GD2 CAR-T cell co-expressing IL-15 and PD-1 antibodies and application of GD2 CAR-T cell

Abstract

The invention discloses a GD2CAR-T cell co-expressing IL-15 and PD-1 antibodies and application of the GD2CAR-T cell, and relates to the technical field of biological medicine. According to the invention, GD2 is taken as a target spot, IL-15 and PD-1 antibodies are tried to be combined for design, and an optimal combination mode of the GD2, the IL-15 and the PD-1 antibodies is found, so that the glioma inhibition effect is exerted to the greatest extent. Finally, it is found that an scfv region of the GD2 antibody, a transmembrane region of CD8, a CD137 intracellular signal region and a CD3zeta intracellular signal region are connected in series and connected with scfv sequences of IL15 and PD1 antibodies, and the optimal multi-target co-expression CAR molecule is obtained. CAR-T cells constructed on the basis of the CAR molecules express IL-15 and PD1 antibodies while killing brain glioma cells in a targeted mode, the brain glioma tumor microenvironment can be effectively resisted, the in-vivo amplification and killing capacity of the CAR-T cells is improved, the treatment effect is improved, and a new strategy is provided for treatment of the brain glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a GD2CAR-T cell co-expressing IL-15 and PD-1 antibodies and applications thereof. Background Art

[0002] Glioblastoma is the most common primary intracranial tumor, with clinical characteristics of high disability rate, high recurrence rate and high mortality rate. It is a recognized difficult disease in the field of tumor treatment. Among them, glioblastoma is highly invasive and has a very poor prognosis. Traditional treatment methods such as surgical resection, radiotherapy and chemotherapy have made certain progress in improving patient survival rates, but there are still serious limitations. Therefore, finding new treatment strategies has become one of the current focuses of research on the treatment of glioma. In recent years, breakthroughs have been made in immunotherapy research. Among them, chimeric antigen receptor T cell (CAR-T) therapy has attracted widespread attention as a novel and promising treatment strategy.

[0003] Chimeric antigen receptors (CARs) are artificial receptors that mimic the functions of T cell receptors, combining the recognition and binding specificity of antigens and antibodies, or ligands and receptors, with the ability of effector T cells to kill recognized tumor cells. CARs are composed of a CD8a guide peptide, an antigen recognition region (ligand or single-chain antibody or Fab fragment), a transmembrane region, and a series of T cell signaling domains (CD28, CD3, and CD137 intracellular signaling domains) linked in sequence. After T cell modification, the surface-expressed CAR first binds to tumor cell surface antigens via the antigen recognition region. Its signaling domain then transmits activation signals intracellularly, activating the T cell's tumor cell-killing activity in a targeted manner. The DNA sequence expressing the CAR is cloned into a lentiviral expression vector and used to infect T cells isolated from the patient's blood, causing them to express the corresponding CAR on their surface. These modified T cells are then infused back into the patient, where they can then target and kill tumor cells expressing the relevant antigen, effectively eliminating tumor cells or related target cells. CAR-T cells can specifically eliminate target cells without causing damage to other tissues and organs of the body.

