WO2020052675A1 - 含单株抗体或其抗原结合片段的医药组合物及其用途 - Google Patents

含单株抗体或其抗原结合片段的医药组合物及其用途 Download PDF

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WO2020052675A1
WO2020052675A1 PCT/CN2019/105824 CN2019105824W WO2020052675A1 WO 2020052675 A1 WO2020052675 A1 WO 2020052675A1 CN 2019105824 W CN2019105824 W CN 2019105824W WO 2020052675 A1 WO2020052675 A1 WO 2020052675A1
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Prior art keywords
monoclonal antibody
antigen
ptx3
binding fragment
seq
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PCT/CN2019/105824
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English (en)
French (fr)
Inventor
王育民
李尹甄
萧郁韦
纪智瑛
杜军毅
梁馨尹
郑朝峻
柯琼媛
陈丰炜
刘芷妘
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王育民
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Priority claimed from PCT/CN2018/105733 external-priority patent/WO2019056991A1/zh
Priority to EP19861063.6A priority Critical patent/EP3862363A4/en
Priority to BR112021004586-4A priority patent/BR112021004586A2/pt
Priority to CN202311402782.7A priority patent/CN117417443A/zh
Priority to JP2021538885A priority patent/JP2022500503A/ja
Priority to CN201980058876.2A priority patent/CN112739714B/zh
Application filed by 王育民 filed Critical 王育民
Priority to MX2021003032A priority patent/MX2021003032A/es
Priority to SG11202102514QA priority patent/SG11202102514QA/en
Priority to KR1020217010919A priority patent/KR20210062036A/ko
Priority to CA3112678A priority patent/CA3112678A1/en
Priority to AU2019337248A priority patent/AU2019337248A1/en
Priority to US17/274,960 priority patent/US20220119507A1/en
Publication of WO2020052675A1 publication Critical patent/WO2020052675A1/zh
Priority to PH12021550529A priority patent/PH12021550529A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to an antibody and use thereof, in particular to a single antibody or antigen-binding fragment thereof that specifically inhibits or slows down the C-terminal specific sequence of PTX3 from binding to the PTX3 receptor, and its application to detection reagents, suppression or slowdown.
  • cancer cells are known to stimulate the microenvironment surrounding the tumor to produce various inflammatory factors, white blood cells, excessive proliferation of blood vessels, and proteases.
  • the chronic inflammatory response of cancer is also related to the growth, metastasis, and invasion of cancer cells.
  • its formation and details There are still many unknowns in the mechanism.
  • the tumor microenvironment is closely related to tumor metastasis (metastasis) and chemotherapy resistance (chemoresistance).
  • the tumor microenvironment is composed of a variety of stromal cells and other cells of different forms, which not only protects the tumor, allows the tumor cells to escape and resist immune cells, and causes the drug resistance of the tumor cells.
  • CEBPD secreted factor-pentraxin-related protein 3
  • PTX3 secreted factor-pentraxin-related protein 3
  • activation of CEBPD in cancer surrounding tissue cells may also promote cancer metastasis and even promote the generation of drug-resistant cancer cells during chemotherapy. These drug-resistant cancer cells will grow faster and more easily metastasize.
  • small molecule anticancer drugs such as cis-diammine dichloroplatinum (II); CDDP; trade name Cisplatin; paclitaxel; trade name Taxol; and 5-Fluorouracil (5-FU), etc.
  • II cis-diammine dichloroplatinum
  • CDDP trade name Cisplatin
  • paclitaxel trade name Taxol
  • the above-mentioned small molecule anticancer drugs not only activate CEBPD expression in cancer cells, but also activate CEBPD expression in macrophages and fibroblasts. Instead, it promotes cancer cells to develop resistance and metastasize quickly, resulting in poor cancer treatment.
  • PTX3 and PTX3 receptor binding are related to fibrotic diseases and / or fibrotic symptoms in addition to the cancers described above.
  • an embodiment of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to the C-terminal specific sequence of one or more pentraxin-related proteins (PTX3). .
  • Another embodiment of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region sequence and a light chain variable region sequence of a specific sequence.
  • kits for detecting PTX3 which comprises the above-mentioned monoclonal antibody or an antigen-binding fragment thereof.
  • Another embodiment of the present invention is to provide a method for detecting PTX3 in vitro, which uses the above kit to detect PTX3.
  • Yet another embodiment of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a monoclonal antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, and the foregoing monoclonal antibody or an antigen-binding fragment thereof As an active ingredient.
  • Yet another embodiment of the present invention provides the use of a monoclonal antibody or an antigen-binding fragment thereof for preparing a pharmaceutical composition that specifically inhibits or slows the binding of PTX3 to a PTX3 receptor, wherein the monoclonal antibody or its antigen binds
  • the fragment is an active ingredient, and the monoclonal antibody or antigen-binding fragment thereof has an effective dose to inhibit or slow down diseases or symptoms related to PTX3 and PTX3 receptor binding.
  • Still another embodiment of the present invention is to provide a method for inhibiting or slowing the activity of tumor cells in vitro, which comprises administering to the tumor cells an effective dose of the above-mentioned pharmaceutical composition to inhibit or slow the activity of tumor cells.
  • Still another embodiment of the present invention is to provide a method for inhibiting or slowing fibrotic diseases and / or fibrotic symptoms in vitro, comprising administering an effective amount of the above to an organ affected by fibrotic diseases and / or fibrotic symptoms.
  • a monoclonal antibody or an antigen-binding fragment thereof is proposed.
  • the above monoclonal antibodies or antigen-binding fragments thereof can specifically bind the non-denaturing amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11.
  • the non-denaturing amino acid sequence may include, but is not limited to, the amino acid sequence listed in any one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 or any combination thereof.
  • the aforementioned non-denaturing amino acid sequence may include, but is not limited to, the amino acid sequence listed in any one of SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID ID NO: 11 or any combination thereof.
  • a monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region sequence and a light chain variable region sequence
  • the heavy chain variable region sequence may be, for example, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and / or amino acid sequences listed in SEQ ID NO: 21
  • the light chain variable region sequence may be, for example, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO : 24 and / or the amino acid sequence listed in SEQ ID NO: 25.
  • the heavy chain variable region sequences of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may be, for example, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and / or SEQ ID NO: 29.
  • the light chain variable region sequence can be, for example, the amino acids encoded by the nucleic acid sequences listed in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and / or SEQ ID NO: 33. sequence.
  • the heavy chain variable region sequence of the above monoclonal antibody or antigen-binding fragment thereof may be, for example, the amino acid sequence listed in SEQ ID NOs: 34 or 35, and the light chain variable region sequence may be, for example, SEQ ID NOs: 36 Or the amino acid sequence listed in 37.
  • the heavy chain variable region sequence of the above-mentioned monoclonal antibody or antigen-binding fragment thereof may be, for example, the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NOs: 38 or 39
  • the light chain variable region sequence may be, for example, SEQ ID NOs: the amino acid sequences encoded by the nucleic acid sequences listed in 40 or 41.
  • the monoclonal antibody or the antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
  • the monoclonal antibody or the antigen-binding fragment thereof is a murine antibody, a human-mouse chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.
  • the single antibody or the antigen-binding fragment thereof may be, for example, a single-chain variable fragment (scFv), a single-chain variable region fragment dimer sc (scFv) 2 ⁇ , Single-chain variable region fragment trimer (scFv) 3 ⁇ , variable fragment (Fv), Fab fragment, Fab 'fragment, F (ab') 2 fragment, nanobody, or the above random combination.
  • scFv single-chain variable fragment
  • scFv single-chain variable region fragment dimer sc
  • scFv Single-chain variable region fragment trimer
  • the monoclonal antibody or antigen-binding fragment thereof is modified through conjugation or binding, saccharification, tag attachment, or any combination thereof.
  • the monoclonal antibody or the antigen-binding fragment thereof is an antibody-drug complex (ADC) or an antigen-binding fragment thereof.
  • ADC antibody-drug complex
  • the above-mentioned monoclonal antibody or antigen-binding fragment thereof is a bifunctional monoclonal antibody (BsAb) or an antigen-binding fragment thereof.
  • BsAb bifunctional monoclonal antibody
  • the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody (antifunctional) or an antigen-binding fragment thereof.
  • the monoclonal antibody or the antigen-binding fragment thereof belongs to the IgG class, the IgM class, the IgA class, the IgD class, or the IgE class. In another embodiment, the monoclonal antibody or the antigen-binding fragment thereof belongs to the IgG class and has the IgG1, IgG2, IgG3, or IgG4 isotype.
  • the above monoclonal antibodies or antigen-binding fragments thereof are inert antibodies or antagonist antibodies.
  • the monoclonal antibody or the antigen-binding fragment thereof specifically inhibits or slows down the binding of PTX3 receptor recognition to one or more C-terminal specific sequences of PTX3.
  • the above monoclonal antibodies or antigen-binding fragments thereof inhibit or slow down the activity of one or more PTX3.
  • the above-mentioned monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the interaction of PTX3 receptor with one or more PTX3, inhibits or slows the transmission of PTX3 information, or any combination thereof.
  • a kit for detecting PTX3 which comprises a monoclonal antibody or an antigen-binding fragment thereof according to any one of the above, wherein the monoclonal antibody or the antigen-binding fragment thereof can specifically bind to non-denaturing Amino acid sequence, and this non-denaturing amino acid sequence may include, but is not limited to, the amino acid sequence as listed in any one of SEQ ID NO: 1 to SEQ ID NO: 11.
  • a method for detecting PTX3 in vitro uses the above-mentioned kit for detecting PTX3 to detect PTX3, wherein the detection of a single antibody or an antigen-binding fragment thereof contained in the kit for detecting PTX3
  • the sensitivity may be, for example, not less than 0.0016 pM.
  • a pharmaceutical composition which comprises a monoclonal antibody or an antigen-binding fragment thereof with an effective dose and a pharmaceutically acceptable carrier, and the above-mentioned monoclonal antibody or an antigen-binding fragment thereof. Is the active ingredient.
  • the pharmaceutical composition may further include an active pharmaceutical ingredient.
  • a monoclonal antibody or an antigen-binding fragment thereof for preparing a pharmaceutical combination that specifically inhibits or slows down the binding of pentraxin-related protein (PTX3) to the PTX3 receptor.
  • PTX3 pentraxin-related protein
  • the monoclonal antibody or the antigen-binding fragment thereof is an active ingredient, and the monoclonal antibody or the antigen-binding fragment thereof has an effective dose to inhibit or slow down diseases or symptoms related to PTX3 and PTX3 receptor binding.
  • the above-mentioned pharmaceutical composition is used to inhibit or slow down a disease or symptom associated with PTX3 and PTX3 receptor binding.
  • the disease or symptom may include epithelial cell carcinoma, adenocarcinoma, and glioblastoma. multiforme (GBM) and fibrosis.
  • epithelial cancer may include lung cancer, breast cancer, and nasopharyngeal carcinoma (NPC).
  • NPC nasopharyngeal carcinoma
  • the aforementioned adenocarcinoma may be, for example, colorectal cancer.
  • the organ affected by the fibrotic disease or symptom may include, but is not limited to, lung, liver, kidney, and skin.
  • the above-mentioned pharmaceutical composition may be injected via subcutaneous (sc), intramuscular, intravenous, intraperitoneal (ip), orthotopic, oral, or oral and inhaled Way to vote.
  • sc subcutaneous
  • ip intravenous
  • ip intraperitoneal
  • orthotopic oral
  • oral and inhaled Way to vote may be injected via subcutaneous (sc), intramuscular, intravenous, intraperitoneal (ip), orthotopic, oral, or oral and inhaled Way to vote.
  • a method for suppressing or slowing the activity of tumor cells in vitro comprises administering an effective dose of the above-mentioned pharmaceutical composition to tumor cells, thereby suppressing or slowing the activity of tumor cells.
  • a method for suppressing or slowing the activity of tumor cells in vitro comprises administering an effective dose of the above-mentioned pharmaceutical composition to tumor cells, thereby suppressing or slowing the activity of tumor cells.
  • a method for suppressing or slowing fibrotic diseases and / or fibrotic symptoms in vitro comprises administering an effective dose of the above to an organ affected by fibrotic diseases and / or fibrotic symptoms.
  • a pharmaceutical composition thereby suppressing or slowing fibrotic diseases and / or fibrotic symptoms of the aforementioned organs.
  • the single antibody or antigen-binding fragment of the present invention is used to specifically inhibit or slow down the binding of PTX3 to the PTX3 receptor using a specific PTX3 single-body antibody or antigen-binding fragment thereof, and is not only applicable to the kit for detecting PTX3 in vitro
  • the method of group and in vitro detection of PTX3 content can also be applied to a pharmaceutical composition that inhibits or slows down PTX3 receptor recognition of PTX3-related diseases or symptoms and uses thereof.
  • FIG. 1 A graph showing the affinity curve of a PTX3 monoclonal antibody to a PTX3 recombinant protein according to an embodiment of the present invention.
  • FIG. 2 A graph showing an affinity curve of a PTX3 monoclonal antibody to a PTX3 recombinant protein according to another embodiment of the present invention.
  • FIG. 3 and [Fig. 4] are epitope mapping maps showing the binding of a PTX3 monoclonal antibody to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention.
  • FIG. 5 shows the results of binding of a PTX3 monoclonal antibody or a commercially available antibody to different kinds of PTX3 recombinant proteins according to an embodiment of the present invention.
  • FIG. 6 A diagram showing a competitive inhibition of the PTX3 monoclonal antibody inhibiting the binding of the PTX3 recombinant protein to the PTX3 receptor according to an embodiment of the present invention.
  • FIG. 7A shows the number of transitional cells (Fig. 7A), invasive cells (Fig. 7B), and cell pellets showing that the PTX3 monoclonal antibody inhibits the breast cancer cell line MDA-MB231 according to an embodiment of the present invention Number ( Figure 7C).
  • FIG. 8A] to [Fig. 8C] shows the number of transitional cells (Fig. 8A), invading cells (Fig. 8B), and cell pellets (Fig. 8B) of lung cancer cell line A549 inhibited by the PTX3 monoclonal antibody according to an embodiment of the present invention.
  • Figure 8C shows the number of transitional cells (Fig. 8A), invading cells (Fig. 8B), and cell pellets (Fig. 8B) of lung cancer cell line A549 inhibited by the PTX3 monoclonal antibody according to an embodiment of the present invention. Figure 8C).
  • FIG. 9A to [FIG. 9C] It is shown that the PTX3 monoclonal antibody inhibits the number of transitional cells (FIG. 9A), invasive cells (FIG. 9B), and cell pellets of nasopharyngeal carcinoma cell line HONE1 according to an embodiment of the present invention ( Figure 9C).
  • FIG. 10A shows the number of transitional cells (Fig. 10A), invasion cells (Fig. 10B), cells of the glioblastoma cell line U87MG inhibited by the PTX3 monoclonal antibody according to an embodiment of the present invention Results of the number of pellets (Fig. 10C).
  • FIG. 11A] to [Fig. 11B] shows that the PTX3 monoclonal antibody or the commercially available antibody inhibits the number of transitional cells (Fig. 11A) and the number of invasive cells (Fig. 11B) of the breast cancer cell line MDA-MB231 according to an embodiment of the present invention. the result of.
  • FIG. 12A results showing that the PTX3 monoclonal antibody or a commercially available antibody inhibits the number of transitional cells (Fig. 12A) and the number of invasive cells (Fig. 12B) of the lung cancer cell line A549 according to an embodiment of the present invention. .
  • FIG. 13A to [Fig. 13B] It is shown that a PTX3 single antibody or a commercially available antibody inhibits the number of transitional cells (Fig. 13A) and invasive cells (Fig. 13B) of nasopharyngeal carcinoma cell line HONE1 according to an embodiment of the present invention. the result of.
  • FIG. 14A] to [FIG. 14C] shows that a PTX3 monoclonal antibody or a commercially available antibody inhibits a breast cancer cell line MDA-MB231 (FIG. 14A), a lung cancer cell line A549 (FIG. 14B), and a nasopharynx according to an embodiment of the present invention. Results of the number of cell pellets of the cancer cell line HONE1 (FIG. 14C).
  • FIG. 15A to [Fig. 15B] It is shown that a PTX3 monoclonal antibody or a control group antibody inhibits tumor volume (Fig. 15A) and tumor metastasis of mouse orthotopically transplanted breast cancer cell MDA-MB231 according to an embodiment of the present invention. ( Figure 15B).
  • a PTX3 monoclonal antibody or an isotype control antibody inhibits tumor volume (Fig. 16A) and tumor metastasis (Fig. 16B).
