NRP1-targeting single-chain antibody and application thereof in CAR-T immunotherapy of fibrosis

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a single-chain antibody targeting NRP1 and application of the single-chain antibody in CAR-T immunotherapy of fibrosis. Four types of high-affinity single-chain antibodies targeting mouse NRP1 antigens are obtained by immunizing mice, the single-chain antibodies are applied to second-generation CAR, T cells of mouse spleen sources are infected after virus packaging, and CAR-T cells are constructed. The CAR-T cells of the targeted NRP1 can be used for effectively killing NRP1 positive cells. In-vivo experiments show that the CAR-T cells can inhibit the growth of NRP1 positive tumor cells. The CAR-T cell can improve the mouse liver fibrosis condition, collagen in the mouse liver is reduced through treatment, and the molecular level of fibrosis markers SMA and NRP1 is reduced, it is indicated that the CAR-T cell recognizes and eliminates activated HSCs through targeting NRP1, and the fibrosis degree is reduced.

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 NRP1 and its application in CAR-T immunotherapy for fibrosis. 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] Fibrosis is a common disease that can occur in many tissues and organs of the human body. Under normal circumstances, fibrosis is mainly caused by excessive deposition of extracellular matrix. Among them, various diseases from the liver, kidney, lung, and heart are often accompanied by fibrosis or further deteriorate into serious diseases through the fibrosis stage. Liver fibrosis often develops due to chronic liver damage, and its occurrence mechanism is diverse. Among them, myofibroblasts and hepatic stellate cells (HSCs) are the main cells causing liver fibrosis. Liver fibrosis is a common pathological process of many chronic liver diseases. If not controlled in time, it can lead to the gradual loss of normal liver function and eventually progress to cirrhosis or even liver cancer, which seriously endangers people's health. There is currently no effective treatment. The typical pathological characteristics of liver fibrosis include abnormal deposition of fibrous connective tissue and pathological angiogenesis. In severe cases, it can develop into cirrhosis or even liver cancer.

[0004] HSCs are an important type of non-parenchymal cell in the liver, and together with myofibroblasts, they play a core role in the pathogenesis of liver fibrosis. So far, no drugs or therapies that directly treat liver fibrosis have been approved for marketing. Many studies have shown that some external or internal factors can cause quiescent HSCs to become activated HSCs, leading to an increase in extracellular matrix, which can cause the liver to show fibrosis, cirrhosis, and even cancer. Various preclinical studies have shown that eliminating activated HSCs can effectively alleviate liver fibrosis. Activated HSCs highly express neuropilin 1 (NRP1), and targeting NRP1 to eliminate HSCs is an effective and feasible strategy to improve liver fibrosis.

[0005] Neuropilin 1 (NRP1) is a transmembrane protein with a molecular weight of 130 kD. It is highly conserved in vertebrates and consists of three parts: the extracellular region, the transmembrane region, and the intracellular region. The extracellular region is relatively long and includes three domains. However, the intracellular region of NRP1 is relatively short and lacks catalytic activity, and cannot independently cause intracellular signal transduction. Studies have found that NRP1, as a co-receptor on the HSCs membrane, induces HSCs activation and promotes abnormal deposition of fibrous tissue in the liver. In the process of fibrosis, NRP1, as a co-receptor of TGFR and PDGFR on the surface of HSCs, can promote the binding of TGFR on the membrane surface with TGF-β, activate HSCs, and secrete a large amount of extracellular matrix; on the other hand, it can promote the binding of PDGFR on the membrane surface with PDGF in the extracellular matrix, promote the proliferation and migration of HSCs, and further aggravate the degree of liver cirrhosis.

[0006] Chimeric antigen receptor (CAR) is a technology based on T cells as a platform that expresses multiple fusion proteins and can specifically recognize, bind and kill target cells. Specifically, CAR consists of an antibody part that recognizes the surface antigen of the target cell, and is fused with the co-stimulatory molecules CD28 or 4-1BB and CD3ζ that can activate T cells through the transmembrane region. After the fusion, the activation signal is transmitted to the T cell through CD28 or 4-1BB and CD3ζ. The part that recognizes the antigen outside the cell is usually a ligand, a receptor and a single-chain antibody (scFv), which can recognize specific antigens. A large number of studies have shown that CAR-T cells with 4-1BB as a co-stimulatory molecule have very strong and persistent anti-tumor ability. Clinical studies have shown that 4-1BB CAR-T cells can survive in vivo for more than 3 months or even up to one year; while CAR-T cells with CD28 as a co-stimulatory molecule have a strong proliferation ability in the short term, but poor persistence, and the survival time in vivo generally does not exceed one month. So far, CAR-T has not only achieved remarkable results in the treatment of tumor diseases, but has also begun to emerge in the research of treating non-tumor diseases. For example, CAR-T cells have a significant improvement effect in the treatment of liver aging and myocardial fibrosis, but there are no reports on the study of CAR-T therapy for the treatment of liver fibrosis by targeting HSCs. Summary of the invention

