CN118146393A

CN118146393A - Chimeric antigen receptor targeting CD19 with optimized signal peptide sequence and application thereof

Info

Publication number: CN118146393A
Application number: CN202410378450.8A
Authority: CN
Inventors: 张同存
Original assignee: Wuhan Bio Raid Biotechnology Co ltd
Current assignee: Wuhan Bio Raid Biotechnology Co ltd
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2024-06-07
Also published as: CN116514992B; CN116514992A

Abstract

The invention discloses signal peptide optimization of chimeric antigen taking CD19 as a target point and application thereof, wherein the optimized signal peptide can obviously improve the expression efficiency and stability of the chimeric antigen receptor taking CD19 receptor as the target point in T cells, thereby better playing the killing effect on the target cells.

Description

Chimeric antigen receptor targeting CD19 with optimized signal peptide sequence and application thereof

The application relates to a chimeric antigen receptor targeting CD19 and application thereof, which are applied for the patent application with the application date of 2022, 11, 7, the application number of 202211383577.6 and the application name of 'a chimeric antigen receptor targeting CD19 with optimized signal peptide sequence'.

Technical Field

The invention relates to the field of biological medicine, in particular to a chimeric antigen receptor targeting CD19 with optimized signal peptide sequence and application thereof.

Background

Chimeric antigen Receptor T (CHIMERIC ANTIGEN Receptor T, CAR-T) cell technology is a technique of expressing a single-chain antibody variable region (SINGLE CHAIN antibody variable fragments, scFvs) sequence directed against a specific antigen on the surface of T cells of a patient by in vitro genetic engineering, thereby targeting and killing tumor cells. The basic design of a CAR includes a tumor-associated antigen binding region (typically an scFv fragment derived from an antibody antigen binding region), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The outer part of the cell membrane is the antigen targeting moiety purified from monoclonal antibodies consisting of single chain variable fragments (scFv), fusion proteins of the heavy and light chain variable regions, and furthermore the signal peptide in scFvs is particularly important for its expression.

In 1975, professor Gunter Bloble of cell biology at the university of Rockfield proposed a "signal hypothesis" in an experimental study to solve the problem of going after protein synthesis. He believes that the newly synthesized protein has an endogenous signal, the signal peptide (SIGNAL PEPTIDE, SP). The signal peptide is a short peptide chain existing at the N-end of the precursor protein, and can regulate the folding and transferring processes of the precursor protein. Studies show that in a prokaryotic system, after the signal peptide is attached, the exogenous gene can be successfully expressed and secreted in the prokaryotic expression system, such as bacillus, lactobacillus, L-type bacteria, escherichia coli and the like. In addition, signal peptides are widely used in eukaryotic expression systems, such as insect baculovirus expression systems and pichia expression systems. Therefore, the signal peptide plays an extremely important role in the secretion process of the protein, and the optimization of the signal peptide sequence by the molecular biology technology has important practical significance for improving the secretion amount and activity of the exogenous protein.

The N-terminal signal peptide of scFv in CAR structure has a triple structure and has the same characteristics as the signal peptide of secreted protein: hydrophobic alpha helical cores (H regions) are flanked at their N-terminus by polar amino acid residues (N regions). The C-terminal side contains helix-cleaving proline and glycine residues at positions-1 and-3 of the cleavage site (C region) and small uncharged residues. Once bound to a tumor antigen, the scFv triggers T cell activation and leads to cytokine release and T cell proliferation. Studies show that the signal peptide can promote the expression of CAR on the surface of T cell membrane, and in addition, the research reports that increasing the positive charge number of the N end of the signal peptide or increasing the hydrophobicity or length of the hydrophobic core H region of the signal peptide is beneficial to improving the processing efficiency of the signal peptide. Whereas mutations in conserved fragments nearer to the N-terminus of a signal peptide, as well as in certain conserved sequence amino acid fragments nearby, have a greater impact on the affinity of the precursor protein for proteins associated with the secretory pathway. Therefore, optimization and selection of signal peptide structure is an important study to increase CAR expression levels.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a chimeric antigen receptor targeting CD19 with optimized signal peptide sequence, which can enable the chimeric antigen receptor targeting CD19 to be expressed more efficiently and stably in T cells, and CAR-T cells carrying ScFv sequences targeting CD19 can effectively kill tumor cells with surfaces expressing CD 19.