  • FIG. 17A] to [Fig. 17C] are images showing the tumor volume of the mouse orthotopically transplanted breast cancer cell 4T1 with or without PTX3 monoclonal antibody according to an embodiment of the present invention, respectively.
  • FIG. 18A is a schematic diagram showing an experimental procedure for evaluating mouse orthotopically transplanted breast cancer cells 4T1 with or without PTX3 monoclonal antibody according to an embodiment of the present invention.
  • FIG. 18B] to [Fig. 18D] shows the tumor volume and the tumor volume of orthotopically transplanted breast cancer cells 4T1 in mice with or without paclitaxel according to another embodiment of the present invention, respectively.
  • Image of metastases Figure 18B
  • tumor volume line chart (18C)
  • mouse survival rate Figure 18D
  • 19A is a schematic flow chart showing an experimental procedure for inhibiting the implantation of a mouse colon cancer cell line MC38 by a PTX3 monoclonal antibody or a control group antibody according to an embodiment of the present invention.
  • FIG. 19B A line graph showing tumor volume of a mouse that is inhibited from implanting a colon cancer cell line MC38 by a PTX3 monoclonal antibody or a control group antibody according to an embodiment of the present invention.
  • FIG. 20A and FIG. 20B are line graphs showing tumor volume of a mouse with a human glioblastoma cell line U87MG inhibited by a PTX3 monoclonal antibody or a control group antibody according to an embodiment of the present invention (FIG. 20A) And mouse survival rates (Figure 20B).
  • FIG. 21A is a schematic diagram showing an experimental procedure for evaluating the improvement effect of acute liver fibrosis mice using a PTX3 monoclonal antibody according to an embodiment of the present invention.
  • FIG. 21B] to [Fig. 21D] are diagrams showing left livers stained with hematoxylin and eosin (H & E) in mice with acute liver fibrosis according to an embodiment of the present invention.
  • Leaf tissue sections Figure 21B
  • liver necrosis area ratio Figure 21C
  • liver weight / weight ratio Figure 21D
  • FIG. 22A is a schematic diagram showing an experimental flow for evaluating an improvement effect on mice with chronic liver fibrosis using a PTX3 monoclonal antibody according to an embodiment of the present invention.
  • FIG. 22B] to [Fig. 22D] are left liver lobe tissue sections (Picture-Sirius Red) stained with PTX3 monoclonal antibody according to an embodiment of the present invention, respectively, in mice with chronic liver fibrosis (Fig. 22B). 22B), liver fibrosis area ratio ( Figure 22C) and liver weight / weight ratio ( Figure 22D).
  • FIG. 23A to [Fig. 23C] The results of Western blot analysis (Fig. 23A) showing the expression of fibrosis-related proteins of renal fibroblasts by PTX3 monoclonal antibodies according to an embodiment of the present invention, and Cell staining image ( Figure 23B) and its histogram ( Figure 23C).
  • FIG. 24A to [Fig. 24D] Schematic diagrams (Fig. 24A and Fig. 24C) and kidney tissue section staining images (Fig. 24A and Fig. 24C) of renal fibrosis experiments of UUO mice according to an example of the present invention, respectively. 24B and FIG. 24D).
  • FIG. 25A The Western blot analysis results (Fig. 25A) and the number of transitional cells showing the expression of fibrosis-related proteins of lung fibroblasts by the PTX3 monoclonal antibody according to an embodiment of the present invention, respectively.
  • Bar chart Figure 25B
  • cell staining image of cell nodules Figure 25C
  • its bar chart Figure 25D
  • FIG. 26A to [Fig. 26D] Schematic diagrams (Fig. 26A), body weight change curves (Fig. 26B), and experimental procedures showing the PTX3 monoclonal antibody according to an embodiment of the present invention on BLM-induced pulmonary fibrosis mice, respectively. Lung appearance and tissue section images ( Figures 26C and 26D).
  • FIG. 27A to [Fig. 27D] Schematic diagrams (Fig. 27A), body weight change curves (Fig. 27B), and experimental procedures showing the PTX3 monoclonal antibody according to an embodiment of the present invention on BLM-induced pulmonary fibrosis mice, respectively. Lung appearance and tissue section images ( Figures 27C and 27D).
  • FIG. 28A to [Fig. 28C] The results of Western blot analysis (Fig. 28A) and cell nodules showing the expression of fibrosis-related proteins of embryonic fibroblasts by the PTX3 monoclonal antibody according to an embodiment of the present invention, respectively.
  • Cell staining image Figure 28B
  • Figure 28C histogram
  • FIG. 29A] to [Fig. 29B] are Western blot analysis results showing the expression of fibrosis-related proteins of hepatic fibroblasts treated with PTX3 monoclonal antibodies according to an embodiment of the present invention, respectively.
  • the present invention provides a pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof and use thereof, which utilizes a monoclonal antibody or an antigen-binding fragment thereof to specifically inhibit a pentaxin-related protein (pentraxin- Related protein (PTX3) binding to PTX3 receptors can be applied to a kit for detecting PTX3 and its detection method, as well as a pharmaceutical composition that inhibits or slows down diseases or symptoms related to the binding of PTX3 and PTX3 receptors, and uses thereof.
  • a pentaxin-related protein pentraxin- Related protein (PTX3) binding to PTX3 receptors
  • the monoclonal antibodies or antigen-binding fragments thereof referred to in the present invention may include a heavy chain variable region sequence and a light chain variable region sequence of a specific sequence to specifically inhibit or slow down the C-terminal specific sequences of PTX3 and PTX3 receptors. Combination.
  • the aforementioned monoclonal antibody or antigen-binding fragment thereof can specifically bind to the C-terminal amino acid sequence of human PTX3, and the sequence range is not limited.
  • NO: 1 to SEQ ID NO: 17 The non-denaturing amino acid sequences listed are preferably specifically combined with the non-denaturing amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11 and are specifically combined with SEQ ID NO: 1 to SEQ ID
  • the non-denaturing amino acid sequences listed in NO: 5 and 11 are preferable, and the non-denaturing amino acid sequences listed in SEQ ID NO: 2 to SEQ ID NO: 4 are specifically combined.
  • the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11 correspond to the 200th amino acid to the 236th amino acid of the non-denatured amino acid sequence of the human PTX3 recombinant protein.
  • the non-denaturing amino acid sequences listed in the aforementioned SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID ID NO: 11 correspond to the 200th amino acid to the 200th amino acid of the non-denaturing amino acid sequence of the human PTX3 recombinant protein. 220 amino acids.
  • non-denatured amino acid sequences listed in the aforementioned SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID ID NO: 11 correspond to the 203rd amino acid to the 203rd amino acid sequence of the non-denatured amino acid sequence of the human PTX3 recombinant protein 217 amino acids.
  • the above monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the sequence of CDR1 of the heavy chain variable region has the sequence shown in SEQ ID NO: 18 Amino acid sequence.
  • the amino acid sequence of CDR2 of the heavy chain variable region can be RIDPANX 1 X 2 TKYDPX 3 FQG, where X 1 represents G or D, X 2 represents D or N, and X 3 represents K or M.
  • the specific amino acid sequence is as shown in SEQ ID NOs: 19 or 20.
  • the sequence of the CDR3 of the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 21.
  • the sequence of the CDR1 of the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 22.
  • the sequence of the CDR2 of the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 23.
  • the amino acid sequence of CDR3 of the light chain variable region may be HQX 4 QRSPLT, where X 4 represents F or Y, and the specific amino acid sequence is as listed in SEQ ID NOs: 24 or 25.
  • the heavy chain variable region sequences of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may have SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and / or SEQ ID NO: 29
  • the amino acid sequence encoded by the listed nucleic acid sequence, and the light chain variable region sequence may have a nucleic acid sequence such as SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and / or SEQ ID NO: 33 Encoded amino acid sequence.
  • the heavy chain variable region sequence of the above monoclonal antibody or antigen-binding fragment thereof may have an amino acid sequence as set forth in SEQ ID NOs: 34 or 35, and the light chain variable region sequence may have a sequence such as SEQ ID NOs: amino acid sequences listed in 36 or 37.
  • the heavy chain variable region sequence of the above monoclonal antibody or antigen-binding fragment thereof may have the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NOs: 38 or 39, and the light chain variable region sequence may It has an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NOs: 40 or 41.
  • the type of the monoclonal antibody or the antigen-binding fragment thereof is not limited, and may be, for example, a chimeric antibody or an antigen-binding fragment thereof.
  • the monoclonal antibody or the antigen-binding fragment thereof may be, for example, a murine antibody, a human-mouse chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.
  • the structure of the above-mentioned monoclonal antibody or its antigen-binding fragment is not limited, and it can be a complete antibody structure, or simplified, under the premise of considering the stability of the complementarity-determining region (CDR) structure.
  • CDR complementarity-determining region
  • Antibody structure such as single-chain variable region (scFv), single-chain variable region fragment dimer (scFv) 2 ⁇ , single-chain variable region fragment trimer sc (scFv) 3 ⁇ , variable fragment (Fv), Fab fragment, Fab 'fragment, F (ab') 2 fragment, nanobody [nanobody, also known as single domain antibody (sdAb) or heavy chain antibody (heavy-chain antibody)] or any combination of the above to simplify the process of recombinant antibodies.
  • the above monoclonal antibodies or antigen-binding fragments thereof may be performed by means of fusion tumor cells or recombinant gene expression, which is well known to those skilled in the technical field to which the present invention pertains and will not be repeated here.
  • the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may be further modified through conjugation or binding, saccharification, tag attachment, or any combination thereof, depending on actual needs.
  • the above-mentioned monoclonal antibody or antigen-binding fragment thereof can further form an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof with the drug.
  • ADC antibody-drug conjugate
  • the above-mentioned monoclonal antibodies or antigen-binding fragments thereof can also bind to specific information peptides to enter specific sites, for example, through the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the above-mentioned monoclonal antibody or antigen-binding fragment thereof may be, for example, a bifunctional monoclonal antibody (BsAb), a trifunctional monoclonal antibody, or an antigen-binding fragment thereof.
  • BsAb bifunctional monoclonal antibody
  • a trifunctional monoclonal antibody or an antigen-binding fragment thereof.
  • the single antibody or the antigen-binding fragment thereof is modified through conjugation or binding, saccharification, tag attachment, or any combination thereof.
  • the single antibody or the antigen-binding fragment thereof is an antibody-drug complex (ADC) or an antigen-binding fragment thereof.
  • ADC antibody-drug complex
  • the monoclonal antibody or the antigen-binding fragment thereof is a bifunctional monoclonal antibody (BsAb) or an antigen-binding fragment thereof.
  • BsAb bifunctional monoclonal antibody
  • the monoclonal antibody or the antigen-binding fragment thereof is a trifunctional monoclonal antibody (antifunctional) or an antigen-binding fragment thereof.
  • the monoclonal antibody or the antigen-binding fragment thereof may belong to the IgG class, the IgM class, the IgA class, the IgD class, or the IgE class.
  • the monoclonal antibody or the antigen-binding fragment thereof belongs to the IgG class and has the IgG1, IgG2, IgG3, or IgG4 isotype.
  • the monoclonal antibody or the antigen-binding fragment thereof has an IgG1 isotype, for example, an IgG1k isotype.
  • the above monoclonal antibodies or antigen-binding fragments thereof belong to inert antibodies or antagonist antibodies.
  • the above monoclonal antibodies or antigen-binding fragments thereof can specifically inhibit or slow down the activity of one or more PTX3. In other specific examples, the above-mentioned monoclonal antibodies or antigen-binding fragments thereof can specifically inhibit or slow down the interaction between the PTX3 receptor and one or more PTX3, inhibit or slow down the transmission of PTX3 information, or any combination thereof.
  • the above-mentioned monoclonal antibodies or antigen-binding fragments thereof can be used in the kits and methods for detecting PTX3.
  • the detection can be improved.
  • the form of the biological sample referred to herein is not limited, and may include, but is not limited to, cells, tissues, blood, urine, lymph fluid, tissue fluid, body fluid, and the like.
  • the above kit for detecting PTX3 can be applied to various existing detection components / equipment, such as flow cytometer, enzyme linked immunosorbent assay (ELISA) detection reagent kit, biochip, etc .; or applied to Existing detection methods such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining, and Western blotting analysis )Wait.
  • ELISA enzyme linked immunosorbent assay
  • Existing detection methods such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining, and Western blotting analysis
  • the analytical sensitivity of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may also be referred to as a lower limit of detection (LLOD), which is generally not lower than 0.0016 pM.
  • LLOD lower limit of detection
  • the aforementioned pharmaceutical composition may optionally include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier, diluent, adjuvant, and / or vehicle that is not itself an active ingredient, but is used to deliver the active ingredient to an individual, or Added to the above composition to improve the handling or storage properties of the composition, or to allow or facilitate the dosage unit of the composition to form an excipient or any substance suitable for a pharmaceutical composition and convenient for administration.
  • the aforementioned pharmaceutically acceptable carriers should not disrupt the pharmacological activity of the active ingredient and should be non-toxic when delivering a sufficient therapeutic dose of the active ingredient.
  • suitable pharmaceutically acceptable carriers may be well known to those generally familiar with the general knowledge of manufacturing pharmaceutical compositions, and include but are not limited to buffers, diluents, disintegrants, adhesives, adhesives, wetting agents, Polymers, lubricants, slippers, substances added to mask or counteract bad taste or odor, dyes, fragrances, and substances added to improve the appearance of the composition.
  • aforementioned pharmaceutically acceptable carrier may include, but are not limited to, citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, oxidation Sodium and calcium salts of magnesium, phosphoric acid and sulfuric acid, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose Substances (such as cellulose esters and cellulose alkyl esters of alkanoic acids), low melting waxes, cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (such as serum albumin), ethylenediaminetetraacetic acid (EDTA ), Dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, glycerol or powder, polymers (such as polyvinylpyrrolidone
  • the diseases or symptoms associated with inhibiting or slowing down the binding of PTX3 and PTX3 receptors as referred to herein may include epithelial cell carcinoma, glioblastoma multiforme (GBM), adenocarcinoma and fibrosis.
  • Examples of the aforementioned epithelial cell carcinoma include lung cancer, breast cancer, and nasopharyngeal cancer.
  • the aforementioned adenocarcinoma may be, for example, colorectal cancer.
  • the aforementioned organs affected by fibrotic diseases or symptoms may include, but are not limited to, lungs, livers (eg, acute liver fibrosis, chronic liver fibrosis), kidneys, and skin.
  • the above-mentioned monoclonal antibody or antigen-binding fragment thereof can be used to administer an effective amount of the above-mentioned monoclonal antibody or antigen-binding fragment thereof or the above-mentioned pharmaceutical composition to a target cell or a subject, thereby inhibiting or slowing down the interaction with PTX3 Diseases or symptoms associated with PTX3 receptor binding.
  • the aforementioned "effective dose" in the present invention refers to the administration of 2 mg to 10 mg of PTX3 monoclonal antibody or antigen-binding fragment thereof per kilogram of body weight, and this dose is administered once a week.
  • the effective dose of the aforementioned PTX3 monoclonal antibody or antigen-binding fragment thereof may be, for example, 5 mg / kg body weight to 10 mg / kg body weight, and more preferably 6 mg / kg body weight to 9 mg / kg body weight. As for application to other subjects, it can be converted into a suitable effective dose according to the biological equality. It is explained here that if the effective dose of the PTX3 monoclonal antibody is less than 2 mg / kg of body weight, it cannot effectively reduce or inhibit or slow down the binding of PTX3 to the PTX3 receptor within a predetermined time.
  • the above monoclonal antibodies or antigen-binding fragments thereof can inhibit or slow down the activity of tumor cells, such as proliferation, cancer stemness, migration, invasion, and metastasis (metastasis), tumor volume, or drug resistance.
  • Fibrosis is defined as the formation of excess fibrous tissue in organs or tissues, such as fibrous connective tissue, or fibrous tissue formed during inflammation, repair or reaction.
  • the fibrous tissue may be reactive, benign, or pathological.
  • fibrosis can be used to describe the pathological state of excessive deposition of fibrous tissue and the process of connective tissue deposition during wound healing. For example, scars formed by wounds, fibroids formed by a single type of cells, or pathological conditions caused by excessive deposition of fibrous tissue.
  • the aforementioned monoclonal antibodies or antigen-binding fragments thereof can inhibit or slow down fibrosis-related diseases or symptoms, such as acute liver fibrosis, chronic liver fibrosis, and the like.
  • the above-mentioned pharmaceutical composition can be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, orthotopic injection, oral administration, oral and nasal inhalation, thereby specifically inhibiting Endogenous PTX3 activity, which in turn inhibits or slows the activity of cancer cells.
