Human PDGFR beta-targeted scFV antibody and application thereof in anti-fibrosis immunotherapy for removing extracellular matrix cells

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a human PDGFR beta targeting scFV antibody and application thereof in anti-fibrosis immunotherapy for removing extracellular matrix cells. Specifically, the invention relates to a single-chain antibody with higher affinity, which is obtained through AI-assisted optimization screening based on a single-chain antibody targeting human PDGFR beta, and application of the single-chain antibody in CAR-T immunotherapy of fibrotic diseases. Based on the original and optimized single-chain antibody sequences, constructing a second-generation CAR of a targeted human PDGFR beta antigen; the CAR-T cells are found to be capable of effectively killing PDGFR beta antigen positive cells, and a foundation is laid for CAR-T treatment of chronic kidney diseases and other fibrosis related diseases with the main characteristic of kidney injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and molecular biology, and specifically relates to a scFV antibody targeting human PDGFRbeta and application thereof in anti-fibrosis immunotherapy for clearing extracellular matrix-producing cells. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] Fibrotic diseases are a group of diseases that seriously endanger human health, and their main feature is the excessive deposition of extracellular matrix (ECM) in tissues and organs, which leads to structural damage and dysfunction of organs. In the occurrence and development of fibrotic diseases, extracellular matrix-producing cells play a key role. These cells are abnormally activated under the stimulation of pathological factors, proliferate in large numbers, and synthesize and secrete excessive ECM components such as collagen and fibronectin, thus driving the progression of fibrosis. For example, kidney fibrosis is a common pathological feature of chronic kidney disease (CKD) caused by various causes, and fibroblasts, pericytes, and myofibroblasts are the main ECM-producing cells.

[0004] Platelet-derived growth factor receptor beta (PDGFRbeta) is a transmembrane tyrosine kinase receptor that is a specific surface antigen of extracellular matrix-producing cells and is highly expressed in various fibrotic diseases. Taking CKD as an example, studies have found that the expression of PDGFRbeta in the renal tubulointerstitium increases with the severity of kidney fibrosis, and the cells expressing PDGFRbeta are mainly PDGFRalpha-positive fibroblasts, NG2-positive pericytes, and alpha-SMA-positive myofibroblasts. After binding with its ligand, PDGFRbeta can activate downstream signaling pathways, further promoting the proliferation, migration, and ECM synthesis of extracellular matrix-producing cells, and exacerbating the degree of fibrosis. Due to the specific expression of PDGFRbeta on extracellular matrix-producing cells, it becomes an ideal target for the treatment of fibrotic diseases; the cell surface localization of PDGFRbeta is very suitable for being used as a target antigen for chimeric antigen receptor (CAR) to develop a treatment method for fibrotic diseases.

[0005] CAR immunotherapy is a promising immunotherapy technology. Taking the most commonly used CAR-T cell therapy as an example, its principle is to fuse the antibody fragment capable of specifically recognizing the target cell surface antigen (in this invention, the extracellular matrix cell surface antigen) with the T cell activation signal domain through genetic engineering technology, construct a CAR molecule, and introduce it into the patient's own T cells, so that the T cells acquire the ability to specifically recognize and kill target cells. CAR-T therapy has achieved remarkable efficacy in the treatment of hematological malignancies, but its application in solid tumors and fibrosis diseases still faces many challenges, one of which is the lack of high-affinity, high-specificity and safe and effective antigen-binding molecules. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide an scFV antibody targeting human PDGFRβ and its application in anti-fibrosis immunotherapy for eliminating extracellular matrix-producing cells. Specifically, the present application is based on a single-chain antibody targeting human PDGFRβ, a single-chain antibody with higher affinity obtained by AI-assisted optimization screening, and its application in CAR-T immunotherapy for fibrosis diseases. Based on the original and optimized single-chain antibody sequences, a second-generation CAR targeting human PDGFRβ antigen is constructed; it is found that these CAR-T cells can effectively kill cells positive for PDGFRβ antigen, 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 application is completed.

[0007] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

[0008] In a first aspect of the present application, an scFV antibody targeting human PDGFRβ is provided, which comprises a heavy chain V H and a light chain V L ;

[0009] The heavy chain V H comprises:

[0010] (a1) the amino acid sequence shown in any one of SEQ ID NO. 1, 3, 5, 7, 9 and 11;

[0011] (a2) an amino acid sequence having at least 85% (such as 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NO. 1, 3, 5, 7, 9 and 11, respectively.

