Anti-LRG1 antibody and application thereof

Abstract

The invention relates to an anti-LRG1 antibody and application thereof. The invention provides an anti-LRG1 antibody or an antigen binding fragment thereof. The anti-LRG1 antibody or the antigen binding fragment thereof has a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3. The invention also provides a preparation method of the anti-LRG1 antibody or the antigen binding fragment thereof and application of the anti-LRG1 antibody or the antigen binding fragment thereof in inhibiting macrophage activation and treating and / or preventing diseases related to macrophage activation.

Description

Technical Field

[0001] The present invention relates to an anti-LRG1 antibody, and more particularly to an anti-LRG1 antibody or an antigen-binding fragment thereof and use thereof in inhibiting macrophage activation and treating and / or preventing diseases associated with macrophage activation. Background Art

[0002] LRG1 is a glycoprotein secreted by the liver and is also a highly conserved member of the leucine-rich repeat (LRR) family of proteins. It is mainly involved in cell adhesion, migration, and signal transduction. It is a newly discovered angiogenic factor in recent years. Wang et al. found that [1], compared with normal mice, LRG1 expression was significantly increased in the retinal neovascularization model. LRG1 activated the TGF-β1 signal transduction pathway by binding to its co-receptor ENG, shifting the TGF-β signal from maintaining vascular stability to a pathological angiogenic state, activating the angiogenic signal TβR-Ⅱ, and then recruiting endothelial ALK-1, initiating the phosphorylation of transcription factors Smad1 / 5 / 8, promoting VEGF transcription upregulation and expression, and then promoting endothelial cell migration and proliferation, and playing its role in promoting pathological angiogenesis. Therefore, LRG1 promotes angiogenesis by regulating the TGFβ signal transduction pathway. Relevant studies have confirmed that increased LRG1 is a predictor of arterial stiffness, endothelial function, and peripheral arterial disease in patients with type 2 diabetes. In addition, increased LRG1 is significantly associated with atherosclerosis and is an independent predictor of cardiovascular disease. In addition, previous studies have found that LRG1-rich extracellular vesicles secreted by lipotoxic renal tubular epithelial cells can activate macrophages. However, whether LRG1 promotes the progression of atherosclerosis by activating macrophages has not been studied yet. Summary of the invention

[0003] The present application aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the main purpose of the present invention is to provide an anti-LRG1 antibody or an antigen-binding fragment thereof and a preparation method thereof, wherein the anti-LRG1 antibody or an antigen-binding fragment thereof can inhibit macrophage activation and related inflammatory responses.

[0004] The present invention also aims to provide the use of the anti-LRG1 antibody or the antigen-binding fragment thereof for inhibiting macrophage activation.

[0005] In addition, the present invention also aims to provide the use of the anti-LRG1 antibody or the antigen-binding fragment thereof in treating and / or preventing diseases related to macrophage activation.

[0006] In one aspect, the present invention provides an anti-LRG1 antibody or an antigen-binding fragment thereof, wherein the anti-LRG1 antibody or an antigen-binding fragment thereof has a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3, and wherein,

[0007] VLCDR1 comprises the amino acid sequence at positions 27-37 of SEQ ID NO.1;

[0008] VLCDR2 comprises the amino acid sequence at positions 55-57 of SEQ ID NO.1;

[0009] VLCDR3 comprises the amino acid sequence at positions 94-102 of SEQ ID NO.1;

[0010] VHCDR1 comprises the amino acid sequence at positions 26-33 of SEQ ID NO.2;

[0011] VHCDR2 comprises the amino acid sequence at positions 51 to 58 of SEQ ID NO.2; and

[0012] VHCDR3 comprises the amino acid sequence at positions 97 to 117 in SEQ ID NO.2.

[0013] In another aspect, the present invention provides a polynucleotide encoding the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention.

[0014] In still another aspect, the present invention provides a recombinant expression vector comprising the polynucleotide according to the present invention.

