A single-chain antibody targeting NRP1 and its application in CAR-T immunotherapy for fibrosis

Abstract

The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to a single-chain antibody targeting NRP1 and its application in CAR-T immunotherapy for fibrosis. In the present invention, four single-chain antibodies with high affinity targeting mouse NRP1 antigen are obtained by immunizing mice, and are applied to the second-generation CAR. After viral packaging, they are used to infect T cells derived from mouse spleens to construct CAR-T cells. CAR-T cells targeting NRP1 can effectively kill NRP1-positive cells. In vivo experiments show that CAR-T cells can inhibit the growth of NRP1-positive tumor cells. CAR-T cells can improve the condition of liver fibrosis in mice. After treatment, the collagen in the livers of mice decreases, and the molecular levels of fibrosis markers SMA and NRP1 decrease, indicating that CAR-T cells recognize and eliminate activated HSCs by targeting NRP1, reducing the degree of fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly 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 art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Fibrosis is a common disease, and fibrosis may occur in various tissues and organs of the human body. Under normal circumstances, fibrosis is mainly caused by excessive deposition of extracellular matrix. Among them, various diseases derived from the liver, kidney, lung, and heart often accompany fibrosis or further deteriorate into severe diseases through the fibrosis stage. Liver fibrosis often develops from chronic liver injury, 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, seriously endangering people's health. Currently, there is no effective treatment method. The typical pathological features of liver fibrosis include abnormal deposition of fibrous connective tissue and pathological angiogenesis, and in severe cases, it will develop into cirrhosis or even liver cancer.

[0004] HSCs are an important type of non-parenchymal cells in the liver, and they and myofibroblasts jointly play a core role in the pathogenesis of liver fibrosis. So far, no drugs or therapies for directly treating liver fibrosis have been approved for marketing. A number of studies have shown that some external or internal factors can cause quiescent HSCs to become activated HSCs, resulting in an increase in extracellular matrix, thereby causing the liver to show fibrosis, cirrhosis, or even canceration. A variety of preclinical studies have shown that removing activated HSCs can effectively relieve liver fibrosis. Activated HSCs highly express neuropilin 1 (NRP1), and it is an effective and feasible strategy to improve liver fibrosis by targeting NRP1 to remove HSCs.

[0005] Neuropilin 1 (NRP1) is a transmembrane protein with a molecular weight of 130 kD, which is highly conserved in vertebrates. It consists of three parts: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region is relatively long and includes three domains. However, the intracellular region of NRP1 is short and lacks catalytic activity, so it cannot independently initiate intracellular signal transduction. Studies have found that NRP1, as a co-receptor on the surface of hepatic stellate cells (HSCs), induces the activation of HSCs and promotes the abnormal deposition of fibrous tissue in the liver. During the process of fibrosis, NRP1, as a co-receptor for TGF-β receptor (TGFR) and platelet-derived growth factor receptor (PDGFR) on the surface of HSCs, on the one hand, can promote the binding of TGFR on the cell membrane to 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 cell membrane to platelet-derived growth factor (PDGF) in the extracellular matrix, promote the proliferation and migration of HSCs, and further exacerbate the degree of liver cirrhosis.

[0006] Chimeric antigen receptor (CAR) is a technology based on T cells that expresses multiple fusion proteins and can specifically recognize, bind to, and kill target cells. Specifically, CAR consists of an antibody part that recognizes antigens on the surface of target cells, a co-stimulatory molecule CD28 or 4-1BB that can be activated through transmembrane region fusion, and a CD3ζ part. Through the interaction with antigens after fusion, the activation signal is transmitted to T cells through components such as CD28 or 4-1BB and CD3ζ. The part that plays a role in antigen recognition outside the cell is usually a ligand, a receptor, or 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 the co-stimulatory molecule have very strong persistent anti-tumor ability. Clinical studies have shown that 4-1BB CAR-T cells can survive in the body for more than 3 months and even up to one year; while CAR-T cells with CD28 as the co-stimulatory molecule have strong proliferation ability in the short term, but poor persistence, and their survival time in the body generally does not exceed one month. So far, CAR-T has not only achieved remarkable results in the treatment of tumor diseases, but also begun to show its potential 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. However, there is no report on the research of CAR-T therapy for treating 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, for the NRP1 molecule, through monoclonal antibody design, immunizing mice, cloning and fusion, titer determination, and sequencing, the murine single-chain antibody sequence is finally obtained, and it is constructed 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. In vivo experiments show that NRP1-CAR-T cells can effectively improve liver fibrosis in mice. In addition, NRP1 CAR-T cells can effectively inhibit the growth of NRP1+ tumor cells. This research 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] To achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0009] In the first aspect of the present invention, a single-chain antibody targeting NRP1 is provided, and the single-chain antibody comprises complementary determining region (CDR) sequences 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 amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the complementary determining region sequences respectively.

