WO2023036305A1 - 抗periostin人源化单克隆抗体及其制备方法与应用 - Google Patents

抗periostin人源化单克隆抗体及其制备方法与应用 Download PDF

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WO2023036305A1
WO2023036305A1 PCT/CN2022/118162 CN2022118162W WO2023036305A1 WO 2023036305 A1 WO2023036305 A1 WO 2023036305A1 CN 2022118162 W CN2022118162 W CN 2022118162W WO 2023036305 A1 WO2023036305 A1 WO 2023036305A1
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periostin
monoclonal antibody
nucleic acid
humanized monoclonal
antibody
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French (fr)
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李雪娇
何伟
张朔
赵航
冯铃
刘辉
赵洪礼
何向东
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Shenyang Eye Industry Technology Institute Ltd
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Shenyang Eye Industry Technology Institute Ltd
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Priority to CN202280054341.XA priority Critical patent/CN117957248B/zh
Priority to US18/691,316 priority patent/US20240400658A1/en
Priority to JP2024539906A priority patent/JP7751338B2/ja
Priority to EP22866766.3A priority patent/EP4403569A4/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention relates to the technical field of antibody drugs, in particular to an anti-periostin humanized monoclonal antibody and its preparation method and application.
  • Proliferative diabetic retinopathy proliferative diabetic retinopathy, PDR
  • proliferative vitreoretinopathy proliferative vitreoretinopathy
  • AMD age-related macular degeneration
  • other vitreoretinal diseases are currently leading to the corresponding population
  • the main cause of vision loss and loss is also a worldwide medical problem. Therefore, finding new biological markers and therapeutic targets for vitreoretinal diseases is crucial for early detection, early diagnosis and early treatment of diseases.
  • Periostin is a cell adhesion protein discovered in mouse embryonic osteoblast precursor cells in 1993, named osteoblast-specific factor-2 (OSF-2), because of its role in adult mouse periodontal Membrane and periosteum can be specifically expressed, so it was renamed as periostin.
  • Periostin is a unique extracellular matrix protein that participates in the corresponding immune and inflammatory responses 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.
  • the research and production of drugs targeting periostin has become a hot spot, and a large number of marketed drugs have achieved remarkable results in the treatment of tumors.
  • periostin The main functions of periostin in the eye include promoting cell proliferation, differentiation, migration and adhesion, inducing fibrogenesis and promoting new blood vessels. More and more domestic and foreign studies have proved that periostin is a key factor in various vitreoretinal diseases, and periostin is expected to become a new biological marker for vitreoretinal diseases, thereby providing a new treatment strategy. 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 neovascular hyperplasia and macular edema in many fields of ophthalmology.
  • Periostin antibodies were all mouse-derived monoclonal antibodies.
  • Mouse-derived antibodies used as heterologous proteins in the human body will cause immune rejection against heterologous proteins, induce human anti-mouse antibodies, and shorten the half-life of antibodies in the body. treatment effect. Therefore, on the basis of maintaining high affinity for specific antigenic epitopes, the humanization of Periostin antibodies can reduce the immunogenicity of heterologous antibodies, improve the specificity and affinity of binding to antigens, and thus play a better role. therapeutic effect.
  • the technical problem to be solved by the present invention is to provide anti-periostin humanized monoclonal antibody and its preparation method and application.
  • its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2 .
  • the constant region of its heavy chain is human IgG1; the constant region of its light chain is human ⁇ type.
  • 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 is shown in SEQ ID NO: 4.
  • the present invention uses periostin mouse monoclonal antibody to humanize it, successfully constructs a eukaryotic expression plasmid, transfects mammalian cell CHO for secreted expression, and conducts affinity and binding in vitro through SDS-PAGE, ELISA, WB, etc.
  • the evaluation of biological functions such as activity provides a theoretical basis for the large-scale expression of anti-periostin humanized monoclonal antibody in CHO cells.
  • 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 functional than chimeric antibodies or other mouse-derived periostin monoclonal antibodies in the prior art. more significant. After testing, the humanized antibody has good specificity to human periostin. In view of the fact that Periostin antibody has been proven to play a key role in the treatment of age-related macular degeneration and diabetic retinopathy in many fields of ophthalmology, such as neovascular hyperplasia and macular edema inside the eye. Therefore, the antibody provided by the present invention has the potential to treat tissue fibrosis.
  • the present invention also provides nucleic acid encoding the monoclonal antibody. Specifically, the present invention provides nucleic acid encoding the heavy chain of the monoclonal antibody, nucleic acid encoding the light chain of the monoclonal antibody, nucleic acid encoding the variable region of the heavy chain of the monoclonal antibody, and/or encoding the monoclonal antibody heavy chain Nucleic acid of an antibody light chain variable region.
  • the nucleic acid encoding the antibody heavy chain 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.
  • the 5' end of the coding nucleic acid provided by the 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 the nucleic acid sequence shown in SEQ ID NO: 7; the light chain nucleic acid encoding the anti-periostin humanized monoclonal antibody has Nucleic acid sequence as shown in SEQ ID NO:8.
  • the present invention also provides an expression vector, including the nucleic acid encoding the monoclonal antibody.
  • the expression vector also includes a backbone vector; in some embodiments, the backbone vector is pCDNA3.4.
  • the present invention also provides host cells transformed or transfected with the expression vector.
  • the host cell is a mammalian cell, and in some specific embodiments, the host cell is a CHO-K1 cell.
  • the preparation method of the anti-periostin humanized monoclonal antibody of the present invention comprises: cultivating the host cell of the present invention, and inducing the expression of the anti-periostin humanized monoclonal antibody.
  • the present invention also provides the application of the anti-periostin humanized monoclonal antibody in the preparation of medicine or detection reagent, and the medicine is medicine for treating tissue fibrosis and/or malignant tumor.
  • the reagent is a reagent for diagnosing tissue fibrotic lesions and/or malignant tumors.
  • the tissue fibrotic lesion is retinal fibrosis.
  • the invention provides a medicine, which comprises the anti-periostin humanized monoclonal antibody of the invention.
  • the present invention also provides a kit, which contains an anti-periostin humanized monoclonal antibody, or comprises a combination of the antibody and a chemical label or a biological label, or comprises the anti-periostin humanized monoclonal antibody and The conjugate prepared by solid medium or semi-solid medium coupling.
  • the chemical label in the present invention is isotope, immunotoxin and/or chemical drug; the biological label is biotin, avidin or 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, lotus root toxin, PAP, herbaceous factor, gelonin or loofah toxin.
  • the solid 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 immunological detection methods, such as ELISA, western blot, immunofluorescence technology, immunohistochemical detection and the like.
  • the present invention also provides a method for treating tissue fibrosis and/or malignant tumors, which comprises administering the drug described in the present invention.
  • the present invention also provides a method for diagnosing tissue fibrosis lesions and/or malignant tumors, which is to diagnose with the diagnostic reagents described in the present invention
  • the present invention provides an anti-periostin humanized monoclonal antibody, which only retains the complementarity determining region that binds to the antigen in the variable region of the murine McAb, and prepares the CDR-grafted antibody. It is less immunogenic than chimeric antibodies. Compared with other antibodies in the experiment, the antibody obtained by screening in the present invention has a higher expression level, better binding specificity and affinity to the antigen, and can better participate in the immune response of the human body.