[0015] In yet another aspect, the present invention provides a recombinant cell comprising the polynucleotide according to the present invention or the recombinant expression vector according to the present invention.

[0016] In another aspect, the present invention provides a method for preparing the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention, the method comprising culturing the recombinant cell according to the present invention, and recovering the antibody or antigen-binding fragment thereof.

[0017] In yet another aspect, the present invention provides a pharmaceutical composition comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier.

[0018] In yet another aspect, the present invention provides a kit comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention.

[0019] In another aspect, the present invention provides use of the anti-LRG1 antibody or antigen-binding fragment thereof, polynucleotide, recombinant expression vector, recombinant cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for inhibiting macrophage activation.

[0020] In another aspect, the present invention provides use of the anti-LRG1 antibody or antigen-binding fragment thereof, polynucleotide, recombinant expression vector, recombinant cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for treating and / or preventing LRG1-related diseases.

[0021] In the present invention, by using the anti-LRG1 antibody or its antigen-binding fragment according to the present invention, the activation of macrophages can be significantly inhibited, especially the differentiation of macrophages into the pro-inflammatory M1 phenotype, thereby providing a new method for inhibiting macrophage activation and inhibiting inflammatory response, and providing new ideas for the development of drugs for diseases related to macrophage activation, such as coronary heart disease and atherosclerosis, so that more patients can benefit early. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a flow cytometry analysis graph and a bar graph showing that the LRG1 antibody according to one embodiment of the present invention intervenes in macrophages and their differentiation into the M1 phenotype.

[0023] Figure 2 The figure is a flow cytometry graph and a bar graph showing the results of the LRG1 antibody according to one embodiment of the present invention affecting the proportion of the M2 phenotype in an in vitro macrophage assay.

[0024] Figure 3 The photographs show the results of the LRG1 antibody according to one embodiment of the present invention inhibiting the progression of atherosclerosis in a mouse atherosclerosis model. DETAILED DESCRIPTION

[0025] Hereinafter, the embodiments of the present invention will be described in more detail to help understand the present invention. It should be understood that the description of these embodiments is for illustrative purposes only and is not intended to limit the scope of protection claimed by the present invention in any way.

[0026] As used in the present invention, an antibody refers to any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that can bind to a specific antigen. A complete antibody is usually composed of two pairs of polypeptide chains, each pair comprising a light (L) chain and a heavy (H) chain, and the light chain can be divided into kappa (κ) type or lambda (λ) type, and antibodies can be defined as IgM, IgD, IgG, IgA and IgE according to the type of heavy chain, μ, δ, γ, α and ε. The heavy chain contains a heavy chain constant region (CH) and a heavy chain variable region (VH). The CH of different types of heavy chains contains 3 to 4 different domains (CH1, CH2, CH3 and CH4). The light chain contains a light chain constant region (CL) and a light chain variable region (VL). Each of VH and VL contains a highly variable complementary determining region (CDR) and a framework region (FR) interspersed therebetween, and each VH and VL contains FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence from the amino terminus to the carboxyl terminus. Antigen-binding fragments refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, specifically, including but not limited to Fab (composed of VL, VH, CL and CH1), Fab' (Fab containing a portion of the hinge region), F(ab')2 (two Fabs connected by a disulfide bridge in the hinge region), Fv (composed of VL and VH), scFv (single-chain Fv), etc.

[0027] As used in the present invention, "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, specifically including but not limited to at least one of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a sc-Fv and a diabody.

[0028] As used herein, "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH domain and the VL domain of an antibody, wherein these domains are present in the form of a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which enables the scFv to form a desired structure for antigen binding.

[0029] As used in the present invention, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of identity (%) can then be obtained.