[0007] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a single-chain antibody targeting NRP1 and its application in CAR-T immunotherapy for fibrosis. Specifically, the present invention targets the NRP1 molecule, and finally obtains the mouse single-chain antibody sequence through monoclonal antibody design, immunization of mice, cloning fusion, titer determination, and sequencing, and constructs it into CAR-T cells targeting NRP1. In vitro experiments show that CAR-T cells targeting NRP1 can effectively eliminate NRP1+ stellate cells and tumor cells, and in vivo experiments show that NRP1-CAR-T cells can effectively improve mouse liver fibrosis. In addition, NRP1 CAR-T cells can effectively inhibit the growth of NRP1+ tumor cells. This study provides new ideas for the clinical treatment of diseases such as liver fibrosis, and provides new targets for CAR-T immunotherapy in the treatment of cardiovascular diseases and even solid tumors. Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0009] In the first aspect of the present invention, a single-chain antibody targeting NRP1 is provided, wherein the single-chain antibody comprises a complementary determining region (CDR) sequence selected from the following: SEQ ID NO.1-6, SEQ ID NO.9-14, SEQ ID NO.17-22, SEQ ID NO.25-30; or comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the complementary determining region sequence, respectively.

[0010] Furthermore, in the single-chain antibody targeting NRP1, the complementary determining region comprises a heavy chain complementary determining region and a light chain complementary determining region;

[0011] Wherein, the heavy chain complementarity determining region is:

[0012] Comprising CDR1 with the amino acid sequence shown in SEQ ID NO.1, 9, 17, 25, CDR2 with the amino acid sequence shown in SEQ ID NO.2, 10, 18, 26, and CDR3 with the amino acid sequence shown in SEQ ID NO.3, 11, 19, 27;

[0013] The light chain complementarity determining region is:

[0014] It comprises CDR1 with the amino acid sequence shown as SEQ ID NO.4, 12, 20, 28, CDR2 with the amino acid sequence shown as SEQ ID NO.5, 13, 21, 29, and CDR3 with the amino acid sequence shown as SEQ ID NO.6, 14, 22, 30.

[0015] Furthermore, the single-chain antibody targeting NRP1 comprises a heavy chain antibody V selected from the following H Amino acid sequence and light chain antibody V L Amino acid sequence: SEQ ID NO.7-8, 15-16, 23-24, 31-32, or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the heavy chain antibody amino acid sequence and the light chain antibody amino acid sequence, respectively.

[0016] Furthermore, the single-chain antibody light chain V L Single-chain antibody heavy chain V H Can be directly connected or connected through a linker; the linker can be (G 4 S) n, wherein n is a positive integer, for example, can be 1, 2, 3, 4, 5 or 6, etc. In some specific embodiments of the present invention, the n is 3.

[0017] Furthermore, the single-chain antibody is composed of a light chain antibody, a linker and a heavy chain antibody in series.

[0018] The second aspect of the present invention provides a chimeric antigen receptor targeting NRP1, which comprises at least an antigen binding domain, wherein the antigen binding domain comprises the above-mentioned single-chain antibody targeting NRP1.

[0019] Furthermore, the chimeric antigen receptor targeting NRP1 is composed of a signal peptide, an antigen binding domain, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain in series.

[0020] The signal peptide may be a CD8 signal peptide (SP), and its amino acid sequence is shown in SEQ ID NO.34.

[0021] The transmembrane region may be a CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.35.

[0022] The co-stimulatory signal transduction domain can be a 4-1BB co-stimulatory signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.36.

[0023] The signal transduction domain may be a CD3ζ signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO.37.

[0024] The third aspect of the present invention provides a nucleic acid molecule encoding the single-chain antibody targeting NRP1 or the chimeric antigen receptor targeting NRP1.

