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

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, Lentiviral Vector, Expression Cell Containing the C3aR Intracellular Domain and drugs

technical field

The invention belongs to the technical field of cellular immunotherapy of tumors, in particular to a chimeric antigen receptor comprising a C3aR intracellular domain, and also to nucleic acids and applications comprising a chimeric antigen receptor encoding the C3aR intracellular domain.

Background technique

Chimeric Antigen Receptor (CAR, Chimeric Antigen Receptors) T cells are a milestone event expected to cure tumors. CAR-T technology is the application of genetic modification technology to combine a single chain fragment variable (scFv) that recognizes tumor antigens. The internal activation motif recombinant gene is transfected into T lymphocytes to achieve better recognition and killing of tumors. CAR-T molecules usually include extracellular hinge region, transmembrane region and intracellular signal region. The extracellular hinge region is formed by connecting the heavy chain and light chain variable regions of a single-chain antibody through a peptide segment; the transmembrane region is mainly derived from the transmembrane region of molecules such as CD8, CD28 or 4-1BB; The intracellular signal region mainly comes from the intracellular segment chimera of T cell signaling molecules such as CD28, 4-1BB, CD3zeta, CD27 or OX-40. The scFv with high affinity and high specificity determines that CAR-T targets a certain antigen. Once bound to the antigen, the intracellular segment of the CAR will transmit activation and co-stimulatory signals to T cells, which will activate T cells and effectively target and kill tumor cells. The activation of T cells requires two activation signals, that is, the combination of TCR-CD3 on the surface of T cells and MHC-I molecules is the first signal of activation, which determines the killing activity of T cells on tumor cells; costimulatory molecules on the surface of T cells are related to the corresponding Ligand binding is the second signal of activation and determines T cell proliferation. In short, CAR-T cells recognize specific molecules on the surface of tumor cells through the antigen-antibody recognition mode, and then activate, proliferate and exert cell killing functions through their intracellular signaling.

The structural design of CAR molecules has undergone many generations of research and development. The structure of the first-generation CAR molecules contains an scFv that recognizes tumor cell surface antigens, a transmembrane domain, and an intracellular domain of CD3zeta, a TCR complex that activates T cells. Because the signaling domain of the first-generation CAR is a single signaling molecule and lacks a costimulatory signal, the first-generation CAR-T cells have a short survival time in vivo, have weak killing power to tumor cells, and have an unsatisfactory curative effect. In order to fully simulate the dual-signal model of T lymphocyte activation in the human body and enhance the ability of the first-generation CAR molecules to activate T cells, the second-generation CAR was developed. The second-generation CAR mainly added a costimulator to the intracellular segment domain. Signaling domain, the costimulatory ligands that have been reported so far mainly focus on antigen-presenting cells that specifically bind to associated costimulatory molecules on T cells, including CD7, B7-1 (CD80), B7-2 (CD86), PD-L1 , PD-L2, 4-1BB, OX-40, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HEVM, lymphotoxin beta receptor, ILT3, ILT4, HVEM, and then Enhance its ability to recognize tumor antigens and kill T lymphocytes (MaQ, GomesEM, LoAS, et al. Advanced generation anti-prostate specific membrane antigen designer Tcells for prostate cancer immunotherapy [J]. Prostate, 2014, 74(3): 286–296). Savoldo et al. constructed 2 anti-CD19-CAR-T cells for 6 patients with B-cell lymphoma. One CAR has only a single signaling molecule ζ chain (first-generation CAR-T) in the signaling domain, and the other is added at the same time. ζ chain and co-stimulatory molecule CD28 (second-generation CAR-T). The results of in vitro culture and testing in this study showed that the second-generation CAR-T cells containing the CD28 signal chain showed significant advantages in terms of in vitro proliferation and cytokine secretion levels. . These two types of cells were injected back into the patient at the same time, and the number of cells was regularly detected. The results showed that the in vivo proliferation rate and survival time of the second-generation CAR-T cells were better than those of the first-generation CAR-T cells (SavoldoB, RamosCA, LiuE, eta1.CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients [J]. ClinInvest, 2011, 121(5): 1822–1826). The same study also confirmed that the second-generation CAR-T containing the CD137 (4-1BB) signal chain has stronger killing effect and persistence than the first-generation CAR-T (Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in Leukemia. NEngL J M. 2014;371:1507-1517). Although CAR-T cell clinical trials have achieved efficacy, there is still room for further improvement, such as further enhancing its activation and cell killing functions, thereby improving the efficacy of CAR-T therapy. The third generation of CAR-T integrates two or more costimulatory molecular signals on the basis of the first generation. Different combinations of costimulatory signals may affect the function and efficacy of CAR-T cells. Studies have shown that not all The third generation CAR-T is better than the second generation. The third-generation CAR-T has been used in clinical trials of mantle cell lymphoma (MCL) and follicular non-Hodgkin lymphoma (NHL), but it has not shown better clinical outcomes than the second-generation. At present, the most successful clinical application in acute leukemia is mainly the second-generation CAR-T technology. One of the co-stimulatory molecules transfected is mainly CD28 or CD137 (4-1BB), and the other co-stimulatory molecule is CD3zeta. It can be seen that the structural design of CAR in the existing technology is not very mature, and it is necessary to further improve the CAR molecule in terms of enhancing the killing function of T cells, enhancing the Th17 cell subset, and eliminating the immunosuppression of regulatory T cells.