[0010] Furthermore, for 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] Among them, the heavy-chain complementary determining region is:

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

[0013] The light-chain complementary determining region is:

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

[0015] Further, the single-chain antibody targeting NRP1 comprises a heavy-chain antibody V selected from the following H amino acid sequence and a 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.

[0016] Further, the light-chain V of the single-chain antibody L and the heavy-chain V of the single-chain antibody H can be directly linked or linked 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 of the present invention, n is 3.

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

[0018] In the second aspect of the present invention, there is provided a chimeric antigen receptor targeting NRP1, which at least comprises an antigen-binding domain, and the antigen-binding domain comprises the above-mentioned single-chain antibody targeting NRP1.

[0019] Further, 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] Among them, the signal peptide can be the CD8 signal peptide (Signal Peptide, SP), and its amino acid sequence is as shown in SEQ ID NO.34.

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

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

[0023] The signal transduction domain can be the CD3ζ signal transduction domain, and its amino acid sequence is as shown in SEQ ID NO.37.

[0024] In the third aspect of the present invention, there is provided a nucleic acid molecule encoding the single-chain antibody targeting NRP1 or the chimeric antigen receptor targeting NRP1.

[0025] In the fourth aspect of the present invention, a recombinant expression vector is provided, and the recombinant expression vector contains the above nucleic acid molecule.

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

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

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

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

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

[0031] Furthermore, the drug can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0032] Furthermore, the administration object of the drug can be a human or a non-human mammal, and the non-human mammal includes mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc., which are not specifically limited herein.

[0033] Furthermore, the present invention has been verified through research that the CAR-T cells targeting NRP1 of the present invention can effectively kill HEK293T cells with positive NRP1 expression and hepatocellular carcinoma cell lines in vitro, and animal models have proven that they can effectively improve liver fibrosis and the growth of hepatocellular carcinoma cells. Therefore, the diseases mediated by high NRP1 expression include, but are not limited to, liver diseases, kidney diseases, and neoplastic diseases, especially fibrosis-mediated liver diseases, kidney diseases, and neoplastic diseases.

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

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

[0036] The detection product can be used to qualitatively or quantitatively detect the expression of NRP1, and thus can be used for basic research related to NRP1 or applied to actual clinical practice, such as screening, (assistant) diagnosis, monitoring or predicting the progression of diseases mediated by high NRP1 expression, etc., which is not specifically limited herein.

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

[0038] The above technical solution first immunizes mice by retro-venous injection of the extracellular domain protein of NRP1, extracts mouse spleen B cells and fuses them with SP2 / 0 mouse myeloma cells, screens out positive cell lines, and sequences multiple positive cell lines to obtain four scFv sequences. The scFv sequences are fused with the CD8 transmembrane region, 4-1BB, and CD3ζ to construct a second-generation CAR, which is packaged into a virus and then infects T cells derived from mouse spleens to construct 4 types of CAR-T cells.

[0039] It can effectively kill HEK293T cells expressing NRP1 and mouse hepatocellular carcinoma cell line (Hepa1-6) in vitro, and the designed 4 types of CAR-T cells can effectively improve mouse liver fibrosis in vivo. In the experiment of the present invention, a mouse liver fibrosis model was established by intraperitoneal injection of CCL4. After the model was successfully established 2 months later, NRP1 CAR-T cells were retro-venously injected, and it was found that NRP1 CAR-T cells could effectively reduce liver fibrosis. In addition, a subcutaneous tumor model was established using Hepa1-6 cells, and it was found that all 4 types of NRP1 CAR-T cells could effectively inhibit the growth of mouse hepatocellular carcinoma cells. This research provides a novel treatment method for CAR-T immunotherapy to treat various fibrotic diseases including liver fibrosis, cardiovascular diseases, and various neoplastic diseases with high NRP1 expression. Therefore, it has good practical application value. Description of the Drawings

[0040] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0041] Figure 1 It is a strategic diagram for obtaining a supernatant containing numerous antibody sequences by immunizing mice with the present invention.

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

[0043] Figure 2 It is a diagram of the purification of the extracellular region antigen of mouse NRP1 of the present invention.

