CN119371538B

CN119371538B - A single-chain antibody targeting NRP1 and its application in CAR-T immunotherapy for fibrosis

Info

Publication number: CN119371538B
Application number: CN202411669250.4A
Authority: CN
Inventors: 赵松柏; 朱强; 隋明皓; 冯跃民; 李德军; 牟成志; 王新宇; 丁翰; 孙大玮; 王景鸿; 王晓庆; 唐辉; 丁南南; 刘慧敏; 薛雨婵
Original assignee: Shandong Provincial Hospital
Current assignee: Shandong Provincial Hospital
Priority date: 2024-11-21
Filing date: 2024-11-21
Publication date: 2025-06-24
Anticipated expiration: 2044-11-21
Also published as: CN120230215A; CN119371538A

Abstract

The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to a single-chain antibody targeting NRP1 and application thereof in CAR-T immunotherapy fibrosis. According to the invention, four high-affinity targeted mouse NRP1 antigen single-chain antibodies are obtained by immunizing a mouse, are applied to a second-generation CAR, and are used for infecting mouse spleen-derived T cells after virus packaging to construct CAR-T cells. NRP 1-targeted CAR-T cells can all kill NRP1 positive cells effectively. In vivo experiments show that CAR-T cells can inhibit NRP1 positive tumor cell growth. The CAR-T cells can improve the liver fibrosis condition of mice, collagen in the liver of the treated mice is reduced, and the levels of fibrosis markers SMA and NRP1 molecules are reduced, which indicates that the CAR-T cells recognize and clear activated HSCs by targeting NRP1, and the degree of fibrosis is reduced.

Description

NRP 1-targeted single-chain antibody and application thereof in CAR-T immunotherapy fibrosis

Technical Field

The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to a single-chain antibody targeting NRP1 and application thereof in CAR-T immunotherapy fibrosis.

Background

The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Fibrosis is a common disease in which various tissues and organs of the human body are likely to undergo fibrosis, which is normally caused mainly by excessive extracellular matrix deposition. Many diseases of liver, kidney, lung, heart and the like are often accompanied by fibrosis or further malignant into serious diseases through fibrosis stages. Liver fibrosis often develops due to chronic liver injury, the mechanism of which is diverse, with myofibroblasts and astrocytes (HEPATIC STELLATE CELLS; HSCs) being the primary cells responsible for liver fibrosis. Liver fibrosis is a common pathological process of many chronic liver diseases, if not controlled in time, normal liver functions can be gradually lost, and finally liver cirrhosis and even liver cancer are progressed, so that the health of people is seriously endangered, and no effective treatment method exists at present. Typical pathological features of liver fibrosis include abnormal deposition of fibrous connective tissue and pathological angiogenesis, and in severe cases, cirrhosis and even liver cancer develop.

HSCs are an important non-parenchymal cell in the liver that together with myofibroblasts play a central role in the pathogenesis of liver fibrosis. So far, no drugs or therapies for directly treating liver fibrosis are marketed in batches. Several studies have shown that some external or internal factors can cause resting HSCs to become activated HSCs, resulting in an increase in extracellular matrix, which can lead to the liver exhibiting fibrosis, cirrhosis and even cancer. Various preclinical studies have shown that removal of activated HSCs is effective in alleviating liver fibrosis. Activated HSCs highly express neuropilin 1 (Neuropilin; NRP 1), and the improvement of liver fibrosis by targeting NRP1 to clear HSCs is an effective and viable strategy.

Neuropilin 1 (Neuropilin; NRP 1) is a transmembrane protein of 130kD molecular weight, highly conserved in vertebrates, and consists of three parts, the extracellular region, the transmembrane region and the intracellular region, wherein the extracellular region is longer and comprises three domains. However, the intracellular region of NRP1 is short, lacks catalytic activity, and cannot cause intracellular signaling independently. NRP1 was found to act as a membrane co-receptor for HSCs, inducing HSCs activation, promoting abnormal deposition of fibrous tissue in the liver. NRP1 is used as a synergistic receptor of TGFR and PDGFR on the surface of HSCs in the fibrosis process, so that on one hand, the TGFR and TGF-beta on the surface of the membrane can be promoted to be combined, the HSCs can be activated, and a large amount of extracellular matrixes are secreted, and on the other hand, the PDGFR on the surface of the membrane and PDGF in the extracellular matrixes can be promoted to be combined, the proliferation and migration of the HSCs can be promoted, and the degree of liver cirrhosis can be further increased.

