Single-chain antibody targeting human pdgfrβ and use thereof in car-t immunotherapy

Abstract

A single-chain antibody targeting human PDGFRβ and the use thereof in CAR-T immunotherapy. An scFv sequence targeting a human PDGFRβ antigen is obtained by immunizing a mouse, on the basis of which a second-generation CAR is constructed. Then, a CAR-T cell is obtained by lentiviral infection. The CAR-T cell can effectively kill PDGFRβ antigen-positive 293T cells. The present invention provides insights for treating chronic kidney diseases, chronic liver diseases and cardiovascular diseases, including various types of organ fibrosis, and multiple neoplastic diseases, and thus has further development value and application prospects.

Description

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

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Application No. 202410952787.5, filed on July 16, 2024, entitled "A Single-Chain Antibody Targeting Human PDGFRβ and Its Application in CAR-T Immunotherapy", the entire contents of which are incorporated herein by reference and constitute a part of this invention for all purposes. Technical Field

[0003] 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. Background Technology

[0004] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0005] Kidney disease is a major global health problem that seriously threatens human health. With the increasing severity of problems such as obesity and population aging, the prevalence of chronic kidney disease is as high as 10%. Studies show that by the end of this century, chronic kidney disease is projected to become the second leading cause of death in countries with longer life expectancies and the fifth leading cause of death globally. Renal fibrosis is a common pathological feature in chronic kidney disease caused by various factors. This pathological change is mainly caused by the activation of fibroblasts, pericytes, and myofibroblasts. Currently, there are no effective and feasible treatments. Treating renal fibrosis and reversing renal function loss are urgent kidney problems that need to be addressed.

[0006] Studies have shown that PDGFRβ antigen is highly expressed on the surface of cells such as fibroblasts, pericytes, and myofibroblasts. Targeting PDGFRβ antigen to eliminate these cells is an effective and feasible approach. Chimeric antigen receptor-modified T-cell therapy (CAR-T) has achieved significant success in treating hematological malignancies. Therefore, CAR-T immunotherapy is also applicable to non-cancerous diseases, such as autoimmune diseases, fibrosis, and cardiovascular diseases. 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 clearing senescent cells. However, the inventors have discovered that there is currently no single-chain antibody (scFv) targeting human PDGFRβ antigen that can be used to construct CARs. Summary of the Invention

[0007] To address the aforementioned limitations of existing technologies, the present invention aims 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 this CAR-T cell can effectively kill PDGFRβ antigen-positive cells, laying the foundation for CAR-T therapy of chronic kidney disease and other fibrosis-related diseases characterized primarily by kidney damage. Based on the above research findings, this invention is thus completed.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a single-chain antibody (scFv) targeting human PDGFRβ, said single-chain antibody comprising 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 Contains 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 contains an amino acid sequence as shown in SEQ ID NO.2 or a functional variant thereof.

[0010] In this invention, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function as the single-chain antibody or chimeric antigen receptor and has at least 85% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100%) sequence identity. In some specific embodiments, the variant of the amino acid sequence has substantially the same function.

[0011] Furthermore, the single-chain antibody heavy chain V H With single-chain antibody light chain V L It can be directly linked or linked via a linker; the linker can be (G4S)n, where n is a positive integer, for example, it can be 1, 2, 3, 4, 5, or 6. In some specific embodiments, n is 3. Further, the (G4S)3 amino acid sequence is shown in SEQ ID NO.3.

[0012] A second aspect of the present invention provides a chimeric antigen receptor (CAR) targeting human PDGFRβ, comprising at least the aforementioned single-chain antibody targeting human PDGFRβ, wherein the single-chain antibody targeting human PDGFRβ is used as an antigen-binding domain in the chimeric antigen receptor.

[0013] Furthermore, the chimeric antigen receptor targeting human PDGFRβ is formed by a series of components including a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and a signal transduction domain.