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

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

[0046] Among them: A is the diagram of four NRP1 CAR structures and elements; 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 detection of the positive rates of four constructed NRP1 CAR-T cells;

[0047] Figure 4 It is the construction of mouse NRP1 overexpressing cell lines and the detection of the killing of cell lines by CAR-T cells of the present invention.

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

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

[0050] Among them: A is a strategic diagram for subcutaneous tumor modeling in mice and the reinfusion of NRP1 CAR-T cells; B is a diagram of the change in the volume of mouse tumor cells; C is a survival curve of mice;

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

[0052] Among them: A shows the detection of liver fibrosis in mice by WB one week after the infusion of CAR-T cells; B shows the detection of liver tissue fibrosis in mice by H.E.; C shows the detection of liver tissue fibrosis in mice by Masson. Among them, the mice in the Sham group were not in the fibrotic model group. Detailed implementation manners

[0053] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0054] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, 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.

[0055] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0056] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions indicated in the following examples are usually carried out under conventional conditions.

[0057] Examples

[0058] The process of immunizing mice:

[0059] For mouse NRP1 immunization, the amino acid sequence of the extracellular region (NRP1 ECD) was used, and the specific sequence is shown in SEQ ID NO.38. The specific process from immunizing mice to obtaining monoclonal cell lines is shown in Figure 1. First, obtain the required CDS sequence of the extracellular region of NRP1 from the company (Qingdao Tsingke Co., Ltd.). Then, construct the optimized DNA into the pcDNA3.0 vector through molecular cloning technology. After protein expression and purification, immunize mice multiple times. Through sequencing, obtain multiple antibody sequences with relatively high affinity. Construct the four NRP1 scFvs obtained after sequencing onto the CAR vector, and then verify the effect by constructing CAR-T cells.

[0060] Obtaining the NRP1 extracellular region protein:

[0061] Transform the pcDNA3.0 vector loaded with the partial sequence of the extracellular region of NRP1 into BL21 competent bacteria for protein expression. After lysing the bacterial solution, extract the protein. Perform SDS-PAGE gel electrophoresis analysis on the obtained protein, and evaluate that the molecular weight of the protein is approximately 98 kDa according to the number of amino acid residues ( Figure 2 A). Transfer the induced and activated bacteria to the LB medium containing ampicillin resistance, and obtain the purified protein after cultivation ( Figure 2 B).

[0062] Screening of high-affinity NRP1 scFv:

[0063] Blood sampling and detection: In this study, immunization was carried out according to the mature immune process, and indicators such as titer determination were completed. Due to the large amount of detected data, this patent only summarizes and summarizes the mice corresponding to the optimal cell line and antibody results obtained effectively and their relevant data, and the rest of the data are not reflected in this patent. After the first, second, and third blood sampling and detections, select the target mice with higher titers that meet the experimental requirements for fusion. In this study, a total of 1 mouse (No. 5) was selected from 5 mice for fusion. Among them, the blood sampling and detection results are incorporated in Table 1.

[0064] Table 1 Blood sampling and detection results

[0065]

[0066] Fusion and cloning: Use SP2 / 0 mouse myeloma cells and the spleen cells of the selected mouse (No. 5) for cell fusion. After fusion, screen the positive wells that meet the conditions through cultivation, observation, detection, and indirect ELISA method (the OD value of the well to be tested ≥ 2.00 is positive; the OD value of the well to be tested < 1.00 is negative). Clone the positive wells with high sensitivity, and finally obtain a batch of hybridoma cells that meet the experimental requirements, and continue to culture and select them.

[0067] 5# Mouse monoclonal cell line determination and sequencing: After the mice were fused, cloned once by the limiting dilution method, and the supernatants were detected, as shown in Table 2, a total of 5 cell lines with 13 meeting the project requirements were obtained. Through comparison, one hybridoma with high titer and optimal affinity was sequenced to obtain the full-length NRP1 antibody sequence. The heavy / light chain variable region genes of the antibody were obtained by PCR to obtain NRP1 scFv.

[0068] Table 2

[0069]

[0070] Structure of CAR and 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 this CAR is: CD8SP-VL-(G4S)3linker-VH-CD8TM-4-1BB-CD3ζ( Figure 3 A). Four CARs targeting NRP1 were respectively constructed into the lentiviral vector pCDH-CMV-MCS-EF1-CopGFP( Figure 3 B), and packaged into lentiviruses by 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, which were purchased from Nanjing Model Organisms Inc. The C57BL / 6J mice were sacrificed by cervical dislocation and soaked in 75% alcohol for about 5 min. The spleens of the mice were removed in a biosafety cabinet and ground into a single cell suspension under sterile conditions. After sorting by a kit (EasySep Mouse TCell Isolation Kit, STEMCELL), mouse T cells with a purity greater than 99% were obtained( Figure 3 C), CD3 / CD28 activation beads (Miltenyi Biotec) were added for activation, and RPMI1640 medium containing 10% fetal bovine serum, 50 IU mL -L IL-7 (Proteintech) and 100 IU mL -L IL-15 (Proteintech) was used for culture.