[0004] Gangliosides are a class of sialic acid-rich glycosphingolipids found on neuroblastoma cell membranes. They are primarily synthesized in the endoplasmic reticulum and Golgi apparatus and can be divided into four series: o-, a-, b-, and c-series. Normal tissues typically express a-series gangliosides, while neuroblastomas express the b-series ganglioside GD2. GD2 isoforms have limited expression in normal tissues but are widely expressed in glioma tumor tissues. GD2 can be considered a tumor-associated antigen and a promising therapeutic target for glioma. GD2 contributes to tumor progression and malignant phenotypes by enhancing cell proliferation, motility, migration, adhesion, and invasion. The development of anti-GD2 monoclonal antibodies and other therapeutic approaches has provided a rationale for targeting the diganglioside GD2 in cancer therapy. Anti-GD2 monoclonal antibodies target GD2-expressing tumor cells, leading to phagocytosis and destruction through antibody-dependent cell-mediated cytotoxicity, lysis through complement-dependent cytotoxicity, and direct cell death through apoptosis and necrosis. Although dactylomab, dactylomab beta, and nasituximab have improved the event-free survival of patients with high-risk, relapsed, or refractory NB, approximately 40% of patients still relapse during or after treatment. GD2CAR-T cells have brought new hope to patients with gliomas. However, cellular immunotherapy using CAR-T cells and ex vivo expanded cytotoxic lymphocytes has not yet achieved significant success in gliomas, with only a small number of patients achieving observable responses. The main reason is that the immunosuppressive microenvironment of GBM and its complex immune escape mechanisms pose many challenges to the implementation of immunotherapy. The increase in the immunosuppressive cell population and cytokine imbalance in the GBM microenvironment severely inhibit the function and expansion of CAR-T cells, weakening the effectiveness of CAR-T cell therapy. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a GD2-targeted chimeric antigen receptor that co-expresses IL-15 and PD-1 antibodies. The CAR-T cells prepared based on this CAR molecule can target tumor cells while also expressing IL-15 cytokines and PD1 antibodies. Under the synergistic effect, they can efficiently target and kill tumor cells, and can effectively resist the tumor microenvironment of brain glioma, thereby improving the treatment effect of brain glioma.

[0006] The first object of the present invention is to provide a chimeric antigen receptor, which comprises a GD2 targeting structure, an IL-15 coding region, and a PD-1 antibody coding region connected together; wherein:

[0007] The GD2 targeting structure comprises a chimeric antigen receptor targeting GD2, wherein the chimeric antigen receptor targeting GD2 comprises a GD2 antibody scFv region (anti-GD2 scFv), a hinge region, a transmembrane region and an intracellular signaling region.

[0008] The IL-15 coding region contains an IL-15 coding sequence, and the PD-1 antibody coding region contains a PD-1 antibody scFv (anti-PD-1 scFv) coding sequence,

[0009] The IL-15 coding region is located between the GD2 targeting structure and the PD-1 antibody coding region, and the GD2 targeting structure is located before the PD-1 antibody coding region.

[0010] In the present invention, a chimeric antigen receptor targeting GD2 was constructed based on GD2 as an effective target for glioma treatment. Furthermore, after multiple explorations and experiments, the inventors discovered that combining it with IL-15 and PD-1 antibodies can produce a synergistic effect, achieving the most effective glioma cell-killing effect. The chimeric antigen receptor of the present invention comprises an extracellular peptide, a transmembrane peptide, and an intracellular domain peptide, connected in series, as well as the scFv of the IL-15 and PD-1 antibodies connected by a 2A peptide. The extracellular peptide comprises a signal peptide and an extracellular antigen-binding region capable of binding to the GD2 antigen. The transmembrane peptide is a CD8 transmembrane peptide, the intracellular domain is CD3zeta, and the costimulatory signaling domain is 4-1BB. The extracellular peptide and the transmembrane peptide are connected by a hinge peptide. When this structure is used to treat gliomas, no significant increase in tumor volume is observed, indicating a significant inhibitory effect. At the same time, the present invention optimizes the combination of scFv of IL-15 and PD1 antibodies with GD2 CAR in T cells to obtain GD2 CAR-T cells that co-express IL-15 and PD1 antibodies. The therapeutic effect is significantly better than GD2 CAR-T, or GD2 CAR-T combined with IL-15 or GD2 CAR-T combined with PD1 antibodies.

[0011] Furthermore, the GD2 targeting structure (the intracellular signaling region) and the IL-15 coding region, and / or the IL-15 coding region and the PD-1 antibody coding region are connected via a self-cleaving peptide.

[0012] Furthermore, the self-cleaving peptide includes 2A peptide, such as P2A peptide, T2A peptide, etc., and the most preferred is P2A peptide (SEQ ID NO. 9).

