A method for purifying an anti-lilrb4 monoclonal antibody

Abstract

The present application relates to the field of biological medicine, and specifically provides a kind of purification method of anti-LILRB4 monoclonal antibody, the method includes cell clarification, affinity chromatography, cation exchange chromatography and anion exchange chromatography.The present application provides a purification method specially suitable for anti-LILRB4 monoclonal antibody, the anti-LILRB4 monoclonal antibody screened can effectively inhibit the binding of LILRB4 antigen and its ligand complex, so as to block its interaction with ligand ApoE complex, then prevent the activation of downstream NF-κB signal pathway and the release of ARG1, prevent the inhibition of T cell proliferation and the promotion of tissue infiltration, can be used for treating cancer, and the method effectively improves the purification efficiency and recovery rate of anti-LILRB4 monoclonal antibody.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a purification method of anti-LILRB4 monoclonal antibody. BACKGROUND

[0002] Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by the proliferation of abnormal myeloblasts, a type of white blood cell, in the bone marrow, which is clinically manifested as anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. Most cases are critically ill and have a poor prognosis, and if not treated in time, it can often endanger life. As a common hematological malignancy, AML accounts for about 70% of all acute leukemias and is one of the top ten high-incidence malignancies in China. Despite progress in treatment, less than 30% of AML patients are still alive within five years of first diagnosis. CMML is a cancer that begins in the bone marrow and invades the blood. This condition is rare, with about 1,100 cases in the United States each year.

[0003] Leukocyte immunoglobulin-like receptor subfamily B protein (LILRBs) is a group of type I transmembrane glycoproteins, which are considered to be immune checkpoint proteins for myeloid cells due to their immune suppression function. LILRB4 mainly changes the immune-suppressive microenvironment, thereby inhibiting T cell activity, leading to the occurrence of immune escape and adverse consequences such as tumor cell proliferation and metastasis. Through in vitro cell experiments and humanized mouse model experiments, researchers found that mononuclear acute myeloid leukemia cells with positive LILRB4 expression can significantly inhibit T cell proliferation. Therefore, the development of antibodies against LILRB4 can effectively alleviate T cell inhibition, thereby effectively killing tumor cells.

[0004] Currently, in the process of developing anti-LILRB4 monoclonal antibodies, many problems are often encountered, such as the difficulty in separating the degradation products and the physicochemical properties of the target protein, and the instability of the process samples, which requires high purification of the process. The classical antibody drug preparation process mainly includes two stages of antibody capture and fine purification, generally using a three-step chromatography method. After capturing the cell culture supernatant by affinity chromatography, the collected sample components are still complex, containing multiple impurities such as polymers, degradation products, host DNA and host cell proteins, which generally need to be further removed by anion exchange chromatography, cation exchange chromatography and hydrophobic chromatography to obtain products with quality meeting the requirements. However, after each step of chromatography, the buffer needs to be changed or adjusted to adapt to the purification conditions of the next step, which not only wastes time, but also affects the yield, increases the cost, and may face the risk of introducing new impurities. Therefore, there is an urgent need to develop a purification method specifically applicable to the anti-LILRB4 monoclonal antibody provided by the present application. SUMMARY

[0005] In order to solve the above problems existing in the existing purification method, the application discloses a purification method of anti-LILRB4 monoclonal antibody.

[0006] The specific technical scheme of the application is as follows:

[0007] The application provides a purification method of anti-LILRB4 monoclonal antibody, which comprises the following steps:

[0008] S1, cell clarification: the cell liquid containing the anti-LILRB4 monoclonal antibody is clarified by centrifugation or deep filtration;

[0009] S2, affinity chromatography: the cell liquid is preliminarily purified and concentrated by using an affinity chromatography column, a protein solution is collected, and the protein solution is subjected to virus inactivation in an acid solution;

[0010] S3, cation exchange chromatography: the protein solution is refined by a cation exchange chromatography column, and an eluate is collected;

[0011] S4, anion exchange chromatography: the eluate in step S3 is purified by using an anion exchange chromatography column, and the protein solution after purification is collected;

[0012] The anti-LILRB4 monoclonal antibody comprises a heavy chain complementarity determining region HCDR1 with an amino acid sequence as shown in SEQ ID No: 1, a heavy chain complementarity determining region HCDR2 with an amino acid sequence as shown in SEQ ID No: 2, a heavy chain complementarity determining region HCDR3 with an amino acid sequence as shown in SEQ ID No: 3, a light chain complementarity determining region LCDR1 with an amino acid sequence as shown in SEQ ID No: 4, a light chain complementarity determining region LCDR2 with an amino acid sequence as shown in SEQ ID No: 5, and a light chain complementarity determining region LCDR3 with an amino acid sequence as shown in SEQ ID No: 6.

[0013] The beneficial effects of the present application are as follows: the present application provides a purification method specially suitable for anti-LILRB4 monoclonal antibody, firstly, the screened anti-LILRB4 monoclonal antibody can effectively inhibit the binding of LILRB4 antigen and its ligand complex, thereby blocking the interaction with the ligand ApoE complex, and further preventing the activation of the downstream NF-kappa B signaling pathway and the release of ARG1, preventing the inhibition of T cell proliferation and promoting tissue infiltration; in addition, the screened anti-LILRB4 monoclonal antibody of the present application can be used for treating cancer, including but not limited to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), blastic plasmacytoid dendritic cell neoplasm (BPDCN), breast cancer, lung cancer or prostate cancer; secondly, the present application screens the conditions and chromatography fillers required for the three-step purification method through a large number of experiments, effectively improves the purification efficiency and recovery rate of the anti-LILRB4 monoclonal antibody, and the elution conditions of the cation exchange chromatography and the flow-through conditions of the anion exchange chromatography are basically the same, so the sample collected by the cation exchange chromatography only needs to be fine-tuned for pH and conductivity value, and can directly enter the anion exchange chromatography, without the need for sample treatment or ultrafiltration liquid change in the middle, not only greatly saving the purification time, but also avoiding the introduction of impurities in the purification process, improving the purity of the purified protein, and having high practicability. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the plasmid map of the pScFv-Disb-HS vector in the embodiment 2 of the present application;

[0015] Figure 2 It is the comparison chart of the relative affinity of the gradient dilution ELISA anti-LILRB4 phage monoclonal antibody in the embodiment 3 of the present application;

[0016] Figure 3 It is the map of the vector pTSE in the embodiment 5 of the present application;

[0017] Figure 4 It is the denatured polyacrylamide gel electrophoresis chart of the murine antibody molecule in the embodiment 5 of the present application;

[0018] Figure 5 It is the comparison chart of the binding ability of the murine antibody and LILRB4 in the embodiment 6 of the present application;

[0019] Figure 6 It is the comparison chart of the binding ability of the murine antibody and the surface LILRB4 of the human monocytic leukemia cell (THP-1) in the embodiment 8 of the present application;

[0020] Figure 7 It is the comparison chart of the experiment of the murine antibody and ApoE competing to bind LILRB4 in the embodiment 9 of the present application;

[0021] Figure 8 Figure for comparison of experiments of the mouse-derived antibody inhibiting ARG1 secretion by THP-1 in Example 10 of the present application;

[0022] Figure 9 Figure for denaturing polyacrylamide gel electrophoresis of the chimeric antibody molecule in Example 12 of the present application;

[0023] Figure 10 Figure for denaturing polyacrylamide gel electrophoresis of the humanized antibody molecule in Example 15 of the present application;

[0024] Figure 11 Figure for experiments of binding of the humanized antibody to LILRB4 in Example 16 of the present application;

[0025] Figure 12 Figure for comparison of experiments of binding of the humanized antibody to LILRB4 on the surface of THP-1 cells in Example 17 of the present application;

[0026] Figure 13 Figure for comparison of experiments of competition of the humanized antibody with ApoE for binding to LILRB4 in Example 18 of the present application;

[0027] Figure 14 Figure for comparison of experiments of the humanized antibody inhibiting ARG1 secretion by THP-1 in Example 19 of the present application;

[0028] Figure 15 Figure for comparison of experiments of biological activity detection (reporter gene) of the humanized antibody molecule in Example 20 of the present application. DETAILED DESCRIPTION

[0029] The present application will be further described in detail below in combination with the following examples.