In the process of innate immune response and adaptive humoral immune response, the complement system has important biological effects. The intrinsic components of complement in the body exist in the form of pro-enzymes under physiological conditions, and when activated, they can produce active molecular fragments, such as C3a, C4a, and C5a, thereby exerting biological immunological effects. During this activation process, membrane attack complexes are eventually synthesized to attack microorganisms or other target cells, resulting in the lytic rupture of target cells. These complement cleavage products mainly exert immunological functions by binding with complement receptors (CR): C3aR and C5aR. There is a close mutual regulation relationship between complement and CD4+T cells. The C3aR complement pathway promotes Th17 differentiation and inhibits the production of regulatory T cells. Using C3aR in combination with other costimulatory molecules for the construction of chimeric antigen receptors, it is even more unpredictable how effective the constructed chimeric antigen receptors will be.

SUMMARY OF THE INVENTION

In view of this, one of the objects of the present invention is to provide a chimeric antigen receptor comprising a C3aR intracellular domain cell;

Another object of the present invention is to provide a lentiviral vector comprising a chimeric antigen receptor in the intracellular domain of C3aR;

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

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

The fifth object of the present invention is to provide the chimeric antigen receptor comprising the intracellular domain of C3aR or the lentiviral vector for the preparation of the chimeric antigen receptor comprising the intracellular domain of C3aR targeting tumor cells. applications in .

The present invention achieves the above objects through the following technical solutions: In the first aspect, the present invention provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and at least one intracellular domain capable of binding an antigen, so The intracellular domain is selected from the C3aR intracellular domain or the intracellular domain of a signaling region in tandem with the C3aR intracellular domain. Here, "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 biological processes. The at least one intracellular domain refers to the intracellular domain of C3aR, or the intracellular domain of other signaling regions connected in series with the intracellular domain of C3aR, such as CD3ζ, CD28, 4-1BB, and the like.

For the above-mentioned CAR molecule, preferably, the antigen can be a tumor antigen, such as: 5T4, α5β1-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, CDK4/ m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnTV, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, Myosin/m, MUC1, MUM-1, MUM-2, MUM3, NA88-A, PAP, Protease-3, p190minor bcr-abl, Pml/RARα, PRAME, PSA , PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, NY-Eso-1 or NY-Eso-B, etc.; further preferably, the tumor antigen is CD19. The antigens described in the patent of the present invention can also be inflammatory cell surface molecules that appear in autoimmune diseases or TCRs that cause autoimmunity.

