A single-chain antibody targeting human PDGFRβ and its application in CAR-T immunotherapy

Abstract

This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a single-chain antibody targeting human PDGFRβ and its application in CAR-T immunotherapy. The invention first obtains an scFv sequence targeting the human PDGFRβ antigen by immunizing mice, then constructs a second-generation CAR based on this sequence. Additionally, CAR-T cells are obtained through lentiviral infection, and these CAR-T cells can effectively kill PDGFRβ antigen-positive 293T cells. This invention provides a novel approach for treating chronic kidney disease, chronic liver disease, cardiovascular disease, and various oncological diseases, including organ fibrosis, by eliminating PDGFRβ-positive cells, and possesses extremely promising potential for further development and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a single-chain antibody targeting human PDGFRβ and its application in CAR-T immunotherapy. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Kidney disease is a major global health problem that seriously threatens human health. With the increasing severity of obesity, aging population and other problems, the prevalence of chronic kidney disease is as high as over 10%. Studies have shown that by the end of this century, chronic kidney disease is expected to become the second leading cause of death in countries with longer life expectancy, and chronic kidney disease will become the fifth most common cause of death worldwide. Renal fibrosis is a common pathological feature of chronic kidney disease caused by various factors. This pathological change is mainly caused by the activation of fibroblasts, pericytes and myofibroblasts. There is currently no effective and feasible treatment. Treating renal fibrosis and reversing kidney function loss are kidney problems that need to be urgently addressed.

[0004] Studies have shown that PDGFRβ antigen is highly expressed on the surface of cells such as fibroblasts, pericytes and myofibroblasts. It is an effective and feasible way to eliminate cells such as fibroblasts, pericytes and myofibroblasts by targeting PDGFRβ antigen. Chimeric antigen receptor-modified T cell therapy (CAR-T) has achieved great success in the treatment of hematological tumors. In view of this, CAR-T immunotherapy is also applicable to non-tumor diseases, such as autoimmune diseases, fibrosis, cardiovascular diseases, etc. Studies have shown that CAR-T cells targeting FAP molecules can effectively inhibit myocardial fibrosis, and CAR-T cells targeting uPAR molecules can alleviate liver fibrosis by removing senescent cells. However, the inventors found that there is currently no single-chain antibody (scFv) targeting human PDGFRβ antigen that can be used to construct CAR. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a single-chain antibody targeting human PDGFRβ and its application in CAR-T immunotherapy. Specifically, a single-chain antibody targeting human PDGFRβ antigen was obtained by immunizing mice, and a second-generation CAR targeting human PDGFRβ antigen was constructed. It was found that the CAR-T cells can effectively kill PDGFRβ antigen-positive cells, laying the foundation for CAR-T treatment of chronic kidney disease and other fibrosis-related diseases characterized by kidney damage. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] In a first aspect of the present invention, a single-chain antibody (scFv) targeting human PDGFRβ is provided, wherein the single-chain antibody comprises a single-chain antibody heavy chain V H and single-chain antibody light chain V L ; wherein the single-chain antibody heavy chain V H comprising an amino acid sequence as shown in SEQ ID NO.1 or a functional variant thereof; the single-chain antibody light chain V L It comprises an amino acid sequence as shown in SEQ ID NO.2 or a functional variant thereof.

[0008] In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the single-chain antibody or chimeric antigen receptor) and having at least 85% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100%) sequence identity therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith.

[0009] Furthermore, the single-chain antibody heavy chain V H Single chain antibody light chain V L The two may be directly connected or connected via a linker; the linker may be (G4S)n, wherein n is a positive integer, for example, 1, 2, 3, 4, 5 or 6. In some embodiments, n is 3. Further, the amino acid sequence of (G4S)3 is shown in SEQ ID NO.3.

[0010] The second aspect of the present invention provides a chimeric antigen receptor (CAR) targeting human PDGFRβ, which at least comprises the above-mentioned single-chain antibody targeting human PDGFRβ, and the single-chain antibody targeting human PDGFRβ is used as an antigen binding domain in the chimeric antigen receptor.

[0011] Furthermore, the chimeric antigen receptor targeting human PDGFRβ is formed by a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain in series;

[0012] Wherein, the signal peptide can guide the antigen binding domain and hinge region to transfer to the cell membrane surface. Any suitable signal peptide or combination of signal peptides can achieve the purpose of the present invention. In a specific embodiment of the present invention, the signal peptide can be a CD8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.4.