[0025] The fourth aspect of the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0026] Among them, the recombinant expression vector can be a viral vector, and the viral vector includes any one or both of a retroviral vector and a lentiviral vector; further preferably, it is a lentiviral vector, and the recombinant expression vector is a recombinant viral expression vector expressing the single-chain antibody or chimeric antigen receptor by inserting a nucleic acid molecule encoding the above-mentioned single-chain antibody or chimeric antigen receptor into a virus.

[0027] A fifth aspect of the present invention provides a CAR-T cell, which is a T lymphocyte modified by the chimeric antigen receptor targeting NRP1.

[0028] The CAR-T cells can be obtained by infecting T cells with lentivirus; wherein the lentivirus is obtained by transfecting a recombinant lentivirus expression vector into a lentivirus packaging cell and then culturing the cell; the recombinant lentivirus expression vector is obtained by inserting the coding gene of the above-mentioned chimeric antigen receptor into a lentivirus expression vector.

[0029] The seventh aspect of the present invention provides a drug for preventing and / or treating diseases mediated by high expression of NRP1, which comprises the single-chain antibody, chimeric antigen receptor, CAR-T cell; or the nucleic acid molecule and recombinant expression vector.

[0030] Furthermore, the drug may also contain a pharmaceutically acceptable carrier, which may be a buffer, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant, a dispersant or a defoaming agent, etc., which are not specifically limited herein.

[0031] Further, the drug can be administered in vivo in a known manner. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary to a great extent depending on a variety of factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0032] Furthermore, the drug can be administered to humans or non-human mammals, including mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc., without specific limitation herein.

[0033] Furthermore, the present invention has confirmed through research that the CAR-T cells targeting NRP1 of the present invention can effectively improve liver fibrosis and the growth of liver cancer cells by in vitro treatment of HEK293T cells and hepatocellular carcinoma cell lines that positively express NRP1, and animal models have proved that it can effectively improve liver fibrosis and the growth of liver cancer cells. Therefore, the diseases mediated by high expression of NRP1 include but are not limited to liver diseases, kidney diseases and tumor diseases, especially fibrosis-mediated liver diseases, kidney diseases and tumor diseases.

[0034] The eighth aspect of the present invention provides a detection product, which comprises the single-chain antibody, chimeric antigen receptor, CAR-T cell, drug; or the nucleic acid molecule and recombinant expression vector.

[0035] The detection product may be a detection kit, a detection device or equipment, which is not limited here.

[0036] The detection product can be used to qualitatively or quantitatively detect the expression of NRP1, and can be used for NRP1-related basic research or applied in actual clinical practice, such as screening, (auxiliary) diagnosis, monitoring or prediction of the progression of diseases mediated by high NRP1 expression, etc., without specific limitation here.

[0037] Beneficial technical effects of one or more of the above technical solutions:

[0038] The above technical scheme firstly infuses NRP1 extracellular domain protein into mice through the tail vein, extracts mouse spleen B cells and fuses them with SP2 / 0 mouse myeloma cells, screens positive cell lines, takes multiple positive cell lines for sequencing, and obtains four scFv sequences. The scFv sequence is fused with CD8 transmembrane region, 4-1BB and CD3ζ to construct the second-generation CAR, and the virus is packaged to infect mouse spleen-derived T cells to construct four types of CAR-T cells.

[0039] It can kill HEK293T cells and mouse hepatocellular carcinoma cell lines (Hepa1-6) expressing NRP1 in vitro, and the designed 4 CAR-T cells can effectively improve mouse liver fibrosis in vivo. In the experiment of the present invention, a mouse liver fibrosis model was created by intraperitoneal injection of CCL4. After the model was successful 2 months later, NRP1 CAR-T cells were reinfused into the tail vein, and it was found that NRP1 CAR-T cells can effectively reduce liver fibrosis. In addition, using Hepa1-6 cells to create a subcutaneous tumor model, it was found that all 4 NRP1 CAR-T cells can effectively inhibit the growth of mouse liver cancer cells. This study provides a novel treatment method for CAR-T immunotherapy for various fibrotic diseases including liver fibrosis, cardiovascular diseases, and various tumor diseases with high expression of NRP1. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 A diagram of the strategy for obtaining supernatants containing numerous antibody sequences for immunizing mice according to the present invention.