  • the monoclonal antibody or the antigen-binding fragment thereof and the pharmaceutical composition containing the same can inhibit or slow down the cancer cell after being used for a preset time, for example, 4 weeks to 11 weeks. active.
  • This example uses the existing fusion tumor method or recombinant protein expression method to prepare a PTX3 monoclonal antibody that specifically recognizes the C-terminal amino acid sequence of the PTX3 recombinant protein.
  • a PTX3 recombinant protein with a non-denaturing amino acid sequence as set forth in SEQ ID NO: 13 was used as an immunogen and injected into the abdominal cavity of Balb / C mice at a dose of 50 ⁇ g per mouse by ip injection. After 2 weeks, the mice were supplemented with a dose of 50 ⁇ g per mouse, once every two weeks for a total of four times. Next, the activated splenocytes are fused with a bone marrow cancer cell to produce a fused tumor cell line.
  • a fusion tumor cell line with high binding affinity to the recombinant protein of the non-denatured amino acid sequence listed in SEQ ID NO: 11 was selected, and the PTX3 monoclonal antibody produced by the obtained fusion tumor cell line
  • the non-denaturing amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11 can be specifically bound.
  • the heavy chain variable region sequence may, for example, have the amino acid sequences listed in SEQ ID NO: 18, SEQ ID NO: 19, and / or SEQ ID NO: 21, and the light chain variable region sequence may be, for example, SEQ ID NO: 22, SEQ ID The amino acid sequence listed in NO: 23 and / or SEQ ID NO: 24.
  • the amino acid sequence of the variable region of the heavy chain has the amino acid sequence listed in SEQ ID NO: 34, or the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 38.
  • the amino acid sequence of the light chain variable region has the amino acid sequence as set forth in SEQ ID ID: 36, or the amino acid sequence encoded by the nucleic acid sequence as set forth in SEQ ID NO: 40.
  • This example uses the existing ELISA kit to evaluate the affinity of the PTX3 recombinant protein and the PTX3 monoclonal antibody of Example 1.
  • PTX3 recombinant protein such as the non-denaturing amino acid sequence listed in SEQ ID NO: 14
  • BSA bovine serum albumin
  • a blocking solution a phosphate buffer solution (PBS) containing 3% skim milk powder
  • PBS phosphate buffer solution
  • the primary antibody is the serially diluted PTX3 monoclonal antibody of Example 1 (concentration is 2.44 ⁇ 10 -4 ⁇ g / mL to 1.00 ⁇ g / mL). Then, unbound PTX3 monoclonal antibody in each well was washed away with PBS, and a secondary antibody was added to react at room temperature (4 ° C to 40 ° C). After that, tetramethyl benzidine (TMB) was added to each well for a period of time, and then 0.1 M sulfuric acid (H 2 SO 4 ) was added for 10 minutes to terminate the reaction.
  • TMB tetramethyl benzidine
  • the secondary antibody was bound horseradish hydrogen peroxide Anti-mouse horse peroxidase (HRP) anti-mouse IgG (IgG-HRP).
  • HRP horseradish hydrogen peroxide Anti-mouse horse peroxidase
  • IgG-HRP horse peroxidase
  • ELISA reader enzyme immunoassay
  • FIG. 1 is a graph showing the affinity curve of a PTX3 monoclonal antibody to a PTX3 recombinant protein according to an embodiment of the present invention, wherein the curve indicated by the graph number represents the affinity curve of a PTX3 monoclonal antibody to a PTX3 recombinant protein, FIG.
  • the curve marked by ⁇ represents the affinity curve of BSA to PTX3 recombinant protein.
  • the PTX3 monoclonal antibody of one embodiment of the present invention has high affinity for the PTX3 recombinant protein.
  • 10 ⁇ g / mL of PTX3 recombinant protein (such as the non-denaturing amino acid sequence listed in SEQ ID NO: 14, dissolved in PBS at pH 7.2) was coated in each well of a 96-well cell culture plate (same as above). , Reaction at 4 ° C until overnight.
  • a blocking solution PBS containing 3% bovine serum albumin (BSA) was added to the well, and the blocking reaction was performed at room temperature (4 ° C to 40 ° C) for 1 hour.
  • BSA bovine serum albumin
  • the primary antibody is the serially diluted PTX3 monoclonal antibody of Example 1 (concentration is 0.01 ng / mL to 1000 ng / mL). Then, each well was rinsed several times with PBST to remove unbound PTX3 monoclonal antibody from each well, and a secondary antibody was added to react at room temperature (4 ° C to 40 ° C), where the secondary antibody was anti-mouse bound to HRP IgG (IgG-HRP).
  • FIG. 2 is a graph showing an affinity curve of a PTX3 monoclonal antibody to a PTX3 recombinant protein according to another embodiment of the present invention.
  • the dissociation constant (KD) of the PTX3 monoclonal antibody for the PTX3 recombinant protein is 85 pM, which represents that the PTX3 monoclonal antibody has a higher affinity for the PTX3 recombinant protein, so it can be applied to the PTX3 detection kit. group.
  • the PTX3 monoclonal antibody can specifically bind the 200th to 359th amino acids of the PTX3 recombinant protein (such as the non-denaturing amino acid sequence listed in SEQ ID NO: 13; not shown in the figure) or The 200th amino acid to the 236th amino acid (such as the non-denaturing amino acid sequence listed in SEQ ID NO: 12; as shown in Figure 3).
  • This example uses the existing ELISA kit to evaluate the epitope mapping region of the PTX3 monoclonal antibody binding to PTX3.
  • This example uses the same method as in Example 1 to evaluate the small binding region of PTX3 monoclonal antibody to PTX3. The difference is that this example uses 200 ⁇ g / mL PTX3 recombinant protein (such as SEQ ID ID NOs : The non-denaturing amino acid sequences listed in 12, 16, and 17 are dissolved in 0.1M sodium bicarbonate aqueous solution, pH 8.3) or BSA (as a control group) and coated in the wells of a 96-well cell culture plate, and reacted at 4 ° C to Overnight. Next, a blocking solution (PBS containing 1% BSA) was added to the wells, and a blocking reaction was performed at room temperature (4 ° C to 40 ° C) for 1 hour.
  • a blocking solution PBS containing 1% BSA
  • each well was rinsed with PBS, and then the PTX3 monoclonal antibody (concentration: 125ng / mL) of Example 1 was added and reacted at room temperature (4 ° C to 40 ° C) for 2 hours. Then, unbound PTX3 monoclonal antibody in each well was washed away with PBS, and a secondary antibody (anti-mouse IgG-HRP, diluted 1: 5000) was added to react at room temperature (4 ° C to 40 ° C) for 1 hour. After that, the TMB reaction was added to each well for a period of time, and 0.1 M sulfuric acid was added for 10 minutes to terminate the reaction. The absorbance at 450 nm was read with a commercially available enzyme immunoassay analyzer. The results are shown in FIG. 3. Each value is a triplicate.
  • FIG. 3 is an epitope mapping map showing the binding of a PTX3 monoclonal antibody to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention, where RI37 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 12 KT44 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 16, GI40 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 17, and the figure number "***" represents a comparison with the control group (that is, BSA group) was statistically significant (p ⁇ 0.001).
  • FIG. 4 is an epitope mapping map showing the binding of a PTX3 monoclonal antibody to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention, where the horizontal axis represents the sequence as listed in SEQ ID NO: 1-10, respectively.
  • PTX3 recombinant protein fragment is an epitope mapping map showing the binding of a PTX3 monoclonal antibody to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention, where the horizontal axis represents the sequence as listed in SEQ ID NO: 1-10, respectively.
  • the affinity of the PTX3 monoclonal antibody for the PTX3 recombinant protein fragments as listed in SEQ ID NOs: 1 to 5 or as listed in SEQ ID ID NOs: 2 to 4 is much higher than other fragments, of which SEQ ID ID NOs:
  • the PTX3 recombinant protein fragments listed in 2 to 4 correspond to the 203 to 217 amino acids of PTX3, such as the amino acid sequence listed in SEQ ID NO: 11, and represent the epitope localization region of the PTX3 monoclonal antibody that binds to PTX3 in Example 1. It is located within the range of the amino acid sequence as listed in SEQ ID NOs: 2 to 4 or as listed in SEQ ID NO: 11.
  • Example 5 the PTX3 monoclonal antibody of Example 1 and a commercially available PTX3 monoclonal antibody (model: ab90806; abcam plc., UK) were used to evaluate the epitope mapping region of PTX3 binding in the same manner as in Example 3. The results are shown in Figure 5. Each value is a triplicate.
  • FIG. 5 is an epitope mapping map showing the binding of PTX3 monoclonal antibodies and commercially available PTX3 monoclonal antibodies to different fragments of PTX3 recombinant proteins according to an embodiment of the present invention
  • PTX3 / FL stands for SEQ ID ID NO : PTX3 recombinant protein fragment listed in: 15
  • RI37 represents the PTX3 recombinant protein fragment listed in SEQ ID NO: 12
  • KT44 represents the PTX3 recombinant protein fragment as listed in SEQ ID ID NO: 16
  • GI40 represents the sequence as in SEQ ID ID NO: 17
  • the listed PTX3 recombinant protein fragments, and the figure "***" represents statistical significance compared to the control group (ie, the BSA group) (p ⁇ 0.001).
  • Example 4 Evaluating the Effect of PTX3 Monoclonal Antibody on the Binding of PTX3 to the PTX3 Receptor in Vitro
  • PTX3 monoclonal antibodies can competitively bind to the PTX3 receptor binding region of PTX3 or its adjacent regions, thereby specifically inhibiting or slowing the chance of PTX3 binding to the PTX3 receptor.
  • This example uses CD44 as an example of the PTX3 receptor, and uses a competitive binding test to evaluate the effect of a PTX3 monoclonal antibody to competitively inhibit the binding of PTX3 to the PTX3 receptor.
  • Example 1 demonstrates that the PTX3 monoclonal antibody of Example 1 can neutralize PTX3, making it unable to bind to the PTX3 receptor (eg, CD44) binding region or its adjacent region.
  • PTX3 receptor eg, CD44
  • the competitive binding analysis method used in this example is similar to that in Example 1, except that this example uses a 10 ⁇ g / mL PTX3 receptor [such as the CD44N-terminated recombinant protein (CD44N-terminus The sequence of the 1st to 220th amino acid residues was dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing, China)] coated in the wells of a 96-well cell culture plate, and reacted at 4 ° C overnight. Next, a blocking solution (PBS containing 3% skim milk powder) was added to the wells, and a blocking reaction was performed at room temperature (4 ° C to 40 ° C) for 1 hour.
  • a 10 ⁇ g / mL PTX3 receptor such as the CD44N-terminated recombinant protein (CD44N-terminus
  • CD44N-terminus The sequence of the 1st to 220th amino acid residues was dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing
  • a PTX3 recombinant protein that binds HRP such as the non-denaturing amino acid sequence listed in SEQ ID NO: 14, HRP-PTX3, with a concentration of 5 ⁇ g / mL
  • HRP-PTX3 a different concentration of the PTX3 monomer of Example 1
  • Strain antibody concentration of 1 ⁇ g / mL or 2 ⁇ g / mL
  • each well was rinsed with PBS, and the pre-reactant was added, and the reaction was performed at room temperature (4 ° C to 40 ° C) for 2 hours. Then, the unbound pre-reactants in each well were washed away with PBS. After adding TMB to each well for a period of time, 0.1 M sulfuric acid was added for 10 minutes to stop the reaction, and the absorbance at 450 nm was read using a commercially available enzyme immunoassay analyzer. The results are shown in Figure 6. Each value is a quadruplicate.
  • FIG. 6 is a graph showing the competitive inhibition of the PTX3 monoclonal antibody hindering the binding of the PTX3 recombinant protein to the PTX3 receptor according to an embodiment of the present invention, wherein the vertical axis represents the competitive inhibition rate (%), and the horizontal axis
  • the figure number "+” or "-” below indicates the presence or absence of specific components added during the binding reaction.
  • the first bar on the left side of Figure 6 represents the control group (that is, the group of PTX3 recombinant protein without PTX3 monoclonal antibody added). ), And the figure "***" represents statistical significance compared to the control group (p ⁇ 0.001).
  • the value of the first lane without added PTX3 monoclonal antibody in FIG. 6 is taken as a 0% competitive inhibition rate.
  • the PTX3 monoclonal antibody pre-reacts with the PTX3 recombinant protein, it then reacts with the CD44 receptor.
  • the obtained competitive inhibition rate (%) is dose-dependent with the concentration of the PTX3 monoclonal antibody, and has statistical significance. It represents that the PTX3 monoclonal antibody of Example 1 can indeed neutralize PTX3 and prevent it from binding to CD44. Area or its adjacent areas.
  • Breast cancer, lung cancer, nasopharyngeal carcinoma, and glioblastoma multiforme are malignant tumors, and the cancer cells have activities such as migration, invasion, and cancer stemness.
  • This embodiment uses human breast cancer cell line (MDA-MB231, deposit number: BCRC 60425, ATCC HTB-26; triple negative breast cancer cell line, hereinafter referred to as MB231), human lung cancer cell line A549 (registration number: BCRC 60074; ATCC CCL-185), human nasopharyngeal carcinoma cell line HONE1 (Int. J. Cancer. 1990 Jan 15; 45 (1): 83-9; Proc. Natl. Acad. Sci. USA, Vol. 86, pp.
  • the above cancer cells were seeded at a cell density of 1 ⁇ 10 5 cells / well in the upper layer (bottom pore size: 8 ⁇ m) of a 24-well Boyden cell and cultured for 3 hours.
  • the upper layer of the cell culture solution was replaced with a serum-free cell culture solution, and 0.2 ⁇ g / mL of a PTX3 recombinant protein (such as the non-s listed in SEQ ID NO: 14) was added to the lower serum-free cell culture medium.
  • a PTX3 recombinant protein such as the non-s listed in SEQ ID NO: 14
  • FIG. 7A, FIG. 8A, FIG. 9A and FIG. 10A respectively show the inhibition of breast cancer cell MB231 (FIG. 7A), lung cancer cell A549 (FIG. 8A), and nasopharyngeal carcinoma using the PTX3 monoclonal antibody of Example 1 of the present invention.
  • the data of the above embodiments are obtained by triplicate experimental data of each time point and each sample, plus or minus the average standard deviation thereof, and all values are analyzed by one-way ANOVA.
  • the figure "***” in the above embodiment represents data having statistical significance (P ⁇ 0.01), and the figure "***” represents data having statistical significance (P ⁇ 0.001).
  • the PTX3 monoclonal antibody can significantly inhibit or slow down breast cancer cell MB231, lung cancer cell A549, nasopharyngeal cancer cell HONE1, and neuroglia.
  • the number of transitional cells of the cell tumor cell line U87MG, and this difference is statistically significant.
  • the upper layer (bottom pore size: 8 ⁇ m) of the 24-well Boyden chamber was coated with a matrigel (purchased from BD Bioscience) in advance. a cell density of 105 cells / well, were seeded in 24-well cell migration Borden's upper and incubated for 3 hours.
  • the upper layer of the cell culture solution was replaced with a serum-free cell culture solution, and 0.2 ⁇ g / mL of a PTX3 recombinant protein (such as the non-s listed in SEQ ID NO: 4) was added to the lower serum-free cell culture medium.
  • a PTX3 recombinant protein such as the non-s listed in SEQ ID NO: 4
  • Denatured amino acid sequence 0.4 ⁇ g / mL PTX3 monoclonal antibody or 0.4 ⁇ g / mL control group antibody (IgG1k).
  • the cells on the inside of the upper layer were scraped off with a cotton swab, and the cells that migrated to the outside of the bottom of the upper layer were treated with 4 ', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole (DAPI; Invitrogen) staining, and the number of cells moving to the outside of the bottom of the upper layer was calculated in a field of view of 200 times magnification of a fluorescence microscope, and the results are shown in FIG. 7B, FIG. 8B, FIG. 9B, and FIG. 10B.
  • DAPI 6-diamidino-2-phenylindole
  • FIG. 7B, FIG. 8B, FIG. 9B, and FIG. 10B respectively show the inhibition of breast cancer cell MB231 (FIG. 7B), lung cancer cell A549 (FIG. 8B), and nasopharyngeal carcinoma by using the PTX3 monoclonal antibody of Example 1 of the present invention.
  • the PTX3 monoclonal antibody can significantly inhibit or slow down breast cancer cells MB231, lung cancer cells A549, nasopharyngeal cancer cells HONE1, and neuroglia.
  • the number of invading cells of the cell tumor cell line U87MG, and this difference is statistically significant.
  • the cancer cells have cancer stemness, and the addition of PTX3 recombinant protein can cause the cancer cells to form cell spheres.
