CN117343191A - EGFRvIII CAR-T cell secreting PD1 and CTLA4 double-single-domain antibodies, and preparation method and application thereof - Google Patents

Abstract

The invention relates to EGFRvIII CAR-T cells secreting PD1 and CTLA4 double-single-domain antibodies, a preparation method and application thereof, wherein chimeric antigen receptors expressed by the CAR-T cells comprise extracellular antigen recognition region EGFRvIII single-domain antibody sequences, PD1 single-domain antibody sequences, CTLA4 single-domain antibody sequences and the like, and are capable of taking PD1 single-domain antibodies and CTLA4 single-domain antibodies out of cells. The preparation method comprises the steps of constructing a recombinant lentiviral vector, packaging lentivirus, infecting T cells by the lentivirus, and the like. The CAR-T cell has excellent killing effect on EGFRvIII positive tumor cells, has enhanced targeting specificity, and is suitable for adoptive immunotherapy of tumors.

Description

EGFRvⅢCAR-T cells secreting PD1 and CTLA4 dual single domain antibodies and preparation method thereof Laws and Applications

Technical field

The invention belongs to the field of tumor biological products and relates to EGFRvⅢCAR-T cells (EGFRvⅢ/PD1/CTLA4 Nb CAR-T cells) secreting PD1 and CTLA4 dual single domain antibodies in adoptive immunotherapy and their preparation methods and applications.

Background technique

T cells can target tumor cells by expressing CAR. Chimeric antigen receptor (CAR) artificially fuses a single-chain antibody that can specifically recognize tumor antigens with the receptor intracellular domain "immunoreceptor tyrosine activation motif" that can activate T cells. Prepared 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 tumor microenvironment. In addition, most of the antigen-recognizing parts of CAR are composed 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- Reasons for the poor efficacy of T treatment. 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 PD1 single domain antibody and a CTLA4 single domain antibody and a preparation method thereof. Another purpose is to provide an engineered CAR-T cell that targets EGFRvIII and secretes Application of CAR-T cells with PD1 single domain antibody and CTLA4 single domain antibody in preparing preparations for treating tumors.

Therefore, in the first aspect, the present invention provides an EGFRvIII CAR-T cell that secretes PD1 and CTLA4 dual single domain antibodies, the CAR-T cell expresses a chimeric antigen receptor, and the chimeric antigen receptor includes 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, PD1 single domain antibody sequence and CTLA4 single domain antibody sequence, and can Transfer PD1 single domain antibody and CTLA4 single domain antibody to the outside of the cell.

In a second aspect, the present invention provides a method for preparing the CAR-T cells described in the first aspect of the present invention. The method includes the following steps:

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

(2) Clone 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 PD1 single domain antibody out of the cells.

In a third aspect, the present invention provides an application of the CAR-T cells according to the first aspect of the present invention in preparing a preparation for treating tumors.

Preferably, the tumor is an EGFRvIII-positive tumor.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising the CAR-T cells according to the first aspect of the present invention or the CAR-T cells prepared according to the method of the second aspect of the present invention and Pharmaceutically acceptable carrier.

In a fifth aspect, the present invention provides a nucleotide sequence encoding a chimeric antigen receptor, wherein the nucleotide sequence encodes a CAR-T cell according to the first aspect of the present invention or a CAR-T cell according to the second aspect of the present invention. The chimeric antigen receptor expressed by the CAR-T cells prepared by the method described in this aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor is a CAR-T cell according to the first aspect of the present invention or prepared according to the method of the second aspect of the present invention. The chimeric antigen receptor expressed by CAR-T cells.

The inventor has discovered through research that in the immunotherapy of EGFRvIII-positive tumors, CAR-T cells that target EGFRvIII and secrete PD1 single domain antibodies and CTLA4 single domain antibodies can bring significant advantages over existing technologies. Effect: Enhance specific targeting, that is, enhance the targeted recognition of tumor antigens by immune cells and reduce toxic side effects on normal tissues; the CAR-T cells (sometimes called EGFRvIII/PD1/CTLA4 Nb CAR-T cells) have These characteristics mainly originate from the functional structure of the chimeric antigen receptor designed in the present invention, which includes an antigen recognition region and an intracellular activation region, and the intracellular activation region can prolong the survival time of cells in the body; in addition, the present invention Lentivirus infection is used to prepare CAR-T cells, providing a method for efficiently preparing EGFRvIII/PD1/CTLA4 Nb CAR-T cells. The CAR-T cells prepared by this method can stably express chimeric antigen receptors and have good Application prospects.

