An injection preparation of anti-lilrb4 monoclonal antibody

Abstract

The present application relates to the field of biological medicine, and specifically provides an anti-LILRB4 monoclonal antibody injection preparation, which comprises an anti-LILRB4 monoclonal antibody, a buffer salt, a protein protective agent, and a surfactant, wherein the pH of the injection preparation is 5.5-6.5. The injection preparation provided by the present application provides a good storage environment for the anti-LILRB4 monoclonal antibody through the synergistic interaction of different contents of the buffer salt, the protein protective agent, and the surfactant, and guarantees the biological activity of the anti-LILRB4 monoclonal antibody. The anti-LILRB4 monoclonal antibody can effectively inhibit the combination of the LILRB4 antigen and its ligand complex, thereby blocking the interaction with the ligand ApoE complex, preventing the activation of the downstream NF-kappa B signal pathway and the release of ARG1, preventing the inhibition of T cell proliferation and the promotion of tissue infiltration, and can be used for treating cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an injectable formulation of an anti-LILRB4 monoclonal antibody. Background Technology

[0002] Acute myeloid leukemia (AML) includes all acute leukemias of non-lymphocyte origin. AML is a clonal malignant proliferative disorder of myeloid progenitor cells in the hematopoietic system. It is a highly heterogeneous group of diseases that can result from the malignant transformation of hematopoietic progenitor cells at different stages of normal myeloid cell differentiation and development.

[0003] Leukocyte immunoglobulin-like receptor subfamily B (LILRB) is a group of type I transmembrane glycoproteins primarily expressed in myeloid cells. They are characterized by an extracellular immunoglobulin-like domain for ligand binding and an intracellular immunoreceptor tyrosine repressor motif (ITIM). Upon activation by the corresponding ligand binding of LILRB receptors, the ITIM domain recruits tyrosine phosphatases SHP-1 and SHP-2, or inositol phosphatase SHIP, thereby regulating related signaling pathways to inhibit the expression of cytokines, chemokines, and co-stimulatory factors, specifically suppressing T cell activation. Due to its immunosuppressive function, LILRB is considered an immune checkpoint protein of myeloid cells. Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), also known as ILT3, is an immunosuppressive transmembrane protein on the surface of myeloid monocytes (including dendritic cells). LILRB4 can inhibit the activation of antigen-presenting cells, leading to immune tolerance. LILRB4 is also expressed on the surface of myeloid monocytes in specific hematologic malignancy cells and solid tumor microenvironments.

[0004] Currently, Yiming Bio, Engem Biopharmaceutical, and Huaxia Yingtai (Beijing) Biotechnology Co., Ltd. are all developing monoclonal antibody drugs targeting LILRB4. As the first T-cell activator that is expected to be used in AML, the research on anti-LILRB4 monoclonal antibodies has great market potential.

[0005] Biological macromolecules possess complex structures, such as primary, secondary, and tertiary higher-order structures. Proteins, especially their higher-order structures, are highly fragile and prone to conformational changes, such as denaturation, aggregation, and precipitation. Like all monoclonal antibodies, anti-LILRB4 monoclonal antibodies are unstable and subject to various chemical and physical degradation processes. Maintaining the higher-order structure of proteins is fundamental to their biological activity. These degradation and aggregation products can significantly impact the safety of biopharmaceuticals. In particular, some protein aggregates can trigger immune responses in the human body, potentially reducing the efficacy of biological drugs or even causing death in severe cases. Furthermore, monoclonal antibody drugs not only require high-purity products during production but also structural stability during transportation, storage, and use. Therefore, the development of anti-LILRB4 monoclonal antibodies necessitates conditional exploration of their formulations to develop an injectable formulation more suitable for anti-LILRB4 monoclonal antibodies. Summary of the Invention

[0006] To address the instability of anti-LILRB4 monoclonal antibodies in existing technologies and to ensure their stability during transportation, storage, and use, it is necessary to explore formulation conditions specifically for anti-LILRB4 monoclonal antibodies. Therefore, this invention discloses an injectable formulation of anti-LILRB4 monoclonal antibody.

[0007] The specific technical solution of this invention is as follows:

[0008] This invention provides an injectable formulation of an anti-LILRB4 monoclonal antibody, which comprises the following components:

[0009]

[0010] The pH of the injectable formulation is 5.5-6.5.

[0011] The beneficial effects of this invention are as follows: The injectable formulation provided by this invention, through the synergistic interaction of different amounts of buffer salts, protein protectants, and surfactants, provides a favorable storage environment for the anti-LILRB4 monoclonal antibody, ensuring its biological activity. The anti-LILRB4 monoclonal antibody provided by this invention can effectively inhibit the binding of LILRB4 antigen to its ligand complex, thereby blocking its interaction with the ApoE ligand complex, and thus preventing the activation of the downstream NF-κB signaling pathway and the release of ARG1, preventing the inhibition of T cell proliferation and the promotion of tissue infiltration; in addition, the anti-LILRB4 monoclonal antibody screened by this invention... B4 monoclonal antibodies can be used to treat cancers, including but not limited to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), blastic plasmacytoid dendritic cell tumor (BPDCN), breast cancer, lung cancer, or prostate cancer. This injectable formulation effectively reduces the rate of antibody aggregation and degradation during storage and transportation, improves the physical stability of the antibody, ensures activity while reducing potential safety risks. The preparation process of this formulation is simple, low-cost, has higher concentration, better stability, and is easy to mass-produce. Attached Figure Description

[0012] Figure 1 This is a plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

[0013] Figure 2 This is a comparison chart of the relative affinities of the serially diluted ELISA anti-LILRB4 phage monoclonal antibody in Example 3 of the present invention;

[0014] Figure 3 This is a spectrum of the carrier pTSE in Embodiment 5 of the present invention;

[0015] Figure 4 This is a denaturing polyacrylamide gel electrophoresis image of the mouse antibody molecule in Example 5 of the present invention;

[0016] Figure 5 This is a comparison diagram of the binding ability of the mouse antibody to LILRB4 in Example 6 of the present invention;