  • fetal calf containing 0.2 ⁇ g / mL PTX3 recombinant protein, 0.4 ⁇ g / mL PTX3 monoclonal antibody or 0.4 ⁇ g / mL control group antibody (IgG1k).
  • RPMI-1640 cell culture solution of serum contains 10% fetal bovine serum (FBS), 50 to 100 ⁇ g / mL of streptomycin and 50 to 100 U / mL of penicillin, It is cultured at a concentration of 5% carbon dioxide with a humidity of 37 ° C. This is any person skilled in the art to which this invention belongs, so it will not be repeated here.
  • FIG. 7C, FIG. 8C, FIG. 9C and FIG. 10C respectively show the use of the PTX3 monoclonal antibody of Example 1 to inhibit breast cancer cell MB231 (FIG. 7C), lung cancer cell A549 (FIG. 8C), and nasopharyngeal carcinoma, respectively.
  • HONE1 Figure 9C
  • the cell pellet number bar graph of the glioblastoma cell line U87MG Figure 10C
  • the PTX3 monoclonal antibody of Example 1 can significantly inhibit or slow down breast cancer cells MB231, lung cancer cells A549, nasopharyngeal cancer cells HONE1 and The number of cell pellets of the glioblastoma cell line U87MG, and this difference is statistically significant.
  • This example evaluates the inhibitory effect of the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (model: ab90806; abcam plc., UK) on the activity of cancer cells in the same manner as in Example 2. As shown in FIGS. 11A to 14C.
  • FIG. 11A, FIG. 12A, and FIG. 13A respectively show the inhibition of breast cancer cell MB231 (FIG. 11A) and lung cancer cell A549 (FIG. 12A) using the PTX3 monoclonal antibody of Example 1 of the present invention or with a commercially available PTX3 monoclonal antibody, respectively.
  • PTX3 monoclonal antibody of Example 1 of the present invention or with a commercially available PTX3 monoclonal antibody, respectively.
  • transitional cell number histogram of nasopharyngeal carcinoma cell HONE1 Figure 13A
  • the figure number "*” represents data with statistical significance (P ⁇ 0.05)
  • the figure number "**” represents statistical significance ( P ⁇ 0.01)
  • the figure "***” represents data with statistical significance (P ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or slow down breast cancer cells MB231 and lung cancer cells.
  • the number of A549 and nasopharyngeal cancer cell HONE1 transitional cells, but the effect of the PTX3 monoclonal antibody of Example 1 in inhibiting or slowing the migration of cancer cells was significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and it was statistically significant.
  • FIG. 11B, FIG. 12B, and FIG. 13B respectively show the inhibition of breast cancer cell MB231 (FIG. 11B) and lung cancer cell A549 (FIG. 12B) using the PTX3 monoclonal antibody of Example 1 of the present invention or the commercially available PTX3 monoclonal antibody.
  • Figure 13B the number of invasive cells of nasopharyngeal carcinoma cell HONE1 ( Figure 13B), where the figure number "*” represents data with statistical significance (P ⁇ 0.05), and the figure number "***” represents statistical significance ( P ⁇ 0.01), and the figure "***” represents data with statistical significance (P ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or slow down breast cancer cells MB231 and lung cancer cells.
  • the number of invading cells of A549 and nasopharyngeal carcinoma cell HONE1 was significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and was statistically significant.
  • FIG. 14A, FIG. 14B and FIG. 14C respectively show the inhibition of breast cancer cell MB231 (FIG. 14A) and lung cancer cell A549 (FIG. 14B) by using the PTX3 monoclonal antibody of Example 1 of the present invention or with a commercially available PTX3 monoclonal antibody, respectively.
  • PTX3 monoclonal antibody of Example 1 of the present invention or with a commercially available PTX3 monoclonal antibody, respectively.
  • Figure 14C histogram of the number of cell pellets of nasopharyngeal carcinoma cell HONE1
  • the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or slow down breast cancer cells MB231 and lung cancer cells.
  • the number of cell pellets of A549 and nasopharyngeal carcinoma cell HONE1 but the effect of the PTX3 monoclonal antibody of Example 1 on inhibiting or slowing the number of cell pellets is still significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and it is statistically significant Sex.
  • Example 6 Evaluating the effects of PTX3 monoclonal antibodies on tumor growth and metastasis in vivo
  • the above human cancer cell line is injected in situ into a mammary fat pad of an immunodeficient mouse, and after tumor formation, the PTX3 monoclonal antibody of Example 1 is applied to evaluate the PTX3 individual strain of Example 1. Antibodies inhibit or slow the effects of tumors.
  • breast cancer cells MB231-Luc2 [MB231 is a human breast cancer cell that does not express estrogen receptor (ER) ⁇ and ER ⁇ ; Luc2 is a gene expressing luciferase] was inoculated in situ into NOD-SCID mice ( (Buy from Lesco Biotechnology Co., Ltd.) in a mammary fat pad. After the average tumor volume reached 80 mm 3 , the experimental mice were administered the PTX3 antibody (8 mg / kg body weight) or the control group antibody (IgG 1 k, 8 mg / kg body weight) of Example 1 to the experimental mice once a week. The results are shown in Figure 15A Up to FIG. 15B. FIG.
  • 15B is the in vivo imaging results of breast cancer cell MB231-Luc2 at week 11 after being inoculated, wherein the in vivo imaging is using a commercially available in vivo bioluminescent images system [eg, a non-invasive 3D in vivo molecular imaging system (IVIS system ), PerkinElmer] observe the size of the tumor, and the luminous image represents the tumor in the mouse with breast cancer cells MB231-Luc2. Then, all mice were sacrificed, the tumor size in vivo was measured, and the tumor volume was calculated using the following formula (I):
  • V (w ⁇ l 2 ) ⁇ 0.52 (I)
  • l represents the tumor length and w represents the tumor width.
  • FIG. 15A to FIG. 15B respectively show that a PTX3 monoclonal antibody or a control group antibody inhibits tumor volume (FIG. 15A) and tumor of a mouse orthotopic xenograft breast cancer cell MDA-MB231 according to an embodiment of the present invention.
  • Results of transfer ( Figure 15B).
  • the data in FIG. 15A is obtained by taking six positive and negative average standard deviations of each time point and each sample, and the figure number "*" indicates that it is statistically significant compared to the control group antibody (IgG1k). Sex (p ⁇ 0.05).
  • the PTX3 monoclonal antibody of Example 1 can significantly inhibit or slow down the tumor volume and tumor metastasis of orthotopically transplanted breast cancer cell MB231, and this The item differences are statistically significant.
  • the above mouse cancer cell lines are injected in situ into the mammary fat pads of normal immune system mice. After tumor formation and establishment of a mouse tri-negative breast cancer (TNBC) model, the administration is performed.
  • TNBC mouse tri-negative breast cancer
  • the PTX3 monoclonal antibody of Example 1 was used to evaluate the tumor suppressing effect of the PTX3 monoclonal antibody of Example 1.
  • FIG. 16A to FIG. 16B respectively show that a PTX3 monoclonal antibody or a control group antibody inhibits tumor volume of orthotopically transplanted breast cancer cell 4T1 in mice (FIG. 16A) and Results of tumor metastasis (Figure 16B).
  • FIG. 16B is the in vivo imaging result at the 5th week after 4T1 inoculation of breast cancer cells, where the luminescence image represents a tumor formed by breast cancer cells 4T1 in the mouse, and the data in FIG. 16A is the six replicates of each time point and each sample The experimental data was obtained by taking the positive and negative average standard deviations, and the figure number "***" represents statistical significance compared to the control group antibody (IgG1k) (p ⁇ 0.01).
  • the PTX3 monoclonal antibody of Example 1 can significantly inhibit or slow down the tumor volume and tumor metastasis of orthotopically transplanted breast cancer cell 4T1, and This difference is statistically significant.
  • Example 6 This example was evaluated in the same manner as in point 2 of Example 6, except that in this example, the mouse breast cancer cell line 4T1 was inoculated in situ into BALB / C female mice (6-8 weeks). Age, purchased from the breast fat pad of Luxco Biotechnology Co., Ltd.). After the average tumor volume reached 50 mm 3 , the PTX3 monoclonal antibody of Example 1 was administered [2.5 mg / kg body weight (also known as mpk), 5.0 mg / kg body weight, or 10.0 mg / kg body weight of PTX3Ab.
  • the PTX3 monoclonal antibody of Example 1 was administered [2.5 mg / kg body weight (also known as mpk), 5.0 mg / kg body weight, or 10.0 mg / kg body weight of PTX3Ab.
  • Control group (10mg / kg body weight IgG1k, model 10101, Wei Qiao Shengyi) or combined with paclitaxel (Taxol or Paclitaxel, 30.0mg / kg body weight), administered to mice by intraperitoneal injection once a week Voted for six weeks. After the sacrifice of the rats in the sixth week, the size and metastasis of the tumors were observed with the fluorescence imaging system of live animals.
  • FIG. 17A to FIG. 17C respectively show tumor volume and tumor volume of mouse orthotopically transplanted breast cancer cell 4T1 inhibited by PTX3 monoclonal antibody with or without paclitaxel according to an embodiment of the present invention.
  • Image images of metastases Figure 17A
  • line graphs of tumor volume changes Figures 17B to 17C.
  • the data in Fig. 17B to Fig. 17C are obtained by taking six repeated experimental data of each time point and each sample, and taking the positive and negative average standard deviations thereof, where the figure number "***" represents that compared with the control group antibody (IgG1k ) Has statistical significance (p ⁇ 0.01).
  • the PTX3 monoclonal antibody or paclitaxel of Example 1 can inhibit or slow down the tumor volume and metastasis of orthotopically transplanted breast cancer cell 4T1, such as 17A and 17B.
  • the combination of PTX3 monoclonal antibody and paclitaxel in mice can increase the tumor volume and metastasis of breast cancer cell 4T1, which is allografted in situ, and the combined effect is far more than the combined effect of separate treatment effects, as shown in the figure. 17A and 17C.
  • the above differences are statistically significant.
  • This embodiment is based on the experimental flow diagram of FIG. 18A, and is evaluated in the same manner as in the third point of the sixth embodiment. The results are shown in FIGS. 18B to 18D.
  • FIG. 18B to FIG. 18D respectively show the tumor volume and the tumor volume of orthotopically transplanted breast cancer cells 4T1 in mice with or without paclitaxel according to another embodiment of the present invention.
  • Image of metastases Figure 18B
  • tumor volume line chart Figure 18C
  • mouse survival rate Figure 18D
  • the data in FIGS. 18C to 18D are obtained by taking six positive and negative average standard deviations of each time point and each sample, and the figure number “*” represents the antibody (IgG1k) compared to the control group. It is statistically significant (p ⁇ 0.05), and the figure "***" indicates that it is statistically significant (p ⁇ 0.01) compared to the control group antibody (IgG1k).
  • the PTX3 monoclonal antibody or paclitaxel of Example 1 can inhibit or slow the tumor volume and metastasis of orthotopically transplanted breast cancer cell 4T1, such as 18B and 18C.
  • the combined use of PTX3 monoclonal antibody and paclitaxel in mice can increase the tumor volume of tumor cells that inhibit or slow down orthotopic transplantation of breast cancer cells 4T1, and increase the survival rate of mice by more than 80%.
  • the synergy that exceeds the effect of the individual treatment is shown in Figure 18D. The above differences are statistically significant.
  • This example is based on the experimental flow diagram of FIG. 19A, and is evaluated in the same manner as in the third point of Example 6. The difference is that this example is the subcutaneous (sc) injection of the above-mentioned mouse colorectal cancer cell line MC38 to C57BL / 6J male mice (6-8 weeks of age, purchased from Rosco Biotechnology Co., Ltd.).
  • mice were administered intraperitoneally (ip) once a week for a total of three replicates. Tumor size was observed weekly using a live animal fluorescence imaging system. After day 23, all mice were sacrificed, the tumor size in vivo was measured, and the tumor volume was calculated using the above formula (I).
  • FIG. 19B shows the results of inhibiting tumor volume of mice implanted with colorectal cancer cell line MC38 by a PTX3 monoclonal antibody or a control group antibody according to an embodiment of the present invention.
  • the data of FIG. 19B is obtained by taking four replicate experiments at each time point and each sample, and taking the positive and negative average standard deviations, where the figure number "*" represents statistical significance compared to the control group antibody (IgG1k). (p ⁇ 0.05), and the figure "***” represents statistical significance compared to the control group antibody (IgG1k) (p ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 can inhibit or slow down the tumor volume of the colorectal cancer cell line MC38 compared to the control group antibody (IgG1k), and this difference is statistically significant.
  • This embodiment is evaluated in the same manner as in point 3 of the sixth embodiment, except that in this embodiment, the above-mentioned human glioblastoma multiforme (GBM) cell line U87MG is injected subcutaneously (sc) to NOD-SCID male mice (6-8 weeks old, purchased from Rosco Biotechnology Co., Ltd.).
  • the PTX3 monoclonal antibody of Example 1 [10.0mg / kg body weight of PTX3Ab, Zhenyi Biotechnology] or the control group antibody (10mg / kg body weight IgG1k, model 10101, Wei Qiao Shengyi) was administered.
  • the mice were administered by intraperitoneal (ip) injection once a week for a total of four weeks. After 20 days, tumor size was measured and tumor volume was calculated using the above formula (I).
  • FIG. 20A and FIG. 20B are line graphs showing tumor volume inhibition of PTX3 monoclonal antibody or control group antibody implantation into a human neuroglioblastoma cell line U87MG according to an embodiment of the present invention (FIG. 20A) And mouse survival rates (Figure 20B).
  • the PTX3 monoclonal antibody of Example 1 can inhibit or slow down the tumor volume of xenograft neuroglioma cell U87MG (FIG. 20A). And can improve the survival rate of mice by 75% (Figure 20B). The above differences are statistically significant.
  • Example 7 Evaluating the Effect of PTX3 Monoclonal Antibody to Slow or Reverse Fibrosis in Vivo
  • FIG. 21A This embodiment is performed according to the experimental flow diagram of FIG. 21A, which is designed according to the guidelines of the Animal Center of Yida Hospital, Yishou University.
  • C57BL / 6J male mice (8 weeks old, purchased from Lesco Biotechnology Co., Ltd.) were intramuscularly injected with 1 mL / kg of tetrachloromethane (CCl 4 and olive oil mixed in a 1: 1 volume ratio).
  • tetrachloromethane CCl 4 and olive oil mixed in a 1: 1 volume ratio.
  • Acute liver fibrosis caused by tetrachloromethane can cause apoptosis and necrosis of liver cells within 12 hours.
  • mice were administered the PTX3 monoclonal antibody of Example 1 [10 mg / kg body weight of PTX3Ab, by the way of intraperitoneal (ip) injection].
  • Or control group antibody IgG1k at 10 mg / kg body weight, model 10101, Wei Qiao Shengyi. All mice were sacrificed on the second day, and liver tissue section changes, liver necrosis area ratio, and liver weight / weight ratio were observed. The results are shown in Figs. 21B to 21D.
  • FIG. 21B to FIG. 21D respectively show the left liver stained with hematoxylin and eosin (H & E) in a mouse with acute liver fibrosis according to an embodiment of the present invention.
  • Leaf tissue sections Fig. 21B, magnification: 20 times, observation of hepatocyte apoptosis
  • proportion of liver necrosis area Fig. 21C
  • liver weight / weight ratio Fig. 21D
  • FIG. 21C is a function of automatically detecting a threshold using a commercially available image analysis software ImageJ (W.S. Rasband, NIH, Bethesda, Maryland, USA) to measure the percentage of liver necrosis area in the total scanning area.
  • ImageJ W.S. Rasband, NIH, Bethesda, Maryland, USA
  • the PTX3 monoclonal antibody of Example 1 can slow the apoptosis (Figure 21B) and necrosis of acute liver fibrosis induced by tetrachloromethane (Figure 21B). 21C), and can slow the increase in liver weight in mice due to acute liver fibrosis ( Figure 21D).
  • the above differences are statistically significant.
  • This embodiment is performed according to the experimental flow diagram of FIG. 22A, which is designed in the same manner as the first point of the seventh embodiment.
  • the difference is that, in this example, C57BL / 6J male mice (8 weeks old, purchased from Lesco Biotechnology Co., Ltd.) were injected intramuscularly with 1 mL / kg of tetrachloromethane (CCl 4 and olive oil to 1: 1 volume ratio mixing), injected twice a week for a total of eight weeks.
  • mice were administered the PTX3 monoclonal antibody of Example 1 [10 mg / kg body weight of PTX3Ab, a biotechnology] or controlled by intraperitoneal (ip) injection.