Chimeric antigen receptor (CHIMERIC ANTIGEN receptor; CAR) is a technology based on T cell as platform that expresses a variety of fusion proteins and is capable of specifically recognizing, binding and killing target cells. Specifically, the CAR consists of an antibody part recognizing a target cell surface antigen, a co-stimulatory molecule CD28 or 4-1BB and a CD3 zeta part which can activate T cells through transmembrane region fusion, and an activation signal is transmitted to the T cells through elements such as CD28 or 4-1BB, CD3 zeta and the like through interaction with the antigen after fusion. The extracellular portions that function to recognize antigens are typically ligands, receptors, and single chain antibodies (scFv) that recognize specific antigens. A large number of researches show that the CAR-T cells taking 4-1BB as a co-stimulatory molecule have very strong sustainable anti-tumor capability, and clinical researches show that the survival time of the CAR-T cells taking 4-1BB in vivo can exceed 3 months and even reach one year, while the CAR-T cells taking CD28 as the co-stimulatory molecule have strong proliferation capability in a short period, but poor persistence, and the survival time in vivo is generally not more than one month. So far, CAR-T has not only achieved significant success in the treatment of neoplastic diseases, but also began to show a significant improvement in the treatment of non-neoplastic diseases, such as CAR-T cells in the treatment of liver aging and myocardial fibrosis, but no study of CAR-T therapy for the treatment of liver fibrosis by targeting HSCs has been reported.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, it is an object of the present invention to provide a single chain antibody targeting NRP1 and its use in CAR-T immunotherapy of fibrosis. Specifically, the invention aims at NRP1 molecules, and finally obtains a murine single-chain antibody sequence through monoclonal antibody design, immune mouse, clone fusion, potency determination and sequencing, and constructs the murine single-chain antibody sequence into NRP 1-targeted CAR-T cells. In vitro experiments show that NRP 1-targeted CAR-T cells can effectively eliminate NRP1+ stellate cells and tumor cells, in vivo experiments show that NRP1-CAR-T cells can effectively improve liver fibrosis of mice, and in addition, NRP1 CAR-T cells can effectively inhibit growth of NRP1+ tumor cells. The research provides a new thought for clinically treating diseases such as liver fibrosis and the like, and provides a new target for CAR-T immunotherapy in treating cardiovascular diseases, even solid tumors and the like. Based on the above results, the present invention has been completed.

In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:

In a first aspect of the invention there is provided a single chain antibody targeting NRP1 comprising a Complementarity Determining Region (CDR) sequence selected from the group consisting of SEQ ID NO.1-6, SEQ ID NO.9-14, SEQ ID NO.17-22, SEQ ID NO.25-30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity respectively to said complementarity determining region sequence.

Further, the single chain antibody targeting NRP1, the complementarity determining region comprising a heavy chain complementarity determining region and a light chain complementarity determining region;

Wherein the heavy chain complementarity determining regions are:

CDR1 comprising the amino acid sequences shown as SEQ ID nos. 1, 9, 17, 25, CDR2 comprising the amino acid sequences shown as SEQ ID nos. 2, 10, 18, 26, and CDR3 comprising the amino acid sequences shown as SEQ ID nos. 3, 11, 19, 27;

the light chain complementarity determining regions are:

CDR1 comprising the amino acid sequences shown as SEQ ID nos. 4, 12, 20, 28, CDR2 comprising the amino acid sequences shown as SEQ ID nos. 5, 13, 21, 29, and CDR3 comprising the amino acid sequences shown as SEQ ID nos. 6, 14, 22, 30.

Further, the single chain antibody targeting NRP1 comprises a heavy chain antibody V _H amino acid sequence and a light chain antibody V _L amino acid sequence selected from SEQ ID NO.7-8, 15-16, 23-24, 31-32, or comprises an amino acid sequence having 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.

Further, the single-chain antibody light chain V _L and the single-chain antibody heavy chain V _H may be directly connected or connected through a linker, and the linker may be (G ₄ S) n, where n is a positive integer, for example, 1, 2, 3, 4, 5 or 6, etc. In some embodiments of the invention, n is 3.

Further, the single chain antibody is composed of a light chain antibody, a linker and a heavy chain antibody in tandem.

