CN117343196A - CAR-T cell for secreting CTLA4 single domain antibody by targeting EGFRvIII, and preparation method and application thereof - Google Patents

Abstract

The invention mainly relates to a target EGFRv III secretion CTLA4 single domain antibody CAR-T cell, a preparation method and application thereof. The CAR-T cells are chimeric antigen receptor-modified T cells, including extracellular antigen recognition region egfrvlll single domain antibody sequences, CTLA4 single domain antibody sequences, and the like. The method comprises the steps of constructing a recombinant lentiviral vector, packaging lentivirus, infecting T cells by the lentivirus so as to obtain the engineering CAR-T cells which are based on single domain antibody targeting EGFRvIII and secrete CTLA4 single domain antibody, and the cells have better killing effect on EGFRvIII positive tumor cells.

Description

CAR-T cells targeting EGFRvIII and secreting CTLA4 single domain antibodies and preparation methods thereof and application

Technical field

The invention belongs to the field of tumor biological products and relates to CAR-T cells (EGFRvIII/CTLA4 Nb CAR-T cells) that target EGFRvIII and secrete CTLA4 single domain antibodies in adoptive immunotherapy and their preparation methods and applications.

Background technique

T cells can target tumor cells by expressing chimeric antigen receptors (CAR). Chimeric antigen receptor (CAR) artificially combines a single-chain antibody that can specifically recognize tumor antigens with the receptor intracellular domain "immune receptor tyrosine activation motif" that can activate T cells. Prepared by fusion into recombinant genes. CAR-T cell therapy is currently the most popular and research-worthy treatment method in tumor immunotherapy. At present, CAR-T therapy has made good progress in hematological tumors. However, due to the complex microenvironment of solid tumors, the killing effect of CAR-T on solid tumors is limited. Therefore, new methods need to be explored to improve the effectiveness of CAR-T in the treatment of solid tumors. active.

The basic design of CAR includes tumor-associated antigen binding region, intracellular signaling region, transmembrane region, extracellular hinge region, etc. CAR-T has now developed into the fourth generation and has made good progress in blood tumors. However, for the treatment of solid tumors, CAR-T cell therapy has been ineffective. An important reason is that tumor-specific T cells are difficult to break through. Immunosuppressive effects of the solid tumor microenvironment. In addition, most of the antigen-recognizing parts of CAR currently consist of single-chain antibodies. Due to mismatches between the VL and VH of different single-chain antibodies, the affinity of the expressed single-chain antibodies decreases, which also causes CAR -The reason why T treatment is not effective. Therefore, new methods need to be explored to improve the activity of CAR-T in treating solid tumors.

Contents of the invention

The purpose of the present invention is to provide an engineered CAR-T cell based on a single domain antibody that targets EGFRvIII and secretes a CTLA4 single domain antibody and a preparation method thereof. Another purpose of the present invention is to provide an engineered CAR-T cell that targets EGFRvIII and secretes CTLA4. Application of single domain antibody CAR-T cells in the preparation of preparations for treating tumors.

In order to achieve the above objects, the present invention provides in a first aspect a chimeric antigen receptor for expression in CAR-T cells, wherein the chimeric antigen receptor includes: at least one signal peptide sequence, extracellular antigen Recognition region EGFRvⅢ single domain antibody sequence, hinge region CD8 and transmembrane region CD8 tandem sequence, intracellular co-stimulatory domain CD137 sequence, intracellular signal transduction domain CD3ζ sequence, CTLA4 single domain antibody sequence, at least one flexible peptide sequence and at least one Self-cleaving peptide sequences.

In a second aspect, the present invention provides a nucleotide sequence encoding the chimeric antigen receptor described in the first aspect of the present invention.

In a third aspect, the present invention provides a CAR-T cell expressing the chimeric antigen receptor described in the first aspect of the present invention.

In a fourth aspect, the present invention provides a method for preparing the CAR-T cells according to the first aspect of the present invention, wherein the method includes the following steps:

(1) Obtain the nucleotide sequence encoding the chimeric antigen receptor;

(2) Recombine the nucleotide sequence into a lentiviral vector and express it in E. coli to obtain a recombinant lentiviral vector including the nucleotide sequence;

(3) Use the recombinant lentiviral vector to package the lentivirus by transfecting 293T cells to obtain the packaged lentivirus;

(4) Co-culture the packaged lentivirus with T cells to obtain CAR-T cells that express the chimeric antigen receptor and can secrete the CTLA4 single domain antibody outside the cells.

In a fifth aspect, the present invention provides a use of the CAR-T cells described in the third aspect of the present invention or the CAR-T cells prepared according to the method described in the fourth aspect of the present invention in the preparation of preparations for treating tumors.

In a sixth aspect, the present invention provides a pharmaceutical composition, which comprises: the CAR-T cells described in the third aspect of the present invention or the CAR-T cells prepared according to the method described in the fourth aspect of the present invention; and a pharmaceutically acceptable carrier.