[0030] The inventors found through research that LRG1 is significantly elevated in people with coronary heart disease and is significantly correlated with inflammatory markers in the patient's plasma. The inventors further found that LRG1 is significantly co-localized with macrophages and macrophage-related inflammatory factors such as TNFα and VCAM-1, and after in-depth research, they found that LRG1 interferes with the differentiation of macrophages into a pro-inflammatory M1 phenotype. Based on the above findings, the inventors prepared and screened a series of LRG1 antibodies, and found that the LRG1 antibodies of the present invention can significantly inhibit the differentiation of macrophages, and have great potential in developing new drugs for inhibiting macrophage activation and related diseases.

[0031] Therefore, in one aspect, the present invention provides an anti-LRG1 antibody or an antigen-binding fragment thereof, wherein the anti-LRG1 antibody or an antigen-binding fragment thereof has a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3, and wherein,

[0032] VLCDR1 comprises the amino acid sequence at positions 27-37 of SEQ ID NO.1: QSLLHSNGYNY;

[0033] VLCDR2 comprises the amino acid sequence at positions 55-57 of SEQ ID NO.1: LGS;

[0034] VLCDR3 comprises the amino acid sequence at positions 94-102 of SEQ ID NO.1: MQALQTPWT;

[0035] VHCDR1 comprises the amino acid sequence at positions 26-33 of SEQ ID NO. 2: GFTFSSYA;

[0036] VHCDR2 comprises the amino acid sequence at positions 51 to 58 of SEQ ID NO. 2: ISSNGGST; and

[0037] VHCDR3 comprises the amino acid sequence at positions 97-117 in SEQ ID NO.2: VKGPHLGYSSGWYSPFYGMDV.

[0038] In some specific embodiments, the anti-LRG1 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region having an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO.1, and a heavy chain variable region having an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO.2. In a preferred embodiment, the anti-LRG1 antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region having a sequence of SEQ ID NO.1 and a heavy chain variable region having a sequence of SEQ ID NO.2.

[0039] In some embodiments, the neutralizing antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, scFv, diabody, single-chain antibody, fusion antibody and intact antibody.

[0040] In another aspect, the present invention provides a polynucleotide encoding the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention.

[0041] In some embodiments, the polynucleotides of the present invention comprise:

[0042] A nucleotide sequence encoding the VLCDR1 comprising the sequence at positions 79 to 111 in SEQ ID NO.3;

[0043] A nucleotide sequence encoding the VLCDR2 comprising the sequence at positions 163-171 in SEQ ID NO.3;

[0044] A nucleotide sequence encoding the VLCDR3 comprising the sequence at positions 280-306 in SEQ ID NO.3;

[0045] A nucleotide sequence encoding the VHCDR1 comprising the sequence at positions 76-99 in SEQ ID NO.4;

[0046] A nucleotide sequence encoding the VHCDR2 comprising the sequence at positions 151-174 in SEQ ID NO.4; and

[0047] A nucleotide sequence encoding the VHCDR3 comprising the sequence at positions 289-351 in SEQ ID NO.4.

[0048] In some specific embodiments, the sequence of the polynucleotide of the present invention has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the above-mentioned polynucleotide sequence.

[0049] In some embodiments, the sequence of the polynucleotide of the present invention comprises:

[0050] A nucleotide sequence encoding the VLCDR1, which has a sequence identity of more than 90% with the sequence at positions 79 to 111 in SEQ ID NO.3;

[0051] A nucleotide sequence encoding the VLCDR2, which has a sequence identity of more than 90% with the sequence at positions 163-171 in SEQ ID NO.3;

[0052] A nucleotide sequence encoding the VLCDR3, which has a sequence identity of more than 90% with the sequence at positions 280-306 in SEQ ID NO.3;

[0053] A nucleotide sequence encoding the VHCDR1, which has a sequence identity of more than 90% with the sequence at positions 76 to 99 in SEQ ID NO.4;

[0054] A nucleotide sequence encoding the VHCDR2, which has a sequence identity of more than 90% with the sequence at positions 151 to 174 in SEQ ID NO.4; and

[0055] The nucleotide sequence encoding the VHCDR3 has a sequence identity of more than 90% with the sequence at positions 289-351 in SEQ ID NO.4.