In a second aspect of the invention, there is provided a NRP 1-targeting chimeric antigen receptor comprising at least an antigen binding domain comprising the above-described NRP 1-targeting single chain antibody.

Furthermore, the chimeric antigen receptor targeting NRP1 is formed by connecting a signal peptide, an antigen binding domain, a transmembrane region, a costimulatory signaling domain and a signaling domain in series.

Wherein, the signal peptide can be CD8 signal peptide (SIGNAL PEPTIDE, SP), and the amino acid sequence is shown as SEQ ID NO. 34.

The transmembrane region may be a CD8 transmembrane region, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO. 35.

The costimulatory signaling domain can be a 4-1BB costimulatory signaling domain, the amino acid sequence of which is shown in SEQ ID NO. 36.

The signal transduction domain can be a CD3 zeta signal transduction domain, and the amino acid sequence of the signal transduction domain is shown as SEQ ID NO. 37.

In a third aspect of the invention, there is provided a nucleic acid molecule encoding said NRP 1-targeting single chain antibody or said NRP 1-targeting chimeric antigen receptor.

In a fourth aspect of the invention, there is provided a recombinant expression vector comprising a nucleic acid molecule as described above.

The recombinant expression vector can be any one or two of a retrovirus vector and a lentiviral vector, and is more preferably a lentiviral vector, and the recombinant expression vector is obtained by inserting a nucleic acid molecule encoding the single-chain antibody or chimeric antigen receptor into a virus.

In a fifth aspect of the invention, there is provided a CAR-T cell that is a T lymphocyte modified by the NRP 1-targeting chimeric antigen receptor.

The CAR-T cells can be obtained by infecting T cells with a lentivirus, wherein the lentivirus is obtained by transfecting a lentivirus packaging cell with a recombinant lentivirus expression vector and then culturing the cell, and the recombinant lentivirus expression vector is obtained by inserting the encoding gene of the chimeric antigen receptor into the lentivirus expression vector.

In a seventh aspect, the invention provides a medicament for preventing and/or treating NRP1 high expression mediated diseases, which comprises the single chain antibody, chimeric antigen receptor, CAR-T cell, or the nucleic acid molecule and recombinant expression vector.

Further, the medicament may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant, a dispersing agent, an antifoaming agent, or the like, and is not particularly limited herein.

Further, the drug may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

Further, the administration subject of the drug may be a human or non-human mammal including mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc., without specific limitation.

Furthermore, the research proves that the NRP 1-targeted CAR-T cell can effectively improve liver fibrosis and liver cancer cell growth in vitro on HEK293T cells and liver cell cancer cell lines positively expressed by NRP 1. Thus, the NRP1 high expression mediated diseases include, but are not limited to, liver diseases, kidney diseases and neoplastic diseases, especially fibrosis mediated liver diseases, kidney diseases and neoplastic diseases.

In an eighth aspect of the invention, there is provided an assay product comprising the single chain antibody, chimeric antigen receptor, CAR-T cell, drug, or the nucleic acid molecule and recombinant expression vector.

The detection product may be a detection kit, a detection device or an apparatus, which is not limited herein.

The detection product can be used for qualitatively or quantitatively detecting the expression condition of NRP1, and further can be used for NRP1 related basic research or applied to actual clinic, such as screening, (auxiliary) diagnosis, monitoring or predicting the progress of NRP1 high expression mediated diseases, and the like, and is not particularly limited.

The beneficial technical effects of one or more of the technical schemes are as follows:

according to the technical scheme, NRP1 extracellular region protein is infused back to a mouse through a tail vein mode to immunize the mouse, spleen B cells of the mouse are extracted to be fused with SP2/0 mouse myeloma cells, positive cell strains are screened out, a plurality of positive cell strains are taken for sequencing, and four scFv sequences are obtained. The scFv sequence is fused with a CD8 transmembrane region, 4-1BB and CD3 zeta to form a second generation CAR, and after virus packaging, mice spleen-derived T cells are infected to form 4 CAR-T cells.

HEK293T cells expressing NRP1 and a mouse liver cell cancer cell line (Hepa 1-6) can be killed well in vitro, and 4 CAR-T cells can be designed to effectively improve mouse liver fibrosis in vivo. In the experiment of the invention, a mouse liver fibrosis model is created by intraperitoneal injection of CCL4, and after 2 months of success of the model, NRP1 CAR-T cells are infused back into tail veins, so that the NRP1 CAR-T cells can be found to effectively alleviate liver fibrosis. In addition, using the 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 liver cancer cells in mice. The study provides a novel therapeutic approach for CAR-T immunotherapy for the treatment of various fibrotic diseases including liver fibrosis, cardiovascular diseases, and various neoplastic diseases with high expression of NRP 1. Therefore, has good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a schematic representation of the immunization of mice according to the present invention to obtain an supernatant comprising a plurality of antibody sequences.

