Chimeric antigen receptor targeting CD45 and application of chimeric antigen receptor in hematopoietic stem cell transplantation

Abstract

The invention discloses a CD45-targeted chimeric antigen receptor and application thereof in hematopoietic stem cell transplantation, and relates to the technical field of biological medicines. The spatial epitope of the CD45 antigen is taken as a target spot, an scfv region, a transmembrane region and an intracellular signal structural domain of the CD45 antibody are connected in series, and the structure is optimized and modified, so that the immunogenicity of CAR molecules is greatly reduced, the in-vivo duration of CAR-T cells is prolonged, hematopoietic stem cells in host bone marrow can be effectively removed, and the immunogenicity of the CAR molecules is improved. The method can be used for medullary removal pretreatment before hematopoietic stem cell transplantation, and a new strategy is provided for transplantation pretreatment. Correspondingly, due to the advantages of low immunogenicity and the like, an effective prevention strategy is provided for hematopoietic stem cell transplantation related complications such as graft versus host disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor targeting CD45 and its application in hematopoietic stem cell transplantation. Background Art

[0002] Hematopoietic stem cell transplantation involves a pre-transfusion conditioning regimen, including whole-body irradiation and / or high-dose chemotherapy, to eliminate abnormal cells, eradicate underlying disease, and suppress the immune system. Pre-collected pluripotent stem cells (from bone marrow, peripheral blood, or fetal umbilical cord blood) are then infused intravenously into the patient to repopulate the bone marrow and restore hematopoietic and immune function, ultimately achieving a cure. Hematopoietic stem cell transplantation (HSCT) is a major milestone in modern medicine and has become an established treatment for many potentially curative blood cancers, immune disorders, and genetic diseases, with an increasing number of patients surviving the procedure. By 2012, the number of hematopoietic stem cell transplants performed worldwide had reached one million.

[0003] Before HSCT for patients with malignant diseases, they must undergo a course of chemotherapy or high-dose radiotherapy to provide adequate immunosuppression to prevent graft rejection and reduce tumor burden, while also creating space for new hematopoietic stem cells to enter the bone marrow. Regimens include myeloablative, reduced-intensity, and nonmyeloablative regimens. The choice of conditioning regimen is influenced by multiple factors, including the type and stage of the disease, physical condition, and donor availability. Myeloablative regimens, consisting of alkylating agents and total body irradiation (TBI), can maximally deplete the recipient's hematopoietic cells but often result in severe, irreversible pancytopenia and, in most cases, are fatal unless hematopoiesis is restored through HSCT. High-dose TBI-based MAC regimens can reduce relapse rates in patients with hematologic malignancies, particularly acute myeloid leukemia (AML). However, adverse reactions and increased treatment-related mortality (TRM) significantly impact long-term overall survival (OS). Too high a TBI dose will increase the patient's risk of serious complications, such as fatal gastrointestinal, liver, and lung damage, secondary tumors, and growth and developmental disorders in children, while too low a TBI dose will increase the risk of transplant rejection and disease recurrence. In addition, the patient's age and complications limit the widespread use of TBI; patients who have received TBI treatment should not receive TBI-based MAC regimens because the cumulative radiation dose to the target organs increases the risk of TBI-related adverse reactions. In addition, a large number of studies have shown that tissue damage caused by TBI increases the risk of GVHD (graft-versus-host disease). Non-myeloablative regimens reduce the side effects of pretreatment to a certain extent, but the risk of transplant failure and tumor recurrence is greatly increased.

[0004] Limited by traditional conditioning regimens, hematopoietic stem cell transplantation faces many challenges. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a chimeric antigen receptor targeting CD45. The chimeric antigen receptor T cells targeting CD45 prepared based on the CAR molecule can target and kill host hematopoietic stem cells and are used for myeloablative treatment before hematopoietic stem cell transplantation.

[0006] The first object of the present invention is to provide a chimeric antigen receptor targeting CD45 for use in the preparation of a pretreatment drug for hematopoietic stem cell transplantation. The chimeric antigen receptor comprises a CD45 antibody scFv region (as an extracellular antigen binding region), a transmembrane region, and an intracellular signaling domain. The heavy chain sequence of the CD45 antibody scFv region is shown in SEQ ID NO.1, and the light chain sequence is shown in SEQ ID NO.2.