  • Group antibodies IgG1k at 10 mg / kg body weight, model 10101, Wei Qiao Shengyi), once a week.
  • All mice were sacrificed, and changes in liver tissue sections, the proportion of liver necrosis areas, and liver weight / weight ratio were observed. The results are shown in Figs. 22B to 22D.
  • FIG. 22B to FIG. 22D are left liver lobe tissue sections (Picture-Sirius Red) stained with PTX3 monoclonal antibody in chronic liver fibrosis mice according to an embodiment of the present invention (FIG. 22B, magnification: 20 times, observation of liver fibrosis), liver fibrosis area ratio (Fig. 22C) and liver weight / weight ratio (Fig. 22D).
  • FIG. 22C is a function of automatically detecting a threshold using a commercially available image analysis software ImageJ (W.S. Rasband, NIH, Bethesda, Maryland, USA) to measure the percentage of the liver necrosis area in the total scanning area.
  • Figs. 22B to 22D From the results in Figs. 22B to 22D, it can be seen that compared with the control group antibody (IgG1k), the PTX3 monoclonal antibody of Example 1 can alleviate chronic liver fibrosis (Fig. 22B) and area ratio (Fig. 22C) caused by tetrachloromethane. , And can slow the increase of liver weight in mice due to chronic liver fibrosis ( Figure 22D). The above differences are statistically significant.
  • This example uses rat kidney fibroblast cell line (NRK49F, deposit number: BCRC 60084, CRL-1570 TM ), and the effect of the PTX3 monoclonal antibody of Example 1 on renal fibrosis was evaluated by the following test.
  • kidney fibroblast cell line NRK49F cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (12800-082, Gibco) containing 10% FBS and 100 ⁇ g / mL of streptomycin. And 100 U / mL penicillin tincture.
  • DMEM Dulbecco's Modified Eagle Medium
  • NRK49F cells were treated with 0.4 ⁇ g / mL IgG1k (model 10101, Wei Qiao Shengyi), 0.4 ⁇ g / mL PTX3 antibody (Zhenyi Biotechnology), and then treated with 200 ng / mL PTX3 for 6 hours.
  • modified radioimmunoprecipitation buffer [modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% deoxycholic acid Sodium (deoxycholate), 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1mg / ml), and leupeptin (1mg) / ml)]] lysed NRK49F cells.
  • modified RIPA buffer modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% deoxycholic acid Sodium (deoxycholate), 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF), apro
  • NRK49F cells were seeded into a 24-well cell culture plate, and treated with 0.4 ⁇ g / mL IgG1k (model 10101, Weiqiao Biomedical), PTX3 antibody (Zhenyi Biotechnology), or 200ng / mL PTX3 for 24 hours.
  • IgG1k model 10101, Weiqiao Biomedical
  • PTX3 antibody Zhenyi Biotechnology
  • 200ng / mL PTX3 200ng / mL PTX3 for 24 hours.
  • NRK49F cells were fixed at -20 ° C to overnight with methanol. The next day, using a Sirius Red Solution (Picro-Sirius Red Solution, model ab246832, Abcam) for 20 minutes at room temperature, rinse with acetic acid twice. The number of nodules in the cells was determined using a light microscope in a field of view with a magnification of 200 times. Then, these cells were lysed with
  • FIGS. 23A to 23C are Western blot analysis results (FIG. 23A), cell nodules showing the expression of fibrosis-related proteins of mouse renal fibroblasts by a PTX3 monoclonal antibody according to an embodiment of the present invention
  • FIGS. 23A to 23C Cell staining image ( Figure 23B) and its histogram ( Figure 23C).
  • the figure number "***" represents statistical significance compared with the control group (p ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 can reduce the expression of fibrosis-related proteins of renal fibroblasts (Figure 23A) and reduce the number of cell nodules. ( Figure 23B, Figure 23C), and this difference is statistically significant.
  • the evaluation is performed according to the experimental flow diagrams of FIG. 24A and FIG. 24C, respectively.
  • male mice of the C57BL / 6J strain purchased from Luxco Biotechnology Co., Ltd.
  • a unilateral ureteral obstruction (UUO) operation was performed from the left incision in the mouse.
  • the left kidney was taken out to expose the ureter, and the ureter was sutured with a silk thread (4-O Silk).
  • the wound was closed with a surgical stapler.
  • on the 0th and 7th days after the operation pretreatment, as shown in Figs. 24A and 24B
  • posttreatment as shown in Figs.
  • mice used 10 mg / kg IgG1k (model 10101, Wei Qiao Shengyi), 10 mg / kg PTX3 antibody (Zhenyi Biotechnology) treatment, mice were euthanized on the 14th day after surgery. Half of the kidneys were fixed in formalin and embedded in paraffin. Tissue sections were stained with Haematoxylin-Eosin (HE) or Sirius Red Solution (Picro-Sirius Red Solution, model ab246832, Abcam), covered with glass slides, and mounted on coverslips. The images were observed at 20x magnification The results are shown in Figures 24B and 24D. The scale in the figure represents 100 ⁇ m.
  • FIG. 24B and FIG. 24D respectively show the staining images of kidney tissue sections of UUO mice by the PTX3 monoclonal antibody according to an embodiment of the present invention.
  • the PTX3 monoclonal antibody of Example 1 can indeed slow down renal fibrosis in UUO animals.
  • This example uses a human lung fibroblast cell line (HFL1, deposit number: BCRC 60299, CCL-153 TM ), and the effect of the PTX3 monoclonal antibody of Example 1 on pulmonary fibrosis was evaluated by the following test.
  • HFL1 human lung fibroblast cell line
  • HFL1 cells were cultured in Han's F-12K (F12K) medium [Kaighn's improvement] (21127-022, Gibco), containing 10% FBS, 100 ⁇ g / mL streptomycin, and 100 U / mL. Penicillin tincture.
  • F12K Han's F-12K
  • Gibco Han's F-12K
  • HFL1 cells were treated with 0.4 ⁇ g / mL IgG1k (model 10101, Wei Qiao Shengyi), 0.4 ⁇ g / mL PTX3 antibody (Zhenyi Biotechnology), and then treated with 200 ng / mL PTX3 for 6 hours. Next, HFL1 cells were lysed with a modified RIPA buffer.
  • HFL1 cells were seeded into a 24-well cell culture plate [inserted culture dish with a pore size of 8- ⁇ m (353097, BD Biosciences)], and the lower culture wells were with or without 0.4 ⁇ g / mL IgG1k (model 10101, Weiqiao Biomedical), PTX3 antibody (Zhenyi Biotechnology) or 200ng / mL PTX3 treatment. After 16 hours of incubation, the cells in the insert culture dish were scraped off with a cotton swab.
  • the total number of cells attached to the lower surface of the polycarbonate film attached to the insert culture dish migrated to the cells outside the bottom of the insert culture dish, and then DAPI staining was used to determine the number of cells under a 200x magnification field of view using a fluorescence microscope. The results are shown in Fig. 25B.
  • HFL1 cells were seeded into a 24-well cell culture plate, and treated with or without 0.4 ⁇ g / mL IgG1k (model 10101, Weiqiao Biomedical), PTX3 antibody (Zhenyi Biotechnology), or 200ng / mL PTX3 for 24 hours.
  • HFL1 cells were fixed with methanol at -20 ° C to overnight.
  • the next day using a Sirius Red Solution (Picro-Sirius Red Solution, model ab246832, Abcam) for 20 minutes at room temperature, rinse with acetic acid twice.
  • the number of nodules in the cells was determined using a light microscope in a field of view with a magnification of 200 times.
  • these cells were lysed with 0.1N NaOH, and the absorbance at 490 nm was measured using a commercially available ELISA reader. The results are shown in Figs. 25C and 25D.
  • FIG. 25A to FIG. 25D are Western blot analysis results (FIG. 25A) showing the expression of fibrosis-related proteins of lung fibroblasts by a PTX3 monoclonal antibody according to an embodiment of the present invention, and a column of the number of transition cells Figure (Figure 25B), cell staining image of the cell nodule ( Figure 25C) and its bar graph ( Figure 25D).
  • the figure number "***" represents statistical significance compared with the control group (p ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 can slow down the fibrosis-related protein expression, migration, and cell nodule number of PTX3-treated lung fibroblasts compared to the control group antibody (IgG1k). .
  • mice of the C57BL / 6J strain purchased from Luxco Biotechnology Co., Ltd.
  • Mice were administered intratracheally instilled (I.T.) to PBS or 2 mg / kg bleomycin (bleomycin, BLM, model ap302, Znzo) to induce fibrosis.
  • mice were administered 10 mg / kg IgG1k (model 10101, Wei Qiao Shengyi) or 10 mg / kg PTX3 antibody (Zhenyi Biotechnology) on the 14th and 21st days by intraperitoneal injection. Euthanasia.
  • mice C57BL / 6 mice were weighed on days 7, 14, 21, or 28 after bleomycin was administered by tracheal infusion, and then 10 mg / kg IgG1k (control group) or 10 mg / kg of PTX3 antibody was administered, and the results are shown in Fig. 26B.
  • the lung tissues of the mice were visually inspected at 7, 14, 21, or 28 days after tracheal perfusion of PBS (ie, the health control group) or 2 mg / kg of bleomycin (ie, BLM). After perfusion of the left lung lobe, it was fixed with paraformaldehyde and embedded with paraffin. After the tissue sections were stained with HE, the slides were covered with coverslips, and the images were observed at a magnification of 20 times. The results are shown in Figure 26C. The scale bar in the figure represents 100 ⁇ m.
  • the lung tissues of the mice on day 28 after bleomycin-induced fibrosis were administered to the naked eye and administered with 10 mg / kg IgG1k or 10 mg / kg PTX3 antibody. After perfusion of the left lung lobe, it was fixed with paraformaldehyde and embedded with paraffin. After the tissue sections were stained with HE, the slides were covered with coverslips, and the images were observed at a magnification of 20 times. The results are shown in Figure 26D. The scale bar in the figure represents 100 ⁇ m.
  • FIG. 26B to FIG. 26D are graphs showing the weight changes of the PLM3 monoclonal antibody against BLM-induced pulmonary fibrosis mice (FIG. 26B), lung appearance, and tissue section images (FIG. 26B) according to an embodiment of the present invention. 26C and FIG. 26D).
  • the PTX3 monoclonal antibody of Example 1 can reverse the weight loss and lung fibers caused by pulmonary fibrosis in mice induced by pulmonary fibrosis. Degree of change.
  • mice of the C57BL / 6J strain purchased from Luxco Biotechnology Co., Ltd.
  • Mice were administered intratracheally instilled (I.T.) to PBS or 2 mg / kg bleomycin (bleomycin, BLM, model ap302, Znzo) to induce fibrosis.
  • mice were administered 10 mg / kg IgG1k (model 10101, Wei Qiao Shengyi) or 10 mg / kg PTX3 antibody (Zhenyi Biotechnology) by intraperitoneal injection on day 21 and euthanized on day 28.
  • mice C57BL / 6 mice were weighed on days 7, 14, 21, or 28 after bleomycin administration by tracheal perfusion, and then 10 mg / kg IgG1k (control) was administered intraperitoneally on day 21. Group) or 10 mg / kg PTX3 antibody was administered, and the results are shown in Fig. 27B.
  • the lung tissues of the mice were visually inspected at 7, 14, 21, or 28 days after tracheal perfusion of PBS (ie, the health control group) or 2 mg / kg of bleomycin (ie, BLM). After perfusion of the left lung lobe, it was fixed with paraformaldehyde and embedded with paraffin. After the tissue sections were stained with HE, the slides were covered with coverslips, and the images were observed at a magnification of 20 times. The results are shown in FIG. 27C. The scale bar in the figure represents 100 ⁇ m.
  • the lung tissues of the mice on day 28 after fibrosis induced by bleomycin and administered with 10 mg / kg IgG1k or 10 mg / kg PTX3 antibody were visually inspected. After perfusion of the left lung lobe, it was fixed with paraformaldehyde and embedded with paraffin. After the tissue sections were stained with HE, the slides were covered with coverslips, and the images were observed at a magnification of 20 times. The results are shown in Figure 27D. The scale bar in the figure represents 100 ⁇ m.
  • FIG. 27B to FIG. 27D are graphs showing the weight changes of the PLM3 monoclonal antibody against BLM-induced pulmonary fibrosis mice (FIG. 27B), lung appearance, and tissue section images (FIG. 27B) according to an embodiment of the present invention. 27C and Figure 27D).
  • the PTX3 monoclonal antibody of Example 1 can reverse the weight loss and lung fibers caused by pulmonary fibrosis in mice induced by pulmonary fibrosis. Degree of change.
  • This example uses a mouse embryo fibroblast cell line (NIH-3T3, deposit number: BCRC 60008, CCL-1658 TM ), and the effect of the PTX3 monoclonal antibody of Example 1 on fibrosis was evaluated by the following test.
  • NIH-3T3 cells were cultured in DMEM (12800-082, Gibco) ⁇ containing 10% FBS, 100 ⁇ g / mL streptomycin and 100 U / mL penicillin ⁇ .
  • NIH-3T3 cells were treated with 0.4 ⁇ g / mL PTX3 antibody (Zhenyi Biotechnology), they were treated with 200 ng / mL PTX3 for 6 hours.
  • the cells were lysed with RIPA buffer.
  • Western blotting was performed using specific antibodies to detect ⁇ -tubulin (model T6199, Sigma), collagen type 1 (Collagen I, model 14695-1-AP, ProteinTech) and ⁇ -smooth muscle sctin ( ⁇ -SMA, model GTX 100904, GeneTex), and the expression of ⁇ -tubulin as the loading control group.
  • the results are as follows: Figure 28A.
  • NIH-3T3 cells were seeded into a 24-well cell culture plate and treated with or without 0.4 ⁇ g / mL IgG1k (model 10101, Weiqiao Biomedical), PTX3 antibody (Zhenyi Biotechnology) or 200ng / mL hour. After that, the NIH-3T3 cells were fixed at -20 ° C to overnight with methanol. The next day, using a Sirius Red Solution (Picro-Sirius Red Solution, model ab246832, Abcam) for 20 minutes at room temperature, rinse with acetic acid twice. The number of nodules in the cells was determined using a light microscope in a field of view with a magnification of 200 times. Then, these cells were lysed with 0.1 N NaOH, and the absorbance at 490 nm was measured using a commercially available ELISA reader. The results are shown in Figs. 28B and 28C.
  • FIG. 28A to FIG. 28C are Western blot analysis results (FIG. 28A) showing the expression of fibrosis-related proteins of embryonic fibroblasts by a PTX3 monoclonal antibody according to an embodiment of the present invention, and cells with nodules Stained image (Figure 28B) and its histogram ( Figure 28C).
  • the figure number "***" represents statistical significance compared with the control group (p ⁇ 0.001).
  • the PTX3 monoclonal antibody of Example 1 can slow down the expression of fibrosis-related proteins and the number of nodules in NIH-3T3 cells compared to the control group antibody (IgG1k).
  • human cancer-associated fibroblasts / liver cells [cancer associated fibroblast / F28 (CAF / F28) cells] were cultured in DMEM (12800-082, Gibco) containing 10% FBS, 100 ⁇ g / mL streptomycin, and 100U / mL penicillin tincture.
  • CAF / F28 cells were exposed to a radiation dose of 8 gray (Gray, Gy), and then treated with 0.4 ⁇ g / ml PTX3 antibody (Zhenyi Biotechnology) for 6 hours.
  • a modified radioimmunoprecipitation buffer (modified RIPA) buffer [modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% deoxycholic acid Sodium (deoxycholate), 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1mg / ml), and leupeptin (1mg) / ml)]] lysed cells.
  • modified RIPA modified RIPA buffer
  • CAF / F28 cells were treated with 0.4 ⁇ g / mL PTX3 antibody (Zhenxi Biotechnology), and then treated with 200 ng / mL PTX3 for 6 hours.
  • the cells were lysed with RIPA buffer.
  • specific antibodies were used to perform western blotting to detect ⁇ -tubulin (Model T6199, Sigma), Fibronectin (Model 15613-1-AP, ProteinTech), Expression of collagen type 1 (Collagen I, model 14695-1-AP, ProteinTech) and ⁇ -smooth muscle sctin ( ⁇ -SMA, model GTX100904, GeneTex), and ⁇ -tubulin
  • the performance amount is taken as the loading control group, and the result is shown in FIG. 29B.
  • FIG. 29A to FIG. 29B are Western blot analysis results showing the expression of fibrosis-related proteins of liver fibroblasts treated with PTX3 monoclonal antibodies according to an embodiment of the present invention.