[0013] The hinge region may be a CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO.5.

[0014] The transmembrane region may be a CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.6.

[0015] The co-stimulatory signal transduction domain may be a 4-1BB co-stimulatory signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.7.

[0016] The signal transduction domain may be a CD3ζ signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.8.

[0017] In addition, any peptide chain can be inserted as a spacer at a suitable position between the above-mentioned antigen recognition region, hinge region, transmembrane region and intracellular signal region. The peptide chain can be an oligopeptide or a polypeptide, which is not specifically limited here.

[0018] According to the present invention, the chimeric antigen receptor is composed of a CD8 signal peptide, an antigen binding domain (V L -(G4S)3-V H ), CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory signaling domain and CD3ζ signaling domain are connected in series.

[0019] The third aspect of the present invention provides an isolated nucleic acid molecule encoding the above-mentioned single-chain antibody or chimeric antigen receptor targeting human PDGFRβ.

[0020] The fourth aspect of the present invention provides a vector, wherein the vector comprises the above-mentioned nucleic acid molecule.

[0021] According to the present invention, the vector is a viral vector, and the viral vector may be a retroviral vector or a lentiviral vector; further preferably, it is a lentiviral vector, and the vector is a recombinant viral vector expressing the single-chain antibody or chimeric antigen receptor by inserting a nucleic acid molecule encoding the single-chain antibody or chimeric antigen receptor into a virus.

[0022] The fifth aspect of the present invention provides an immunocompetent cell, which expresses any of the above-mentioned single-chain antibodies or chimeric antigen receptors; or, contains a nucleic acid molecule encoding any of the above-mentioned single-chain antibodies or chimeric antigen receptors; preferably, the immunocompetent cell is selected from: T cells, NK cells, monocytes, macrophages, dendritic cells or mast cells; among which T cells are preferred, and further, when they express chimeric antibody receptors or contain nucleic acid molecules encoding chimeric antigen receptors, the immunocompetent cell is a CAR-T cell.

[0023] The sixth aspect of the present invention provides a method for preparing the immune active cells (especially CAR-T cells), which comprises: infecting T cells with a lentivirus; the lentivirus is obtained by transfecting a recombinant lentiviral vector into a lentiviral packaging cell and then culturing the cells; the recombinant lentiviral vector is prepared by inserting a nucleic acid molecule encoding the above-mentioned chimeric antigen receptor into a lentiviral vector.

[0024] The seventh aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or a combination of: any of the above-mentioned single-chain antibodies; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune-competent cells; or any of the above-mentioned nucleic acid molecules, vectors, or products prepared by any of the above-mentioned methods, and a pharmaceutically acceptable carrier.

[0025] The eighth aspect of the present invention provides the use of any of the above-mentioned single-chain antibodies; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune-competent cells; or any of the above-mentioned nucleic acid molecules, vectors, or products or pharmaceutical compositions prepared by any of the above-mentioned methods in the preparation of drugs for preventing and / or treating diseases mediated by high PDGFRβ expression.

[0026] Specifically, the present invention has confirmed through research that the PDGFRβCAR-T cells of the present invention can inhibit PDGFRβ in vitro. + Cells have a strong killing ability, so the diseases mediated by high PDGFRβ expression include but are not limited to kidney disease, liver disease, cardiovascular disease and tumor disease, especially fibrosis-mediated chronic kidney disease, liver disease, cardiovascular disease and tumor disease.

[0027] The ninth aspect of the present invention provides a method for preventing and / or treating diseases mediated by high expression of PDGFRβ, the method comprising administering to a subject a therapeutically effective amount of any of the above-mentioned single-chain antibodies; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune-competent cells; or any of the above-mentioned nucleic acid molecules, vectors, or products or pharmaceutical compositions prepared by any of the above-mentioned methods.

[0028] The tenth aspect of the present invention provides a kit comprising any of the above-mentioned single-chain antibodies; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune-competent cells; or any of the above-mentioned nucleic acid molecules, vectors, or products or pharmaceutical compositions prepared by any of the above-mentioned methods.

[0029] The eleventh aspect of the present invention provides any of the above-mentioned single-chain antibodies; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune-competent cells; or any of the above-mentioned nucleic acid molecules, vectors, or the products, pharmaceutical compositions or kits prepared by any of the above-mentioned methods for use in detecting PDGFRβ expression or preparing products for detecting PDGFRβ expression.