  • Fig. 1 shows mouse hybridoma cell (45-2-G3-1-G7-B7) VH and VL PCR amplification result
  • Figure 2 shows the PCR results after cloning
  • Figure 3-1 and Figure 3-2 show the plasmid maps of the heavy chain and light chain
  • Fig. 4 shows purification effect
  • Figure 5 shows the ELISA results of 18 humanized antibodies
  • Fig. 6 shows expression vector electrophoresis detection result
  • Fig. 7 shows the electrophoresis detection result of the linearized expression vector
  • Figure 8 shows the protein expression and purification results after pressure screening
  • Figure 9 shows the results of purification of cell-expressed proteins obtained through screening
  • Figure 10 shows the identification of humanized anti-periostin antibodies 5-G4, 5-G6, and 5-G9;
  • Figure 11 shows the results of microtubule inhibition experiments, wherein the Periostin antibody is a humanized monoclonal antibody, and the control Periostin is a mouse monoclonal antibody;
  • Figure 12 shows the results of cell migration experiments
  • Figure 13 shows the cell mobility relative to the blank control group
  • Figure 14 shows the results of cell invasion experiments
  • Figure 15 shows the cell invasion rate relative to the blank control group
  • Fig. 16 shows the result of cell scratch experiment
  • Figure 17 shows the relative migration distance (%) of cells
  • Figure 18 shows the expression WB detection figure of VEGFA, ⁇ -SMA, Col I, Col III, Fibronectin protein in different groups of RPE cells;
  • Figure 19 shows the expression levels of VEGFA, ⁇ -SMA, Col I, Col III, Fibronectin protein in different groups of RPE cells;
  • Figure 20 shows the mRNA expression levels of ⁇ v ⁇ 3, ⁇ v ⁇ 5 integrin, VEGFR-2, FAK, PI3K, p-PI3K, AKT, p-AKT on the vascular endothelial cell membrane of each group;
  • Figure 21 shows the Western blot detection chart of each protein in different groups of HUVEC cells
  • Figure 22 shows the expression levels of each protein in different groups of HUVEC cells
  • Figure 23 shows that IF detects the expression of FAK protein in HUVEC cells
  • Figure 24 shows that IF detects the expression of VEGFR2 protein in HUVEC cells
  • Figure 25 shows IF detection of ⁇ v ⁇ 3 protein expression in HUVEC cells
  • Figure 26 shows IF detection of ⁇ v ⁇ 5 protein expression in HUVEC cells.
  • the present invention provides an anti-periostin humanized monoclonal antibody and its preparation method and application.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • Antibody refers to a protein composed of one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer, which consists 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 and heavy chains combine to be responsible for antigen binding, while the constant regions are responsible for the antibody's effector functions.
  • the term “monoclonal antibody” refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.
  • the "variable region" of an antibody heavy or light chain is the N-terminal mature region of that chain.
  • Currently known antibody classes include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or their other class equivalents.
  • Full-length immunoglobulin "light chains” (about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus, 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), likewise comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, such as gamma (about 330 amino acids).
  • Antibody includes 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 A fusion protein comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • the invention provides a humanized monoclonal antibody of periostin.
  • variable region gene of anti-Periostin mouse monoclonal antibody replaces the framework region of the variable region gene of anti-Periostin mouse monoclonal antibody with the framework region of human antibody, humanize the variable region gene fragment of the antibody, and recombine it into a vector containing regulatory sequences and human antibody constant region gene
  • the complete gene of anti-Periostin humanized monoclonal antibody and the eukaryotic expression vector containing the gene were constructed, and then transfected into CHO-K1 cells.
  • CHO-K1 cells that continuously and stably secrete humanized antibodies were obtained through pressurized screening by the GS system.
  • amino acid sequence of the heavy chain variable region of the anti-periostin humanized monoclonal antibody of the present invention is:
  • amino acid sequence of the light chain variable region of the anti-periostin humanized monoclonal antibody of the present invention is:
  • amino acid sequence of the heavy chain constant region of the anti-periostin humanized monoclonal antibody of the present invention is:
  • amino acid sequence of the light chain constant region of the anti-periostin humanized monoclonal antibody of the present invention is:
  • amino acid sequence of the heavy chain of the anti-periostin humanized monoclonal antibody of the present invention is:
  • amino acid sequence of the light chain of the anti-periostin humanized monoclonal antibody of the present invention is:
  • linker fragments were added to the N-terminals of the heavy chain fragment and the light chain fragment during the process of vector construction and protein expression. This fragment was removed during subsequent purification.
  • the amino acid sequence of the linker added to the N-terminus of the heavy chain of the anti-periostin humanized monoclonal antibody is MKHLWFFFLLLVAAPRWVLS
  • the linker added to the N-terminus of the light chain of the anti-periostin humanized monoclonal antibody is The amino acid sequence is MVLQTQVFISLLLWISGAYG.
  • Antibodies can be labeled and detected, for example, with radioisotopes, enzymes that produce detectable substances, fluorescent proteins, biotin, and the like. Antibodies can also be bound to solid supports, including but not limited to polystyrene plates or beads, and the like.
  • the medicament of the present invention contains at least one functional component, and also includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffer saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid Magnesium, 0.3% glycerin, hyaluronic acid, alcohol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.
  • test materials used in the present invention are all common commercially available products, which can be purchased in the market. Below in conjunction with embodiment, further set forth the present invention:
  • mouse hybridoma cells (45-2-G3-1-G7-B7, see "Anti-human periostin monoclonal chimeric antibody and its application” for the construction method, application number: 202010788885.1, from Shenyang He's Eye Industry Group Co., Ltd. or Shenyang Eye Industry Technology Research Institute Co., Ltd.) to isolate all the mRNA, as a template for cDNA synthesis, from the cDNA to separate heavy chain variable region DNA fragments and light chain variable region DNA fragments, and then clone these obtained DNA fragments into Carrier and sequencing to obtain the sequence results are as follows:
  • amino acid sequences of the three CDR regions in the light chain variable region are: SSASY, DTS, QQWSSNPPT.
  • amino acid sequences of the three CDR regions in the heavy chain variable region are: GYSFTDYF, INPYSGDT, GRSGVSGLDY.
  • Igblast developed by NCBI (https://www.ncbi.nlm.nih.gov/igblast/) to search the IMGT mouse and human V gene database, input the mouse antibody variable region sequence, and determine the homology after sequence alignment The highest human antibody variable region, and then transplant the mouse antibody CDR region to the human framework acceptor region to complete the CDR grafting.
  • variable region sequence Link the variable region sequence with the human constant region DNA sequence, insert it into the pcDNA3.4 expression vector, and 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 as follows shown.
  • Nucleic acid sequence information includes:
  • the 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%. Take part of the cells to collect the culture supernatant, purify it with protein G, run SDS-PAGE gel, and use the antigen protein to perform ELISA to detect the activity of the antibody.
  • 3Transfection Add 3ml of transfection mixture to 27ml of cell suspension, mix gently, and culture at 37°C, 5% CO 2 , 130rpm until the cell viability drops below 50% and collect the sample.
  • the expression level of antibody No. 18 551-VHB/139-VLA is the highest, and the binding ability can meet the requirements.
  • -VLA is 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 results of agarose gel electrophoresis run are shown in Figure 6.
  • the target fragment was digested with double enzymes (EcoR I/Not I), ligated and subcloned into the expression vector pATX-GS2, then stably transfected into CHO cells, subjected to MSX pressurized screening, and ELISA limiting dilution to obtain stable cell lines.
  • the supernatant was collected after culturing in a shaker at 37° C. and 5% carbon dioxide for 7 days, and the humanized antibody in the supernatant was purified with agarose gel Protein A chromatography filler.
  • CHO-K1 cells were transfected with PVUI linearized plasmid pATX-GS2-139-VLA-551-VHB, and transfected by chemical method (FectoCHO TM Expression System Transfection Reagent box).