[0017] Figure 6 This is a comparison diagram of the binding ability of the murine antibody to LILRB4 on the surface of human monocytic leukemia cells (THP-1) in Example 8 of the present invention;

[0018] Figure 7 Comparison of mouse antibody and ApoE in competitive binding to LILRB4 in Example 9 of this invention;

[0019] Figure 8 This is a comparative diagram of the experiment in Example 10 of the present invention on the inhibition of THP-1 secretion of ARG1 by murine antibody;

[0020] Figure 9 This is a denaturing polyacrylamide gel electrophoresis image of the chimeric antibody molecules in Example 12 of this invention;

[0021] Figure 10 This is a denaturing polyacrylamide gel electrophoresis image of the humanized antibody molecule in Example 15 of this invention;

[0022] Figure 11 This is a diagram of the binding experiment between the humanized antibody and LILRB4 in Example 16 of the present invention;

[0023] Figure 12 This is a comparative diagram of the binding experiment between the humanized antibody and LILRB4 on the surface of THP-1 cells in Example 17 of the present invention;

[0024] Figure 13 This is a comparison diagram of the competitive binding of humanized antibody and ApoE to LILRB4 in Example 18 of the present invention;

[0025] Figure 14 This is a comparative graph showing the humanized antibody inhibiting THP-1 secretion of ARG1 in Example 19 of this invention.

[0026] Figure 15 This is a comparative diagram of the biological activity detection (reporter gene) experiment of humanized antibody molecules in Example 20 of the present invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the following embodiments.

[0028] Example 1

[0029] Example 1 of this invention provides an injectable formulation of an anti-LILRB4 monoclonal antibody, which comprises the following components:

[0030]

[0031] The pH of the injectable formulation is 5.5-6.5.

[0032] The anti-LILRB4 monoclonal antibody comprises: the heavy chain complementarity-determining region HCDR1 as shown in SEQ ID No: 1, the heavy chain complementarity-determining region HCDR2 as shown in SEQ ID No: 2, the heavy chain complementarity-determining region HCDR3 as shown in SEQ ID No: 3, the light chain complementarity-determining region LCDR1 as shown in SEQ ID No: 4, the light chain complementarity-determining region LCDR2 as shown in SEQ ID No: 5, and the light chain complementarity-determining region LCDR3 as shown in SEQ ID No: 6.

[0033] Heavy chain complementarity determinant HCDR1 Heavy chain complementarity determinant region HCDR2 Heavy chain complementarity determinant HCDR3 Light chain complementarity determination region LCDR1 Light chain complementarity determination region LCDR2 Light chain complementarity determination region LCDR3 SYTMS TISSGGTYTYYPDSVKG DGYDGFDY RSSQSLAHHSNGNTYLH KVSNRFS SQSTLVFr SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID No: 4 SEQ ID NO: 5 SEQ ID NO: 6

[0034] Example 2: Screening of mouse antibody molecules

[0035] This invention optimizes the immunization method by immunizing mice with LILRB4 antigen (the extracellular domain of LILRB4 protein; subsequent experiments used both LILRB4 antigen and protein as the extracellular domain of LILRB4) and creates a phage display library. The specific construction, screening, and identification of the phage display library are as follows:

[0036] Step 1: Immunize mice with LILRB4 antigen

[0037] 1. Laboratory animals: Species and strain: BALB / c, female, mouse; weight: 18-20g;

[0038] Laboratory animal provider: Yikang (Beijing) Pharmaceutical Technology Co., Ltd.

[0039] 2. Immunization: Mice were immunized with human LILRB4 (a gene synthesized by Nanjing Genscript Biotech Co., Ltd., whose vector was constructed and purified by our company).

[0040] Step 2: Construction of phage antibody library: Mouse spleen cells with high titers were collected, and total RNA was extracted from mouse spleen cells using Trizol reagent (purchased from Ambion, catalog number: 15596026). cDNA was obtained by RT-PCR. Using cDNA as a template, PCR amplification was performed using degenerate primers (the degenerate primers used are referenced in Journal of Immunological Methods 233 (2000) 167-177) to obtain the heavy chain variable region (VH) gene library and the light chain variable region (VL) gene library of immunized mouse antibodies. The light and heavy chains were double-digested with enzymes and ligated into a vector that had been digested in the same steps to construct the pScFv-Disb-HS-VH-VL gene library. The pScFv-Disb-HS vector was modified by a series of gene cloning methods to modify the pComb3 vector (purchased from the China Plasmid Vector Strains Cell Line Gene Preservation Center) for the construction and expression of phage single-chain antibody library. The modified vector was named pScFv-Disb-HS, and its plasmid map was obtained as follows. Figure 1 As shown, a mouse immune phage antibody library was constructed based on this vector.

[0041] Step 3: Coat the immunotubes with LILRB4 antigen at a rate of 5 μg / 500 μL / tube, and incubate overnight at 4°C. Then, block the immunotubes and the immunophage antibody library separately with 4% skim milk powder / PBST at room temperature for 1 hour. Add the blocked immunophage antibody library to the immunotubes for antigen-antibody binding; the phage dosage is approximately 10 μg / 500 μL. 9 ~10 12 After reacting at room temperature for 1 hour, unbound phages were washed away with PBST-PBS, followed by elution with 0.1M pH 2.2 Glycine-HCl. Finally, the eluted phage antibody solution was neutralized to approximately pH 7.0 with 1.5M pH 8.8 Tris-HCl.

[0042] Step 4: Infect 10 mL of TG1 bacterial culture grown to the logarithmic phase with the neutralized phage. Incubate at 37°C for 30 minutes. Take a portion of the bacterial culture and perform serial dilutions, then spread it on 2YTAG plates to calculate the phage yield. Centrifuge the remaining bacterial culture, discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate it, and spread it on a large 2YTAG plate to prepare for the next round of screening.

[0043] Step 5: Scrape the infected bacterial cells from the large plate, inoculate them into 2YTAG liquid medium, shake until the logarithmic phase, add M13KO7 helper phage for superinfection, and incubate overnight at 220 rpm at 28°C to prepare phages. Purify the phages by PEG / NaCl precipitation for the next round of screening, and perform one round of phage library enrichment screening.