Compared with the existing technology, the present invention has the following technical advantages:

(1) Strong targeting specificity. The CAR-T cells of the present invention can express chimeric antigen receptors containing EGFRvⅢ single domain antibody sequences, can target EGFRvⅢ-positive tumors, and have enhanced ability of immune cells to target and recognize tumor antigens, so they can significantly reduce the impact on normal tissues or Cell toxic side effects.

(2) Strong anti-tumor ability. Since the CAR-T cells can also express CTLA4 single domain antibodies and secrete them out of the cells, the killing or killing ability of tumor cells is further improved.

(3) Cells have a long survival time in vivo. Since the chimeric antigen receptor contains an antigen recognition region and an intracellular activation region, these regions can prolong the survival time of cells in the body.

(4) The method of the present invention uses lentivirus infection to prepare the CAR-T cells. Experiments have proven that this method can prepare CAR-T cells that can stably express the chimeric antigen receptor, so it has very good application prospects.

Description of drawings

Figure 1 is a gel electrophoresis diagram of the EGFRvIII/CTLA4 single domain antibody CAR gene sequence synthesized by the PCR method of the present invention.

Figure 2 is a schematic diagram of the recombinant lentiviral vector in Example 2 of the present invention.

Figure 3 is a PCR identification chart of DH5α-positive clone colonies in Example 2 of the present invention.

Figure 4 shows the RT-PCR method in Example 3 of the present invention to identify the expression of EGFRvIII/CTLA4 single domain antibody CAR in cells.

Figure 5 shows the recombinant lentiviral vector packaging in Example 3 of the present invention.

Figure 6 shows the fluorescence expression of T cells after lentiviral transfection in Example 4 of the present invention.

Figure 7 shows the detection of GFP expression on CAR after lentiviral transfection of T cells in Example 4 of the present invention.

Figure 8 shows that T cell surface activation molecules CD25 and CD69 increased after co-culture of EGFRvIII/CTLA4 Nb CART and EGFRvIII positive tumor cells in Example 5 of the present invention.

Figure 9 shows that T cell proliferation increased after co-incubation of EGFRvIII/CTLA4 Nb CAR-T cells and EGFRvIII-positive tumor cells in Example 6 of the present invention.

Figure 10 shows that after the EGFRvIII/CTLA4 Nb CAR T cells in Example 7 of the present invention were co-incubated with EGFRvIII-positive tumor cells, the levels of cytokines IFN-γ, TNF-α, and IL-2 secreted by the CAR-T cells increased.

Figure 11 shows that the EGFRvIII/CTLA4 Nb CAR T in Example 8 of the present invention specifically kills EGFRvIII-positive tumor cells.

Figure 12 shows the tumor growth slowdown after the EGFRvIII/CTLA4 Nb CAR T cells in Example 9 of the present invention treat EGFRvIII-positive mice bearing human tumor cells.

Figure 13 shows that after the EGFRvIII/CTLA4 Nb CAR T cells in Example 9 of the present invention treated EGFRvIII-positive mice bearing human tumor cells, the survival time of the mice was prolonged.

Detailed ways

Specific embodiments of the present invention will be further described below.

In the first aspect, the present invention provides a chimeric antigen receptor for expression in CAR-T cells. The chimeric antigen receptor includes: at least one signal peptide sequence, an extracellular antigen recognition region EGFRvⅢ single domain antibody sequence , hinge region CD8 and transmembrane region CD8 tandem sequences, intracellular costimulatory domain CD137 sequence, intracellular signal transduction domain CD3ζ sequence, CTLA4 single domain antibody sequence, at least one flexible peptide sequence and at least one self-cleaving polypeptide sequence.

In some preferred embodiments, the at least one signal peptide sequence includes a first signal peptide sequence and a second signal peptide sequence; preferably, the first signal peptide sequence is encoded by SEQ ID NO.1, and the first signal peptide sequence is encoded by SEQ ID NO.1. The second signal peptide sequence is encoded by SEQ ID NO. 11.

In other preferred embodiments, the extracellular antigen recognition region EGFRvIII single domain antibody sequence is encoded by SEQ ID NO.4.

In other preferred embodiments, the hinge region CD8 and transmembrane region CD8 tandem sequences are encoded by SEQ ID NO.5.

In other preferred embodiments, the intracellular costimulatory domain CD137 sequence is encoded by SEQ ID NO.6.

In other preferred embodiments, the intracellular signal transduction domain CD3ζ sequence is encoded by SEQ ID NO.7.

In other preferred embodiments, the CTLA4 single domain antibody sequence is encoded by SEQ ID NO. 14.

In other preferred embodiments, the at least one flexible peptide sequence is a G4S flexible peptide sequence; more preferably, the G4S flexible peptide sequence includes a first G4S flexible peptide sequence, a second G4S flexible peptide sequence, a third G4S flexible peptide sequence and the fourth G4S flexible peptide sequence; further preferably, the first G4S flexible peptide sequence, the second G4S flexible peptide sequence, the third G4S flexible peptide sequence and the fourth G4S flexible peptide sequence are selected from SEQ. Sequence coding of ID NO.3, 8, 12 and 15.