[0030] Example 1

[0031] The present application discloses a method for purifying an anti-LILRB4 monoclonal antibody, which comprises the following steps:

[0032] S1, cell clarification: the cell liquid containing the anti-LILRB4 monoclonal antibody is clarified by centrifugation or deep filtration;

[0033] S2, affinity chromatography: the cell liquid is preliminarily purified and concentrated by using an affinity chromatography column, the protein solution is collected, and the protein solution is inactivated by viruses in an acid solution;

[0034] S3, cation exchange chromatography: the protein solution is refined by a cation exchange chromatography column, and the eluate is collected;

[0035] S4, anion exchange chromatography; purifying the eluate in step S3 using an anion exchange chromatography column, and collecting the purified protein solution;

[0036] The anti-LILRB4 monoclonal antibody comprises: a heavy chain complementarity determining region HCDR1 having an amino acid sequence as shown in SEQ ID No: 1, a heavy chain complementarity determining region HCDR2 having an amino acid sequence as shown in SEQ ID No: 2, a heavy chain complementarity determining region HCDR3 having an amino acid sequence as shown in SEQ ID No: 3, a light chain complementarity determining region LCDR1 having an amino acid sequence as shown in SEQ ID No: 4, a light chain complementarity determining region LCDR2 having an amino acid sequence as shown in SEQ ID No: 5, and a light chain complementarity determining region LCDR3 having an amino acid sequence as shown in SEQ ID No: 6.

[0037] Heavy chain complementarity determining region HCDR1 Heavy chain complementarity determining region HCDR2 Heavy chain complementarity determining region HCDR3 Light chain complementarity determining region LCDR1 Light chain complementarity determining region LCDR2 Light chain complementarity determining region LCDR3 SYTMS TISSGGTYTYYPDSVKG DGYDGFDY RSSQSLAHSNGNTYLH KVSNRFS SQSTLVPT SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6

[0038] Example 2: Screening of mouse-derived antibody molecules

[0039] The present application immunizes mice with LILRB4 antigen (LILRB4 protein extracellular segment, LILRB4 antigen and protein used in subsequent experiments are LILRB4 extracellular segment), optimizes the immunization method, and creates a phage display library. The construction, screening and identification of the specific phage display library are as follows:

[0040] Step one: immunizing mice with LILRB4 antigen

[0041] 1. Experimental animals: species and strain: BALB / c, female, mice; body weight: 18-20 g;

[0042] The experimental animal provider is Yikang (Beijing) Pharmaceutical Technology Co., Ltd.

[0043] 2. Immunization: immunize mice with human LILRB4 as the immunogen (synthetic gene by Nanjing Kingsriver Biological Technology Co., Ltd., vector construction and expression and purification by the company).

[0044] Step two: Construction of phage antibody library: Take the mouse spleen cells with high titer, use Trizol reagent (purchased from Ambion, item number: 15596026) to extract total RNA from mouse spleen cells, obtain cDNA by RT-PCR, use degenerate primers (the used degenerate primers are described in the reference: Journal of Immunological Methods 233 (2000) 167-177) for PCR amplification, thereby obtaining the immune mouse antibody heavy chain variable region (VH) gene library and light chain variable region (VL) gene library, double enzyme digestion of light and heavy chains respectively, and then connect to the same step-by-step enzyme treatment vector to construct pScFv-Disb-HS-VH-VL gene library. The pScFv-Disb-HS vector is modified by a series of gene cloning methods on the pComb3 vector (purchased from China Plasmid Vector Strain Cell Strain Gene Preservation Center) to be used for the construction and expression of phage single-chain antibody library. The modified vector is named pScFv-Disb-HS vector, and its plasmid map is shown in Figure 1 , and the mouse immune phage antibody library is constructed based on this vector.

[0045] Step three: Coat the immunotube with LILRB4 as the antigen, the antigen coating amount is 5 μg / 500 μL / tube, coat overnight at 4°C, then use 4% skim milk / PBST to block the immunotube and the immune phage antibody library respectively, block at room temperature for 1 hour. After blocking, the immune phage antibody library is added to the immunotube for antigen-antibody binding, the phage input amount is about 10 9 ~10 12 After reaction at room temperature for 1 hour, the unbound phage is washed away with PBST-PBS, eluted by 0.1M pH 2.2 Glycine-HCl, and finally the eluted phage antibody solution is neutralized to about pH 7.0 using 1.5M pH 8.8 Tris-HCl.

[0046] Step four: Infect 10 mL of TG1 bacteria liquid grown to the logarithmic phase with the above neutralized phage, and let it stand in a 37°C incubator for 30 minutes. Take part of the bacterial liquid for gradient dilution and spread on 2YTAG plates for calculating the phage output. The remaining bacterial liquid is centrifuged to discard the supernatant, and the bacterial pellet is resuspended in a small amount of culture medium, then aspirated and spread on a 2YTAG large plate for the next round of screening.

[0047] Step five: Scrape the bacteria from the large plate after the above infection and spread, inoculate into 2YTAG liquid medium, shake to the logarithmic phase, then add M13KO7 helper phage superinfection, culture at 28°C, 220 rpm overnight to prepare phage, and PEG / NaCl sedimentation is used to purify the phage for the next round of screening.

[0048] Step six: screening of LILRB4 phage single-chain antibody positive clones: after one round of screening, well-separated single colonies were picked and inoculated into 2YTAG liquid medium in 96-well deep plates, and cultured at 37°C and 220 rpm until the logarithmic growth phase, about 10 10 Helper phage M13KO7 was added, and the mixture was incubated at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were resuspended and precipitated in 2YTAK. The mixture was cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was collected for ELISA identification. Finally, two LILRB4 mouse antibody candidates with high affinity were obtained, named MA-I and MB-I, respectively. The above monoclonal antibodies were sequenced to determine the correct antibody sequence. After sequencing, the sequences of the two monoclonal antibodies screened above were as follows:

[0049] Murine antibody molecule Heavy chain variable region sequence Light chain variable region sequence MA-I SEQ ID No: 7 SEQ ID No: 8 MB-I SEQ ID No: 9 SEQ ID No: 10

[0050] Specifically, SEQ ID No: 7 (amino acid sequence of the heavy chain variable region of MA-I):

[0051] EVQLQQSGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDGYDGFDYWGQGTTLTVSS;

[0052] SEQ ID No: 8 (amino acid sequence of the light chain variable region of MA-I):

[0053] DIVMTQTTLSLPVSPGDQASISCRSSQSLAHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTLVPTFGGGTKLEIK;

[0054] SEQ ID No: 9 (amino acid sequence of the heavy chain variable region of MB-I):

[0055] QVQLQESGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWGQLGLREGYYAVDYWGQGTSVTVSS;

[0056] SEQ ID No: 10 (amino acid sequence of the light chain variable region of MB-I):

[0057] DIVMTQSPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK.

[0058] Example 3 Gradient dilution ELISA to compare the affinity of anti-LILRB4 phage monoclonal antibodies

[0059] The two murine antibody molecules (MA-I and MB-I) obtained in Example 2 were subjected to monoclonal phage display and purification, and then subjected to phage gradient dilution ELISA to identify the affinity, the specific method is as follows: LILRB4 antigen was coated with pH 9.6 carbonate buffer, 100 ng / well / 100 μL, coated overnight at 4°C, washed three times with PBST, the two phage monoclonal antibodies screened in Example 2 were diluted four times with PBST, 100 μL of diluted sample was added to each well, and incubated at room temperature for 1 hour. Wash the ELISA plate with PBST, add HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with PBST to the ELISA plate, and incubate at room temperature for 1 hour. Color development with TMB color development kit, color development at room temperature for 10 minutes, stop with 2M H2SO4, and read at 450nm / 630nm with a microplate reader, and calculate the corresponding half maximal effective concentration (EC50) value, the specific data are as follows:

[0060] Clone MA-I MB-I EC50 1.635 1.936

[0061] From the above data and as shown in Figure 2 , the two different murine antibody candidate molecules screened in Example 2 can bind to LILRB4.