Preferably, the extracellular domain capable of binding an antigen refers to a single-chain variable fragment that binds an antibody targeting the antigen. In a specific embodiment, the above-mentioned CAR molecule may only use the C3aR intracellular domain as its intracellular domain, and may also include one or more (eg 2 or 3) other cells in addition to the C3aR intracellular domain internal domain. For example, in a preferred embodiment, in addition to the C3aR intracellular domain, the intracellular domain also includes 4-1BB, CD3ζ intracellular domain; further preferably, the C3aR intracellular domain is configured in CD3ζ C-terminal side of the intracellular domain.

In a specific embodiment, the intracellular domain is a 4-1BB intracellular domain, a CD3ζ intracellular domain and a C3aR intracellular domain linked in order from the N-terminal side. Furthermore, the chimeric antigen receptor of the present invention also encompasses the case where its intracellular domain comprises two or more intracellular domains connected in tandem; and, alternatively, the C3aR intracellular domain may be Arranged on the N-terminal side of the chimeric antigen receptor intracellular domain. In a preferred embodiment, the chimeric antigen receptor includes, from the N-terminal side, the single-chain variable region of an anti-tumor antigen antibody as the extracellular domain, the transmembrane domain of the CD28 molecule, and the 4-1BB cytoplasmic domain. Intradomain, CD3ζ intracellular domain, C3aR intracellular domain.

The C3aR intracellular domain contains the nucleotide sequence shown in Sequence Listing 1. The nucleic acid encoding the chimeric antigen receptor contains the sequence described in Sequence Table 1, but is not limited to this series, and should also include other nucleic acids encoding C3aR. The intracellular domain of the signaling region in tandem with the C3aR intracellular domain is selected from one or more of CD3ζ, CD28, 4-1BB.

In a second aspect, the present invention provides a lentiviral vector expressing the above-mentioned chimeric antigen receptor.

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 cell population containing T cells .

The present invention provides a method for preparing 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 the cell; preferably, the cell T cells or cell populations containing T cells.

In a fourth aspect, the present invention provides a chimeric antigen receptor according to the first aspect, or a lentiviral vector according to the second aspect, or a chimeric antigen receptor expressing cell according to the third aspect Use in the preparation of a medicament for treating tumors.

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

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

The chimeric antigen receptor of the present invention can improve T17 cell subsets, eliminate the immunosuppressive effect of regulatory T cells, can significantly improve the in vitro killing efficiency of tumor target cells, and can significantly improve the killing effect of second-generation CAR T cells on tumors , providing a new idea and option for the field of CAR-T cell therapy.

Description of drawings

Figure 1a is a schematic diagram of a viral vector containing a combination of C3aR intracellular domains.

Figure 1b is a schematic diagram of a viral vector that does not contain a combination of C3aR intracellular domains.

Figure 2 shows the results of the in vitro killing effect of GFP T, CAR19T and CAR19C3aR T cells on NALM6-GL cells expressing CD19. The results show that CAR19C3aR T is superior to GFP T and CAR19T, and NALM6 in the figure represents NALM6-GL cells.

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

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

Figure 5 shows the load results of GFP T, CAR19T and CAR19C3aR T cells on NALM6 tumor cells (CD19+) in NCG mice. The results show that CAR19C3aR T is superior to GFP T and CAR19T. ^* P<0.05.

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

Figure 7 shows the effect of GFP T, CAR19T and CAR19C3aR T cells on the growth phase of NCG mice. The results show that CAR19C3aR T can significantly prolong the growth phase of NCG mice compared with GFP T and CAR19T. ^* P<0.05.

Detailed ways

The content of the present invention will be described in more detail below with reference to the embodiments. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention in any form fall into the protection scope of the present invention. The implementation method that does not specify specific conditions in the examples is usually in accordance with the conditions described in the conventional conditions or in accordance with the conditions suggested by the manufacturer.

