Anti-periostin humanized monoclonal antibody and preparation method and application thereof

Abstract

The present application provides an anti-periostin humanized monoclonal antibody and a preparation method and application thereof. The anti-periostin humanized monoclonal antibody has a high expression level and good specificity and affinity for binding to an antigen.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on September 13, 2021, with application number 202111068177.1 and invention name “Anti-periostin humanized monoclonal antibodies and preparation methods and applications thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of antibody drugs, and in particular to an anti-periostin humanized monoclonal antibody and a preparation method and application thereof. Background Art

[0003] Vitreoretinal diseases such as proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and age-related macular degeneration (AMD) are the main causes of vision loss and loss in the corresponding population at this stage, and are also a global medical problem. Therefore, finding new biological markers and therapeutic targets for vitreoretinal diseases is crucial for early detection, early diagnosis and early treatment of the disease.

[0004] Periostin is a cell adhesion protein found in mouse embryonic osteoblast precursor cells in 1993 and named osteoblast-specific factor-2 (OSF-2). Because it can be specifically expressed in the periodontal membrane and periosteum of adult mice, it was renamed periostin. Periostin is a unique extracellular matrix protein that participates in the corresponding immune inflammatory response of various tissues and organs in the human body, such as remodeling after myocardial infarction, bone marrow fibrosis, periodontal tissue regeneration and repair, skin wound healing, tumor cell metastasis, kidney damage, and can induce chronic allergic diseases. In recent years, the research and production of drugs targeting periostin has become a hot topic, and a large number of marketed drugs have achieved remarkable results in the treatment of tumors.

[0005] The main effects of periostin in the eye include promoting cell proliferation, differentiation, migration and adhesion, inducing fiber formation and promoting angiogenesis. More and more domestic and foreign studies have shown that periostin is a key factor in various vitreoretinal diseases. Periostin is expected to become a new biological marker for vitreoretinal diseases, thereby providing new treatment strategies. For example, it has been reported that Periostin antibodies have been proven to play a key role in the treatment of age-related macular degeneration and diabetic retinopathy-induced neovascularization and macular edema in many fields of ophthalmology.

[0006] However, all previous Periostin antibodies were mouse monoclonal antibodies. Mouse antibodies, as heterologous proteins, will cause immune rejection reactions against heterologous proteins when applied to the human body, induce the production of human anti-mouse antibodies, shorten the half-life of antibodies in the body, and affect the therapeutic effect of antibodies. Therefore, humanization of Periostin antibodies while maintaining high affinity for specific antigen epitopes can reduce the immunogenicity of heterologous antibodies, improve the specificity and affinity of binding to antigens, and thus achieve better therapeutic effects. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an anti-periostin humanized monoclonal antibody and a preparation method and application thereof.

[0008] The anti-periostin humanized monoclonal antibody provided by the present invention has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2.

[0009] The anti-periostin humanized monoclonal antibody provided by the present invention has a heavy chain constant region of human IgG1 and a light chain constant region of human κ type.

[0010] In some specific embodiments, the amino acid sequence of the heavy chain of the humanized antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain thereof is shown in SEQ ID NO:4.

[0011] The present invention utilizes periostin mouse monoclonal antibody, performs humanized transformation on it, successfully constructs eukaryotic expression plasmid, transfects mammalian CHO cells for secretory expression, and performs in vitro biological function evaluations such as affinity and binding activity by SDS-PAGE, ELISA, WB, etc., providing a theoretical basis for the study of large-scale expression of anti-periostin humanized monoclonal antibody in CHO cells.

[0012] The anti-periostin humanized monoclonal antibody provided by the present invention is composed of the above-mentioned heavy chain and light chain. After humanization, it is safer, more reliable and more functional than the chimeric antibody or other mouse-derived periostin monoclonal antibody in the prior art. After testing, the humanized antibody has good specificity for human periostin. In view of the fact that Periostin antibodies have been proven to play a key role in the treatment of age-related macular degeneration and diabetic retinopathy in many fields of ophthalmology, neovascularization and hyperplasia in the eye, macular edema, etc. Therefore, the antibody provided by the present invention has the potential to treat tissue fibrosis.

[0013] The present invention also provides nucleic acids encoding the monoclonal antibodies. Specifically, the present invention provides nucleic acids encoding the heavy chain of the monoclonal antibody, nucleic acids encoding the light chain of the monoclonal antibody, nucleic acids encoding the heavy chain variable region of the monoclonal antibody, and / or nucleic acids encoding the light chain variable region of the monoclonal antibody. In some embodiments, the nucleic acid encoding the heavy chain of the antibody has a nucleic acid sequence as shown in SEQ ID NO:5. The nucleic acid encoding the light chain of the antibody has a nucleic acid sequence as shown in SEQ ID NO:6. In order to facilitate protein expression, the 5' end of the encoding nucleic acid provided by the present invention also includes a linker sequence. Therefore, in some specific embodiments, the nucleic acid encoding the heavy chain of the anti-periostin humanized monoclonal antibody has a nucleic acid sequence as shown in SEQ ID NO:7; the light chain nucleic acid encoding the anti-periostin humanized monoclonal antibody has a nucleic acid sequence as shown in SEQ ID NO:8.

[0014] The present invention also provides an expression vector, comprising a nucleic acid encoding the monoclonal antibody. In the present invention, the expression vector also includes a backbone vector; in some embodiments, the backbone vector is pCDNA3.4.

[0015] The present invention also provides a host cell transformed or transfected with the expression vector. In the present invention, the host cell is a mammalian cell, and in some specific embodiments, the host cell is a CHO-K1 cell.

[0016] The method for preparing the anti-periostin humanized monoclonal antibody of the present invention comprises: culturing the host cell of the present invention and inducing the expression of the anti-periostin humanized monoclonal antibody.

[0017] The present invention also provides the use of anti-periostin humanized monoclonal antibodies in the preparation of drugs or detection reagents, wherein the drugs are drugs for treating tissue fibrosis and / or malignant tumors. The reagents are reagents for diagnosing tissue fibrosis and / or malignant tumors. In some embodiments, the tissue fibrosis is retinal fibrosis.

[0018] The present invention provides a medicine, which comprises the anti-periostin humanized monoclonal antibody of the present invention.

[0019] The present invention also provides a kit, which contains an anti-periostin humanized monoclonal antibody, or a conjugate formed by the antibody and a chemical marker or a biological marker, or a conjugate prepared by coupling the anti-periostin humanized monoclonal antibody with a solid medium or a semi-solid medium.

[0020] The chemical label in the present invention is an isotope, an immunotoxin and / or a chemical drug; the biological label is biotin, avidin or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe agglutinin, radix fasciatus toxin, PAP, herbivores, gelonin or luffa toxin. The solid medium or non-solid medium in the conjugate is selected from colloidal gold, polystyrene plates or beads. The kit can be used for the diagnosis of tissue fibrosis and / or malignant tumors. The diagnostic method adopts an immunological detection method, such as ELISA, western blot, immunofluorescence technology, immunohistochemistry detection, etc.