[0062] Example 4

[0063] The mouse antibody molecule of the embodiment 4 is further limited based on the embodiment 2, and further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region is selected from one of the constant regions of mouse IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region is a mouse C k The amino acid sequence of the constant region of the IgG1 is shown as SEQ ID No: 11, the amino acid sequence of the constant region of the IgG2a is shown as SEQ ID No: 12, the amino acid sequence of the constant region of the IgG2b is shown as SEQ ID No: 13, and the amino acid sequence of the constant region of the IgG3 is shown as SEQ ID No: 14, and the specific sequences are as follows.

[0064] SEQ ID No: 11 (amino acid sequence of the heavy chain constant region of mouse IgG1):

[0065] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0066] SEQ ID No: 12 (amino acid sequence of the heavy chain constant region of mouse IgG2a):

[0067] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0068] SEQ ID No: 13 (amino acid sequence of heavy chain constant region of mouse IgG2b type):

[0069] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0070] SEQ ID No: 14 (amino acid sequence of heavy chain constant region of mouse IgG3 type):

[0071] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA;

[0072] SEQ ID No: 15 (murine C k type light chain constant region amino acid sequence):

[0073] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0074] Example 5 Preparation of anti-LILRB4 murine antibody molecules

[0075] Example 5 of the present application is based on the preferred definition of the murine antibody molecules comprising murine IgG1 type heavy chain constant region (the amino acid sequence of which is shown in SEQ ID No: 11) and murine C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 15). The antibody preparation method is as follows:

[0076] 1. The genes encoding the VH and VL of the two monoclonal antibodies screened in Example 2 are respectively cloned into the vector pTSE (as shown in Figure 3 , which is loaded with heavy and light chain constant region genes, preferably the heavy chain constant region is murine IgG1 type heavy chain constant region (the amino acid sequence of which is shown in SEQ ID No: 11), and the light chain constant region is murine C k type light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 15), and the structure of the pTSE vector is as follows:Figure 3 The pTSE vector preparation process is shown (see CN103525868A specification page 3

[0019] paragraph).

[0077] 2, transient transfection of HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number GNHu43), antibody expression, 4 monoclonal antibodies were obtained by protein A affinity column purification using AKTA instrument, and protein concentration was determined using BCA kit (purchased from Beijing Huitian Dongfang Technology Co., Ltd., item number: BCA0020), and then the protein size was identified by SDS-PAGE, and the results are shown as Figure 4 From left to right, non-reducing MA-I, reducing MA-I, non-reducing MB-I, reducing MB-I mouse anti-LILRB4 monoclonal antibody and protein molecular weight marker, the molecular weight of each band is consistent with the theory.

[0078] Example 6 Binding experiment of mouse antibody with LILRB4

[0079] Coat LILRB4 with pH 9.6 carbonate buffer, 100 ng / well / 100 μL, coat overnight at 4°C. Wash five times with 300 μL / well PBST, then add 1% BSA-PBST and block at 37°C for 1 hour, add different dilution concentrations of MA-I, MB-I mouse antibodies, the starting highest concentration of the two antibodies is 50 μg / mL, respectively, 5-fold gradient dilution, each antibody is diluted for 12 gradients, incubate at 37°C for 1 hour. Wash five times with 300 μL / well PBST, then add Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, incubate at 37°C for 1 hour. TMB color developing kit, 100 μL / well, color development at room temperature for 8 minutes, then stop color development with 2M H2SO4. Read at 450nm / 630nm with a microplate reader, and calculate the corresponding EC50 value, the specific data are as follows:

[0080] Clone MA-I MB-I EC50 (ng / mL) 8.347 12.80

[0081] From the above data and as shown in Figure 5 , the two different mouse antibodies MA-I and MB-I selected can bind to LILRB4.

[0082] Example 7 Binding experiment of mouse antibody with LILR family proteins

[0083] LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5 were coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, overnight at 4 °C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST to block for 1 hour at 37 °C, followed by the addition of 100 μL MA-I and MB-I mouse antibody molecules, both at a concentration of 50 μg / mL. Incubate for 1 hour at 37 °C. Washed five times with 300 μL / well PBST, then added Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, incubate for 1 hour at 37 °C. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 8 minutes, then stop color development with 2M H2SO4. Readings were taken on a microplate reader at 450 nm / 630 nm, and the specific data are as follows:

[0084]

[0085] From the above data, it can be concluded that the two different mouse antibodies MA-I and MB-I selected can specifically bind to LILRB4 and do not bind to other proteins in the LILR family.

[0086] Example 8 Binding experiment of mouse antibodies to LILRB4 on the surface of human monocytic leukemia cells (THP-1)

[0087] Take 50 μL of different dilution concentrations of MA-I and MB-I mouse antibodies, with an initial working concentration of 30 μg / mL, 3-fold gradient dilution, a total of 10 gradients, added to 96-well V-bottom plates. Then add 50 μL of THP-1 cell suspension to the wells, at a concentration of 2 x 10 6 cells / mL, mix well. Incubate at 4 °C for 1 hour. Then add 100 μL PBS buffer per well, centrifuge at 3000 rpm for 5 minutes and discard the supernatant. Add 100 μL / well fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG (purchased from Beijing Zhongshanjingqiao Biotechnology Co., Ltd., item number: ZF-0312) (1:100 dilution). Mix well, then incubate at 4 °C in the dark for 30 minutes. Add 100 μL PBS buffer per well, centrifuge at 3000 rpm for 5 minutes and discard the supernatant. Resuspend the cells by adding 100 μL PBS buffer, and detect on a flow cytometer. Collect the data and calculate the corresponding EC50 values, and the specific data are as follows:

[0088] Clone MA-I MB-I EC50 (ng / mL) 305.9 351.0

[0089] As shown by the above data and as shown in Figure 6, the two different mouse antibodies MA-I and MB-I screened can both bind to LILRB4 on the surface of THP-1 cells. Figure 6

[0090] Example 9 Experiment of Mouse Antibodies Competing with ApoE for Binding to LILRB4

[0091] THP-1 cells were taken, with a concentration of 2 x 10 6 cells / mL, and were plated in a V-bottom 96-well plate, with 50 μL of cell suspension added to each well. Then 50 μL of MA-I and MB-I mouse antibodies at different dilution concentrations were added to the wells, with a starting working concentration of 400 μg / mL, 5-fold gradient dilution, for a total of 10 gradients. In addition, 100 μL of 0.4 μg / mL FITC-labeled ApoE protein was added to the wells. Incubation was performed at 4°C in the dark for 1.5 hours. Then 200 μL of PBS buffer was added to each well, and centrifugation was performed at 3000 rpm for 5 minutes, with the supernatant discarded. 100 μL of PBS buffer was again added to resuspend the cells, and flow cytometry was performed. Data were collected and the corresponding IC50 values were calculated, with the specific data as follows:

[0092] Clone MA-I MB-I IC50 (μg / mL) 0.2037 0.4399

[0093] As shown by the above data and as shown in Figure 6, the two different mouse antibodies MA-I and MB-I screened can both bind to LILRB4 on the surface of THP-1 cells. Figure 7

[0094] Example 10 Inhibition of ARG1 Secretion by THP-1 by Mouse Antibodies

[0095] THP-1 cells were taken, with a concentration of 1 x 10 7 ​​Cells / mL were seeded into 96-well plates with a V-bottom bottom, and 50 μL of cell suspension was added to each well. First, 50 μL each of different concentrations of MA-I, MB-I mouse antibodies, and ApoE were mixed thoroughly at a 1:1 ratio. The initial working concentration of mouse antibody was 400 μg / mL, serially diluted 2-fold for a total of 8 gradients, with a final working concentration of ApoE of 0.5 μg / mL. Then, 50 μL of the mouse antibody and ApoE mixture was added to each well. After incubation at 37°C for 20 hours, the plates were centrifuged at 3000 rpm for 5 minutes. 40 μL of supernatant was added to each well of the 96-well plate. The reaction substrate was then prepared and preheated according to the arginase activity assay kit (purchased from Sigma-Aldrich, catalog number: MAK112-1KT). 10 μL of the reaction substrate was added to each well and mixed thoroughly. The plate was incubated at 37°C for 1 hour. Finally, 200 μL of stop solution was added to each well, and the absorbance was read at 450 nm using a multi-plate reader. Collect data and calculate the corresponding IC50 values. The specific data is as follows:

[0096] Clone MA-I MB-I IC50 (μg / mL) 23.86 45.97

[0097] Based on the above data and as follows Figure 8 As shown, the two different murine antibodies MA-Ⅰ and MB-Ⅰ screened out can both inhibit the secretion of ARG1 by THP-1 cells.