In order to achieve the technical effects, the invention provides the following technical scheme:

In a first aspect of the present invention, there is provided a chimeric antigen receptor targeting CD19 with optimized signal peptide sequence, wherein the chimeric antigen receptor is spliced in sequence from N-terminal to C-terminal: optimized signal peptide, anti-CD 19 antibody single chain variable region, CD8hinge, CD28 transmembrane region, CD28 intracellular domain, intracellular co-stimulatory domain 4-1BB and CD3 zeta chain.

In one embodiment, the optimized signal peptide is SP4 (unmutated), SP4K (mutated tyrosine Y to lysine K), SP4R (mutated tyrosine Y to arginine R), respectively, the nucleotide sequence of which is selected from the sequences set forth in any one of SEQ ID nos. 2,4, 6, respectively.

The amino acid sequence of the single-chain variable region of the anti-CD 19 antibody is shown as SEQ ID NO.7, the nucleotide sequence of CD8hinge is shown as SEQ ID NO.9, the amino acid sequences of the CD28 transmembrane region and the CD28 intracellular domain are shown as SEQ ID NO.11 and SEQ ID NO.13 respectively, the amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.15, and the amino acid sequence of CD3 zeta is shown as SEQ ID NO. 17; in addition, the nucleotide sequence of the single-chain variable region of the anti-CD 19 antibody is shown as SEQ ID NO.8, the nucleotide sequence of CD8 range is shown as SEQ ID NO.10, the nucleotide sequences of the CD28 transmembrane region and the CD28 intracellular domain are shown as SEQ ID NO.12 and SEQ ID NO. 14 respectively, the nucleotide sequence of the intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.16, and the nucleotide sequence of CD3 zeta is shown as SEQ ID NO. 18.

In another embodiment, the nucleotide sequence of the chimeric antigen receptor is selected from the sequences set forth in any one of SEQ ID NOS.20, 22, 24.

In a second aspect of the invention there is provided an immune cell expressing a chimeric antigen receptor, characterised in that the cell is obtained from an immune cell of a human or other mammal transfected with a chimeric antigen receptor as described above.

In one embodiment, the immune cells of the human or other mammal are selected from the group consisting of umbilical cord blood, peripheral blood or IPSC derived T cells, NK cells, NKT cells, αβ T cells, γδ T cells, cd4+ T cells, or cd8+ T cells. Preferably, the immune cells of the human or other mammal are T cells of peripheral blood origin.

In a third aspect, the present invention provides an application of the chimeric antigen receptor or the chimeric antigen receptor-expressing immune cell in preparing a medicament for treating or preventing tumor.

In one embodiment, the tumor is B-cell acute lymphoblastic leukemia or other disease in which CD19 is the target of treatment. Preferably, the other CD19 targeted disease is B cell lymphoma.

Compared with the prior art, the invention has the following beneficial technical effects:

1. In the invention, after the CD19 chimeric antigen receptor sequence is optimized by the signal peptide sequence, the expression in T cells is more stable and efficient, as shown by a flow immunofluorescence staining chart result, the expression efficiency of CAR is obviously improved after the signal peptide is optimized, and the CD19 chimeric antigen receptor sequence can play a better role in treating CD19 positive B cell leukemia or lymphoma;

2. The invention also provides a preparation method of immune cells expressing the chimeric antigen receptor, which activates the isolated immune cells for 2-10 days and then infects the lentivirus expressing the chimeric antigen receptor, so that the original immune cells cannot influence the tumor killing effect of the transfected immune cells expressing the chimeric antigen receptor, and furthermore, when the immune cells expressing the chimeric antigen receptor are subjected to in vitro function detection, the selected cell line is a cell line with a CD19 target point expressed outside cell membranes, so that the tumor killing effect evaluation of the immune cells expressing the chimeric antigen receptor is more scientific.