  • the present invention selects the GS screening system (glutamine synthetase Gene Expression System).
  • the cells were pressurized and treated with MSX at a concentration of 30um: the natural resistance of the CHO-K1 cell line to MSX was measured using a 6-well plate standard batch culture method, and the MSX effect on the untransfected cell line CHO-K1 was measured. The minimum killing concentration is 20uM. Add MSX at a concentration of 30um, and replace the new screening medium every 3 to 4 days until stable cells with resistance appear. After 2 to 3 weeks of MSX screening, 3 stable pools are generated.
  • Cells were cultured in 30 ml selective media in 125 ml shake flasks under standard conditions (37°C, 5% CO 2 , 130 rpm). When the cells were in good condition in shake flasks, a 30 ml fed-batch expression assay was performed, adding feed medium on D3, D5, D7 and D9 and monitoring blood glucose throughout. Continuously cultured 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, wherein reduced shows the results of reduced SDS-PAGE electrophoresis, and non-reduced shows the results of non-reduced SDS-PAGE electrophoresis.
  • Antibody Concentration Specification Quantity Yield 18-stable 1 0.86mg/ml 1.75ml/vial 4vials 6.02mg/30ml 18-stable 2 0.94mg/ml 1.80ml/vial 4vials 6.76mg/30ml 18-stable 3 1.62mg/ml 1.80ml/vial 4vials 11.66mg/30ml
  • the antibody of the 5-G6 stable transfected strain was used for the transwell migration test of fibroblasts in vitro and in vivo, the study of the number of migrating cells, and the immunohistochemical analysis of the migration of fibroblasts in mice; and the observation of mouse tissues by HE and Masson staining Pathological changes, the study of fibrosis area ratio; fluorescence quantitative polymerase chain reaction (PCR) method to detect the expression of type I collagen (Col1a1 and Col1a2) and type III collagen (Col3a1) mRNA in the mouse fibrosis model;
  • PCR fluorescence quantitative polymerase chain reaction
  • the Wilcoxon rank sum test compared the fibrosis area ratio, the number of positive infiltrating fibroblasts, and the median of the fibroblast ratio and the number of migrating fibroblasts.
  • SPSS statistical software was used for experimental data processing, and analysis of variance between groups was used. In all statistical analysis, significance was defined as p ⁇ 0.05.
  • the fibrosis area ratio was 28dpi. Compared with the blank control group mice, the fibrosis area in the Postn nAb group mice was significantly reduced.
  • HUVEC cells were cultured in vitro, plated in 12-well plates at 50,000 cells/well (24h), cultured in 5% CO2 overnight at 37°C, and then treated with drugs for 16h, and angiogenesis experiments were performed, observed and photographed at appropriate time points, and recorded. The results of the statistical experiment are shown in the following table and Figure 11.
  • aBlank control group no recombinant periostin protein or antibody
  • periostin group only periostin
  • control IgG group containing periostin + control IgG
  • periostin antibody group containing periostin+Postn nAb
  • Postn nAb periostin humanized antibody prepared by the present invention
  • the experimental results showed that, compared with the blank control group (Control), the cell migration rate and the number of migrated cells in the periostin treatment group increased significantly (P ⁇ 0.001), while the periostin+Postn nAb treatment group was significantly lower than the blank control group (P ⁇ 0.001 ), the degree of cell migration in the periostin+IgG treatment group was slightly smaller than that in the periostin treatment group, indicating that the periostin humanized monoclonal antibody can inhibit the migration of RPE cells induced by periostin.
  • the Transwell chamber was pre-coated with Matrigel, added a certain concentration of cell suspension, and added FBS-containing medium to the lower chamber, and cultured in a 37°C, 5% CO 2 incubator for 48 hours and then observed and photographed ( Figure 14). The number of migrated cells was counted ( Figure 15).
  • the experimental results showed that compared with the blank control group at 48 hours of culture, the relative migration distance of cells in the periostin treatment group was significantly increased (P ⁇ 0.01), while the periostin+Postn nAb treatment group was significantly lower than that in the blank control group (P ⁇ 0.001).
  • the migration of RPE cells in the +IgG treatment group was slightly less than that in the periostin treatment group, and the results showed that the humanized monoclonal antibody of periostin could inhibit the effect of periostin-induced RPE cells.
  • VEGFA VEGFA
  • ⁇ -SMA Col I
  • Col III Fibronectin proteins
  • VEGFA vascular endothelial growth factor A: VEGFA induces an angiogenic response and promotes angiogenesis.
  • ⁇ -SMA smooth muscle actin
  • ColI, Col III type I collagen and type III collagen: closely related to the skin damage repair process and repair quality. Promoting the proliferation of fibroblasts in the dermis, mainly in connective tissues such as skin, tendons, ligaments, blood vessels, etc., forming an extracellular matrix network structure, supporting organs and protecting the body, and is also related to cell attachment and cell migration.
  • Fibronectin promotes cell adhesion growth, affects cell adhesion, migration or tumor metastasis, embryonic development, growth and differentiation, etc.
  • Periostin can promote the formation and fibrosis of retinal blood vessels through the integrin (Integrin)-focal adhesion kinase (FAK)-phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT/PKB) signaling pathway.
  • Integrin integrin-focal adhesion kinase
  • PI3K phosphoinositide 3-kinase
  • AKT/PKB protein kinase B
  • Human HUVEC cells were cultured in vitro and divided into groups:
  • aBlank control group no recombinant periostin protein or antibody
  • periostin group only periostin
  • control IgG group containing periostin + control IgG
  • periostin antibody group containing periostin+Postn nAb
  • Postn nAb periostin humanized antibody prepared by the present invention
  • Human periostin protein and antibody were incubated for 1 hour, then added to the cells and treated for another 24 hours, and the following detection was performed.
  • the working concentration of control IgG was the same as that of anti-periostin humanized monoclonal antibody.
  • the normal cultured HUVEC cells were taken, plated at 10 6 cells/dish (6 cm dish), cultured overnight at 37° C. in 5% CO 2 . After the cells adhered to the wall overnight, they were processed according to the grouping; BCA kit was used to detect the total protein content, after SDS-PAGE electrophoresis, the membrane was transferred, and the blocking solution was blocked. After washing, the primary antibody was refrigerated overnight at 4°C, the membrane was washed, and the secondary antibody was incubated at room temperature for 1 hour. , color. 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 each protein.
  • the periostin protein was expressed in the cytoplasm of HUVEC cells, and the cytoplasm of the blank control group with ⁇ v ⁇ 3, ⁇ v ⁇ 5, VEGFR-2, and FAK proteins showed weak fluorescence; the cytoplasm of the cells treated with periostin showed strong fluorescence; periostin+Postn nAb treatment Compared with the periostin treatment group, the fluorescence signal of the cells in the group was significantly weakened, and there was no significant difference compared with the blank control group, which indicated that the periostin humanized monoclonal antibody could block the Integrin-FAK-PI3K-AKT/PKB signaling pathway The role of periostin, thereby inhibiting retinal fibrosis.