[0021] The present invention also provides a method for treating tissue fibrosis and / or malignant tumors, which comprises administering the drug of the present invention.

[0022] The present invention also provides a method for diagnosing tissue fibrosis and / or malignant tumors, which is to use the diagnostic reagent of the present invention to diagnose

[0023] The present invention provides an anti-periostin humanized monoclonal antibody, which retains only the complementary determining region in the variable region of mouse-derived McAb that binds to the antigen, and prepares a CDR-transplanted antibody. Compared with chimeric antibodies, the immunogenicity thereof is lower. Compared with other antibodies in the experiment, the antibody obtained by the screening of the present invention has a higher expression amount, better specificity and affinity for binding to the antigen, and can better participate in the human immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown are the results of PCR amplification of VH and VL of mouse hybridoma cells (45-2-G3-1-G7-B7);

[0025] Figure 2 Shows the PCR results after cloning;

[0026] Figure 3-1 and Figure 3-2 The plasmid maps of heavy and light chains are shown;

[0027] Figure 4 Show purification effect;

[0028] Figure 5 ELISA results of 18 humanized antibodies are shown;

[0029] Figure 6 The electrophoresis test results of the expression vector are shown;

[0030] Figure 7 The figure shows the electrophoresis detection result of the linearized expression vector;

[0031] Figure 8 Shows the results of protein expression and purification after pressure screening;

[0032] Fig. 9 The results of cell expression protein purification obtained by screening are shown;

[0033] Fig.10 Shows the identification of humanized anti-periostin antibodies 5-G4, 5-G6, and 5-G9;

[0034] Fig.11 The results of microtubule inhibition experiments are shown, wherein the Periostin antibody is a humanized monoclonal antibody, and the control Periostin is a mouse monoclonal antibody;

[0035] Fig.12 Show the results of cell migration experiment;

[0036] Fig.13 Shows the cell migration rate relative to the blank control group;

[0037] Fig.14 Show the results of cell invasion assay;

[0038] Fig.15 Shows the cell invasion rate relative to the blank control group;

[0039] Fig.16 Shows the results of cell scratch test;

[0040] Fig.17 Shows the relative migration distance of cells (%);

[0041] Fig.18 The WB detection diagrams of the expression of VEGFA, α-SMA, Col I, Col III, and Fibronectin proteins in RPE cells of different groups are shown;

[0042] Fig.19 Show the expression levels of VEGFA, α-SMA, Col I, Col III, and Fibronectin proteins in RPE cells of different groups;

[0043] Fig. 20 The mRNA expression levels of αvβ3, αvβ5 integrins, VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT on the vascular endothelial cell membrane of each group are shown;

[0044] Fig.21 Shown are Western blot detection images of various proteins in HUVEC cells of different groups;

[0045] Fig. 22 Shows the expression levels of each protein in HUVEC cells of different groups;

[0046] Fig.23 The expression of FAK protein in HUVEC cells was detected by IF;

[0047] Fig.24 IF detection of VEGFR2 protein expression in HUVEC cells;

[0048] Fig.25 The expression of αvβ3 protein in HUVEC cells was detected by IF;

[0049] Fig.26 Figure 5 shows the expression of αvβ5 protein in HUVEC cells detected by IF. DETAILED DESCRIPTION

[0050] The present invention provides anti-periostin humanized monoclonal antibodies and preparation methods and applications thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in this area, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0052] "Antibody" refers to a protein composed of one or more polypeptides that can specifically bind to an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light chain and the heavy chain are jointly responsible for binding to the antigen, while the constant region is responsible for the effector function of the antibody. The "monoclonal antibody" refers to a preparation of antibody molecules with a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a specific epitope.

[0053] The "variable region" of an antibody heavy or light chain is the N-terminal mature region of the chain. Currently known antibody types include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or their equivalents. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) comprises an NH 2 -terminal variable region of about 110 amino acids, and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids), also comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, such as gamma (about 330 amino acids).

[0054] "Antibodies" include antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. The present invention provides a humanized monoclonal antibody to periostin. Total RNA was extracted from cultured hybridoma cells 45-2-G3-1-G7-B7, and cDNA was synthesized by reverse transcription using oligonucleotide primers. VH and VL fragments were amplified using IgG degenerate primers and kappa-specific primers, respectively, and the results were observed by gel electrophoresis ( Figure 1 ). The PCR product was then subcloned into a standard vector for clone picking and PCR verification ( Figure 2 ), and finally sequence the positive clones. Using gene synthesis, the framework region of the variable region gene of the anti-Periostin mouse monoclonal antibody was replaced with the framework region of the human antibody, and the humanized antibody variable region gene fragment was recombined into a vector containing a regulatory sequence and a human antibody constant region gene, to construct the complete gene of the anti-Periostin humanized monoclonal antibody and a eukaryotic expression vector containing the gene, and then transfected into CHO-K1 cells. After GS system pressure screening, CHO-K1 cells that continuously and stably secrete humanized antibodies were obtained.

[0055] The amino acid sequence of the heavy chain variable region of the anti-periostin humanized monoclonal antibody of the present invention is: EVQLVQSGAEVKKPGESLKISCKASGYSFTDYFMNWVRQMPGKGLEWIGRINPYSGDTLYNQRLQGQVTLSADKSISTAYLQLSSLKASDTAMYYCGRSGVSGLDYWGQGTLVTVSS (SEQ ID NO: 1).

[0056] The amino acid sequence of the light chain variable region of the anti-periostin humanized monoclonal antibody of the present invention is: DIQLTQSPSSLSASVGDRVTITCSASSSASYMHWYQQKPGKAPKNWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPPTFGGGTKVEIK (SEQ ID NO: 2).

[0057] The amino acid sequence of the heavy chain constant region of the anti-periostin humanized monoclonal antibody of the present invention is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0058] The amino acid sequence of the light chain constant region of the anti-periostin humanized monoclonal antibody of the present invention is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0059] The amino acid sequence of the heavy chain of the anti-periostin humanized monoclonal antibody of the present invention is: MKHLWFFLLLVAAPRWVLSEVQLVQSGAEVKKPGESLKISCKASGYSFTDYFMNWVRQMPGKGLEWIGRINPYSGDTLYNQRLQGQVTLSADKSISTAYLQLSSLKASDTAMYYCGRSGVSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3).