Description of drawings

Figure 1 is a gel electrophoresis diagram of the EGFRvIII/PD1/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/PD1/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 that T cells in Example 4 of the present invention express fluorescence after lentiviral transfection;

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-incubation of EGFRvIII/PD-1/CTLA-4Nb CAR-T cells 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/PD1/CTLA4 Nb CAR-T cells and EGFRvIII-positive tumor cells in Example 6 of the present invention;

Figure 10 shows that after co-incubation of EGFRvIII/PD1/CTLA4 Nb CAR-T cells and EGFRvIII-positive tumor cells in Example 7 of the present invention, the levels of cytokines IFN-γ, TNF-α, and IL-2 secreted by CART cells increased;

Figure 11 shows that the EGFRvIII/PD1/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/PD1/CTLA4 Nb CAR-T in Example 9 of the present invention treated mice bearing EGFRvIII-positive human tumor cells; and

Figure 13 shows that the EGFRvIII/PD1/CTLA4 Nb CAR-T in Example 9 of the present invention prolongs the survival time of mice bearing EGFRvIII-positive human tumor cells.

Detailed ways

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

In a first aspect, the present invention provides an engineered CAR-T cell based on a single domain antibody targeting EGFRvIII and secreting a PD1 single domain antibody and a CTLA4 single domain antibody. The CAR-T cell expresses a chimeric antigen receptor, which includes an extracellular antigen recognition region EGFRvIII single domain antibody sequence, a hinge region CD8 and a transmembrane region CD8 tandem sequence, and an intracellular costimulatory domain CD137 sequence. , intracellular signal transduction domain CD3ζ sequence, PD1 single domain antibody sequence and CTLA4 single domain antibody sequence, and can transport PD1 single domain antibody and CTLA4 single domain antibody to the outside of the cell.

Preferably, the extracellular antigen recognition region EGFRvIII single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO.4.

Alternatively or further preferably, the hinge region CD8 and transmembrane region CD8 tandem sequences are encoded by the nucleotide sequence shown in SEQ ID NO.5.

Alternatively or further preferably, the intracellular costimulatory domain CD137 sequence is encoded by the nucleotide sequence shown in SEQ ID NO.6.

Alternatively or further preferably, the intracellular signal transduction domain CD3ζ sequence is encoded by the nucleotide sequence shown in SEQ ID NO.7.

Alternatively or further preferably, the PD1 single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO. 14.

Alternatively or further preferably, the CTLA4 single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO. 21.

In some preferred embodiments, the chimeric antigen receptor further includes at least one signal peptide sequence, at least one tag sequence, at least one flexible peptide sequence and/or at least one self-cleaving polypeptide sequence.

Preferably, the at least one signal peptide sequence may include three signal peptide sequences, which may be sequentially referred to as a first signal peptide sequence, a second signal peptide sequence and a third signal peptide sequence, which may be sequentially represented by SEQ ID NO.1 , 11 and 18 encode the nucleotide sequences.

Alternatively or further preferably, the at least one tag sequence may be four tag sequences, for example, may be encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 2, 9, 13, 16, 19 and 23 . Specifically, the at least one tag sequence may include two Flag tag sequences (eg, a first Flag tag sequence and a second Flag tag sequence), two Myc tag sequences (eg, a first Myc tag sequence and a second Flag tag sequence). Two Myc tag sequences) and two His tag sequences (for example, it can be a first His tag sequence and a second His tag sequence) for later detection or confirmation. The first Flag tag sequence may be encoded by the nucleotide sequence shown in SEQ ID NO.2; the second Flag tag sequence may be encoded by the nucleotide sequence shown in SEQ ID NO.9; the first The Myc tag sequence can be encoded by the nucleotide sequence shown in SEQ ID NO.13; the second Myc tag sequence can be encoded by the nucleotide sequence shown in SEQ ID NO.16; the first His tag sequence can It is encoded by the nucleotide sequence shown in SEQ ID NO.19; the second His tag sequence may be encoded by the nucleotide sequence shown in SEQ ID NO.23.