[0098] Example 11

[0099] Example 11 of this invention further specifies that the monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule. The chimeric antibody molecule includes a heavy chain variable region of a murine antibody molecule, a light chain variable region of a murine antibody molecule, and a human antibody constant region. The humanized antibody constant region includes a humanized antibody heavy chain constant region and a humanized antibody light chain constant region. The humanized antibody heavy chain constant region is selected from one of the constant regions of human IgG1, IgG2, or IgG4 types. The humanized antibody light chain constant region has an amino acid sequence as shown in SEQ ID No:19, representing human C... k The constant region of the heavy chain of the IgG1 type is shown in SEQ ID No:16, the amino acid sequence of the heavy chain constant region of the IgG2 type is shown in SEQ ID No:17, and the amino acid sequence of the heavy chain constant region of the IgG4 type is shown in SEQ ID No:18.

[0100] SEQ ID No:16 (Amino acid sequence of the heavy chain constant region of human IgG1):

[0101] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0102] SEQ ID No: 17 (Amino acid sequence of the heavy chain constant region of human IgG2):

[0103] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0104] SEQ ID No: 18 (Amino acid sequence of the heavy chain constant region of human IgG4):

[0105] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0106] SEQ ID No: 19 (amino acid sequence of light chain constant region of human Ck):

[0107] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0108] Preparation of chimeric antibody molecules

[0109] The embodiment 12 of the present application further limits the humanized antibody constant region to include a heavy chain constant region of human IgG1 type (the amino acid sequence of which is shown as SEQ ID No: 16) and a light chain constant region of human Ck type (the amino acid sequence of which is shown as SEQ ID No: 19) based on the embodiment 11. k

[0110] Specific preparation method: the heavy chain variable region VH (SEQ ID No: 7) and light chain variable region VL gene (SEQ ID No: 8) of the antibody molecule MA-I and the heavy chain variable region VH (SEQ ID No: 9) and light chain variable region VL gene (SEQ ID No: 10) of MB-I obtained by screening the immunized phage antibody library in embodiment 2 are kept as murine sequences, and are respectively cloned into the vector pTSE (as shown in Figure 3 k ​​Chimeric antibodies CA-I and CB-I were obtained by transient transfection of HEK293 cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number: GNHu43) for antibody expression. The results of identification of chimeric antibody CA-I and CB-I proteins by SDS-PAGE are shown in Figure 6, from left to right: protein molecular weight marker, reduced CA-I, non-reduced CA-I, reduced CB-I and non-reduced CB-I anti-LILRB4 monoclonal antibody. The molecular weight of each band is consistent with the theory. Figure 9

[0111] Example 13 Humanization of murine antibody molecules MA-I and MB-I

[0112] First, the sequences of murine antibody molecules MA-I and MB-I in Example 2 were compared with the human antibody germline database (v-base) to find higher homologous human antibody light and heavy chain germlines as candidate sequences, and then the CDR sequences of murine antibody molecules MA-I and MB-I were transplanted onto the human candidate sequences for homology modeling. Then, by three-dimensional structure simulation calculation, the key framework amino acid residues that may play an important role in maintaining the CDR loop structure were designed for the back mutation of the humanized antibodies. The designed light and heavy chain variable regions of the humanized antibodies containing back mutations were synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and then connected to the transient expression vector. The light and heavy chains obtained by humanization were analyzed to obtain the following humanized antibody molecules: HA-I, HA-II, HA-III, HB-I, HB-II, HB-III. The sequences of the six monoclonal antibodies screened above are as follows:

[0113] Monoclonal antibody Heavy chain variable region Light chain variable region HA-I SEQ ID No: 20 SEQ ID No: 21 HA-II SEQ ID No: 22 SEQ ID No: 23 HA-III SEQ ID No: 24 SEQ ID No: 25 HB-I SEQ ID No: 26 SEQ ID No: 27 HB-II SEQ ID No: 26 SEQ ID No: 28 HB-III SEQ ID No: 26 SEQ ID No: 29

[0114] Specifically, SEQ ID No: 20 (amino acid sequence of the heavy chain variable region of HA-I):

[0115] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSTISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYDGFDYWGQGTTVTVSS;

[0116] SEQ ID No: 21 (amino acid sequence of the light chain variable region of HA-I):

[0117] ​DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPTFGGGTKVEIK;

[0118] SEQ ID No: 22 (amino acid sequence of the variable region of the heavy chain of HA-II):

[0119] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYDGFDYWGQGTTVTVSS;

[0120] SEQ ID No: 23 (amino acid sequence of the variable region of the light chain of HA-II):

[0121] DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPTFGGGTKVEIK;

[0122] SEQ ID No: 24 (amino acid sequence of the variable region of the heavy chain of HA-III):

[0123] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDGYDGFDYWGQGTTVTVSS;

[0124] SEQ ID No: 25 (amino acid sequence of the variable region of the light chain of HA-III):

[0125] DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPTFGGGTK VEIK;

[0126] SEQ ID No: 26 (amino acid sequence of the variable region of the heavy chain of HB-I, HB-II, HB-III):

[0127] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWIG EINPSNGRTNYNEKFKTRATLTVDTSTSTAYMELSSLRSEDTAVYYCARWGQLGL REGYYAVDYWGQGTLVTVSS;

[0128] SEQ ID No:27 (amino acid sequence of the variable region of the light chain of HB-I):

[0129] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0130] SEQ ID No:28 (amino acid sequence of the variable region of the light chain of HB-II):

[0131] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0132] SEQ ID No:29 (amino acid sequence of the variable region of the light chain of HB-III):

[0133] DIVMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK.

[0134] Example 14

[0135] The embodiment 14 of the present application further limits the constant region of the humanized antibody to include the heavy chain constant region of the humanized antibody and the light chain constant region of the humanized antibody, the heavy chain constant region of the humanized antibody is selected from one of the constant regions of the IgG1 type, the IgG2 type or the IgG4 type of human, and the light chain constant region of the humanized antibody is the human C kThe amino acid sequence of the heavy chain constant region of IgG1 type is shown as SEQ ID No: 16, the amino acid sequence of the heavy chain constant region of IgG2 type is shown as SEQ ID No: 17, and the amino acid sequence of the heavy chain constant region of IgG4 type is shown as SEQ ID No: 18.

[0136] The specific sequence of the constant region of the humanized antibody is the same as that in Example 11.

[0137] Example 15 Preparation of humanized antibody molecules

[0138] In Example 15 of the present application, the constant region of the humanized antibody is further defined based on Example 14 to include a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 16) and a human C k light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 19).

[0139] The coding genes of the heavy chain VH and light chain VL of the 6 humanized antibody molecules obtained in Example 13 above were respectively cloned into the vector pTSE (as shown in Figure 3 SEQ ID No: 16) and a human C k light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 19).

[0140] The humanized antibody molecules HA-I, HA-II, HA-III, HB-I, HB-II, and HB-III were respectively transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the item number GNHu43), and the expression of the antibodies was carried out. The monoclonal antibodies were purified by protein A affinity column using AKTA instrument, and the protein concentration was determined using a BCA kit (purchased from Beijing Huitian Dongfang Science and Technology Co., Ltd., with the item number BCA0020). Then the protein size was identified by SDS-PAGE, and the results are shown in Figure 10 from left to right, non-reduced HA-I, reduced HA-I, non-reduced HA-II, reduced HA-II, non-reduced HA-III, reduced HA-III, protein molecular weight marker, non-reduced HB-I, non-reduced HB-I, non-reduced HB-II, non-reduced HB-II, non-reduced HB-III, and non-reduced HB-III anti-LILRB4 monoclonal antibodies. The molecular weight of each band is consistent with the theory.