[0007] In the present invention:

[0008] Hematopoietic stem cell transplantation (HSCT) has achieved remarkable results in the treatment of hematologic malignancies, but challenges such as graft-versus-host disease (GVHD) and relapse remain. To ensure successful transplantation, high-dose chemoradiotherapy and immunosuppressive therapy are required, which can delay the recovery of the recipient's immune function, predisposing them to infection, relapse, and even death in severe cases. Therefore, exploring new immunotherapy and gene therapy technologies to further improve the success rate of HSC transplantation and reduce complications and relapse rates is a hot topic in this field.

[0009] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function 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 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 signal transduction regions (CD28, CD3, and CD137 intracellular signaling domains) connected in sequence. After T cells are modified, the surface-expressed CAR first binds to the target cell surface antigen through the antigen recognition region, and then transmits the activation signal into the cell through its signal transduction region, specifically activating the T cell's killing activity against the target cell. 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 (of course, commercial cell lines or allogeneic cell lines can also be used, but autologous T cells are preferred, depending on the specific application). This allows the T cells to express the corresponding CAR on their surface. These modified T cells are then infused back into the patient's body. The modified T cells can then target and kill cells expressing the relevant antigen in the patient's body, achieving the effect of eliminating the relevant target cells. CAR-T cells can specifically eliminate target cells without causing damage to other tissues and organs in the body.

[0010] CD45, also known as protein tyrosine phosphatase receptor C (PTPRC), is also known as leukocyte common antigen (LCA). It is expressed by all hematopoietic lineage cells except mature red blood cells. It is a large glycoprotein of 180-220 kDa, composed of two intracellular phosphatase domains, a transmembrane domain, and an extracellular domain. Expression of different CD45 isoforms in hematopoietic lineage cells. The gene encoding the CD45 protein, PTPRC, was cloned in the 1980s. It is located in the 1q31-32 region of the human chromosome and has a total of 35 exons. Four of these exons (exons 4 / 5 / 6 / 7) can undergo alternative splicing, generating multiple isoforms of the CD45 protein family. Different isoforms are expressed on the surface of different immune cells.

[0011] The present invention constructs CD45-CAR-T cells as a pretreatment solution for hematopoietic stem cells to create space for transplantation of new donor cells. Currently, there are no reports of applying CD45-targeted CAR-T therapy to hematopoietic stem cell transplantation. At the same time, the present invention has modified and screened different isomers and different CAR molecular structures and found that only the CAR-T cells provided by the present invention have greater potential for application in myeloablative pretreatment before recipient hematopoietic stem cell transplantation. Compared with T cells and CAR-T cells with other structures, the CAR-T cells of the present invention can efficiently eliminate target cells.

[0012] Furthermore, the amino acid sequence of the scFv region of the CD45 antibody is shown in SEQ ID NO.3.

[0013] Furthermore, the chimeric antigen receptor contains a CD45 antibody scFv region, a hinge region, a transmembrane region, and an intracellular signaling region connected in series.

[0014] Furthermore, the chimeric antigen receptor contains a CD45 antibody scFv region, a CD8 hinge region, a CD8 transmembrane region, a CD137 intracellular signal region, and a CD3zeta intracellular signal region connected in series.

[0015] Furthermore, the chimeric antigen receptor also contains an extracellular signal peptide structure before the scFv region of the CD45 antibody.

[0016] Furthermore, the extracellular signal peptide may be CD8a signal peptide.

[0017] Furthermore, the amino acid sequences of the CD8a signal peptide, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signal region, and CD3zeta intracellular signal region are shown in SEQ ID NOs. 4-8, respectively.

[0018] A second object of the present invention is to provide a chimeric antigen receptor targeting CD45, wherein the chimeric antigen receptor contains the scFv region, transmembrane region and intracellular signaling domain of the CD45 antibody, the heavy chain sequence of the scFv region of the CD45 antibody is shown in SEQ ID NO.1, and the light chain sequence is shown in SEQ ID NO.2.

[0019] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.9.

[0020] The third object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor.

[0021] Furthermore, the nucleic acid sequence is shown as SEQ ID NO.10.

[0022] The fourth object of the present invention is to provide an expression vector containing the nucleic acid molecule.

[0023] The fifth object of the present invention is to provide a recombinant cell containing the chimeric antigen receptor.

[0024] Furthermore, the starting cells of the recombinant cells include but are not limited to immune cells, such as T cells.

[0025] The sixth object of the present invention is to provide the use of the recombinant cells in the preparation of pretreatment drugs for hematopoietic stem cell transplantation.