Alternatively or further preferably, the at least one flexible peptide sequence is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 3, 8, 12, 15, 20 and 22. For example, the at least one flexible peptide sequence may include 6 flexible peptide sequences as needed (for example, encoded by nucleotide sequences selected from the group consisting of SEQ ID NO. 3, 8, 12, 15, 20 and 22).

Alternatively or further preferably, the at least one self-cleaving polypeptide sequence may be selected from a P2A coding sequence or a T2A coding sequence. The P2A coding sequence may be as shown in SEQ ID NO.10; and/or the T2A coding sequence may be as shown in SEQ ID NO.17.

In some preferred embodiments, the chimeric antigen receptor is encoded by the nucleotide sequence formed by SEQ ID NO. 1 to 23 in series. In some most preferred embodiments, the chimeric antigen receptor is encoded by the nucleotide sequence shown in SEQ ID NO. 24.

For ease of reference, the numbering sequence mentioned above is as shown in the table below:

In some embodiments, the chimeric antigen receptor expressed by the CAR-T cell (in the case of containing the corresponding sequence) can be expressed as flag-Nb(EGFRvIII)-2ndCART-P2A-Nb(PD1)-myc- T2A-6his-Nb (CTLA4), the chimeric antigen receptor is composed of an extracellular antigen recognition region EGFRvIII single domain antibody, hinge region CD8, transmembrane region CD8, intracellular costimulatory domain CD137 and intracellular signal transduction domain in series and can secrete PD1 single domain antibodies and CTLA4 single domain antibodies outside the cells.

The chimeric antigen receptor expressed by the CAR-T cells of the present invention includes a single domain antibody. 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, single-domain antibodies have the advantages of good stability, high affinity, weak immunogenicity, easy genetic modification, and do not suffer from the problems of mispairing that traditional antibodies are prone to and the need to optimize the heavy chain and light chain connection sequences, etc., in tumors. Immunotherapy and other aspects have shown broad application prospects.

The chimeric antigen receptor expressed by the CAR-T cells of the present invention includes an epidermal growth factor receptor III mutant (epidermal growth factor receper variant III, EGFRvⅢ) single domain antibody. EGFRvIII is an epidermal growth factor receptor mutant, 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.

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 receptors expressed by the CAR-T cells of the present invention include programmed death-1 (Programmed death-1, PD1 or PD-1) single domain antibodies and cytotoxic T lymphocyte-associated antigen-4 (CytotoxicT -lymphocyte-associated protein 4, CTLA4 or CTLA-4) single domain antibody. PD1 and CTLA4 are negative regulatory proteins for T cell activation and are two representative immune checkpoints. Monoclonal antibody drugs for PD1 and CTLA4 can improve the anti-tumor effect of T cells and have achieved good results in clinical applications.

This study integrated the functions of a CAR targeting EGFRvIII and secreting PD1 and CTLA4 single domain antibodies into a CAR structure to achieve stronger anti-tumor efficacy of CAR-T cells. CAR-T cells that self-express PD1 single domain antibodies and CTLA4 single domain antibodies are T lymphocytes that are genetically engineered so that while expressing specific chimeric antigen receptor T cells, they can autosecret PD1 single domain while recognizing tumor cells. Antibodies and CTLA4 single domain antibodies. When CAR-T cells contact tumor cells, CAR-T cells are activated to kill tumor cells. At the same time, autocrine immune checkpoint inhibitors (PD1 single domain antibodies and CTLA4 single domain antibodies) can weaken the tumor cells' response to CAR-T cells. The inhibitory effect of T cells can also stimulate endogenous tumor-infiltrating T cells, thereby collaboratively killing tumors and maximizing the anti-tumor effect.

In a second aspect, the present invention provides a method for preparing the CAR-T cells described in the first aspect of the present invention. The method includes the following steps:

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

(2) Clone 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 PD1 single domain antibody out of the cells.