[0042] Among them: C57 mice were immunized 5 times with the coating solution coated with NRP1 extracellular domain antigen, 5 mice were immunized, once every 14 days, and blood was collected during the period to detect the immune effect.

[0043] Figure 2 This is a diagram showing the purification of mouse NRP1 extracellular domain antigen of the present invention.

[0044] Among them: A is the detection of unpurified antigen after induction, 1 is uninduced bacteria, 2, 3 are induced bacteria, 4 is the precipitate after induction, 5 is the supernatant after induction, and M is the marker; B is the detection of purified antigen, 1, 2 are purified proteins, and M is the marker;

[0045] Figure 3 The present invention is the construction and detection of mouse CAR-T cells.

[0046] Among them: A is the structure and original diagram of four NRP1 CARs; B is the lentiviral vector pCDH-CMV-MCS-EF1-CopGFP used for construction; C is the flow cytometry identification of mouse T cells after sorting; D is the positive rate detection of the four constructed NRP1 CAR-T cells;

[0047] Figure 4 This is the construction of the NRP1 overexpressing cell line of the present invention and the detection of CAR-T cells killing the cell line.

[0048] Among them: Figure A is a mouse NRP1 overexpression cell line constructed using HEK293T cells as a platform; Figure B is a mouse NRP1 overexpression cell line constructed using Hepa1-6 cells as a platform; Figure C is a diagram showing four types of CAR-T cells killing HEK293T-NRP1 cells, with an effector-target ratio of 1:1; Figure D is a diagram showing four types of CAR-T cells killing Hepa1-6-NRP1 cells, with an effector-target ratio of 1:1;

[0049] Figure 5 This is an in vivo anti-tumor experiment of NRP1 CAR-T cells of the present invention.

[0050] Among them: A is a diagram of mouse subcutaneous tumor modeling and NRP1 CAR-T cell reinfusion strategy; B is a diagram of mouse tumor cell volume changes; C is a diagram of mouse survival curve;

[0051] Figure 6 This is the effect of the NRP1 CAR-T cells of the present invention on liver fibrosis in vivo.

[0052] Among them: A is the WB detection of mouse liver fibrosis one week after CAR-T cell infusion; B is the HE detection of mouse liver fibrosis; C is the Masson detection of mouse liver fibrosis, among which the Sham group mice are the non-fibrosis model group. DETAILED DESCRIPTION

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Example

[0058] The process of mouse immunization:

[0059] The amino acid sequence used for mouse NRP1 immunization is the extracellular region (NRP1 ECD), and the specific sequence is shown in SEQ ID NO.38. The specific process of immunizing mice to obtaining monoclonal cell lines is shown in Figure 1First, the required NRP1 extracellular region CDS sequence was obtained from the company (Qingdao Qingke Co., Ltd.), and then the optimized DNA was constructed into the pcDNA3.0 vector through molecular cloning technology. After protein expression, it was purified, and then mice were immunized multiple times. After sequencing, a variety of antibody sequences with high affinity were obtained. The four NRP1 scFvs obtained after sequencing were constructed into the CAR vector, and then the effect was verified by constructing CAR-T cells.

[0060] Acquisition of NRP1 extracellular domain protein:

[0061] The pcDNA3.0 vector loaded with the partial sequence of the extracellular region of NRP1 was transformed into BL21 competent bacteria for protein expression. After the bacterial solution was lysed, the protein was extracted and analyzed by SDS-PAGE gel electrophoresis. The molecular weight of the protein was estimated to be approximately 98 kDa based on the number of amino acid residues ( Figure 2 A), the induced activated bacteria were transferred to LB medium containing ampicillin resistance, and purified protein was obtained after culture ( Figure 2 B).

[0062] Screening of high affinity NRP1 scFv:

[0063] Blood collection test: This study followed the mature immune process for immunization and completed the titer determination and other indicator tests. Due to the large amount of data detected, this patent only summarizes and summarizes the mice and related data corresponding to the effective optimal cell lines and antibody results. The remaining data are not reflected in this patent. After the first, second, and third blood collection tests, target mice with higher titers that meet the experimental requirements are selected for fusion. In this study, one mouse 5# was selected from 5 mice for fusion, and the blood collection test results are shown in Table 1.