[0012] The light chain V L comprises:

[0013] (b1) An amino acid sequence as shown in any one of SEQ ID NO. 2, 4, 6, 8, 10 and 12;

[0014] (b2) An amino acid sequence that is at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to any one of SEQ ID NO. 2, 4, 6, 8, 10 and 12.

[0015] Furthermore, the heavy chain V H With light chain V L It can be connected directly or through 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, etc. In some specific embodiments, n is 3.

[0016] A second aspect of the present invention provides a chimeric antigen receptor (CAR) comprising at least an antigen-binding domain that recognizes a human PDGFRβ antigen, said antigen-binding domain comprising the aforementioned single-chain antibody.

[0017] In this invention, the chimeric antigen receptor can be any one of the first to fifth generation chimeric antigen receptors.

[0018] Specifically, the chimeric antigen receptor comprises a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and a signal transduction domain.

[0019] In one specific embodiment of the present invention, the signal peptide may be a CD8 signal peptide, the amino acid sequence of which is shown in SEQ ID NO.14.

[0020] The hinge region can be a CD8 hinge region, the amino acid sequence of which is shown in SEQ ID NO.15.

[0021] The transmembrane region may be the CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.16.

[0022] 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.17.

[0023] The signal transduction domain may be the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.18.

[0024] In one specific embodiment of the present invention, the chimeric antigen receptor comprises a CD8 signal peptide, an antigen-binding domain (V) that binds to the 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.

[0025] A third aspect of the invention provides an isolated nucleic acid molecule that encodes the aforementioned single-chain antibody or chimeric antigen receptor.

[0026] In a fourth aspect, the present invention provides a carrier comprising the nucleic acid molecule described in the third aspect.

[0027] The vector can be an expression vector or a cloning vector. In some embodiments, the vector is a viral vector. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives.

[0028] A fifth aspect of the invention provides a host cell comprising the vector described in the fourth aspect.

[0029] Suitable host cells for cloning or expressing DNA are prokaryotic cells, yeast cells, or higher eukaryotic cells. Common examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. Common examples of eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

[0030] A sixth aspect of the present invention provides an immune effector cell that expresses the above-mentioned scFV or chimeric antigen receptor targeting human PDGFRβ;

[0031] In this invention, "immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, the immune effector cells are selected from at least one of: immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (same as T cells). In some embodiments, T cells can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or combinations thereof. In some embodiments, T cells produce IL-2, IFN, and / or TNF when expressing a chimeric antigen receptor and binding to target cells. In some implementations, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.

[0032] This invention provides a method for preparing the aforementioned immune effector cells, comprising infecting immune effector cells with the isolated nucleic acid or the vector described in this invention. Preferably, this invention prepares genetically modified immune effector cells by introducing a chimeric antigen receptor into immune effector cells (such as T cells).

[0033] A seventh aspect of the invention provides the use of a reagent in the preparation of compositions, pharmaceuticals, formulations, or kits for the prevention and / or treatment of diseases associated with positive PDGFRβ expression, said reagent comprising: scFV according to a first aspect of the invention, or a chimeric antigen receptor according to a second aspect, or an immune effector cell according to a sixth aspect;

[0034] Specifically, this invention demonstrates through research that the CAR-T cells targeting human PDGFRβ of this invention exhibit in vitro efficacy against PDGFRβ. + The cells have a strong killing ability, therefore the diseases associated with positive PDGFRβ expression include, but are not limited to, kidney diseases, liver diseases, cardiovascular diseases and neoplastic diseases, especially fibrosis-related chronic kidney diseases, liver diseases, cardiovascular diseases and neoplastic diseases.

[0035] An eighth aspect of the invention provides the use of the scFV according to the first aspect of the invention, or the chimeric antigen receptor according to the second aspect, or the immune effector cells according to the seventh aspect, in combination with other drugs. These other drugs include, but are not limited to, diagnostic agents, preventative agents, and / or therapeutic agents.