[0073] The NRP1 CAR virus was obtained by self-packaging in our laboratory, and a third-generation packaging system was used for virus packaging. Specifically, 293T cells with an appropriate density were plated 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 the 293T cells at a ratio of 6.5:2.5:3.5 by the liposome wrapping method. After 6 hours, the medium was changed to fresh medium, and the virus was collected at 48 and 72 hours respectively, and the virus titer was detected after concentration.

[0074] Take a virus amount with MOI = 100 to infect an appropriate amount of T cells, centrifuge for infection in a 12-well plate at 800 g for 60 minutes, transfer the infected T cells to fresh medium for culture, and detect the positive rate by flow cytometry after culturing for about 3 days to obtain a positive rate greater than 50%( Figure 3 D) of mouse NRP1 CAR-T cells and transfuse them into mice within one week. At the same time, use lentiviral empty vector packaged virus to infect T cells as a control, named T-Vec (T-Vector).

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

[0076] From the above experiments and their results, the following conclusions can be drawn:

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

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

[0079] To explore the in vitro killing ability of the four constructed mouse NRP1 CAR-T cells, the inventors co-cultured the four NRP1 CAR-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), the killing effector-to-target ratio was 1:1, the killing time was 12 h, and an LDH kit was used for detection.

[0080] The results showed that: the target cell mortality rate in the Vec-T (Vector transduced T cell) cell group was very low, while the target cell mortality rate in the four NRP1 CAR-T cell groups was relatively high( Figure 4 C, D).

[0081] From the above experiments and their results, the following conclusions can be drawn:

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

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

[0084] Using C57 mice to establish a model, subcutaneously implant 1×10 6 mouse NRP1 overexpressing cell line Hepa1-6 cells. One week later, inject 5×10 6 NRP1 CAR-T cells into each group ( Figure 5 A), measure the tumor size of the mice every two days, observe the survival of the mice, and count the survival period of the mice.

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

[0086] From the above experiments and their results, the following conclusions can be drawn:

[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 the NRP1 antigen after being expressed as a protein in vivo, and eliminate NRP1-positive tumor cells.

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

[0089] Using C57 mice to establish a fibrosis model, intraperitoneally inject CCL4 (CCL4:Oil = 1:3), the injection dose is 5 mL / kg, and the injection frequency is twice a week for eight consecutive weeks to successfully establish the model. Eight weeks after modeling, inject CAR-T cells into the tail vein, and the injection dose is 5×10 6 / mouse. One week later, euthanize the mice and collect liver tissues for detection.

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

[0091] From the above experiments and their results, the following conclusions can be drawn:

[0092] NRP1 CAR-T cells can alleviate 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 scFv-1 heavy chain sequence is:

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

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

[0108] DIVITQTPLSLPVSLGDQASISCRSSQNIIHVNGNTFLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSYVPPTFGGGTKLEIK(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 scFv-2 heavy chain sequence is:

[0122] EVQLEESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQSPGNKLEWMGHIHYSGYTDYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCASRRGYDEDYWGQGTTLTVSS(SEQ ID NO.15)

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

[0124] DIVITQTTASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYTASNQGSGVPARFSGSGSGTDFSLNIHPMEADDTAMYFCQQDKEVPWTFGGGTKLEIK(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] QVQLEQSGAELAKPGASVKLSCKASGYTFTRYWMNWVKQRPGQGLEWIGEINPSNGRTDYNEKLKNKATLTVDKSSSTAYMQLSRLTSEDSAVYYCARGNWDFYGMDYWGQGTSVIVSS(SEQ ID NO.23)

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

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

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

[0142] SYWMH(SEQ ID NO.25)

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

[0144] DIYPGGDNILYNEKFKT(SEQ ID NO.26)

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

[0146] GPPYDRYDYWYFDV(SEQ ID NO.27)

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

[0148] KSSQSLLNSRNQKNYLA(SEQ ID NO.28)

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

[0150] FASTRDS(SEQ ID NO.29)

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

[0152] QQHYRTPLT(SEQ ID NO.30)