[0044] Step Six: Screening of LILRB4 phage single-chain antibody-positive clones: After one round of screening, well-separated single-clone colonies were picked and inoculated into 96-well deep-well plates containing 2 YTAG liquid medium. The plates were incubated at 37°C and 220 rpm until the logarithmic growth phase. Approximately 10 μL of the medium was added to each well. 10 The helper phage M13KO7 was used for static infection at 37°C for 30 minutes. After centrifugation at 4000 rpm for 15 minutes, the supernatant was discarded, and the bacterial cells were resuspended in 2YTAK solution and cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 minutes, the amplified phage supernatant was subjected to ELISA identification. Two candidate anti-LILRB4 murine antibodies with high affinity were finally screened and named MA-Ⅰ and MB-Ⅰ, respectively. The obtained monoclonal antibodies were sequenced to confirm their correct antibody sequences. The sequences of the two selected monoclonal antibodies are as follows:

[0045] Mouse antibody molecules 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

[0046] Specifically, SEQ ID No:7 (amino acid sequence of the heavy chain variable region of MA-Ⅰ):

[0047] EVQLQQSGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDGYDGFDYWGQGTTLTVSS;

[0048] SEQ ID No:8 (Amino acid sequence of the light chain variable region of MA-Ⅰ):

[0049] DIVMTQTTLSLPVSPGDQASISCRSSQSLAHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTLVPTFGGGTKLEIK;

[0050] SEQ ID No:9 (Amino acid sequence of the heavy chain variable region of MB-Ⅰ):

[0051] QVQLQESGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWGQLGLREGYYAVDYWGQGTSVTVSS;

[0052] SEQ ID No:10 (Amino acid sequence of the light chain variable region of MB-I):

[0053] DIVMTQSPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK.

[0054] Example 3: Comparison of affinity of anti-LILRB4 phage monoclonal antibody using serially diluted ELISA

[0055] The two murine antibody molecules (MA-Ⅰ and MB-Ⅰ) obtained in Example 2 were displayed and purified as monoclonal phages, and then affinity was identified by a phage serial dilution ELISA experiment. The specific method is as follows: LILRB4 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at 4°C. The sample was washed three times with PBST. The two phage monoclonal antibodies screened in Example 2 were serially diluted four-fold with PBST, and 100 μL of the diluted sample was added to each well. The plate was incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and the PBST-diluted HRP-anti-M13 (purchased from Bio-viewshine, catalog number: GE27-9421-01) monoclonal antibody was added to the ELISA plate and incubated at room temperature for 1 hour. The TMB chromogenic kit was used for color development. Color development was performed at room temperature for 10 minutes, followed by termination with 2M H₂SO₄. Readings were then taken using a microplate reader at 450nm / 630nm, and the corresponding half-maximum effect concentration (EC50) values ​​were calculated. Specific data are as follows:

[0056] clone MA-Ⅰ MB-Ⅰ EC50 1.635 1.936

[0057] Based on the above data and as follows Figure 2 As shown, both of the two different murine antibody candidate molecules screened in Example 2 were able to bind to LILRB4.

[0058] Example 4

[0059] Example 4 of this invention further specifies, based on Example 2, that the murine antibody molecule also 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 amino acid sequences of the constant regions of the IgG1 type are shown in SEQ ID No:11, the amino acid sequences of the constant regions of the IgG2a type are shown in SEQ ID No:12, the amino acid sequences of the constant regions of the IgG2b type are shown in SEQ ID No:13, and the amino acid sequences of the constant regions of the IgG3 type are shown in SEQ ID No:14. The specific sequences are as follows:

[0060] SEQ ID No:11 (Amino acid sequence of the heavy chain constant region of mouse IgG1 type):

[0061] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0062] SEQ ID No:12 (Amino acid sequence of the heavy chain constant region of mouse IgG2a type):

[0063] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0064] SEQ ID No:13 (Amino acid sequence of the heavy chain constant region of murine IgG2b):

[0065] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0066] SEQ ID No:14 (Amino acid sequence of the heavy chain constant region of murine IgG3):

[0067] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGK EFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSW LQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA;

[0068] SEQ ID No:15 (Rat C) k (Amino acid sequence of the light chain constant region):

[0069] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0070] Example 5: Preparation of anti-LILRB4 murine antibody molecules

[0071] Example 5 of the present invention, based on Example 4, preferably specifies that the murine antibody molecule includes the heavy chain constant region of murine IgG1 (its amino acid sequence is shown in SEQ ID No: 11) and murine C k The light chain constant region of type [type] (its amino acid sequence is shown in SEQ ID No:15). The specific antibody preparation method is as follows:

[0072] 1. The encoding genes for VH and VL of the two monoclonal antibodies screened in Example 2 were cloned into the vector pTSE (e.g., ...) containing the heavy chain and light chain constant region genes, respectively. Figure 3 As shown in SEQ ID No: 11), the preferred heavy chain constant region is the mouse IgG1 type heavy chain constant region (amino acid sequence shown in SEQ ID No: 11), and the light chain constant region is the mouse C... k The light chain constant region of type (amino acid sequence as shown in SEQ ID No:15), pTSE vector structure as shown Figure 3 As shown (for the preparation process of the pTSE vector, please refer to paragraph

[0019] on page 3 of the instruction manual CN103525868A).

[0073] 2. HEK293 cells were transiently transfected (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Four monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 4 As shown, from left to right, the images represent non-reduced MA-I, reduced MA-I, non-reduced MB-I, reduced MB-I murine anti-LILRB4 monoclonal antibodies and protein molecular weight markers. The molecular weight of each band is consistent with the theoretical values.