[0064] Table 1 Blood test results

[0065]

[0066] Fusion and cloning: SP2 / 0 mouse myeloma cells were used for cell fusion with spleen cells of the preferred mouse (5#). After fusion, the positive wells that met the conditions were screened out through culture, observation, detection and indirect ELISA detection (OD value of the tested well ≥2.00 was positive; OD value of the tested well <1.00 was negative). The screened positive wells with high sensitivity were cloned to finally obtain a batch of hybridoma cells that met the experimental requirements, and they were further cultured and selected.

[0067] 5# Mouse strain identification and sequencing: After fusion, primary cloning by limiting dilution method and supernatant detection, as shown in Table 2, 5 cell lines with 13 cell lines that met the project requirements were obtained. By comparison, a hybridoma with high titer and optimal affinity was sequenced to obtain the full-length sequence of NRP1 antibody. The antibody heavy chain / light chain variable region genes were obtained by PCR to obtain NRP1 scFv.

[0068] Table 2

[0069]

[0070] The structure of CAR and the construction of CAR-T cells:

[0071] The CAR used in the present invention is a second-generation CAR structure, and the specific molecular structure sequence of the CAR is: CD8SP-VL-(G 4 S) 3 linker-VH-CD8TM-4-1BB-CD3ζ( Figure 3 A). Four CARs targeting NRP1 were constructed into the lentiviral vector pCDH-CMV-MCS-EF1-CopGFP ( Figure 3 B), packaged into lentivirus using a third-generation lentiviral packaging system.

[0072] Obtaining mouse T cells and constructing CAR-T cells: The T cells used were from C57BL / 6J mice purchased from Nanjing Model Organisms Inc. The C57BL / 6J mice were sacrificed by cervical vertebrae and soaked in 75% alcohol for about 5 minutes. The spleen of the mice was removed in a biosafety cabinet and ground under sterile conditions to form a single cell suspension. Mouse T cells with a purity greater than 99% were obtained after sorting using a kit (EasySep Mouse TCell Isolation Kit, STEMCELL) ( Figure 3 C), add CD3 / CD28 activation magnetic beads (Miltenyi Biotec) for activation, use a medium containing 10% fetal bovine serum, 50 IU mL -L IL-7 (Proteintech) and 100 IU mL -L The cells were cultured in RPMI1640 medium supplemented with IL-15 (Proteintech).

[0073] NRP1 CAR virus was packaged by our laboratory, and the third-generation packaging system was used for packaging virus. Specifically, 293T cells of appropriate density were laid in a 10 cm dish. When the cell density was about 70%, the three packaging plasmids of △R, Rev and VSV-G were added to 293T cells at a ratio of 6.5:2.5:3.5 using the liposome encapsulation method. After 6 hours, the culture medium was replaced with fresh medium, and the virus was collected at 48 and 72 hours respectively, and the virus titer was detected after concentration.

[0074] Take the virus with MOI=100 to infect an appropriate amount of T cells, centrifuge the infection in a 12-well plate, 800g×60 minutes, transfer the infected T cells to fresh culture medium, and culture for about 3 days. Flow cytometry test the positive rate, and obtain a positive rate greater than 50% ( Figure 3 D) of mouse NRP1 CAR-T cells were infused back into mice within one week. At the same time, T cells obtained by virus infection using lentiviral empty vector packaging were used as a control and named T-Vec (T-Vector).

[0075] The results showed that four CARs targeting NRP1 antigen were successfully constructed in the present invention, which were named NRP1 CAR1, NRP1 CAR2, NRP1 CAR3 and NRP1 CAR4 ( Figure 3 A), after lentivirus infection of high-purity T cells ( Figure 3 C), and obtained CAR-T cells with a positive rate higher than 50% ( Figure 3 D).

[0076] From the above experiments and their results, we can draw the following conclusions:

[0077] Through molecular experiments, four mouse NRP CARs were successfully constructed and verified by sequencing. After obtaining mouse spleen-derived T cells, mouse CAR-T cells were successfully constructed, laying the foundation for subsequent functional exploration of CAR-T cells and the smooth progress of in vivo and in vitro experiments.

[0078] Mouse NRP1 CAR-T cells can effectively kill NRP1-positive cells in vitro:

[0079] In order to explore the in vitro killing ability of the four constructed mouse NRP1 CAR-T cells, the inventors co-cultured the four NRP1CAR-T cells with HEK293T cells expressing mouse NRP1 and Hepa1-6 cells expressing NRP1, respectively. HEK293T cells expressing mouse NRP1 and Hepa1-6 cells expressing NRP1 were artificially constructed (4A, B), with a killing effect-target ratio of 1:1 and a killing time of 12 hours. The LDH kit was used for detection.