In other preferred embodiments, the at least one self-cleaving polypeptide sequence is a P2A sequence, preferably encoded by SEQ ID NO. 10.

In some preferred embodiments, the chimeric antigen receptor further includes a Flag tag sequence and/or a Myc tag sequence.

Preferably, the Flag tag sequence includes a first Flag tag sequence and a second Flag tag sequence. It is also preferred or further preferred that the Myc tag sequence includes a first Myc tag sequence and a second Myc tag sequence.

More preferably, the Flag tag sequence is encoded by SEQ ID NO.2 or SEQ ID NO.9; and/or the Myc tag sequence is encoded by SEQ ID NO.13 or SEQ ID NO.16.

In some preferred embodiments, the chimeric antigen receptor is composed of the following sequences in series: a first signal peptide sequence, a first Flag tag sequence, a first G4S flexible peptide sequence, and an extracellular antigen recognition region EGFRvIII single domain. Antibody sequence, hinge region CD8 and transmembrane region CD8 tandem sequence, intracellular costimulatory domain CD137 sequence, intracellular signal transduction domain CD3ζ sequence, second G4S flexible peptide sequence, second Flag tag sequence, self-cleaving polypeptide sequence, The second signal peptide sequence, the third G4S flexible peptide sequence, the first Myc tag sequence, the CTLA4 single domain antibody sequence, the fourth G4S flexible peptide sequence, and the second Myc tag sequence;

In some particularly preferred embodiments, the chimeric antigen receptor is a sequence encoded by the nucleotide shown in SEQ ID NO. 17.

In a second aspect, the present invention provides a nucleotide sequence encoding the chimeric antigen receptor described in the first aspect of the present invention. In some particularly preferred embodiments, the nucleotide sequence is as shown in SEQ ID NO. 17.

In a third aspect, the present invention provides a CAR-T cell expressing the chimeric antigen receptor described in the first aspect of the present invention. The CAR-T cell is an engineered CAR-T cell based on a single domain antibody targeting EGFRvIII and capable of secreting the CTLA4 single domain antibody into the extracellular space. The chimeric antigen receptor expressed by the cell includes the corresponding sequence. In the case of , it can be written as CART(2flag-VH(E3)-2ndCART-P2A-VH(C4)-2myc), for example. In some embodiments, the chimeric antigen receptor includes tandem extracellular antigen recognition region EGFRvIII single domain antibody, hinge region CD8, transmembrane region CD8, intracellular costimulatory domain CD137 and intracellular signal transduction domain CD3ζ sequence . The above-mentioned method can secrete CTLA4 single domain antibody to the outside of cells.

In some particularly preferred embodiments, the chimeric antigen receptor CART(2flag-VH(E3)-2ndCART-P2A-VH() modified in the CAR-T cells targeting EGFRvIII and secreting CTLA4 single domain antibodies The nucleotide sequence of C4)-2myc) can be shown as SEQ ID NO. 17 in the sequence listing.

In a fourth aspect, the present invention provides a method for preparing the CAR-T cells according to the first aspect of the present invention, wherein the method includes the following steps:

(1) Obtain the nucleotide sequence encoding the chimeric antigen receptor;

(2) Recombine the nucleotide sequence into a lentiviral vector and express it in E. coli to obtain a recombinant lentiviral vector including the nucleotide sequence;

(3) Use the recombinant lentiviral vector to package the lentivirus by transfecting 293T cells to obtain the packaged lentivirus;

(4) Co-culture the packaged lentivirus with T cells to obtain CAR-T cells that express the chimeric antigen receptor and can secrete the CTLA4 single domain antibody outside the cells.

In some specific embodiments, the method includes the following steps: designing a CAR target sequence, and obtaining a coding chimeric antigen receptor such as CART(2flag-VH(E3)-2ndCART-P2A-VH(C4)-2myc by PCR) ) of the CAR target sequence fragment. Specifically, the enzyme-digested viral vector, such as a lentiviral vector (such as GV538), can be recombined with the CAR target sequence obtained by PCR. For detection purposes, the recombinant lentiviral vector can also be transformed into DH5α E. coli and positive clones can be screened. The positive clones were sequenced to verify whether the lentiviral vector containing the EGFRvIII/CTLA4 single domain antibody CAR sequence was successfully constructed. The successfully sequenced recombinant positive clone strains were extracted with plasmids, transfected into 293T cells, and then RT-PCR was used to identify the expression of the EGFRvIII/CTLA4 single domain antibody CAR sequence in the cells. After verifying that the recombinant lentiviral vector can be expressed normally, a three-plasmid system is used to package the lentivirus and the titer is tested. Lentiviral infection technology is used to construct CAR-T cells, so that the T cells express the chimeric antigen receptor.