[0153] The scFv-4 heavy chain sequence 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 4-1BB intracellular region sequence 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] MERGLPLLCATLALALALAGAFRSDKCGGTIKIENPGYLTSPGYPHSYHPSEKCEWLIQAPEPYQRIMINFNPHFDLEDRDCKYDYVEVIDGENEGGRLWGKFCGKIAPSPVVSSGPFLFIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTAPTGVIKSPGFPEKYPNSLECTYIINRP1KMSEIILEFESFDLEQDSNPPGGMFCRYDRLEI WDGFPEVGPHIGRYCGQKTPGRIRSSSGVLSMVFYTDSAIAKEGFSANYSVLQSSISEDFKCMEALGMESGEIHSDQITASSQYGTNWSVERSRLNYPENGWTPGEDSYKEWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYRVDISSNGEDWISLKEGNKAIIFQGNTNPTDVVLGVFSKPLITRFVRIKPVSWETGISMRFEVYGCKITDYPCSGMLGMVSGLISDSQITASNQADRNWMPENIRLVTSRTGWALPPSPHPYTNEWLQVDLGDEKIVRGVIIQGGKHRENKVFMRKFKIAYSNNGSDWKTIMDDSKRKAKSFEGNNNYDTPELRTFSPLSTRFIRIYPERATHSGLGLRMELLGCEVEAPTAGPTTPNGNPVDECDDDQANCHSGTGDDFQLTGGTTVLATEKPTIIDSTIQSEFPTYGFNCEFGWGSHKTFCHWEHDSHAQLRWSVLTSKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQSSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVWMVVGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLGGIAVDDISINNHISQEDCAKPTDLDKKN(SEQ ID NO.38)

[0169] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A single-chain antibody targeting NRP1, characterized in that: The single-chain antibody comprises the following complementary determining region (CDR) sequences: SEQ ID NO. 1-6.

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 complementary determining region is: CDR1 of the amino acid sequence shown in SEQ ID NO.1, CDR2 of the amino acid sequence shown in SEQ ID NO.2, and CDR3 of the amino acid sequence shown in SEQ ID NO.3; The light chain complementarity determining region is: CDR1 of the amino acid sequence shown in SEQ ID NO.4, CDR2 of the amino acid sequence shown in SEQ ID NO.5, and CDR3 of the amino acid sequence shown in SEQ ID NO.

6.

3. The single-chain antibody according to claim 2, characterized in that The single-chain antibody targeting NRP1 comprises the following heavy chain antibody V H Amino acid sequence and light chain antibody V L Amino acid sequence: SEQ ID NO.7-8.

4. The single-chain antibody according to claim 3, characterized in that 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.

5. The single-chain antibody according to claim 4, characterized in that The n is 3.

6. A chimeric antigen receptor targeting NRP1, characterized in that It at least comprises an antigen binding domain, and the antigen binding domain comprises the single-chain antibody targeting NRP1 according to any one of claims 1 to 5.

7. The chimeric antigen receptor targeting NRP1 according to claim 6, wherein 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; in, 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 a CD3ζ signal transduction domain, and its amino acid sequence is shown in SEQ ID NO.

37.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single-chain antibody targeting NRP1 according to any one of claims 1 to 5 or the chimeric antigen receptor targeting NRP1 according to claim 6 or 7.

9. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 8.

10. A CAR-T cell, characterized in that: The CAR-T cell is a T lymphocyte modified by the chimeric antigen receptor targeting NRP1 according to claim 6 or 7.

11. A drug for treating diseases mediated by high expression of NRP1, characterized in that: It comprises any one or more of the single-chain antibody according to any one of claims 1 to 5, the chimeric antigen receptor according to claim 6 or 7, the nucleic acid molecule according to claim 8, the recombinant expression vector according to claim 9, and the CAR-T cell according to claim 10; The diseases mediated by high NRP1 expression are liver cancer and liver fibrosis.

12. The drug according to claim 11, characterized in that The medicament further comprises a pharmaceutically acceptable carrier.

13. A detection product, characterized in that: Comprising any one or more of the single-chain antibody according to any one of claims 1 to 5, the chimeric antigen receptor according to claim 6 or 7, the nucleic acid molecule according to claim 8, the recombinant expression vector according to claim 9, the CAR-T cell according to claim 10, and the drug according to any one of claims 11 to 12.

14. The detection product according to claim 13, characterized in that: The detection product is a detection kit or a detection device; the detection product is used to qualitatively or quantitatively detect the expression of NRP1.

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