Drawings

FIG. 1 is a graph showing the results of the titers of SP4 and its mutant lentiviruses;

FIG. 2 is a graphical representation of the results of transfection efficiency of SP4 and its mutant CAR-T cells;

FIG. 3 (middle) is a graphical representation of the in vitro killing results of SP4 and its mutant CAR-T cells against cell line K562-CD 19;

FIG. 3 (right) is a graph showing the in vitro killing results of SP4 and its mutant CAR-T cells against cell line NALM 6;

FIG. 3 (left) is a graph showing in vitro killing results of SP4 and its mutant CAR-T cells against negative cell line K562;

FIG. 4A is a schematic representation of the results of cytokine IFN-gamma release following in vitro co-incubation of SP4 and its mutant CAR-T cells with the cell line NALM 6;

Figure 4B is a schematic representation of the results of cytokine TNF- α release following in vitro co-incubation of SP4 and its mutant CAR-T cells with the cell line NALM 6.

Detailed Description

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: acquisition of chimeric antigen receptor lentiviral vectors targeting CD19 (PTK-EF 1 alpha-SP 4-CAR, PTK-EF1 alpha-SP 4K-CAR, PTK-EF1 alpha-SP 4R-CAR)

1. The SP4 sequence is a signal peptide specific to the secreted protein itself, and according to previous experimental studies by the inventors, it was found that SP4 can significantly enhance the function of CD19-CAR compared to other natural signal peptide sequences (Li Fan et al, J.cancer in China, 2022). On the basis of the invention, tyrosine in the sequence is mutated into lysine and arginine respectively to improve the positive charge number of the N end of the signal peptide, and the mutated tyrosine is named as SP4K and SP4R.

2. SP4-CAR, SP4K-CAR, SP4R-CAR fragments with cleavage sites EcoRI and BamHI were obtained by OverlapPCR amplification using SP (SP 4, SP4K, SP 4R), SCFV and CD8 range-CD28 TM+ICD-4-1BB-CD3 zeta as templates, respectively, wherein the sequences of SP (SP 4, SP4K, SP R), anti-CD 19 SCFV, CD8 range, CD28TM+ICD, 4-1BB, CD3 zeta are shown in Table 1 below:

TABLE 1

Complete CAR structural sequences were obtained for SP4-CAR, SP4K-CAR, SP4R-CAR as shown in table 2:

TABLE 2

3. The plasmid PTK-EF 1. Alpha. -Kan (the plasmid construction method is described in the inventors' prior patent ZL 201611246081.9) was subjected to double digestion with EcoRI and BamHI restriction enzymes, the product was subjected to 0.8% agarose gel electrophoresis and the gel cut was recovered in an Eppendorf tube, the corresponding fragment was recovered with an Axygen agarose gel recovery kit, and the purity and concentration of the product were determined.

4. Fragments were taken as 1: adding an Eppendorf tube in a molar ratio of 2, adding Exnase ligase (Vazyme) and 5 XCE buffer of homologous recombinase, and reacting for 0.5 hours at 37 ℃; taking 10 mu L of the connecting solution, adding 100 mu LDH5 alpha competent cells into the connecting solution, carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃, and adding 500 mu Lsoc culture medium for culturing for 2 hours at 220rpm at 37 ℃ after the completion of the heat shock; after 2 hours, 4000g of the Eppendorf tube was centrifuged for 1min to remove 400. Mu.L of excess liquid. Coating the residual liquid on an LB plate for culturing for 12 hours at 37 ℃; single colonies were picked up on plates and inoculated into 5mL of LB liquid medium for 12 hours at 37℃and 220 rpm.

5. Extracting plasmids by using an Axygen small extraction kit to obtain plasmids PTK-EF1 alpha-SP 4-CAR, PTK-EF1 alpha-SP 4K-CAR and PTK-EF1 alpha-SP 4R-CAR; after the first generation sequencing verification of the technology company of the Shanghai Co., ltd, the E.coli DH5 alpha strain containing plasmids PTK-EF1 alpha-SP 4-CAR, PTK-EF1 alpha-SP 4K-CAR and PTK-EF1 alpha-SP 4R-CAR is preserved.