[0014] The signal peptide can guide the translocation of the antigen-binding domain and hinge region to the cell membrane surface. Any suitable signal peptide or combination of signal peptides can achieve the purpose of this invention. In one specific embodiment of this invention, the signal peptide can be the CD8 signal peptide, whose amino acid sequence is shown in SEQ ID NO.4.

[0015] The hinge region may be a CD8 hinge region, the amino acid sequence of which is shown in SEQ ID NO.5.

[0016] The transmembrane region may be a CD8 transmembrane region, the amino acid sequence of which is shown in SEQ ID NO.6.

[0017] The co-stimulatory signal transduction domain can be a 4-1BB co-stimulatory signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.7.

[0018] The signal transduction domain can be the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.8.

[0019] In addition, any peptide chain can be inserted as a spacer at appropriate positions between the antigen recognition region, hinge region, transmembrane region and intracellular signaling region. The peptide chain can be an oligopeptide or a polypeptide, and no specific limitation is made here.

[0020] According to the present invention, the chimeric antigen receptor comprises a CD8 signal peptide, an antigen-binding domain (V) that binds to human PDGFRβ antigen, and a CD8 signal peptide. L -(G4S)3-V HIt is composed of the CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory signal transduction domain and CD3ζ signal transduction domain connected in series.

[0021] A third aspect of the present invention provides an isolated nucleic acid molecule that encodes the aforementioned single-chain antibody or chimeric antigen receptor targeting human PDGFRβ.

[0022] In a fourth aspect, the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0023] According to the present invention, the vector is a viral vector, which may be a retroviral vector or a lentiviral vector; more preferably, it is a lentiviral vector, wherein the vector is obtained by inserting a nucleic acid molecule encoding the above-mentioned single-chain antibody or chimeric antigen receptor into a virus to obtain a recombinant viral vector expressing the above-mentioned single-chain antibody or chimeric antigen receptor.

[0024] A fifth aspect of the present invention provides an immune-active cell that expresses any of the aforementioned single-chain antibodies or chimeric antigen receptors; or, contains a nucleic acid molecule encoding any of the aforementioned single-chain antibodies or chimeric antigen receptors; preferably, the immune-active cell is selected from: T cells, NK cells, monocytes, macrophages, dendritic cells, or mast cells; wherein T cells are preferred, and further, when it expresses a chimeric antibody receptor or contains a nucleic acid molecule encoding a chimeric antigen receptor, the immune-active cell is a CAR-T cell.

[0025] A sixth aspect of the present invention provides a method for preparing the immune-active cells (particularly CAR-T cells), comprising: infecting T cells with a lentivirus; wherein the lentivirus is obtained by transfecting a recombinant lentiviral vector into lentiviral packaging cells and then culturing the cells; wherein the recombinant lentiviral vector is prepared by inserting a nucleic acid molecule encoding the chimeric antigen receptor into a lentiviral vector.

[0026] A seventh aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or a combination of: a single-chain antibody of any of the above; a chimeric antigen receptor of any of the above; or an immune-active cell of any of the above; or a nucleic acid molecule, a carrier of any of the above; or a product prepared by any of the methods described above; and a pharmaceutically acceptable carrier.

[0027] An 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-active cells; or any of the above-mentioned nucleic acid molecules, carriers, or products or pharmaceutical compositions prepared by any of the above-mentioned methods in the preparation of medicaments for the prevention and / or treatment of PDGFRβ-mediated diseases.

[0028] Specifically, this invention demonstrates through research that the PDGFRβ CAR-T cells of this invention exhibit in vitro efficacy against PDGFRβ. + The cells have a strong killing ability, therefore the diseases mediated by the high expression of PDGFRβ include, but are not limited to, kidney diseases, liver diseases, cardiovascular diseases and neoplastic diseases, especially fibrosis-mediated chronic kidney diseases, liver diseases, cardiovascular diseases and neoplastic diseases.

[0029] A ninth aspect of the present invention provides a method for preventing and / or treating PDGFRβ-mediated diseases, 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-active cells; or any of the above-mentioned nucleic acid molecules, carriers, or products or pharmaceutical compositions prepared by any of the above-mentioned methods.