[0030] Specifically, qualitative or quantitative detection of PDGFRβ expression in a subject sample can be used for screening, (auxiliary) diagnosis or prediction of the progression of a disease mediated by high PDGFRβ expression.

[0031] Beneficial technical effects of one or more of the above technical solutions:

[0032] The above technical scheme first obtains the scFv sequence targeting the human PDGFRβ antigen by immunizing mice, and then constructs the second-generation CAR on this basis. In addition, CAR-T cells are obtained by lentivirus infection. The CAR-T cells can effectively kill PDGFRβ antigen-positive 293T cells.

[0033] The above technical solution provides a new idea for eliminating PDGFRβ-positive cells to treat chronic kidney disease, liver disease, cardiovascular disease and various tumor diseases including various organ fibrosis, and has extremely attractive value for further development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 The results after protein purification in the examples of the present invention;

[0036] Among them: A is the detection of PDGFRβ extracellular domain protein before purification, each band is 1: uninduced bacteria, 2-4: induced bacteria, 5: induced precipitation, 6: induced supernatant, M: marker; B is the detection of PDGFRβ extracellular domain protein after purification, each band is 1: purified protein, M: marker, BSA: bovine serum albumin.

[0037] Figure 2This is the process of mouse immunization and monoclonal antibody cell line acquisition in the embodiments of the present invention.

[0038] Figure 3 This is the PDGFRβCAR structure and CAR-T cell construction in the examples of the present invention.

[0039] Among them: A is the PDGFRβCAR structure; B is the lentiviral vector used to construct CAR; C is the PDGFRβCAR-T cell positive rate detection.

[0040] Figure 4 This is the PDGFRβCAR killing experiment in the embodiment of the present invention;

[0041] Among them: A is the flow cytometry diagram of 293T-PDGFRβ cells; B is the killing curve of PDGFRβCAR-T cells. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0044] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0045] In the present invention, the term "specific binding" means that an antigen-binding molecule (e.g., an antibody) typically binds specifically to an antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is usually expressed in terms of equilibrium dissociation constant (K D ) to reflect that the lower K D Indicates higher affinity.

[0046] In the present invention, the term "antibody" is used in the broadest sense, and refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the heavy chain of an immunoglobulin and / or sufficient sequence from the variable region of the light chain of an immunoglobulin, so as to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen-binding activity.

[0047] In the present invention, the term "monoclonal antibody" refers to an antibody molecule preparation of a single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.

[0048] In the present invention, the term "antibody" can be derived from any animal, including but not limited to humans and non-human animals, wherein the non-human animal can be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (such as sharks).

[0049] In the present invention, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, antigen-binding fragments, naked antibodies, conjugated antibodies, humanized antibodies or fully human antibodies.

[0050] In the present invention, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0051] In the present invention, the terms "identity" and "consistency" are interchangeable and are calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0052] In the present invention, the term "immunoactive cells" refers to cells that are responsible for immune functions in a living body. As immunoactive cells, for example, lymphocytes such as T cells, natural killer cells (NK cells), B cells, etc.; antigen presenting cells such as monocytes, macrophages, dendritic cells, etc.; granulocytes such as neutrophils, eosinophils, basophils, mast cells, etc. Specifically, preferably, T cells or NK cells from mammals such as humans, dogs, cats, pigs, mice, etc. are listed, preferably T cells or NK cells from humans. In addition, T cells can be separated and purified from body fluids such as blood, bone marrow fluid, and tissues such as spleen, thymus, lymph nodes, or immunoactive cells infiltrated into cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites, and T cells made from ES cells and iPS cells can also be used. It should be noted that the source of immunoactive cells and the subject of administration may be the same or different. Furthermore, when the subject of administration is a human, as immunoactive cells, autologous cells collected from the patient himself as the subject of administration can be used, or allogeneic cells collected from others can be used. That is, the donor and the acceptor may or may not be identical, but are preferably identical.