Example 2 preparation and sequencing of plasmids

1. Preparation of plasmids

Coli DH 5. Alpha. Strain containing plasmids PTK-EF 1. Alpha. -SP4-CAR, PTK-EF 1. Alpha. -SP4K-CAR and PTK-EF 1. Alpha. -SP4R-CAR were inoculated into 250mL of LB medium containing 100. Mu.g/mL kanamycin, and cultured overnight at 37℃and 220rpm, respectively. The culture broth was centrifuged at 6000g for 20min at 4℃and the supernatant was discarded.

BuffersP1 in Endo FREE PLASMID MEGA KIT (Qiagen) was removed, 120mL of pre-chilled Buffers P1 was added to the E.coli pellet obtained by centrifugation, the centrifuge bottle cap was closed, and the centrifuge bottle was vigorously shaken to completely disperse the E.coli pellet in Buffers P.

120ML Buffers P2 was added to the centrifuge flask, the flask was capped and placed on a roller mixer, slowly accelerated to 50rpm, thoroughly mixed and then left at room temperature for 5min.

120MLBuffers P3 are added into a centrifugal bottle, a bottle cover is covered and placed on a roller mixer, the speed is slowly increased to 70rpm of the maximum rotation speed of the roller mixer, and the mixture is thoroughly mixed until the mixture is white, non-sticky and fluffy. Centrifuge at 9000g for 15min at 4 ℃.

50: 50MLBuffer FW was poured into QIA FILTER CARTRIDGE, and the supernatant obtained by centrifugation was poured into QIA FILTER CARTRIDGE, and the mixture was gently stirred and mixed. And filtering the mixed solution into a marked corresponding glass bottle.

20ML Buffer ER was added to each flask, mixed up and down 6 times and incubated at-20℃for 30min.

The labeled mega columns were placed on corresponding shelves and 35mL Buffers QBT equilibrated and gravity drained into each mega column.

The liquid in the glass bottle was poured into the corresponding labeled mega column in batches, and after the liquid in the column was drained, 200mL Buffer QC was added to each mega column in batches for washing. After the liquid in the column is drained, the waste liquid in the waste liquid collecting tray is poured into a 50mL clean centrifuge tube.

Then 40mL Buffer QN is added into each mega column, the effluent is collected by using a 50mL clean centrifuge tube, the mixture is mixed up and down for 6 times, and 20mL is packed into another clean marked 50mL centrifuge tube.

14ML of isopropyl alcohol (normal temperature) was added to each 50mL centrifuge tube, and the mixture was mixed by reversing the above for 6 times. Centrifuge at 15000g for 50min at 4 ℃.

The supernatant was removed in an ultra clean bench, and each tube was rinsed with 3.5mL Endotoxin-freewater without flushing the bottom pellet. Centrifuge at 15000g for 30min at 4 ℃. The Buffer TE in Endo FREE PLASMID MEGA KIT was placed in an oven for preheating.

The supernatant after centrifugation is sucked up in an ultra-clean workbench, and is dried (residual absolute ethyl alcohol is volatilized for about 10 min) in the ultra-clean workbench.

Taking out Buffer TE in an oven, adding 1mL Buffer TE into each tube in an ultra-clean workbench, blowing 10 times by using a gun, and putting into the oven at 65 ℃ while continuously knocking the tube wall to promote the precipitation to be completely dissolved. Centrifuging at 4000g for 1min at 4deg.C, throwing the liquid on the pipe wall to the bottom of the pipe, and blowing and mixing.

The liquid was transferred in its entirety into endotoxin-free, heat-source-free nuclease-free correspondingly labeled EP tubes in a super clean bench. 2. Mu.L was aspirated, the plasmid concentration was measured with a micro-spectrophotometer and labeled on the corresponding EP tube to obtain plasmids PTK-EF 1. Alpha. -SP4-CAR, PTK-EF 1. Alpha. -SP4K-CAR, PTK-EF 1. Alpha. -SP4R-CAR.

2. Sequencing of genes of interest

And respectively taking 20 mu L (500 ng) of plasmid DNA, carrying out outward sequencing, checking whether the target gene of a product obtained by plasmid production is changed according to the original seed sequence, and under the stable process, the target gene is not changed in the process of fermentation culture amplification of working seeds, so that the method can be used for the production of the next link and the correct expression of protein.