[0030] In a tenth aspect, 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-active cells; or any of the above-mentioned nucleic acid molecules, carriers, or products or pharmaceutical compositions prepared by any of the above-mentioned methods.

[0031] In an eleventh aspect of the present invention, 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-active cells; or any of the above-mentioned nucleic acid molecules, vectors, or products, pharmaceutical compositions, or kits prepared by any of the above-mentioned methods in detecting PDGFRβ expression or preparing products for detecting PDGFRβ expression is provided.

[0032] Specifically, qualitative or quantitative detection of PDGFRβ expression in subject samples can be used for screening, (aiding) diagnosis, or prediction of disease progression mediated by high PDGFRβ expression.

[0033] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0034] The above technical solution first obtains the scFv sequence targeting the human PDGFRβ antigen by immunizing mice, and then constructs a second-generation CAR based on this. In addition, CAR-T cells are obtained by lentivirus infection, and these CAR-T cells can effectively kill PDGFRβ antigen-positive 293T cells.

[0035] The above-mentioned technical solution provides a completely new approach for the treatment of chronic kidney disease, liver disease, cardiovascular disease, and various tumor diseases, including fibrosis of various organs, by clearing PDGFRβ-positive cells. It has extremely attractive potential for further development and application. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figures 1A and 1B show the results of protein purification in the embodiments of the present invention;

[0038] Figure 1A shows the detection of PDGFRβ extracellular protein before purification. The bands are 1: uninduced cells, 2-4: induced cells, 5: precipitate after induction, 6: supernatant after induction, and M: marker. Figure 1B shows the detection of PDGFRβ extracellular protein after purification. The bands are 1: purified protein, M: marker, and BSA: bovine serum albumin.

[0039] Figure 2 illustrates the mouse immunization and monoclonal antibody cell line acquisition process in an embodiment of the present invention.

[0040] Figures 3A to 3C illustrate the PDGFRβCAR structure and CAR-T cell construction in an embodiment of the present invention.

[0041] Figure 3A shows the structure of PDGFRβCAR; Figure 3B shows the lentiviral vector used to construct CAR; Figure 3C shows the PDGFRβCAR-T cell positivity rate.

[0042] Figures 4A and 4B illustrate the PDGFRβCAR killing experiment in an embodiment of the present invention;

[0043] Figure 4A shows the flow cytometry diagram of 293T-PDGFRβ cell construction; Figure 4B shows the killing curve of PDGFRβCAR-T cells. Detailed Implementation

[0044] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0046] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0047] In this invention, the term "specific binding" refers to the ability of antigen-binding molecules (e.g., antibodies) to specifically bind antigens and substantially the same antigens with high affinity, but not to unrelated antigens with high affinity. Affinity is typically expressed as an equilibrium dissociation constant (K0). D This is reflected in the lower K. D It indicates a high degree of affinity.

[0048] In this invention, the term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain, thereby enabling specific binding to an antigen. The term "antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0049] In this invention, the term "monoclonal antibody" refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies exhibit single-molecule binding specificity and affinity for a specific epitope.

[0050] In this invention, the term "antibody" can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents, and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0051] In this 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.

[0052] In this invention, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody originates from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) originates from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance immune responses.

[0053] In this invention, the terms "identity" and "consistency" are interchangeable and are calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid 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 that position.

[0054] In this invention, the term "immunoactive cells" refers to cells that perform immune functions in an organism. Examples of immunoactive cells include: lymphocytes such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells. Specifically, T cells or NK cells from mammals such as humans, dogs, cats, pigs, and mice are preferred, with human T cells or NK cells being the most preferred. Furthermore, T cells can be isolated and purified from immunoactive cells in bodily fluids such as blood and bone marrow fluid, as well as tissues such as the spleen, thymus, and lymph nodes, or from cancerous tissues such as primary tumors, metastatic tumors, and cancerous ascites. T cells made from ES cells or iPS cells can also be used. It should be noted that the source of the immunoactive cells and the target population can be the same or different. Furthermore, when the target population is human, the immunoactive cells can be autologous cells collected from the patient or allogeneic cells collected from another person. That is, the donor and the recipient can be the same or different, but they are preferred to be the same.

