CN107903326A - Chimeric antigen receptor, slow virus carrier, expression cell and medicine comprising C3aR intracellular domain - Google Patents

Abstract

The present invention provides a chimeric antigen receptor comprising a C3aR intracellular domain, a lentiviral vector, an expression cell, and a drug. The chimeric antigen receptor comprising a C3aR intracellular domain comprises a cellular antigen capable of binding an antigen. An ectodomain, a transmembrane domain and at least one intracellular domain selected from a C3aR intracellular domain or an intracellular domain of a signaling region in tandem with a C3aR intracellular domain. The chimeric antigen receptor of the present invention can increase T17 cell subgroups, 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 , to provide a new idea and choice for the field of CAR‑T cell therapy.

Description

Chimeric antigen receptors containing C3aR intracellular domains, lentiviral vectors, expressing cells and drugs

technical field

The invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a chimeric antigen receptor comprising a C3aR intracellular domain, and also relates to a nucleic acid and application of the chimeric antigen receptor encoding a C3aR intracellular domain.

Background technique

Chimeric Antigen Receptors (CAR, Chimeric Antigen Receptors) T cells are a milestone event that is expected to cure tumors. CAR-T technology uses genetic modification technology to combine a single chain fragment variable (scFv) that recognizes tumor antigens with cells. The internal activation motif recombinant gene is transfected to T lymphocytes to achieve better tumor recognition and killing effects. CAR-T molecules usually include an extracellular hinge region, a transmembrane region, and an intracellular signal region. Among them, the extracellular hinge region is formed by connecting the heavy chain and light chain variable regions of the single-chain antibody through a peptide; the transmembrane region mainly comes from the transmembrane region of molecules such as CD8, CD28 or 4-1BB; The intracellular signaling region mainly comes from the intracellular mosaic of T cell transduction signaling molecules such as CD28, 4-1BB, CD3zeta, CD27 or OX-40. The high-affinity and high-specificity scFv determines that CAR-T targets a certain antigen. Once the antigen is bound, the intracellular segment of CAR will transmit activation and co-stimulatory signals to T cells, 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 against tumor cells; co-stimulatory molecules on the surface of T cells and corresponding Ligand binding is the second signal of activation, which determines the proliferation of T cells. 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 intracellular signal transduction.

The structural design of CAR molecules has undergone multiple generations of research and development. The structure of the first-generation CAR molecule includes scFv that recognizes tumor cell surface antigens, a transmembrane domain, and the intracellular domain of the TCR complex CD3zeta that activates T cells. Since the signaling domain of the first-generation CAR is a single signaling molecule and lacks co-stimulatory signals, the survival time of the first-generation CAR-T cells in the body is short, the lethality to tumor cells is weak, and the curative effect is not ideal. 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 molecule to activate T cells, the second-generation CAR was developed. The second-generation CAR mainly added a co-stimulator to the intracellular domain In the signaling domain, costimulatory ligands that have been reported so far are mainly concentrated 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 Enhance its ability to recognize tumor antigens and kill T cells (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 two types of anti-CD19-CAR-T cells for 6 patients with B-cell lymphoma. The signaling domain of one CAR only had a single signaling molecule ζ chain (first-generation CAR-T), and the other added both ζ chain and co-stimulatory molecule CD28 (second-generation CAR-T). The in vitro culture and detection results of 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 detected regularly. The results showed that the proliferation speed and survival time of the second-generation CAR-T cells in vivo were better than those of the first-generation CAR-T cells (SavoldoB, RamosCA, LiuE, eta1. CD28costimulation 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 a 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 curative effect, there is still room for further improvement, such as further enhancing its activation and cell killing functions, so as to improve the efficacy of CAR-T therapy. The third-generation CAR-T integrates two or more co-stimulatory molecular signals on the basis of the first generation. Different combinations of co-stimulatory 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's lymphoma (NHL), but it has not shown better clinical results than the second-generation CAR-T. At present, the more successful clinical application of acute leukemia is mainly the second-generation CAR-T technology. One costimulatory molecule transfected is mainly CD28 or CD137 (4-1BB), and the other costimulatory molecule is CD3zeta. It can be seen that the structural design of CAR in the prior art is not very mature, and CAR molecules need to be further improved in terms of enhancing T cell killing function, enhancing Th17 cell subsets, and eliminating immunosuppression of regulatory T cells.