EXAMPLE 3 preparation of PTK-EF1 alpha-SP 4-CAR, PTK-EF1 alpha-SP 4K-CAR, PTK-EF1 alpha-SP 4R-CAR lentiviral vector and live-drop detection

1. Preparation of lentiviral vectors

A multilayer cell culture flask (Hyperflash) was inoculated with 130.0 to 140.0X10 ⁶ numbers of 293T cells (Takara), a total of 560mL of DMEM complete medium (50 mL of fetal bovine serum, 5mL Antibiotic-Antimycotic (100X), and incubated in a 37℃incubator containing 5% CO ₂ for 24 hours. 320. Mu.g of plasmid (PTK-EF 1. Alpha. -SP4-CAR/PTK-EF 1. Alpha. -SP4R-CAR: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid = 12:10:6:5) was added to 960. Mu.g PEI tube, vortexed, and equilibrated at room temperature for 10 minutes, and the above 35mL of PEI and plasmid mixture was mixed with 525mL of DMEM complete medium, respectively, and the multilayer cell culture flask was replaced, and the cell culture flask was incubated in a 35℃incubator containing 5% CO for 3 days, and the cell culture supernatant was collected.

After centrifugation at 4000rpm (or 3000 g) for 30min, cryonase enzyme (Takara) was added to the supernatant after centrifugation and placed at 4 ℃. After 6 hours, the lentiviral supernatant was suction filtered using a 0.22 μm filter and centrifuged at 30000g for 2.5h at 4 ℃. The supernatant was removed and 1mL of T cell medium was added to resuspend the pellet. After resuspension, 20 mu L of the slow virus concentrated solution is reserved for virus activity titer detection, and the rest slow virus concentrated solution is subpackaged, marked as Lenti3-SP4-CAR, lenti3-SP4K-CAR and Lenti3-SP4R-CAR and placed at the temperature of minus 80 ℃ for standby.

2. Lentiviral vector activity titer assay

Principle of: the PE-Labeled Human CD19 Protein is marked with fluorescein, and the PE-Labeled Human CD19 Protein can be specifically combined with ScFv in the CAR, and the fluorescent signal detected by a flow cytometry indirectly reflects the expression condition of the CAR in 293T cells.

The method comprises the following steps: 5.0X10 ⁵ cells/well 293T cells were plated in 6 well plates, 0.5. Mu.L, 1. Mu.L, 2. Mu.L, 5. Mu.L, 10. Mu.L, respectively, of lentiviral concentrate was added to each well, and 1 negative control was set. Culturing in incubator containing 5% CO ₂ at 37deg.C. After three days, 293T cell flow cytometry was collected with Versene solution (Gibco) to detect CAR positive 293T cell proportion and converted to Lenti3-SP4-CAR, lenti3-SP4K-CAR, lenti3-SP4R-CAR lentiviral concentrate active titers.

The active titer of the current lentiviral concentrate is in the range of 1X 10 ⁸～10×10⁸ (TU/mL), and the detection and analysis results are shown in FIG. 1 and Table 2. The result shows that each lentiviral vector can obtain higher activity titer and can be used for the subsequent preparation of chimeric antigen receptor immune cells.

TABLE 2 results of lentiviral Activity titre assay

Sample numbering	Activity titre (TU/mL)
		Lenti3-SP4-CAR	(3.2±0.14)×10⁸
Lenti3-SP4K-CAR	(3.6±0.10)×10⁸
		Lenti3-SP4R-CAR	(3.8±0.13)×10⁸

Example 4 preparation of SP4-CAR-T, SP4K-CAR-T, SP R-CAR-T cells

1. CAR-T cell preparation:

100mL of peripheral blood of a healthy donor is collected, and mononuclear cells are separated by Ficoll lymphocyte separation liquid. After counting, CD3 positive cells were sorted using an appropriate amount of CD3 microblades, human (Meitian. Also known as "Meitian"), and cultured at a density of 1.0 to 2.0X10 ⁶/mL in IL-2 (Shuanglu pharmaceutical industry) of T cell complete broth (OpTmizer^TMCTS^TMT-Cell Expansion Basal Medium,OpTmizer^TMCTST-Cell Expansion Supplement(Invitrogen),500IU/mL, while activating T cells by adding 25. Mu. L Dynabeads HumanT-Activator CD3/CD28 (Invitrogen) per 10 ⁶ cells.