[0060] The amino acid sequence of the light chain of the anti-periostin humanized monoclonal antibody of the present invention is: MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCSASSSASYMHWYQQKPGKAPKNWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0061] In order to facilitate the expression and purification of the antibody, during the vector construction and protein expression process, a linker fragment is added to the N-terminus of the heavy chain fragment and the light chain fragment, respectively. The fragment is removed during the subsequent purification process. In some embodiments, the amino acid sequence of the linker added to the N-terminus of the heavy chain of the anti-periostin humanized monoclonal antibody is MKHLWFFLLLVAAPRWVLS, and the amino acid sequence of the linker added to the N-terminus of the light chain of the anti-periostin humanized monoclonal antibody is MVLQTQVFISLLLWISGAYG.

[0062] The antibody can be labeled and detected, for example, by radioisotopes, enzymes that can produce detectable substances, fluorescent proteins, biotin, etc. The antibody can also be bound to a solid support, including but not limited to polystyrene plates or beads, etc.

[0063] The medicine of the present invention contains at least one functional ingredient and also includes a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycol such as polypropylene glycol, triglyceride, etc. The type of pharmaceutically acceptable carrier used depends in particular on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0064] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0065] Example 1

[0066] 1. Humanized antibody design

[0067] (1) CDR region sequence identification

[0068] All mRNA was isolated from mouse hybridoma cells (45-2-G3-1-G7-B7, for the construction method, see "Anti-human periostin monoclonal chimeric antibody and application", application number: 202010788885.1, from Shenyang He's Eye Industry Group Co., Ltd. or Shenyang Eye Industry Technology Research Institute Co., Ltd.), and used as a template for synthesizing cDNA, and the heavy chain variable region DNA fragment and the light chain variable region DNA fragment were separated from the cDNA, and then the obtained DNA fragments were cloned into the vector and sequenced to obtain the sequence results as follows:

[0069] Amino Acid Sequence:

[0070] >45-2-G3-1-G7-B7-VL1

[0071] DIVLTQSPAIMSASPGDKVTMTCSASSSASYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK

[0072] >45-2-G3-1-G7-B7-VL2 and VL3

[0073] QIVLTQSPVIMSASPGDKVTMTCSASSSASYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIK

[0074] >45-2-G3-1-G7-B7-VH

[0075] EVQLQQSGPELVKPGASVRISCKASGYSFTDYFMNWVKQSHGRSLEWIGRINPYSGDTLYNQRLKGKATLTVDKSSSTAHMELLSLTSEDSAVYYCGRSGVSGLDYWGQGTSVTVSS

[0076] The amino acid sequences of the three CDR regions in the light chain variable region are: SSASY, DTS, and QQWSSNPPT.

[0077] The amino acid sequences of the three CDR regions in the heavy chain variable region are: GYSFTDYF, INPYSGDT, GRSGVSGLDY.

[0078] (2) Sequence alignment and mouse antibody CDR transplantation

[0079] Use Igblast (https: / / www.ncbi.nlm.nih.gov / igblast / ) developed by NCBI to search the IMGT mouse and human V gene databases, input the mouse antibody variable region sequence, and determine the human antibody variable region with the highest homology after sequence alignment. Then transplant the mouse antibody CDR region to the human framework acceptor region to complete the CDR transplantation.

[0080] 2. Construction and expression of 18 humanized antibody vectors

[0081] (1) Gene synthesis

[0082] The variable region sequence was linked to the human constant region DNA sequence and inserted into the pcDNA3.4 expression vector to obtain the plasmid map ( Figure 3-1 and Figure 3-2 ), the heavy chain and light chain nucleic acid sequences and amino acid sequences of the periostin antibody are shown below.

[0083] (2) Subcloning into expression vector

[0084] After gene synthesis, the CHO system codons were optimized and subcloned into the expression vector PATX1.

[0085] Nucleotide sequence information includes:

[0086] DNA sequence

[0087] >12-VHA-1423bp

[0088]

[0089] >12-VHB-1423bp

[0090]

[0091] >459-VHA-1423bp

[0092] gaattcgccgccaccATGAAGCACCTGTGGTTTTTCCTGCTGCTGGTGGCCGCCCCCCGGTGG

[0093] GTGCTGTCCCAGGTGCAGCTGCAGGAGAGCGGCCCTGGCCTGGTGAAGCCCAGCGA

[0094] GACCCTGAGCCTGACCTGCACCGCTTCCGGCTACAGCTTCACCGATTACTTCATGAAC

[0095] TGGGTGCGGCAGCCCCCTGGCAAGGGCTTGGAGTGGATCGGCAGGATCAACCCTTAC

[0096] AGCGGCGACACCCTGTATAATCAGCGGCTGAAGGGCAGGGTGACCCTGAGCGTGGAT

[0097] AAGAGCAAGAACCAGGCCAGCCTGAAGCTGAGCAGCGTGACCGCTGCCGATACCGC

[0098] CGTGTATTATTGTGGCCGGTCCGGCGTGAGCGGCCTGGATTACTGGGGCCAGGGCAC

[0099] CCTGGTGACCGTGAGCAGCgctagcACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTT

[0100] CTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTT

[0101] CCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATAC

[0102] TTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTG

[0103] CCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCA

[0104] ACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGT

[0105] CCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTA

[0106] AGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCG

[0107] ATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAG

[0108] TGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTG

[0109] GTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGC

[0110] AAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAA

[0111] GGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGAC

[0112] AAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGC

[0113] CGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGT

[0114] CCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAG

[0115] GTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCA

[0116] CTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC

[0117] >459-VHB-1423bp

[0118]

[0119] >459-VHC-1423bp

[0120] gaattcgccgccaccATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCCCCCAGGTGGGTTCTGAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTAGCGAGACCCTGTCCCTGACCTGCACCGTGTCCGGCGGCTCCATCACCGATTATTTCATGAACTGGATCAGGCAGCCCCCTGGCAAGGGCCTGGAGTGGATCGGCCGGATCAATCCCTATAGCGGCGACACCCTGTACAATCAGCGGCTGAAGAGCAGGGTGACCCTGAGCGTGGATAAGTCCAAGAATCAGGCCAGCCTGAAGCTGTCCTCCGTGACCGCCGCCGACACCGCTGTGTACTACTGCGGCCGGTCCGGCGTGAGCGGCTTGGATTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCgctagcACCAAGGGACCTTCTGTGTTCCCTCTGGCTCCTTCTTCTAAGTCCACTTCCGGTGGTACAGCAGCTCTGGGTTGTCTGGTGAAGGATTACTTCCCAGAACCAGTGACTGTGTCCTGGAACTCCGGAGCTCTGACTTCTGGAGTGCATACTTTCCCAGCAGTGCTGCAATCTAGCGGACTGTACTCTCTGTCTTCCGTGGTGACTGTGCCTTCTTCTTCCCTGGGGACTCAAACTTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCAAAGAGCTGCGATAAGACCCACACCTGTCCACCTTGTCCAGCTCCAGAACTGCTGGGTGGGCCTTCTGTGTTTCTGTTCCCACCTAAGCCAAAGGATACCCTGATGATCTCTAGGACCCCAGAAGTGACCTGTGTGGTCGTCGATGTGTCTCATGAAGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGGGTGGAAGTGCATAACGCAAAGACCAAGCCCAGGGAAGAGCAATACAACTCCACCTACAGGGTGGTCTCCGTCCTGACAGTCCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAATAAAGCCCTGCCTGCCCCTATCGAGAAAACCATTAGCAAAGCCAAAGGCCAGCCCAGGGAGCCCCAGGTCTATACACTGCCCCCCAGCAGGGAGGAGATGACAAAAAATCAGGTCAGCCTGACATGCCTGGTCAAAGGCTTTTATCCCAGCGACATTGCCGTCGAGTGGGAGTCCAATGGCCAGCCCGAGAATAATTATAAAACAACACCCCCCGTCCTGGACAGCGACGGCAGCTTTTTTCTGTATAGCAAACTGACAGTCGATAAAAGCAGGTGGCAGCAGGGCAATGTCTTTTCCTGCAGCGTCATGCACGAGGCCCTGCACAATCACTATACTCAGAAAAGCCTGAGCCTGTCCCCCGGGAAATGAGCGGCCGC