Wherein, C57 mice are immunized 5 times by using a coating liquid for coating NRP1 extracellular region antigen, 5 mice are immunized once every 14 days, and the immune effect is detected by blood sampling during the period.

FIG. 2 is a diagram showing purification of the mouse NRP1 extracellular domain antigen according to the present invention.

Wherein A is the condition of detecting the antigen after induction, 1 is the non-induced thallus, 2,3 is the induced thallus, 4 is the induced sediment, 5 is the induced supernatant, M is Marker;

FIG. 3 is a diagram of the construction and detection of mouse CAR-T cells of the invention.

Wherein, A is four NRP1 CAR structures and original pictures, B is a slow virus vector pCDH-CMV-MCS-EF1-CopGFP used for construction, C is a flow identification condition after the separation of mouse T cells, D is a positive rate detection condition of four constructed NRP1 CAR-T cells;

FIG. 4 shows the construction of NRP1 overexpressing cell lines and detection of killing of cell lines by CAR-T cells according to the present invention.

The method comprises the steps of (a) constructing a mouse NRP1 over-expression cell strain by taking HEK293T cells as a platform, B constructing a mouse NRP1 over-expression cell strain by taking HEpa1-6 cells as a platform, C killing HEK293T-NRP1 cells by four CAR-T cells, wherein the effective target ratio is 1:1, and D killing HEpa1-6-NRP1 cells by four CAR-T cells, wherein the effective target ratio is 1:1;

FIG. 5 is an in vivo anti-tumor assay for NRP1 CAR-T cells according to the present invention.

Wherein, A is a mouse subcutaneous tumor model and NRP1 CAR-T cell feedback strategy diagram, B is a mouse tumor cell volume change diagram, C is a mouse survival diagram;

FIG. 6 is an in vivo effect of NRP1 CAR-T cells of the present invention on liver fibrosis.

Wherein, A is the liver fibrosis condition of mice detected by WB means after one week of CAR-T cell reinfusion, B is the liver fibrosis condition of mice detected by H.E, and C is the liver fibrosis condition of mice detected by Pinus massoniana, wherein, the Sham group mice are non-fibrotic model groups.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, 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 belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. The reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified, and are usually used under conventional conditions or under conditions recommended by the reagent company.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.

Examples

The process of mouse immunization:

The mouse NRP1 immunization uses an extracellular region (NRP 1 ECD) with an amino acid sequence, and the specific sequence is shown in SEQ ID NO. 38. The specific immunization of mice to the acquisition of monoclonal cell lines is shown in FIG. 1. Firstly, a company (Qingdao Optimaceae Co., ltd.) obtains a required NRP1 extracellular region CDS sequence, then the optimized DNA is constructed into a pcDNA3.0 vector through a molecular cloning technology, the protein is purified after being expressed, then mice are immunized for multiple times, a plurality of antibody sequences with higher affinity are obtained through sequencing, four NRP1 scFv obtained after sequencing are constructed onto a CAR vector, and then the CAR-T cell verification effect is constructed.

Acquisition of NRP1 keep-out region protein:

The pcDNA3.0 vector loaded with the NRP1 extracellular region partial sequence was transformed into BL21 competent bacteria, protein expression was performed, the bacterial solution was lysed, the protein was extracted, the obtained protein was analyzed by SDS-PAGE gel electrophoresis, the molecular weight of the protein was estimated to be about 98kDa according to the number of amino acid residues (FIG. 2A), the activation-induced bacteria was transferred into LB medium containing ampicillin resistance, and purified protein was obtained after culturing (FIG. 2B).

Screening of high affinity NRP1 scFv:

Blood sampling detection, namely, the study performs immunization according to a mature immunization flow and completes index detection such as titer measurement, and the like, and the patent only gathers and summarizes mice and related data thereof corresponding to the optimal cell strain and antibody result effectively because of the large detected data volume, and the rest data are not reflected in the patent. And selecting target mice with higher titers meeting experimental requirements for fusion after primary, secondary and tertiary blood sampling detection. In this study, 1 mouse # 5 was selected from 5 mice to fuse, and the blood collection test results were shown in Table 1.