[0036] A ninth aspect of the present invention provides a method for preventing and / or treating diseases associated with positive PDGFRβ expression, the method comprising administering to a subject a therapeutically effective amount of any of the above-mentioned scFV; any of the above-mentioned chimeric antigen receptors; or any of the above-mentioned immune effector cells; or any of the above-mentioned nucleic acid molecules, vectors, or compositions, pharmaceuticals, formulations, or kits prepared by any of the above-mentioned methods.

[0037] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0038] The above technical solution first obtains the scFv sequence targeting the human PDGFRβ antigen by immunizing mice, and then obtains five optimized single-chain antibody scFV sequences with higher affinity through AI optimization; on this basis, a second-generation CAR is constructed, and CAR-T cells are obtained by lentivirus infection. These CAR-T cells can effectively kill PDGFRβ antigen-positive 293T cells.

[0039] The above-mentioned technical solution provides a completely new approach for eliminating PDGFRβ-positive ECM-producing cells and treating chronic kidney disease, liver disease, cardiovascular disease, and various tumor diseases, including organ fibrosis. It has extremely attractive potential for further development and application. Attached Figure Description

[0040] 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.

[0041] Figure 1 The results are those after protein purification in the embodiments of the present invention;

[0042] Where: A represents the detection of PDGFRβ extracellular protein before purification, with each band representing 1: uninduced cells, 2-4: induced cells, 5: precipitate after induction, 6: supernatant after induction, and M: marker; B represents the detection of PDGFRβ extracellular protein after purification, with each band representing 1: purified protein, M: marker, and BSA: bovine serum albumin.

[0043] Figure 2 This describes the mouse immunization and monoclonal antibody cell line acquisition process in this embodiment of the invention.

[0044] Figure 3 This is a three-dimensional model and interface analysis of the target antigen protein-antibody complex in the embodiments of the present invention;

[0045] Wherein: A represents the AI ​​optimization strategy used in this invention; B is a three-dimensional model of the human PDGFRβ protein-original single-chain antibody complex, in which PDGFRβ exists in a dimer form, green and yellow represent the three-dimensional structure of human PDGFRβ, and blue represents the three-dimensional structure of the original single-chain antibody; C is the interface analysis of the human PDGFRβ protein-original single-chain antibody complex.

[0046] Figure 4 The binding free energy of the original complex and the optimized complex in the embodiments of the present invention;

[0047] Among them, the binding free energy of the top five optimized complexes, which are higher than the original complex, is listed.

[0048] Figure 5 This describes the PDGFRβCAR structure and CAR-T cell construction in this embodiment of the invention.

[0049] Where: A is the PDGFRβCAR structure; B is the lentiviral vector used to construct the CAR; C is the PDGFRβCAR-T cell positivity rate detection.

[0050] Figure 6 This is a PDGFRβCAR killing experiment in an embodiment of the present invention;

[0051] Where: A is the flow cytometry diagram of 293T-PDGFRβ cell construction; B is the killing curve of PDGFRβCAR-T cells. Detailed Implementation

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Example

[0057] method:

[0058] Mouse immune process

[0059] The human PDGFRβ antigen amino acid sequence is shown in SEQ ID NO. 19. The extracellular region sequence of the human PDGFRβ antigen (SEQ ID NO. 20) was constructed into the pcDNA3.0 vector, preceded by a membrane localization signal peptide (SEQ ID NO. 21). 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 incubated 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 using 12% SDS-PAGE gel electrophoresis showed expression of the fusion protein, approximately 80 kDa, primarily in the form of inclusion bodies. Protein expression identification techniques revealed the constructed band (…). Figure 1 A), and then relatively pure PDGFRβ antigen extracellular region protein was obtained through protein purification technology. Figure 1 B). The immune process, such as Figure 2 As shown, 4-week-old Balb / c mice were immunized with PDGFRβ extracellular protein purified from eukaryotic cells via tail vein transfusion. The immunization dose was 100 micrograms per mouse per immunization, and a total of 4 immunizations were performed. Fusion was performed 13 days after the end of immunization.

[0060] PDGFRβscFv screening

[0061] 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.

[0062] 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.

[0063] #2 Mouse cell line identification and sequencing: After fusion and limiting dilution cloning of mice and analysis of the supernatant, a total of 14 cell lines with 31 cells meeting the project requirements were obtained. By comparison, the hybridoma cell lines with high titer and the top three affinity were 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 (the csFv amino acid sequence of the original single-chain antibody of this invention comes from one of the top three hybridoma cell lines).