[0141] Example 16 Binding experiment of humanized antibody with LILRB4

[0142] LILRB4 was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST to block for 1 hour at 37°C, added different dilution concentrations of humanized antibodies HA-I, HA-II, HA-III, HB-I, HB-II, HB-III and chimeric antibodies CA-I and CB-I prepared in Example 12, the starting highest concentration of the eight antibodies was 50 μg / mL, each antibody was diluted 12 gradients after 5-fold dilution, incubated for 1 hour at 37°C. Washed five times with 300 μL / well PBST, then added Goat Anti Human IgG-HRP (purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., item number: ZB-2304) diluted 1:5000 with 1% BSA-PBST, incubated for 1 hour at 37°C. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 5 minutes, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm on a microplate reader, and the corresponding EC50 values were calculated, and the specific data are as follows:

[0143] Clone HA-I HA-II HA-III HB-I HB-II HB-III CA-I CB-I EC50 (ng / mL) 17.37 15.36 14.01 342.8 917.8 74.32 17.83 65.46

[0144] As shown in the above data and experimental results, Figure 11 all six different humanized antibody molecules can bind to LILRB4. Among them, the affinities of the three humanized monoclonal antibodies HA-I, HA-II and HA-III are relatively close. Among the three humanized monoclonal antibodies HB-I, HB-II and HB-III, HB-III has the lowest EC50 value, indicating that it has the best binding ability and the highest affinity to LILRB4. At the same time, the EC50 values of HA-I, HA-II and HA-III are similar to those of chimeric antibody CA-I, and the EC50 value of HB-III is similar to that of chimeric antibody CB-I, indicating that humanized HA-I, HA-II, HA-III and HB-III retain the high affinity of mouse parent antibodies MA-I and MB-I to LILRB4.

[0145] Example 17 Binding experiment of humanized antibodies to LILRB4 on THP-1 cell surface

[0146] Take 50 μL of different dilution concentrations of HA-I, HA-II, HA-III, HB-III, CA-I and CB-I antibodies, the starting working concentration is 32 μg / mL, 2-fold gradient dilution, a total of 12 gradients, added to 96-well V-bottom plates. Then add 50 μL of THP-1 cell suspension to the wells, the concentration is 2×10 6Cells / mL, mix well. Incubate at 4°C for 1 hour. Then add 100 μL of PBS buffer to each well, centrifuge at 3000 rpm for 5 minutes and discard the supernatant. Add 100 μL / well of FITC-labeled goat anti-mouse IgG (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZF-0312) (1:100 dilution). Mix well and incubate at 4°C in the dark for 30 minutes. Add 100 μL of PBS buffer to each well, centrifuge at 3000 rpm for 5 minutes and discard the supernatant. Resuspend cells in 100 μL of PBS buffer and analyze by flow cytometry. Collect data and calculate the corresponding EC50 values. Specific data are as follows:

[0147] Clone HA-I HA-II HA-III HB-III CA-I CB-I EC50 (ng / mL) 181.5 178.7 185.7 385.9 200.3 445.3

[0148] Based on the above data and as follows Figure 12 As shown, all four humanized antibodies screened were able to bind to LILRB4 on the surface of THP-1 cells. Furthermore, the EC50 values ​​of these four humanized antibody molecules were similar to those of their corresponding chimeric antibodies, indicating that the humanized HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, and HB-Ⅲ retained the high affinity of the murine parental antibodies MA-Ⅰ and MB-Ⅰ for LILRB4.

[0149] Example 18: Humanized Antibody Competitively Binding to LILRB4 with ApoE

[0150] THP-1 cells were collected at a concentration of 2 × 10⁻⁶. 6 Cells were seeded at a density of 100 μL / mL in 96-well plates with a V-bottom, with 50 μL of cell suspension added to each well. Then, 50 μL of humanized antibodies at different dilutions (HA-I, HA-II, HA-III, and HB-III) were added to each well, starting at a working concentration of 400 μg / mL and serially diluted 5-fold (12 dilutions in total). Additionally, 100 μL of 0.4 μg / mL FITC-labeled ApoE protein was added to each well, and the cells were incubated at 4°C in the dark for 1.5 hours. Next, 200 μL of PBS buffer was added to each well, and the cells were centrifuged at 3000 rpm for 5 minutes, discarding the supernatant. The cells were resuspended in 100 μL of PBS buffer, and the cells were analyzed by flow cytometry. Data were collected, and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0151] Clone HA-I HA-II HA-III HB-III IC50 (μg / mL) 0.4797 2.752 2.559 0.4265

[0152] Based on the above data and as follows Figure 13 As shown, all four humanized antibodies screened were able to competitively bind to LILRB4 on the surface of THP-1 cells against ApoE. Furthermore, HA-Ⅰ and HB-Ⅲ exhibited the lowest IC50 values, indicating that they effectively inhibited the binding of ApoE to LILRB4.

[0153] Example 19: Humanized antibody inhibits THP-1 secretion of ARG1

[0154] THP-1 cells were collected at a concentration of 1×10⁻⁶. 7 Cells / mL were seeded into 96-well plates with a V-bottom bottom. 50 μL of cell suspension was added to each well. First, 50 μL each of different concentrations of humanized antibodies (HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, HB-Ⅲ) and ApoE were mixed thoroughly at a 1:1 ratio. The initial working concentration of the humanized antibody was 400 μg / mL, serially diluted 2-fold for a total of 8 dilutions. The working concentration of ApoE was 0.5 μg / mL. Then, 50 μL of the humanized antibody and ApoE mixture was added to each well. After incubation at 37°C for 20 hours, the plates were centrifuged at 3000 rpm for 5 minutes. 40 μL of supernatant was added to each well of the 96-well plate. The reaction substrate was then prepared and preheated according to the arginase activity assay kit (purchased from Sigma-Aldrich, catalog number: MAK112-1KT). 10 μL of the reaction substrate was added to each well and mixed thoroughly. The plates were incubated at 37°C for 1 hour. Add 200 μL of stop solution to each well and read the absorbance at 450 nm using a multi-plate reader. Collect the data and calculate the corresponding IC50 values. The specific data are as follows:

[0155] Clone HA-I HA-II HA-III HB-III IC50 (μg / mL) 31.20 249.5 98.54 30.57

[0156] Based on the above data and as follows Figure 14 As shown, all four different humanized antibodies screened inhibited ARG1 secretion from THP-1 cells. Furthermore, among the four humanized antibody molecules provided by this invention, HA-Ⅰ and HB-Ⅲ had the lowest IC50 values, indicating that they could effectively inhibit ARG1 secretion.

[0157] Example 20: Detection of the biological activity of humanized antibody molecules (reporter gene)

[0158] THP-1-NF-κB-Luc engineered cells were used at a concentration of 2×10⁻⁶. 6The cells were diluted to 1.5*10<5> cells / mL and added to a V-bottom 96-well plate, 50 μL of cell suspension per well. Then 100 μL of four humanized antibody molecules HA-I, HA-II, HA-III and HB-III at different dilution concentrations were added to the wells, and the initial working concentration of the antibodies was 200 μg / mL, 5-fold gradient dilution, a total of 8 gradients. After incubation at 37 DEG C for 2 hours, 20 μg / mL ApoE protein was added, 50 μL per well. The cell culture plate was gently mixed and incubated in a 37 DEG C incubator for 5 hours. Centrifugation at 1000 rpm for 5 minutes, after discarding the supernatant, 50 μL of Lysis Buffer (purchased from Promega, product number: E2661) was added to each well according to the instructions, and it was allowed to act at room temperature for 10 minutes for complete lysis. 10 μL was transferred to a 384-well plate per well, and an equal volume of fluorescent substrate (purchased from Promega, product number: E2610) was added to each well, and the reaction was carried out at room temperature for 5 minutes. The fluorescence value was read on an enzyme-labeled instrument, and the corresponding IC50 value was calculated, and the specific data are as follows:

[0159] Clone HA-I HA-II HA-III HB-III IC50 (μg / mL) 0.7557 2.678 3.437 0.8302

[0160] As shown by the above data and Figure 15 , the four humanized antibody molecules screened can block the binding of ApoE to LILRB4, thereby inhibiting the conduction of the downstream NF-κB signal pathway. In addition, the IC50 values of HA-I and HB-III among the four humanized antibody molecules provided by the present application are the lowest, indicating that they can effectively inhibit the conduction of the downstream signal pathway.