[0056] In some specific embodiments, the polynucleotide of the present invention comprises a nucleotide sequence encoding the light chain variable region having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO.3, and a nucleotide sequence encoding the heavy chain variable region having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO.4. In a preferred embodiment, the polynucleotide of the present invention comprises a nucleotide sequence encoding the light chain variable region having a sequence of SEQ ID NO.3 and a nucleotide sequence encoding the heavy chain variable region having a sequence of SEQ ID NO.4.

[0057] In still another aspect, the present invention provides a recombinant expression vector comprising the polynucleotide according to the present invention.

[0058] As used in the present invention, "expression vector" refers to a nucleic acid delivery vehicle that can express the protein encoded by the inserted polynucleotide after being introduced into a host cell by transformation, transduction, transfection, etc. Expression vectors include, but are not limited to, plasmids, bacteriophages, lentiviruses, adenoviruses, adeno-associated viruses, etc. It is understood that the vector may also contain elements that control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, and reporter genes.

[0059] In yet another aspect, the present invention provides a recombinant cell comprising the polynucleotide according to the present invention or the recombinant expression vector according to the present invention.

[0060] In another aspect, the present invention provides a method for preparing the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention, the method comprising culturing the recombinant cell according to the present invention, and recovering the antibody or antigen-binding fragment thereof.

[0061] In yet another aspect, the present invention provides a pharmaceutical composition comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier.

[0062] In yet another aspect, the present invention provides a kit comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to the present invention.

[0063] In another aspect, the present invention provides use of the anti-LRG1 antibody or antigen-binding fragment thereof, polynucleotide, recombinant expression vector, recombinant cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for inhibiting macrophage activation.

[0064] In another aspect, the present invention provides use of the anti-LRG1 antibody or antigen-binding fragment thereof, polynucleotide, recombinant expression vector, recombinant cell, or pharmaceutical composition according to the present invention in the preparation of a medicament for treating and / or preventing LRG1-related diseases.

[0065] Hereinafter, the present invention will be described in more detail by examples. However, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and variations without departing from the scope and spirit of the present invention, and the modifications and variations also fall within the scope of the present invention.

[0066] Example

[0067] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0068] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0069] Example 1. Preparation and screening of anti-LRG1 antibodies

[0070] LRG1 protein purification

[0071] Some materials and reagents are described as follows:

[0072] Experimental materials: Mouse and other species LRG1 gene sequences and LRG1 amino acid sequences were obtained from the NCBI database; human embryonic kidney cell line (expi293) was a gift from Zheng Sanduo's laboratory. pCAGGS-mouse LRG1-ECD-HisAvi plasmid was constructed by the laboratory.

[0073] Reagents: plasmid extraction kit (Vazyme); nickel column Ni-NTAAgarose (QIAGEN); restriction endonucleases DraIII and BspEI (Thermo); 2YT agar, 2YT medium; culture medium: SMM 293-TII (Sino Biological); Opti-MEM (Gibco).

[0074] method:

[0075] 1. Primer design and synthesis: Primers were designed using the mouse LRG1 sequence as a template. The vector contained His Avi, which was located at the C-terminus of the fragment, as shown in Table 1. The primers used in the experiment were commissioned to be synthesized by Ruibo Co., Ltd.

[0076] Table 1. LRG1 primers

[0077] Primer name Primer sequence (5'-3') His-LRG1 upstream CCTGCACGGCGTGCGCGGCCTCAAGGAATGCCTGATA Downstream of His-LRG1 GCCTCCGGACAGGGACCCCAGCTCTGC

[0078] 2. Construction of pCAGGS-mouse LRG1-ECD-HisAvi eukaryotic expression vector

[0079] The eukaryotic expression vector was used as a template, and the target gene was obtained, identified and purified by agarose gel. The purified product and PCAGGS vector were digested by DraIII and BspEI, and the target gene was subcloned into PCAGGS, clones were picked, and sequenced.