[0064] Table 1 Blood Collection and Testing Results

[0065]

[0066] Feeder cell preparation

[0067] 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.

[0068] Preparation of spleen cells and myeloma cells

[0069] 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.

[0070] Fusion

[0071] 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.

[0072] AI optimization for human PDGFRβ single-chain antibody:

[0073] To obtain a single-chain antibody against human PDGFRβ with stronger binding affinity, this invention uses Alphafold3 to predict the structure of a human PDGFRβ-PDGFRβ single-chain antibody complex, then analyzes the complex binding interface to identify hotspot residues, uses the Rosetta platform to optimize the design of hotspot residues, and evaluates the affinity of the newly generated antibody sequence. Figure 3 A).

[0074] (1) Structural prediction of the target antigen protein-antibody complex: The amino acid sequence of the target antigen protein—human PDGFRβ (Uniprot ID: P09619) and the heavy and light chain amino acid sequences of the single-chain antibody are used as inputs. Preferably, the AlphaFold3 structure prediction platform is used for calculation to obtain multiple three-dimensional models of the target antigen protein-antibody complex. Further, the obtained complex models are sorted according to prediction confidence and overall energy function, and models with reasonable structures and clear interface binding mechanisms are selected. Figure 3 B), serving as the foundational structure for subsequent analysis.

[0075] (2) Interface analysis of the complex binding mode: After obtaining the complex model, a systematic analysis of its interface interaction modes is performed. This analysis includes identifying interface residues, determining the number and distribution of hydrogen bonds, salt bridges, and hydrophobic interactions, and calculating the buried surface area and inter-residue atomic distances. Figure 3 C). Preferably, the analysis results are used to determine whether the distance between the monoclonal antibody and the target protein is less than [a certain value]. The key residues, as well as the important functional residues at the target protein interface, are identified to clarify the action sites that can be used for subsequent optimization.

[0076] (3) Targeted residue optimization of the antibody CDR region: After determining the key action sites, the CDR region of the single-chain antibody was optimized using the Rosetta molecular design platform. Furthermore, mild backbone relaxation can be introduced during the optimization process to alleviate local spatial conflicts and obtain a conformationally reasonable complex structure. This optimization step can explore multiple candidate residue combinations and generate several modified antibody complex models.

[0077] (4) Calculation and evaluation of the binding free energy of the optimized complex: The binding free energy of the optimized antibody-target protein complex model was calculated using Rosetta's interface analysis module. Preferably, the calculation of the binding free energy includes minimizing the energy of the antibody and target protein after separation, reassembling the complex, and calculating the binding energy (ΔG), while simultaneously measuring the change in embedded surface area (ΔSASA), the number of hydrogen bonds, the number of salt bridges, and the number of hydrophobic interactions. Further, the binding free energy of the optimized complex was compared with that of the original complex, and optimized single-chain antibody sequences with significantly better binding free energies than the original single-chain antibody were selected. The top five optimized single-chain antibodies were selected for further research. Figure 4 ).

[0078] CAR structure

[0079] 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. After synthesizing this sequence, it was constructed into the specified vector pCDH-EF1-MCS-T2A-copGFP( Figure 5 B) The restriction enzyme sites before and after the reaction are XbaI and BamHI, respectively, and the sequences were found to be identical after sequencing comparison. Lentiviral virus was packaged using a second-generation viral packaging system (packaging plasmids PSPAX2 and PM2G).

[0080] Human T-cell acquisition

[0081] 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.

[0082] Construction of CAR-T cells

[0083] Human PDGFRβCAR virus was packaged by our research team using a second-generation lentivirus packaging system, and 1×10⁻⁶ viruses with an MOI of 20 were used to infect 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.

[0084] Flow cytometry

[0085] 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. After washing twice with pre-chilled PBS, analysis was performed using a flow cytometer (Beckman). PDGFRβ-overexpressing 293T cells were constructed.

[0086] The human PDGFRβ antigen sequence was found on the Uniprot website, synthesized, and then constructed into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP. The restriction enzyme sites before and after the sequence were XbaI and BamHI, 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.