[0053] In the present invention, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and the like that are physiologically compatible. Generally, the nature of the carrier depends on the specific mode of administration adopted. For example, parenteral preparations generally include an injectable fluid as a vehicle, and the injectable fluid includes a pharmaceutically and physiologically acceptable fluid, such as water, physiological saline, balanced salt solution, aqueous glucose solution, glycerol, etc. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered can also include a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0054] In the present invention, the term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic or preventive result at the necessary dosage and time period. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0055] The term "treatment and / or prevention" refers to an attempt to alter the natural course of a disease in a treated individual, and may be a clinical intervention performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a condition.

[0056] In the present invention, "individual" or "subject" is preferably a mammal. The mammal includes humans and non-human mammals, and the non-human mammals include mice, rats, guinea pigs, cattle, sheep, cats, dogs, horses, monkeys, gorillas, etc., wherein the individual or subject is preferably a human.

[0057] Specifically, the cells designed by the present invention are human T cells.

[0058] Among them: human T cells are derived from peripheral blood and activated by stimulation with CD3 / CD28 magnetic beads.

[0059] The CAR-T cells used in the present invention are constructed by lentiviral infection.

[0060] Wherein: the lentiviral vector is pCDH-EF1-MCS-T2A-copGFP, and the relevant lentivirus is packaged by using the second-generation lentiviral packaging method, using auxiliary plasmids PSPAX2 and PM2G, and the infection coefficient MOI=20.

[0061] The CAR-T cells targeting human PDGFRβ of the present invention can effectively kill cells expressing PDGFRβ in vitro. + of 293T cells.

[0062] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0063] Example

[0064] method:

[0065] Mouse immunization process

[0066] The amino acids of human PDGFRβ antigen are shown in SEQ ID NO.9. The gene synthesis design constructs the extracellular region sequence of human PDGFRβ antigen (SEQ ID NO.10) into the pcDNA3.0 vector, and adds a membrane localization signal peptide (SEQ ID NO.11) in front. Add 2μL of plasmid to 50μL BL21 competent bacteria and place on ice for 30min. Heat shock at 42℃ for 90s, quickly place on ice for 5min, and add 500μL LB culture medium. Shake at 37℃, 220rpm for 1h, spread on an LB plate containing ampicillin resistance, and invert and culture at 37℃ overnight. Pick a single clone on the plate and inoculate it in a test tube containing 5mL LB culture medium containing ampicillin resistance. Shake at 37℃ and 200rpm until the culture medium OD reaches 0. 600 0.6-0.8. Add IPTG to a final concentration of 0.1mM, and set a control without IPTG. Shake at 16℃200rpm for 16h to induce the expression of fusion protein. Take out 1mL of culture, centrifuge at 12000g for 10min at room temperature, discard the supernatant, resuspend the bacterial pellet with 50μL 1×PBS buffer and add 25μL 3×Loading Buffer. Analyze by 12% SDS-PAGE gel electrophoresis, the fusion protein is expressed, about 80kDa, mainly expressed in the form of inclusion bodies. The constructed band ( Figure 1 A), and then a relatively pure PDGFRβ antigen extracellular domain protein was obtained by protein purification technology ( Figure 1 B) Immune process such as Figure 2 As shown, 4-week-old Balb / c cells were immunized with PDGFRβ extracellular domain protein purified from eukaryotic cells via tail vein infusion. The immunization dose was 100 μg / mouse / time, for a total of 4 immunizations. Fusion was performed 13 days after the end of immunization.

[0067] Screening of high affinity PDGFRβ scFv

[0068] Blood collection and testing: Immunization was performed according to the mature immunization process and titer determination and other index tests were completed. In this example, the mice and their related data corresponding to the optimal cell lines and antibody results were effectively obtained were summarized and summarized. As shown in Table 1, multiple rounds of blood collection and testing data showed that the antibody titer of 2# mouse was the highest, so 2# mouse was selected for hybridoma fusion.

[0069] Fusion and cloning: Use SP2 / 0 mouse myeloma cells and spleen cells of preferred mice (2#) for cell fusion. After fusion, culture, observation, detection and positive and negative control tests are performed to obtain a batch of hybridoma cells that meet the experimental requirements, and continue to culture and select them.

[0070] 2# Mouse strain identification and sequencing: After fusion, primary cloning by limiting dilution method and supernatant detection, 14 cell lines with 31 cells that met the project requirements were obtained. By comparison, a hybridoma with high titer and optimal affinity was sequenced to obtain the full-length PDGFRβ antibody sequence. The antibody heavy chain / light chain variable region genes were obtained by PCR to obtain PDGFRβscFv.