In other specific embodiments, the method includes the following steps:

(1) Construction of a recombinant lentiviral vector containing the target CAR sequence: obtain a CAR fragment such as CART (2flag-VH(E3)-2ndCART-P2A-VH(C4)-2myc) by PCR, and subclone the synthesized fragment into the BamHI/NheI double-digested lentiviral vector GV538, transform the recombinant lentiviral vector into DH5α E. coli, and use the primers on the lentiviral vector and the primers on the target gene to screen the colonies after ampicillin antibiotics screening. PCR identification. The successfully identified bacterial fluids were sequenced. After successful sequencing, plasmid extraction was performed using Plasmid Mini Kit.

(2) Lentiviral packaging: A three-plasmid system can be used to co-transfect 293T cells with the recombinant lentiviral vector containing the CAR sequence through two helper vector plasmids (such as Helper 1.0 and Helper 2.0). Observe the cells under a fluorescence microscope after 48 hours. Green fluorescent expression (GFP). After successfully expressing fluorescence, the 293T cell supernatant was collected, concentrated and purified, and the virus concentration was measured using the fluorescence dilution method. The number of GFP-positive cells was counted. The virus titer was calculated according to the formula: virus concentration = number of fluorescent cells/volume of virus stock solution in the well.

(3) T cell preparation: Isolate mononuclear cells from peripheral blood and culture them to obtain T cells.

(4) Preparation of EGFRvIII/CTLA4 Nb CAR-T cells: Use special T cells containing human CD3 monoclonal antibody, human CD28 monoclonal antibody (purchased from Thermo Company, USA) and human IL-2 cytokine (purchased from PEPROTECH Company, USA). Cell culture medium (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) was used to culture T cells for 24 hours. The next day, add the lentiviral solution to the stimulated T cells at MOI 30 and culture them in a 37°C CO ₂ incubator. At 48 hours, place the CAR-T cells under a common microscope to observe the fluorescence expression (GFP) of the CAR-T cells.

In step 4), when the T cells after lentivirus transfection account for 80-90% of the culture flask, the cells can be collected and replaced with a complete culture medium of IL-2 with a final concentration of 100 U/mL. Expansion culture was performed, and infected cells were identified using flow cytometry.

In some of the methods, the CAR-T cells are prepared using lentiviral methods.

In a fifth aspect, the present invention provides a use of the CAR-T cells described in the third aspect of the present invention or the CAR-T cells prepared according to the method described in the fourth aspect of the present invention in the preparation of preparations for treating tumors. The tumor refers to an EGFRvIII-positive tumor.

In a sixth aspect, the present invention provides a pharmaceutical composition, which comprises: the CAR-T cells described in the third aspect of the present invention or the CAR-T cells prepared according to the method described in the fourth aspect of the present invention; and a pharmaceutically acceptable carrier.

The chimeric antigen receptor of the present invention includes an epidermal growth factor receptor III mutant (epidermalgrowthfactor receper variant III, EGFRvIII) sequence. EGFRvIII is a tumor-specific receptor that is only expressed on the surface of glioblastoma (GBM) and other tumor cells, but does not exist in normal tissues. The expression rate of EGFRvIII in newly diagnosed GBM cases is about 30%, making it an ideal target for the treatment of glioblastoma (GBM). The expression of EGFRvIII in tumors is often associated with poor prognosis. Antibody drugs targeting EGFRvIII have great therapeutic value in inhibiting the progression and invasion of GBM.

The chimeric antigen receptors of the present invention include single domain antibody sequences. Single domain antibody (Nanobody, Nb) is currently the smallest functional antigen-specific binding natural fragment, consisting of approximately 120 amino acids, with a length of 4 nm and a diameter of 2.5 nm. Compared with traditional monoclonal antibodies and Fab fragments (55 × 10 ³ ) or scFv (28×10 ³ ), single domain antibodies have a smaller molecular weight, so they have stronger and faster tissue penetration capabilities and can reach dense tissues such as solid tumors to exert their effects. In addition, the single-domain antibody of the present invention has good stability, high affinity, weak immunogenicity, easy genetic modification, and does not have the problems of traditional antibodies prone to mispairing and the need to optimize the heavy chain and light chain connection sequences. Advantages, showing broad application prospects in tumor immunotherapy and other aspects.

Immune checkpoints are regulatory molecules that play a suppressive role in the immune system. They are usually highly expressed on the surface of tumor cells. They inhibit the function of T cells by binding to corresponding receptors/ligands on T cells, which is one of the causes of tumor immune escape. . Immune checkpoint inhibitors can specifically bind to immune checkpoint molecules to restore immune function to immune cells represented by T cells. The chimeric antigen receptor of the present invention includes a cytotoxic T-lymphocyte-associated antigen-4 (Cytotoxic T-lymphocyte-associated protein 4, CTLA4 or CTLA-4) single domain antibody. CTLA4 is a negative regulatory protein for T cell activation and a representative immune checkpoint. CTLA4 monoclonal antibody drugs can improve the anti-tumor effect of T cells and have achieved good results in clinical applications.