Example 1 Preparation of Plasmids Containing Anti-CD19 ScFv-4-1BB-CD3ζ-C3aR (CAR19C3aR)

The plasmid carrying the chimeric antigen receptor gene containing the C3aR intracellular domain of the present invention is prepared according to the following steps:

(1) Plasmid pUC57-CAR19 containing anti-CD19ScFv-4-1BB-CD3ζ(CAR19) was obtained by means of gene synthesis, molecular cloning and other means , namely CD19ScFv-4-1BB-CD3ζ.

(2) The obtained pUC57-CAR19 plasmid was digested by endonuclease Pmel and Sep1 to obtain the CAR19 gene, and then the CAR19 gene was connected to the lentiviral vector pWPXLd-GFP to construct pWPXLd-CAR19-GFP.

(3) The obtained pWPXLd-CAR19-GFP plasmid was digested with endonucleases NotI and SpeI to obtain the intracellular fragment 4-1BB-CD3ζ of the CAR19 gene.

(4) Using the cDNA of the tandem C3aR intracellular signaling domain of 4-1BB-CD3ζ as a template, four primers were constructed, and 4-1BB-CD3ζ-C3aR was obtained by overlapping extension PCR.

(5) 4-1BB-CD3ζ in pUC57-CAR19 was replaced with 4-1BB-CD3ζ-C3aR by digestion with NotI and SpeI to obtain pUC57-CAR19C3aR plasmid.

(6) Finally, CAR19 in pWPXLD-CAR19-GFP was replaced with CAR19C3aR by endonuclease PmeI and SpeI digestion to obtain pWPXLd-CAR19C3aR-GFP plasmid. (figure 1)

Example 2 Lentiviral Packaging of CAR Plasmids

Three lentiviruses expressing GFP (blank control), CAR19-GFP (control) and CAR19C3aR-GFP were obtained by lentivirus packaging using the CAR plasmid of the present invention prepared in Example 1 and related control plasmids. In Examples 2 and 3, the CAR-containing plasmid is uniformly described as pWPXLd-CAR-GFP plasmid, and the CAR-overexpressing lentivirus is uniformly described as CAR lentivirus.

Specific steps are as follows:

(1) Cultivate 293T cells in a 10cm petri dish, the medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

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

(3) After replacing the medium and culturing for 2-6 hours, the two plasmids pWPXLd-CAR-GFP (that is, containing CAR19 and CAR19C3aR, respectively) or the blank control plasmid pWPXLd-GFP and the lentiviral packaging helper plasmid pMD2. G, psPAX2 were co-transfected into 293T cells, and the reagents and doses were added as shown in Table 1:

Table 1

reagent dose pWPXLd-CAR-GFP two plasmids or control plasmid 9ug pMD2.G helper plasmid 3ug psPAX2 12ug PEI 72ug

(4) 24, 48 and 72 hours after the transformation, respectively, collect the medium supernatant, and add fresh medium (DMEM high glucose medium + 1% FBS + 1% double antibody);

(5) After the medium supernatant was collected, the supernatant was centrifuged at 2500g for 0.5 hours;

(6) Take the centrifugal supernatant, filter with a 0.45um filter, and centrifuge at 28,000 rpm for 1.5 hours using an ultra-high-speed centrifuge;

(7) After ultracentrifugation, gently remove the supernatant, add 200ul PBS, and dissolve at 4 degrees for 12-16 hours to obtain two CAR lentiviruses or blank control GFP lentiviruses;

(8) After the virus is dissolved, the virus is collected and aliquoted into cryopreservation tubes, and frozen at -80°C for later use.

Example 3 Infection of human T cells using packaged CAR virus

(1) Separation and purification of T cells: Mononuclear cells in blood were separated by Ficoll density gradient method, and red blood cells were removed by lysing with red blood cell lysate, and then T cells were sorted by MACS Pan-T magnetic beads;

(2) Dilute the sorted T cells with medium (AIM-V medium + 5% FBS + 100 U/ml of penicillin + 0.1 mg/ml of streptomycin) to a cell concentration of 2.5×10 ⁶ cells/ml for use;

(3) T cells are stimulated by magnetic beads coated with CD2, CD3 and CD28 antibodies (product source: Miltenyi, Germany), that is, the coated magnetic beads and T cells are mixed in a ratio of 1:2, and the final density of T cells should be 5 ×10 ⁶ /ml/cm2. After mixing, the cells were placed in a 37°C, 5% CO2 incubator for stimulation for 48 hours.