[0080] 3. Cell Culture and Transfection

[0081] Expi293 cells were cultured in a 37°C, 5% CO2 incubator containing SMM 293-TII. The recombinant plasmid and PEI were mixed with Opti-MEM and then left to stand for 5 minutes. The PEI mixture was then added to the plasmid mixture and mixed. After standing for 20 minutes, it was added to the expi293 cells. After 5 days of culture, the cell supernatant was collected.

[0082] The cell fluid overexpressing LRG1-His protein was centrifuged to obtain the supernatant, Ni Beads was added to the supernatant and shaken at room temperature for 15 minutes before passing through a column, impurities were washed away with ten times the column volume of PBS, low concentration of imidazole was added to wash away non-specifically bound impurities, and then eluted with high concentration of imidazole, the eluted protein was ultrafiltered with an ultrafiltration tube, and the eluate was replaced with PBS to obtain purified LRG1-His protein.

[0083] Antibody preparation and screening

[0084] Some reagent descriptions are as follows:

[0085] M-280Dynabeads (Thermo Fisher, Waltham, MA)

[0086] Phage input: 1*10 13

[0087] mLRG1-His6-Av Bio antigen dosage: 10ug

[0088] Triethanolamine: Beijing Yili Fine Chemicals Co., Ltd.

[0089] In order to recover high-affinity antibodies from magnetic beads and increase the diversity of recovered phage-scfvs, phage-scfvs from the human non-immune antibody library were incubated twice with M-280 magnetic beads. The library after two subtractions was subjected to two rounds of panning with mLRG1 antigen. phage-scfvs specifically bind to mLRG1. The selected anti-mLRG1 antibodies were eluted with alkaline triethanolamine solution. The eluted phage-scfvs were transfected into TG1 Escherichia coli and plated. About 480 monoclonal clones were randomly selected to produce phage-scfvs in the bacterial culture supernatant.

[0090] The specific binding of these clones to mLRG1-Bio was detected by ELISA. 96 clones with higher positive values ​​were selected for antibody gene sequencing. The variable heavy chain (VH) and light chain (VL) gene sequences were obtained. After analysis, a total of 25 clones with unique sequences were identified. The antibody was constructed as a full-length mIgG1 format and expressed using expi-293F. The binding to mLRG1-Bio was then analyzed again by ELISA. Finally, the top 15 clones were screened based on their affinity, and the clone with the highest affinity was selected and purified to obtain the antibody E4C12.

[0091] Example 2. LRG1 neutralizing antibody inhibits macrophage M1 differentiation

[0092] In this example, LRG1-induced intervention in macrophages and polarization induction of M1 phenotype and M2 phenotype were measured, and the effect of the LRG1 antibody E4C12 of the present invention on inhibiting macrophage M1 differentiation was evaluated.

[0093] 1. Bone Marrow-derived Macrophages (BMDM) Culture

[0094] 1) 6-12 week old C57BL / 6c mice were killed by cervical dislocation and sprayed with 70% medical alcohol for 3-5 minutes;

[0095] 2) Pour 5 ml of D-Hanks solution into a 35 mm diameter culture dish and place the dish on an ice pack to cool;

[0096] 3) Take out the sterilized mice and place them in a culture dish;

[0097] 4) Open the sterile microsurgical instrument package, take out the ophthalmic scissors and tweezers, adjust the mouse to a supine position, cut the skin along the line of the mouse's hind paw at the hip joint of the mouse's thigh, and use tweezers to peel off and remove subcutaneous fat, muscle, nerve and other tissues in sequence to fully expose the femur and tibia and fibula. Use scissors to carefully shorten the connection between nerves, muscles and bones, and separate the femur and tibia and fibula at the hip joint and ankle joint respectively. Be careful not to expose the bone marrow cavity here;

[0098] 5) Take an ice box, soak the removed femur and tibia and fibula in a 15 ml centrifuge tube containing 70% medical alcohol, and place it on the ice box;