[0121] >551-VHB-1423bp

[0122]

[0123] >621-VLA-725bp

[0124] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCAGCGGCGCCTATGGCGAGATCGTGCTGACCCAGTCCCCTGATTTCCAGAGCGTGACCCCTAAGGAGAAGGTGACCATCACCTGCAGCGCCAGCAGCTCCGCCAGCTATATGCACTGGTACCAGCAGAAGCCCGACCAGTCCCCTAAGCGGTGGATCTATGACACCAGCAAGCTGGCTAGCGGCGTGCCTAGCAGGTTCTCCGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAACTCCCTGGAGGCTGAGGACGCCGCCACCTATTACTGCCAGCAGTGGAGCTCCAACCCCCCTACCTTTGGCGGCGGCACCAAGGTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0125] >311-VLA-725bp

[0126] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCAGCGGCGCCTACGGCGAGATCGTGCTGACCCAGTCCCCCGCCACCCTGTCCCTGTCCCCAGGAGAGAGGGCTACCCTGAGCTGCTCCGCCAGCTCCAGCGCCTCCTACATCCACTGGTACCAGCAGAAGCCTGGCCAGGCCCCTCGGAGATGGATGTACGATACCTCCAAGCTGGCCTCCGGCATCCCCGCCAGATTCAGCGGCAGCGGCAGCGGAACCGATTACACCCTGACCATCAGCTCCCTGGAGCCTGAGGACGCCGCCGTGTACTACTGCCAGCAGTGGAGCAGCAACCCTCCTACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0127] >139-VLA-725bp

[0128] gaattcgccgccaccATGGTGCTGCAGACCCAGGTGTTTATCAGCCTGCTGCTGTGGATCAGCGGCGCTTACGGCGACATCCAGCTGACCCAGTCCCCCTCCAGCCTGAGCGCTAGCGTGGGCGACCGGGTGACCATCACCTGCTCCGCCTCCAGCTCCGCCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAACTGGATCTATGATACCAGCAAGCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGATTACACCCTGACCATCAGCTCCCTGCAGCCCGAGGATGCTGCCACCTACTACTGCCAGCAGTGGTCCTCCAACCCCCCTACCTTTGGCGGCGGCACCAAGGTGGAGATCAAGcgtacgGTGGCTGCACCTTCTGTGTTCATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGAACCGCATCTGTCGTCTGTCTGCTGAACAACTTTTACCCCAGGGAGGCTAAGGTCCAATGGAAGGTGGACAACGCCCTGCAGTCTGGTAATAGCCAGGAAAGCGTGACCGAACAGGATTCCAAGGACTCCACCTACTCCCTGTCCTCCACACTGACACTGAGCAAAGCCGACTATGAAAAGCACAAAGTGTATGCCTGCGAGGTCACTCATCAGGGCCTGTCCAGCCCCGTGACTAAAAGCTTTAATAGGGGGGAGTGCTGAGCGGCCGC

[0129] >12-VHA

[0130] MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFMNWVRQAPGQGLEWIGRINPYSGDTLYNQRLKGRATLTVDKSISTAYMELSRLRSDDTAVYYCGRSGVSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEKPKSCDK THTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK;SIP:[1:19],Human IgG1 constant region:[137:466]

[0131] >12-VHB

[0132] MKHLWFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFMNWVRQAPGQGLEWIGRINPYSGDTLYNQKLQGRVTMTVDKSISTAYMELSRLRSDDTAVYYCGRSGVSGLDYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;SIP:[1:19],Human IgG1 constant region:[137:466]

[0133] >459-VHA

[0134] MKHLWFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTASGYSFTDYFMNWVRQPPGKGLEWIGRINPYSGDTLYNQRLKGRVTLSVDKSKNQASLKLSSVTAADTAVYYCGRSGVSGLDYWGQGTLVTSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;SIP:[1:19],Human IgG1 constant region:[137:466]

[0135] >459-VHB

[0136] MKHLWFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTASGYSFTDYFMNWIRQPPGKGLEWIGRINPYSGDTLYNQRLKSRVTLSVDKSKNQASLKLSSVTAADTAVYYCGRSGVSGLDYWGQGTLVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;SIP:[1:19],Human IgG1 constant region:[137:466]

[0137] >459-VHC

[0138] MKHLWFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSITDYFMNWIRQPPGKGLEWIGRINPYSGDTLYNQRLKSRVTLSVDKSKNQASLKLSSVTAADTAVYYCGRSGVSGLDYWGQGTLVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,SIP:[1:19,Human IgG1 constant region:[137:466]

[0139] >551-VHB

[0140] MKHLWFFLLLVAAPRWVLSEVQLVQSGAEVKKPGESLKISCKASGYSFTDYFMNWVRQMPGKGLEWIGRINPYSGDTLYNQRLQGQVTLSADKSISTAYLQLSSLKASDTAMYYCGRSGVSGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,SIP:[1:19],Human IgG1 constant region:[137:466]

[0141] >621-VLA

[0142] MVLQTQVFISLLLWISGAYGEIVLTQSPDFQSVTPKEKVTITCSASSSASYMHWYQQKPDQSPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTINSLEAEDAATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,SIP:[1:20],Human kappa constant region:[127:233]

[0143] >311-VLA

[0144] MVLQTQVFISLLLWISGAYGEIVLTQSPATLSLSPGERATLSCSASSSASYIHWYQQKPGQAPRRWMYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSNPPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,SIP:[1:20],Human kappa constant region:[127:233]

[0145] >139-VLA

[0146] MVLQTQVFISLLLWISGAYGDIQLTQSPSSSLSASVGDRVTITCSASSSASYMHWYQQKPGKAPKNWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPPTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,SIP:[1:20],Human kappa constant region:[127:233]

[0147] (3) Instantaneous transfer

[0148] Endotoxin-free plasmid DNA was extracted, and the constructed vector was co-transfected into CHO-K1 cells and cultured until the survival rate dropped below 50%. The culture supernatant was collected from some cells, purified with protein G, and then run on SDS-PAGE gel, and the activity of the antibody was detected by ELISA using antigen protein.