  • the PTX3 monoclonal antibody of Example 1 can slow down the expression of fibrosis-related proteins caused by radiation or PTX3 in CAF / F28 liver fibroblasts.
  • the PTX3 monoclonal antibody of Example 1 of the present invention has good affinity and sensitivity for PTX3 recombinant protein, and can be applied to the set and method for detecting PTX3 to detect the PTX3 content in biological samples in vitro.
  • applicable biological samples methods, kits, components / equipment, etc. suitable for detecting PTX3, it is as described above, without further ado.
  • the pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof and uses thereof according to the present invention utilize a PTX3 single-antibody or an antigen-binding fragment thereof with high affinity and sensitivity as an active ingredient to effectively inhibit or slow down PTX3 and
  • the binding of PTX3 receptors, thereby inhibiting or slowing down the diseases or symptoms related to the binding of PTX3 and PTX3 receptors, can be used as a cross-disease broad-spectrum drug.
  • the present invention uses a specific sequence of a PTX3 monoclonal antibody, a specific analysis mode, or a specific evaluation method as examples, the pharmaceutical composition containing the monoclonal antibody or an antigen-binding fragment thereof of the present invention and its use are described, and Any person skilled in the art to which the present invention pertains may know that the present invention is not limited thereto, and without departing from the concept and scope of the present invention, the pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof of the present invention and uses thereof, also It can be performed using other analysis modes or other evaluation methods.
  • the pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof and the use thereof according to the present invention have the advantage of using a specific PTX3 monoclonal antibody or an antigen-binding fragment thereof as an active ingredient to specifically inhibit or Slowing down the binding of PTX3 to the PTX3 receptor can be applied to kits and methods for detecting PTX3, and to inhibit or slow down diseases or symptoms related to the binding of PTX3 to the PTX3 receptor.

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Abstract

本发明提供了一种PTX3单株抗体或其抗原结合片段及其用途。前述单株抗体或其抗原结合片段专一性抑制或减缓PTX3与PTX3受体的结合，可应用于检测PTX3的套组及其检测方法，以及抑制或减缓PTX3与PTX3受体结合相关的疾病或症状的医药组合物及其用途。

Description

含单株抗体或其抗原结合片段的医药组合物及其用途 技术领域
本发明涉及一种抗体及其用途，特别涉及一种专一性抑制或减缓PTX3的C端特定序列与PTX3受体结合的单株抗体或其抗原结合片段及其应用于检测试剂、抑制或减缓PTX3与PTX3受体结合相关的疾病或症状的医药组合物及其用途。
背景技术
目前已知癌细胞会刺激肿瘤周边微环境产生各种发炎因子、白血球、血管过度增生及蛋白酶等，而癌症的慢性发炎反应也与癌细胞的生长、转移与侵袭有关，然而其形成原因及详细机制，仍有诸多未明之处。
肿瘤微环境除了与发炎反应有关之外，其它研究也指出，肿瘤微环境与肿瘤转移(metastasis)及化疗抗药性(chemoresistance)也息息相关。肿瘤微环境是由多种的基质细胞(stromal cells)及其它不同形态的细胞所构成，不仅可保护肿瘤，使肿瘤细胞得以逃脱和抵抗免疫细胞，而造成肿瘤细胞的抗药性。
在肿瘤周边基质组织中的纤维母细胞及巨噬细胞受到CEBPD活化后，会诱导产生分泌型因子-正五聚蛋白相关蛋白3(pentraxin-related protein 3；PTX3)，其具有促进血管新生的活性，且可增加鼻咽癌细胞的移行及侵入组织(或称侵袭)的能力。另外，过去研究也证实癌周边组织细胞中CEBPD受到活化，也可能促使癌转移，甚至促使在化疗过程中产生抗药性癌细胞，这些抗药性癌细胞会生长的更快并且更容易转移。
目前市面上虽有一些小分子抗癌药物，例如顺-双氨双氯铂(cis-diammine dichloroplatinum(II)；CDDP；商品名顺铂(Cisplatin))、太平洋紫杉醇(paclitaxel；商品名Taxol)以及5-氟尿嘧啶(5-Fluorouracil；5-FU)等，然而最近的研究发现，上述小分子抗癌药物不仅活化癌细胞中的CEBPD表现，也可活化巨噬细胞及纤维母细胞内CEBPD的表现，反而促使癌细胞产生抗药性并快速转移，导致癌症治疗效果不佳。
PTX3与PTX3受体结合相关的疾病或症状除了上述的癌症外，也涉及纤维化疾病和/或纤维化症状。
有鉴于此，亟需发展一种专一性结合PTX3的抗体，以检测生物样本中的PTX3含量、 克服现有药物对于癌症及纤维化的治疗效果不佳等问题。
发明内容
因此，本发明的一实施方式是提供一种单株抗体或其抗原结合片段，其是专一性结合一或多种正五聚蛋白相关蛋白(pentraxin-related protein；PTX3)的C端特定序列。
本发明的另一实施方式是提供一种单株抗体或其抗原结合片段，其包含特定序列的重链可变区序列以及轻链可变区序列。
本发明的又一实施方式是提供一种检测PTX3的套组，包含上述的单株抗体或其抗原结合片段。
本发明的再一实施方式是在提供一种体外检测PTX3的方法，其利用上述套组检测PTX3。
本发明的又一实施方式是提供一种医药组合物，其包含具有有效剂量的单株抗体或其抗原结合片段及医药学上可接受的载剂，且上述的单株抗体或其抗原结合片段作为有效成分。
本发明的又另一实施方式是提供一种单株抗体或其抗原结合片段用于制备专一性抑制或减缓PTX3与PTX3受体结合的医药组合物的用途，其中单株抗体或其抗原结合片段为活性成分，且单株抗体或其抗原结合片段具有有效剂量，以抑制或减缓与PTX3及PTX3受体结合相关的疾病或症状。
本发明的再一实施方式是提供一种用于体外抑制或减缓肿瘤细胞的活性的方法，包含对肿瘤细胞投予具有有效剂量的上述医药组合物，以抑制或减缓肿瘤细胞的活性。
本发明的又一实施方式是提供一种用于体外抑制或减缓纤维化疾病和/或纤维化症状的方法，包含对受纤维化疾病和/或纤维化症状影响的器官投予有效剂量的上述医药组合物，以抑制或减缓器官的纤维化疾病和/或纤维化症状。
根据本发明的上述实施方式，提出一种单株抗体或其抗原结合片段。在一实施例中，上述单株抗体或其抗原结合片段可专一性结合如SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列。
在一实施例中，上述非变性氨基酸序列可包含但不限于由SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11任一者所列的氨基酸序列或上述任意组合。在其它实施例中，上述非变性氨基酸序列可包含但不限于由SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11任一者所列的氨基酸序列或上述任意组合。
根据本发明的另一实施方式，提供一种单株抗体或其抗原结合片段，其包含重链可变区序列以及轻链可变区序列，其中重链可变区序列可例如SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20和/或SEQ ID NO：21所列的氨基酸序列，轻链可变区序列可例如SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24和/或SEQ ID NO：25所列的氨基酸序列。
在一些实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和/或SEQ ID NO：29所列的核酸序列编码的氨基酸序列，轻链可变区序列可例如SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32和/或SEQ ID NO：33所列的核酸序列编码的氨基酸序列。
在其它实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NOs：34或35所列的氨基酸序列，轻链可变区序列可例如SEQ ID NOs：36或37所列的氨基酸序列。
在另一些实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NOs：38或39所列的核酸序列编码的氨基酸序列，轻链可变区序列可例如SEQ ID NOs：40或41所列的核酸序列编码的氨基酸序列。
在一实施例中，上述单株抗体或其抗原结合片段为嵌合抗体或其抗原结合片段。在一例示中，上述单株抗体或其抗原结合片段为鼠源抗体、人鼠嵌合抗体、人源化抗体或其抗原结合片段。
在一实施例中，上述单株抗体或其抗原结合片段可例如为单链可变区片段(single-chain variable fragment；scFv)、单链可变区片段二聚体﹝(scFv) 2﹞、单链可变区片段三聚体﹝(scFv) 3﹞、可变区片段(variable fragment；Fv)、Fab片段、Fab'片段、F(ab') 2片段、纳米抗体(nanobody)或上述的任意组合。
在一实施例中，上述单株抗体或其抗原结合片段是经由复合(conjugation)或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。
在一实施例中，上述单株抗体或其抗原结合片段为抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。
在一实施例中，上述单株抗体或其抗原结合片段为双功能单株抗体(bispecific monoclonal antibody；BsAb)或其抗原结合片段。
在一实施例中，上述单株抗体或其抗原结合片段为三功能单株抗体(trifunctional monoclonal antibody)或其抗原结合片段。
在一实施例中，上述单株抗体或其抗原结合片段属于IgG类、IgM类别、IgA类别、 IgD类别或IgE类别。在另一实施例中，上述单株抗体或其抗原结合片段属于IgG类且具有IgG1、IgG2、IgG3或IgG4同型。
在一实施例中，上述单株抗体或其抗原结合片段属于惰性抗体或拮抗剂抗体。
在一实施例中，上述单株抗体或其抗原结合片段是专一性抑制或减缓PTX3受体识别与一或多种PTX3的C端特定序列的结合。在一些例示中，上述单株抗体或其抗原结合片段是抑制或减缓一或多种PTX3的活性。在另一些例示中，上述单株抗体或其抗原结合片段是专一性抑制或减缓PTX3受体与一或多种PTX3的交互作用、抑制或减缓PTX3信息传递或上述的任意组合。
根据本发明的又一实施方式，提出一种检测PTX3的套组，包含如上述任一者的单株抗体或其抗原结合片段，其中单株抗体或其抗原结合片段可专一性结合非变性氨基酸序列，且此非变性氨基酸序列可包括但不限于如SEQ ID NO：1至SEQ ID NO：11任一者所列的氨基酸序列。
根据本发明的更另一实施方式，提出一种体外检测PTX3的方法，其是利用上述检测PTX3的套组检测PTX3，其中检测PTX3的套组所含的单株抗体或其抗原结合片段的分析灵敏度可例如不低于0.0016pM。
根据本发明的其它实施方式，提出一种医药组合物，其包含具有有效剂量的单株抗体或其抗原结合片段及医药学上可接受的载剂，且上述的单株抗体或其抗原结合片段为有效成分。
依据本发明一实施例，上述医药组合物还可包含活性药物成分。
根据本发明的其它实施方式，提出一种单株抗体或其抗原结合片段用于制备专一性抑制或减缓正五聚蛋白相关蛋白(pentraxin-related protein；PTX3)与PTX3受体结合的医药组合物的用途。在一实施例中，前述单株抗体或其抗原结合片段为活性成分，且单株抗体或其抗原结合片段具有有效剂量，以抑制或减缓与PTX3及PTX3受体结合相关的疾病或症状。
依据本发明一实施例，上述医药组合物用于抑制或减缓与PTX3与PTX3受体结合相关的疾病或症状，其中此疾病或症状可包括上皮细胞癌、腺癌、神经胶母细胞瘤(glioblastoma multiforme；GBM)及纤维化。在一些例示中，上皮细胞癌可包括肺癌(lung cancer)、乳癌(breast cancer)及鼻咽癌(nasopharyngeal carcinoma；NPC)。前述腺癌可例如大肠癌。
依据本发明一实施例，上述受纤维化的疾病或症状影响的器官可包括但不限于肺、肝、 肾及皮肤。
依据本发明一实施例，上述医药组合物可经由皮下(subcutaneous，s.c.)注射、肌肉注射、静脉注射、腹腔(intraperitoneal，i.p.)注射、原位(orthotopic)注射、经口投予或口鼻吸入的方式投予。
根据本发明的其它实施方式，提出一种用于体外抑制或减缓肿瘤细胞的活性的方法，包括对肿瘤细胞投予有效剂量的上述医药组合物，借此抑制或减缓肿瘤细胞的活性。
根据本发明的其它实施方式，提出一种用于体外抑制或减缓肿瘤细胞的活性的方法，包括对肿瘤细胞投予有效剂量的上述医药组合物，借此抑制或减缓肿瘤细胞的活性。
根据本发明的其它实施方式，提出一种用于体外抑制或减缓纤维化疾病和/或纤维化症状的方法，包括对受纤维化疾病和/或纤维化症状影响的器官投予有效剂量的上述医药组合物，借此抑制或减缓前述器官的纤维化疾病和/或纤维化症状。
应用本发明的单株抗体或其抗原结合片段，其是利用特定的PTX3单株抗体或其抗原结合片段专一性抑制或减缓PTX3与PTX3受体的结合，不仅可应用于体外检测PTX3的套组及体外检测PTX3含量的方法，还可应用于抑制或减缓PTX3受体识别PTX3相关的疾病或症状的医药组合物及其用途。
附图说明
〔图1〕是示出根据本发明一实施例的PTX3单株抗体对PTX3重组蛋白的亲和力曲线图。
〔图2〕是示出根据本发明另一实施例的PTX3单株抗体对PTX3重组蛋白的亲和力曲线图。
〔图3〕与〔图4〕是示出根据本发明一实施例的PTX3单株抗体与不同片段的PTX3重组蛋白结合的表位定位图谱。
〔图5〕是示出根据本发明一实施例的PTX3单株抗体或市售抗体对于不同种PTX3重组蛋白的结合的结果。
〔图6〕是示出根据本发明一实施例的PTX3单株抗体阻碍PTX3重组蛋白与PTX3受体的结合的竞争性抑制图。
〔图7A〕至〔图7C〕是示出根据本发明一实施例的PTX3单株抗体抑制乳癌细胞株MDA-MB231的移行细胞数(图7A)、侵袭细胞数(图7B)、细胞球团数(图7C)的结果。
〔图8A〕至〔图8C〕是示出根据本发明一实施例的PTX3单株抗体抑制肺癌细胞株 A549的移行细胞数(图8A)、侵袭细胞数(图8B)、细胞球团数(图8C)的结果。
〔图9A〕至〔图9C〕是示出根据本发明一实施例的PTX3单株抗体抑制鼻咽癌细胞株HONE1的移行细胞数(图9A)、侵袭细胞数(图9B)、细胞球团数(图9C)的结果。
〔图10A〕至〔图10C〕是示出根据本发明一实施例的PTX3单株抗体抑制神经胶母细胞瘤细胞株U87MG的移行细胞数(图10A)、侵袭细胞数(图10B)、细胞球团数(图10C)的结果。
〔图11A〕至〔图11B〕是示出根据本发明一实施例的PTX3单株抗体或市售抗体抑制乳癌细胞株MDA-MB231的移行细胞数(图11A)及侵袭细胞数(图11B)的结果。
〔图12A〕至〔图12B〕是示出根据本发明一实施例的PTX3单株抗体或市售抗体抑制肺癌细胞株A549的移行细胞数(图12A)及侵袭细胞数(图12B)的结果。