[0074] Example 6: Binding experiment of mouse antibody with LILRB4

[0075] LILRB4 was coated with carbonate buffer (pH 9.6), 100 ng / well / 100 μL, overnight at 4°C. The wells were washed five times with 300 μL / well PBST, then blocked with 1% BSA-PBST at 37°C for 1 hour. Different concentrations of MA-Ⅰ and MB-Ⅰ mouse antibodies were added, with an initial maximum concentration of 50 μg / mL for both antibodies, serially diluted 5-fold (12 dilutions total for each antibody), and incubated at 37°C for 1 hour. The wells were washed five times with 300 μL / well PBST, then Goat Anti-Mouse IgG-HRP (purchased from SolarBio, catalog number: SE131) diluted 1:2000 with 1% BSA-PBST was added, and incubated at 37°C for 1 hour. The TMB chromogenic kit was used for color development, 100 μL / well, incubated at room temperature for 8 minutes, then stopped with 2M H2SO4. The microplate reader was used to read values ​​at 450nm and 630nm, and the corresponding EC50 values ​​were calculated. The specific data are as follows:

[0076] clone MA-Ⅰ MB-Ⅰ EC50 (ng / mL) 8.347 12.80

[0077] Based on the above data and as follows Figure 5 As shown, the two different murine antibodies MA-Ⅰ and MB-Ⅰ selected can both bind to LILRB4.

[0078] Example 7: Binding experiment of murine antibody to LILR family proteins

[0079] LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRA1, LILRA2, LILRA3, LILRA4, and LILRA5 were coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and incubated overnight at 4°C. The cells were washed five times with 300 μL / well PBST, then blocked with 1% BSA-PBST at 37°C for 1 hour. Subsequently, 100 μL of MA-Ⅰ and MB-Ⅰ mouse antibody molecules (both at a concentration of 50 μg / mL) were added. The cells were incubated at 37°C for 1 hour. The cells were washed five times with 300 μL / well PBST, then Goat Anti-Mouse IgG-HRP (purchased from SolarBio, catalog number: SE131) diluted 1:2000 with 1% BSA-PBST was added, and the cells were incubated at 37°C for 1 hour. The TMB chromogenic kit was used for color development. 100 μL / well was incubated at room temperature for 8 minutes, then the color development was stopped with 2M H₂SO₄. Readings were taken using a microplate reader at 450 nm / 630 nm. Specific data are as follows:

[0080]

[0081] Based on the above data, it can be concluded that the two different murine antibodies MA-Ⅰ and MB-Ⅰ screened out can specifically bind to LILRB4 and do not bind to other proteins in the LILR family.

[0082] Example 8: Binding experiment of murine antibody to LILRB4 on the surface of human monocytic leukemia cells (THP-1)

[0083] Take 50 μL of MA-Ⅰ and MB-Ⅰ mouse antibodies at different dilutions, starting at a working concentration of 30 μg / mL, and perform 3-fold serial dilutions for a total of 10 dilutions. Add the diluted solutions to 96-well V plates. Then add 50 μL of LHP-1 cell suspension to each well, at a concentration of 2 × 10⁻⁶. 6 Cells / 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:

[0084] clone MA-Ⅰ MB-Ⅰ EC50 (ng / mL) 305.9 351.0

[0085] Based on the above data and as follows Figure 6 As shown, the two different murine antibodies MA-Ⅰ and MB-Ⅰ selected can both bind to LILRB4 on the surface of THP-1 cells.

[0086] Example 9: Mouse antibody competitively binding to LILRB4 with ApoE

[0087] THP-1 cells were collected at a concentration of 2 × 10⁻⁶. 6 Cells were seeded at 50 μL / mL in 96-well plates with a V-bottom, and 50 μL of cell suspension was added to each well. Then, 50 μL of different concentrations of MA-I and MB-I mouse antibodies were added to each well, starting at a working concentration of 400 μg / mL and serially diluted 5-fold for a total of 10 gradients. Additionally, 100 μL of 0.4 μg / mL FITC-labeled ApoE protein was added to each well. The cells were incubated at 4°C in the dark for 1.5 hours. Then, 200 μL of PBS buffer was added to each well, and the cells were centrifuged at 3000 rpm for 5 minutes, discarding the supernatant. Cells were resuspended in 100 μL of PBS buffer and analyzed by flow cytometry. Data were collected and the corresponding IC50 values ​​were calculated. Specific data are as follows:

[0088] clone MA-Ⅰ MB-Ⅰ IC50 (μg / mL) 0.2037 0.4399

[0089] Based on the above data and as follows Figure 7 As shown, the two different murine antibodies MA-Ⅰ and MB-Ⅰ selected can both competitively bind to LILRB4 on the surface of THP-1 cells with ApoE.

[0090] Example 10: Mouse antibody inhibits THP-1 secretion of ARG1

[0091] THP-1 cells were collected at a concentration of 1×10⁻⁶. 7Cells / 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:

[0092] clone MA-Ⅰ MB-Ⅰ IC50 (μg / mL) 23.86 45.97

[0093] 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.

[0094] Example 11

[0095] 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.

[0096] SEQ ID No:16 (Amino acid sequence of the heavy chain constant region of human IgG1):

[0097] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0098] SEQ ID No:17 (Amino acid sequence of the heavy chain constant region of human IgG2):

[0099] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0100] SEQ ID No:18 (Amino acid sequence of the heavy chain constant region of human IgG4):

[0101] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0102] SEQ ID No:19 (human C k (Amino acid sequence of the light chain constant region):

[0103] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0104] Example 12 Preparation of chimeric antibody molecules

[0105] Example 12 of the present invention further defines the constant region of the human antibody as including the heavy chain constant region of human IgG1 (whose amino acid sequence is shown in SEQ ID No: 16) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:19).

[0106] Specific preparation method: The heavy chain variable region VH (SEQ ID No:7) and light chain variable region VL genes (SEQ ID No:8) of antibody molecules MA-Ⅰ obtained from the phage antibody library screening in Example 2, and the heavy chain variable region VH (SEQ ID No:9) and light chain variable region VL genes (SEQ ID No:10) of MB-Ⅰ, were cloned into the vector pTSE (e.g., containing heavy chain constant region and light chain constant region genes, while keeping the mouse-derived sequences unchanged. Figure 3 As shown in SEQ ID NO:16, the heavy chain constant region is human IgG1 type, and the light chain constant region is human C. kType (amino acid sequence as shown in SEQ ID NO:19). HEK293 cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: GNHu43) were transiently transfected to express antibodies, yielding chimeric antibodies CA-Ⅰ and CB-Ⅰ, respectively. The results of SDS-PAGE identification of chimeric antibodies CA-Ⅰ and CB-Ⅰ proteins are shown below. Figure 9 As shown, from left to right, the bands represent protein molecular weight markers, reduced CA-I, non-reduced CA-I, reduced CB-I, and non-reduced CB-I anti-LILRB4 monoclonal antibodies. The molecular weight of each band is consistent with the theoretical values.