The complement system plays an important biological role in the process of innate immune response and adaptive humoral immune response. The inherent components of complement in the body exist in the form of precursor 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 finally synthesized to attack microorganisms or other target cells, resulting in the lysis and rupture of target cells. These complement cleavage products mainly exert immunological functions by binding to complement receptors (CR): C3aR and C5aR. There is a close mutual regulatory relationship between complement and CD4+ T cells, and the C3aR complement pathway promotes Th17 differentiation while inhibiting the generation of regulatory T cells. The combination of C3aR and other co-stimulatory molecules is used for the construction of chimeric antigen receptors, and it is impossible to predict the effect of the constructed chimeric antigen receptors.

Contents of the invention

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

The second object of the present invention is to provide a lentiviral vector comprising a chimeric antigen receptor containing a C3aR intracellular domain;

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

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

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

The present invention achieves the above object through the following technical solutions: In the first aspect, the present invention provides a chimeric antigen receptor, which comprises an extracellular domain capable of binding antigen, a transmembrane domain and at least one intracellular domain, wherein The intracellular domain is selected from a C3aR intracellular domain or an intracellular domain of a signaling region in tandem with a C3aR intracellular domain. Here, "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain in cells 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 signal transmission regions connected in series with the intracellular domain of C3aR, such as CD3ζ, CD28, 4-1BB, etc.

For the above-mentioned CAR molecule, preferably, the antigen may be a tumor antigen, and the tumor antigen includes, for example: 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.; more preferably, the tumor antigen is CD19. The antigen described in the patent of the present invention may also be an inflammatory cell surface molecule appearing in an autoimmune disease or a TCR leading to autoimmunity.

Preferably, the extracellular domain capable of binding to an antigen refers to a single-chain variable fragment of an antibody that binds to an antigen. In a specific embodiment, the above-mentioned CAR molecule may only use the C3aR intracellular domain as its intracellular domain, and may also contain one or more (for example, 2 or 3) other cellular domains other than 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 the 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 sequentially connected from the N-terminal side. In addition, 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 can be It is arranged on the N-terminal side of the intracellular domain of the chimeric antigen receptor. In a preferred specific embodiment, the chimeric antigen receptor includes the single-chain variable region of an anti-tumor antigen antibody as an extracellular domain, the transmembrane domain of a CD28 molecule, and the 4-1BB cellular domain in order from the N-terminal side. endodomain, CD3ζ endodomain, C3aR endodomain.

The intracellular domain of C3aR contains the nucleotide sequence shown in Sequence Table 1. The coding nucleic acid of the chimeric antigen receptor contains the sequences described in Sequence Table 1, but is not limited to this series, and should also include other coding nucleic acids of C3aR. The intracellular domain of the signaling region connected in series with the C3aR intracellular domain is selected from one or more of CD3ζ, CD28, and 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 as described in the first aspect is introduced; preferably, the cell is a T cell or a cell population containing a T cell .

The present invention provides a method for preparing the chimeric antigen receptor expressing cell as described in the third aspect, which includes the step of introducing the chimeric antigen receptor as described in the first aspect into the cell; preferably, the cell T cells or cell populations containing T cells.

In the fourth aspect, the present invention provides a chimeric antigen receptor as described in the first aspect, or a lentiviral vector as described in the second aspect, or a chimeric antigen receptor expressing cell as described in the third aspect Use in the preparation of medicines 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 comprises the C3aR intracellular domain as its intracellular domain. The C3aR intracellular domain includes variants thereof that have the same function. The term "variant" refers to any variant comprising a substitution, deletion or addition of one or a few to several amino acids, provided that the variant retains substantially the same function as that of the original sequence.