[0013] Furthermore, the GD2 antibody scfv region contains a GD2 antibody scfv coding sequence, the heavy chain sequence of the GD2 antibody scfv is shown in SEQ ID NO.1, and the light chain sequence of the GD2 antibody scfv is shown in SEQ ID NO.2.

[0014] Furthermore, the hinge region includes a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.5.

[0015] Furthermore, the transmembrane region includes a CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.6.

[0016] Furthermore, the intracellular signaling region contains a costimulatory molecule region and / or a signal transduction molecule region; the costimulatory molecule region includes a CD137 intracellular signaling region, and the signal transduction molecule region includes a CD3zeta intracellular signaling region.

[0017] Furthermore, the amino acid sequence of the CD137 intracellular signaling region is shown as SEQ ID NO.7, and the amino acid sequence of the CD3zeta intracellular signaling region is shown as SEQ ID NO.8.

[0018] Furthermore, according to actual needs, the chimeric antigen receptor can be designed with an extracellular signal peptide region before the GD2 antibody scFv region. Preferably, the signal peptide in the extracellular signal peptide region can be CD8a signal peptide (SEQ ID NO. 4).

[0019] Furthermore, the GD2 antibody scFv region is located before the hinge region, the transmembrane region and the intracellular signaling region are located after the hinge region, and the intracellular signaling region is located at one end of the transmembrane region away from the hinge region.

[0020] Furthermore, the amino acid sequence of IL-15 is shown in SEQ ID NO.10, and the amino acid sequence of PD-1 antibody scfv is shown in SEQ ID NO.11.

[0021] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.12.

[0022] The second object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor.

[0023] Furthermore, the sequence of the nucleic acid molecule is shown as SEQ ID NO.13.

[0024] The third object of the present invention is to provide an expression vector containing the nucleic acid molecule.

[0025] The fourth object of the present invention is to provide a recombinant cell containing the chimeric antigen receptor.

[0026] Furthermore, the starting cells of the recombinant cells include but are not limited to immune cells, such as T cells, chimeric antigen receptor (CAR)-T cells, T cell receptor (TCR)-T cells, tumor infiltrating lymphocytes (TIL), NK cells, NK-T cells, CAR-NK cells, CAR-NKT cells, dendritic cells, macrophages, CAR-macrophages or any genetically modified tumor-specific immune cells.

[0027] A fifth object of the present invention is to provide a CAR-T cell containing the nucleic acid molecule or expression vector.

[0028] The sixth object of the present invention is to provide the use of the chimeric antigen receptor, nucleic acid molecule, expression vector, recombinant cell or CAR-T cell in the preparation of anti-tumor drugs.

[0029] Furthermore, the anti-tumor drug is used to prevent or treat solid tumors, including but not limited to gliomas.

[0030] The seventh object of the present invention is to provide an anti-tumor drug (glioma treatment drug) containing the chimeric antigen receptor, nucleic acid molecule, expression vector, recombinant cell or CAR-T cell.

[0031] By means of the above solution, the present invention has at least the following advantages:

[0032] This invention innovatively incorporates genes expressing IL-15 and the PD-1 antibody scFv into a GD2-targeting CAR structure. This allows CAR-T cells to simultaneously express IL-15 and PD-1 antibodies at high levels, significantly enhancing the tumor-killing ability of CAR-T cells. Our experimental results demonstrate that when the CAR structure targets GD2, GD2 CAR-T cells co-expressing IL-15 and the PD-1 antibody scFv exhibit highly potent cytotoxicity and specificity both in vitro and in vivo. The synergistic effects of the three selected targets, combined with structural optimization, yield superior anti-tumor effects unattainable with other structures, significantly outperforming second-generation CAR-T cells in in vivo testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0034] Figure 1 is a schematic diagram of the chimeric antigen receptor of the present invention.

[0035] Figure 2 This is a comparison of the killing abilities of different CAR-T cells against target cells CHLA255.