[0055] In this invention, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delayers, and the like. Generally, the nature of the carrier depends on the specific route of administration. For example, parenteral preparations typically contain injectable fluids as the vehicle, which include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, glucose solutions, glycerol, etc. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0056] In this invention, the term "therapeuticly effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dosage and time period. A therapeutically effective amount of an agent may eliminate, reduce, delay, minimize, or prevent adverse effects of a disease.

[0057] The term "treatment and / or prevention" refers to an attempt to alter the natural course of a disease in an individual, and may be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of symptoms.

[0058] In this invention, the "individual" or "subject" is preferably a mammal. The mammal includes humans and non-human mammals, including mice, rats, guinea pigs, cattle, sheep, cats, dogs, horses, monkeys, and orangutans, etc., wherein the individual or subject is preferably a human.

[0059] Specifically, the cells designed in this invention are human T cells.

[0060] Among them, human T cells are derived from peripheral blood and are activated by stimulation with CD3 / CD28 magnetic beads.

[0061] The CAR-T cells used in this invention are constructed via lentiviral infection.

[0062] The lentiviral vector was pCDH-EF1-MCS-T2A-copGFP. The lentivirus was packaged using a second-generation lentiviral packaging method. The helper plasmids PSPAX2 and PM2G were used, and the infection coefficient MOI was 20.

[0063] The CAR-T cells targeting human PDGFRβ of this invention can effectively kill PDGFRβ-expressing cells in vitro. + 293T cells.

[0064] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0065] Example

[0066] method:

[0067] Mouse immune process

[0068] The amino acid sequence of the human PDGFRβ antigen is shown in SEQ ID NO. 9. The extracellular region sequence of the human PDGFRβ antigen (SEQ ID NO. 10) was constructed into the pcDNA3.0 vector, preceded by a membrane localization signal peptide (SEQ ID NO. 11). 2 μL of plasmid was added to 50 μL of BL21 competent bacteria and incubated on ice for 30 min. The mixture was then heat-shocked at 42℃ for 90 s, immediately placed on ice for 5 min, and 500 μL of LB medium was added. The mixture was incubated at 37℃ with shaking at 220 rpm for 1 h, and then plated onto LB agar plates containing ampicillin. The plates were incubated upside down at 37℃ overnight. Single colonies were picked from the plates and inoculated into test tubes containing 5 mL of ampicillin-containing LB medium. The plates were incubated at 37℃ with shaking at 200 rpm until the culture medium reached OD. 600 The concentration was 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, with a control group not receiving IPTG. The mixture was shaken at 16℃ and 200 rpm for 16 h to induce fusion protein expression. 1 mL of culture was centrifuged at 12000 g for 10 min at room temperature, the supernatant was discarded, and the bacterial pellet was resuspended in 50 μL of 1×PBS buffer with 25 μL of 3×Loading Buffer. Analysis by 12% SDS-PAGE gel electrophoresis showed expression of the fusion protein, approximately 80 kDa, primarily in the form of inclusion bodies. The constructed band was identified using protein expression identification technology (Figure 1A), and then purified to obtain a relatively pure extracellular region of the PDGFRβ antigen (Figure 1B). The immunization process is shown in Figure 2. Four-week-old Balb / c mice were immunized via tail vein transfusion with eukaryotically purified PDGFRβ extracellular region protein at a dose of 100 μg / mouse / immunization, for a total of four immunizations. Fusion was performed 13 days after the immunization period.

[0069] Screening for high-affinity PDGFRβscFv

[0070] Blood sampling and testing: Immunization was carried out according to the mature immunization procedure and indicators such as titer determination were completed. In this embodiment, the mice corresponding to the optimal cell line and antibody results and their related data were summarized and compiled, as shown in Table 1. Multiple rounds of blood sampling and testing data showed that mouse #2 had the highest antibody titer, so mouse #2 was selected for hybridoma fusion.

