimulating effect of the stimulating cell expressing the stimulating cell expressing the stimulating effect of leukemia cell expressing the stimulating effect of the stimulating cell expressing the stimulating cell expressing the stimulating cell expressing the stimulating cell expressing the stimulating cell.

The complement system has important biological effects during the innate immune response and the adaptive humoral immune response. The inherent components of complement in vivo exist in the form of precursor enzyme under physiological conditions, and after being activated, active molecular fragments such as C3a, C4a and C5a can be generated, thereby exerting biological immunological effect. During this activation, eventually a membrane-attacking complex is synthesized, attacking the microorganism or other target cell, causing lytic rupture of the target cell. These complement lysates are mainly mediated by Complement Receptors (CR): c3aR and C5aR bind to exert immunological functions. The complement has a close mutual regulation relation with CD4+ T cells, and the C3aR complement pathway promotes Th17 differentiation and simultaneously inhibits the generation of regulatory T cells. The C3aR is used for constructing the chimeric antigen receptor together with other co-stimulatory molecules, and how the effect of the constructed chimeric antigen receptor is expected is further unexpected.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a chimeric antigen receptor comprising a C3aR endodomain cell;

it is another object of the present invention to provide a lentiviral vector comprising a chimeric antigen receptor comprising a C3aR endodomain cell;

the third invention is to provide the application of the chimeric antigen receptor containing the C3aR intracellular domain cells or the lentiviral vector in preparing immune cells of the chimeric antigen receptor containing the C3aR intracellular domain cells;

the fourth object of the present invention is to provide an immune cell expressing a chimeric antigen receptor comprising a C3aR endodomain cell, prepared using the chimeric antigen receptor comprising a C3aR endodomain cell or the lentiviral vector;

the fifth purpose of the present invention is to provide an application of the chimeric antigen receptor containing the intracellular domain cell of C3aR or the lentiviral vector in the preparation of a drug for targeting the chimeric antigen receptor containing the intracellular domain of C3aR to tumor cells.

The invention realizes the aim through the following technical scheme: in a first aspect, the present invention provides a chimeric antigen receptor comprising an ectodomain capable of binding an antigen, a transmembrane domain and at least one endodomain selected from the intracellular domain of C3aR or a signaling region in tandem with the intracellular domain of C3 aR. As used herein, "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process. And the at least one intracellular domain refers to the intracellular domain of C3aR, or other signaling region in tandem with the intracellular domain of C3aR, such as the intracellular domains of CD3 ζ, CD28, 4-1BB, etc.

Preferably, the above CAR molecule is a tumor antigen, such as 5T4, α 5 β 1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β -catenin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK 80/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB 80, ELF 280, EMIN, EpCam, ETV 80-AML 80, AMG 250, GAGE, MPRT, Gp100, HARAR, HER-2/new, HLA-A0201-80, MUNL-80, MUTRPR-80, MUT-80, MAGNER-80, MAGN-80, VEGF-MAG-80, VEGF-7, VEGF-19, VEGF-7, VEGF-80, VEGF-19, VEGF-80, VEGF-3, VEGF-80, VEGF-36.

Preferably, the extracellular domain capable of binding an antigen refers to a single chain variable fragment of an antibody that binds to a targeted antigen. In particular embodiments, the CAR molecule described above may have as its intracellular domain only the C3aR intracellular domain, and may further comprise one or more (e.g. 2 or 3) other intracellular domains in addition to the C3aR intracellular domain. For example, in a preferred embodiment, the intracellular domain comprises, in addition to the C3aR intracellular domain, a 4-1BB, CD3 ζ intracellular domain; more preferably, the intracellular domain of C3aR is disposed at the C-terminal side of the intracellular domain of CD3 ζ.

In a specific embodiment, the intracellular domain is a 4-1BB intracellular domain, a CD3 ζ intracellular domain, and a C3aR intracellular domain which are connected in this order from the N-terminal side. Furthermore, the chimeric antigen receptor of the present invention also encompasses the case where the intracellular domain thereof comprises two or more intracellular domains connected in series; and, alternatively, the C3aR endodomain may be disposed on the N-terminal side of the chimeric antigen receptor endodomain. In a preferred embodiment, the chimeric antigen receptor comprises, in order from the N-terminal side, a single chain variable region of an anti-tumor antigen antibody as an extracellular domain, a transmembrane domain of CD28 molecule and an intracellular domain of 4-1BB, an intracellular domain of CD3 ζ and an intracellular domain of C3 aR.