(4) Lentivirus-transfected T cells: remove the magnetic beads in the activated T cell-magnetic bead mixture by magnetic field, centrifuge at 300g for 5 min, remove the supernatant, resuspend in fresh medium, and add expressing CAR and GFP respectively (blank control) After lentivirus (the amount of virus added is MOI=10), 8 μg/ml polybrene and 300 IU/ml IL-2 were added. After culturing at 37°C in a 5% CO2 incubator for 24h, centrifuge at 300g for 5min, remove the supernatant, and resuspend in fresh medium containing 300IU/ml IL-2 to obtain CAR-overexpressing T cells.

(5) CAR T cell expansion: maintain the CAR T cell density at about 1×10 ⁶ cells/ml, and perform half-volume medium changes every 2-3 days. After two weeks, the number of CAR T cells could expand 100-fold. GFP-positive cells are successfully transfected cells, and the GFP-positive ratio is detected by flow cytometry, that is, the ratio of two types of CAR T cells (referred to as CAR19-GFP, CAR19C3aR-GFP) or blank control T cells (GFP-T) is obtained. . The effects on Th17 cell subsets (CD4+IL17A+) and regulatory T cell subsets (CD4+CD25+FoxP3+) were detected.

Detection method of regulatory T cell subsets 1. Prepare effector cells (GFP-T, CAR19-GFP or CAR19C3aR-GFP T cells); add surface antibodies 5ul CD4-PERCP, 5ul CD25-PE and corresponding isotype antibodies, and incubate at 4°C in the dark 30min, add 1ml PBS for washing, centrifuge at 300g for 5min, remove the supernatant, add 1ml of permeabilizing fixative (membrane permeation agent: diluent = 1:3, freshly prepared), and incubate at 4°C for 35min in the dark. After 2.35 minutes, add 2 ml of membrane breaking buffer (buffer: pure water = 1:9), mix well, and centrifuge at 300 g for 5 minutes. 3. Remove the supernatant, add 5ul of FoxP3 antibody, and incubate at 4°C for 30min in the dark. After 4.30min, add 2ml PBE to wash, centrifuge at 300g for 5min. 5. Add 400ul PBS to each tube to resuspend and test on the machine.

Th17 cell subset detection method: 1. Prepare effector cells (GFP-T, CAR19-GFP or CAR19C3aR-GFP T cells), add surface antibody 5ul CD4-PERCP, mix well, protect from light, and incubate for 30min. 2. Add 100ul of Membrane Breaker A and incubate at room temperature for 15min. 3. Add 3ml PBS+0.1%NaN3+5%FBS.4.300g, centrifuge for 5min, remove the supernatant, add buffer B, mix well, add 5ul IL-17A APC-Cy7, and incubate for 20min. 5. Add 3ml PBS+0.1%NaN3+5%FBS.300g, centrifuge for 5min, remove the supernatant, resuspend in 400-500ul PBE and test on the machine.

Example 4 The effect of CAR T cells on tumor-killing recognition in vitro

The GFP T (blank control), CAR19-GFP T (control), and CAR19C3aR-GFP T cells prepared in Example 3 were mixed with 5× ^{10 4} tumor cells in different proportions and added to a 96-well U-shaped plate. 3 duplicate wells, and the tumor cell group alone was set as a positive control. After centrifugation at 250g for 5 min, the cells were placed in a 37-degree 5% CO2 incubator for co-cultivation for 24 h; the comparison of GFP T, CAR19-GFP T, and CAR19C3aR-GFP T cells in vitro was compared. In the identification and killing function of hematological tumors, two leukemia or lymphoma cell lines, NALM6-GL and Raji, were selected for tumor cells.