[0071] Table 1 Blood test results

[0072]

[0073] The structure of CAR

[0074] The sequence of CAR in this example is CD8 signal peptide-VL-(G4S)3-VH-CD8 hinge region-CD8 transmembrane region-4-1BB-CDζ. This sequence was synthesized by Qingke Biotechnology Co., Ltd. and constructed into the specified vector pCDH-EF1-MCS-T2A-copGFP ( Figure 3 B), the front and rear restriction sites are XbaI and BamHI respectively, and the sequences are found to be consistent after sequencing. The second-generation virus packaging system is used to package into lentivirus (the packaging plasmids are PSPAX2 and PM2G).

[0075] Feeder cell preparation

[0076] BALB / C mice were killed by pulling the neck, and then disinfected by soaking in 75% alcohol for 5-10 minutes. A small cut was made in the mouse abdomen with surgical scissors, and the skin was peeled off to expose the abdominal cavity. The peritoneal membrane was clamped with hemostatic forceps and lifted up (do not clamp the abdominal organs), and 4ml-5ml DMEM solution was injected into the abdominal cavity with a syringe. The mouse legs were clamped with forceps and the mouse was shaken back and forth for about 1 minute. The peritoneal fluid was still withdrawn with the syringe and added to the centrifuge tube. Repeat 2-3 times. Centrifuge at 1000r / min for 10 minutes. The supernatant was discarded, and the cells were made into a suspension with HAT selection medium.

[0077] Preparation of spleen cells and myeloma cells

[0078] Kill the mice by pulling their necks, soak them in 75% alcohol for 5-10 minutes for disinfection, remove them for dissection, and remove the spleen under sterile conditions. Rinse the spleen in 5ml DMEM solution to wash away the red blood cells on the spleen. Use a 5ml syringe to absorb DMEM and blow the spleen to form a spleen cell suspension. Transfer the suspension into a 50ML centrifuge tube and repeat several times until the spleen becomes transparent. Discard the culture medium of SP2 / 0 cultured in advance, blow off the SP2 / 0 with new DMEM preheated at 37℃, transfer it to the centrifuge tube containing spleen cells in the above step, and mix the two cells. Place the centrifuge tube in a normal temperature centrifuge and centrifuge at 1000rpm for 5-10 minutes.

[0079] Fusion

[0080] After the above cells are centrifuged, the supernatant is discarded, the wall of the centrifuge tube is wiped dry with high-pressure filter paper, the tube cap is tightened, the precipitated cells at the bottom of the tube are knocked evenly, and the cells are evenly spread on the bottom of the centrifuge tube. 1ml PEG is slowly added, and the centrifuge tube is rotated while adding, 1ml / min, and the centrifuge tube is placed flat on the biosafety counter to expand the contact area between PEG and cells. After standing for 30s-1min, preheated DMEM is added in advance, 1ml for the first minute, 2ml for the second minute, 3ml for the third minute, 4ml for the fourth minute, and 5ml for the fifth minute, until it is greater than 20ml to dilute the PEG, terminate the fusion, and centrifuge the above cell mixture at 1000rpm for 5-10min. Discard the supernatant, resuspend the precipitate with the prepared HAT-containing culture medium, mix it with the feeder cells prepared in the first step, and spread it in a 96-well cell plate, 300ul / well. Observe the cell growth every day, change the culture medium when the cells grow to the 5th-6th day, and culture for 7-10 days for detection.

[0081] Human T cell acquisition

[0082] 5 ml of peripheral blood was drawn from a healthy person, and the operation was performed in a clean bench. The peripheral blood was slowly added to 5 to 10 ml of Ficoll (GE) to prevent the blood from mixing completely with the Ficoll, and then centrifuged at 800g×30min, with the centrifuge set to 5 for the speed increase and 0 for the speed decrease. After the centrifugation, the buffy coat layer was aspirated, and washed 1 to 2 times with PBS, 600g×10min. The obtained precipitate was placed in a 24-well plate for culture, and cultured in X-VIVO 15 (Lonza) medium containing 10% fetal bovine serum and 50 IU / ml recombinant IL-2. 10 microliters of CD3 / CD28 activation antibody was added to the well plate for activation (STEMCELL), and the T cells obtained after 48 hours of culture were obtained.