[0107] Example 13 Humanization of mouse antibody molecules MA-I and MB-I

[0108] First, the sequences of the murine antibody molecules MA-I and MB-I from Example 2 were compared with the human antibody lineage database (v-base) to identify human antibody light and heavy chain lineages with high homology as candidate sequences. Then, the CDR sequences of the murine antibody molecules MA-I and MB-I were transplanted onto the human candidate sequences for homology modeling. Next, three-dimensional structural simulations were used to calculate key framework amino acid residues that might play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for humanized antibodies. The light and heavy chain variable regions of the designed humanized antibodies containing reversion mutations were optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated into a transient expression vector. Analysis of the humanized light and heavy chain combinations yielded the following humanized antibody molecules: HA-I, HA-II, HA-III, HB-I, HB-II, and HB-III. The six monoclonal antibody sequences screened above are as follows:

[0109] Monoclonal antibodies 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

[0110] Specifically, SEQ ID No:20 (amino acid sequence of the HA-I heavy chain variable region):

[0111] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVSTISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYDGFDYWGQGTTVTVSS;

[0112] SEQ ID No:21 (Amino acid sequence of the HA-Ⅰ light chain variable region):

[0113] DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPTFGGGTKVEIK;

[0114] SEQ ID No:22 (Amino acid sequence of the HA-II heavy chain variable region):

[0115] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYDGFDYWGQGTTVTVSS;

[0116] SEQ ID No:23 (Amino acid sequence of the variable region of HA-II light chain):

[0117] DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPTFGGGTKVEIK;

[0118] SEQ ID No:24 (Amino acid sequence of the HA-Ⅲ heavy chain variable region):

[0119] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDGYDGFD YWGQGTTVTVSS;

[0120] SEQ ID No:25 (Amino acid sequence of the HA-Ⅲ light chain variable region):

[0121] DIVMTQTPLSLSVTPGQPASISCRSSQSLAHSNGNTYLHWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPTFGGGTK VEIK;

[0122] SEQ ID No:26 (Amino acid sequence of the variable region of the HB-Ⅰ, HB-Ⅱ, HB-Ⅲ heavy chain):

[0123] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWIG EINPSNGRTNYNEKFKTRATLTVDTSTSTAYMELSSLRSEDTAVYYCARWGQLGL REGYYAVDYWGQGTLVTVSS;

[0124] SEQ ID No:27 (Amino acid sequence of the variable region of the HB-Ⅰ light chain):

[0125] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPPTFGGGTKVEIK;

[0126] SEQ ID No:28 (Amino acid sequence of the variable region of the HB-II light chain):

[0127] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK;

[0128] SEQ ID No:29 (Amino acid sequence of the variable region of HB-Ⅲ light chain):

[0129] DIVMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGGAVKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPPTFGGGTKVEIK.

[0130] Example 14

[0131] Example 14 of this invention further defines the humanized antibody constant region as including a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, based on Example 13. The humanized antibody heavy chain constant region is selected from one of the constant regions of human IgG1, IgG2, or IgG4 types, and the humanized antibody light chain constant region has an amino acid sequence as shown in SEQ ID No:19. kThe 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.

[0132] The specific sequence of the constant region of the above-mentioned human antibody is the same as that in Example 11.

[0133] Example 15 Preparation of humanized antibody molecules

[0134] Example 15 of the present invention further defines the constant region of the human antibody, based on Example 14, as including the constant region of the human IgG1 heavy chain (whose amino acid sequence is shown in SEQ ID No: 16) and human C k Type light chain constant region (its amino acid sequence is shown in SEQ ID No:19).

[0135] The heavy chain VH and light chain VL encoding genes of the six humanized antibody molecules obtained in Example 13 above were cloned into the vector pTSE (e.g., ...) containing the heavy chain constant region and light chain constant region genes, respectively. Figure 3 As shown in SEQ ID NO:16), the heavy chain constant region is human IgG1 type (amino acid sequence shown in SEQ ID NO:16), and the light chain constant region is C. k Chain (amino acid sequence as shown in SEQ ID NO:19).

[0136] Humanized antibody molecules HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, HB-Ⅰ, HB-Ⅱ, and HB-Ⅲ were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. Results are shown below. Figure 10 As shown, from left to right, the following are the molecular weight markers for anti-LILRB4 monoclonal antibodies: 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. The molecular weight of each band is consistent with the theoretical values.

[0137] Example 16: Humanized Antibody Binding Experiment with LILRB4

[0138] LILRB4 was coated with carbonate buffer (pH 9.6), 100 ng / well / 100 μL, overnight at 4°C. The wells were washed five times with 300 μL / well PBST, then blocked with 1% BSA-PBST at 37°C for 1 hour. 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 were added at different dilutions. The initial maximum concentration of all eight antibodies was 50 μg / mL. Each antibody was diluted 5-fold, and then diluted 12 times. The wells were incubated at 37°C for 1 hour. The wells were washed five times with 300 μL / well PBST, then Goat Anti Human IgG-HRP (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZB-2304) diluted 1:5000 with 1% BSA-PBST was added, and the wells were incubated at 37°C for 1 hour. The TMB chromogenic kit was used for color development. 100 μL / well was incubated at room temperature for 5 minutes, then the development was stopped with 2M H₂SO₄. Readings were taken at 450 nm and 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0139] 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

[0140] Based on the above data and experimental results, as follows: Figure 11 As shown, all six different humanized antibody molecules can bind to LILRB4. Among them, the three humanized monoclonal antibodies HA-Ⅰ, HA-Ⅱ, and HA-Ⅲ have relatively similar affinities. Of 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. Meanwhile, the EC50 values ​​of HA-Ⅰ, HA-Ⅱ, and HA-Ⅲ are similar to those of the chimeric antibody CA-Ⅰ, and the EC50 value of HB-Ⅲ is similar to that of the chimeric antibody CB-Ⅰ, indicating that the humanized HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, and HB-Ⅲ retain the high affinity of the murine parent antibodies MA-Ⅰ and MB-Ⅰ for LILRB4.