[0161] Example 21

[0162] The anti-LILRB4 monoclonal antibody of the present application is further limited in the purification method based on the embodiment 1, and the specific method is as follows: S1, cell clarification: the cell liquid containing the anti-LILRB4 monoclonal antibody is clarified by centrifugation or deep filtration, wherein the amino acid sequence of the anti-LILRB4 monoclonal antibody is the same as that of the embodiment 1;

[0163] S2, affinity chromatography: the cell liquid is preliminarily purified and concentrated by using an affinity chromatography column, the protein solution is collected, and the protein solution is inactivated in an acid solution;

[0164] S201, using a buffer B1 with a concentration of 30-60 mM to balance the affinity chromatography column, the filler of the affinity chromatography column is selected from MabSelect Sure LX, MabSelect PrismA, Eshmuno A or NMab Pro; the buffer B1 is selected from Tris-HCl buffer, HEPES buffer or phosphate buffer, the pH of the buffer B1 is 7-8, and the conductivity of the buffer B1 is 10-30 mS / cm;

[0165] S202, loading the cell liquid containing the anti-LILRB4 monoclonal antibody onto the affinity chromatography column at a flow rate of 50-300 cm / h, the loading capacity being 20-70 mg of protein per ml of packing, and the bed height of the affinity chromatography column being 18-22 cm;

[0166] S203, after the loading is completed, rebalancing is performed using buffer B1, and after the ultraviolet signal is stable, at least one intermediate washing elution is performed using buffer B2 with a concentration of 30-60 mM until the curve of the ultraviolet absorption value is reduced to be stable, then the flushing is stopped, the buffer B2 is selected from Tris-HCl buffer, HEPES buffer, phosphate buffer, citrate buffer or acetate buffer, and the pH of the buffer B2 is 5-6;

[0167] S204, eluting the affinity chromatography column using buffer B3 with a concentration of 100-500 mM, starting to collect the protein solution when the ultraviolet absorption value rises to 100 mAU, and ending the collection when the ultraviolet absorption value falls to 100 mAU, and reserving, the buffer B3 is selected from glycine-hydrochloride buffer, citrate buffer, acetate buffer or arginine salt buffer, and the pH of the buffer B3 is 3-4;

[0168] S205, adjusting the pH of the collected protein solution to 3.5-3.7 using an acid solution, and incubating at room temperature for 0.5-2 h under this pH condition, and finally adjusting the pH of the protein solution to 5-7 using 1 M Tris buffer, the acid solution being selected from citric acid, acetic acid or hydrochloric acid.

[0169] S3, cation exchange chromatography: purifying the protein solution through a cation exchange chromatography column, and collecting the eluate;

[0170] S301, balancing: balancing the cation exchange chromatography column using buffer B4 with a concentration of 10-50 mM, the packing of the cation exchange chromatography column being selected from Capto SP, Capto MMC, Eshmuno S or NanoGel50SP, the buffer B4 being selected from citric acid buffer, acetate buffer or phosphate buffer, the pH of the buffer B4 being 5.5-6.5, and the conductivity being 2-5 mS / cm;

[0171] S302, loading: loading the filtered protein solution in step S2 onto the cation exchange chromatography column at a flow rate of 100-200 cm / h, the loading capacity being 20-85 mg of protein per ml of packing, and the bed height of the cation exchange chromatography column being 18-22 cm;

[0172] S303, rebalancing: after the end of loading, rebalance again using the buffer B4, and stop flushing when the ultraviolet absorption value is stable;

[0173] S304, elution: using buffer B5 to elute the cation exchange chromatography column, when the ultraviolet absorption value rises to 100 mAU, start collecting the eluate, and when the ultraviolet absorption value drops to 100 mAU, end the collection; buffer B5 is citric acid buffer, acetate buffer or phosphate buffer containing 90-150 mM sodium chloride.

[0174] S4, anion exchange chromatography; using an anion exchange chromatography column to purify the eluate in step S3, and collecting the purified protein solution;

[0175] S401, using 7-10 CV of buffer B6 to balance the anion exchange chromatography column at a flow rate of 100-200 cm / h, and loading the eluate collected in step S3 at a flow rate of 100-200 cm / h, the filler of the anion exchange chromatography column is selected from Capto Q, Capto adhere, POROS XQ, Eshmuno Q or NanoGel50Q, buffer B6 is citric acid buffer, acetate buffer or phosphate buffer containing 100-300 mM sodium chloride, and the pH value of buffer B6 is 6.0-6.5;

[0176] S402, after the end of loading, flush again using buffer B6, and when the ultraviolet absorption value rises to 100 mAU, start collecting the flow-through, and when the ultraviolet absorption value drops to 200 mAU, end the collection.

[0177] Example 22

[0178] The embodiment 22 of the present application further limits a method for purifying an anti-LILRB4 monoclonal antibody based on the embodiment 1, and specifically comprises the following method: S1, cell clarification: using centrifugation or depth filtration to clarify the cell liquid containing the anti-LILRB4 monoclonal antibody, wherein the amino acid sequence of the anti-LILRB4 monoclonal antibody is the same as that in the embodiment 1;

[0179] S2, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate the cell liquid, collecting the protein solution, and inactivating viruses in the protein solution in an acid solution;

[0180] S201, equilibrate the affinity chromatography column with buffer B1 with a concentration of 50 mM, the filler of the affinity chromatography column is NMab Pro; buffer B1 is Tris-HCl buffer containing 150 mM sodium chloride, the pH of buffer B1 is 7.4, and the conductivity of buffer B1 is 18 mS / cm;

[0181] S202, load the cell liquid containing the anti-LILRB4 monoclonal antibody onto the affinity chromatography column at a flow rate of 150 cm / h, the loading capacity is 59 mg of protein per ml of filler, and the bed height of the affinity chromatography column is 20 cm;

[0182] S203, after the loading is completed, re-equilibrate with buffer B1, and when the ultraviolet signal is stable, perform at least one intermediate washing elution with buffer B2 with a concentration of 50 mM until the curve of ultraviolet absorption value decreases to be stable, then stop the washing, buffer B2 is acetate buffer, and the pH of buffer B2 is 5.5;

[0183] S204, elute the affinity chromatography column with buffer B3 with a concentration of 300 mM, start collecting the protein solution when the ultraviolet absorption value rises to 100 mAU, and end the collection when the ultraviolet absorption value decreases to 100 mAU, and reserve, buffer B3 is arginine salt buffer, and the pH of buffer B3 is 3.6; S205, adjust the pH of the collected protein solution to 3.5-3.7 by using an acid solution, and incubate at room temperature for 1.5 h under the pH condition, and finally adjust the pH of the protein solution to 5-7 by using 1M Tris buffer, and the acid solution is selected from citric acid.

[0184] S3, cation exchange chromatography: purify the protein solution by a cation exchange chromatography column, and collect the eluate;

[0185] S301, balancing: a cation exchange chromatography column is balanced using a buffer B4 with a concentration of 20 mM, the filler of the cation exchange chromatography column is NanoGel 50SP, the buffer B4 is a phosphate buffer, the pH value of the buffer B4 is 6.0, and the conductivity of the buffer B4 is 2.5 mS / cm; S302, sample loading: the protein solution filtered in the step S2 is loaded into the cation exchange chromatography column at a flow rate of 150 cm / h, the sample loading capacity is 68 mg of protein per milliliter of filler, and the bed height of the cation exchange chromatography column is 20 cm; S303, rebalancing: after the sample loading is completed, the buffer B4 is used again for rebalancing, and the washing is stopped after the ultraviolet absorption value is stable; S304, elution: the cation exchange chromatography column is eluted using a buffer B5, the eluate is collected when the ultraviolet absorption value rises to 100 mAU, and the collection is ended when the ultraviolet absorption value drops to 100 mAU; the buffer B5 is a phosphate buffer containing 110 mM of sodium chloride.