In some preferred embodiments, the T cells are obtained by culturing mononuclear cells isolated from peripheral blood; preferably, the T cells are cultured using human CD3, human CD28 monoclonal antibodies and human IL-2 cytokines. cultured in T cell culture medium.

It is also preferred that the lentiviral vector is lentiviral vector GV538.

In some specific embodiments, the method may include the following steps: designing a CAR target sequence, and obtaining, for example, flag-Nb(EGFRvIII)-2ndCART-P2A-Nb(PD1)-myc-T2A-6his-Nb( CTLA4) CAR target sequence fragment. The digested lentiviral vector GV538 was recombined with the CAR target sequence obtained by PCR. The recombinant lentiviral vector was transformed into DH5α E. coli, and positive clones were screened. Positive clones were sequenced to verify whether the lentiviral vector containing the EGFRvIII/PD1/CTLA4 single domain antibody CAR sequence was successfully constructed. The recombinant positive clone strains that were successfully sequenced were extracted and plasmids were transfected into 293T cells and then RT-PCR was used to identify the expression of the EGFRvIII/PD1/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 may include the following steps:

(1) Construction of recombinant lentiviral vector containing the target CAR sequence: Obtain the CAR fragment such as flag-Nb(EGFRvIII)-2ndCART-P2A-Nb(PD1)-myc-T2A-6his-Nb(CTLA4) by PCR, and The synthesized fragment was subcloned into the BamHI/NheI double-digested lentiviral vector GV538, and 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 pair ampicillin antibiotics. The screened colonies were identified by PCR. The successfully identified bacterial fluids were sequenced. After successful sequencing, plasmid extraction was performed using Plasmid Mini Kit.

(2) Lentivirus packaging: Using a three-plasmid system, the recombinant lentiviral vector containing the CAR sequence is co-transfected into 293T cells through two helper vector plasmids (Helper 1.0 and Helper 2.0). After 48 hours, the green fluorescence of the cells is observed under a fluorescence microscope. 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/PD1/CTLA4 Nb CAR-T cells: Culture T cells for 24 hours in a special T cell culture medium containing human CD3 monoclonal antibody, human CD28 monoclonal antibody and human IL-2 cytokine. 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.

Preferably, in step (4), when the T cells after lentivirus transfection account for 80-90% of the culture flask, the cells are collected and the medium is changed, and IL-2 with a final concentration of 100 U/mL is added. The complete culture medium was used for amplification and culture, and the infected cells were identified using flow cytometry.

It is also preferred that the CAR-T cells are prepared using a lentiviral method.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising the CAR-T cells according to the first aspect of the present invention or the CAR-T cells prepared according to the method of the second aspect of the present invention and Pharmaceutically acceptable carrier.

In a fifth aspect, the present invention provides a nucleotide sequence encoding a chimeric antigen receptor, wherein the nucleotide sequence encodes a CAR-T cell according to the first aspect of the present invention or a CAR-T cell according to the second aspect of the present invention. The chimeric antigen receptor expressed by the CAR-T cells prepared by the method described in this aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor is a CAR-T cell according to the first aspect of the present invention or prepared according to the method of the second aspect of the present invention. The chimeric antigen receptor expressed by CAR-T cells.

Example

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 lymphocytes. Pour the cell 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, the tube can be 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. Add 4 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 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 PRMI 1640 (purchased from ThermoFisher) containing human CD3, human CD28 monoclonal antibody (purchased from ThermoFisher) and human IL-2 cytokine (purchased from PEPROTECH) as a special T cell culture medium. Culture T cells for 24 hours.