[0071] Fusion and cloning: SP2 / 0 mouse myeloma cells were fused with spleen cells from selected mice (2#). After fusion, the cells were cultured, observed, tested, and subjected to positive and negative control experiments to obtain a batch of hybridoma cells that met the experimental requirements. These cells were then further cultured and selected.

[0072] #2 Mouse cell line identification and sequencing: After cloning and supernatant analysis using the fusion and limiting dilution method, 14 cell lines with 31 cells meeting the project requirements were obtained from mice. By comparison, the hybridoma with the highest titer and the best affinity was sequenced to obtain the full-length PDGFRβ antibody sequence. The antibody heavy chain / light chain variable region gene was obtained by PCR, resulting in PDGFRβscFv.

[0073] Table 1 Blood sampling test results

[0074] CAR structure

[0075] In this embodiment, the CAR sequence is as follows: 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 3B). The restriction enzyme sites before and after the restriction are XbaI and BamHI, respectively, and the sequences were found to be identical after sequencing alignment. Lentiviral virus was packaged using a second-generation viral packaging system (packaging plasmids PSPAX2 and PM2G).

[0076] Feeder cell preparation

[0077] BALB / c mice were euthanized by cervical dislocation. After disinfection by immersion in 75% alcohol for 5-10 minutes, a small incision was made in the abdomen using surgical scissors, and the skin was peeled back to expose the peritoneal cavity. The peritoneal membrane was lifted using hemostatic forceps (avoiding the abdominal organs). 4-5 ml of DMEM solution was injected into the peritoneal cavity using a syringe. The mouse's legs were held by forceps and the mouse was shaken back and forth for about 1 minute. The peritoneal fluid was then aspirated using the same syringe and added to a centrifuge tube. This process was repeated 2-3 times. The cells were centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in HAT selection medium.

[0078] Preparation of spleen cells and myeloma cells

[0079] Mice were euthanized by cervical dislocation. The mice were then disinfected by immersing them in 75% alcohol for 5-10 minutes. After dissection, the spleen was harvested under aseptic conditions. The spleen was rinsed in 5 ml of DMEM solution to remove red blood cells. DMEM was aspirated into the spleen using a 5 ml syringe to create a spleen cell suspension. This suspension was transferred to a 50 ml centrifuge tube and repeated several times until the spleen became transparent. The pre-cultured SP2 / 0 cells were discarded. The SP2 / 0 cells were then transferred to a new, pre-warmed (37°C) DMEM solution and added to the centrifuge tube containing the spleen cells from the previous step. The two cell types were thoroughly mixed. The centrifuge tube was then centrifuged at 1000 rpm for 5-10 minutes at room temperature.

[0080] Fusion

[0081] After centrifugation, discard the supernatant. Wipe the centrifuge tube walls dry with autoclaved filter paper, tighten the cap, and gently tap the cell pellet at the bottom of the tube to ensure even cell distribution. Slowly add 1 ml of PEG while rotating the centrifuge tube at a rate of 1 ml / min. Place the centrifuge tube flat on a biosafety counter to maximize the contact area between the PEG and cells. Let it stand for 30 seconds to 1 minute, then add preheated DMEM at a rate of 1 ml for the first minute, 2 ml for the second minute, 3 ml for the third minute, 4 ml for the fourth minute, and 5 ml for the fifth minute, continuing until the effect of more than 20 ml of diluted PEG is achieved, thus stopping the confluence. Centrifuge the cell mixture at 1000 rpm for 5-10 minutes. Discard the supernatant, resuspend the pellet in prepared HAT-containing medium, and mix it with the feeder cells prepared in the first step. Plate the mixture in 96-well plates at 300 μL / well. Observe cell growth daily. Change the medium on day 5-6 and perform analysis after 7-10 days of culture.