The inventors integrated the functions of EGFRvIII-targeting CAR and CTLA4 single-domain antibody secretion into a CAR structure to achieve stronger anti-tumor efficacy of CAR-T cells. CAR-T cells that self-express CTLA4 single domain antibodies are T lymphocytes that are genetically engineered so that they can autosecret CTLA4 single domain antibodies while expressing specific chimeric antigen receptor T cells that recognize tumor cells. When CAR-T cells contact tumor cells, CAR-T cells are activated to kill tumor cells. At the same time, autocrine immune checkpoint inhibitors (CTLA4 single domain antibodies) can weaken the inhibitory effect of tumor cells on CAR-T cells. , and can stimulate endogenous tumor-infiltrating T cells, thereby collaboratively killing tumors and maximizing the anti-tumor effect.

Example

The present invention will be described in further detail below with reference to the examples. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

The experimental methods in the following examples are all conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1 Preparation and activation of T cells

(1) Isolation of peripheral blood T lymphocytes by density gradient centrifugation

Use blood collection tubes with anticoagulants to collect fresh peripheral blood from healthy volunteers, mix equal volumes of 37°C PBS and peripheral blood, use density gradient centrifugation to separate peripheral blood T lymphocytes, and draw 4 ml of 37°C sterile Pour the lymphocyte separation liquid into a 15 ml centrifuge tube. Take 8 ml of diluted peripheral blood and slowly add it to the surface of the separation liquid along the wall of the centrifuge tube. Centrifuge at 2000rpm for 20 minutes. After centrifugation, it can be seen that the tube is divided into four layers. Use a Pasteur tube to carefully absorb the cloudy buffy coat cells (PBMC) along the edge of the centrifuge tube. Collect the cell suspension into a sterile 50ml centrifuge tube and add four layers. Double the volume of PBS and wash the cells twice. Resuspend the collected PBMC cells in complete cell culture medium RIPM 1640, transfer to a cell culture dish, and culture it in a 37°C cell culture incubator for 2 hours. Take the cells suspended in the cell culture dish to another culture dish for culture, and suspend them. The cells are T lymphocytes. T lymphocytes were cultured in complete RIPM 1640 medium containing 100 U/ml rhIL-2.

(2) Culture of T cells: use a special T cell culture medium (purchased from Zhejiang Meisen) containing human CD3, human CD28 monoclonal antibody (purchased from Thermo Company of the United States) and human IL-2 cytokine (purchased from PEPROTECH Company of the United States). Cell Technology Co., Ltd.) cultured T cells for 24 hours.

Example 2 Construction of recombinant lentiviral vector

The EGFRvIII/ CTLA-4 single domain antibody CAR gene sequence (shown in SEQ ID NO. 17, sometimes called the CAR target sequence) was amplified by PCR, and the gel electrophoresis pattern of the sequence is shown in Figure 1.

The GV538 vector was digested using a double enzyme digestion method, and the digested lentiviral vector was recombined with the CAR target sequence to obtain a recombinant lentiviral vector, as shown in Figure 2. The recombinant lentiviral vector was transformed into DH5α E. coli. The primers on the lentiviral vector and the primers on the target gene were used to conduct PCR identification of the colonies screened with ampicillin antibiotics. The PCR product electrophoresis was shown in Figure 3, indicating that it contained the target gene. The lentiviral recombinant vector plasmid of CAR sequence was successfully constructed. The positive clones were sequenced and compared with the target sequence.

Example 3 Lentivirus packaging

The successfully sequenced recombinant positive clones were extracted with plasmid using Plasmid Mini Kit and transfected into 293T cells. Real-time Quantitative PCR Detecting System was used to quantitatively check and analyze the expression of the EGFRvIII/CTLA4 single domain antibody CAR gene sequence in 293T cells. As shown in Figure 4, the CAR gene sequence in the transfection group The expression level was 7596102.362 times that of the untransfected group, indicating that the EGFRvIII/CTLA4 single domain antibody CAR gene sequence was expressed normally in cells. Using a three-plasmid system, the recombinant lentiviral vector containing the CAR sequence was co-transfected into 293T cells through two helper vector plasmids (Helper 1.0 and Helper 2.0). After 48 hours, obvious green fluorescence (GFP) was observed in the cells under a fluorescence microscope and collected. After the 293T cell supernatant is concentrated and purified, the virus concentration is measured using the fluorescence dilution method, and the number of GFP-positive cells is counted. The virus titer is calculated according to the formula: virus concentration = number of fluorescent cells/volume of the virus stock solution in the well, as shown in Figure 5 Show.