[0036] Figure 3 This is a comparison of the IL-2 secretion capabilities of different CAR-T cells.

[0037] Figure 4 This is a comparison of the ability of different CAR-T cells to secrete IFN-γ.

[0038] Figure 5The figure shows the comparative results of the efficacy of different CAR-T cell animal experiments; compared with the 15P GD2CAR-T group, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0040] Example 1 Construction of Chimeric Antigen Receptor (CAR) Lentiviral Expression Vector

[0041] The intracellular domain of 4-1BB (also known as CD137) and the ITAM region of CD3Zeta were used as activation signals and fused with a single-chain antibody targeting GD2 (Anti-GD2 scFv). IL-15 and Anti-PD1 scFv were linked via P2A to construct a chimeric antigen receptor expression vector and subcloned into the PLVX-EF1a (purchased from clontech) vector. The order of the components in the constructed chimeric antigen receptor lentiviral expression vector is as follows: Figure 1 As shown:

[0042] The amino acid sequences of the various elements in the constructed chimeric antigen receptor are as follows:

[0043] Anti-GD2 scfv VH(SEQ ID NO.1):

[0044] EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS

[0045] Anti-GD2 scfv VL(SEQ ID NO.2):

[0046] EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

[0047] Extracellular antigen binding region sequence (SEQ ID NO.3):

[0048] EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

[0049] CD8a signal peptide (SEQ ID NO.4):

[0050] MALPVTALLLPLALLLHAARP

[0051] CD8 hinge region (CD8a Hinge, SEQ ID NO.5):

[0052] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0053] CD8 transmembrane region (CD8a TM, SEQ ID NO.6):

[0054] IYIWAPLAGTCGVLLLSLVITLYC

[0055] CD137 (4-1BB) intracellular domain (SEQ ID NO.7): <000012​​​​​​​​​​​​​​​​IL-15 sequence (SEQ ID NO.10):

[0062] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0063] Anti-PD1 scfv sequence (SEQ ID NO.11):

[0064] MALPVTALLLPLALLLHAARPQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK

[0065] Full-length sequence of chimeric antigen receptor (CAR) (SEQ ID NO.12):

[0066] MALPVTALLLPLALLLHAARPEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK

[0067] Chimeric antigen receptor (CAR) full-length nucleotide sequence (SEQ ID NO.13):

[0068]

[0069] Example 2 Lentivirus Preparation

[0070] The specific experimental steps are as follows:

[0071] S1, prepare a 15cm dish, inoculate 5*10 6 293T cells (purchased from ATCC) were added with complete culture medium (DMEM high glucose, 10% FBS, double antibody), placed in a 37°C, 5% CO2 incubator, and cultured overnight.

[0072] S2. Remove 100 μM PEI and lentiviral packaging plasmids (PLVX-EF1a-CAR, pGP, and pVSVG) from the refrigerator. Thaw at room temperature and mix thoroughly by pipetting up and down. Remove PBS or HBSS buffer and warm to room temperature. Transfer 2 mL of PBS to one well of a 6-well plate and add 10 μg of PLVX-EF1a-CAR, 4 μg of pGP, and 2 μg of pVSVG, respectively. Mix thoroughly by pipetting up and down. Then, add 18 μL of 100 μM PEI and immediately mix by pipetting up and down. Let stand at room temperature for 10 minutes.

[0073] S3. Add the DNA / PEI complex dropwise to a 15 cm culture dish and gently shake the dish to mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator. After 6-8 hours of incubation, remove the culture medium containing the transfection reagent and replace with fresh complete culture medium.

[0074] After 48 hours of continuous incubation, collect the virus-containing supernatant from the culture dish, filter it through a 0.45 μm filter, add 20% volume of 50% PEG 6000 solution, incubate at 4°C for 2 hours, transfer to a centrifuge tube, balance, and centrifuge at 3000 x g at 4°C for 0.5 hours. After centrifugation, carefully remove the liquid from the centrifuge tube in a biosafety cabinet, resuspend the pellet in 500 μL of PBS buffer, and store the virus at -80°C.