[0082] Human T-cell acquisition

[0083] 5 ml of peripheral blood was drawn from healthy individuals and processed in a laminar flow hood. The peripheral blood was slowly added to a container containing 5-10 ml of Ficoll (GE), avoiding complete mixing of the blood and Ficoll. The container was then centrifuged at 800 g for 30 min with an ascending speed of 5 and a descending speed of 0. After centrifugation, the white membrane layer was aspirated and washed 1-2 times with PBS, then centrifuged at 600 g for 10 min. The resulting pellet was cultured in 24-well plates using X-VIVO 15 (Lonza) medium containing 10% fetal bovine serum and 50 IU / ml recombinant IL-2. Activation (STEMCELL) was performed by adding 10 μL of CD3 / CD28 activation antibody to the wells. T cells were obtained after 48 h of culture.

[0084] Construction of CAR-T cells

[0085] Human PDGFRβCAR virus was packaged by our research team using a second-generation lentivirus packaging system, and a viral load of MOI=20 was used to infect 1×10⁻⁶ cells. 6 One T cell was centrifuged in a 24-well plate for infection at 600g for 60 min. After infection, the T cells were transferred to fresh culture medium for culture.

[0086] Flow cytometry

[0087] Take 2×10 5 Cells (Vec-T and PDGFRβCAR-T) were incubated with PDGFRβ flow cytometry antibody in 500 μL of pre-chilled PBS at room temperature in the dark for 15 min, washed twice with pre-chilled PBS, and then analyzed by flow cytometer (Beckman).

[0088] Construction of PDGFRβ-overexpressing 293T cells

[0089] The human PDGFRβ antigen sequence was found on the Uniprot website and synthesized by Qingke Company. This sequence was then constructed into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP, with XbaI and BamHI restriction enzyme sites before and after digestion, respectively. A second-generation lentiviral packaging system was used to construct PDGFRβ antigen lentivirus, which was then used to infect 293T cells in T25 flasks at an MOI of 10. Flow cytometry was used to detect GFP expression in the cells 48 hours after infection.

[0090] CAR-T cell killing experiment

[0091] This experiment was conducted using a smart cell real-time monitoring system (device model: CM100-α; manufacturer: East China University of Science and Technology, Shanghai; Six Broad Beans). 5 × 10⁵ cells were added to the electronic chip plate. 5 293 T-PDGFRβ cells were placed in the machine for monitoring, and 5 × 10⁶ cells were added after 5 hours. 5 1 T (Vec-T and PDGFRβCAR-T) cells were monitored until 18 hours later.

[0092] result:

[0093] The structure of CAR and the construction of CAR-T cells:

[0094] This embodiment uses a conventional second-generation CAR with 4-1BB as the co-stimulatory molecule. The overall CAR structure sequence is CD8 signal peptide-VL-(G4S)3-VH-CD8 hinge region-CD8 transmembrane region-4-1BB-CDζ (Figure 3A). After synthesizing this CAR structure using Qingke Biotechnology, we constructed it into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP (Figure 3B), with XbaI and BamHI restriction enzyme sites before and after digestion, respectively. Lentiviral virus was packaged using a second-generation viral packaging system (packaging plasmids PSPAX2 and PM2G).

[0095] T cells were derived from peripheral blood mononuclear cells from healthy adults. After isolation, T cells were activated using CD3 / CD28 activation antibody (STEMCELL). The T cells were cultured in X-VIVO 15 medium and infected with MOI=20 to obtain CAR-T cells with a high positive rate (Figure 3C).

[0096] The results showed that the present invention successfully constructed a second-generation CAR targeting human PDGFRβ antigen (Figure 3A). By infecting peripheral blood T cells of healthy individuals with lentivirus, CAR-T cells with a high positive rate were obtained (Figure 3C).

[0097] Based on the above experiments and their results, the following conclusions can be drawn:

[0098] Human PDGFRβCAR was successfully constructed through molecular experiments and verified by sequencing. Human CAR-T cells were also successfully constructed, laying the foundation for subsequent CAR-T cell killing experiments.