[0099] 6) Wash the alcohol off the surface of the femur and tibia and fibula with ice PBS, insert a 10mL syringe connected to a 1mL syringe needle into the bone marrow cavity, and quickly inject the previously drawn ice PBS solution into the bone marrow cavity to flush out the bone marrow. Repeat three times to ensure that the bone marrow tissue of the mouse hind limb is fully obtained;

[0100] 7) Filter the removed bone marrow tissue with a 40 μm filter, and grind the remaining large pieces of bone marrow repeatedly to obtain sufficient bone marrow cells. Use a 10 ml pipette to repeatedly blow the PBS solution containing the bone marrow tissue to mix it evenly.

[0101] 8) Transfer the PBS liquid containing bone marrow cells to a 15 mL centrifuge tube using a pipette, centrifuge at 4°C, 900 g for 4 min, discard the supernatant, add 6-10 mL of red blood cell lysis buffer, resuspend, let stand at room temperature for 3-5 min, and centrifuge at 4°C, 900 g for 4 min.

[0102] 9) Take out the centrifuged cells, discard the supernatant, and resuspend and culture them in DMEM medium containing 10-20% L929 cell culture medium supernatant, 10% FBS, and 1% penicillin / streptomycin antibiotics.

[0103] 2. Stimulation Induction

[0104] The above cultured macrophages were cultured until the 6th day and then divided into plates. The cells were plated at 5*10 5 Up to 1*10 6 The cells were inoculated at a density of 100 μg / mL in a 6-well plate and incubated overnight at 37°C. LPS (1 μg / mL) + IFNγ (50 ng / mL); IL4 (10 μg / mL); LRG1 (100 μg / mL); LRG1 (100 μg / mL) + E4C12 (200 μg / mL) were added to the cells. After 24 hours of stimulation intervention of the above groups on macrophages, the cells were further divided into the following groups: M0 group, LR1 group, M1 group (LPS + IFNγ), M2 (IL-4) group, and the cells were photographed and kept for later use.

[0105] 3. Phenotypic Identification

[0106] The expression of macrophage markers F4 / 80, M1 macrophage marker CD80, and M2 macrophage marker CD206 was detected by flow cytometry (for specific steps, see Section 4 below, with the antibody replaced with CD16 / 32-FITC) to confirm that mature M1 and M2 bone marrow-derived macrophages were obtained and to identify the induced phenotype of M0 macrophages by LRG1.

[0107] 4. Flow cytometry to detect the expression of cell markers F4 / 80 and CD80

[0108] 1) Take out the cells after overnight intervention from the incubator, remove the conditioned medium in the culture dish, wash with PBS, add trypsin to digest for 10 minutes, and use DMEM solution prepared with 100mL / L FBS to terminate the digestion. Use a pipette to transfer the suspended cells to a 15mL centrifuge tube and centrifuge at 500g for 4 minutes.

[0109] 2) After centrifugation, discard the supernatant of the cells, add 1 mL of MACS solution to resuspend and wash 1-2 times, then centrifuge at 4°C, 500 g for 4 min, discard the supernatant, add 100 μL of FC block solution (0.5% FBS + 2 Mm EDTA + PBS) to each sample and block on ice for 5 min.

[0110] 3) Place each sample in a 1.5 mL EP tube, add F4 / 80-APC and CD80-PE antibodies at a concentration of 1:400, dilute the antibodies with FC-block, set up single positive tubes, F4 / 80-APC, CD80-PE, and incubate at 4°C for 30 min-1 h.

[0111] 4) Antibody washing: Centrifuge, discard the supernatant, resuspend the antibody in 100-200 μL MACS buffer (0.5% FBS + 2Mm EDTA + PBS) for 1-2 times, resuspend in 100 μL MACS buffer, add DAPI (1:1000) for nucleus staining. If storing, resuspend in PFA.

[0112] 5) The cells were transferred to a flow cytometry tube and the expression of cell surface markers was detected by flow cytometry.