After 24 hours (Day 1), the Lentiviral vectors of Lenti3-SP4-CAR, lenti3-SP4K-CAR and Lenti3-SP4R-CAR are respectively added according to MOI of 5 for transduction, and after uniform mixing, the mixture is placed in a CO ₂ incubator for incubation, and after 4 hours, a proper amount of complete T cell culture medium is added for culture.

After 24 hours of slow virus transduction, the transduced cells are changed into fresh T cell complete culture solution, the density of the living cells is regulated to be 1.0-2.0X10 ⁶/mL, the cells are continuously cultured and amplified for 10-20 days, the cells are observed and counted every day, and the liquid supplementing and expanding culture is carried out according to the counted cell number, so that the cell culture density is always kept to be the same as the whole cell culture density

1.0-2.0×10⁶/mL。

2. SP4CAR-T, SP4KCAR-T, SP4RCAR-T cell transduction efficiency assay

1.0X10 ⁶ transduced T cells were incubated with 1. Mu.LPE-Labeled Human CD Protein for 30min at room temperature, washed twice with physiological saline, and PE fluorescent signals were detected by flow cytometry to measure PE positive cell ratios reflecting the ratio of CAR-T cells in total cells. The results of the SP4-CAR-T, SP4K-CAR-T, SP4R-CAR-T cell transduction efficiency assay are shown in FIG. 2 and Table 3. Table 3 shows that CAR-T cells are successfully prepared and the expression efficiency of the CAR of the SP4K/SP4R CAR-T cells is obviously higher than that of the SP4-CAR-T cells, and the result shows that the expression influence of different signal peptides on chimeric antigen receptors after optimization is different, and the optimization of the SP4K and SP4R signal peptides can obviously improve the expression of CD19 chimeric antigen receptors in T cells.

TABLE 3 CAR-T cell transduction efficiency assay results

Numbering device	Transduction type	Expression efficiency
			1	SP4CAR-T	33.9％
2	SP4KCAR-T	35.5％
			3	SP4RCAR-T	36.8％

Example 5, SP4CAR-T, SP4KCAR-T, SP4RCAR-T in vitro function assay

1. In vitro tumor killing detection:

And performing in-vitro tumor killing function detection on T, SP-CAR-T, SP4K-CAR-T, SP4R-CAR-T cells by adopting a calcein detection method.

The target cells were K562 (CD 19 negative cell line), K562-CD19, NALM6 tumor cell line. Appropriate amounts of target cells were taken and incubated in a 1X 10 ⁶/mL cell suspension (PBS, 5% fetal bovine serum) with Calcein-acetylhydroxymethyl ester (Calcein-AM) to a final concentration of 25. Mu.M for 30min. Cells were resuspended to 0.5X10 ⁵/mL after washing twice at normal temperature, 0.5X10 ⁵/mL cells were added per well in 96-well plates, 25:1 into normal T cells, SP4-CAR-T, SP4K-CAR-T, SP R-CAR-T cells, and incubating at 37 ℃ for 2-3 hours. The supernatant was taken after the incubation was completed, the fluorescence intensity of calcein therein was measured, and the percent target cell lysis was calculated from the spontaneous release control and the maximum release control.

The in vitro killing and cracking results of the common T cells and the SP4-CAR-T, SP4K-CAR-T, SP4R-CAR-T cells on the CD19 high-expression K562-CDl9 cell line are shown in a graph (in) of fig. 3, the in vitro killing and cracking results of the CD19 high-expression NALM6 cell line (human acute lymphoblastic leukemia cell line) are shown in a graph (right) of fig. 3, the in vitro killing and cracking results of the CD19 non-expression K562 cell line are shown in a graph (left) of fig. 3, and the result shows that the targeting and cracking ability of the SP4-CAR-T, SP4K-CAR-T, SP4R-CAR-T cell is improved compared with that of the T cell, so that the in vitro killing and cracking ability of the CD19 high-expression NALM6 cell line and the NALM6 cell line are shown in the graph (in the left). Moreover, in the co-incubation system with K562-CD19 and NALM6 cell lines, the targeting lysis capacity of SP4R-CAR-T cells is obviously higher than that of SP4-CAR-T, SP4K-CAR-T cells. From the above in vitro tumoricidal results, SP4R-CAR-T cells constructed with SP4 are preferred for use in the treatment of tumors.