[0075] Example 3 Isolation of primary T cells

[0076] The specific experimental steps are as follows:

[0077] S1. Invert the lymphocyte separation solution upside down several times to thoroughly mix the Lymphoprep reagent.

[0078] S2. In a biosafety cabinet, add 15 mL of Lymphoprep reagent to a 50 mL centrifuge tube (or a 15 mL centrifuge tube, depending on the volume of the blood sample to be separated) and set aside.

[0079] S3. Dilute the blood sample with an equal volume of PBS + 2% FBS.

[0080] S4. Use a pipette to carefully add the diluted blood sample slowly along the wall of the tube to the upper layer of the separation reagent to avoid mixing the separation reagent and the blood sample.

[0081] S5. Set the centrifuge to 800 x g, set the speed reduction rate to the slowest, and centrifuge at room temperature for 20 minutes.

[0082] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store at -80℃.

[0083] S7. Gently aspirate the mononuclear cell layer at the interface of serum and separation reagent into a new centrifuge tube and wash the cells once with culture medium.

[0084] S8. Adjust the cell density to 1*10 8 cells / mL (total volume not to exceed 2.5 mL) and resuspend in a 5 mL round-bottom tube.

[0085] S9. Add 100 μl / mL antibody cocktail, mix thoroughly, and incubate at room temperature for 15 minutes.

[0086] S10. Take out the magnetic beads and mix them thoroughly by pipetting up and down at least 5 times.

[0087] S11. Pipette 50 μl of magnetic beads / mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

[0088] S12. Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let it stand at room temperature for 5 minutes.

[0089] S13. After incubation, keep the tube in the magnet and gently invert it to pour out the cells.

[0090] S14. Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U / mL IL-2, 5 ng / mL IL-15 and 10 ng / mL IL-7.

[0091] Example 4 Activation of primary T cells and lentiviral infection

[0092] The specific experimental steps are as follows:

[0093] S1. Adjust the cell density to 1*10 6cells / mL, and cytokine and antibody complexes (final concentration of 300 U / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 500 ng / mL Anti-CD3 (OKT3), 2 ug / mL Anti-CD28) were added and cultured for 48 hours.

[0094] S2. Calculate the required amount of virus based on an MOI of 20. The calculation formula is as follows: Required amount of virus (mL) = (MOI*number of cells) / virus titer.

[0095] S3. After removing the virus from the -80°C freezer, rapidly thaw it in a 37°C water bath. Add the calculated amount of virus to a six-well plate and polybrene to a final concentration of 6 μg / mL. Mix thoroughly, seal the plate with sealing film, and centrifuge at 800 x g for 1 hour.

[0096] S4. After centrifugation, remove the sealing film and place the six-well plate in an incubator at 37°C and 5% CO2 for 24 hours.

[0097] S5. Centrifuge at 250 x g for 10 minutes, remove the virus-containing culture medium supernatant, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days.

[0098] Example 5 CAR-T cells lyse target cells

[0099] Real-time label-free dynamic cell analysis (RTCA) utilizes a specialized process to integrate a microelectronic cell sensor chip into the bottom of a cell assay plate, creating a cell impedance detection sensing system that dynamically and quantitatively tracks changes in cell morphology, proliferation, and differentiation in real time. When cells adherent to the microelectrode surface cause changes in the impedance of the adherent electrode interface, these changes correlate with changes in the cells' real-time functional state. Real-time dynamic electrode impedance detection can provide biological information related to cellular physiological functions, including cell growth, extension, morphological changes, death, and adherence. This method enables real-time monitoring of CAR-T cell killing of target cells.

[0100] The specific experimental steps are as follows:

[0101] S1. Using CHLA255 cells (neuroblastoma cell line) as target cells, remove the target cells from the incubator, discard the supernatant, add PBS to rinse once, and discard the PBS.