[0113] 5. Detection of CD206 Expression on Macrophages by Flow Cytometry

[0114] 1) Take out the cells after overnight intervention from the incubator, remove the conditioned medium in the culture dish, wash with PBS, add trypsin to digest for 10 minutes, and use DMEM solution prepared with 100mL / L FBS to terminate the digestion. Use a pipette to transfer the suspended cells to a 15mL centrifuge tube and centrifuge at 500g for 4 minutes.

[0115] 2) After centrifugation, discard the supernatant of the cells, add 1 mL of MACS solution to resuspend and wash 1-2 times, then centrifuge at 4°C, 500 g for 4 min, discard the supernatant, add 100 μL of FC block solution (0.5% FBS + 2 Mm EDTA + PBS) to each sample and block on ice for 5 min.

[0116] 3) The cells were divided into 4 groups: blank control group; F4 / 80-APC group, CD206-BV421 group, and F4 / 80+CD206-BV421 group. Each group had 5 replicates and placed in 1.5 mL EP tubes. F4 / 80-APC antibody was added to the F4 / 80-APC group and F4 / 80+CD206-BV421 group at a concentration of 1:400. The antibody was diluted with FC-block and incubated at 4°C for 30 min-1 h.

[0117] 4) Antibody washing: centrifuge, discard the supernatant, resuspend the antibody in 100-200 μL MACS buffer (0.5% FBS + 2Mm EDTA + PBS) for 1-2 times, add fix / perm (invitrogen eBioscience TM foxp3 / Transcription) were fixed at room temperature for 1 hour, and then washed 1-2 times with Permeabilization buffer. Antibody was stained with buffer dilution (1:400) for 30 minutes to 1 hour, and then centrifuged. After washing 1-2 times with buffer, the cells were resuspended in 300 μL MACS and loaded onto the machine.

[0118] Figure 1 It is a flow cytometry analysis graph and a proportional bar graph showing that the LRG1 antibody according to one embodiment of the present invention intervenes in macrophages and their differentiation into the M1 phenotype. Figure 2 It is a bar graph showing the flow analysis profile and ratio results of the LRG1 antibody according to one embodiment of the present invention affecting the ratio of inflammatory M2 macrophages in an in vitro macrophage assay. It can be seen that LRG1 interferes with the differentiation of macrophages into the pro-inflammatory M1 phenotype, and the LRG1 antibody of the present invention significantly inhibits the differentiation of macrophages into the pro-inflammatory M1 phenotype.

[0119] Example 3. LRG1 neutralizing antibody inhibits atherosclerotic plaque formation in a mouse atherosclerosis model

[0120] ApoE purchased from Saiye - / - Two mice (one female and one male) were mated and multiplied by themselves. The male ApoE - / - Ten mice were fed a high-fat diet starting at 6-8 weeks old for 16 weeks, and were given an intraperitoneal injection of E4C12 antibody. Based on the titer of the antibody, we gave the mice a therapeutic injection dose of 25 mg / kg, once a week, for 16 consecutive weeks. Figure 3 The photographs show the results of the LRG1 antibody according to one embodiment of the present invention inhibiting the progression of atherosclerosis in a mouse atherosclerosis model. - / - Compared with mice, intraperitoneal injection of E4C12 antibody significantly inhibited the progression of atherosclerosis.

[0121] References

[0122] 1.Wang X,Abraham S,McKenzie JAG,Jeffs N,Swire M,Tripathi VB,LuhmannUFO,Lange CAK,Zhai Z,Arthur HM,Bainbridge J,Moss SE,Greenwood J. TGF-βsignalling.Nature.2013Jul18;499(7458):306-11.