The intracellular domain of C3aR contains the nucleotide sequence shown in the sequence table 1. The encoding nucleic acid of the chimeric antigen receptor contains the sequence described in the sequence table 1, but is not limited to the sequence, and other encoding nucleic acids of C3aR are also included. The intracellular domain of the signaling region in tandem with the intracellular domain of C3aR is selected from one or more of CD3 ζ, CD28, and 4-1 BB.

In a second aspect, the present invention provides a lentiviral vector expressing a receptor comprising a chimeric antigen as described above.

In a third aspect, the present invention provides a chimeric antigen receptor-expressing cell into which the chimeric antigen receptor according to the first aspect is introduced; preferably, the cell is a T cell or a population of cells comprising T cells.

The present invention provides a method for producing the chimeric antigen receptor-expressing cell according to the third aspect, which comprises the step of introducing the chimeric antigen receptor according to the first aspect into a cell; preferably, the cell is a T cell or a population of cells comprising T cells.

In a fourth aspect, the present invention provides a use of the chimeric antigen receptor of the first aspect, or the lentiviral vector of the second aspect, or the chimeric antigen receptor-expressing cell of the third aspect, in the preparation of a medicament for the treatment of a tumor.

Preferably, the tumor is a solid tumor or a hematological tumor.

In a particular embodiment of this use provided herein, the tumor is B-ALL. Notably, the CAR of the invention is characterized in that it comprises as its intracellular domain the C3aR intracellular domain. The C3aR intracellular domain includes variants thereof having the same function. The term "variant" refers to any variant comprising one or several to several amino acid substitutions, deletions or additions, provided that the variant retains substantially the same function as the original sequence.

The chimeric antigen receptor can improve T17 cell subset, eliminate the effect of immunosuppression of regulatory T cells, remarkably improve the in vitro killing efficiency of tumor target cells, remarkably improve the killing effect of second generation CAR T cells on tumors, and provide a new idea and selection for the CAR-T cell treatment field.

Drawings

FIG. 1a is a schematic representation of a viral vector containing a combination of intracellular domains of C3 aR.

FIG. 1b is a schematic representation of a viral vector that does not contain a combination of the intracellular domains of C3 aR.

FIG. 2 is a graph of the results of the in vitro killing effect of GFP T, CAR19T and CAR19C3aR T cells on CD19 expressing NALM6-GL cells, showing that CAR19C3aR T is superior to GFP T, CAR19T, in which NALM6 represents NALM6-GL cells.

Figure 3 is a graph of the results of the killing effect of GFP T, CAR19T and CAR19C3aR T cells on CD19 expressing Raji cells in vitro, showing that CAR19C3aR T is superior to GFP T, CAR 19T.

FIGS. 4a and 4b are graphs showing the effect of GFP T, CAR19T and CAR19C3aR T cells on T17 cell subsets (CD4+ IL17A +), regulatory T cell subsets (CD4+ CD25+ FoxP3+), and the results show that CAR19C3aR T can significantly increase T17 cell subsets and eliminate immunosuppression on regulatory T cells.^*P<0.05。

Figure 5 is a graph of the results of GFP T, CAR19T and CAR19C3aR T cell loading on NALM6 tumor cells (CD19+) in NCG mice, showing that CAR19C3aR T is superior to GFP T, CAR 19T.^*P<0.05。

Figure 6 is a BLI image showing the load and location of NALM6 cells in NCG mice.

FIG. 7 is a graph showing the effect of GFP T, CAR19T and CAR19C3aR T cells on the growth phase of NCG mice, and the results show that CAR19C3aR T significantly prolongs the growth phase of NCG mice more than GFP T and CAR 19T.^*P<0.05。

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that various changes and/or modifications may be made without departing from the scope of the invention. The specific conditions not specified in the examples are usually carried out under the conditions described in the conventional conditions or under the conditions recommended by the manufacturers.

Example 1 preparation of plasmid containing anti-CD 19ScFv-4-1BB-CD3 ζ -C3aR (CAR19C3aR)

A plasmid carrying a chimeric antigen receptor gene containing the intracellular domain of C3aR according to the present invention was prepared as follows:

(1) the plasmid pUC57-CAR19 gene containing anti-CD 19ScFv-4-1BB-CD 3zeta (CAR19) obtained by means of gene synthesis, molecular cloning and the like comprises anti-CD 19 monoclonal antibody ScFv, CD28 transmembrane region, and 4-1BB, CD3zeta intracellular region, namely CD19ScFv-4-1BB-CD3 zeta.