[0149] Transfection method:

[0150] ① One day before transfection, inoculate 27×10 6 The cells were cultured in complete medium (without antibiotics and anti-aggregation agents) so that the cells were in the logarithmic growth phase on the day of transfection.

[0151] ② Preparation of transfection complex

[0152] aDilute DNA into complete medium and mix gently.

[0153] b Dilute PEI transfection reagent into complete culture medium, mix gently, and incubate at room temperature for 5 minutes.

[0154] c. Add diluted PEI to the diluted DNA to a total volume of 3 ml for transfection. Mix gently and incubate at room temperature for 20-30 min.

[0155] ③ Transfection: Add 3 ml of transfection mixture to 27 ml of cell suspension, mix gently, and incubate at 37°C, 5% CO 2 , cultured at 130 rpm until the cell viability dropped below 50% and then collected the samples.

[0156] Transfection information:

[0157]

[0158]

[0159] (4) Purification

[0160] The supernatant was collected by centrifugation and loaded into 18 1ml protein A purification columns. The filler was balanced with PBS pH 7.5. Finally, the protein A resin was mixed with the culture supernatant and incubated overnight. After the incubation, it was purified. Washed with PBS, pH 7.5 buffer, eluted with 20mM citric acid pH 2.7 buffer, and neutralized with 1M Tris HCl, pH 9.0 to 6.0. After the purification was completed, 20ul of each of the IN, FT, washing and elution samples were run on SDS-PAGE. The samples were collected according to SDS-PAGE and dialyzed overnight. The samples were then pooled and buffer exchanged and concentrated. The final results are as follows: Figure 4 shown.

[0161] Sample Information

[0162] Serial number Antibody Concentration Quantity Number Yield 1 12-VHA / 621-VLA 1.05mg / ml 0.50mg / vial 1vial 0.50mg 2 12-VHA / 311-VLA 1.16mg / ml 0.50mg / vial 1vial 0.50mg 3 12-VHA / 139-VLA 1.31mg / ml 0.50mg / vial 1vial 0.50mg 4 12-VHB / 621-VLA 1.01mg / ml 0.50mg / vial 1vial 0.50mg 5 12-VHB / 311-VLA 1.00mg / ml 0.50mg / vial 1vial 0.50mg 6 12-VHB / 139-VLA 1.13mg / ml 0.50mg / vial 1vial 0.50mg 7 459-VHA / 621-VLA 0.96mg / ml 0.50mg / vial 1vial 0.50mg 8 459-VHA / 311-VLA 1.02mg / ml 0.50mg / vial 1vial 0.50mg 9 459-VHA / 139-VLA 1.07mg / ml 0.50mg / vial 1vial 0.50mg 10 459-VHB / 621-VLA 0.80mg / ml 0.50mg / vial 1vial 0.50mg 11 459-VHB / 311-VLA 1.13mg / ml 0.50mg / vial 1vial 0.50mg 12 459-VHB / 139-VLA 1.25mg / ml 0.50mg / vial 1vial 0.50mg 13 459-VHC / 621-VLA 0.69mg / ml 0.50mg / vial 1vial 0.50mg 14 459-VHC / 311-VLA 1.00mg / ml 0.50mg / vial 1vial 0.50mg 15 459-VHC / 139-VLA 1.16mg / ml 0.50mg / vial 1vial 0.50mg 16 551-VHB / 621-VLA 1.78mg / ml 0.50mg / vial 1vial 0.50mg 17 551-VHB / 311-VLA 1.71mg / ml 0.50mg / vial 1vial 0.50mg 18 551-VHB / 139-VLA 1.80mg / ml 0.50mg / vial 1vial 0.50mg

[0163] Western Blot verified the specific binding, where 1-18 are 18 humanized monoclonal antibodies, 19009414P 11 (human IgG1) is a periostin chimeric antibody, and 45-2-G3-1-G7-B7 is a periostin mouse monoclonal antibody. The results are shown in the following table and Figure 5 shown.

[0164]

[0165]

[0166] Humanized antibody ELISA layout

[0167]

[0168] ELISA test results

[0169] 1 2 3 4 5 6 7 8 2.2676 2.1962 2.1325 1.0578 1.0122 1.0485 2.0413 1.7380 2.3149 2.2910 2.1548 1.9094 1.9964 1.9358 1.9902 1.6759 1.8726 1.9203 1.8613 2.0560 1.8604 1.8602 1.9714 1.6779 2.1021 1.9955 1.9839 0.4797 0.4511 0.4934 1.7166 0.7665 2.1828 2.1139 2.0769 1.1582 1.0934 1.0282 1.7625 0.7699 2.0264 2.1278 2.1131 0.6429 0.5133 0.5998 1.7652 0.6939 1.8504 1.9261 1.9378 0.0641 0.0639 0.0626 0.0619 0.0555 2.0901 2.2168 2.1762 2.0171 2.1129 2.0738 0.0554 0.0567

[0170] 3. Construction and expression of humanized antibody stable strains

[0171] (1) Vector construction

[0172] From the results of the transient transfection test in the previous step, it can be seen that the expression level of antibody No. 18 551-VHB / 139-VLA is the highest, and the binding ability can meet the requirements. Therefore, after comprehensively considering the expression level and antibody binding ability, 551-VHB and 139-VLA were used as the heavy chain and light chain of the periostin humanized antibody, subcloned into the expression vector pATX-GS2, and then co-transfected into CHO cells to express the antibody protein. The agarose gel electrophoresis running gel results are shown in Figure 2. Figure 6 .

[0173] (2) Expression of humanized periostin antibody

[0174] The target fragment was double-digested (EcoR I / Not I), ligated, and then subcloned into the expression vector pATX-GS2, and then stably transfected into CHO cells. A stable cell line was obtained by MSX pressure screening and ELISA limiting dilution. After 7 days of culture in a shaker at 37°C and 5% carbon dioxide, the supernatant was collected and the humanized antibody in the supernatant was purified using agarose gel Protein A chromatography filler.

[0175] ①Plasmid linearization

[0176] Take out 10ul of the extracted plasmid PATX-GS2-139-VLA-551-VHB in advance as a backup (to verify whether the linearization is successful), add the enzyme and buffer to the plasmid in turn, and mix well; insert the EP tube into the float and put it in a 37-degree water bath for enzyme digestion for about 1-2 hours. After successful enzyme digestion, it is necessary to recover: add 0.75 times isopropanol according to the volume of the plasmid, mix well; centrifuge at 4 degrees for 30 minutes and discard the supernatant; add 1ml 70% ethanol and centrifuge at 4 degrees for 5 minutes, run the gel to measure the concentration ( Figure 7 ).