TABLE 1 blood collection test results

Fusing and cloning, namely performing cell fusion by utilizing SP2/0 mouse myeloma cells and spleen cells of a preferred mouse (5#), culturing, observing, detecting and detecting by an indirect ELISA method (the OD value of a hole to be detected is more than or equal to 2.00 and is positive, the OD value of the hole to be detected is negative, and the OD value of the hole to be detected is less than 1.00), screening out positive holes meeting the condition, cloning the screened positive holes with high sensitivity, finally obtaining a batch of hybridoma cells meeting the experimental requirement, and continuously culturing and selecting the hybridoma cells.

The 5# mouse is subjected to primary cloning and supernatant detection by fusion and limiting dilution method, as shown in table 2, 5 strains of 13 cell strains meeting the project requirements are obtained, one hybridoma with high titer and optimal affinity is sequenced by comparison, the full length of an NRP1 antibody sequence is obtained, and the heavy chain/light chain variable region genes of the antibody are obtained by PCR, so that the NRP1 scFv is obtained.

TABLE 2

Construction of CAR and construction of CAR-T cells:

The CAR used in the invention is of a second-generation CAR structure, the specific molecular structural sequence of the CAR is CD8SP-VL- (G ₄S)₃ linker-VH-CD8TM-4-1BB-CD3 zeta (figure 3A), four CARs targeting NRP1 are respectively constructed on lentiviral vectors pCDH-CMV-MCS-EF1-CopGFP (figure 3B), and the lentiviruses are packaged by using a third-generation lentiviral packaging system.

Mouse T cell acquisition and CAR-T cell construction using T cells from C57BL/6J mice purchased from Nanjing model organism Inc., cervical sacrificed C57BL/6J mice, immersed in 75% alcohol for about 5min, the mouse spleen removed in a biosafety cabinet, ground under sterile conditions to form a single cell suspension, sorted by kit (EasySep Mouse T Cell Isolation Kit, STEMCELL) to obtain mouse T cells with purity greater than 99% (FIG. 3C), activated by addition of CD3/CD28 activating beads (Mei Tian Liu), and cultured using RPMI1640 medium containing 10% fetal bovine serum, 50IU of ^-L IL-7 (Proteintech) and 100IU of ^-L IL-15 (Proteintech).

NRP1 CAR virus is obtained by self-packaging in the laboratory, and the packaged virus uses a third-generation packaging system. Specifically, 293T cells with proper density are laid in a 10cm dish, when the cell density is about 70%, three packaging plasmids of Delta R, rev and VSV-G are added into the 293T cells in a ratio of 6.5:2.5:3.5 by using a liposome packaging method, fresh culture medium is changed after 6 hours, viruses are respectively collected at 48 hours and 72 hours, and virus titer is detected after concentration.

And (3) taking a proper amount of T cells infected by the virus with MOI=100, centrifugally infecting the T cells in a 12-hole plate for 800g×60 minutes, transferring the infected T cells into a fresh culture medium for culturing, and carrying out flow detection on the positive rate for about 3 days to obtain the NRP1 CAR-T cells of the mice with the positive rate of more than 50% (shown in fig. 3D), and carrying out one week of reinfusion into the bodies of the mice. Meanwhile, the virus infection was packaged using lentiviral empty Vector to obtain T cells as a control, which was designated as T-Vec (T-Vector).

The results show that four NRP1 antigen-targeted CARs are successfully constructed in the invention and are respectively named as NRP1 CAR1, NRP1 CAR2, NRP1 CAR3 and NRP1 CAR4 (figure 3A), and after lentiviruses infect high-purity T cells (figure 3C), CAR-T cells with a positive rate higher than 50% are obtained (figure 3D).

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

four mouse NRP CARs are successfully constructed through molecular experiments, verification is carried out through sequencing, and after the mouse spleen-derived T cells are obtained, the mouse CAR-T cells are successfully constructed, so that a foundation is laid for the function exploration of subsequent CAR-T cells and the smooth in-vitro and in-vivo experiments.