CFSE/PI double-stained cytotoxicity assay to evaluate quantitatively evaluate the killing efficiency: 1. Prepare effector cells (GFP T (blank control), CAR19-GFP T (control), CAR19C3aR-GFP T cells) and target cells, count, and place CAR The GFP % of T cells was calibrated with the same batch of WT cells to make the GFP % of each group similar in size; T cells were diluted 8x10 ⁶ /ml with 1640+10% FBS+1% P/S medium. 2. The required amount of target cells (Nalm6 cells, Raji cells), resuspended in 1ml 1640 medium, add 5umol CSFE, incubate at 37°C for 20min in the dark, add 10ml medium to stop for 5min, centrifuge, remove the supernatant, target cells Dilute to ^5x105 /ml. 3. Dilute the T cells by doubling, and pre-add 100ul of fresh medium to the V-bottom 96-well plate with a row gun. The first row is not added, then 100ul of T cells are added to the first row, and 100ul of T cells are added to the second row. Then use the row gun to blow the second row more than 5 times, suck 100ul into the third row, and so on, dilute the T cells along the row number, add 100ul of target cells to all wells, so that the T cells and the target cells are Mixed in different ratios (16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4). 4. Put the mixed cells in the incubator for 24 hours. 5. Aspirate the cells in the 96-well plate and centrifuge. 6. Wash once with PBS at 4°C, add 100ul 1x IP Buffer, then add 5ul PI, incubate in the dark for 15 min, then add 200ul 1x IP Buffer, and test on the machine within 1 hour. 7. Calculation of killing rate: taking target cell well without T cells as a reference, killing percentage = target well CSFE+PI+%-control well CSFE+PI+%. The results showed that both CAR19C3aRT and CD19-expressing tumor target cells had significantly higher in vitro killing efficiencies than CAR19T cells (see Figures 2 and 3).

Example 5 CAR19C3aR T cells recognize and kill tumors in vivo

In order to compare the effect of CAR19T and CAR19C3aR T cells on recognizing and killing tumors in vivo, the same number (5×10 ⁵ ) of NALM6 cells (expressing luciferase) were passed through the tail vein of 7 NCG (NOD/SCID IL2rg ^-/- ) immunodeficient cells, respectively. In mice; 2 and 8 days after NALM6 cell transplantation (the day of tumor cell transplantation is day 0), 5 × 10 ⁶ T cells (four groups: GFP T, CAR19T, CAR19C3aRT T, 6 in each group were injected mice) intravenously into NSI immunodeficient mice transplanted with NALM6 cells. Since NALM6 expresses luciferase, 200ul of the substrate D-luciferin was injected intraperitoneally. After 5 minutes, it was placed in the anesthesia room attached to the in vivo imaging, and the anesthetized mice were anesthetized with isoflurane. Then the anesthetized mice were placed in the imager for imaging. , imaging was performed in automatic exposure mode, and the mice were put back into the cage after imaging. The fractional data showed that CAR19C3aRT could significantly reduce the burden of NALM6 tumor cells in NCG mice (see Figures 5 and 6), and continued to observe small The mice died directly during the survival period, and the final result was that CAR19C3aRT could significantly prolong the growth period of NCG mice (Figure 7). It is suggested that adding the intracellular domain of C3aR can significantly improve the killing effect of second-generation CAR T cells (CAR19T) on tumors. Through the above experimental results, comparing the strength of CAR T's ability to recognize and kill tumors in the experimental group and the control group, it was verified that the intracellular signaling domain of C3aR improved the tumor-killing function of CAR T in vivo and in vitro.

The applicant declares that the products, uses and usages of the present invention are illustrated by the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed purposes or usages, that is, it does not mean that the present invention must rely on the above-mentioned detailed purposes and usages in order to be tested . Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention and the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

sequence listing

<110> Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences)

<120> Chimeric antigen receptor, lentiviral vector, expression cell and drug containing C3aR intracellular domain

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Claims (4)

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.

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