[0177] Enzyme digestion system:

[0178] Reagent name Volume(ul) Enzyme digestion buffer 10 Plasmids all Enzymes 6-10

[0179] ② Transfection

[0180] To obtain a stable pool of transfected cells expressing recombinant antibodies, CHO-K1 cells were transfected with the PVUI-linearized plasmid pATX-GS2-139-VLA-551-VHB and chemically transfected (FectoCHO TM Expression system transfection kit).

[0181] (3) Pressurized screening

[0182] 48 hours after transfection, the transfected host cells are subjected to corresponding selection pressure screening. The present invention uses the GS screening system (glutamine synthetase Gene Expression System). The cells are subjected to pressure treatment and treated with 30um concentration of MSX: the natural resistance of CHO-K1 cell line to MSX is determined by the 6-well plate standard batch culture method, and the minimum killing concentration of MSX for the untransfected cell line CHO-K1 is 20uM. Add 30um concentration of MSX, replace new screening medium every 3 to 4 days until resistant stable cells appear, and produce 3 stable pools after 2 to 3 weeks of MSX screening.

[0183] Cells were cultured in 30 ml of selective medium in 125 ml shake flasks under standard conditions (37°C, 5% CO 2 When the cells were in good condition in the shake flask, a 30 ml fed-batch expression experiment was performed. Feed medium was added on D3, D5, D7 and D9 and blood glucose was monitored throughout the process. The culture was continued for 3 generations until the cell viability dropped below 50%. The culture was terminated and the culture supernatant was collected for purification analysis. The purification results are shown in Figure 8 As shown, reduced shows the results of reduced SDS-PAGE electrophoresis, and non-reduced shows the results of non-reduced SDS-PAGE electrophoresis.

[0184] Sample Information:

[0185] Antibody Concentration Specification Quantity Yield 18-stable1 0.86mg / ml 1.75ml / vial 4vials 6.02mg / 30ml 18-stable2 0.94mg / ml 1.80ml / vial 4vials 6.76mg / 30ml 18-stable3 1.62mg / ml 1.80ml / vial 4vials 11.66mg / 30ml

[0186] (4) Isolation of monoclonal antibodies by limiting dilution method: Select the Stable pool 3 polyclonal cell line for limited dilution monoclonal pressure plating, dilute the cell suspension of the stable transfection pool to a very low density, so that an average of 1 cell is inoculated in each well. Use ELISA to screen out 7 monoclonal cell lines with high expression levels, including 5-G4, 5-E6, 5-G6, 5-D7, 5-G9, 2-H3, and 2-C7, for subsequent expansion culture. With a 1:3 dilution gradient, the antibody concentrations are 1, 0.33, 0.11, 0.037, 0.012, 0.004, 0.0014ug / ml, and a blank control, for a total of 8 gradients.

[0187] Monoclonal clones were observed under a microscope and positive monoclonal clones were detected by ELSIA method. The results are shown in the table below.

[0188] Antibody ELISA results:

[0189]

[0190] Cells of clones 5-G4, 2-C7, 5-E6, 5-G9, 2-H3, 5-D7, and 5-G6 were cultured in 30 ml selective medium in a 125 ml shake flask and cultured under standard conditions (37°C, 5% carbon dioxide, 130 rpm) for 3 generations. When the cells were in good condition in the shake flask, a 30 ml fed-batch expression experiment was performed. Feed medium was added on D3, D5, D7, and D9. Blood glucose was monitored throughout the process. The culture medium was collected until the cell viability dropped below 50%. The purification results are shown in Figure 2. Fig. 9 :

[0191] Sample Information:

[0192] Antibody Concentration Volume Quantity Yield Yield 5-E6 4.49mg / ml 1.63ml / vial 7vials 51.23mg / 30ml 1.70g / L 5-G4 7.13mg / ml 1.64ml / vial 7vials 81.86mg / 30ml 2.72g / L 2-H3 3.91mg / ml 1.67ml / vial 7vials 45.71mg / 30ml 1.52g / L 5-D7 4.63mg / ml 1.55ml / vial 6vials 43.06mg / 30ml 1.44g / L 2-C7 3.64mg / ml 1.63ml / vial 7vials 41.53mg / 30ml 1.38g / L 5-G6 5.07mg / m 1.65ml / vial 7vials 58.56mg / 30ml 1.95g / L 5-G9 6.14 mg / m 1.61ml / vial 7vials 69.20mg / 30ml 2.31g / L

[0193] (5) Identification of humanized anti-periostin antibodies

[0194] Based on the expression level and ELISA screening results, three stable strains 5-G4, 5-G6, and 5-G9 were selected for WB detection to verify the binding specificity.

[0195] Experimental conditions: antigen 1ug, primary antibody 1:1000 dilution, incubation 1h, secondary antibody 1:10000 dilution, incubation 45min. Fig.10 .

[0196] Depend on Fig.10 It can be seen that the three stable transfectants 5-G4, 5-G6 and 5-G9 all specifically bind to the periostin antigen protein.

[0197] 4. Study on the anti-fibrosis effect of humanized anti-periostin antibody

[0198] 4.1 Research Methods

[0199] The antibodies of the 5-G6 stable strain were used in vitro and in vivo for fibroblast transwell migration test, study of the number of migrating cells, and immunohistochemical analysis of mouse fibroblast migration; HE and Masson staining were used to observe the pathological changes in mouse tissues and study the fibrosis area ratio; fluorescent quantitative polymerase chain reaction (PCR) was used to detect the expression of type I collagen (Col1a1 and Col1a2) and type III collagen (Col3a1) mRNA in the mouse fibrosis model. The experiment used the conditioned medium containing periostin + control IgG as the control IgG group, and the conditioned medium containing periostin + humanized periostin antibody as the test group recorded as the Postn nAb group.

[0200] The Wilcoxon rank-sum test was used to compare the fibrosis area ratio, the number of positive infiltrating fibroblasts, and the median values ​​of the fibroblast ratio and the number of migrating fibroblasts.

[0201] Finally, statistical methods: SPSS statistical software was used to process experimental data, and variance analysis was performed between groups. In all statistical analyses, significance was defined as p < 0.05.

[0202] 4.2 Research Results

[0203] 4.2.1. In the trans-well migration experiment, the number of fibroblasts in the blank control group was much larger than that in the PostnnAb group, that is, the PostnnAb group could effectively inhibit the migration of fibroblasts. This result proved the effectiveness of PostnnAb in vitro.

[0204] 4.2.2. Compared with the blank control group, at 14 dpi, immunohistological analysis showed that PostnnAb reduced the number of infiltrating fibroblasts.