[0186] S4, anion exchange chromatography; the eluate in the step S3 is purified using an anion exchange chromatography column, and the protein solution after purification is collected;

[0187] S401, the anion exchange chromatography column is balanced using 8 CV of a buffer B6 at a flow rate of 150 cm / h, and the eluate collected in the step S3 is loaded into the anion exchange chromatography column at a flow rate of 150 cm / h, the filler of the anion exchange chromatography column is Capto adhere, the buffer B6 is a phosphate buffer containing 200 mM of sodium chloride, and the pH value of the buffer B6 is 6.2; S402, after the sample loading is completed, the buffer B6 is used again for washing, and the flow-through is collected when the ultraviolet absorption value rises to 100 mAU, and the collection is ended when the ultraviolet absorption value drops to 200 mAU.

[0188] Embodiment 23

[0189] The anti-LILRB4 monoclonal antibody purification method in the embodiment 23 is further limited that the acid solution in the inactivation treatment process in the step S2 is preferably acetic acid based on the embodiment 22.

[0190] Embodiments 24-27

[0191] The anti-LILRB4 monoclonal antibody purification method in the embodiments 24-27 is further limited that the salt concentration and the pH value of the buffer B5 in the cation exchange chromatography step based on the embodiment 23, and other methods and parameters are all the same as those in the embodiment 23, and are specifically as follows.

[0192] Example pH value NaCl (mM) Example 24 5.0 150 Example 25 5.5 120 Example 26 6.0 110 Example 27 6.5 90

[0193] Embodiments 28-31

[0194] Embodiment 8-31 of the present application is based on embodiment 26, further limits the anion exchange chromatography filler and buffer B6 in step S4 of the purification method of anti-LILRB4 monoclonal antibody, and other methods and parameters are the same as embodiment 26. The chromatography filler is Capto adhere, and the buffer B6 is as follows.

[0195]

[0196] Comparative example 1

[0197] The present application provides a purification method of anti-LILRB4 monoclonal antibody. The method is different from embodiment 21 in that the first step is affinity chromatography, the filler of the affinity chromatography column is MabSelect SuRe LX, the second step is cation exchange chromatography, the filler of the cation exchange chromatography column is Capto Q, and the third step is hydrophobic chromatography, the filler of the hydrophobic chromatography column is Captol Butyl. Other methods and parameters are the same as embodiment 21.

[0198] Comparative example 2

[0199] The present application provides a purification method of anti-LILRB4 monoclonal antibody. The method is different from embodiment 21 in that the first step is affinity chromatography, the filler of the affinity chromatography column is MabSelect SuRe LX, the second step is cation exchange chromatography, the filler of the cation exchange chromatography column is Capto Q, and the third step is hydrophobic chromatography, the filler of the hydrophobic chromatography column is Captol Butyl. Other methods and parameters are the same as embodiment 21.

[0200] Experimental example 1, physical and chemical detection of anti-LILRB4 monoclonal antibody

[0201] The anti-LILRB4 monoclonal antibody obtained by the purification method provided in the above embodiments and comparative examples of the present application is detected for purity by high performance liquid chromatography technique, and the contents of aggregates, monomers and degradation products in the sample during the purification process are analyzed. In addition, ion chromatography technique is used to analyze the contents of acid-base peaks of charge isomers, and the total recovery rate is calculated by the following formula: total recovery rate = affinity chromatography protein yield (%) * cation exchange chromatography protein yield (%) * anion exchange chromatography protein yield (%). The specific data are as follows:

[0202]

[0203] As can be seen from the above table, the anti-LILRB4 monoclonal antibody obtained by the purification method provided in the above embodiments of the present application has a protein purity of more than 99%, and the total protein recovery rate can reach more than 60%. The protein purity and recovery rate of the sample obtained by the purification method provided in embodiment 31 are the highest, and the total protein recovery rate reaches more than 70%, which can significantly improve the yield and reduce the production cost in antibody production.

[0204] Example 23 compared with Example 22, when the acid solution is preferably acetic acid, the protein purity and recovery rate are significantly improved.

[0205] Experimental Example 24-27, compared with the composition screening of buffer B5 (eluent) in cation exchange chromatography, the pH value of the buffer provided in Example 26 is 6.0, and buffer B5 is a phosphate buffer containing 110 mM sodium chloride, which can effectively elute the target protein anti-LILRB4 monoclonal antibody, and protect the target protein from forming aggregates, thereby improving the purity of the sample.

[0206] Examples 28-31 further limit the chromatography filler used in step S4, the composite mode filler Capto adhere, which can effectively remove degradation products, aggregates and other impurities, and has better purity and yield than pure anion filler. Therefore, Example 31 is the optimal embodiment of the present application;

[0207] Example 31 and Control Example 1, compared with Example 31, is a three-step purification process, and the third step of purification is a composite mode chromatography, which replaces the hydrophobic chromatography in Control Example 1. There is no significant difference in the purity of the final sample between the two process routes of hydrophobic chromatography and composite mode chromatography, but the composite mode chromatography can remove acidic isomers at the same time, thereby increasing the content of the main peak. Hydrophobic chromatography does not have this purification function. The loading capacity of the hydrophobic filler in Control Example 1 is 20-30 mg, and the loading capacity of the composite mode filler in Example 31 can reach 150 mg, thereby reducing the cost and improving the competitiveness of the product.

[0208] Example 31 and Control Example 2, compared with Example 31, the yield of the three-step chromatography process is comparable to that of the two-step chromatography process, but for the purification of anti-LILRB4 monoclonal antibody, the final sample of the three-step purification process is better than that of the two-step chromatography process in terms of purity. The cation exchange chromatography in the three-step purification process mainly removes aggregates and a small amount of degradation products, and the composite mode chromatography can also remove aggregates and degradation products. The purification effect is complementary to each other, which ensures the stability of the purification process and always follows the concept of design of quality.

[0209] Experimental Example 2, detection of related impurities of anti-LILRB4 monoclonal antibody

[0210] The anti-LILRB4 monoclonal antibody purified by the purification method provided in the above embodiments of the present application is detected for the content of process-related impurities by using a special kit.

[0211]

[0212] The experimental data show that, compared with Comparative Example 1, the three-step chromatography process of Example 31 has advantages in removing process-related impurities such as HCP, DNA, Protein A, etc., and the filler used in the application has excellent alkali resistance and pressure-flow rate characteristics, significantly reducing the cost.

[0213] Compared with Comparative Example 2, the three-step chromatography process of Example 31 can not only effectively remove process-related impurities such as HCP, DNA, Protein A, and aggregates, so that the final purity of the sample is more than 99%, but also has smooth process connection, and eliminates the intermediate desalting or ultrafiltration steps. Although Comparative Example 2 is a two-step chromatography process, the residual DNA and Protein A meet the quality requirements, but the residual HCP is significantly higher than that of Example 31, which obviously has the risk of unqualified quality. Since the application adds a step of cation exchange chromatography to remove HCP and DNA impurities, it not only does not reduce the recovery rate, but also improves the robustness of the process, ensuring that the purified product meets the quality requirements.

[0214] Through the comparison between Example 31 and other examples, it is found that the composition, content and pH of buffer B1, buffer B2, buffer B3, buffer B4, buffer B5 and buffer B6 defined in Example 11 based on Example 21 can significantly remove HCP, DNA and Protein A and other process-related impurities, and the detection results meet the quality requirements. At the same time, the aggregates and degradation products significantly improve the purity of the protein, so Example 31 of the application is the best embodiment and can be more suitable for the purification of anti-LILRB4 monoclonal antibodies.

[0215] Experimental Example 3: Detection of binding activity and biological activity of anti-LILRB4 monoclonal antibody

[0216] For the anti-LILRB4 monoclonal antibody purified by the purification method provided in the above embodiments of the application, the application uses ELISA to analyze the specific binding ability of the purified anti-LILRB4 monoclonal antibody to LILRB4, so as to evaluate the activity of the purified antibody. The detection results are as follows:

[0217] Example Binding activity (%) Biological activity (%) Example 21 90 89 Example 22 115 107 Example 23 100 98 Example 24 104 115 Example 25 76 85 Example 26 88 79 Example 27 97 100 Example 28 108 102 Example 29 91 87 Example 30 82 88 Example 31 117 109

[0218] As can be seen from the above table, the anti-LILRB4 monoclonal antibody purified by the method provided in the above embodiments of the application has good binding activity and biological activity, which is within the range of 100%±30%, indicating that the anti-LILRB4 monoclonal antibody purified under the conditions defined by the method provided in the application has good biological activity through a large number of experimental condition screening.