[0083] Construction of CAR-T cells

[0084] The human PDGFRβCAR virus was packaged by our research team using the second-generation lentiviral packaging system. The virus volume of MOI=20 was used to infect 1×10 6 T cells were infected by centrifugation in a 24-well plate at 600 g for 60 min. After infection, the T cells were transferred to fresh culture medium for culture.

[0085] Flow cytometry

[0086] Take 2×10 5The cells (Vec-T and PDGFRβCAR-T) were placed in 500 μL pre-cooled PBS and PDGFRβ flow cytometry antibody, incubated for 15 min at room temperature in the dark, washed twice with pre-cooled PBS, and then analyzed by flow cytometer (Beckman).

[0087] Construction of PDGFRβ-overexpressing 293T cells

[0088] The human PDGFRβ antigen sequence was searched on the Uniprot website, and the sequence was synthesized by Qingke Company and constructed into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP, with the front and back restriction sites XbaI and BamHI respectively. The PDGFRβ antigen lentivirus was constructed using the second-generation lentiviral packaging system, and 293T cells were infected in T25 flasks with MOI=10. 48 hours after infection, the expression of GFP in the cells was detected by flow cytometry.

[0089] CAR-T cell killing experiment

[0090] The experiment was completed using an intelligent real-time cell monitor (equipment model: CM100-α; manufacturer: Shanghai East China University of Science and Technology, Six Broad Beans). 5×10 5 293T-PDGFRβ cells were placed in the machine for monitoring. After 5 h, 5×10 5 T (Vec-T and PDGFRβCAR-T) cells were monitored until 18h.

[0091] result:

[0092] The structure of CAR and the construction of CAR-T cells:

[0093] This example uses a second-generation CAR with a traditional structure, with 4-1BB as a co-stimulatory molecule, and the overall CAR structure sequence is CD8 signal peptide-VL-(G4S)3-VH-CD8 hinge region-CD8 transmembrane region-4-1BB-CDζ( Figure 3 A). After the CAR structure was synthesized by Qingke Biotechnology, we constructed the CAR structure into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP ( Figure 3 B), the front and rear restriction sites are XbaI and BamHI respectively. The second generation virus packaging system is used to package into lentivirus (the packaging plasmids are PSPAX2 and PM2G).

[0094] T cells were derived from peripheral blood mononuclear cells of healthy adults. After separation, the T cells were activated with CD3 / CD28 activating antibodies (STEMCELL). T cells were cultured with X-VIVO 15 medium at an infection dose of MOI = 20 to obtain high-positive rate CAR-T cells ( Figure 3 C).

[0095] The results showed that the present invention successfully constructed a second-generation CAR targeting human PDGFRβ antigen ( Figure 3 A), by using lentivirus to infect healthy human peripheral blood T cells, a high positive rate of CAR-T cells was obtained ( Figure 3 C).

[0096] From the above experiments and their results, we can draw the following conclusions:

[0097] Through molecular experiments, human PDGFRβCAR was successfully constructed and verified by sequencing. At the same time, human CAR-T cells were successfully constructed, laying the foundation for subsequent CAR-T cell killing experiments.

[0098] Human PDGFRβCAR-T cells can effectively kill PDGFRβ in vitro + cell:

[0099] In order to explore the killing ability of human CAR-T cells on target cells, we constructed human PDGFRβ antigen overexpressing 293T cells ( Figure 4 A), killing experiment was performed on 293T-PDGFRβ cells using an intelligent cell real-time monitor (Shanghai East China University of Science and Technology, Six Broad Beans; CM100-α), the effector-target ratio was 1:1, and the killing time was 18 h.

[0100] The results showed that the target cell death rate in the Vec-T (Vector transduced T cell) cell group was very low, while the target cell death rate in the PDGFRβCAR-T cell group was relatively high ( Figure 4 B).

[0101] From the above experiments and their results, we can draw the following conclusions:

[0102] PDGFRβCAR-T cells inhibit PDGFRβ in vitro + Cells have strong killing ability.