〔图13A〕至〔图13B〕是示出根据本发明一实施例的PTX3单株抗体或市售抗体抑制鼻咽癌细胞株HONE1的移行细胞数(图13A)及侵袭细胞数(图13B)的结果。
〔图14A〕至〔图14C〕是示出根据本发明一实施例的PTX3单株抗体或市售抗体抑制乳癌细胞株MDA-MB231(图14A)、肺癌细胞株A549(图14B)以及鼻咽癌细胞株HONE1(图14C)的细胞球团数的结果。
〔图15A〕至〔图15B〕是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠原位异种移植的乳癌细胞MDA-MB231的肿瘤体积(图15A)及肿瘤转移(图15B)的结果。
〔图16A〕至〔图16B〕是示出根据本发明一实施例的PTX3单株抗体或同型对照抗体抑制小鼠原位同种移植乳癌细胞4T1的肿瘤体积(图16A)及肿瘤转移(图16B)的结果。
〔图17A〕至〔图17C〕是分别示出根据本发明一实施例的PTX3单株抗体在并用或未并用紫杉醇的情形下，抑制小鼠原位异种移植的乳癌细胞4T1的肿瘤体积的影像图(图17A)及肿瘤体积变化折线图(图17B至图17C)。
〔图18A〕是示出根据本发明一实施例的PTX3单株抗体在并用或未并用紫杉醇的情形下，评估小鼠原位同种移植的乳癌细胞4T1的实验流程示意图。
〔图18B〕至〔图18D〕是分别示出根据本发明另一实施例的PTX3单株抗体在并用或未并用紫杉醇的情形下，小鼠原位同种移植的乳癌细胞4T1的肿瘤体积与转移的影像图(图18B)、肿瘤体积折线图(18C)及小鼠存活率(图18D)。
〔图19A〕是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠植入大肠癌细胞株MC38的实验流程示意图。
〔图19B〕是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠植入大肠癌细胞株MC38的肿瘤体积折线图。
〔图20A〕及〔图20B〕是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠植入人类神经胶母细胞瘤细胞株U87MG的肿瘤体积折线图(图20A)及小鼠存活率(图20B)。
〔图21A〕是示出根据本发明一实施例的是示出利用本发明一实施例的PTX3单株抗体评估对急性肝纤维化小鼠改善效果的实验流程示意图。
〔图21B〕至〔图21D〕是分别示出根据本发明一实施例的PTX3单株抗体在急性肝纤维化小鼠体内以苏木精-伊红(hematoxylin and eosin，H&E)染色的左肝叶组织切片(图21B)、肝坏死区域比例(图21C)及肝脏重/体重比(图21D)。
〔图22A〕是示出利用本发明一实施例的PTX3单株抗体评估对慢性肝纤维化小鼠改善效果的实验流程示意图。
〔图22B〕至〔图22D〕是分别示出根据本发明一实施例的PTX3单株抗体在慢性肝纤维化小鼠体内以天狼星红(Picro-Sirius Red)染色的左肝叶组织切片(图22B)、肝纤维化区域比例(图22C)及肝脏重/体重比(图22D)。
〔图23A〕至〔图23C〕是分别示出根据本发明一实施例的PTX3单株抗体对肾纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图23A)、细胞结节的细胞染色影像(图23B)及其柱形图(图23C)。
〔图24A〕至〔图24D〕是分别示出根据本发明一实施例的PTX3单株抗体对UUO小鼠的肾纤维化实验流程示意图(图24A及图24C)及肾脏组织切片染色影像(图24B及图24D)。
〔图25A〕至〔图25D〕是分别示出根据本发明一实施例的PTX3单株抗体对肺纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图25A)、移行细胞数的柱形图(图25B)、细胞结节的细胞染色影像(图25C)及其柱形图(图25D)。
〔图26A〕至〔图26D〕是分别示出根据本发明一实施例的PTX3单株抗体对BLM诱发肺纤维化小鼠的实验流程示意图(图26A)、体重变化曲线图(图26B)、肺部外观及组织切片影像(图26C及图26D)。
〔图27A〕至〔图27D〕是分别示出根据本发明一实施例的PTX3单株抗体对BLM诱发肺纤维化小鼠的实验流程示意图(图27A)、体重变化曲线图(图27B)、肺部外观及组织切片影像(图27C及图27D)。
〔图28A〕至〔图28C〕是分别示出根据本发明一实施例的PTX3单株抗体对胚胎纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图28A)、细胞结节的细胞染色影像(图28B)及其柱形图(图28C)。
〔图29A〕至〔图29B〕是分别示出根据本发明一实施例的PTX3单株抗体对不同处理的肝纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果。
具体实施方式
为让本发明的上述和其它目的、特征、优点与实施例能更明显易懂，说明书附图的详细说明如下。
承前所述，本发明提供一种含单株抗体或其抗原结合片段的医药组合物及其用途，其是利用单株抗体或其抗原结合片段专一性抑制正五聚蛋白相关蛋白(pentraxin-related protein；PTX3)与PTX3受体的结合，可应用于检测PTX3的套组及其检测方法，以及抑制或减缓PTX3与PTX3受体结合相关的疾病或症状的医药组合物及其用途。
本发明此处所称的单株抗体或其抗原结合片段可包含特定序列的重链可变区序列以及轻链可变区序列，以专一性抑制或减缓PTX3与PTX3受体的C端特定序列的结合。
申言之，在一实施例中，前述单株抗体或其抗原结合片段可专一性结合人类PTX3的C端氨基酸序列，其序列范围不限，可例如NO：1至SEQ ID NO：17所列的非变性氨基酸序列，然以专一性结合如SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列为佳，又以专一性结合如SEQ ID NO：1至SEQ ID NO：5以及11所列的非变性氨基酸序列为优选，又以专一性结合如SEQ ID NO：2至SEQ ID NO：4所列的非变性氨基酸序列为优选。在上述实施例中，SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列的对应于人类PTX3重组蛋白的非变性氨基酸序列的第200个氨基酸至第236个氨基酸。在另一个例示中，前述SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的非变性氨基酸序列的对应于人类PTX3重组蛋白的非变性氨基酸序列的第200个氨基酸至第220个氨基酸。在又一个例示中，前述SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的非变性氨基酸序列的对应于人类PTX3重组蛋白的非变性氨基酸序列的第203个氨基酸至第217个氨基酸。
在一实施例中，上述单株抗体或其抗原结合片段包含重链可变区序列以及轻链可变区序列，其中重链可变区的CDR1的序列具有如SEQ ID NO：18所列的氨基酸序列。重链可变区的CDR2的氨基酸序列可为RIDPANX 1X 2TKYDPX 3FQG，其中X 1代表G或D， X 2代表D或N，X 3代表K或M，其具体的氨基酸序列如SEQ ID NOs：19或20所列。重链可变区的CDR3的序列具有如SEQ ID NO：21所列的氨基酸序列。轻链可变区的CDR1的序列具有如SEQ ID NO：22所列的氨基酸序列。轻链可变区的CDR2的序列具有如SEQ ID NO：23所列的氨基酸序列。轻链可变区的CDR3的氨基酸序列可为HQX 4QRSPLT，其中X 4代表F或Y，其具体的氨基酸序列如SEQ ID NOs：24或25所列。
在其它实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可具有如SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和/或SEQ ID NO：29所列的核酸序列编码的氨基酸序列，而轻链可变区序列可具有如SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32和/或SEQ ID NO：33所列的核酸序列编码的氨基酸序列。
在其它实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可具有具有如SEQ ID NOs：34或35所列的氨基酸序列，而轻链可变区序列可具有如SEQ ID NOs：36或37所列的氨基酸序列。
在其它实施例中，上述单株抗体或其抗原结合片段的重链可变区序列可具有如SEQ ID NOs：38或39所列的核酸序列编码的氨基酸序列，而轻链可变区序列可具有如SEQ ID NOs：40或41所列的核酸序列编码的氨基酸序列。
在一些实施例中，单株抗体或其抗原结合片段的种类不限，可例如嵌合抗体或其抗原结合片段。在其它例示中，上述单株抗体或其抗原结合片段可例如为鼠源抗体、人鼠嵌合抗体、人源化抗体或其抗原结合片段。
在一些实施例中，上述单株抗体或其抗原结合片段的结构不限，在兼顾互补决定区(complementarity-determining region；CDR)结构稳定性的前提下，可为完整的抗体结构，或是简化的抗体结构，例如单链可变区片段(single-chain variable fragment；scFv)、单链可变区片段二聚体﹝(scFv) 2﹞、单链可变区片段三聚体﹝(scFv) 3﹞、可变区片段(variable fragment；Fv)、Fab片段、Fab'片段、F(ab') 2片段、纳米抗体〔nanobody，又称单域抗体(single domain antibody，sdAb)或重链抗体(heavy-chain antibody)〕或上述的任意组合，借此简化重组抗体的制程。上述单株抗体或其抗原结合片段可利用融合瘤细胞或重组基因表现等方式进行，此为本发明所属技术领域中技术人员所熟知，此处不再赘述。
在一些实施例中，上述单株抗体或其抗原结合片段可视实际需求，进一步经由复合(conjugation)或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。举例而言，上述单株抗体或其抗原结合片段可与药物进一步形成抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。在其它例子中，上述单株抗体或其抗原结合片段也 可与特定的信息胜肽结合，以进入特定部位，例如通过血脑屏障(blood-brain barrier，BBB)。
在一些实施例中，上述单株抗体或其抗原结合片段可例如为双功能单株抗体(bispecific monoclonal antibody；BsAb)、三功能单株抗体(trifunctional monoclonal antibody)或其抗原结合片段。
依据本发明一实施例，上述单株抗体或其抗原结合片段是经由复合(conjugation)或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。
依据本发明一实施例，上述单株抗体或其抗原结合片段为抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。
依据本发明一实施例，上述单株抗体或其抗原结合片段为双功能单株抗体(bispecific monoclonal antibody；BsAb)或其抗原结合片段。
依据本发明一实施例，上述单株抗体或其抗原结合片段为三功能单株抗体(trifunctional monoclonal antibody)或其抗原结合片段。
在一实施例中，上述单株抗体或其抗原结合片段可属于IgG类、IgM类别、IgA类别、IgD类别或IgE类别。在一具体实施例中，上述单株抗体或其抗原结合片段属于IgG类且具有IgG1、IgG2、IgG3或IgG4同型。在一例示中，上述单株抗体或其抗原结合片段具有IgG1同型，例如具有IgG1k同型。在一些具体例示中，上述单株抗体或其抗原结合片段属于惰性抗体或拮抗剂抗体。在一些具体例示中，上述单株抗体或其抗原结合片段可专一性抑制或减缓一或多种PTX3的活性。在另一些具体例示中，上述单株抗体或其抗原结合片段可专一性抑制或减缓PTX3受体与一或多种PTX3的交互作用、抑制或减缓PTX3信息传递或上述的任意组合。
在应用时，上述单株抗体或其抗原结合片段可用于检测PTX3的套组及方法，通过与SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列专一性结合，提高检测生物样本中PTX3的分析灵敏度。本发明此处所称的生物样本之形式不限，可包括但不限于例如细胞、组织、血液、尿液、淋巴液、组织液、体液等。上述检测PTX3的套组可应用于各种现有检测用的组件/设备，例如流式细胞仪、酵素连结免疫吸附分析(enzymelinked immunosorbent assay；ELISA)检测试剂套组、生物芯片等；或应用于现有检测用的方法例如直接ELISA(direct ELISA)、间接ELISA(indirect ELISA)、三明治ELISA(sandwich ELISA)、竞争性ELISA(competitive ELISA)、免疫组织化学染色及西方墨点分析法(Western bloting analysis)等。在一具体例中，上述单株抗体或其抗原结合片段的分析灵敏度(analytical sensitivity)，也可称为检测下限值(lower limit of detection；LLOD)，一般而言不 低于0.0016pM。
上述的单株抗体或其抗原结合片段可作为有效成分，添加于医药组合物中。在一实施例中，前述医药组合物可选择性包含医药学上可接受的载剂。本发明此处所称的「医药学上可接受的载剂」是指本身非属活性成分，而是用以将活性成分传递至个体的载剂、稀释剂、佐剂和/或媒剂，或添加至上述组合物中以改善组合物的处理或存储性质，或允许或有助于组合物的剂量单位形成适于医药组合物并方便投予的赋形剂或任何物质。前述医药学上可接受的载剂不应破坏活性成分的药理学活性，且在传递足够治疗剂量的活性成分时应无毒性。
前述适用的医药学上可接受的载剂可为一般熟悉制造医药组合物之通常知识者所熟知，且包括但不限于缓冲剂、稀释剂、崩解剂、黏合剂、黏着剂、湿润剂、聚合物、润滑剂、滑动剂、为遮蔽或抵消不良味道或气味而添加的物质、染料、芳香剂及为改善组合物之外观而添加的物质。前述医药学上可接受的载剂之具体例可包括但不限于柠檬酸盐缓冲剂、磷酸盐缓冲剂、乙酸盐缓冲剂、碳酸氢盐缓冲剂、硬脂酸、硬脂酸镁、氧化镁、磷酸及硫酸之钠盐及钙盐、碳酸镁、滑石、明胶、阿拉伯胶、海藻酸钠、果胶、糊精、甘露糖醇、山梨糖醇、乳糖、蔗糖、淀粉、明胶、纤维素物质(诸如烷酸之纤维素酯及纤维素烷基酯)、低熔点蜡、可可脂、氨基酸、尿素、醇类、抗坏血酸、磷脂、蛋白质(例如血清白蛋白)、乙二胺四乙酸(EDTA)、二甲亚砜(DMSO)、氯化钠或其它盐、脂质体、甘油或粉末、聚合物(诸如聚乙烯吡咯啶酮、聚乙烯醇及聚乙二醇)及其它医药学上可接受的物质。
本发明此处所称的抑制或减缓与PTX3与PTX3受体结合相关的疾病或症状可包括上皮细胞癌、神经胶母细胞瘤(glioblastoma multiforme；GBM)、腺癌及纤维化。前述上皮细胞癌可例如肺癌、乳癌及鼻咽癌。前述腺癌可例如大肠癌。前述受纤维化的疾病或症状影响的器官可包括但不限于肺、肝(例如急性肝纤维化、慢性肝纤维化)、肾及皮肤等。
在应用时，上述单株抗体或其抗原结合片段可用于对目标细胞或受试对象投予有效剂量的上述单株抗体或其抗原结合片段或上述的医药组合物，借此抑制或减缓与PTX3与PTX3受体结合相关的疾病或症状。以小鼠为例，本发明前述所称的「有效剂量」是指每公斤体重投以2mg至10mg的PTX3单株抗体或其抗原结合片段，以此剂量一周一次投予。在另一例示中，前述的PTX3单株抗体或其抗原结合片段的有效剂量可例如5mg/kg体重至10mg/kg体重为优选，然以6mg/kg体重至9mg/kg体重为优选。至于应用到其它对象时，则可根据生体相等性换算成适合的有效剂量。在此说明的是，倘若PTX3单株抗体的有效剂量低于2mg/kg体重，则无法于预设时间内有效减少或抑制或减缓PTX3与 PTX3受体结合。
对于肿瘤而言，上述单株抗体或其抗原结合片段可抑制或减缓肿瘤细胞的活性，例如增生(proliferation)、癌干原细胞性(cancer stemness)、移行(migration)、侵袭(invasion)、转移(metastasis)、肿瘤体积或抗药性(drug resistance)。
本发明所述的纤维化定义为在器官或组织中形成过量的纤维组织，例如纤维结缔组织，或在发炎、修复或反应过程中形成的纤维组织。上述纤维组织可为反应性、良性或病理状态。在生理学上，纤维化可用于描述纤维组织的过度沉积的病理状态，以及伤口愈合中结缔组织沉积的过程。例如由伤口形成的瘢痕，或由单一种类细胞形成之纤维瘤，或由纤维组织过度沉积的病理状态。对于受纤维化的疾病或症状影响的器官而言，上述单株抗体或其抗原结合片段可抑制或减缓纤维化相关的疾病或症状，例如急性肝纤维化、慢性肝纤维化等。
上述的医药组合物可经由皮下注射(subcutaneous injection)、肌肉注射、静脉注射、腹腔注射、原位注射(orthotopic injection)、经口投予、口鼻吸入等方式投予，借此专一性抑制内生性PTX3活性，进而抑制或减缓癌细胞的活性。具体而言，经体外细胞实验证实，本发明的单株抗体或其抗原结合片段及含此的医药组合物经使用达预设时间，例如4周至11周后，即可抑制或减缓癌细胞的活性。
以下利用数个实施例以说明本发明的应用，然其并非用以限定本发明，本发明技术领域中技术人员，在不脱离本发明的构思和范围内，当可作各种的变动与修饰。
实施例1、PTX3单株抗体的制备
此实施例是利用现有融合瘤法或重组蛋白表现法，制备专一性识别PTX3重组蛋白的C端氨基酸序列的PTX3单株抗体。
简言之，将如SEQ ID NO：13所列的非变性氨基酸序列的PTX3重组蛋白作为免疫原，以每头小鼠50μg之剂量以腹腔注射(i.p.)的方式注入Balb/C小鼠腹腔内，2周后再以每头小鼠50μg之剂量补强免疫，二周一次，共四次。接着，将活化的脾细胞与骨髓癌细胞融合后，产生融合瘤细胞株。
从上述融合瘤细胞株中，筛选出对SEQ ID NO：11所列的非变性氨基酸序列的重组蛋白的结合亲和力较高的融合瘤细胞株，其中所得的融合瘤细胞株产生的PTX3单株抗体可专一性结合如序列识别编号(SEQ ID NO：)1至SEQ ID NO：11所列的非变性氨基酸序列。