[0080] 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 four NRP1 CAR-T cell groups was relatively high ( Figure 4 C, D).

[0081] From the above experiments and their results, we can draw the following conclusions:

[0082] NRP1 CAR-T cells have a strong killing ability against NRP1-positive cells in vitro.

[0083] NRP1 CAR-T cells can effectively inhibit tumor growth in vivo:

[0084] C57 mice were used for modeling, and 1×10 6 One week later, 5×10 6 NRP1 CAR-T cells ( Figure 5 A), measure the tumor size of mice every two days, observe the survival of mice, and calculate the survival time of mice.

[0085] The results showed that NRP1 CAR-T cells can effectively inhibit the growth of tumor cells ( Figure 5 B) and prolong the survival of mice ( Figure 5 C).

[0086] From the above experiments and their results, we can draw the following conclusions:

[0087] T cells expressing NRP1 CAR have strong anti-tumor function in vivo, indicating that the NRP1 scFv we screened can effectively recognize and bind to NRP1 antigen after being expressed as protein in vivo, and eliminate NRP1-positive tumor cells.

[0088] NRP1 CAR-T cells can effectively alleviate CCL4-induced liver fibrosis:

[0089] C57 mice were used to establish a fibrosis model. CCL4 (CCL4: Oil = 1:3) was injected intraperitoneally at a dose of 5 mL / kg and twice a week for eight consecutive weeks. The model was successfully established. After eight weeks of model establishment, CAR-T cells were reinfused into the tail vein at a dose of 5×10 6 / mouse, and the mice were euthanized one week later, and the liver tissues were removed for detection.

[0090] It was found that compared with the Sham group (control group; unmodeled mice) and the T-Vec group, the expression level of NRP1 molecules in the liver of the NRP1CAR-T cell-transfused mice was significantly lower than that of the T-Vec-transfused group, and the expression levels of fibrosis-related molecules α-sma, collagen-1, and fibronectin decreased ( Figure 6 A). At the same time, HE staining and Masson staining showed that the degree of liver fibrosis in mice in the NRP1CAR-T cell infusion group was reduced ( Figure 6 B, C).

[0091] From the above experiments and their results, we can draw the following conclusions:

[0092] NRP1 CAR-T cells can relieve liver fibrosis by clearing activated HSCs in the liver. Amino acid sequence information used in the examples:

[0093] The scFv-1 heavy chain CDR1 sequence is:

[0094] NYWMN (SEQ ID NO. 1)

[0095] The scFv-1 heavy chain CDR2 sequence is:

[0096] NIYPDTGNTNYDEKFNS(SEQ ID NO.2)

[0097] The scFv-1 heavy chain CDR3 sequence is:

[0098] SEFFFYGSDWFFDV (SEQ ID NO. 3)

[0099] The scFv-1 light chain CDR1 sequence is:

[0100] RSSQNIIHVNGNTFLE (SEQ ID NO. 4)

[0101] The scFv-1 light chain CDR2 sequence is:

[0102] KVSNRFS (SEQ ID NO.5)

[0103] The scFv-1 light chain CDR3 sequence is:

[0104] FQGSYVPPT (SEQ ID NO. 6)

[0105] The heavy chain sequence of scFv-1 is:

[0106] QVQLEQSGSELVRPGASVRLSCKASGYPFTNYWMNWVKQSHGQGLEWI GNIYPDTGNTNYDEKFNSKATLTVDTSSSTAYIHLSSLTSEDSAVYYCTRSEFFF YGSDWFFDVWGAGTTVTVSS(SEQ ID NO.7)

[0107] The sequence of the scFv-1 light chain is:

[0108] DIVITQTPLSLPVSLGDQASISCRSSQNIIHVNGNTFLEWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSYVPPTFGG GTKLEIK(SEQ ID NO.8)

[0109] The scFv-2 heavy chain CDR1 sequence is:

[0110] SGYSWH (SEQ ID NO. 9)

[0111] The scFv-2 heavy chain CDR2 sequence is:

[0112] HIHYSGYTDYNPSLKS(SEQ ID NO.10)

[0113] The scFv-2 heavy chain CDR3 sequence is:

[0114] WGQGTTLTVSS (SEQ ID NO. 11)

[0115] The scFv-2 light chain CDR1 sequence is:

[0116] RASESVDNYGISFMN (SEQ ID NO.12)

[0117] The scFv-2 light chain CDR2 sequence is:

[0118] TASNQGS (SEQ ID NO. 13)

[0119] The scFv-2 light chain CDR3 sequence is:

[0120] QQDKEVPWT (SEQ ID NO. 14)

[0121] The heavy chain sequence of scFv-2 is:

[0122] EVQLEESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQSPGNKLEWMG HIHYSGYTDYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCASRRGYDE DYWGQGTTLTVSS(SEQ ID NO.15)

[0123] The scFv-2 light chain sequence is:

[0124] DIVITQTTASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLL IYTASNQGSGVPARFSGSGSGTDFSLNIHPMEADDTAMYFCQQDKEVPWTFG GGTKLEIK(SEQ ID NO.16)

[0125] The scFv-3 heavy chain CDR1 sequence is:

[0126] RYWMN (SEQ ID NO. 17)

[0127] The scFv-3 heavy chain CDR2 sequence is:

[0128] EINPSNGRTDYNEKLKN(SEQ ID NO.18)

[0129] The scFv-3 heavy chain CDR3 sequence is:

[0130] GNWDFYGMDY (SEQ ID NO. 19)

[0131] The scFv-3 light chain CDR1 sequence is:

[0132] TASSSVSSSYLA (SEQ ID NO. 20)

[0133] The scFv-3 light chain CDR2 sequence is:

[0134] SASNLAS (SEQ ID NO. 21)

[0135] The scFv-3 light chain CDR3 sequence is:

[0136] HQFHRST (SEQ ID NO. 22)

[0137] The scFv-3 heavy chain sequence is:

[0138] QVQLEQSGAELAKPGASVKLSCKASGYTFTRYWMNWVKQRPGQGLEW IGEINPSNGRTDYNEKLKNKATLTVDKSSSTAYMQLSRLTSEDSAVYYCARGN WDFYGMDYWGQGTSVIVSS(SEQ ID NO.23)

[0139] The scFv-3 light chain sequence is:

[0140] DIVITQSTAIMSASLGERVTMTCTASSSVSSSYLAWFQQKPGSSPKIWIYSA SNLASGVPARLSGSGSGTSYSLTINSMEAEDAATYYCHQFHRSTFGAGTKLEL K(SEQ ID NO.24)

[0141] The scFv-4 heavy chain CDR1 sequence is:

[0142] SYWMH (SEQ ID NO. 25)

[0143] The scFv-4 heavy chain CDR2 sequence is:

[0144] DIYPGGDNILYNEKFKT(SEQ ID NO.26)

[0145] The scFv-4 heavy chain CDR3 sequence is:

[0146] GPPYDRYDYWYFDV (SEQ ID NO. 27)

[0147] The scFv-4 light chain CDR1 sequence is:

[0148] KSSQSLLNSRNQKNYLA(SEQ ID NO.28)

[0149] The scFv-4 light chain CDR2 sequence is:

[0150] FASTRDS (SEQ ID NO. 29)

[0151] The scFv-4 light chain CDR3 sequence is:

[0152] QQHYRTPLT (SEQ ID NO. 30)

[0153] The heavy chain sequence of scFv-4 is:

[0154] QVQLQESGAELVKPGTSVKMSCKTSGYTFTSYWMHWVKQRPGRGLEWI GDIYPGGDNILYNEKFKTRATLSVDTSSSTAYMQLSSLTSEDSAVYFCARGPPY DRYDYWYFDVWGAGTTVTVSS(SEQ ID NO.31)

[0155] The scFv-4 light chain sequence is:

[0156] DIVMTQSPSSLTMSVGQKVTMNCKSSQSLLNSRNQKNYLAWYQQKPGQ SPKLLVYFASTRDSGVPDRFRGSGSGTNFTLTITSVQVEDLADYFCQQHYRTPL TFGTGTKLELK(SEQ ID NO.32)

[0157] The G4S linker sequence is:

[0158] GGGGSGGGGSGGGGS (SEQ ID NO. 33)

[0159] The CD8 signal peptide (SP) sequence is:

[0160] MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.34)

[0161] The CD8 transmembrane region sequence is:

[0162] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSR(SEQ ID NO.35)

[0163] The sequence of the 4-1BB intracellular region is:

[0164] KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL(SEQ ID NO.36)