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Abstract

本申请提供了抗periostin人源化单克隆抗体及其制备方法与应用，所述抗periostin人源化单克隆抗体的表达量较高，与抗原结合的特异性和亲和性较好。

Description

抗periostin人源化单克隆抗体及其制备方法与应用
本申请要求于2021年09月13日提交中国专利局、申请号为202111068177.1、发明名称为“抗periostin人源化单克隆抗体及其制备方法与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及抗体药物技术领域，尤其涉及抗periostin人源化单克隆抗体及其制备方法与应用。
背景技术
增生型糖尿病性视网膜病变(proliferative diabetic retinopathy，PDR)、增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)和年龄相关性黄斑变性(age-related macular degeneration，AMD)等玻璃体视网膜疾病是现阶段导致相应人群视力下降和丧失的主要原因，也是世界性医学难题。因此，寻玻璃体视网膜疾病的新的生物学标记物和治疗靶点对疾病的早期发现、早期诊断及早期治疗至关重要。
骨膜蛋白(periostin)是于1993年发现于小鼠胚胎成骨细胞前体细胞中的细胞黏附蛋白，命名为成骨细胞特异性因子-2(OSF-2)，因其在成年小鼠牙周膜和骨膜可以特异性表达，故更名为骨膜蛋白。骨膜蛋白(periostin)是一种独特的细胞外基质蛋白，参与人体各组织器官的相应免疫炎症反应，如心肌梗死后重塑、骨髓纤维化、牙周组织再生修复、皮肤伤口愈合、肿瘤细胞转移、肾脏损伤、以及可诱导慢性过敏性疾病等。近年以periostin为靶点的药物研究与生产成为热点，大量上市药物在肿瘤治疗过程中取得了令人瞩目的成绩。
骨膜蛋白在眼部的主要作用包括促进细胞增殖、分化、迁移和粘附，诱导纤维形成以及促进新生血管等。越来越多的国内外研究证明，骨膜蛋白是各玻璃体视网膜疾病中的关键因子，骨膜蛋白有望成为玻璃体视网膜疾病的新的生物学标志物，以此提供新的治疗策略。例如，有报道称：Periostin抗体已被证实对眼科诸多领域对年龄相关性黄斑变以及糖尿病视网膜病变所引发的眼内部的新生血管性增生、黄斑水肿等治疗起到关键作用。
但此前的Periostin抗体皆为鼠源单抗，鼠源性抗体作为异源蛋白应用于人体会引起针对异源蛋白的免疫排斥反应，诱发产生人抗鼠抗体，缩短抗体在体内的半衰期影响抗体的治疗效果。因此，在保持对特异性抗原表位高亲和力的基础上对Periostin抗体进行人源化改造，能够减少异源抗体的免疫原性，提高与抗原结合的特异性和亲和性，从而起到更好的治疗作用。
发明内容
有鉴于此，本发明要解决的技术问题在于提供抗periostin人源化单克隆抗体及其制备方法与应用。
本发明提供的抗periostin人源化单克隆抗体，其重链可变区具有如SEQ ID NO：1所示的氨基酸序列；其轻链可变区具有如SEQ ID NO：2所示的氨基酸序列。
本发明提供的抗periostin人源化单克隆抗体，其重链的恒定区为人IgG1；其轻链的恒定区为人κ型。
一些具体实施例中，所述人源化抗体的重链的氨基酸序列如SEQ ID NO：3所示， 其轻链的氨基酸序列如SEQ ID NO：4所示。
本发明利用periostin鼠源单抗，对其进行人源化改造，成功构建真核表达质粒，转染哺乳动物细胞CHO进行分泌表达，通过SDS-PAGE、ELISA、WB等在体外进行了亲和力、结合活性等生物学功能评价，为CHO细胞大规模表达抗periostin人源化单克隆抗体研究提供了理论基础。
本发明提供的抗periostin人源化单克隆抗体以上述重链及轻链组成，经人源化后，比现有技术中的嵌合抗体或其他鼠源periostin单克隆抗体更加安全可靠的、功能更显著。经检测，该人源化抗体对人periostin具有良好的特异性。鉴于Periostin抗体已被证实对眼科诸多领域对年龄相关性黄斑变以及糖尿病视网膜病变所引发的眼内部的新生血管性增生、黄斑水肿等治疗起到关键作用。因此，本发明提供的抗体具有潜在的治疗组织纤维化病变的作用。
本发明还提供了编码所述的单克隆抗体的核酸。具体的，本发明提供了编码所述单克隆抗体重链的核酸、编码所述单克隆抗体轻链的核酸、编码所述单克隆抗体重链可变区的核酸和/或编码所述单克隆抗体轻链可变区的核酸。一些实施例中，编码所述抗体重链的核酸具有如SEQ ID NO：5所示的核酸序列。编码所述抗体轻链的核酸具有如SEQ ID NO：6所示的核酸序列。为了方面蛋白的表达，本发明提供的编码核酸的5’端还包括接头序列。因此，一些具体实施例中，编码所述抗periostin人源化单克隆抗体重链的核酸具有如SEQ ID NO：7所示的核酸序列；编码抗periostin人源化单克隆抗体的轻链核酸具有如SEQ ID NO：8所示的核酸序列。
本发明还提供了一种表达载体，包括编码所述的单克隆抗体的核酸。本发明中，所述表达载体中还包括骨架载体；一些实施例中，所述骨架载体为pCDNA3.4。
本发明还提供了转化或转染所述表达载体的宿主细胞。本发明中，所述宿主细胞为哺乳动物细胞，一些具体实施例中，所述宿主细胞为CHO-K1细胞。
本发明所述抗periostin人源化单克隆抗体的制备方法包括：培养本发明宿主细胞、诱导抗periostin人源化单克隆抗体的表达。
本发明还提供了抗periostin人源化单克隆抗体在制备药物或检测试剂中的应用，所述药物为治疗组织纤维纤维化病变和/或恶性肿瘤的药物。所述试剂为诊断组织纤维纤维化病变和/或恶性肿瘤的试剂。一些实施例中，所述组织纤维化病变为视网膜纤维化。
本发明提供了一种药物，其包括本发明所述的抗periostin人源化单克隆抗体。
本发明还提供了一种试剂盒，其含有抗periostin人源化单克隆抗体，或包括将该抗体与化学标记或生物标记形成的结合物，或包括所述抗periostin人源化单克隆抗体与固体介质或半固体介质偶联制得的偶联物。
本发明中所述化学标记为同位素、免疫毒素和/或化学药物；所述生物标记为生物素、亲和素或酶标记。所述酶标记优选为辣根过氧化物酶或碱性磷酸酶。所述免疫毒素优选为黄曲霉毒素、白喉毒素、绿脓杆菌外毒素、蓖麻毒蛋白、相思子毒蛋白、槲寄生凝集素、蒴莲根毒素、PAP、造草素、白树毒素或丝瓜毒素。所述偶联物中的固体介质或非固体介质选自胶体金、聚苯乙烯平板或珠粒。该试剂盒能够用于组织纤维纤维化病变和/或恶性肿瘤的诊断。所述诊断的方法采用免疫学检测方法，例如ELISA、western blot、免疫荧光技术、免疫组化检测等。
本发明还提供了一种治疗组织纤维纤维化病变和/或恶性肿瘤的方法，其为给予本发明所述的药物。
本发明还提供了一种诊断组织纤维纤维化病变和/或恶性肿瘤的方法，其为以本 发明所述的诊断试剂进行诊断
本发明提供提供了抗periostin人源化单克隆抗体，该抗体仅保留鼠源性McAb可变区中与抗原结合的互补决定区，制备CDR移植抗体。与嵌合抗体相比，其免疫原性更低。相对于实验中的其他抗体，本发明筛选获得的抗体表达量更高，与抗原结合的特异性和亲和性更好，能更好地参与人体免疫反应。
附图说明
图1示鼠杂交瘤细胞(45-2-G3-1-G7-B7)VH和VL PCR扩增结果；
图2示克隆后PCR结果；
图3-1和图3-2示重链和轻链的质粒图谱；
图4示纯化效果；
图5示18个人源化抗体的ELISA结果；
图6示表达载体电泳检测结果；
图7示线性化表达载体电泳检测结果；
图8示加压筛选后蛋白表达纯化结果；
图9示筛选获得细胞表达蛋白纯化结果；
图10示人源化抗periostin抗体5-G4、5-G6、5-G9鉴定；
图11示微管抑制实验结果，其中，Periostin抗体为人源化单抗，对照Periostin为小鼠单抗；
图12示细胞迁移实验结果；
图13示相对于空白对照组的细胞迁移率；
图14示细胞侵袭实验结果；
图15示相对于空白对照组的细胞侵袭率；
图16示细胞划痕实验结果；