The chimeric antigen receptor of the present invention can increase T17 cell subgroups, 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 , to provide a new idea and choice 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 in vitro killing effect of GFP T, CAR19T and CAR19C3aR T cells on CD19-expressing NALM6-GL cells. 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 CD19-expressing Raji cells. The results show that CAR19C3aR T is superior to GFP T and CAR19T.

Figure 4a and Figure 4b show the effects 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 can significantly increase Subpopulation of T17 cells, eliminating immunosuppression of regulatory T cells. ^* P<0.05.

Figure 5 is a graph showing the burden 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 burden and location of NALM6 cells in NCG mice.

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

Detailed ways

Below in conjunction with embodiment, the content of the present invention is described more specifically. 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 fall within the protection scope of the present invention. For the implementation methods not indicated in the specific conditions in the examples, the conditions described in the general conditions or the conditions suggested by the manufacturer are generally followed.

Example 1 Preparation of plasmid containing anti-CD19ScFv-4-1BB-CD3ζ-C3aR (CAR19C3aR)

Prepare the plasmid of the present invention carrying the chimeric antigen receptor gene containing the intracellular domain of C3aR as follows:

(1) The plasmid pUC57-CAR19 containing anti-CD19ScFv-4-1BB-CD3ζ (CAR19) was obtained by means of gene synthesis and molecular cloning, including anti-CD19 monoclonal antibody ScFv, CD28 transmembrane region and 4-1BB, CD3ζ intracellular region , namely CD19ScFv-4-1BB-CD3ζ.

(2) The resulting pUC57-CAR19 plasmid was digested with endonucleases Pmel and Sepl to obtain the CAR19 gene, and then the CAR19 gene was connected to the lentiviral vector pWPXLd-GFP to construct pWPXLd-CAR19-GFP.

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

(4) Using the cDNA of 4-1BB-CD3ζ tandem C3aR intracellular signaling domain as a template, construct 4 primers, and obtain 4-1BB-CD3ζ-C3aR by overlap extension PCR.

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

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

Lentiviral packaging of embodiment 2CAR plasmid

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

Specific steps are as follows:

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

(2) When the 293T cell density 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 were respectively combined with the lentiviral packaging helper plasmid pMD2. G. psPAX2 was co-transduced into 293T cells, and the reagents and dosages 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 transformation respectively, the medium supernatant was collected, and fresh medium (DMEM high glucose medium + 1% FBS + 1% double antibody) was added;

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

(6) Take the centrifuged supernatant, filter it with a 0.45um filter, and then centrifuge at 28000rpm for 1.5 hours in an ultrahigh-speed centrifuge;

(7) After ultra-high-speed centrifugation, gently remove the supernatant, add 200ul PBS, and dissolve at 4 degrees for 12-16 hours to obtain 2 kinds of CAR lentiviruses or blank control GFP lentiviruses;

(8) After the virus is dissolved, the collected virus is divided into cryopreservation tubes and stored at -80°C until use.

Example 3 Using packaged CAR virus to infect human T cells

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

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

(3) Stimulate T cells 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 at a ratio of 1:2, and the final density of T cells should be 5 ×10 ⁶ pieces/ml/cm2. After mixing, place them in a 37°C, 5% CO2 incubator to incubate and stimulate for 48 hours.