[0219] The present application is not limited to the above best mode, anyone under the inspiration of the present application can derive other various forms of products, but regardless of any changes in its shape or structure, as long as it has the same or similar technical solutions as this application, it falls within the scope of the present application.

Claims

1. A method for purifying anti-LILRB4 monoclonal antibody, characterized in that, The method includes: S1. Cell Clarification: The cell solution containing anti-LILRB4 monoclonal antibody is clarified by centrifugation or deep filtration. S2. Affinity chromatography: The cell slurry is initially purified and concentrated using an affinity chromatography column, the protein solution is collected, and the protein solution is inactivated against the virus in an acidic solution. The packing material for the affinity chromatography column is NMab Pro. S3. Cation exchange chromatography: The protein solution is purified by cation exchange chromatography column and the eluent is collected. The packing material of the cation exchange chromatography column is NanoGel 50SP. S4. Anion exchange chromatography: The eluent from step S3 is purified using an anion exchange chromatography column, and the purified protein solution is collected. The packing material for the anion exchange chromatography column is Capto adhere. The anti-LILRB4 monoclonal antibody comprises: a heavy chain complementarity-determining region HCDR1 as shown in SEQ ID No:1, a heavy chain complementarity-determining region HCDR2 as shown in SEQ ID No:2, a heavy chain complementarity-determining region HCDR3 as shown in SEQ ID No:3, a light chain complementarity-determining region LCDR1 as shown in SEQ ID No:4, a light chain complementarity-determining region LCDR2 as shown in SEQ ID No:5, and a light chain complementarity-determining region LCDR3 as shown in SEQ ID No:

6.

2. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 1, characterized in that, The anti-LILRB4 monoclonal antibody is a murine antibody molecule, which includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID No:7 and a light chain variable region with an amino acid sequence as shown in SEQ ID No:

8.

3. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 2, characterized in that, The murine antibody molecule further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is selected from one of the constant regions of mouse IgG1, IgG2a, IgG2b, or IgG3 types. The light chain constant region is a murine C amino acid sequence as shown in SEQ ID No:

15. k The constant regions of the IgG1 type are shown in SEQ ID No:11, the constant regions of the IgG2a type are shown in SEQ ID No:12, the constant regions of the IgG2b type are shown in SEQ ID No:13, and the constant regions of the IgG3 type are shown in SEQ ID No:

14.

4. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 1, characterized in that, The anti-LILRB4 monoclonal antibody is a humanized antibody molecule, and the humanized antibody molecule is selected from any one of the following: HA-Ⅰ: The heavy chain variable region with amino acid sequence as shown in SEQ ID No:20 and the light chain variable region with amino acid sequence as shown in SEQ ID No:21; HA-II: The heavy chain variable region with amino acid sequence as shown in SEQ ID No:22 and the light chain variable region with amino acid sequence as shown in SEQ ID No:23; HA-Ⅲ: Heavy chain variable region with amino acid sequence as shown in SEQ ID No:24, and light chain variable region with amino acid sequence as shown in SEQ ID No:

25.

5. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 4, characterized in that, The humanized antibody molecule further includes a humanized antibody heavy chain constant region and a humanized antibody light chain constant region. The humanized antibody heavy chain constant region is selected from one of the constant regions of human IgG1, IgG2, or IgG4 types. The humanized antibody light chain constant region has an amino acid sequence as shown in SEQ ID No:19, representing human C. k The constant region of the heavy chain of the IgG1 type; the amino acid sequence of the heavy chain constant region of the IgG2 type is shown in SEQ ID No:16, the amino acid sequence of the heavy chain constant region of the IgG4 type is shown in SEQ ID No:17, and the amino acid sequence of the heavy chain constant region of the IgG4 type is shown in SEQ ID No:

18.

6. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 1, characterized in that, In step S2, affinity chromatography: the cell slurry is initially purified and concentrated using an affinity chromatography column, the protein solution is collected, and the protein solution is then subjected to virus inactivation in an acidic solution. Specifically... Including the following methods: S201. Equilibrate the affinity chromatography column with a 30-60 mM buffer solution B1. The packing material of the affinity chromatography column is NMab Pro. The buffer solution B1 is selected from Tris-HCl buffer, HEPES buffer, or phosphate buffer. The pH of the buffer solution B1 is 7-8, and the conductivity of the buffer solution B1 is 10-30 mS / cm. S202. The cell solution containing anti-LILRB4 monoclonal antibody is loaded onto the affinity chromatography column at a flow rate of 50-300 cm / h, with a loading capacity of 20-70 mg protein / ml packing material, and the column height of the affinity chromatography column is 18-22 cm. S203. After the sample loading is completed, use the buffer B1 to reequilibrate. After the UV signal stabilizes, use the buffer B2 with a concentration of 30-60mM to perform at least one intermediate rinse until the UV absorbance curve drops to a stable level. Stop rinsing. The buffer B2 is selected from Tris-HCl buffer, HEPES buffer, phosphate buffer, citrate buffer or acetate buffer, and the pH of the buffer B2 is 5-6. S204. Elute the affinity chromatography column using 100-500mM buffer B3. When the UV absorbance rises to 100mAU, start collecting the protein solution. Stop collecting when the UV absorbance drops to 100mAU. Set aside for later use. Buffer B3 is selected from glycine-hydrochloride buffer, citrate buffer, acetate buffer, or arginine buffer. The pH of buffer B3 is 3-4. S205. Adjust the pH of the collected protein solution to 3.5-3.7 using an acid solution, and incubate at room temperature for 0.5-2 hours under this pH condition. Finally, adjust the pH of the protein solution back to 5-7 using 1M Tris buffer. The acid solution is selected from citric acid, acetic acid, or hydrochloric acid.

7. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 6, characterized in that, In step S3, cation exchange chromatography: the protein solution is purified using a cation exchange chromatography column, and the eluent is collected. This specifically includes the following steps: S301. Equilibration: The cation exchange chromatography column is equilibrated with buffer B4 at a concentration of 10-50 mM. The packing material of the cation exchange chromatography column is NanoGel 50SP. The buffer B4 is selected from citrate buffer, acetate buffer or phosphate buffer. The pH value of the buffer B4 is 5.5-6.5 and its conductivity is 2-5 mS / cm. S302, Sample loading: The protein solution filtered in step S2 is loaded onto the cation exchange chromatography column at a flow rate of 100-200 cm / h. The loading amount is 20-85 mg protein / ml packing material. The column height of the cation exchange chromatography column is 18-22 cm. S303. Rebalancing: After the sample loading is completed, use the buffer solution B4 again for rebalancing. Stop rinsing after the UV absorbance value stabilizes. S304, Elution: The cation exchange chromatography column is eluted using buffer B5. The eluent is collected when the UV absorbance rises to 100 mAU and is collected when the UV absorbance drops to 100 mAU. The buffer B5 is a citrate buffer, acetate buffer, or phosphate buffer containing 90-150 mM sodium chloride.

8. The purification method for the anti-LILRB4 monoclonal antibody as described in claim 7, characterized in that, In step S4, anion exchange chromatography is performed; the eluent from step S3 is purified using an anion exchange chromatography column, and the purified protein solution is collected. This specifically includes the following steps: S401. Equilibrate the anion exchange chromatography column using 7-10 CV buffer B6 at a flow rate of 100-200 cm / h. Load the eluent collected in step S3 at a flow rate of 100-200 cm / h. The packing material of the anion exchange chromatography column is Capto adhere. The buffer B6 is a citrate buffer, acetate buffer, or phosphate buffer containing 100-300 mM sodium chloride. The pH of the buffer B6 is 6.0-6.

5. S402. After the sample loading is completed, rinse again with the buffer solution B6. When the UV absorbance value rises to 100mAU, start collecting the flow-through solution. When the UV absorbance value drops to 200mAU, stop collecting.

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