[0099] Human PDGFRβ CAR-T cells can effectively kill PDGFRβ in vitro. + cell:

[0100] To investigate the killing ability of human CAR-T cells against target cells, we constructed human PDGFRβ antigen-overexpressing 293T cells (Figure 4A) and conducted killing experiments on 293T-PDGFRβ cells using a smart cell real-time monitoring instrument (Shanghai East China University of Science and Technology, Six Broad Beans; CM100-α). The effector-to-target ratio was 1:1, and the killing time was 18 hours.

[0101] The results showed that the target cell mortality rate was very low in the Vec-T (Vector transduced T cell) group, while it was relatively high in the PDGFRβCAR-T cell group (Figure 4B).

[0102] Based on the above experiments and their results, the following conclusions can be drawn:

[0103] PDGFRβ CAR-T cells in vitro against PDGFRβ + The cells have a strong killing ability.

[0104] Amino acid sequence information used in the examples:

[0105] Single-chain antibody heavy chain sequence:

[0106] Single-chain antibody light chain sequence:

[0107] G4S linker sequence:

[0108] CD8 signal peptide sequence:

[0109] CD8 Hinge Sequence:

[0110] CD8 transmembrane region (TM) sequence:

[0111] 4-1BB intracellular region sequence:

[0112] CD3ζ chain sequence:

[0113] Human PDGFRβ antigen amino acid sequence:

[0114] PDGFRβ antigen extracellular amino acid sequence:

[0115] Signal peptide sequence:

[0116] MRLPGAMPALALKGELLLLSLLLLLEPQISQG(SEQ ID NO.11)

[0117] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection 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 a single-chain antibody light chain V L ; wherein the single-chain antibody heavy chain V H has an amino acid sequence as shown in SEQ ID NO. 1; and the single-chain antibody light chain V L has an amino acid sequence as shown in SEQ ID NO.

2.

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

3. The chimeric antigen receptor targeting human PDGFRβ according to claim 2, wherein, The chimeric antigen receptor targeting human PDGFRβ is formed in series with a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, a costimulatory signaling domain and a signaling domain; The signal peptide is a CD8 signal peptide, and its amino acid sequence is shown as SEQ ID NO. 4; The hinge region is a CD8 hinge region, and its amino acid sequence is shown as SEQ ID NO. 5; The transmembrane region is a CD8 transmembrane region, and its amino acid sequence is shown as SEQ ID NO. 6; The costimulatory signaling domain is a 4-1BB costimulatory signaling domain, and its amino acid sequence is shown as SEQ ID NO. 7; The signaling domain is a CD3ζ signaling domain, and its amino acid sequence is shown as SEQ ID NO.

8.

4. The chimeric antigen receptor targeting human PDGFRβ according to claim 2, wherein, The chimeric antigen receptor is composed in tandem of a CD8 signal peptide, an antigen binding domain V L -(G4S)3-V H , a CD8 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain.

5. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the single-chain antibody targeting human PDGFRβ according to claim 1 or the chimeric antigen receptor according to any one of claims 2-4.

6. A vector, 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 according to claim 1 or the chimeric antigen receptor according to any one of claims 2-4; or comprises a nucleic acid molecule encoding the single-chain antibody according to claim 1 or the chimeric antigen receptor according to any one of claims 2-4.

8. The immunocompetent cell of claim 7, wherein The immunocompetent cell is selected from the group consisting of T cells, NK cells, monocytes, macrophages, dendritic cells and mast cells.

9. A method of preparing the immunocompetent cells according to any one of claims 7-8, characterized in that, It comprises: infecting T cells with The lentivirus is obtained by transfecting a recombinant lentiviral vector into lentivirus packaging cells, and then culturing the cells.

10. A pharmaceutical composition, characterized by, 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, the product prepared by the method according to claim 5, the vector according to claim 6 or the method according to claim 9, and a pharmaceutically acceptable carrier.

11. A kit characterized in that, It comprises the product prepared by the method 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 method according to claim 9, or the pharmaceutical composition according to 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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