Example 4 Construction of CAR-T cells targeting EGFRvIII and secreting CTLA4 single domain antibody (EGFRvIII/CTLA4 single domain antibody CAR-T)

(1) Lentiviral method to infect T lymphocytes: Plate T cells into a 24-well plate, adjust the cell density in each well to 1×10 ⁷ cells, and use a method containing 10% FBS, human CD3 monoclonal antibody, and human CD28 monoclonal The antibody-human IL-2 cytokine-specific medium was cultured for 24 hours. The lentivirus was added to the T cells after stimulation at MOI 30. After 48 hours, the expression of GFP was observed with a fluorescence microscope, as shown in Figure 6.

(2) Detection of CAR expression in EGFRvIII/CTLA4 Nb CAR-T cells by flow cytometry

Collect 1×10 ⁶ lentivirus-transfected EGFRvⅢ/CTLA4 Nb CAR-T cells, CAR-T cells without extracellular recognition receptors (Mock) and untransfected T cells respectively, wash them three times with PBS, and Flow cytometry simultaneously detected the expression of GFP on the surface of CAR-T cells in each group. The results showed that chimeric antigen receptors were expressed in T cells, and the expression rates were all higher than 40%, as shown in Figure 7.

Example 5 Expression of surface activation molecules CD25 and CD69

Prepare each group of effector T cells (Utd group, Mock group, EGFRvⅢNb-CAR T cell group, EGFRvⅢ/CTLA4Nb-CAR T cell group), and add U87 that highly expresses EGFRvⅢ to each cell culture well containing the above effector T cells. tumor cells and co-cultured in complete medium. Collect effector T cells from each group and centrifuge, discard the supernatant, collect the cell pellet, resuspend it in PBS, and centrifuge again to collect the cell pellet. Add PBS to each group to resuspend the cell pellet, add fluorescent antibody CD25 or CD69, mix gently by pipetting, incubate in the dark, and then add PBS to wash the cells. Resuspend the cells in PBS to adjust the corresponding cell concentration, add the filter to the flow tube, keep on ice to protect from light, and put it on the flow cytometer for detection as soon as possible. As shown in Figure 8, after EGFRvⅢ/CTLA4 Nb CART cells were co-cultured with high-expressing EGFRvⅢ tumor cells, T cell surface activation molecules CD25 and CD69 increased. After contact with EGFRvⅢ-positive tumor cells, EGFRvⅢ/CTLA-4Nb CAR-T cells were replaced. Effective activation.

Example 6 Determination of Proliferation Ability

Effector T cells in each group were stained with eFluor 670. The stained effector T cells were mixed with tumor cells that highly expressed EGFRvIII and cultured in an incubator for 5 days. After culture, the effector T cells of each group were collected and washed with PBS. After resuspending in PBS, the fluorescence attenuation of the effector T cells of each group was detected by flow cytometry. The greater the fluorescence attenuation (the flow cytometry result chart moves to the left), the better the cell proliferation is. As shown in Figure 9, after EGFRvIII/CTLA4 Nb CAR T cells were co-cultured with tumor cells that highly expressed EGFRvIII, the T cell proliferation level was significantly higher than that of T cells in other groups.

Example 7 Determination of cytokine levels secreted by T cell proliferation cells

Effector T cells in each group were mixed with tumor cells that highly expressed EGFRvIII in a certain proportion. The cells were cultured in an incubator for 24 hours. After the culture was completed, the supernatant was collected. The levels of cytokines in the supernatant were measured using TNF-α, IFN-γ, and IL-2 enzyme-linked immunosorbent assay kits. As shown in Figure 10, after EGFRvIII/CTLA4 Nb CAR T cells were co-cultured with EGFRvIII-positive tumor cells, the levels of cytokines IFN-γ, TNF-α, and IL-2 secreted by CAR T cells increased significantly.

Example 8 Cytotoxicity analysis of the killing effect of EGFRvIII/CTLA4 Nb CAR T cells on human tumor cells

Use eFluor670 to stain tumor cells that highly express EGFRvIII. Take the EGFRvIII/CTLA4 Nb CAR T cells prepared in Example 4 and the T cells cultured in Example 1, respectively, according to the effective-to-tumor ratio (E:T) of 1:1 and 10, respectively. :1 and 20:1, add the corresponding number of effector T lymphocytes and tumor cells to the 24-well plate. After culturing for 16 hours, collect the above cells, add 100 μl of 1× buffer and 5 μl of AnnexinV and 7-AAD staining solutions at room temperature. Incubate in the dark for 15 minutes. Centrifuge at 2000 rpm for 6 minutes in a high-speed centrifuge, remove the supernatant, and add 400 μl of PBS to resuspend the cells. AnnexinV+7-AAD+ are dead cells. Flow cytometry detects the proportion of AnnexinV+7-AAD+ cells, and calculates CAR T according to the formula Cell killing efficiency of different cells. Killing rate = 100% × (apoptosis rate of experimental wells - apoptosis rate of natural release holes) / (100 - apoptosis rate of natural release holes). The natural release holes are tumor cells without effector cells. As shown in Figure 11, EGFRvIII/CTLA4 Nb CAR T cells have specific killing activity against tumor cells that highly express EGFRvIII.