2. In vitro cytokine detection:

Appropriate amounts of target cells were taken, and after washing twice at room temperature with 1X 10 ⁶/mL of cell suspension (PBS, 5% fetal bovine serum), the cells were resuspended to 0.5X 10 ⁵/mL, 0.05X 10 ⁵/mL of target cells were added to each well of a 96-well plate, and the ratio was 25:1 effective target ratio T, SP4-CAR-T, SP4K-CAR-T, SP4R-CAR-T cells were added, centrifuged at 200g for 30 seconds and incubated at 37 ℃ for 18 hours. The supernatant was taken after the completion of the incubation, and the concentration of IFN-gamma and TNF-alpha therein was measured.

The results of IFN-gamma and TNF-alpha secretion of common T cells, SP4-CAR-T, SP4K-CAR-T, SP4R-CAR-T cells and CD19 high-expression NALM6 tumor cell line after in vitro incubation are shown in figure 4, and are consistent with the tumor killing results, and the concentration results of IFN-Y and TNF-alpha show that the IFN-gamma and TNF-alpha secreted by the SP4-CAR-T, SP4K-CAR-T, SP R-CAR-T cells are obviously improved compared with the common T cells, thereby further illustrating the in vitro killing function of the SP4-CAR-T, SP4K-CAR-T, SP R-CAR-T cells on acute lymphoblastic leukemia.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A chimeric antigen receptor targeting CD19 optimized by a signal peptide, characterized by the sequential splicing from N-terminus to C-terminus: optimized signal peptide, anti-CD 19 antibody single chain variable region, CD8hinge, CD28 transmembrane region, CD28 intracellular domain, intracellular co-stimulatory domain 4-1BB and CD3 zeta chain; the amino acid sequence of the optimized signal peptide is shown as SEQ ID NO.3, and the amino acid sequence of the single-chain variable region of the anti-CD 19 antibody is shown as SEQ ID NO. 7.

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of CD8hinge is shown in SEQ ID No.9, the amino acid sequences of the CD28 transmembrane region and the CD28 intracellular domain are shown in SEQ ID No.11 and SEQ ID No.13, respectively, and the amino acid sequence of intracellular co-stimulatory domain 4-1BB is shown in SEQ ID No.15 and the amino acid sequence of CD3 zeta is shown in SEQ ID No. 17.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the nucleotide sequence of the optimized signal peptide is shown in SEQ ID NO.6, the nucleotide sequence of the single chain variable region of the anti-CD 19 antibody is shown in SEQ ID NO.8, the nucleotide sequence of CD8 range is shown in SEQ ID NO.10, the nucleotide sequences of the CD28 transmembrane region and the CD28 intracellular domain are shown in SEQ ID NO.12 and 14, respectively, the nucleotide sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.16, and the nucleotide sequence of CD3 zeta is shown in SEQ ID NO. 18.

4. The chimeric antigen receptor according to claim 1 or 2, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.23 and the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID No. 24.

5. An immune cell expressing a chimeric antigen receptor, wherein the cell is obtained by transfecting an immune cell of a human or other mammal with the chimeric antigen receptor of any one of claims 1 to 4.

6. The immune cell of claim 5, wherein the human or other mammalian immune cell is selected from the group consisting of umbilical cord blood, apheresis, peripheral blood, or IPSC-derived T cells, NK cells, NKT cells, αβ T cells, γδ T cells, CD4 ⁺ T cells, and CD8 ⁺ T cells.

7. The immune cell of claim 6, wherein the human or other mammalian immune cell is a peripheral blood-derived T cell.

8. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the chimeric antigen receptor-expressing immune cell according to claim 5 or 6 for the preparation of a medicament for the treatment or prevention of a tumor, which is a B-cell acute lymphoblastic leukemia or lymphoma.