[0103] Amino acid sequence information used in the examples:

[0104] SCAV heavy chain sequence:

[0105] QVQLQQSGPDLVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIG WIYPGNVNTKYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRDYA INFDYWGQGTILTVSS(SEQ ID NO.1)

[0106] SCAV light chain sequence:

[0107] DIMMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHFPWTF GGGTKLEIK(SEQ ID NO.2)

[0108] G4S linker sequence:

[0109] GGGGSGGGGSGGGGS (SEQ ID NO. 3)

[0110] CD8 signal peptide sequence:

[0111] MALPVTALLLPLALLLHAARP(SEQ ID NO.4)

[0112] CD8 hinge sequence:

[0113] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.5)

[0114] CD8 transmembrane region (TM) sequence:

[0115] MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.6)

[0116] 4-1BB intracellular region sequence:

[0117] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.7)

[0118] CD3 ζ chain sequence:

[0119] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR(SEQ ID NO.8)

[0120] Human PDGFRβ antigen amino acid sequence:

[0121]

[0122] PDGFRβ antigen extracellular domain amino acid sequence:

[0123] LVVTPGPELVLNVSSTFVLTCSSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCR VTDPQLVVTLHEKKGDVALVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDM PYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRV KVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHV(SEQ ID NO.10)

[0124] Signal peptide sequence:

[0125] MRLPGAMPALALKGELLLLSLLLLLEPQISQG(SEQ ID NO.11)

[0126] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A single-chain antibody targeting human PDGFRβ, characterized in that: The single-chain antibody comprises a single-chain antibody heavy chain V H and single-chain antibody light chain V L ; wherein the single-chain antibody heavy chain V H The amino acid sequence is shown in SEQ ID NO.1; the single-chain antibody light chain V L The amino acid sequence is shown in SEQ ID NO.

2.

2. A chimeric antigen receptor targeting human PDGFRβ, characterized in that: It at least comprises the single-chain antibody targeting human PDGFRβ according to claim 1.

3. The chimeric antigen receptor targeting human PDGFRβ according to claim 2, characterized in that The chimeric antigen receptor targeting human PDGFRβ is formed by a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain in series; The signal peptide is a CD8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.4; The hinge region is a CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO.5; The transmembrane region is the CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.6; The co-stimulatory signal transduction domain is a 4-1BB co-stimulatory signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.7; The signal transduction domain is a CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.

8.

4. The chimeric antigen receptor targeting human PDGFRβ according to claim 2, characterized in that The chimeric antigen receptor is composed of a CD8 signal peptide, an antigen binding domain V binding to human PDGFRβ antigen, L -(G4S)3-V H , CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory signaling domain and CD3ζ signaling domain are connected in series.

5. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single-chain antibody targeting human PDGFRβ as described in claim 1 or the chimeric antigen receptor as described in any one of claims 2-4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 5.

7. An immunocompetent cell, characterized in that: It expresses the single-chain antibody of claim 1 or the chimeric antigen receptor of any one of claims 2-4; or, comprises a nucleic acid molecule encoding the single-chain antibody of claim 1 or the chimeric antigen receptor of any one of claims 2-4.

8. The immunocompetent cell according to claim 7, wherein The immunocompetent cells are selected from the group consisting of: T cells, NK cells, monocytes, macrophages, dendritic cells or mast cells.

9. A method for preparing the immunocompetent cells according to any one of claims 7 to 8, characterized in that: It includes: The lentivirus infects T cells; the lentivirus is obtained by transfecting a recombinant lentiviral vector into a lentiviral packaging cell and then culturing the cell; the recombinant lentiviral vector is prepared by inserting a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 2 to 4 into a lentiviral vector.

10. A pharmaceutical composition, characterized in that It comprises a therapeutically effective amount of one or a combination of: the single-chain antibody according to claim 1, the chimeric antigen receptor according to any one of claims 2-4, the immunocompetent cell according to any one of claims 7-8 or the nucleic acid molecule according to claim 5, the vector according to claim 6 or the product prepared by the method according to claim 9, and a pharmaceutically acceptable carrier.

11. A kit, characterized in that: It comprises the single-chain antibody of claim 1, the chimeric antigen receptor of any one of claims 2-4, the immunocompetent cell of any one of claims 7-8 or the nucleic acid molecule of claim 5, the vector of claim 6 or the product prepared by the method of claim 9, or the pharmaceutical composition of claim 10.

12. Use of the single-chain antibody according to claim 1, the chimeric antigen receptor according to any one of claims 2-4, the immunocompetent cell according to any one of claims 7-8, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the product prepared by the method according to claim 9, the pharmaceutical composition according to claim 10, or the kit according to claim 11 in the preparation of a product for detecting PDGFRβ expression.

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