图17示细胞相对迁移距离(％)；
图18示不同组别RPE细胞中VEGFA、α-SMA、Col I、Col III、Fibronectin蛋白的表达WB检测图；
图19示不同组别RPE细胞中VEGFA、α-SMA、Col I、Col III、Fibronectin蛋白的表达水平；
图20示各组血管内皮细胞膜上的αvβ3、αvβ5整合素，VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT的mRNA表达量；
图21示不同组别HUVEC细胞中各个蛋白的Western blot检测图；
图22示不同组别HUVEC细胞中各个蛋白的表达水平；
图23示IF检测HUVEC细胞中FAK蛋白的表达；
图24示IF检测HUVEC细胞中VEGFR2蛋白的表达；
图25示IF检测HUVEC细胞中αvβ3蛋白的表达；
图26示IF检测HUVEC细胞中αvβ5蛋白的表达。
具体实施方式
本发明提供了抗periostin人源化单克隆抗体及其制备方法与应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的 方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
“抗体”是指由能特异结合抗原的一种或多种多肽构成的蛋白质。抗体的一种形式构成了抗体的基本结构单元。这种形式是四聚物，它由两对完全相同的抗体链构成，每一对都有一个轻链和一个重链。在每对抗体链中，轻链和重链的可变区联合在一起共同负责结合抗原，而恒定区则负责抗体的效应器功能。所述“单克隆抗体”系指具有单一分子组成的抗体分子的制备物。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和性。
抗体重链或轻链的“可变区”是该链的N端成熟区域。目前已知的抗体类型包括κ和λ轻链，以及α，γ(IgG1，IgG2，IgG3，IgG4)，δ，ε和μ重链或它们的其它类型等价物。全长的免疫球蛋白“轻链”(大约25kDa或大约214个氨基酸)包含一个由NH2-末端上大约110个氨基酸形成的可变区，以及一个COOH-末端上的κ或λ恒定区。全长的免疫球蛋白“重链”(大约50kDa或大约446个氨基酸)，同样包含一个可变区(大约116个氨基酸)，以及重链恒定区之一，例如γ(大约330个氨基酸)。
“抗体”包括任何同型体的抗体或免疫球蛋白，或保持与抗原特异结合的抗体片段，包括但不限于Fab，Fv，scFv和Fd片段、嵌合抗体、人源化抗体、单链抗体以及包含抗体的抗原结合部分和非抗体蛋白质的融合蛋白质。本发明提供的为periostin的人源化单克隆抗体。从培养的杂交瘤细胞45-2-G3-1-G7-B7中提取总RNA，用寡核苷酸引物反转录合成cDNA，用IgG简并引物和kappa特异性引物分别扩增VH和VL片段，用凝胶电泳观察到结果(图1)。然后将PCR产物亚克隆到标准载体中，进行克隆采摘和PCR验证(图2)，最后对阳性克隆进行测序。采用基因合成法，将抗Periostin鼠单克隆抗体的可变区基因的框架区替换为人抗体的框架区，人源化抗体可变区基因片段，重组入含有调控序列和人抗体恒定区基因的载体中，构建抗Periostin人源化单克隆抗体的完整基因及包含所述基因的真核表达载体，再转染CHO-K1细胞。经GS系统加压筛选，获得持续稳定分泌人源化抗体的CHO-K1细胞。
本发明所述抗periostin人源化单克隆抗体的重链可变区的氨基酸序列为：
Figure PCTCN2022118162-appb-000001
本发明所述抗periostin人源化单克隆抗体的轻链可变区的氨基酸序列为：
Figure PCTCN2022118162-appb-000002
本发明所述抗periostin人源化单克隆抗体的重链恒定区的氨基酸序列为：
Figure PCTCN2022118162-appb-000003
Figure PCTCN2022118162-appb-000004
本发明所述抗periostin人源化单克隆抗体的轻链恒定区的氨基酸序列为：
Figure PCTCN2022118162-appb-000005
本发明所述抗periostin人源化单克隆抗体的重链的氨基酸序列为：
Figure PCTCN2022118162-appb-000006
本发明所述抗periostin人源化单克隆抗体的轻链的氨基酸序列为：
Figure PCTCN2022118162-appb-000007
为了方便抗体的表达和纯化，在载体构建和蛋白表达的过程中，分别在重链片段和轻链片段的N端添加接头片段。该片段在后续纯化过程中被去掉。一些实施例中，所述抗periostin人源化单克隆抗体的重链的N端添加的接头的氨基酸序列为MKHLWFFLLLVAAPRWVLS，所述抗periostin人源化单克隆抗体的轻链的N端添加的接头的氨基酸序列为MVLQTQVFISLLLWISGAYG。
抗体可以被标记和检测，例如，可以通过放射性同位素、能产生可检测物的酶、荧光蛋白质、生物素等等进行标记并被检测。抗体还可以结合于固相载体，包括但不限于聚苯乙烯平板或珠粒等等。
本发明所述药物中含有至少一种功能成分，还包括可药用的载体。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1
1、抗体人源化设计
(1)、CDR区序列鉴定
由鼠杂交瘤细胞(45-2-G3-1-G7-B7，构建方法参见《抗人periostin单克隆嵌合 抗体及应用》，申请号：202010788885.1，来自沈阳何氏眼产业集团有限公司或沈阳眼产业技术研究院有限公司)分离出全部mRNA，以此作为合成cDNA的模板，从cDNA中分离重链可变区DNA片段及轻链可变区DNA片段，再将这些得到的DNA片段克隆进载体并测序得到序列结果如下：
氨基酸序列：
>45-2-G3-1-G7-B7-VL1
Figure PCTCN2022118162-appb-000008
>45-2-G3-1-G7-B7-VL2 and VL3
Figure PCTCN2022118162-appb-000009
>45-2-G3-1-G7-B7-VH
Figure PCTCN2022118162-appb-000010
轻链可变区中的3个CDR区的氨基酸序列依次为：SSASY、DTS、QQWSSNPPT。
重链可变区中的3个CDR区的氨基酸序列依次为：GYSFTDYF、INPYSGDT、GRSGVSGLDY。
(2)序列比对及鼠源抗体CDR移植
使用NCBI开发的Igblast(https：//www.ncbi.nlm.nih.gov/igblast/)搜索IMGT小鼠和人类V基因数据库，输入小鼠抗体可变区序列，序列比对后确定同源性最高的人抗体可变区，然后将小鼠抗体CDR区域移植到的人类框架受体区域，以此完成CDR移植。
2、18个人源化抗体载体构建及表达
(1)基因合成
将可变区序列与人恒定区DNA序列链接，插人pcDNA3.4表达载体，得到质粒图谱(图3-1和图3-2)，periostin抗体的重链和轻链核酸序列、氨基酸序列如下所示。
(2)亚克隆到表达载体
基因合成后对CHO系统密码子优化，并亚克隆到表达载体PATX1。
核酸序列信息包括：
DNA序列
>12-VHA-1423bp
Figure PCTCN2022118162-appb-000011
Figure PCTCN2022118162-appb-000012
>12-VHB-1423bp
Figure PCTCN2022118162-appb-000013
Figure PCTCN2022118162-appb-000014
>459-VHA-1423bp
Figure PCTCN2022118162-appb-000015
>459-VHB-1423bp
Figure PCTCN2022118162-appb-000016
Figure PCTCN2022118162-appb-000017
>459-VHC-1423bp
Figure PCTCN2022118162-appb-000018