[0087] CAR-T cell killing experiment

[0088] This experiment uses CytoTox The Assay kit (Promega, USA) measures lactate dehydrogenase (LDH) released by target cells to assess target cell killing activity. PDGFRβ-overexpressing 293T cells (1×10⁻⁶) were seeded in cell culture medium. 5 The cells were incubated with CAR-T cells at a 1:1 effector-to-target cell ratio (E:T) for 15 hours, with a total volume of 500 μL. 50 μL of cell supernatant was placed in each well of a 96-well plate, and 50 μL of CytoTox 96 reagent was added to each well. The plates were incubated at room temperature in the dark for 30 minutes. Then, 50 μL of stop solution was added to each well, and the absorbance at 492 nm was recorded using a multi-functional microplate reader (TECAN, infiniteM200pro). The spontaneous LDH release from effector and target cells, as well as the maximum LDH release from target cells, were measured.

[0089] Result Calculation

[0090] (1) All absorbance values ​​measured in experimental wells, target cell LDH spontaneous release wells and effector cell LDH spontaneous release wells were reduced by the average absorbance of the culture substrate.

[0091] (2) The absorbance value of the target cell maximum LDH release control should be reduced by the average absorbance value of the volume-corrected control.

[0092] (3) Calculate the cytotoxicity percentage based on the following formula and the correction values ​​obtained in steps 1 and 2.

[0093] Kill rate (%) = (Experimental value - Spontaneous release value of effector cells - Spontaneous release value of target cells) / (Maximum release value of target cells - Spontaneous release value of target cells) × 100%

[0094] result:

[0095] AI optimization for human PDGFRβ single-chain antibody:

[0096] This embodiment uses an optimization strategy that includes complex structure prediction, interface analysis, hotspot residue optimization design, and optimized single-chain antibody affinity assessment. Figure 3 A) A three-dimensional model of the human PDGFRβ-original single-chain antibody complex with a reasonable structure and a clear interface binding mode was obtained using the AlphaFold3 structure prediction platform. Figure 3 B); Conduct a systematic analysis of its interface interaction modes, identify interface residues, and define atomic distances less than [missing information]. The amino acid residues are key residues that directly participate in the binding of target proteins, as well as important functional residues at the target protein interface. Figure 3 C); The CDR region of the single-chain antibody was optimized using the Rosetta molecular design platform to generate multiple modified antibody complex models; the binding free energy was calculated using Rosetta's interface analysis module, and the top five optimized single-chain antibody sequences with significantly better binding free energies than the original single-chain antibody were selected. Figure 4 ).

[0097] The structure of CAR and the construction of CAR-T cells:

[0098] This embodiment uses a conventional second-generation CAR with 4-1BB as a 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 5 A). After synthesizing the CAR structure using Qingke Biotechnology, we constructed this CAR structure into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP ( Figure 5B) The restriction enzyme sites before and after the restriction are XbaI and BamHI, respectively. Lentiviral viruses were packaged using a second-generation viral packaging system (packaging plasmids were PSPAX2 and PM2G).

[0099] T cells were derived from peripheral blood mononuclear cells of healthy adults. After isolation, T cells were activated using a CD3 / CD28 activation antibody (STEMCELL). The T cells were cultured in X-VIVO 15 medium and infected with an MOI of 20 to obtain CAR-T cells with a high positive rate. Figure 5 C).

[0100] The results show that this invention successfully constructed a second-generation CAR targeting the human PDGFRβ antigen. Figure 5 A) By infecting peripheral blood T cells of healthy individuals with lentivirus, CAR-T cells with a high positive rate were obtained. Figure 5 C).

[0101] Based on the above experiments and their results, the following conclusions can be drawn:

[0102] 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.

[0103] Human PDGFRβ CAR-T cells can effectively kill PDGFRβ in vitro. + cell:

[0104] To investigate the killing ability of human CAR-T cells against target cells, we constructed human PDGFRβ antigen-overexpressing 293T cells (… Figure 6 A) Killing experiments were conducted on 293T-PDGFRβ cells. The lactate dehydrogenase (LDH) release method was used to detect the killing activity of CAR-T cells against target cells. The efficacy of five optimized single-chain antibodies in CAR-T immunotherapy was evaluated. The effector-to-target ratio was 1:1 and the killing time was 15h.

[0105] The results showed that the target cell mortality rate was very low in the Vec-T (vector transduced T cell) group, while the target cell mortality rate was relatively high in the five PDGFRβCAR-T cell groups. Figure 6 B).