[0026] Furthermore, the recombinant cell is a T cell containing the chimeric antigen receptor, and the pretreatment drug is used for pretreatment of myeloablative hematopoietic stem cell transplantation or non-myeloablative hematopoietic stem cell transplantation.

[0027] The sixth object of the present invention is to provide a hematopoietic stem cell transplantation therapeutic drug containing the chimeric antigen receptor, nucleic acid molecule, expression vector or recombinant cell.

[0028] Furthermore, the hematopoietic stem cell transplantation treatment drug is used to eliminate hematopoietic stem cells in the recipient's bone marrow. After pretreatment, low-dose chemotherapy and radiotherapy are performed before HSCT transplantation to eliminate remaining CAR-T cells. The present invention uses CD45-targeted CAR-T cells to reduce the pretreatment chemotherapy and radiotherapy dose.

[0029] By means of the above solution, the present invention has at least the following advantages:

[0030] The present invention transforms and screens antibodies targeting CD45 to obtain a chimeric antigen receptor targeting CD45 with reduced immunogenicity and long in vivo retention time, including an extracellular antigen binding region, a transmembrane region and an intracellular domain that can bind to the CD45 antigen, the transmembrane region peptide segment is a CD8 transmembrane region peptide segment, the intracellular domain is CD3zeta, and the co-stimulatory signal domain is 4-1BB, wherein the extracellular region peptide segment and the transmembrane region peptide segment are connected by a hinge region peptide segment. The present invention targets CD45 and targets hematopoietic stem cell surface markers. Compared with other structures, the CAR-T molecule of the present invention performs better in targeting and killing hematopoietic stem cells of recipients of hematopoietic stem cell transplantation, realizing effective pretreatment of hematopoietic stem cell transplantation, replacing radiotherapy and chemotherapy regimens in traditional pretreatment, reducing the toxic side effects of pretreatment, and improving the quality of life and therapeutic effect of patients after hematopoietic stem cell transplantation.

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 is a schematic diagram of the chimeric antigen receptor of the present invention;

[0034] Figure 2 This is a comparison of the killing ability of CD45-CAR-T cells on target cells CD34+HSC;

[0035] Figure 3 This is a comparison of the ability of CD45-CAR-T cells to secrete IL-2;

[0036] Figure 4 This is a comparison of the ability of CD45-CAR-T cells to secrete IFN-γ. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1 Construction of Chimeric Antigen Receptor (CAR) Lentiviral Expression Vector

[0039] 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 CD45 to construct a chimeric antigen receptor expression vector and subcloned into the PLVX-EF1a (purchased from clontech) vector. The combination order of each element in the constructed chimeric antigen receptor lentiviral expression vector is as follows Figure 1 As shown:

[0040] The amino acid sequences of the various elements in the constructed chimeric antigen receptor are as follows:

[0041] VH: SEQ NO.1

[0042] EVQLVESGAEVKKPGASVKVSCKASGYTFTNYIHWVKQEPGQGLEWIGYFNPYNHGTKYNEKFKGRATLTANKSISTAYMELSSLRSEDTAVYYCARSGPYWFDTWGQGTTVTVSS

[0043] VL: SEQ NO.2

[0044] DILLTQSPATLSLSPGERATFSCRASQNIGTSIQWYQQKTNGAPRLLIRSSSESISGIPSRFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNTWPFTFGQGTKLEIK

[0045] Extracellular antigen binding region sequence: SEQ NO.3

[0046] EVQLVESGAEVKKPGASVKVSCKASGYTFTNYIHWVKQEPGQGLEWIGYFNPYNHGTKYNEKFKGRATLTANKSISTAYMELSSLRSEDTAVYYCARSGPYWFDTWGQGTTVTVSSGGG GSGGGGSGGGGSDILLTQSPATLSLSPGERATFSCRASQNIGTSIQWYQQKTNGAPRLLIRSSSESISGIPSRFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNTWPFTFGQGTKLEIK

[0047] Signal peptide: SEQ NO.4

[0048] MALPVTALLLPLALLLHAARP

[0049] CD8 hinge region: SEQ NO.5

[0050] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0051] CD8 transmembrane region: SEQ NO.6

[0052] IYIWAPLAGTCGVLLLSLVITLYC

[0053] CD137 (4-1BB) intracellular domain: SEQ NO.7

[0054] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

[0055] CD3Zeta: SEQ NO.8

[0056] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0057] Amino acid sequence of the CAR molecule: SEQ NO.9