The mouse NRP1 CAR-T cells are able to kill NRP1 positive cells effectively in vitro:

In order to explore the in vitro killing capacity of the four constructed mouse NRP1 CAR-T cells, the inventor respectively co-cultures the four NRP1 CAR-T cells with HEK293T cells expressing mouse NRP1 and Hepa1-6 cells expressing NRP1, the HEK293T cells expressing mouse NRP1 and the Hepa1-6 cells expressing NRP1 are constructed by human (4A, B), the killing efficiency target ratio is 1:1, the killing time is 12h, and the detection is carried out by using an LDH kit.

The results showed that the Vec-T (Vector transduced T cell) cell group target cell mortality was very low, while the four NRP1 CAR-T cell groups target cell mortality was relatively high (FIGS. 4C, D).

NRP1 CAR-T cells have a strong killing capacity against NRP1 positive cells in vitro.

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

Using C57 mice modeling, 1X 10 ⁶ mice NRP1 over-expressed cell lines Hepa1-6 cells were inoculated subcutaneously, 5X 10 ⁶ NRP1 CAR-T cells were returned per group after one week (FIG. 5A), the tumor size of the mice was measured once every two days, survival of the mice was observed, and survival of the mice was counted.

The results showed that NRP1 CAR-T cells were effective in inhibiting tumor cell growth (fig. 5B), prolonging survival of mice (fig. 5C).

The T cells expressing the NRP1 CAR have stronger anti-tumor function in vivo, which shows that the NRP1 scFv screened by us can effectively recognize and bind with NRP1 antigen after expressing the protein in vivo, and eliminate the NRP1 positive tumor cells.

NRP1 CAR-T cells can effectively alleviate CCL 4-induced liver fibrosis:

A fibrosis model was constructed using C57 mice, and CCL4 (CCL 4: oil=1:3) was injected intraperitoneally at a dose of 5mL/kg with an injection frequency of twice weekly for eight consecutive weeks with successful molding. The CAR-T cells were infused back into the tail vein eight weeks after molding at a dose of 5X 10 ⁶/mouse, and after one week mice were euthanized and liver tissue removed for detection.

Mice from the Sham (control; non-model mice) group and mice from the reinfusion NRP1 CAR-T cell group were found to express significantly lower levels of NRP1 molecules than mice from the reinfusion T-Vec group and the fibrosis-associated molecules α -sma, collagen-1, fibrinectin were expressed at reduced levels compared to the T-Vec group (FIG. 6A). At the same time, h.e. staining and masson staining showed a reduced degree of liver fibrosis in mice reinfused NRP1 CAR-T cell group (fig. 6b, c).

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

The scFv-1 heavy chain CDR1 sequence is:

NYWMN(SEQ ID NO.1)

the scFv-1 heavy chain CDR2 sequence is:

NIYPDTGNTNYDEKFNS(SEQ ID NO.2)

the scFv-1 heavy chain CDR3 sequence is:

SEFFFYGSDWFFDV(SEQ ID NO.3)

The scFv-1 light chain CDR1 sequence is:

RSSQNIIHVNGNTFLE(SEQ ID NO.4)

the scFv-1 light chain CDR2 sequence is:

KVSNRFS(SEQ ID NO.5)

the scFv-1 light chain CDR3 sequence is:

FQGSYVPPT(SEQ ID NO.6)

The scFv-1 heavy chain sequence is:

QVQLEQSGSELVRPGASVRLSCKASGYPFTNYWMNWVKQSHGQGLEWI GNIYPDTGNTNYDEKFNSKATLTVDTSSSTAYIHLSSLTSEDSAVYYCTRSEFFF YGSDWFFDVWGAGTTVTVSS(SEQ ID NO.7)

The scFv-1 light chain sequence is:

DIVITQTPLSLPVSLGDQASISCRSSQNIIHVNGNTFLEWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSYVPPTFGG GTKLEIK(SEQ ID NO.8)

the scFv-2 heavy chain CDR1 sequence is:

SGYSWH(SEQ ID NO.9)

The scFv-2 heavy chain CDR2 sequence is:

HIHYSGYTDYNPSLKS(SEQ ID NO.10)

the scFv-2 heavy chain CDR3 sequence is:

WGQGTTLTVSS(SEQ ID NO.11)

the scFv-2 light chain CDR1 sequence is:

RASESVDNYGISFMN(SEQ ID NO.12)

the scFv-2 light chain CDR2 sequence is:

TASNQGS(SEQ ID NO.13)

the scFv-2 light chain CDR3 sequence is:

QQDKEVPWT(SEQ ID NO.14)