[0165] The CD3ζ chain sequence is:

[0166] FSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQR RRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTY DALHMQTLAPR(SEQ ID NO.37)

[0167] The extracellular region (NRP1 ECD) sequence is:

[0168] MERGLPLLCATLALALALAGAFRSDKCGGTIKIENPGYLTSPGYPHSYHPSEKCEWLIQAPEPYQRIMINFNPHFDLEDRDCKYDYVEVIDGENEGGRLWGKFCGKI APSPVVSSGPFLFIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTAPTGVIKSPGFPEKYPNSLECTYIINRP1KMSEIILEFESFDLEQDSNPPGGMFCRYDRLEI (SEQ IDNO.38)

[0169] 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 NRP1, characterized in that: The single-chain antibody comprises a complementary determining region (CDR) sequence selected from the following: SEQ ID NO.1-6, SEQ ID NO.9-14, SEQ ID NO.17-22, SEQ ID NO.25-30; or or comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the complementary determining region sequence, respectively.

2. The single-chain antibody according to claim 1, characterized in that The single-chain antibody targeting NRP1, wherein the complementary determining region comprises a heavy chain complementary determining region and a light chain complementary determining region; Wherein, the heavy chain complementarity determining region is: Comprising CDR1 with the amino acid sequence shown in SEQ ID NO.1, 9, 17, 25, CDR2 with the amino acid sequence shown in SEQ ID NO.2, 10, 18, 26, and CDR3 with the amino acid sequence shown in SEQ ID NO.3, 11, 19, 27; The light chain complementarity determining region is: Comprising CDR1 with amino acid sequences as shown in SEQ ID NOs. 4, 12, 20, and 28, CDR2 with amino acid sequences as shown in SEQ ID NOs. 5, 13, 21, and 29, and CDR3 with amino acid sequences as shown in SEQ ID NOs. 6, 14, 22, and 30; Furthermore, the single-chain antibody targeting NRP1 comprises a heavy chain antibody V selected from the following H Amino acid sequence and light chain antibody V L Amino acid sequence: SEQ ID NO. 7-8, 15-16, 23-24, 31-32, or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the heavy chain antibody amino acid sequence and the light chain antibody amino acid sequence, respectively; Furthermore, the single-chain antibody heavy chain V H Single chain antibody light chain V L Directly connected or connected through a linker; the linker is (G4S)n, wherein n is a positive integer, and further, n is 3.

3. A chimeric antigen receptor targeting NRP1, characterized in that It at least comprises an antigen binding domain, wherein the antigen binding domain comprises the single-chain antibody targeting NRP1 according to claim 1 or 2; Furthermore, the chimeric antigen receptor targeting NRP1 is composed of a signal peptide, an antigen binding domain, a transmembrane region, a co-stimulatory signal transduction domain and a signal transduction domain in series; Wherein, the signal peptide is a CD8 signal peptide, and its amino acid sequence is shown in SEQ ID NO.34; The transmembrane region is a CD8 transmembrane region, and its amino acid sequence is shown in SEQ ID NO.35; 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.36; The signal transduction domain is the CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.

37.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single-chain antibody targeting NRP1 according to claim 1 or 2 or the chimeric antigen receptor targeting NRP1 according to claim 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 4.

6. A CAR-T cell, characterized in that: The CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting NRP1 as described in claim 3.

7. A drug for preventing and / or treating diseases mediated by high expression of NRP1, characterized in that: It comprises any one or more of the single-chain antibody according to claim 1 or 2, the chimeric antigen receptor according to claim 3, the nucleic acid molecule according to claim 4, the recombinant expression vector according to claim 5, and the CAR-T cell according to claim 6.

8. The drug according to claim 7, characterized in that The drug further comprises a pharmaceutically acceptable carrier; The diseases mediated by high NRP1 expression include liver diseases, kidney diseases and tumor diseases, and further include liver diseases, kidney diseases and tumor diseases mediated by fibrosis.

9. A detection product, characterized in that: Any one or more of the single-chain antibody of claim 1 or 2, the chimeric antigen receptor of claim 3, the nucleic acid molecule of claim 4, the recombinant expression vector of claim 5, the CAR-T cell of claim 6, and the drug of any one of claims 7-8.

10. The detection product according to claim 9, characterized in that: The detection product is a detection kit, a detection device or equipment; the detection product is used to qualitatively or quantitatively detect the expression of NRP1.

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