>551-VHB-1423bp
Figure PCTCN2022118162-appb-000019
>621-VLA-725bp
Figure PCTCN2022118162-appb-000020
>311-VLA-725bp
Figure PCTCN2022118162-appb-000021
>139-VLA-725bp
Figure PCTCN2022118162-appb-000022
>12-VHA
Figure PCTCN2022118162-appb-000023
>12-VHB
Figure PCTCN2022118162-appb-000024
Figure PCTCN2022118162-appb-000025
>459-VHA
Figure PCTCN2022118162-appb-000026
>459-VHB
Figure PCTCN2022118162-appb-000027
>459-VHC
Figure PCTCN2022118162-appb-000028
>551-VHB
Figure PCTCN2022118162-appb-000029
Figure PCTCN2022118162-appb-000030
>621-VLA
Figure PCTCN2022118162-appb-000031
>311-VLA
Figure PCTCN2022118162-appb-000032
>139-VLA
Figure PCTCN2022118162-appb-000033
(3)瞬转
无内毒素质粒DNA提取，将构建的载体共转染CHO-K1细胞，培养至存活率下降到50％以下。取部分细胞收集培养上清，用protein G纯化后跑SDS-PAGE胶，用抗原蛋白进行ELISA检测抗体的活性。
转染方法：
①转染前一天，接种27×10 6个细胞到完全培养基中培养(不含抗生素及抗聚集试剂)，使细胞在转染当天处于对数生长期。
②转染复合物的准备
a稀释DNA至完全培养基中，轻柔混匀。
b稀释PEI转染试剂至完全培养基中，轻柔混匀，室温孵育5min。
c加稀释的PEI到稀释的DNA中，转染混合物总体积为3ml，轻柔混匀，室温孵育20-30min。
③转染：加入3ml转染混合物到27ml细胞悬液中，轻柔混匀，在37℃，5％CO 2，130rpm条件下培养至细胞活率降到50％以下收样。
转染信息：
Figure PCTCN2022118162-appb-000034
Figure PCTCN2022118162-appb-000035
(4)纯化
离心收集上清，装填18个1ml蛋白A纯化柱，填料用PBS pH 7.5平衡，最后将蛋白A树脂与培养上清混匀孵育过夜，孵育结束后纯化。用PBS，pH 7.5的缓冲液清洗，用20mM柠檬酸pH 2.7缓冲液洗脱，中和缓冲液为1M Tris HCl，pH 9.0中和至6.0。纯化完成后取IN、FT、洗涤和洗脱的样各20ul跑SDS-PAGE，根据SDS-PAGE收集样品透析过夜，然后将样品汇集并进行缓冲液交换和浓缩，最终结果如图4所示。
样品信息
序号 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
Western Blot验证特异性结合，其中1-18为18个人源化单抗，19009414P 11(human IgG1)为periostin嵌合抗体，45-2-G3-1-G7-B7为periostin鼠单抗，结果如下表和图5所示。
Figure PCTCN2022118162-appb-000036
Figure PCTCN2022118162-appb-000037
人源化抗体ELISA的排板情况
Figure PCTCN2022118162-appb-000038
ELISA检测结果
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
3、人源化抗体稳转株构建及表达
(1)载体构建
由上一步瞬转测试结果可得，18号抗体551-VHB/139-VLA的表达量最高，且结合能力能够满足要求，因此，综合考虑表达量和抗体结合能力后，以551-VHB和139-VLA作为periostin人源化抗体的重链和轻链，亚克隆到表达载体pATX-GS2中，后共同转染CHO细胞表达抗体蛋白。琼脂糖凝胶电泳跑胶结果如图6。
(2)periostin人源化抗体表达
将目的片段经双酶切(EcoR I/Not I)、连接后亚克隆到表达载体pATX-GS2中，然后稳定转染CHO细胞，经MSX加压筛选、ELISA有限稀释得到稳定的细胞株。在37℃、5％二氧化碳的摇床中培养7d后收集上清液，用琼脂糖凝胶ProteinA层析填料纯化上清液中的人源化抗体。
①质粒线性化
将提取好的质粒PATX-GS2-139-VLA-551-VHB提前取出10ul备份(用于验证线性化是否成功)，将酶，buffer依次加入质粒，并混匀；EP管插入浮漂，放入37度水浴锅中酶切大概1-2h。酶切成功后需进行回收：根据质粒体积加入0.75倍的异丙醇，混匀；4度离心30min后弃上清；加入1ml 70％乙醇4度离心5min，跑胶测浓度(图7)。
酶切体系：
试剂名称 体积(ul)
酶切buffer 10
质粒 全部
酶 6-10
②转染
为了获得稳定的表达重组抗体的转染细胞池，用PVUI线性化的质粒pATX-GS2-139-VLA-551-VHB转染CHO-K1细胞，化学方法进行转染(FectoCHO TM表达系统转染试剂盒)。
(3)加压筛选
转染48小时后，对转染后的宿主细胞施加相应的选择压力筛选，本发明选用GS筛选系统(glutamine synthetase Gene Expression System)。对细胞进行加压处理，并用30um浓度的MSX处理细胞：采用6孔平板标准分批培养法分别测定CHO-K1细胞株对MSX的自然抗性，测定MSX对未转染细胞株CHO-K1的最低杀灭浓度为20uM。加入30um浓度的MSX，每3～4天更换新的筛选培养基，直至有抗性的稳定细胞出现，MSX筛选2～3周后，产生3个稳定池。
在125ml摇瓶中，细胞培养在30ml选择性培养基中，标准条件下培养(37℃、5％CO 2、130rpm)。当细胞在摇瓶中处于良好状态时，进行30ml补料分批表达试验，在D3、D5、D7和D9上添加进料培养基并全程监测血糖。连续培养3代，直到细胞 活力下降到50％以下结束培养，收集培养液上清，进行纯化分析。纯化结果如图8所示，其中reduced示还原SDS-PAGE电泳结果，non-reduced示非还原SDS-PAGE电泳结果。
样品信息：
Antibody Concentration Specification Quantity Yield
18-stable 1 0.86mg/ml 1.75ml/vial 4vials 6.02mg/30ml
18-stable 2 0.94mg/ml 1.80ml/vial 4vials 6.76mg/30ml
18-stable 3 1.62mg/ml 1.80ml/vial 4vials 11.66mg/30ml
(4)有限稀释法分离单克隆抗体：挑选Stable pool 3多克隆细胞株进行有限稀释单克隆加压铺板，将获得的稳定转染池的细胞悬液稀释到很低的密度，使每个孔平均接种1个细胞。用ELISA法筛选出表达量较高的5-G4、5-E6、5-G6、5-D7、5-G9、2-H3、2-C7共7株单克隆细胞株进行后续的扩大培养。以1∶3倍稀释梯度，抗体浓度依次为1、0.33、0.11、0.037、0.012、0.004、0.0014ug/ml，以及空白对照共8个梯度。显微镜下观察单克隆，ELSIA法检测阳性单克隆，结果见下表。
抗体ELISA结果：
Figure PCTCN2022118162-appb-000039
将克隆5-G4、2-C7、5-E6、5-G9、2-H3、5-D7、5-G6的细胞培养于125ml摇瓶中的30ml选择性培养基中，并在标准条件下培养(37℃，5％二氧化碳，130转/分)3代。当细胞在摇瓶中处于良好状态时，进行30ml补料分批表达试验。在D3、D5、D7和D9上添加进料培养基。全程监测血糖。收集培养基，直到细胞活力下降到50％以下。纯化结果如图9：