[0106] Based on the above experiments and their results, the following conclusions can be drawn:

[0107] CAR-T cells carrying original or optimized single-chain antibody scFv targeting human PDGFRβ were used in vitro to target PDGFRβ. + The cells have a strong killing ability.

[0108] Amino acid sequence information used in the examples:

[0109] Original single-chain antibody heavy chain sequence:

[0110] QVQVKQSGPELVKPGASVKISCKASGYVFSGSWMNWVKQRPGQGLEWIGRIYPGDGGINYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVFFCAFITTVSAYWGQGTLVTVSA(SEQ ID NO.1)

[0111] Original single-chain antibody light chain sequence:

[0112] DILMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK(SEQ ID NO.2)

[0113] Optimize the heavy chain sequence of single-chain antibody 1:

[0114] QVQVKQSGPELVKPGASVKISCKASGIAFQGAWMNWVKQRPGQGLEWIGAILPGDGGINYNGKFKGKATLTADRSSSTAYMQLSSLTSVDSAVFFCAFITTTTAYWGQGTLVTVSA(SEQ ID NO.3)

[0115] Optimize the light chain sequence of single-chain antibody 1:

[0116] DILMTQTPLSLPVSLGDKASISCRSSQSIVHDNGKTYLMWFLQKPGQSPKLLIYDVSNLAPGVPSRFSGSGSGTDFTLVISRVEAEDLGVYYCMQGAHVPWTFGGGTKLEIK(SEQ ID NO.4)

[0117] Optimize the heavy chain sequence of single-chain antibodies:

[0118] QVQVKQSGPELVKPGASVKISCKASGIAFQGAWMNWVKQRPGQGLEWIGAILPGDGGINYNGKFKGKATLTADRSSSTAYMQLSSLTSVDSAVFFCAFLTAVSAYWGQGTLVTVSA(SEQ ID NO.5)

[0119] Optimize the light chain sequence of single-chain antibody 2:

[0120] DILMTQTPLSLPVSLGDKASISCRSSQSIVHDNGATYLQWYLQKPGQSPKLLIYKVSNLA PGVPSRFSGSGSGTDFTLVISRVEAEDLGVYYCFQGAHVPWTFGGGTKLEIK(SEQ ID NO.6)

[0121] Optimize the triple chain sequence of single-chain antibodies:

[0122] QVQVKQSGPELVKPGASVKISCKASGIAFQGAWMNWVKQRPGQGLEWIGAILPGDGGINYNGKFKGKATLTADRSSSTAYMQLSSLTSVDSAVFFCAFLTAVAAYWGQGTLVTVSA(SEQ ID NO.7)

[0123] Optimize the light chain sequence of single-chain antibody 3:

[0124] DILMTQTPLSLPVSLGDKASISCRSSQSIVAPNGATYLQWYLQKPGQSPKLLIYKVSNLAPGVPSRFSGSGSGTDFTLVISRVEAEDLGVYYCFQGAHVPWTFGGGTKLEIK(SEQ ID NO.8)

[0125] Optimize the quadruple chain sequence of single-chain antibodies:

[0126] QVQVKQSGPELVKPGASVKISCKASGIAFQGAWMNWVKQRPGQGLEWIGAILPGDGGINYNGKFKGKATLTADRSSSTAYMQLSSLTSVDSAVFFCAFITAVAAYWGQGTLVTVSA(SEQ ID NO.9)

[0127] Optimize the light chain sequence of single-chain antibody 4:

[0128] DILMTQTPLSLPVSLGDKASISCRSSQSIVAPNGATYLMWFLQKPGQSPKLLIYKVSNLAPGVPSRFSGSGSGTDFTLVISRVEAEDLGVYYCMQGAHVPWTFGGGTKLEIK(SEQ ID NO.10)

[0129] Optimize the 5-chain heavy chain sequence of single-chain antibodies:

[0130] QVQVKQSGPELVKPGASVKISCKASGIAFQGAWMNWVKQRPGQGLEWIGAILPGDGGINYNGKFKGKATLTADRSSSTAYMQLSSLTSVDSAVFFCAFITATTAYWGQGTLVTVSA(SEQ ID NO.11)

[0131] Optimize the 5-light chain sequence of single-chain antibody:

[0132] DILMTQTPLSLPVSLGDKASISCRSSQSIVHDNGATYLMWFLQKPGQSPKLLIYKVSNLA PGVPSRFSGSGSGTDFTLVISRVEAEDLGVYYCMQGAHVPWTFGGGTKLEIK(SEQ ID NO.12)

[0133] G4S linker sequence:

[0134] GGGGSGGGGSGGGGS(SEQ ID NO.13)

[0135] CD8 signal peptide sequence:

[0136] MALPVTALLLPLALLLHAARP(SEQ ID NO.14)

[0137] CD8 Hinge Sequence:

[0138] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.15)

[0139] CD8 transmembrane region (TM) sequence:

[0140] MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.16)

[0141] 4-1BB intracellular region sequence:

[0142] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.17)

[0143] CD3ζ chain sequence:

[0144] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.18)

[0145] Human PDGFRβ antigen amino acid sequence:

[0146]

[0147] PDGFRβ antigen extracellular amino acid sequence:

[0148] LVVTPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCR VTDPQLVVTLHEKKGDVALVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDM PYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRV KVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHV(SEQ ID NO.20)

[0149] Signal peptide sequence:

[0150] MRLPGAMPALALKGELLLLSLLLLLEPQISQG(SEQ ID NO.21)

[0151] Matters not covered in this invention are common knowledge.

[0152] 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. An scFV antibody targeting human PDGFRβ, characterized in that, The scFV antibody contains heavy chain V. H and light chain V L ; The heavy chain V H The amino acid sequence is selected from: (a1) An amino acid sequence as shown in any one of SEQ ID NO. 1, 3, 5, 7, 9 and 11; (a2) An amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 1, 3, 5, 7, 9 and 11; The light chain V L The amino acid sequence is selected from: (b1) An amino acid sequence as shown in any one of SEQ ID NO2, 4, 6, 8, 10 and 12; (b2) An amino acid sequence that is at least 85% identical to any one of SEQ ID NO. 2, 4, 6, 8, 10 and 12; Furthermore, the heavy chain V H With light chain V L Direct connection or connection via a connector; the connector is specifically (G4S)n, where n is a positive integer.

2. A chimeric antigen receptor targeting human PDGFRβ, characterized in that, It comprises at least the scFV antibody targeting human PDGFRβ as described in claim 1; The chimeric antigen receptor is any one of the first to fifth generation chimeric antigen receptors; Furthermore, the chimeric antigen receptor is formed by a series connection of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and a signal transduction domain. Furthermore, the signal peptide is a CD8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.14; The hinge region is a CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO.15; The transmembrane region is the CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.16; The co-stimulatory signal transduction domain is a 4-1BB co-stimulatory signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO. 17; The signal transduction domain is the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.

18.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the scFV antibody of claim 1 or the chimeric antigen receptor of claim 2.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, It comprises the carrier as described in claim 4.

6. An immune effector cell, characterized in that, It expresses the scFV antibody targeting human PDGFRβ as described in claim 1 or the chimeric antigen receptor as described in claim 2.

7. The immune effector cells as described in claim 6, characterized in that, The immune effector cells are selected from at least one of the following: immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells, and hematopoietic stem cells; preferably, the immune effector cells are T cells.

8. The use of the reagent in the preparation of compositions, pharmaceuticals, formulations, or kits for the prevention and / or treatment of diseases associated with PDGFRβ expression positivity, characterized in that, The reagent comprises: the scFV antibody of claim 1, or the chimeric antigen receptor of claim 2, or the immune effector cells of any one of claims 6-7; Furthermore, the diseases associated with positive PDGFRβ expression include kidney disease, liver disease, cardiovascular disease, and neoplastic diseases, preferably fibrosis-related diseases, including but not limited to chronic kidney disease, liver disease, and cardiovascular disease.

9. The use of the scFV antibody of claim 1, or the chimeric antigen receptor of claim 2, or the immune effector cells of any one of claims 6-7 in combination with other drugs; characterized in that, Other medications include diagnostic agents, preventative agents, and / or therapeutic agents.

10. A method for preventing and / or treating diseases associated with PDGFRβ expression positivity, characterized in that, The method includes administering to a subject a therapeutically effective amount of the scFV antibody of claim 1; or the chimeric antigen receptor of claim 2; or the immune effector cells of any one of claims 6-7; or the nucleic acid molecule of claim 3, or the carrier of claim 4.

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