[0058] MALPVTALLLPLALLLHAARPEVQLVESGAEVKKPGASVKVSCKASGYTFTNYIHWVKQEPGQGLEWIGYFNPYNHGTKYNEKFKGRATLTANKSISTAYMELSSLRSEDTAVYYCARSGPYWFDTWGQGTTVTVSSGGGGSGGGGSGGGGSDILLTQSPATLSLSPGERATFSCRASQNIGTSIQWYQQKTNGAPRLLIRSSSESISGIPSRFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNTWPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0059] CAR molecule nucleotide sequence: SEQ NO.10

[0060]

[0061] Example 2 Lentivirus Preparation

[0062] The specific experimental steps are as follows:

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Example 3 Isolation of primary T cells

[0068] The specific experimental steps are as follows:

[0069] S1. Invert the lymphocyte separation solution upside down several times to thoroughly mix the Lymphoprep reagent.

[0070] 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.

[0071] S3. Dilute the blood sample with an equal volume of PBS + 2% FBS.

[0072] 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.

[0073] S5. Set the centrifuge to 800 x g, set the speed reduction rate to the slowest, and centrifuge at room temperature for 20 minutes.

[0074] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store at -80℃.

[0075] 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.

[0076] 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.

[0077] S9. Add 100 μl / mL antibody cocktail, mix thoroughly, and incubate at room temperature for 15 minutes.

[0078] S10. Take out the magnetic beads and mix them thoroughly by pipetting up and down at least 5 times.

[0079] S11. Pipette 50 μl of magnetic beads / mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

[0080] 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.

[0081] S13. After incubation, keep the tube in the magnet and gently invert it to pour out the cells.

[0082] 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.

[0083] Example 4 Activation of primary T cells and lentiviral infection

[0084] The specific experimental steps are as follows:

[0085] S1. Adjust the cell density to 1*10 6cells / mL, and cytokine and antibody complexes purchased from R&D Company (final concentration of 300U / mL IL-2, 10ng / mL IL-7, 5ng / mL IL-15, 500ng / mL Anti-CD3 (OKT3), 2ug / mL Anti-CD28) were added and cultured for 48 hours.

[0086] 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.

[0087] 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.

[0088] S4. After centrifugation, remove the sealing film and place the six-well plate in a 37°C, 5% CO2 incubator for 24 hours.

[0089] 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.

[0090] Example 5 Isolation of hematopoietic stem cells from umbilical cord blood

[0091] The specific experimental steps are as follows:

[0092] S1. Invert the lymphocyte separation solution upside down several times to thoroughly mix the Lymphoprep reagent.

[0093] 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.

[0094] S3. Dilute the collected umbilical cord blood sample with an equal volume of PBS + 2% FBS.

[0095] 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.

[0096] S5. Set the centrifuge to 800 x g, set the speed reduction rate to the slowest, and centrifuge at room temperature for 20 minutes.

[0097] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store at -80℃.

[0098] 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.

[0099] 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.

[0100] S9, add 100 μl / ml CD34, mix thoroughly and incubate at 2-8°C for 30 minutes while gently tilting and rotating the tube.

[0101] S10. Pour cold buffer into the test tube until it reaches the height of the magnet (or at least 1 mL), and then resuspend the cell-microbead complex.

[0102] S11. Place the tube in a magnet for 2 minutes and discard the supernatant.

[0103] S12. Resuspend the beads-bound cells in 2 mL of buffer by vortexing or pipetting. Separate on a magnet for 1 minute.

[0104] S13. Resuspend the cells in X-vivo 15 medium for later use.

[0105] Example 6 CAR-T cells lyse target cells

[0106] The specific experimental steps are as follows:

[0107] S1, CD34+ hematopoietic stem cells were used as target cells and centrifuged at 400 g for 10 min;

[0108] 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.

[0109] 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 bring the final volume to 100 μL / well. For both the Maxi lysis and Mini lysis assays, inoculate the same number of target cells but without CAR-T cells. Place the plate in a 5% CO2, 37°C incubator and incubate for 6 hours.

[0110] S4. After the incubation period, remove the well plate from the incubator and add the lysis buffer in the LDH detection kit to the Maxi lysis wells. After the target cells are completely lysed, centrifuge the 96-well plate at 1200xg at room temperature for 5 minutes. Gently remove the plate and transfer 50μL from each well to another new 96-well plate. After adding the LDH detection reagent, read the OD value using a microplate reader.

[0111] The formula for calculating the percentage of target cell lysis is:

[0112] lysis%=(OD eachwell -OD minilysis ) / (OD maxilysis )x100%

[0113] S5. The processed data were plotted using GraphPad 6.0.