Example 2 Construction of recombinant lentiviral vector

The EGFRvIII/PD1/CTLA4 single-domain antibody CAR gene sequence (shown in SEQ ID NO. 24, sometimes called the CAR target sequence) was amplified and synthesized by PCR. The gel electrophoresis pattern of this 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 amplified and synthesized EGFRvIII/PD1/CTLA4 single domain antibody CAR gene sequence to obtain a recombinant lentiviral vector, as shown in Figure 2 shown. 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 examine and analyze the expression of the EGFRvIII/PD1/CTLA4 single domain antibody CAR gene sequence in 293T cells. As shown in Figure 4, the CAR gene in the transfected group The expression level of the sequence was 7089707.881 times that of the untransfected group, indicating that the EGFRvIII/PD1/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, cells were observed under a fluorescence microscope to show obvious green fluorescence (GFP), 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 PD1 single domain antibody and CTLA4 single domain antibody (EGFRvIII/PD1/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/PD1/CTLA4 Nb CAR-T cells by flow cytometry

Collect 1×10 ⁶ lentivirus-transfected EGFRvⅢ/PD1/CTLA4 Nb CAR-T cells, CAR-T cells without extracellular recognition receptors (Mock) and untransfected T cells, and wash them three times with PBS. , using flow cytometry to simultaneously detect the expression of GFP on the surface of CAR-T cells in each group. The results showed that the chimeric antigen receptor was expressed in T cells, and the expression rate was higher than 40%, as shown in Figure 7.

Example 5 After co-culture of EGFRvIII/PD1/CTLA4 Nb CAR-T cells and tumor cells with high expression of EGFRvIII, determination of T cell surface activation molecules CD25 and CD69

Prepare each group of effector T cells (Utd group, Mock group, EGFRvⅢ/CTLA4 Nb-CAR-T cell group, EGFRvⅢ/PD1 Nb-CAR-T cell group and EGFRvⅢ/PD1/CTLA4 Nb-CAR-T cell group). U87 tumor cells that highly express EGFRvIII were added to each cell culture well containing the above-mentioned effector T cells and co-cultured in complete culture 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/PD1/CTLA4 Nb CAR-T cells were co-incubated with EGFRvⅢ-positive tumor cells, the T cell surface activation molecules CD25 and CD69 increased, and the degree of activation was higher than that of other groups, indicating that they were exposed to EGFRvⅢ-positive tumor cells. Afterwards, EGFRvIII/PD1/CTLA4 Nb CAR-T cells were activated more effectively.

Example 6 Determination of T cell proliferation ability after co-culture of EGFRvⅢ/PD1/CTLA4 Nb CAR-T cells and high-expressing EGFRvⅢ tumor cells

Effector T cells from each group were stained with eFluor ^TM 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, the T cell proliferation level after co-culture of EGFRvIII/PD1/CTLA4 Nb CAR-T cells and EGFRvIII-positive tumor cells was significantly higher than that of other T cells.

Example 7 After co-culture of EGFRvⅢ/PD1/CTLA4 Nb CAR-T cells and high-expressing EGFRvⅢ tumor cells, the level of cytokines secreted by T cells was measured.

Effector T cells in each group were mixed with U87 tumor cells that highly expressed EGFRvIII, and 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/PD1/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 CART cells increased significantly and were higher than those in other groups.

Example 8 Cytotoxicity analysis of the killing effect of EGFRvIII/PD1/CTLA4 Nb CAR-T cells on human tumor cells

The target cells that highly express EGFRvIII were stained with eFluor670. The EGFRvIII/PD1/CTLA4 Nb CAR-T cells prepared in Example 4 and the T cells cultured in Example 1 were taken respectively, and the effect-to-target ratio (E:T) was 1: 1. 10:1 and 20:1, add the corresponding number of effector T lymphocytes and target cells to the 24-well plate. After culturing for 16 hours, collect the above cells, add 100 μl of 1×buffer and AnnexinV and 7-AAD staining solutions. 5μl, incubate at room temperature 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- according to the formula The killing efficiency of T cells against 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 target cells without effector cells. As shown in Figure 11, EGFRvIII/PD1/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/PD1/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 in mice was approximately (80-100) ^mm3 , the mice were randomly divided into 6 groups and received different treatments (PBS, Utd, Mock, EGFRvIII/CTLA4 Nb-CAR-T cells, EGFRvIII/PD1Nb-CAR -T cells and EGFRvIII/PD1/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/PD1/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> EGFRvⅢ CAR-T cells secreting PD1 and CTLA4 dual single domain antibodies and their preparation methods and applications