[0205] 4.2.3. Fibrosis area ratio At 28 dpi, the fibrosis area in the PostnnAb group mice was significantly reduced compared with that in the blank control group mice.

[0206] 4.2.4. The mRNA expression levels of type I collagen and type III collagen in the Postn nAb group mice were significantly lower than those in the blank control group mice (P < 0.05).

[0207] Research conclusion: This study successfully prepared anti-periostin humanized monoclonal antibodies, and the prepared anti-periostin humanized monoclonal antibodies can specifically bind to periostin protein in vivo, thereby effectively inhibiting fibrosis in vivo. Therefore, anti-periostin humanized monoclonal antibodies are expected to become a new drug for the treatment of retinal fibrosis.

[0208] 5. In vitro activity experiment of humanized Periostin monoclonal antibody

[0209] 5.1 Microtubule inhibition assay

[0210] HUVEC cells were cultured in vitro, and 50,000 cells / well (24 h) were plated in 12-well plates. After overnight culture at 5% CO2 and 37°C, drugs were added for 16 h. Angiogenesis experiments were performed, and observations were made and photographs were taken at appropriate time points. The experimental results were statistically analyzed and the following table and Fig.11 .

[0211] HUVEC Number of tubes HUVEC Number of tubes Group (μg / mL) 16h Group (μg / mL) 16h Control periostin0 121 Periostin antibody 1 162 Control periostin1 108 Periostin antibody 10 57 Control periostin10 91 Periostin Antibody 100 41 Control periostin100 26 Periostin Antibody 1000 2

[0212] As can be seen from the table, as the antibody concentration increases, the number of microtubules decreases, and at the same concentration, the inhibition rate of microtubules by the humanized monoclonal antibody is greater than that of the mouse monoclonal antibody, which confirms that the above periostin humanized monoclonal antibody can inhibit the effect of periostin on the microtubule formation of human vascular endothelial cells.

[0213] 5.2 RPE cells were cultured in vitro and grouped for cell migration, cell invasion and cell scratch experiments

[0214] aBlank control group (Control): does not contain recombinant periostin protein or antibody

[0215] b periostin group: only contains periostin

[0216] c Control IgG group: containing periostin + control IgG

[0217] dperiostin antibody group: containing periostin + PostnnAb

[0218] PostnnAb: humanized periostin antibody prepared by the present invention

[0219] 5.2.1 Cell migration assay

[0220] Transwell assay

[0221] (1) After the cells are centrifuged, discard the culture medium, wash them 1-2 times with PBS, resuspend them in serum-free DMEM medium, and adjust the cell density to 1×10 5 Pieces / ml;

[0222] (2) Take 200 μl of the cell suspension and add it to the Transwell chamber. Add 600 μl of culture medium containing 10% FBS to the lower chamber of the 24-well plate and culture at 37°C for 16-24 h.

[0223] (3) Remove the transwell chamber, discard the culture medium in the well, and wash twice with PBS;

[0224] (4) Fixation with 4% PFA for 10 min and wash twice with PBS;

[0225] (5) Crystal violet staining for 10 min, and washing twice with PBS;

[0226] (6) Observe the migrating cells under a microscope ( Fig.12 ), randomly selected 3 fields of view to take pictures, and counted them using image J software, and plotted ( Fig.13 ).

[0227] Experimental Results

[0228]

[0229] The experimental results showed that compared with the blank control group (Control), the cell migration rate and the number of migrating cells in the periostin-treated group were significantly increased (P < 0.001), while those in the periostin+Postn nAb-treated group were significantly lower than those in the blank control group (P < 0.001). The degree of cell migration in the periostin+IgG-treated group was slightly smaller than that in the periostin-treated group, indicating that periostin humanized monoclonal antibody can inhibit the migration of RPE cells caused by periostin.

[0230] 5.2.2 Cell invasion assay

[0231] The Transwell chamber was pre-coated with Matrigel matrix gel, and a certain concentration of cell suspension was added. The culture medium containing FBS was added to the lower chamber at 37°C and 5% CO. 2 After 48 hours of culture in the incubator, observe and take photos ( Fig.14 ), and counted the number of migrating cells ( Fig.15 ).

[0232]

[0233] The results showed that compared with the blank control group, the cell migration rate and the number of invasive cells in the periostin-treated group were significantly increased (P < 0.01), while those in the periostin+Postn nAb-treated group were significantly decreased (P < 0.01). At the same time, the degree of RPE cell migration in the periostin+IgG-treated group was slightly smaller than that in the periostin-treated group. The results showed that periostin humanized monoclonal antibody has the effect of inhibiting periostin.

[0234] 5.2.3 Scratch test

[0235] (1) After the cells have been grouped as described above, a 10 μl pipette tip is used to make scratches on the cells perpendicular to the well plate, and the width of each scratch is kept consistent as much as possible;

[0236] (2) Aspirate the cell culture medium, rinse the plate three times with PBS to remove the cell debris produced by the scratch, and add the corresponding serum-free culture medium;

[0237] (3) Place the culture plate in the incubator and take it out and take photos after 0, 6, 24, and 48 hours;

[0238] Analyze the experimental results based on the collected image data ( Figures 16-17 ). Experimental results and analysis: After the cells were grouped as above, a 10 μl pipette tip was used to make scratches on the cells perpendicular to the well plate. The culture plate was placed in an incubator and cultured for 0, 6, 24, and 48 hours, and then taken out and photographed.

[0239]

[0240]

[0241] The experimental results showed that after 48 hours of culture, the relative migration distance of cells in the periostin-treated group was significantly increased compared with the blank control group (P < 0.01), while that in the periostin+Postn nAb-treated group was significantly lower than that in the blank control group (P < 0.001). At the same time, the degree of RPE cell migration in the periostin+IgG-treated group was slightly smaller than that in the periostin-treated group. The results showed that periostin humanized monoclonal antibody can inhibit the effect of periostin-induced RPE cells.

[0242] 5.3 Western Blot results

[0243] The expressions of VEGFA, α-SMA, Col I, Col III and Fibronectin proteins in RPE cells of different groups were detected.

[0244] VEGFA (vascular endothelial growth factor) A: VEGFA induces angiogenic response and promotes blood vessel regeneration.

[0245] α-SMA (α-smooth muscle actin): It exists in the vascular wall, intestinal mucosal muscular layer and propria muscular layer and various tissue interstitium. It is highly expressed in myofibroblasts and myoepithelial cells. Studies have shown that in the study of organ fibrosis, it increases with the severity of fibrosis.

[0246] ColI, Col III (collagen type I and collagen type III): closely related to the skin damage repair process and repair quality. It promotes the proliferation of fibroblasts in the dermis, mainly in connective tissues such as skin, tendons, ligaments, and blood vessels, forming an extracellular matrix network structure that supports organs and protects the body. It is also related to cell attachment and cell migration.