Example 9 Tumor growth curve and mouse survival curve after EGFRvIII/CTLA4 Nb CAR T treatment of mice bearing EGFRvIII-positive tumor cells

EGFRvIII-positive U87 tumor cells in the logarithmic growth phase were collected and inoculated subcutaneously in NOD/SCID mice. When the subcutaneous tumor volume of the mice was approximately (80-100) ^mm3 , the mice were randomly divided into 5 groups and received different treatments (PBS, Utd, Mock, EGFRvⅢNb-CAR T cells, EGFRvⅢ/CTLA4 Nb-CAR T cells) . A total of 2 treatments were given, one week apart. From the beginning of administration, the tumor volume of the mice was measured every 3 days with a vernier caliper. Use the same treatment method to observe the survival time of another group of mice to analyze the survival rate of each group. The results in Figure 12 show that EGFRvIII/CTLA4 Nb CAR T cells can significantly slow down the tumor growth of tumor-bearing mice and significantly extend the survival time of mice.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Guangxi Medical University

<120> CAR-T cells targeting EGFRvIII and secreting CTLA4 single domain antibodies and their preparation methods and applications

<130> GY22100452

<160> 17

<170> PatentIn version 3.5

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agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 8

<211> 15

<212> DNA

<213> Artificial sequence

<400> 8

gggggtgggg gatcc 15

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<400> 9

gactacaagg atgacgatga caag 24

<210> 10

<211> 66

<212> DNA

<213> Artificial sequence

<400> 10

ggcagcggcg ccaccaactt cagcctgctg aagcaggccg gtgacgtgga ggagaatccc 60

ggccct 66

<210> 11

<211> 63

<212> DNA

<213> Artificial sequence

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 12

<211> 15

<212> DNA

<213> Artificial sequence

<400> 12

ggcggtggcggatct 15

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<400> 13

gaacaaaaac tcatctcaga agaggatctg 30

<210> 14

<211> 351

<212> DNA

<213> Artificial sequence

<400> 14

caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggggggtc tctaagactc 60

tcctgtacag cctctggatt cggtgttgat ggcactgaca tgggctggta ccgccaggct 120

ccagggaatg agtgcgagtt ggtctcaagt attagcagta ttggtattgg atactattca 180

gagtccgtga agggccgatt caccatctcc cgagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgagacc tgacgacacg gccgtgtatt actgcggtag acgatggatt 300

gggtaccgat gtggtaactg gggccggggg acccaggtca ccgtctcctc a 351

<210> 15

<211> 15

<212> DNA

<213> Artificial sequence

<400> 15

ggaggtggag gatca 15

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<400> 16

gaacaaaaac tcatctcaga agaggatctg 30

<210> 17

<211> 1746

<212> DNA

<213> Artificial sequence

<400> 17

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggactaca aggatgacga tgacaaggga ggtggaggat cacaggtgca gctgcaggag 120

tctggggggag gctcggtgca ggctggagga tctctgagac tctcctgtgc agtctctgga 180

ttaacctaca gtagcaggtg tacgggctgg ttccgccagg ctccagggaa ggagcgcgag 240

ggggtcgcag ctattcatac tggtggtggt atcacatact ataccgactc cgtgaagggc 300

cgattcaccc tctcccaaga caacgccaag aacacgctgt atctgcaaat gaacaatctg 360

aaacctgaag acactggcat gtactactgt gcggcaaatc ccagtggtta ctgctcctca 420

aattttgctt actggggcca ggggacccag gtcaccgtct cctcaaccac gacgccagcg 480

ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 540

gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 600

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 660

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 720

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 780

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 840

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 900

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 960

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1020

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1080

acagccacca aggacacccta cgacgccctt cacatgcagg ccctgcccccc tcgcgggggt 1140

gggggatccg actacaagga tgacgatgac aagggcagcg gcgccaccaa cttcagcctg 1200

ctgaagcagg ccggtgacgt ggaggagaat cccggcccta tggccttacc agtgaccgcc 1260

ttgctcctgc cgctggcctt gctgctccac gccgccaggc cgggcggtgg cggatctgaa 1320

caaaaactca tctcagaaga ggatctgcag gtgcagctgc aggagtctgg gggaggctcg 1380

gtgcaggctg gggggtctct aagactctcc tgtacagcct ctggattcgg tgttgatggc 1440

actgacatgg gctggtaccg ccaggctcca gggaatgagt gcgagttggt ctcaagtatt 1500

agcagtattg gtattggata ctattcagag tccgtgaagg gccgattcac catctcccga 1560

gacaacgcca agaacacggt gtatctgcaa atgaacagcc tgagacctga cgacacggcc 1620

gtgtattact gcggtagacg atggattggg taccgatgtg gtaactgggg ccgggggacc 1680

caggtcaccg tctcctcagg aggtggagga tcagaacaaa aactcatctc agaagaggat 1740

ctgtaa 1746

Claims (10)