(2) The obtained pUC57-CAR19 plasmid was digested with endonuclease Pmel and Sepl to obtain CAR19 gene, and then CAR19 gene was ligated to lentiviral vector pWPXld-GFP to construct pWPXld-CAR 19-GFP.

(3) The obtained pWPXld-CAR19-GFP plasmid was digested with the endonucleases NotI and SpeI to obtain the intracellular fragment 4-1BB-CD3 ζ of CAR19 gene.

(4) 4-1BB-CD3 ζ -C3aR was obtained by overlap extension PCR using 4-1BB-CD3 ζ -tandem cDNA of the intracellular signaling domain of C3aR as a template to construct 4 primers.

(5) The pUC57-CAR19C3aR plasmid was obtained by digesting with NotI and SpeI, replacing 4-1BB-CD 3zeta in pUC57-CAR19 with 4-1BB-CD3 zeta-C3 aR.

(6) Finally, by the restriction of endonuclease PmeI and SpeI, the CAR19 in pWPXLD-CAR19-GFP is replaced by CAR19C3aR, and the pWPXLD-CAR19C3aR-GFP plasmid is obtained. (FIG. 1)

Example 2 lentiviral packaging of CAR plasmid

Three lentiviruses expressing GFP (blank control), CAR19-GFP (control), CAR19C3aR-GFP, respectively, were obtained by lentivirus packaging using the inventive CAR plasmid prepared in example 1 and the related control plasmid. In this examples 2 and 3, the CAR-containing plasmid was collectively described as a pwxld-CAR-GFP plasmid, and the CAR-overexpressing lentivirus was collectively described as a CAR lentivirus.

The method comprises the following specific steps:

(1) culturing 293T cells in a 10cm culture dish in a DMEM high-sugar medium, 10% FBS (fetal bovine serum) and 1% double antibody (100 × penicillin-streptomycin mixed solution);

(2) when the density of 293T cells in a 150mm culture dish reaches 80-90%, the culture medium is replaced: DMEM high-glucose medium + 1% FBS + 1% double antibody;

(3) after 2-6 hours of medium change culture, pwxld-CAR-GFP plasmid (i.e., CAR19, CAR19C3aR, respectively) or the blank plasmid pwxld-GFP, respectively, were co-transfected with PEI into 293T cells along with lentiviral packaging helper plasmids pmd2.g, psPAX2, respectively, and the reagents and doses as given in table 1 below were added:

TABLE 1

Reagent	Dosage form
		pWPXld-CAR-GFP two plasmids or control plasmid	9ug
pMD2.G helper plasmid	3ug
		psPAX2	12ug
PEI	72ug

(4) At 24, 48 and 72 hours after transformation, respectively, culture medium supernatants were collected and fresh medium (DMEM high-glucose medium + 1% FBS + 1% diabody) was added;

(5) after collecting the culture medium supernatant, centrifuging 2500g of the supernatant for 0.5 hour;

(6) taking the centrifugal supernatant, filtering the centrifugal supernatant by using a 0.45um filter, and centrifuging the centrifugal supernatant for 1.5 hours by using a super high speed centrifuge at 28000 rpm;

(7) after ultra-high speed centrifugation, gently removing the supernatant, adding 200ul PBS, and dissolving at 4 ℃ for 12-16 hours to obtain 2CAR lentiviruses or blank control GFP lentiviruses;

(8) after the virus is dissolved, collecting the virus and subpackaging the virus in a freezing tube, and freezing and storing the virus at the temperature of minus 80 ℃ for later use.

Example 3 infection of human T cells with packaged CAR Virus

(1) Separation and purification of T cells: separating mononuclear cells in blood by a Ficoll density gradient method, removing red blood cells by cracking a red blood cell lysate, and then separating T cells by MACS Pan-T magnetic beads;

(2) the sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100U/ml + streptomycin 0.1mg/ml) to a cell concentration of 2.5 × 10⁶Each ml is ready for use;

(3) t cells are stimulated by magnetic beads (product source: America and Germany) coated with CD2, CD3 and CD28 antibodies, namely the coated magnetic beads are mixed with the T cells in a ratio of 1:2, and the final density of the T cells is 5 × 10⁶Pieces/ml/cm 2. After mixing, the mixture was cultured and stimulated in a 5% CO2 incubator at 37 ℃ for 48 hours.