The scFv-2 heavy chain sequence is:

EVQLEESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQSPGNKLEWMG HIHYSGYTDYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCASRRGYDE DYWGQGTTLTVSS(SEQ ID NO.15)

the scFv-2 light chain sequence is:

DIVITQTTASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLL IYTASNQGSGVPARFSGSGSGTDFSLNIHPMEADDTAMYFCQQDKEVPWTFG GGTKLEIK(SEQ ID NO.16)

the scFv-3 heavy chain CDR1 sequence is:

RYWMN(SEQ ID NO.17)

the scFv-3 heavy chain CDR2 sequence is:

EINPSNGRTDYNEKLKN(SEQ ID NO.18)

the scFv-3 heavy chain CDR3 sequence is:

GNWDFYGMDY(SEQ ID NO.19)

the scFv-3 light chain CDR1 sequence is:

TASSSVSSSYLA(SEQ ID NO.20)

The scFv-3 light chain CDR2 sequence is:

SASNLAS(SEQ ID NO.21)

the scFv-3 light chain CDR3 sequence is:

HQFHRST(SEQ ID NO.22)

The scFv-3 heavy chain sequence is:

QVQLEQSGAELAKPGASVKLSCKASGYTFTRYWMNWVKQRPGQGLEW IGEINPSNGRTDYNEKLKNKATLTVDKSSSTAYMQLSRLTSEDSAVYYCARGN WDFYGMDYWGQGTSVIVSS(SEQ ID NO.23)

the scFv-3 light chain sequence is:

DIVITQSTAIMSASLGERVTMTCTASSSVSSSYLAWFQQKPGSSPKIWIYSA SNLASGVPARLSGSGSGTSYSLTINSMEAEDAATYYCHQFHRSTFGAGTKLEL K(SEQ ID NO.24)

the scFv-4 heavy chain CDR1 sequence is:

SYWMH(SEQ ID NO.25)

the scFv-4 heavy chain CDR2 sequence is:

DIYPGGDNILYNEKFKT(SEQ ID NO.26)

the scFv-4 heavy chain CDR3 sequence is:

GPPYDRYDYWYFDV(SEQ ID NO.27)

the scFv-4 light chain CDR1 sequence is:

KSSQSLLNSRNQKNYLA(SEQ ID NO.28)

the scFv-4 light chain CDR2 sequence is:

FASTRDS(SEQ ID NO.29)

the scFv-4 light chain CDR3 sequence is:

QQHYRTPLT(SEQ ID NO.30)

the scFv-4 heavy chain sequence is:

QVQLQESGAELVKPGTSVKMSCKTSGYTFTSYWMHWVKQRPGRGLEWI GDIYPGGDNILYNEKFKTRATLSVDTSSSTAYMQLSSLTSEDSAVYFCARGPPY DRYDYWYFDVWGAGTTVTVSS(SEQ ID NO.31)

the scFv-4 light chain sequence is:

DIVMTQSPSSLTMSVGQKVTMNCKSSQSLLNSRNQKNYLAWYQQKPGQ SPKLLVYFASTRDSGVPDRFRGSGSGTNFTLTITSVQVEDLADYFCQQHYRTPL TFGTGTKLELK(SEQ ID NO.32)

the G4S linker sequence is:

GGGGSGGGGSGGGGS(SEQ ID NO.33)

The CD8 signal peptide (SIGNAL PEPTIDE, SP) sequence is:

MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.34)

The CD8 transmembrane region sequence is:

TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA GICVALLLSLIITLICYHRSR(SEQ ID NO.35)

the sequence of the intracellular region of the 4-1BB is as follows:

KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL(SEQ ID NO.36)

The CD3 zeta chain sequence is:

FSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQR RRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTY DALHMQTLAPR(SEQ ID NO.37)

The extracellular domain (NRP 1 ECD) sequence is:

MERGLPLLCATLALALALAGAFRSDKCGGTIKIENPGYLTSPGYPHSYHPSEKCEWLIQAPEPYQRIMINFNPHFDLEDRDCKYDYVEVIDGENEGGRLWGKFCGKIAPSPVVSSGPFLFIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTAPTGVIKSPGFPEKYPNSLECTYIINRP1KMSEIILEFESFDLEQDSNPPGGMFCRYDRLEI WDGFPEVGPHIGRYCGQKTPGRIRSSSGVLSMVFYTDSAIAKEGFSANYSVLQSSISEDFKCMEALGMESGEIHSDQITASSQYGTNWSVERSRLNYPENGWTPGEDSYKEWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYRVDISSNGEDWISLKEGNKAIIFQGNTNPTDVVLGVFSKPLITRFVRIKPVSWETGISMRFEVYGCKITDYPCSGMLGMVSGLISDSQITASNQADRNWMPENIRLVTSRTGWALPPSPHPYTNEWLQVDLGDEKIVRGVIIQGGKHRENKVFMRKFKIAYSNNGSDWKTIMDDSKRKAKSFEGNNNYDTPELRTFSPLSTRFIRIYPERATHSGLGLRMELLGCEVEAPTAGPTTPNGNPVDECDDDQANCHSGTGDDFQLTGGTTVLATEKPTIIDSTIQSEFPTYGFNCEFGWGSHKTFCHWEHDSHAQLRWSVLTSKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQSSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVWMVVGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLGGIAVDDISINNHISQEDCAKPTDLDKKN(SEQ ID NO.38)

The above embodiments are provided to illustrate 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 the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.

Claims

1. A single-chain antibody targeting NRP1, which is characterized in that the single-chain antibody comprises Complementarity Determining Region (CDR) sequences of SEQ ID NO.1-6.

2. The single chain antibody of claim 1, wherein the single chain antibody targeting NRP1, the complementarity determining region comprises a heavy chain complementarity determining region and a light chain complementarity determining region;

Wherein the heavy chain complementarity determining regions are:

CDR1 of the amino acid sequence shown as SEQ ID No.1, CDR2 of the amino acid sequence shown as SEQ ID No.2, and CDR3 of the amino acid sequence shown as SEQ ID No. 3;

the light chain complementarity determining regions are:

CDR1 of the amino acid sequence shown as SEQ ID No.4, CDR2 of the amino acid sequence shown as SEQ ID No.5, and CDR3 of the amino acid sequence shown as SEQ ID No. 6.

3. The single chain antibody of claim 2, wherein the single chain antibody targeting NRP1 comprises the heavy chain antibody V _H amino acid sequence and the light chain antibody V _L amino acid sequence of SEQ ID nos. 7-8.

4. The single chain antibody of claim 3, wherein the single chain antibody heavy chain V _H is directly linked to the single chain antibody light chain V _L or linked via a linker, and wherein the linker is (G ₄ S) n, wherein n is a positive integer.

5. The single chain antibody of claim 4, wherein n is 3.

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

7. The NRP 1-targeting chimeric antigen receptor of claim 6, said NRP 1-targeting chimeric antigen receptor being made up of a signal peptide, an antigen binding domain, a transmembrane region, a costimulatory signaling domain, and a signaling domain in tandem;

wherein the signal peptide is CD8 signal peptide, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 34;

The transmembrane region is a CD8 transmembrane region, and the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 35;

the costimulatory signaling domain is a 4-1BB costimulatory signaling domain, and the amino acid sequence of the costimulatory signaling domain is shown as SEQ ID NO. 36;

The signal transduction domain is a CD3 zeta signal transduction domain, and the amino acid sequence of the signal transduction domain is shown as SEQ ID NO. 37.

8. A nucleic acid molecule encoding the NRP 1-targeting single chain antibody of any one of claims 1-5 or the NRP 1-targeting chimeric antigen receptor of claim 6 or 7.

9. A recombinant expression vector comprising the nucleic acid molecule of claim 8.

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

11. A medicament for the treatment of a disease mediated by NRP1 overexpression, characterized in that it comprises any one or more of the single chain antibody of any one of claims 1-5, the chimeric antigen receptor of claim 6 or 7, the nucleic acid molecule of claim 8, the recombinant expression vector of claim 9 and the CAR-T cell of claim 10;

The NRP1 high-expression mediated disease is liver cancer and liver fibrosis.

12. The medicament of claim 11, further comprising a pharmaceutically acceptable carrier.

13. An assay product comprising any one or more of the single chain antibody of any one of claims 1-5, the chimeric antigen receptor of claim 6 or 7, the nucleic acid molecule of claim 8, the recombinant expression vector of claim 9, the CAR-T cell of claim 10, and the drug of any one of claims 11-12.

14. The test product according to claim 13, wherein the test product is a test kit or a test device, and wherein the test product is used for qualitatively or quantitatively detecting NRP1 expression.