[0102] S2. Add 0.25% trypsin for digestion. When the cells are basically detached, add complete culture medium to terminate the digestion. Transfer to a centrifuge tube and mix well. Take 20 μL of cell suspension and mix with 20 μL of AO / PI staining solution and count.

[0103] S3. Take the required amount of cell suspension, centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend in culture medium.

[0104] S4. Take out the 96W20idf electrode plate and add 100 μL of target cell suspension to each well, 40,000 target cells.

[0105] S5. After the cells are added, the electrode plate is placed on the clean bench and allowed to stand for 15 minutes.

[0106] S6. Place the electrode plate into the Station and fasten it. Click Check to check whether the electrode plate and the workstation are connected properly and then click Start to start data acquisition.

[0107] S7. After the cells are added, the electrode plate is placed on a clean bench and allowed to stand for 15 minutes.

[0108] S8. After approximately 24-36 hours of data collection (depending on experimental requirements), the target cells are in a stable cell growth plateau phase.

[0109] S9, CAR-T cells and T cells were blown evenly for counting. 20 μL of cell suspension was mixed with 20 μL of AO / PI staining solution for counting. The required amount of cell suspension was centrifuged at 500 g for 5 min, the supernatant was discarded, and the culture medium was resuspended to adjust the corresponding concentration.

[0110] S10. Click Pause to remove the electrode plate, aspirate the culture medium, add CAR-T cells and T cell suspension according to the effector-target ratio, and replenish the corresponding culture medium to the same final volume in each well. Return the electrode plate to the workstation, click Check, and then click Resume.

[0111] S11. After continuing to collect data for 48 hours (determined by experimental requirements), click Finish to end the experiment.

[0112] Experimental groups:

[0113] (1) NC group: no effector cells added

[0114] (2) T cell group: T cells were used as effector cells and added at an effector-target ratio of 1:1.

[0115] (3) GD2 CAR-T group: GD2 CAR-T cells were used as effector cells, and effector cells were added at an effector-target ratio of 1:1.

[0116] (4) αPD-1GD2 CAR-T group: GD2 CAR-T cells secreting PD-1 antibodies were used as effector cells, and effector cells were added at an effector-target ratio of 1:1.

[0117] (5) 15GD2 CAR-T group: GD2 CAR-T cells that secrete IL-15 were used as effector cells, and effector cells were added at an effector-target ratio of 1:1.

[0118] (6) 15P GD2 CAR-T group: GD2 CAR-T cells co-expressing IL-15 and PD1 antibodies were used as effector cells, and effector cells were added at an effector-target ratio of 1:1.

[0119] in,

[0120] In the above experimental groups, the GD2 CAR structure co-expressing IL-15 and PD1 antibodies is shown in Figure 1 , GD2CAR secreting PD-1 antibody and Figure 1 The difference between the structures shown is that they do not contain IL-15 and the preceding P2A peptide. The GD2 CAR that secretes IL-15 is different from Figure 1 The structures shown differ only in that they do not contain the Anti-PD1 scFv and the P2A peptide preceding it.

[0121] GD2 CAR-T cells are common second-generation CAR-T cells, and their preparation is the same as in Examples 1-4.

[0122] αPD-1GD2 CAR-T is a GD2 CAR-T that secretes PD1 antibodies and was prepared as in Example 1-4.

[0123] 15GD2 CAR-T is a GD2 CAR-T that secretes IL-15 cytokine and is prepared as in Example 1-4.

[0124] 15P GD2 CAR-T is a GD2 CAR-T that co-expresses IL-15 cytokine and PD1 antibody and is prepared as in Example 1-4.

[0125] Data analysis: Open the analysis software (CP96Analyze) for data analysis, open the file, and select the required data to export.

[0126] Experimental results:

[0127] A co-culture system was established using different CAR-T cells or T cells as effector cells and the brain glial cell line CHLA255 as target cells. In a 96-well plate, 40,000 target cells were fixed in each well. The co-culture system was cultured in serum-free medium for continuous culture and data analysis was performed using analysis software (CP96Analyze). Figure 2As shown in the figures, for brain glioma cell lines, the killing effect of the 15P GD2CAR-T cells of the present invention is better than that of other experimental groups.