[0141] Example 17: Binding experiment of humanized antibody to LILRB4 on the surface of THP-1 cells

[0142] Take 50 μL of antibodies of different dilutions (HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, HB-Ⅲ, CA-Ⅰ, and CB-Ⅰ), starting at a working concentration of 32 μg / mL, and perform serial dilutions of 2-fold for a total of 12 dilutions. Add the diluted solutions to 96-well V plates. Then add 50 μL of LHP-1 cell suspension to each well, at a concentration of 2 × 10⁻⁶. 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:

[0143] 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

[0144] 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.

[0145] Example 18: Humanized Antibody Competitively Binding to LILRB4 with ApoE

[0146] 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:

[0147] clone HA-Ⅰ HA-II HA-Ⅲ HB-Ⅲ IC50 (μg / mL) 0.4797 2.752 2.559 0.4265

[0148] 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.

[0149] Example 19: Humanized antibody inhibits THP-1 secretion of ARG1

[0150] 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:

[0151] clone HA-Ⅰ HA-II HA-Ⅲ HB-Ⅲ IC50 (μg / mL) 31.20 249.5 98.54 30.57

[0152] 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.

[0153] Example 20: Detection of the biological activity of humanized antibody molecules (reporter gene)

[0154] THP-1-NF-κB-Luc engineered cells were used at a concentration of 2×10⁻⁶. 6Cells / mL were seeded into 96-well plates with a V-bottom, with 50 μL of cell suspension added to each well. Then, 100 μL of four humanized antibody molecules (HA-Ⅰ, HA-Ⅱ, HA-Ⅲ, and HB-Ⅲ) at different dilutions were added to each well. The starting working concentration of the antibodies was 200 μg / mL, with 5-fold serial dilutions for a total of 8 gradients. After incubating at 37°C for 2 hours, 50 μL of 20 μg / mL ApoE protein was added to each well. The cell culture plate was gently mixed and incubated at 37°C for 5 hours. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and 50 μL of Lysis Buffer (purchased from Promega, catalog number: E2661) was added to each well according to the manufacturer's instructions. The cells were incubated at room temperature for 10 minutes for complete lysis. Transfer 10 μL of the fluorescent substrate (Promega, catalog number: E2610) to each well, and then add an equal volume of the fluorescent substrate to each well. Incubate at room temperature for 5 minutes, read the fluorescence values ​​using a microplate reader, 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) 0.7557 2.678 3.437 0.8302

[0156] Based on the above data and Figure 15 As shown, all four humanized antibody molecules screened can block the binding of ApoE to LILRB4, thereby inhibiting the transduction of the downstream NF-κB signaling pathway. Furthermore, among the four humanized antibody molecules provided by this invention, HA-Ⅰ and HB-Ⅲ have the lowest IC50 values, indicating that they can effectively inhibit the transduction of downstream signaling pathways.

[0157] Example 22

[0158] Example 22 of the present invention provides an injectable formulation of an anti-LILRB4 monoclonal antibody, which comprises the following components:

[0159]

[0160] The pH of the injectable formulation is 5.5-6.5.

[0161] Among them, the anti-LILRB4 monoclonal antibody is the humanized antibody molecule HA-Ⅰ screened in Examples 2-20, and its amino acid sequence is the same as that in Example 1.

[0162] The buffer salt is histidine-histidine hydrochloride buffer or acetate-sodium acetate buffer; the protein protectant is selected from one or more combinations of sorbitol, mannitol, trehalose or sucrose; the surfactant is selected from one or more combinations of polysorbate 20, polysorbate 80 or poloxamer.

[0163] Example 23 Screening of formulation buffer salts

[0164] Preparation method of anti-LILRB4 monoclonal antibody injection formulation: The purified anti-LILRB4 monoclonal antibody HA-Ⅰ was transferred to the target buffer salt via ultrafiltration tube. The concentrated anti-LILRB4 monoclonal antibody HA-Ⅰ sample was then diluted to the required concentration. The sample was aseptically filtered through a 0.22 μm filter and aliquoted into 2 ml vials, 1 ml / vial. After aliquoting, the thermal stability of the protein was tested, and the samples were incubated at 40±2℃ for purity and charge isomer stability testing.

[0165] Analysis and detection methods: Thermal stability: The thermal denaturation temperature (Tm) and aggregation temperature (Tagg) were detected using a multifunctional protein stability analysis system (Uncle);

[0166] Purity determination: Analysis was performed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

[0167] Charge isomers: The content of the main peak of charge isomers was determined by cation exchange chromatography (CEX-HPLC).

[0168] (1) Screening of buffer salt pH range

[0169] Screening was conducted within the pH range commonly used in commercial protein formulations. Protein conformational stability (Tm) and colloidal stability (Tagg) were assessed using a multifunctional protein stability analysis system (Uncle). Protein purity (SEC-HPLC) and charge isomer (CEX-HPLC) peak content changes were evaluated at 40±2℃ to screen for relatively stable pH ranges.

[0170] The buffer salt design consists of the following components:

[0171]

[0172] The thermal stability test results are as follows:

[0173]

[0174] Results of accelerated stability test at 40±2℃

[0175]

[0176] Thermal stability results showed that, within the buffer pH range of 5.0-6.5, the Tm and Tagg values ​​of the anti-LILRB4 monoclonal antibody HA-Ⅰ protein molecule were higher than those of other buffers.