[0247] Fibronectin: Promotes cell adhesion and growth, affects cell adhesion, migration or tumor metastasis, embryonic development, growth and differentiation, etc.

[0248]

[0249]

[0250] The experimental results show that ( Figures 18-19 ), compared with the blank control group (Control), the protein expression of periostin treatment group, periostin+IgG treatment group, and periostin+Postn nAb treatment group was statistically significant (P<0.001). Compared with the blank control group (Control), the protein expression of periostin treatment group was significantly increased (P<0.001), while the protein expression of VEGFA, α-SMA, Col I, Col III, and Fibronectin in periostin+Postn nAb treatment group was significantly decreased (P<0.001), which indicates that periostin monoclonal antibody can inhibit the expression of VEGFA, α-SMA, Col I, ColIII, and Fibronectin proteins. 6HUVEC cell in vitro fluorescence quantitative PCR, WB, and IF detection

[0251] Periostin can promote retinal angiogenesis and fibrosis through the integrin-focal adhesion kinase (FAK)-phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT / PKB) signaling pathway. The mRNA expression levels and protein expression levels of integrins (αvβ3, αvβ5), endothelial growth factor receptor-2 (VEGFR-2), FAK, PI3K, and AKT on the human vascular endothelial cell membrane were determined by fluorescence quantitative PCR, immunofluorescence, and western blotting, confirming that periostin humanized monoclonal antibody has a therapeutic effect on vitreoretinal fibrotic lesions.

[0252] In vitro culture of human HUVEC cells and grouping:

[0253] aBlank control group (Control): does not contain recombinant periostin protein or antibody

[0254] b periostin group: only contains periostin

[0255] c Control IgG group: containing periostin + control IgG

[0256] dperiostin antibody group: containing periostin + PostnnAb

[0257] PostnnAb: humanized periostin antibody prepared by the present invention

[0258] The human periostin protein and antibody were incubated for 1 hour, then added to the cells and treated for another 24 hours before the following tests were performed. The working concentration of the control IgG was the same as the concentration of the anti-periostin humanized monoclonal antibody.

[0259] 6.1 Fluorescence quantitative PCR assay

[0260] Detect the mRNA expression of αvβ3, αvβ5 integrins, VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT on the vascular endothelial cell membrane

[0261] Depend on Fig. 20 It can be seen that compared with the blank control group, the expression of αvβ3, αvβ5, VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT genes in the periostin-treated group increased, while the gene expression levels in the periostin+Postn nAb-treated group decreased, especially the mRNA expression level of VEGFR-2 was significantly lower than that in the blank control group (P<0.001). This shows that periostin humanized monoclonal antibody can inhibit the expression of VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT mRNA genes.

[0262] 6.2 Western blot detection of αvβ3, αvβ5, VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT protein expression in HUVEC cells

[0263] Normally cultured HUVEC cells were taken and 10 6 Each dish (6 cm dish) was plated, 5% CO 2, and cultured at 37℃ overnight. After the cells adhered overnight, they were treated according to the groups; the total protein content was detected by BCA kit, and the membrane was transferred after SDS-PAGE electrophoresis, blocked with blocking solution, washed, and incubated with primary antibody in a refrigerator at 4℃ overnight, washed, and incubated with secondary antibody on a shaker at room temperature for 1h, and color was developed. The ratio of the gray value of each group to the gray value of the internal reference under the same conditions was calculated as the relative expression of various proteins.

[0264] The expression and grayscale analysis of each protein in HUVEC cells of different groups are shown in Figures 21-22 The experimental results showed that compared with the blank control group, the expression of αvβ3, αvβ5, VEGFR-2, FAK, PI3K, p-PI3K, AKT, and p-AKT proteins in the periostin-treated group increased, while the protein expression levels in the periostin+Postn nAb-treated group decreased, and were all lower than those in the periostin-treated group. This shows that periostin humanized monoclonal antibody can inhibit the effect of periostin.

[0265] 6.3IF detection of αvβ3, αvβ5, VEGFR-2, and FAK protein expression in HUVEC cells

[0266] Wash the slides with cells in the culture plate with PBS, fix the slides with 4% paraformaldehyde, permeabilize with 0.5% Triton X-100 at room temperature for 20 minutes, add primary antibody and incubate in a humidified box at 4°C overnight, add fluorescent secondary antibody and incubate in a humidified box at 20-37°C for 1 hour, add DAPI nuclear counterstain, seal the slides and observe under a fluorescence microscope ( Figures 23-26 ).

[0267] Under a fluorescence microscope, periostin protein was expressed in the cytoplasm of HUVEC cells. The cytoplasm of cells in the blank control group with αvβ3, αvβ5, VEGFR-2, and FAK proteins showed weak fluorescence; the cytoplasm of cells in the periostin-treated group showed strong fluorescence; the fluorescence signal of cells in the periostin+Postn nAb-treated group was significantly weakened compared with that in the periostin-treated group, and there was no significant difference compared with the blank control group. This shows that periostin humanized monoclonal antibody can inhibit retinal fibrosis by blocking the Integrin-FAK-PI3K-AKT / PKB signaling pathway and thus blocking the effect of periostin.

[0268] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-periostin humanized monoclonal antibody, characterized in that: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:

2.

2. The monoclonal antibody according to claim 1, characterized in that The constant region of its heavy chain is human IgG1; The constant region of its light chain is of human κ type.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of its heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of its light chain is shown in SEQ ID NO:

4.

4. A nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that The nucleic acid encoding the heavy chain thereof has the nucleic acid sequence shown in SEQ ID NO:5; The nucleic acid encoding the light chain thereof has the nucleic acid sequence shown in SEQ ID NO:

6.

6. The nucleic acid according to claim 5, characterized in that The nucleic acid encoding the heavy chain thereof has the nucleic acid sequence shown in SEQ ID NO:7; The nucleic acid encoding the light chain thereof has the nucleic acid sequence shown in SEQ ID NO:

8.

7. An expression vector comprising the nucleic acid according to any one of claims 4 to 6.

8. The expression vector according to claim 7, characterized in that Its skeleton carrier is PATX-GS2.

9. A host cell transformed or transfected with the expression vector of claim 8.

10. A method for preparing the anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3, comprising: Cultivating the host cell according to claim 9 and inducing the expression of the anti-periostin humanized monoclonal antibody.

11. Use of the anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3 in the preparation of a drug or a detection reagent, wherein the drug is a drug for treating retinal fibrosis, and the reagent is a reagent for diagnosing retinal fibrosis.

12. A drug for treating retinal fibrosis, characterized in that: The invention comprises the anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3.

13. A kit for diagnosing retinal fibrosis, characterized in that: include: The anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3, or comprising a conjugate formed by the anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3 and a chemical marker or a biological marker; Or a conjugate prepared by coupling the anti-periostin humanized monoclonal antibody according to any one of claims 1 to 3 with a solid medium or a semi-solid medium.

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