(4) Lentiviral transfection of T cells: remove the magnetic beads in the activated T cell-magnetic bead mixture by magnetic field, centrifuge at 300g for 5min, remove the supernatant, resuspend with fresh medium, add 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 cultured in a 5% CO2 incubator at 37°C for 24 hours, centrifuge at 300 g for 5 minutes, remove the supernatant, and resuspend with fresh medium containing 300 IU/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 a half-volume medium change every 2-3 days. After two weeks, the number of CAR T cells can be expanded 100-fold. GFP-positive cells are successfully transfected cells, and the GFP-positive ratio is detected by flow cytometry to obtain the ratio of two types of CAR T cells (referred to as CAR19-GFP and CAR19C3aR-GFP) or blank control T cells (GFP-T) . Detect the effect on Th17 cell subsets (CD4+IL17A+) and regulatory T cell subsets (CD4+CD25+FoxP3+).

Regulatory T cell subset detection method 1. Prepare effector cells (GFP-T, CAR19-GFP or CAR19C3aR-GFP T cells); add surface antibodies 5ul CD4-PERCP, 5ulCD25-PE and corresponding isotype antibodies, and incubate at 4°C in the dark After 30 min, add 1 ml of PBS to wash, centrifuge at 300 g for 5 min, remove the supernatant, add 1 ml of membrane breaking fixative (membrane breaking agent: diluent = 1:3, freshly prepared), and incubate at 4°C in the dark for 35 min. After 2.35 min, 2 ml of membrane permeation buffer (buffer: pure water = 1:9) was added, mixed evenly, and centrifuged at 300 g for 5 min. 3. Remove the supernatant, add 5ul of FoxP3 antibody, and incubate at 4°C in the dark for 30min. 4. After 30 minutes, add 2ml of PBE to wash, and centrifuge at 300g for 5 minutes. 5. Add 400ulPBS 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 membrane disrupting agent 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, incubate for 20min. 5. Add 3ml PBS+0.1%NaN3+5%FBS. 300g, centrifuge for 5min, remove the supernatant, resuspend in 400-500ulPBE and test on the machine.

Example 4 The effect of CAR T cells recognizing and killing tumors in vitro

The GFP T (blank control), CAR19-GFP T (control), and CAR19C3aR-GFP T cells prepared in Example 3 were mixed with ^5x104 tumor cells in different proportions, and added to a 96-well U-shaped plate. 3 duplicate wells, and a tumor cell group alone was set as a positive control, centrifuged at 250g for 5 minutes, and placed in a 5% CO2 incubator at 37 degrees for 24 hours; compared GFP T, CAR19-GFP T, and CAR19C3aR-GFP T cells in vitro For the recognition and killing function of hematological tumors, two leukemia or lymphoma cell lines, NALM6-GL and Raji, were used as tumor cells.

Evaluation of CFSE/PI double-stained cytotoxicity detection method Quantitative evaluation of killing efficiency: 1. Prepare effector cells (GFP T (blank control), CAR19-GFP T (control), CAR19C3aR-GFP T cells) and target cells, count, and CAR The GFP% of T cells was calibrated with the same batch of WT cells, so that the GFP% of each group was similar; the T cells were diluted 8x10 ⁶ /ml with 1640+10%FBS+1%P/S medium. 2. Resuspend the required amount of target cells (Nalm6 cells, Raji cells) in 1ml 1640 medium, add 5umol CSFE, incubate at 37°C in the dark for 20min, add 10ml medium to stop for 5min, centrifuge, remove the supernatant, target cells Dilute to 5x10 ⁵ /ml. 3. Doubling dilution of T cells, pre-add 100ul of fresh medium into the V-bottom 96-well plate with a row gun, without adding to the first row, then add 100ul of T cells to the first row, and 100ul of T cells to the second row, Then use a row gun to blow the second row more than 5 times, draw 100ul into the third row, and so on, dilute the T cells along the number of rows, add 100ul target cells to all the wells, so that the T cells and target cells Mixing 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 minutes, then add 200ul 1xIP Buffer, and test on the machine within 1 hour. 7. Calculation of killing rate: Taking target cell wells without adding T cells as a reference, killing percentage=objective well CSFE+PI+%−control well CSFE+PI+%. The results showed that the in vitro killing efficiency of CAR19C3aRT and CD19-expressing tumor target cells was significantly higher than that of CAR19T cells (see Figure 2 and Figure 3).