[0177] Accelerated at 40±2℃ for 1 week, the purity (SEC-HPLC) of each formulation sample showed a decreasing trend with increasing pH. Examples 2 and 3 exhibited the highest content of the charge isomer main peak and significantly higher purity (SEC-HPLC) than other examples. Examples 4 and 5 showed a significant decrease in charge isomer content after pH exceeded 6.5. Therefore, considering both thermal stability and accelerated stability at 40±2℃, the anti-LILRB4 monoclonal antibody HA-Ⅰ protein showed good stability between pH 5.0 and pH 6.5. Future plans include screening for buffer salts between pH 5.0 and pH 6.5 in the formulation.

[0178] (2) Screening of the types of buffer salts used in the formulation

[0179] The selection of buffer salt systems and pH values ​​was carried out within a buffer salt range of approximately 5.0-6.5. The changes in protein purity (SEC-HPLC) and charge isomer (CEX-HPLC) main peak content were accelerated and observed at 40±2℃ to screen suitable buffer salt systems and relatively more stable pH values.

[0180] The buffer solution is designed with the following composition:

[0181]

[0182]

[0183] Results of accelerated stability test at 40±2℃:

[0184]

[0185] Accelerated stability testing showed that after 4 weeks at 40°C, the purity of all samples decreased to varying degrees. Sample 10 showed the smallest decrease in monomer purity (2.9%), while Sample 17 showed the largest decrease (4.4%). The decrease in monomer purity gradually increased with increasing pH, with Samples 9-11 showing a slightly smaller decrease than the others. After 4 weeks at 40±2°C, the acid content of all samples increased, with Sample 10 showing the smallest increase (6.9%) and Sample 12 showing the largest increase (13.2%). The main peak content of all samples decreased, with Sample 10 showing the smallest decrease (9.2%) and Sample 12 showing the largest decrease (18.1%). The alkaline content of all samples increased, with Sample 16 showing the smallest increase (0%) and Sample 12 showing the largest increase (5.0%). The results of the various investigations show that, in the above experimental examples, the protein can effectively maintain the stability of the anti-LILRB4 monoclonal antibody HA-Ⅰ protein under the conditions of histidine-histidine hydrochloride buffer and acetate-sodium acetate buffer at pH 5.5-6.5. Preferably, the pH range is 6.0-6.5, and histidine-histidine hydrochloride buffer is superior to acetate-sodium acetate buffer.

[0186] (3) Screening of protein protectants and surfactants.

[0187] Based on the previous screening of buffer salts and pH values, accelerated stability experiments were conducted at 40±2℃. Protein protectants and surfactants were then screened. The proposed buffer salt was 20mM histidine-histidine hydrochloride buffer, with a pH of 6.0. The concentration of the anti-LILRB4 monoclonal antibody HA-Ⅰ was 80mg / ml. The formulation composition design is shown in the table below.

[0188]

[0189] Results of accelerated stability test at 40±2℃

[0190]

[0191]

[0192] Accelerated stability results showed that after 4 weeks of storage at 40°C, the monomer purity of all experimental samples decreased to varying degrees. Compared to experimental samples 18-19 and 21-29, samples 20 and 30-32 showed the smallest decrease in monomer purity. Regarding charge isomers, the content of the main charge isomer peak decreased in all experimental samples, with sample 31 showing the smallest decrease. As for sub-visible particles, the total number of particles and the number of particles ≥25μm increased to varying degrees across different experimental samples. Samples 30-32 had significantly fewer particles than the other samples, indicating that the addition of surfactants significantly improved the protein particle condition. Thermal stability results showed that, except for samples 26-29, the Tm and Tagg values ​​of the other samples were relatively high, indicating good conformational and colloidal stability.

[0193] The results of the combined investigations showed no significant differences in monomer purity and charge isomer stability among the experimental cases with different protein protectants. However, experimental cases 19-22 with added mannitol, sorbitol, trehalose, and sucrose exhibited better performance in terms of sub-visible particles than those with added glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride, and sodium chloride (cases 23-29). Furthermore, experimental cases 30-32 with added surfactants showed significantly better performance in terms of sub-visible particles than those without surfactants. Therefore, mannitol, sorbitol, trehalose, and sucrose are the preferred protein protectants.

[0194] (4) Confirmation of protein protectants

[0195] Based on the protein protectant selected in the previous round, through B 22 (Second virial coefficient) and k D (Diffusion interaction parameters) were used to confirm the protein protectant. The proposed buffer salt was 20 mM histidine-histidine hydrochloride buffer, pH 6.0, with a protein concentration of 80 mg / ml. The composition design of the formulation experimental cases is shown in the table below:

[0196]

[0197] B 22 &k D result

[0198]

[0199] B in protein samples from each experimental group 22 and k D All values ​​are positive, and the B value of sample 34 in experiment 34 is positive. 22 and k D A higher value indicates a stronger weaker repulsion effect, and sorbitol has a better protective effect on proteins. Therefore, sorbitol has been initially selected as the protein protectant.

[0200] (5) Screening of surfactant content

[0201] Early formulation evaluation and screening revealed that the addition of surfactants significantly improved the effect on subvisible particles. Histidine-histidine hydrochloride buffer was selected as the formulation buffer system, sorbitol as the protein protectant, and polysorbate 20 as the surfactant. The surfactant content was screened through accelerated stability testing at 40±2℃. The formulation composition design is shown in the table below:

[0202] Results of accelerated stability test at 40±2℃

[0203]

[0204]

[0205] Note: In terms of appearance, A indicates "colorless, clear liquid, free of visible foreign matter"; B indicates "colorless, clear liquid, with slight particle content".

[0206] Accelerated stability testing showed that after 4 weeks of storage at 40±2℃, slight particle formation was observed without the addition of the nonionic surfactant polysorbate 20. However, with the addition of different concentrations of the nonionic surfactant polysorbate 20, the anti-LILRB4 monoclonal antibody HA-Ⅰ injection formulation exhibited excellent stability, remaining as a colorless, clear liquid without visible foreign matter. The total particle count and the number of particles ≥25μm were slightly higher with the absence of nonionic surfactant or with very low concentrations of nonionic surfactant, indicating a tendency for protein aggregation. Conversely, excessively high surfactant concentrations may cause side effects and affect the safety of the formulation infusion. Ultimately, a polysorbate 20 surfactant content of 0.01% was selected for the antibody formulation.