[0128] Example 6 Detection of CAR-T cell factor secretion levels

[0129] The specific experimental steps are as follows:

[0130] S1. CHLA255 cells were used as target cells and centrifuged at 400 g for 10 min.

[0131] S2. Resuspend the target cells in culture medium and adjust the cell density to 5*10 5 100 μL / well. Add 100 μL of sterile water to each unused well around the 96-well plate to prevent evaporation of water in the central wells. Place the plate in a 5% CO2, 37°C incubator and incubate overnight.

[0132] S3. Collect the prepared CAR-T cells by centrifugation and resuspend in serum-free 1640 medium. Remove the 96-well plate from the incubator, completely aspirate the medium in the wells, and gently wash the cells with sterile PBS. Then, add CAR-T cells according to the above E / T ratio and make the final volume up to 100 μL / well. Place the plate in a 5% CO2, 37°C incubator and incubate for 6 hours. Simultaneously, establish a control T cell group.

[0133] S4. After the incubation period, remove the well plate from the incubator, centrifuge the 96-well plate at 1200 x g at room temperature for 5 minutes, gently remove the plate, transfer 50 μL of culture supernatant from each well, use ELISA kit to detect the expression of IFN-γ and IL-2, and read the OD value using a microplate reader.

[0134] S5. The data obtained above were plotted using GraphPad 6.0.

[0135] Experimental results:

[0136] Different CAR-T cells were used as effector cells and CHLA255 cells were used as target cells. A co-culture system was established at the same effector-target ratio. That is, in a 96-well plate, the number of fixed target cells in each well was 40,000. Different CAR-T cells were added respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was removed and centrifuged at 1200×g for 10 minutes at room temperature to allow all suspended cells to settle to the bottom of the well plate. Then, 30 μL of supernatant was taken from each well, and the expression levels of IFN-γ and IL-2 secreted by CAR-T cells after activation by tumor cells in the culture medium supernatant were detected by ELISA. The results are shown in FIG. Figure 3 and Figure 4As shown in the figure, after the chimeric antigen receptor targeting GD2 binds to the targeted tumor cells, it can effectively activate primary T cells and cause an increase in the secretion and expression of cytokines. However, when the effector-target ratio is 1:1, the 15P GD2 CAR-T cells can secrete a large amount of IFN-γ and IL-2 after being activated by tumor cells, which is significantly higher than the CAR-T cells and control T cells in other experimental groups. It can be seen that the 15P GD2 CAR of the present invention has an excellent effect in the treatment of brain glioma.

[0137] Example 7 CAR-T cell in vivo efficacy experiment

[0138] S1. CHLA255 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS before inoculation. Tumor cells were purchased from Saiye Biotechnology Co., Ltd. (Suzhou) and inoculated subcutaneously into NKG mice (male, 6 weeks old). Each mouse was inoculated with 1x10 7 cells / 100ul;

[0139] S2. Grouped medication:

[0140] The average tumor volume of the mice was 105.26 mm3. The mice were randomly divided into groups based on tumor volume. Each group consisted of 6 mice, for a total of 36 mice. The day of grouping was defined as D0, and drug treatment was administered on the same day.

[0141] Experimental groups:

[0142] (1) NC group: injection of 100 μl PBS;

[0143] (2) T cell group: 1E06 T cells were injected;

[0144] (3) GD2 CAR-T group: 1E06 GD2 CAR-T cells were injected;

[0145] (4) αPD-1GD2 CAR-T group: 1E06 αPD-1GD2 CAR-T cells were injected;

[0146] (5) 15GD2 CAR-T group: 1E06 15GD2 CAR-T cells were injected;

[0147] (6) 15P GD2 CAR-T group: 1E06 15P GD2 CAR-T cells were injected;

[0148] S3. Experimental observation and data collection:

[0149] After cell inoculation, the effects of the tumor on the animals' normal behavior were routinely monitored weekly. Specific content included the animals' activity, food and water intake, weight gain or loss, and abnormalities of the eyes, fur, and other conditions.