[0123] Sequence Listing

[0124] Anti-LRG1 antibody E4C12 light chain variable region sequence (SEQ ID NO.1):

[0125] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK

[0126] Anti-LRG1 antibody E4C12 heavy chain variable region sequence (SEQ ID NO.2):

[0127] EVQLVQSGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVKGPHLGYSSGWYSPFYGMDVWGQGTTVTVSS

[0128] Nucleotide sequence encoding the light chain variable region of anti-LRG1 antibody E4C12 (SEQ ID NO.3):

[0129] GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0130] Nucleotide sequence encoding the heavy chain variable region of the anti-LRG1 antibody E4C12 (SEQ ID NO.4):

[0131] GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGCTATTAGTAGTAATGGGGGTAGCACATACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAAAGGACCCCACCTCGGGTATAGCAGTGGCTGGTATTCGCCATTTTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Claims

1. An anti-LRG1 antibody or an antigen-binding fragment thereof, wherein: The anti-LRG1 antibody or antigen-binding fragment thereof has a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3, and wherein, VLCDR1 comprises the amino acid sequence at positions 27-37 of SEQ ID NO.1; VLCDR2 comprises the amino acid sequence at positions 55-57 of SEQ ID NO.1; VLCDR3 comprises the amino acid sequence at positions 94-102 of SEQ ID NO.1; VHCDR1 comprises the amino acid sequence at positions 26-33 of SEQ ID NO.2; VHCDR2 comprises the amino acid sequence at positions 51 to 58 of SEQ ID NO.2; and VHCDR3 comprises the amino acid sequence at positions 97 to 117 in SEQ ID NO.

2.

2. The anti-LRG1 antibody or antigen-binding fragment thereof according to claim 1, wherein: The anti-LRG1 antibody or antigen-binding fragment thereof comprises a light chain variable region with a sequence of SEQ ID NO.1 and a heavy chain variable region with a sequence of SEQ ID NO.

2.

3. The anti-LRG1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The anti-LRG1 or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, diabody, single-chain antibody, fusion antibody and complete antibody. 4 . A polynucleotide encoding the anti-LRG1 antibody or antigen-binding fragment thereof according to claim 1 .

5. The polynucleotide according to claim 4, wherein The polynucleotide comprises: A nucleotide sequence encoding the VLCDR1 comprising the sequence at positions 79 to 111 in SEQ ID NO.3; A nucleotide sequence encoding the VLCDR2 comprising the sequence at positions 163-171 in SEQ ID NO.3; A nucleotide sequence encoding the VLCDR3 comprising the sequence at positions 280-306 in SEQ ID NO.3; A nucleotide sequence encoding the VHCDR1 comprising the sequence at positions 76-99 in SEQ ID NO.4; A nucleotide sequence encoding the VHCDR2 comprising the sequence at positions 151-174 in SEQ ID NO.4; and A nucleotide sequence encoding the VHCDR3 comprising the sequence at positions 289-351 in SEQ ID NO.

4.

6. The polynucleotide according to claim 4 or 5, wherein The sequence of the polynucleotide comprises a nucleotide sequence of SEQ ID NO.3 encoding the light chain variable region and a nucleotide sequence of SEQ ID NO.4 encoding the heavy chain variable region.

7. A recombinant expression vector comprising the polynucleotide according to any one of claims 4 to 6. 8 . A recombinant cell comprising the polynucleotide according to claim 4 , or the recombinant expression vector according to claim 7 .

9. A method for preparing the anti-LRG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the method comprising culturing the recombinant cell according to claim 8, and recovering the anti-LRG1 antibody or antigen-binding fragment thereof. 10 . A pharmaceutical composition comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. 11 . A kit comprising the anti-LRG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

12. Use of the anti-LRG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 6, the expression vector according to claim 7, the recombinant cell according to claim 8, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for inhibiting macrophage activation.

13. Use of the anti-LRG1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the polynucleotide according to any one of claims 4 to 6, the expression vector according to claim 7, the recombinant cell according to claim 8, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating and / or preventing a disease associated with LRG1, wherein: The LRG1-related disease is selected from atherosclerosis, coronary heart disease, ischemic stroke and peripheral arterial atherosclerotic disease.

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