1. A chimeric antigen receptor for expression in CAR-T cells, characterized by: The chimeric antigen receptor includes: at least one signal peptide sequence, extracellular antigen recognition region EGFRvⅢ single domain antibody sequence, hinge region CD8 and transmembrane region CD8 tandem sequences, intracellular costimulatory domain CD137 sequence, intracellular signal transduction Domain CD3ζ sequence, CTLA4 single domain antibody sequence, at least one flexible peptide sequence and at least one self-cleaving polypeptide sequence. 2. The chimeric antigen receptor according to claim 1, characterized in that: The at least one signal peptide sequence includes a first signal peptide sequence and a second signal peptide sequence; preferably, the first signal peptide sequence is encoded by SEQ ID NO.1, and the second signal peptide sequence is encoded by SEQ ID NO. .11 encoding; The extracellular antigen recognition region EGFRvIII single domain antibody sequence is encoded by SEQ ID NO.4; The hinge region CD8 and transmembrane region CD8 tandem sequences are encoded by SEQ ID NO.5; The intracellular costimulatory domain CD137 sequence is encoded by SEQ ID NO.6; The intracellular signal transduction domain CD3ζ sequence is encoded by SEQ ID NO.7; The CTLA4 single domain antibody sequence is encoded by SEQ ID NO.14; The at least one flexible peptide sequence is a G4S flexible peptide sequence; preferably, the G4S flexible peptide sequence includes a first G4S flexible peptide sequence, a second G4S flexible peptide sequence, a third G4S flexible peptide sequence and a fourth G4S flexible peptide Sequence; More preferably, the first G4S flexible peptide sequence, the second G4S flexible peptide sequence, the third G4S flexible peptide sequence and the fourth G4S flexible peptide sequence are selected from SEQ ID NO. 3, 8, 12 and 15 sequence encoding; and/or The at least one self-cleaving polypeptide sequence is a P2A sequence, preferably encoded by SEQ ID NO.10. 3. The chimeric antigen receptor according to claim 1 or 2, characterized in that: The chimeric antigen receptor also includes a Flag tag sequence and/or a Myc tag sequence; Preferably, the Flag tag sequence includes a first Flag tag sequence and a second Flag tag sequence; It is also preferred that the Myc tag sequence includes a first Myc tag sequence and a second Myc tag sequence; It is further preferred that the Flag tag sequence is encoded by SEQ ID NO.2 or SEQ ID NO.9; and/or the Myc tag sequence is encoded by SEQ ID NO.13 or SEQ ID NO.16. 4. The chimeric antigen receptor according to claims 1 to 3, characterized in that: The chimeric antigen receptor is composed of the following sequences in series: a first signal peptide sequence, a first Flag tag sequence, a first G4S flexible peptide sequence, an extracellular antigen recognition region EGFRvIII single domain antibody sequence, a hinge region CD8 and a transgene. Membrane region CD8 tandem sequence, intracellular costimulatory domain CD137 sequence, intracellular signal transduction domain CD3ζ sequence, second G4S flexible peptide sequence, second Flag tag sequence, self-cleaving polypeptide sequence, second signal peptide sequence, third G4S Flexible peptide sequence, first Myc tag sequence, CTLA4 single domain antibody sequence, fourth G4S flexible peptide sequence, and second Myc tag sequence; Preferably, the chimeric antigen receptor is the sequence encoded by SEQ ID NO. 17. 5. A nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 4. 6. A CAR-T cell expressing the chimeric antigen receptor according to claims 1 to 4. 7. A method for preparing CAR-T cells according to claim 6, characterized in that: the method includes the following steps: (1) Obtain the nucleotide sequence encoding the chimeric antigen receptor; (2) Recombine the nucleotide sequence into a lentiviral vector and express it in E. coli to obtain a recombinant lentiviral vector including the nucleotide sequence; (3) Use the recombinant lentiviral vector to package the lentivirus by transfecting 293T cells to obtain the packaged lentivirus; (4) Co-culture the packaged lentivirus with T cells to obtain CAR-T cells that express the chimeric antigen receptor and can secrete the CTLA4 single domain antibody outside the cells. 8. The method according to claim 6, characterized in that: The T cells are cultured in a T cell culture medium containing human CD3, human CD28 monoclonal antibody, and human IL-2 cytokine; It is also preferred that the lentiviral vector is lentiviral vector GV538. 9. The use of a CAR-T cell according to any one of claims 1 to 4 or a CAR-T cell prepared according to the method of claim 5 or 6 in the preparation of a preparation for treating tumors; Preferably, the tumor is an EGFRvIII-positive tumor. 10. A pharmaceutical composition comprising the CAR-T cell according to any one of claims 1 to 4 or the CAR-T cell prepared according to the method of claim 5 or 6 and a pharmaceutical Acceptable carrier.