(4) Lentivirus-transfected T cells: the beads in the activated T cell-bead mixture were removed by magnetic field, centrifuged at 300g for 5min, the supernatant removed, resuspended in fresh medium, and added with CAR-and GFP-expressing lentiviruses (virus loading MOI 10) respectively, followed by 8. mu.g/ml polybrene and 300IU/ml IL-2. Culturing at 37 deg.C in 5% CO2 incubator for 24h, centrifuging at 300g for 5min, removing supernatant, and resuspending with fresh culture medium containing 300IU/ml IL-2 to obtain over-expression CAR T cell.

(5) CAR T cell expansion maintaining CAR T cell density at 1 × 10⁶About one/ml, and half liquid change is carried out every 2 to 3 days. After two weeks, CAR T cell numbers can expand 100-fold. GFP positive cells are cells which are transfected successfully, and the GFP positive proportion is detected by flow type, so that the proportion of 2CAR T cells (CAR 19-GFP and CAR19C3aR-GFP respectively) or blank control T cells (GFP-T) is obtained. The effect on Th17 cell subset (CD4+ IL17A +) and regulatory T cell subset (CD4+ CD25+ FoxP3+) was examined.

Regulatory T cell subpopulation detection method 1. preparation of effector cells (GFP-T, CAR19-GFP or CAR19C3aR-GFP T cells); adding surface antibody 5ul CD4-PERCP, 5ul CD25-PE and corresponding isotype antibody, incubating at 4 deg.C in dark for 30min, adding 1ml PBS for washing, centrifuging at 300g for 5min, removing supernatant, adding membrane-breaking fixer (membrane-breaking agent: diluent: 1:3, fresh preparation) 1ml, and incubating at 4 deg.C in dark for 35 min. After 2.35min, 2ml of membrane breaking buffer (buffer: pure water 1:9) was added, mixed well, and centrifuged at 300g for 5 min. 3. The supernatant was removed, 5ul of the FoxP3 antibody was added, and the cells were incubated at 4 ℃ for 30min in the absence of light. After 4.30min, 2ml PBE was added for washing, and the mixture was centrifuged at 300g for 5 min. 5. Each tube was resuspended in 400ul PBS and tested on the machine.

Detection method of Th17 cell subpopulation: 1. effector cells (GFP-T, CAR19-GFP or CAR19C3aR-GFP T cells) were prepared, surface antibody 5ul CD4-PERCP was added, mixed well, protected from light, and incubated for 30 min. 2. 100ul of the membrane breaking agent A was added and incubated at room temperature for 15 min. 3. Adding 3ml PBS + 0.1% NaN3+ 5% FBS.4.300g, centrifuging for 5min, removing supernatant, adding buffer B, mixing, adding 5ul IL-17A APC-Cy7, and incubating for 20 min. 5. 3ml PBS + 0.1% NaN3+ 5% FBS.300g was added, centrifuged for 5min, the supernatant was removed, and 400 pieces of PBE were resuspended in a machine for testing.

Example 4 in vitro recognition of the effects of CAR T cells on killing tumors

GFP T (blank control), CAR19-GFP T (control), CAR19C3aR-GFP T cells prepared in example 3 were mixed with 5X10 at different ratios⁴The tumor cells of (2) were mixed and added to a 96-well U-plate, each groupSetting 3 multiple wells, setting a single tumor cell group as a positive control, centrifuging for 5min at 250g, and CO-culturing in a 37-degree 5% CO2 incubator for 24 h; when the recognition and killing functions of GFP T, CAR19-GFP T and CAR19C3aR-GFP T cells on blood tumors are compared in vitro, the tumor cells are selected from NALM6-GL and Raji leukemia or lymphoma cell lines.