[0150] After the start of drug administration, the tumor size was observed and the mice were weighed. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 );

[0151] S4. Mice were euthanized 65 days after CAR-T cell therapy.

[0152] Experimental results: Figure 5 As shown in the results, GD2 CAR-T cells co-expressing IL-15 and PD1 antibodies have significant efficacy in mouse tumor models and are superior to other CAR-T cells.

[0153] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor contains a GD2 targeting structure, an IL-15 coding region, and a PD-1 antibody coding region that are connected and arranged; The GD2 targeting structure comprises a chimeric antigen receptor targeting GD2, wherein the chimeric antigen receptor targeting GD2 comprises a GD2 antibody scFv region, a hinge region, a transmembrane region and an intracellular signaling region; The IL-15 coding region contains an IL-15 coding sequence, and the PD-1 antibody coding region contains a PD-1 antibody scFv coding sequence, The IL-15 coding region is located between the GD2 targeting structure and the PD-1 antibody coding region, and the GD2 targeting structure is located before the PD-1 antibody coding region.

2. The chimeric antigen receptor according to claim 1, wherein Contains at least one of the following characteristics: (1) The GD2 targeting structure is connected to the IL-15 coding region, and / or the IL-15 coding region is connected to the PD-1 antibody coding region via a self-cleaving peptide; (2) The GD2 antibody scFv region is located before the hinge region, the transmembrane region and the intracellular signaling region are located after the hinge region, and the intracellular signaling region is located at one end of the transmembrane region away from the hinge region; (3) The chimeric antigen receptor also has an extracellular signal peptide region before the scFv region of the GD2 antibody.

3. The chimeric antigen receptor according to claim 2, characterized in that Contains at least one of the following characteristics: (1) Self-cleaving peptides include 2A peptides; (2) The self-cleaving peptide includes a P2A peptide and / or a T2A peptide; the amino acid sequence of the P2A peptide is shown in SEQ ID NO.9; (3) The signal peptide in the extracellular signal peptide region includes a CD8a signal peptide; the amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO.

4.

4. The chimeric antigen receptor according to claim 1, wherein Contains at least one of the following characteristics: (1) The GD2 antibody scFv region contains the GD2 antibody scFv coding sequence, the heavy chain sequence of the GD2 antibody scFv is shown in SEQ ID NO.1, and the light chain sequence of the GD2 antibody scFv is shown in SEQ ID NO.2; (2) The hinge region includes a CD8 hinge region; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO. 5; (3) The transmembrane region includes a CD8 transmembrane region; the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.6; (4) The intracellular signaling region contains a costimulatory molecule region and / or a signal transduction molecule region; the costimulatory molecule region includes a CD137 intracellular signaling region, and the signal transduction molecule region includes a CD3zeta intracellular signaling region; (5) The amino acid sequence of IL-15 is shown in SEQ ID NO. 10; (6) The amino acid sequence of the PD-1 antibody scFv is shown in SEQ ID NO. 11; (7) The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

12.

5. A nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The sequence is shown as SEQ ID NO.

13.

7. An expression vector or recombinant T cell containing the nucleic acid molecule according to claim 5 or 6.

8. A CAR-T cell, characterized in that: The CAR-T cell contains the nucleic acid molecule according to claim 5 or 6 or the expression vector according to claim 7.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 or 6, the expression vector or recombinant T cell according to claim 7, or the CAR-T cell according to claim 8 in the preparation of an anti-tumor drug, characterized in that: The tumor includes a glioma.

10. A drug for treating brain glioma, characterized in that: Containing the chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 or 6, the expression vector or recombinant T cell according to claim 7, or the CAR-T cell according to claim 8.

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