<130> GY22100451

<160> 24

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caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggaggatc tctgagactc 60

tcctgtgcag tctctggatt aacctacagt agcaggtgta cgggctggtt ccgccaggct 120

ccagggaagg agcgcgaggg ggtcgcagct attcatactg gtggtggtat cacatactat 180

accgactccg tgaagggccg attcaccctc tcccaagaca acgccaagaa cacgctgtat 240

ctgcaaatga acaatctgaa acctgaagac actggcatgt actactgtgc ggcaaatccc 300

agtggttatact gctcctcaaa ttttgcttac tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210> 5

<211> 207

<212> DNA

<213>xx

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 6

<211> 126

<212> DNA

<213>xx

<400> 6

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 7

<211> 336

<212> DNA

<213>xx

<400> 7

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>xx

<400> 8

gggggtgggg gatcc 15

<210> 9

<211> 24

<212> DNA

<213>xx

<400> 9

gactacaagg atgacgatga caag 24

<210> 10

<211> 66

<212> DNA

<213>xx

<400> 10

ggcagcggcg ccaccaactt cagcctgctg aagcaggccg gtgacgtgga ggagaatccc 60

ggccct 66

<210> 11

<211> 63

<212> DNA

<213>xx

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 12

<211> 15

<212> DNA

<213>xx

<400> 12

ggcggtggcggatct 15

<210> 13

<211> 30

<212> DNA

<213>xx

<400> 13

gaacaaaaac tcatctcaga agaggatctg 30

<210> 14

<211> 378

<212> DNA

<213>xx

<400> 14

caggtgcagc tgcaggagtc tggaggaggc tcggtgcagt ccggagggtc tctgagactc 60

acctgtgcag cctctggata cacctacagt aactactaca tgggctggtt ccgccaggct 120

ccaggaaagg agcgcgaggg ggtcgcaagt attgatactc ttggttatac aagatacgca 180

gactccgtga aggggcgatt caccatctcc cgtgacaacg ataagaacac gctgtatctg 240

caaatgagca gcctgcaacc ggaggacacg gccatgtatt actgtgcggc ccctcgttct 300

ccttactacc ggggtcagac gttctgggag ggggcgtata actactgggg ccaggggacc 360

caggtcaccg tctcctca 378

<210> 15

<211> 15

<212> DNA

<213>xx

<400> 15

ggaggtggag gatca 15

<210> 16

<211> 30

<212> DNA

<213>xx

<400> 16

gaacaaaaac tcatctcaga agaggatctg 30

<210> 17

<211> 63

<212> DNA

<213>xx

<400> 17

ggcagcggcg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60

cct 63

<210> 18

<211> 63

<212> DNA

<213>xx

<400> 18

atggctctgc ccgtgaccgc tttgttgctg cccctggctt tgctgctcca cgccgccagg 60

ccc 63

<210> 19

<211> 18

<212> DNA

<213>xx

<400> 19

catcatcatc atcatcat 18

<210> 20

<211> 15

<212> DNA

<213>xx

<400> 20

gggggtgggg gatcc 15

<210> 21

<211> 351

<212> DNA

<213>xx

<400> 21

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> 22

<211> 15

<212> DNA

<213>xx

<400> 22

ggcggtggcggatct 15

<210> 23

<211> 18

<212> DNA

<213>xx

<400> 23

catcatcatc atcatcat 18

<210> 24

<211> 2316

<212> DNA

<213>xx

<400> 24

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 aggaggctcg 1380

gtgcagtccg gagggtctct gagactcacc tgtgcagcct ctggataacac ctacagtaac 1440

tactacatgg gctggttccg ccaggctcca ggaaaggagc gcgaggggggt cgcaagtatt 1500

gatactcttg gttatacaag atacgcagac tccgtgaagg ggcgattcac catctcccgt 1560

gacaacgata agaacacgct gtatctgcaa atgagcagcc tgcaaccgga ggacacggcc 1620

atgtattact gtgcggcccc tcgttctcct tactaccggg gtcagacgtt ctgggagggg 1680

gcgtataact actggggcca ggggacccag gtcaccgtct cctcaggagg tggaggatca 1740

gaacaaaaac tcatctcaga agaggatctg ggcagcggcg agggcagagg aagtcttcta 1800

acatgcggtg acgtggagga gaatcccggc cctatggctc tgcccgtgac cgctttgttg 1860

ctgcccctgg ctttgctgct ccacgccgcc aggccccatc atcatcatca tcatgggggt 1920

gggggatccc aggtgcagct gcaggagtct gggggaggct cggtgcaggc tggggggtct 1980

ctaagactct cctgtacagc ctctggattc ggtgttgatg gcactgacat gggctggtac 2040

cgccaggctc cagggaatga gtgcgagttg gtctcaagta ttagcagtat tggtattgga 2100

tactattcag agtccgtgaa gggccgattc accatctccc gagacaacgc caagaacacg 2160

gtgtatctgc aaatgaacag cctgagacct gacgacacgg ccgtgtatta ctgcggtaga 2220

cgatggattg ggtaccgatg tggtaactgg ggccggggga cccaggtcac cgtctcctca 2280