样品信息：
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 6.14mg/m 1.61ml/vial 7vials 69.20mg/30ml 2.31g/L
(5)、人源化抗periostin抗体鉴定
从表达量和ELISA筛选结果，选三株稳转株5-G4、5-G6、5-G9进行WB检测，验证结合特异性。
实验条件：抗原1ug，一抗1∶1000稀释，孵育1h，二抗1∶10000稀释，孵育 45min。结果如图10。
由图10可见，5-G4、5-G6、5-G9三株稳转株均与periostin抗原蛋白特异性结合。
4、抗periostin人源化抗体抗纤维化作用研究
4.1研究方法
将5-G6稳转株的抗体在体内外分别进行成纤维细胞的跨孔迁移试验、迁移细胞数量研究，免疫组化分析小鼠的成纤维细胞的迁移；以及HE和Masson染色观察小鼠组织病理变化，研究纤维化面积比率；荧光定量聚合酶链式反应(PCR)法分别检测小鼠纤维化模型中I型胶原蛋白(Col1a1和Col1a2)和III型胶原蛋白(Col3a1)mRNA的表达情况；实验以含periostin+对照IgG的条件培养基为对照IgG组，将含periostin+人源化periostin抗体的条件培养基受试组记做Postn nAb组。
Wilcoxon秩和检验比较纤维化面积比率、阳性浸润成纤维细胞数量及成纤维细胞比率和迁移成纤维细胞数量的中值。
最后统计学方法：采用SPSS统计软件进行实验数据处理，采用方差对组间分析，在所有统计分析中，显著性定义为p＜0.05。
4.2研究结果
4.2.1、跨孔迁移实验中，空白对照组中的成纤维细胞数量远远大于Postn nAb组，即Postn nAb组能够有效抑制成纤维细胞的迁移，这一结果证明了Postn nAb在体外的有效性。
4.2.2、与空白对照组相比，在14dpi时，免疫组织学分析显示Postn nAb减少浸润成纤维细胞的数量。
4.2.3、纤维化面积比率28dpi，与空白对照组小鼠相比，Postn nAb组小鼠中纤维化面积显著减少。
4.2.4、Postn nAb组小鼠中I型胶原蛋白和III型胶原蛋白mRNA表达量与空白对照组小鼠明显降低(P＜0.05)。
研究结论：本研究成功制备了抗periostin人源化单克隆抗体，且制备的抗periostin人源化单克隆抗体能够特异性结合体内periostin蛋白，从而有效的抑制体内纤维化作用，因此抗periostin人源化单克隆抗体有望成为治疗视网膜纤维化的新型药物。
5、Periostin人源化单抗体外活性实验
5.1微管抑制实验
体外培养HUVEC细胞，分别以50000个/孔(24h)进行12孔板铺板，5％CO2，37℃过夜培养后进行加药处理16h，进行血管形成实验，观察并在适当时间点拍照记录，并统计实验结果得下表和图11。
HUVEC Number of tubes HUVEC Number of tubes
分组(μg/mL) 16h 分组(μg/mL) 16h
对照periostin 0 121 periostin抗体1 162
对照periostin 1 108 periostin抗体10 57
对照periostin 10 91 periostin抗体100 41
对照periostin 100 26 periostin抗体1000 2
由表可知，随着抗体浓度增高，微管数量减少，且在相同浓度下人源化单抗的抑制微管率大于鼠单抗，可以证实上述periostin人源化单抗可抑制periostin对人血管内皮细胞微管形成作用。
5.2体外培养RPE细胞并分组，分别进行细胞迁移、细胞侵袭以及细胞划痕实验
a空白对照组(Control)：不含重组periostin蛋白或抗体
b periostin组：只含periostin
c对照IgG组：含periostin+对照IgG
d periostin抗体组：含periostin+Postn nAb
Postn nAb：本发明制备的periostin人源化抗体
5.2.1细胞迁移实验
Transwell检测
(1)细胞离心完成之后弃去培养液，用PBS洗1-2遍，用无血清DMEM培养基重悬，调整细胞密度至1×10 5个/ml；
(2)取细胞悬液200μl加入Transwell小室，24孔板下室加入600μl含10％FBS的培养基，37℃培养16-24h；
(3)取出transwell小室，弃去孔中培养液，PBS洗2次；
(4)4％PFA固定，10min，PBS洗2次；
(5)结晶紫染色10min，PBS洗2次；
(6)显微镜下观察迁移的细胞(图12)，随机选取3个视野进行拍照，并用image J软件进行计数，作图(图13)。
实验结果
Figure PCTCN2022118162-appb-000040
实验结果显示，与空白对照组(Control)相比，periostin处理组细胞迁移率和迁移细胞数显著增大(P＜0.001)，而periostin+Postn nAb处理组显著低于空白对照组(P＜0.001)，periostin+IgG处理组比periostin处理组细胞迁移程度略小，说明periostin人源化单抗可抑制periostin引起的RPE细胞的迁移。
5.2.2细胞侵袭实验
Transwell小室预先涂好Matrigel基质胶，加入一定浓度的细胞悬液，并于下室中加入含FBS的培养基，37℃、5％CO 2培养箱中培养48h后观察、拍照(图14)，统计迁移细胞数(图15)。
Figure PCTCN2022118162-appb-000041
结果显示，与空白对照组相比，periostin处理组细胞迁移率和侵袭细胞数显著增高(P＜0.01)，而periostin+Postn nAb处理组显著降低(P＜0.01)，同时periostin+IgG处理组比periostin处理组RPE细胞迁移程度略小，结果表明periostin人源化单抗有抑制periostin的作用。
5.2.3划痕实验
(1)细胞经上述分组处理后用10μl枪头垂直于孔板制造细胞划痕，尽量保证各个划痕宽度一致；
(2)吸去细胞培养液，用PBS冲洗孔板三次，洗去划痕产生的细胞碎片，加入 相应无血清培养基；
(3)将培养板放入培养箱培养0、6h、24h、48h分别取出拍照；
根据收集图片数据分析实验结果(图16～17)。实验结果与分析：细胞经上述分组处理后用10μl枪头垂直于孔板制造细胞划痕，将培养板放入培养箱培养0、6h、24h、48h分别取出拍照。
Figure PCTCN2022118162-appb-000042
Figure PCTCN2022118162-appb-000043
实验结果显示，培养48h时与空白对照组相比，periostin处理组细胞相对迁移距离显著增高(P＜0.01)，而periostin+Postn nAb处理组显著低于空白对照组(P＜0.001)，同时periostin+IgG处理组比periostin处理组RPE细胞迁移程度略小，结果表明periostin人源化单抗可抑制periostin诱导的RPE细胞的作用。
5.3 Western Blot检测结果
检测不同组别RPE细胞中VEGFA、α-SMA、Col I、Col III、Fibronectin蛋白的表达。
VEGFA(血管内皮生长因子)A：VEGFA诱导血管生成反应，促进血管再生。
α-SMA(α-平滑肌肌动蛋白)：存在于血管壁、肠粘膜肌层和固有肌层以及各种组织间质中，在肌成纤维细胞和肌上皮细胞中高表达，有研究表明在器官纤维化的研究中随纤维化程度加重而增加。
ColI、Col III(I型胶原和III型胶原蛋白)：与皮肤损伤修复过程和修复质量紧密相关。促使真皮层成纤维母细胞增生，主要存在皮肤、肌腱、韧带、血管等结缔组织，构成细胞外基质网状结构，起到支撑器官、保护机体的作用，还与细胞附着、细胞迁移有关。
Fibronectin纤维粘连素：促进细胞的黏连生长，影响细胞的黏附、迁移或肿瘤转移、胚胎发育、生长和分化等。