[0207] Therefore, through the above component screening, the preferred components of the anti-LILRB4 monoclonal antibody injection formulation provided by the present invention are as follows: 60-100 mg / mL of anti-LILRB4 monoclonal antibody, 20 mM of histidine-histidine hydrochloride buffer, 250 mM of sorbitol and 0.01% (w / v) of polysorbate 20, wherein the pH of the injection formulation is 6.0.

[0208] Among them, the anti-LILRB4 monoclonal antibody is the humanized antibody molecule HA-Ⅰ screened in Examples 2-20, and its amino acid sequence is the same as that in Example 1.

[0209] Example 24: Component identification of the anti-LILRB4 monoclonal antibody injection formulation (investigation of protein concentration)

[0210] Preparation method of protein liquid formulation: The purified anti-LILRB4 monoclonal antibody HA-Ⅰ was transferred to the buffer salt in the experimental case list through an ultrafiltration tube. Various excipients were added according to the requirements of the experimental case list, and the protein was diluted to the required concentration. The sample was aseptically filtered through a 0.22 μm filter and dispensed into 2 ml vials, 1 ml / vial. After dispensing, accelerated testing and influencing factor tests were performed, using purity, charge isomers, and subvisible particles as key indicators to confirm the protein concentration and experimental case. The composition design of the formulation experimental cases is shown in the table below:

[0211]

[0212] Analytical and testing methods:

[0213] Purity determination: Analyzed using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC); Charge isomers: Determined by cation exchange chromatography (CEX-HPLC) for the main peak content of charge isomers. Subvisible particles: Detected using microfluidic imaging. Confirmatory tests included accelerated stability testing (40±2℃), freeze-thaw stability testing (-20℃), shaking stability testing (2-8℃, 120 rpm), and light stability testing (25±2℃, RH 60%, 4500±500 lx).

[0214] 1) Results of accelerated stability test at 40±2℃

[0215]

[0216] Accelerated stability results showed that after 4 weeks of storage at 40±2℃, there were no significant differences in purity, charge isomers, total particle number, and number of sub-visible particles ≥25μm when the concentrations of anti-LILRB4 monoclonal antibody HA-Ⅰ protein were 60mg / ml, 80mg / ml, and 100mg / ml. The purity and charge isomers were slightly worse at a concentration of 120mg / ml.

[0217] 2) Results of freeze-thaw stability study

[0218]

[0219]

[0220] As can be seen from the table above, after the anti-LILRB4 monoclonal antibody HA-Ⅰ protein was frozen and thawed five times at -20℃, there were no significant changes in purity, charge isomer content, and number of subvisible particles under each formulation condition.

[0221] 3) Results of the shaking stability test

[0222]

[0223] As can be seen from the table above, after 7 days of horizontal shaking at 120 rpm under the conditions of 2-8℃, the purity, charge isomer content and sub-visible particle number of the anti-LILRB4 monoclonal antibody HA-Ⅰ protein did not change significantly under the conditions of each experimental example.

[0224] 4) Results of light stability study

[0225]

[0226] As shown in the table above, when the anti-LILRB4 monoclonal antibody HA-Ⅰ protein was placed at 25±2℃ and a light intensity of 4500±500lx for 10 days, there was no significant difference in purity, charge isomers, total particle number, and number of subvisible particles ≥25μm when the concentration of anti-LILRB4 monoclonal antibody HA-Ⅰ protein was 60mg / ml, 80mg / ml, and 100mg / ml; the purity and charge isomers were slightly worse when the concentration was 120mg / ml.

[0227] The above experiments demonstrate that the concentration of the anti-LILRB4 monoclonal antibody HA-Ⅰ protein is very stable in the range of 60-100 mg / ml, with the preferred protein concentration being 60 mg / ml-80 mg / ml.

[0228] Further tests, including accelerated testing at 40℃, freeze-thaw testing, shaking testing, and light exposure testing, verified that the anti-LILRB4 monoclonal antibody HA-Ⅰ with a protein concentration of 60 mg / ml-80 mg / ml exhibited better stability under the conditions of 20 mM histidine-histidine hydrochloride buffer, 250 mM sorbitol, 0.01% polysorbate 20, and pH 6.0.

[0229] This invention is not limited to the preferred embodiments described above. Anyone can derive other products in various forms under the guidance of this invention. However, regardless of any changes in shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. An injectable formulation of an anti-LILRB4 monoclonal antibody, characterized in that, This injectable formulation contains the following components: Anti-LILRB4 monoclonal antibody 60-100 mg / mL; Buffer salt 15-30 mM; Protein protectant 100-250mM; Surfactant 0.01%-0.04% (w / v); The pH of the injectable formulation is 5.5-6.5; 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 anti-LILRB4 monoclonal antibody injection formulation 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 anti-LILRB4 monoclonal antibody injection formulation 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 has 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 anti-LILRB4 monoclonal antibody injection formulation 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 anti-LILRB4 monoclonal antibody injection formulation 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, and 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 anti-LILRB4 monoclonal antibody injection formulation as described in claim 1, characterized in that, The buffer salt is histidine-histidine hydrochloride buffer salt or acetate-sodium acetate buffer salt.

7. The anti-LILRB4 monoclonal antibody injection formulation as described in claim 6, characterized in that, The buffer salt is histidine-histidine hydrochloride buffer salt.

8. The anti-LILRB4 monoclonal antibody injection formulation as described in claim 1, characterized in that, The protein protectant is selected from one or more combinations of sorbitol, mannitol, trehalose, or sucrose.

9. The anti-LILRB4 monoclonal antibody injection formulation as described in claim 8, characterized in that, The protein protectant is sorbitol.

10. The anti-LILRB4 monoclonal antibody injection formulation as described in claim 1, characterized in that, The surfactant is selected from one or more combinations of polysorbate 20, polysorbate 80, or poloxamer.

11. The anti-LILRB4 monoclonal antibody injection formulation as described in claim 10, characterized in that, The surfactant is 0.01-0.02% w / v polysorbate 20.

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