Example 5 CAR19C3aR T cells recognize and kill tumors in vivo

In order to compare the in vivo recognition and killing effects of CAR19T and CAR19C3aR T cells, the same number (5×10 ⁵ ) of NALM6 cells (expressing luciferase) were passed through the tail vein of 7 NCG (NOD/SCID IL2rg ^-/- ) immunodeficient mice. In mice; on the 2nd and 8th days after NALM6 cell transplantation (the day of tumor cell transplantation was Day 0), 5×10 ⁶ T cells (four groups: GFP T, CAR19T, CAR19C3aRT, 6 mice in each group) were injected mice) were injected intravenously into NSI immunodeficient mice transplanted with NALM6 cells. Since NALM6 expresses luciferase, 200ul of the substrate D-luciferin is injected intraperitoneally, and after 5 minutes of timing, it is placed in the anesthesia room attached to the in vivo imaging, anesthetized by isoflurane, and then the anesthetized mouse is placed in the imager for imaging , imaged by automatic exposure mode, and the mice were put back into the cage to raise after imaging. The results of fractional data showed that CAR19C3aRT can significantly reduce the burden of NALM6 tumor cells in NCG mice (see Figures 5 and 6), and continue to observe small The survival period of mice was directly dead, 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 the second-generation CAR T cells (CAR19T) on tumors. Through the above experimental results, comparing the strength of CAR T's tumor recognition and killing function between 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 to illustrate the product, use and use method of the present invention through the above implementation examples, but the present invention is not limited to the above-mentioned detailed use or use method, that is, it does not mean that the present invention must rely on the above-mentioned detailed use and use method to experiment . 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, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

sequence listing

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

<120> Chimeric antigen receptors containing C3aR intracellular domains, lentiviral vectors, expressing cells and drugs

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Claims (10)

1. a kind of Chimeric antigen receptor for including C3aR intracellular domain, includes extracellular domain, the cross-film that can combine antigen Domain and at least one intracellular domain, it is characterised in that the intracellular domain be selected from C3aR intracellular domain or with The intracellular domain in the signal transfer region domain of C3aR intracellular domain series connection.

2. Chimeric antigen receptor as claimed in claim 1, it is characterised in that the signal transmission connected with C3aR intracellular domain One or more of the intracellular domain in region in CD3 ζ, CD28,4-1BB.

3. Chimeric antigen receptor as claimed in claim 2, it is characterised in that the intracellular domain further includes CD3 ζ intracellular knots Structure domain, the C3aR intracellular domain configuration is in the C-terminal side of CD3 ζ intracellular domain.

4. Chimeric antigen receptor as claimed in claim 2, it is characterised in that the intracellular domain is since N-terminal side Sequentially connected 4-1BB intracellular domain, CD3 ζ intracellular domain and C3aR intracellular domain.

5. the Chimeric antigen receptor as described in claim 1-4 any one, it is characterised in that antigen be selected from tumour antigen, The inflammatory cell surface molecular that occurs in autoimmune disease or the TCR for causing autoimmunity.

6. Chimeric antigen receptor as claimed in claim 1, it is characterised in that described to be with reference to the extracellular domain of antigen Refer to the single chain variable fragment of the antibody with reference to targeting antigen.

7. Chimeric antigen receptor as claimed in claim 1, it is characterised in that C3aR intracellular domain contains such as the institute of sequence table 1 The nucleotide sequence shown.

8. a kind of express the slow virus carrier for including the Chimeric antigen receptor as described in claim 7 is any.

A kind of 9. cell for expressing Chimeric antigen receptor, it is characterised in that：It is any described chimeric to introduce claim 1~8 Antigen receptor.

Prevent or the medicine for the treatment of tumour 10. a kind of, comprising just like any Chimeric antigen receptor of claim 1~7 or Slow virus carrier as claimed in claim 8 or cell as claimed in claim 9.