Figure PCTCN2022118162-appb-000044
Figure PCTCN2022118162-appb-000045
Figure PCTCN2022118162-appb-000046
Figure PCTCN2022118162-appb-000047
Figure PCTCN2022118162-appb-000048
Figure PCTCN2022118162-appb-000049
实验结果显示(图18～19)，与空白对照组(Control)相比，periostin处理组、periostin+IgG处理组、periostin+Postn nAb处理组蛋白表达差异均有统计学意义，(P＜0.001)，与空白对照组(Control)相比，periostin处理组蛋白表达量均显著增高(P＜0.001)，而periostin+Postn nAb处理组的VEGFA、α-SMA、Col I、Col III、Fibronectin蛋白表达量均显著降低(P＜0.001)，由此可说明periostin单抗可抑制VEGFA、α-SMA、Col I、Col III、Fibronectin蛋白的表达。
6 HUVEC细胞体外荧光定量PCR、WB、IF检测
periostin可通过整合素(Integrin)-局部黏着斑激酶(FAK)-磷酸肌醇3激酶(PI3K)-蛋白激酶B(AKT/PKB)信号通路促进视网膜血管的形成和纤维化，通过荧光定量PCR技术，免疫荧光和蛋白质印记法测定人血管内皮细胞膜上的整合素(αvβ3、αvβ5)，内皮生长因子受体-2(VEGFR-2，vascular endothelial growth factor receptor-2)、FAK、PI3K、AKT的mRNA表达量及蛋白表达水平的差异变化，从而证实periostin人源化单抗对玻璃体视网膜纤维化性病变具有治疗作用。
体外培养人HUVEC细胞并分组：
a空白对照组(Control)：不含重组periostin蛋白或抗体
b periostin组：只含periostin
c对照IgG组：含periostin+对照IgG
d periostin抗体组：含periostin+Postn nAb
Postn nAb：本发明制备的periostin人源化抗体
人periostin蛋白以及抗体先孵育1h后，再加入到细胞里再次处理24h后进行下述检测，对照IgG的工作浓度同抗periostin人源化单抗的浓度。
6.1荧光定量PCR测定
检测血管内皮细胞膜上的αvβ3、αvβ5整合素，VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT的mRNA表达量
由图20可知，与空白对照组相比，periostin处理组αvβ3、αvβ5、VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT基因表达增多，periostin+Postn nAb处理组的基因表达量均降低，尤其VEGFR-2的mRNA表达量显著低于空白对照组(P＜0.001)，由此可说明periostin人源化单抗可抑制VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT的mRNA基因的表达。
6.2 Western blot检测HUVEC细胞中αvβ3、αvβ5、VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT蛋白的表达
取正常培养的HUVEC细胞，以10 6个/皿(6cm皿)进行铺板，5％CO 2，37℃过夜培养。细胞过夜贴壁后，按照分组进行处理；BCA试剂盒检测总蛋白含量，SDS-PAGE电泳后转膜，封闭液封闭，洗涤后一抗4℃冰箱过夜，洗膜，二抗室温摇床孵育1h，显色。计算各组灰度值与同条件下内参灰度值之比作为各种蛋白的相对表达量。
不同组别HUVEC细胞中各个蛋白的表达及灰度分析见图21～22，实验结果显示，与空白对照组相比，periostin处理组αvβ3、αvβ5、VEGFR-2、FAK、PI3K、p-PI3K、AKT、p-AKT蛋白的表达增多，而periostin+Postn nAb处理组的蛋白表达量降低，且均低于periostin处理组，由此可说明periostin人源化单抗可抑制periostin的作用。
6.3 IF检测HUVEC细胞中αvβ3、αvβ5、VEGFR-2、FAK蛋白的表达
在培养板中将已爬好细胞的玻片用PBS浸洗，4％的多聚甲醛固定爬片，0.5％TritonX-100室温通透20min，加一抗放入湿盒4℃孵育过夜，加荧光二抗，湿盒中20-37℃孵育1h，滴加DAPI核复染，封片并置于荧光显微镜下观察(图23～26)。
荧光显微镜下可见，periostin蛋白在HUVEC细胞胞浆表达，αvβ3、αvβ5、VEGFR-2、FAK蛋白的空白对照组细胞胞浆呈现弱荧光；periostin处理组细胞胞浆显示强荧光；periostin+Postn nAb处理组细胞荧光信号较periostin处理组明显减弱，且与空白对照组相比无明显差异，由此可以说明periostin人源化单抗可通过阻断Integrin-FAK-PI3K-AKT/PKB信号通路进而阻断periostin的作用，从而抑制视网膜纤维化。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 抗periostin人源化单克隆抗体，其特征在于，
    其重链可变区具有如SEQ ID NO：1所示的氨基酸序列；
    其轻链可变区具有如SEQ ID NO：2所示的氨基酸序列。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，
    其重链的恒定区为人IgG1；
    其轻链的恒定区为人κ型。
  3. 根据权利要求1所述的单克隆抗体，其特征在于，其重链的氨基酸序列如SEQ ID NO：3所示，其轻链的氨基酸序列如SEQ ID NO：4所示。
  4. 编码权利要求1～3任一项所述的单克隆抗体的核酸。
  5. 根据权利要求4所述的核酸，其特征在于，
    编码其重链的核酸具有如SEQ ID NO：5所示的核酸序列；
    编码其轻链的核酸具有如SEQ ID NO：6所示的核酸序列。
  6. 根据权利要求5所述的核酸，其特征在于，
    编码其重链的核酸具有如SEQ ID NO：7所示的核酸序列；
    编码其轻链的核酸具有如SEQ ID NO：8所示的核酸序列。
  7. 包括权利要求4～6任一项所述的核酸的表达载体。
  8. 根据权利要求7所述的表达载体，其特征在于，其骨架载体为PATX-GS2。
  9. 转化或转染权利要求8所述的表达载体的宿主细胞。
  10. 权利要求1～3任一项所述的抗periostin人源化单克隆抗体的制备方法，包括：培养权利要求9所述的宿主细胞、诱导抗periostin人源化单克隆抗体的表达。
  11. 权利要求1～3任一项所述抗periostin人源化单克隆抗体在制备药物或检测试剂中的应用，所述药物为治疗组织纤维纤维化病变和/或恶性肿瘤的药物，所述试剂为诊断组织纤维纤维化病变和/或恶性肿瘤的试剂。
  12. 根据权利要求11所述的应用，其特征在于，所述组织纤维化病变为视网膜纤维化。
  13. 一种治疗组织纤维纤维化病变和/或恶性肿瘤的药物，其特征在于，包括权利要求1～3任一项所述抗periostin人源化单克隆抗体。
  14. 一种诊断组织纤维纤维化病变和/或恶性肿瘤的试剂盒，其特征在于，
    包括：权利要求1～3任一项所述的抗periostin人源化单克隆抗体，
    或包括将权利要求1～3任一项所述的抗periostin人源化单克隆抗体与化学标记或生物标记形成的结合物；
    或包括权利要求1～3任一项所述的抗periostin人源化单克隆抗体与固体介质或半固体介质偶联制得的偶联物。
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