ggcggtggcg gatctcatca tcatcatcat cattaa 2316

Claims (10)

1. An EGFRvIII CAR-T cell secreting PD1 and CTLA4 dual single domain antibodies, characterized in that the CAR-T cell expresses a chimeric antigen receptor, and the chimeric antigen receptor includes 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, PD1 single domain antibody sequence and CTLA4 single domain antibody sequence, and can combine PD1 single domain antibodies and CTLA4 single domain antibodies to the outside of cells. 2. The CAR-T cell according to claim 1, characterized in that: The extracellular antigen recognition region EGFRvIII single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO.4; The hinge region CD8 and transmembrane region CD8 tandem sequences are encoded by the nucleotide sequence shown in SEQ ID NO.5; The intracellular costimulatory domain CD137 sequence is encoded by the nucleotide sequence shown in SEQ ID NO.6; The intracellular signal transduction domain CD3ζ sequence is encoded by the nucleotide sequence shown in SEQ ID NO.7; The PD1 single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO. 14; and/or The CTLA4 single domain antibody sequence is encoded by the nucleotide sequence shown in SEQ ID NO. 21. 3. The CAR-T cell according to claim 1 or 2, characterized in that: The chimeric antigen receptor further includes at least one signal peptide sequence, at least one tag sequence, at least one flexible peptide sequence and/or at least one self-cleaving polypeptide sequence; The at least one signal peptide sequence is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 1, 11 and 18; The at least one tag sequence is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 2, 9, 13, 16, 19 and 23; The at least one flexible peptide sequence is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 3, 8, 12, 15, 20 and 22; and/or The at least one self-cleaving polypeptide sequence is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO. 10 and 17. 4. The CAR-T cell according to any one of claims 1 to 3, characterized in that: The chimeric antigen receptor is encoded by the nucleotide sequence formed by SEQ ID NO.1 to 23 in series; preferably, the chimeric antigen receptor is encoded by the nucleotide sequence shown in SEQ ID NO.24 . 5. A method for preparing the CAR-T cells according to any one of claims 1 to 4, characterized in that the method includes the following steps: (1) Obtain the nucleotide sequence encoding the chimeric antigen receptor; (2) Clone 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 PD1 single domain antibody out of the cells. 6. The method according to claim 5, characterized in that: The T cells are cultured from mononuclear cells isolated from peripheral blood; preferably, the T cells are cultured using a T cell culture medium containing human CD3, human CD28 monoclonal antibodies and human IL-2 cytokines; It is also preferred that the lentiviral vector is lentiviral vector GV538. 7. The use of CAR-T cells according to any one of claims 1 to 4 or CAR-T cells prepared according to the method of claims 5 or 6 in the preparation of tumor treatment preparations; preferably, The tumor is an EGFRvIII-positive tumor. 8. 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. 9. A nucleotide sequence encoding a chimeric antigen receptor, characterized by: The nucleotide sequence encodes the chimeric antigen receptor expressed by 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; Preferably, the nucleotide sequence is shown in SEQ ID NO. 24. 10. A chimeric antigen receptor, characterized in that the chimeric antigen receptor is a CAR-T cell according to any one of claims 1 to 4 or prepared according to the method of claims 5 or 6 The chimeric antigen receptor expressed by CAR-T cells.