The CFSE/PI double-staining cytotoxicity detection method is used for evaluating and quantitatively evaluating killing efficiency: 1. preparing effector cells (GFP T (blank), CAR19-GFP T (control), CAR19C3aR-GFP T cells) and target cells, counting, and calibrating GFP% of CAR T cells with the same batch of WT cells to make the GFP% of each group similar in size; dilution of T cells 8X10 with 1640+ 10% FBS + 1% P/S Medium⁶And/ml. 2. Resuspending the desired amount of target cells (Nalm6 cells, Raji cells) in 1ml 1640 medium, adding 5umol CSFE, incubating at 37 deg.C in dark for 20min, adding 10ml medium, terminating for 5min, centrifuging, removing supernatant, and diluting the target cells to 5X10⁵And/ml. 3. Diluting T cells in a multiple ratio, adding 100ul of fresh culture medium into a V-shaped bottom 96-well plate in advance by using a row gun, adding 100ul of T cells into the first row, adding 100ul of T cells into the second row, blowing and beating the second row for more than 5 times by using the row gun, sucking 100ul of T cells, putting the third row, repeating the steps, diluting the T cells in multiple ratios along the rows, adding 100ul of target cells into all the holes, and mixing the T cells and the target cells in different ratios (16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1: 4). 4. The mixed cells were cultured in an incubator for 24 hours. 5. Cells in 96-well plates were aspirated and centrifuged. 6. Washing with PBS at 4 deg.C once, adding 100ul 1xIP Buffer, adding 5ul PI, incubating for 15min in dark place, adding 200ul1xIP Buffer, and detecting in machine within 1 hr. 7. And (3) calculating the killing rate: the target cell wells without T cells were used as reference, and the percentage of killing was target well CSFE + PI +% -control well CSFE + PI +%. The results show that both CAR19C3aR T and the in vitro killing efficiency against CD19 expressing tumor target cells are significantly higher than CAR19T cells (see fig. 2 and 3).

Example 5 recognition of CAR19C3aR T cells in vivo to kill tumors

To compare the in vivo tumor killing recognition effects of CAR19T, CAR19C3aR T cells, an equivalent number (5)×10⁵) The NALM6 cells (expressing luciferase) were isolated by tail vein using 7 NCGs (NOD/SCID IL2 rg)^-/-) In immunodeficient mice, 5 × 10 cells were transplanted on

days

2 and 8 after NALM6 cell transplantation (day 0 on the day of tumor cell transplantation)⁶Individual T cells (four groups: GFP T, CAR19T, CAR19C3aR T, 6 mice per group) were injected intravenously into NSI immunodeficient mice into which NALM6 cells had been transplanted. Since NALM6 expresses luciferase, substrate D-luciferin is injected into the abdominal cavity for 200ul, the time is counted for 5 minutes, the mice are placed into an anesthesia chamber attached to in vivo imaging and are anesthetized through isoflurane, the anesthetized mice are placed into an imaging instrument for imaging, the imaging is carried out through an automatic exposure mode, the mice are placed back into a cage for feeding after imaging, and the score data result shows that CAR19C3aR T can obviously reduce the load of NALM6 tumor cells in NCG mice (see figures 5 and 6), the survival period of the mice is continuously observed and the mice die directly, and finally, the CAR19C3aR T can obviously prolong the growth period of the NCG mice (figure 7). It was suggested that the addition of the intracellular domain of C3aR significantly improved the killing effect of second generation CAR T cells (CAR19T) on tumors. Through the experimental results, the strength of the CAR T in the experimental group and the CAR T in the control group to the tumor recognition killing function is compared, and the improvement of the intracellular signaling domain of C3aR to the in-vivo and in-vitro tumor killing function of the CAR T is verified.

The applicant claims that the product, use and use of the invention are illustrated by the above embodiments, but the invention is not limited to the above detailed use or use, i.e. it is not meant that the invention must rely on the above detailed use and use to be tested. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitution of each raw material and addition of auxiliary components to the product of the present invention, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

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Claims

1. A chimeric antigen receptor comprising a C3aR endodomain, comprising an extracellular domain capable of binding an antigen, a transmembrane domain and an intracellular domain, wherein the intracellular domain is a 4-1BB intracellular domain, a CD3 ζ intracellular domain and a C3aR intracellular domain which are connected in this order from the N-terminal side, the extracellular domain capable of binding an antigen is a CD19ScFv, the transmembrane domain is CD28, and the nucleotide sequence of the C3aR intracellular domain is as shown in sequence Table 1.

2. A lentiviral vector expressing a chimeric antigen receptor of claim 1.

3. A cell expressing a chimeric antigen receptor, characterized by: the chimeric antigen receptor of claim 1 is introduced.

4. A medicament for preventing or treating a tumor comprising the chimeric antigen receptor of claim 1, or the lentiviral vector of claim 2, or the cell of claim 3.