[0114] Experimental results:

[0115] Using CAR-T cells as effector cells and CD34+ hematopoietic stem cells as target cells, a co-culture system was established according to different effector-target ratios. That is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was removed and centrifuged at 1200xg for 10 minutes at room temperature to allow all suspended cells to settle to the bottom of the well plate. Then, 30 microliters of supernatant was taken from each well, and the amount of LDH released in the culture supernatant was detected to reflect the lysis ability of CAR-T cells on target cells. The results are as follows: Figure 2 As shown in the figure, the chimeric antigen receptor targeting CD45 can efficiently mediate T cell killing of target cells when the effector-target ratio is 1:1; as the effector-target ratio increases, the killing effect of CAR-T cells on target cells also increases, reaching the highest level when the effector-target ratio is 8:1.

[0116] Example 7 Detection of CAR-T cell factor secretion levels

[0117] The specific experimental steps are as follows:

[0118] S1, CD34+ hematopoietic stem cells were used as target cells and centrifuged at 400 g for 10 min;

[0119] 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.

[0120] 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 once 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.

[0121] 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.

[0122] S5. The data obtained above were plotted using GraphPad 6.0.

[0123] Experimental results:

[0124] CAR-T cells were used as effector cells and CD34+ hematopoietic stem cells were used as target cells. A co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was removed and centrifuged at 1200xg for 10 minutes at room temperature to allow all suspended cells to settle to the bottom of the well plate. Then 30 microliters of supernatant was taken from each well, and the expression of IFN-γ and IL-2 secreted by CAR-T cells after activation by CD34+ hematopoietic stem cells in the culture medium supernatant was detected by ELISA. The results are shown in the figure. Figure 3 and Figure 4 As shown in the figure, after the chimeric antigen receptor targeting CD45 combines with the targeted CD34+ hematopoietic stem cells, it can effectively activate primary T cells and cause an increase in the secretion and expression of cytokines; when the effector-target ratio is 1:1, CAR-T cells can secrete a large amount of IFN-γ and IL-2 after being activated by CD34+ hematopoietic stem cells, which is significantly higher than that of control T cells; the secretion amount reaches the highest value when the effector-target ratio is 8:1.

[0125] 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. Use of a chimeric antigen receptor targeting CD45 in the preparation of a pretreatment drug for hematopoietic stem cell transplantation, characterized in that: The chimeric antigen receptor contains the CD45 antibody scFv region, the transmembrane region and the intracellular signaling domain. The heavy chain sequence of the CD45 antibody scFv region is shown in SEQ ID NO.1, and the light chain sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The chimeric antigen receptor contains an extracellular signal peptide, a CD45 antibody scFv region, a hinge region, a transmembrane region and an intracellular signal region which are connected in series.

3. The use according to claim 2, characterized in that The extracellular signal peptide includes a CD8a signal peptide, the hinge region includes a CD8 hinge region, the transmembrane region includes a CD8 transmembrane region, and the intracellular signal region includes a CD137 intracellular signal region and / or a CD3zeta intracellular signal region; Preferably, the amino acid sequences of the CD8a signal peptide, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signal region, and CD3zeta intracellular signal region are shown as SEQ ID NOs. 4-8, respectively.

4. A chimeric antigen receptor targeting CD45, characterized in that The chimeric antigen receptor contains a CD8a signal peptide, a CD45 antibody scFv region, a hinge region, a transmembrane region and an intracellular signaling domain. The heavy chain sequence of the CD45 antibody scfv region is shown in SEQ NO.1, the light chain sequence is shown in SEQ NO.2, and the amino acid sequences of the CD8a signal peptide, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signal region, and CD3zeta intracellular signal region are shown in SEQ ID NOs.4-8, respectively.

5. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 4.

6. An expression vector containing the nucleic acid molecule according to claim 5.

7. A recombinant T cell containing the chimeric antigen receptor according to claim 4.

8. Use of the recombinant T cells according to claim 7 in the preparation of a pretreatment drug for hematopoietic stem cell transplantation.

9. The use according to claim 8, characterized in that The pretreatment drug is used for pretreatment of myeloablative hematopoietic stem cell transplantation or non-myeloablative hematopoietic stem cell transplantation; preferably, it is used to eliminate hematopoietic stem cells in the bone marrow of the transplant recipient.

10. A drug for treating hematopoietic stem cell transplantation, characterized in that: Containing the chimeric antigen receptor according to claim 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 or the recombinant T cell according to claim 7.

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