收集上述所得的融合瘤细胞株的培养上清液经由市售管柱纯化出PTX3单株抗体(臻崴生技)后，委由伟乔生医股份公司分析重链可变区及轻链可变区的互补决定区(complementarity-determining region；CDR)的氨基酸序列及对应的核酸序列。重链可变区序列可例如具有SEQ ID NO：18、SEQ ID NO：19和/或SEQ ID NO：21所列的氨基酸序列，轻链可变区序列可例如SEQ ID NO：22、SEQ ID NO：23和/或SEQ ID NO：24所列的氨基酸序列。重链可变区的氨基酸序列具有如SEQ ID NO：34所列的氨基酸序列，或如SEQ ID NO：38所列的核酸序列编码的氨基酸序列。轻链可变区的氨基酸序列具有如SEQ ID Ns：36所列的氨基酸序列，或如SEQ ID NO：40所列的核酸序列编码的氨基酸序列。
另外，上述PTX3单株抗体经市售单株抗体分型套组分析后，确认其抗体分型为IgG1k。
实施例二、评估PTX3单株抗体的亲和力
此实施例利用现有ELISA套组评估PTX3重组蛋白与实施例一的PTX3单株抗体的亲和力。
首先，将5μg/mL的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列)或牛血清白蛋白(bovine serum albumin，BSA；作为控制组)涂布在96孔细胞培养盘(型号：9018，Corning Costar)之各孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含3％脱脂奶粉之磷酸盐缓冲溶液(PBS)﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。在去除阻隔溶液后，利用PBS润洗各孔，再加入初级抗体于室温(4℃至40℃)进行反应1小时，其中初级抗体为经序列稀释的实施例一的PTX3单株抗体(浓度为2.44×10 -4μg/mL至1.00μg/mL)。然后，利用PBS洗去各孔未结合的PTX3单株抗体，并加入二级抗体于室温(4℃至40℃)反应。之后，各孔加入四甲基联苯胺(tetramethyl benzidine；TMB)反应一段时间后，加入0.1M硫酸(H 2SO 4)反应10分钟，以终止反应，其中二级抗体为结合辣根过氧化氢酶(anti-mouse horse peroxidase；HRP)的抗小鼠IgG(IgG-HRP)。接下来，利用市售酵素免疫分析仪(ELISA reader)读取450nm的吸光值，其结果如图1所示。每个数值为三重复。上述二级抗体的反应时间系参照制造商的操作手册进行，此应为本发明所属技术领域中任何技术人员所熟知，故不另赘述。
请参阅图1，其是示出根据本发明一实施例的PTX3单株抗体对PTX3重组蛋白的亲和力曲线图，其中图号●标示的曲线代表PTX3单株抗体对PTX3重组蛋白的亲和力曲线， 图号■标示的曲线代表BSA对PTX3重组蛋白的亲和力曲线。
由图1结果可知，在高达4 6倍的稀释(抗体浓度相当于0.244ng/mL或0.0016pM)后，PTX3单株抗体仍维持对PTX3重组蛋白良好的亲和力。
另外，利用上述ELISA套组也证明，本发明一实施例的PTX3单株抗体对PTX3重组蛋白具有高亲和力。首先，将10μg/mL的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列，溶于pH7.2的PBS中)涂布在96孔细胞培养盘(型号同上)之各孔内，于4℃反应至隔夜。接着，将阻隔溶液﹝含3％牛血清白蛋白(BSA)的PBS﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。在去除阻隔溶液后，利用PBS润洗各孔，再加入初级抗体于室温(4℃至40℃)进行反应1小时，其中初级抗体为经序列稀释的实施例一的PTX3单株抗体(浓度为0.01ng/mL至1000ng/mL)。然后，利用PBST润洗各孔数次，以去除各孔未结合的PTX3单株抗体，并加入二级抗体于室温(4℃至40℃)反应，其中二级抗体为结合HRP的抗小鼠IgG(IgG-HRP)。之后，各孔加入TMB反应反应一段时间后，加入0.1M硫酸(H 2SO 4)反应10分钟，以终止反应。接下来，利用市售酵素免疫分析仪读取450nm的吸光值，其结果如图2所示。每个数值为四重复。上述二级抗体的反应时间应为本发明所属技术领域中任何技术人员所熟知，故不另赘述。
请参阅图2，其是示出根据本发明另一实施例的PTX3单株抗体对PTX3重组蛋白的亲和力曲线图。
由图2结果可知，PTX3单株抗体对于PTX3重组蛋白(即抗原)的解离常数(KD)为85pM，代表PTX3单株抗体对PTX3重组蛋白具有较高的亲和力，故可应用于PTX3检测套组。
另外，在其它实验中，PTX3单株抗体可专一性结合PTX3重组蛋白之第200个氨基酸至第359个氨基酸(如SEQ ID NO：13所列的非变性氨基酸序列；图未示出)或第200个氨基酸至第236个氨基酸(如SEQ ID NO：12所列的非变性氨基酸序列；如图3所示)。
实施例三、评估PTX3单株抗体与PTX3结合的区域
1.评估实施例一的PTX3单株抗体与PTX3结合的表位定位区域
此实施例利用现有ELISA套组评估PTX3单株抗体与PTX3结合的表位定位(epitope mapping)区域。
此实施例是利用与实施例一相同的方式，评估PTX3单株抗体对PTX3的较小结合区域，而其不同的处在于，本实施例是将200μg/mL的PTX3重组蛋白(如SEQ ID NOs：12、 16、17所列的非变性氨基酸序列，溶于0.1M的碳酸氢钠水溶液，pH 8.3)或BSA(作为控制组)涂布在96孔细胞培养盘的孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含1％BSA的PBS﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。在去除阻隔溶液后，利用PBS润洗各孔，再加入实施例一的PTX3单株抗体(浓度为125ng/mL)于室温(4℃至40℃)进行反应2小时。然后，利用PBS洗去各孔未结合的PTX3单株抗体，并加入二级抗体(抗小鼠IgG-HRP，以1：5000稀释)于室温(4℃至40℃)反应1小时。之后，各孔加入TMB反应反应一段时间后，加入0.1M硫酸反应10分钟以终止反应，并利用市售酵素免疫分析仪读取450nm的吸光值，其结果如图3所示。每个数值为三重复。
请参阅图3，其是示出根据本发明一实施例的PTX3单株抗体与不同片段的PTX3重组蛋白结合的表位定位图谱，其中RI37代表如SEQ ID NO：12所列的PTX3重组蛋白片段，KT44代表如SEQ ID NO：16所列的PTX3重组蛋白片段，GI40代表如SEQ ID NO：17所列的PTX3重组蛋白片段，而图号「＊＊＊」则代表相较于控制组(即BSA组)具有统计显著性(p＜0.001)。
由图3的结果可知，PTX3单株抗体对于如SEQ ID NO：12所列的PTX3重组蛋白片段的亲和力，高于其它片段，且具有统计显著性。
请参阅图4，其是示出根据本发明一实施例的PTX3单株抗体与不同片段的PTX3重组蛋白结合的表位定位图谱，其中横轴分别代表如SEQ ID NO：1～10所列的PTX3重组蛋白片段。
由图4的结果可知，PTX3单株抗体对于如SEQ ID NOs：1～5或如SEQ ID NOs：2～4所列的PTX3重组蛋白片段的亲和力，远高于其它片段，其中SEQ ID NOs：2～4所列的PTX3重组蛋白片段的对应于PTX3第203至217个氨基酸，如SEQ ID NO：11所列的氨基酸序列，代表实施例一的PTX3单株抗体与PTX3结合的表位定位区域坐落于如SEQ ID NOs：2～4所列或如SEQ ID NO：11所列的氨基酸序列的范围内。
2.评估实施例一的PTX3单株抗体与市售PTX3单株抗体与PTX3结合的表位定位区域之差异
此实施例是利用与实施例三相同的方式，评估实施例一的PTX3单株抗体与市售PTX3单株抗体(型号：ab90806；abcam plc.，U.K.)对于PTX3结合的表位定位区域，其结果如图5所示。每个数值为三重复。
请参阅图5，其是示出根据本发明一实施例的PTX3单株抗体及市售PTX3单株抗体 与不同片段的PTX3重组蛋白结合的表位定位图谱，其中PTX3/FL代表如SEQ ID NO：15所列的PTX3重组蛋白片段，RI37代表如SEQ ID NO：12所列的PTX3重组蛋白片段，KT44代表如SEQ ID NO：16所列的PTX3重组蛋白片段，GI40代表如SEQ ID NO：17所列的PTX3重组蛋白片段，而图号「＊＊＊」则代表相较于控制组(即BSA组)具有统计显著性(p＜0.001)。
由图5的结果可知，实施例一的PTX3单株抗体与市售PTX3单株抗体(ab90806)对于PTX3/FL(SEQ ID NO：15)的亲和力皆较高，但实施例一的PTX3单株抗体PTX3单株抗体对于RI37(SEQ ID NO：12)的PTX3重组蛋白片段的亲和力高于市售PTX3单株抗体(ab90806)，且具有统计显著性，代表PTX3单株抗体与市售PTX3单株抗体(ab90806)对于PTX3结合的表位定位区域确实具有差异。
实施例四、评估PTX3单株抗体在体外对PTX3与PTX3受体的结合的影响
PTX3单株抗体可竞争性结合至PTX3的PTX3受体结合区或其邻近区域，进而专一性抑制或减缓PTX3与PTX3受体的结合的机会。本实施例是以CD44作为PTX3受体的一个例子，利用竞争性结合试验，评估PTX3单株抗体竞争性抑制PTX3结合至PTX3受体的效果。
本实施例证实，实施例一的PTX3单株抗体可中和PTX3，使其无法与PTX3受体(例如CD44)的结合区域或其邻近区域结合。
申言之，此实施例使用的竞争性结合分析法是与实施例一相似的方式，而不同的处在于，本实施例是将10μg/mL的PTX3受体〔例如CD44N端重组蛋白(CD44N端第1～220个氨基酸残基的序列，溶于PBS，pH 7.2；Sino Biological Inc.，北京，中国)〕涂布在96孔细胞培养盘的孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含3％脱脂奶粉的PBS﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。
在进行竞争性结合试验时，将结合HRP的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列，HRP-PTX3，浓度为5μg/mL)与不同浓度的实施例一的PTX3单株抗体(浓度为1μg/mL或2μg/mL)于室温(4℃至40℃)进行预反应1小时，以形成预反应物。
在去除阻隔溶液后，利用PBS润洗各孔，再加入上述预反应物，于室温(4℃至40℃)进行反应2小时。然后，利用PBS洗去各孔未结合的预反应物，于各孔加入TMB反应一段时间后，加入0.1M硫酸反应10分钟以终止反应，并利用市售酵素免疫分析仪读取450nm的吸光值，其结果如图6所示。每个数值为四重复。
请参阅图6，其是示出根据本发明一实施例的PTX3单株抗体阻碍PTX3重组蛋白与PTX3受体的结合的竞争性抑制图，其中纵轴代表竞争性抑制率(％)，横轴下方的图号「+」或「－」代表进行结合反应时有或无添加特定成分，图6左侧第1道直条代表控制组(即未加PTX3单株抗体的PTX3重组蛋白的组别)，而图号「＊＊＊」则代表相较于控制组具有统计显著性(p＜0.001)。
由图6的结果可知，以图6左侧第1道未加PTX3单株抗体的数值作为0％之竞争性抑制率，当PTX3单株抗体与PTX3重组蛋白预反应后再与CD44受体反应，所得的竞争性抑制率(％)与PTX3单株抗体的浓度呈现剂量依存关系，且具有统计显著性，代表实施例一的PTX3单株抗体确实可中和PTX3，使其无法与CD44的结合区域或其邻近区域结合。
实施例五、评估PTX3单株抗体对癌细胞活性的影响
乳癌、肺癌、鼻咽癌、神经胶母细胞瘤(glioblastoma multiforme；GBM)属于恶性肿瘤，且上述癌细胞具有移行(migration)、侵袭(invasion)及癌干原细胞性(cancer stemness)等活性。此实施例是利用人类乳癌细胞株(MDA-MB231，寄存编号：BCRC 60425，ATCC HTB-26；三阴性乳腺癌细胞株，以下简称MB231)、人类肺癌细胞株A549(寄存编号：BCRC 60074；ATCC CCL-185)、人类鼻咽癌细胞株HONE1(Int.J.Cancer.1990Jan 15；45(1):83-9；Proc.Natl.Acad.Sci.USA,Vol.86,pp.9524-9528,December 1989)、人类GBM癌细胞U87MG(ATCC寄存编号：HTB-14；BCRC寄存编号：60360)等，通过下述试验评估实施例一的PTX3单株抗体对癌细胞活性的影响。
1.评估PTX3单株抗体对癌细胞移行的影响
在进行移行实验时，将上述癌细胞以1×10 5细胞/孔的细胞密度，接种于24孔的博登细胞移行器(Boyden chamber)的上层(底部孔径为8μm)，并培养3小时。接着，将上层的细胞培养液更换为不含血清的细胞培养液，并于下层的不含血清的细胞培养基中添加0.2μg/mL的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列)、0.4μg/mL的PTX3单株抗体或0.4μg/mL的控制组抗体(IgG1k)。
在培养16小时后，以棉棒刮除上层内侧的细胞，移行至上层底部外侧的细胞利用4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI；Invitrogen)进行染色，并于荧光显微镜的放大倍率200倍的视野下，计算移动至上层底部外侧的细胞数，其结果如图7A、 图8A、图9A及图10A所示。
请参阅图7A、图8A、图9A及图10A，其是分别示出利用本发明实施例一的PTX3单株抗体抑制乳癌细胞MB231(图7A)、肺癌细胞A549(图8A)、鼻咽癌细胞HONE1(图9A)及神经胶母细胞瘤细胞株U87MG(图10A)的移行细胞数柱形图。上述实施例的数据皆是将每一时间点及每一样品的三重复实验数据，正负其平均标准偏差而获得，所有的值皆系通过单因子变异数分析(one way ANOVA)而分析。上述实施例的图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图7A、图8A、图9A及图10A的结果可知，相较于控制组抗体IgG1k，PTX3单株抗体可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549、鼻咽癌细胞HONE1及神经胶母细胞瘤细胞株U87MG的移行细胞数，且此项差异具有统计显著性。
2.评估PTX3单株抗体对癌细胞侵袭的影响
在进行侵袭实验时，24孔的博登细胞移行器(Boyden chamber)的上层(底部孔径为8μm)底面预先以基底膜基质(matrigel，购自BD Bioscience)涂覆后，将上述癌细胞以1×10 5细胞/孔的细胞密度，接种于24孔的博登细胞移行器的上层，并培养3小时。接着，将上层的细胞培养液更换为不含血清的细胞培养液，并于下层的不含血清的细胞培养基中添加0.2μg/mL的PTX3重组蛋白(如SEQ ID NO：4所列的非变性氨基酸序列)、0.4μg/mL的PTX3单株抗体或0.4μg/mL的控制组抗体(IgG1k)。
在培养16小时后，以棉棒刮除上层内侧的细胞，移行至上层底部外侧的细胞利用4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI；Invitrogen)进行染色，并于荧光显微镜的放大倍率200倍的视野下，计算移动至上层底部外侧的细胞数，其结果如图7B、图8B、图9B及图10B所示。
请参阅图7B、图8B、图9B及图10B，其是分别示出利用本发明实施例一的PTX3单株抗体抑制乳癌细胞MB231(图7B)、肺癌细胞A549(图8B)、鼻咽癌细胞HONE1(图9B)及神经胶母细胞瘤细胞株U87MG(图10B)的侵袭细胞数柱形图，其中图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图7B、图8B、图9B及图10B的结果可知，相较于控制组抗体IgG1k，PTX3单株抗体可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549、鼻咽癌细胞HONE1及神经胶母细胞瘤细胞株U87MG的侵袭细胞数，且此项差异具有统计显著性。
3.评估PTX3单株抗体对癌细胞移行的影响
上述癌细胞具有癌干原细胞性(cancer stemness)，添加PTX3重组蛋白可引起前述癌细胞形成细胞球团(sphere)。
在进行细胞球团实验时，将上述癌细胞加入含0.2μg/mL的PTX3重组蛋白、0.4μg/mL的PTX3单株抗体或0.4μg/mL的控制组抗体(IgG1k)的含10％胎牛血清(Fetal Bovine Serum；FBS)的RPMI-1640细胞培养液﹝含10％胎牛血清(Fetal Bovine Serum；FBS)，50～100μg/mL的链霉素及50～100U/mL的青霉素﹞中，并在37℃保持湿度之5％二氧化碳浓度下培养，此为本发明所属技术领域中任何技术人员，故不另赘述。
然后，将上述不同处理的癌细胞以5×10 3细胞/孔的细胞密度，接种于超低吸附表面多孔培养皿(multi-well plate with ultra-low attachment surface；Corning Inc.)中，以不含血清(serum-free)的细胞培养基DMEM/F12(Gibco)[含B27(Invitrogen)、20ng/mL的表皮生长因子(epidermal growth factor，EGF；Abcam)以及10ng/mL的碱性纤维母细胞生长因子(basic Fibroblast Growth Factor，bFGF；Peprotech)]，进行共同培养。经培养2周后，以光学显微镜观察细胞球团数，其结果如图7C、图8C、图9C及图10C所示。
请参阅图7C、图8C、图9C及图10C，其是分别示出利用本发明实施例一的PTX3单株抗体抑制乳癌细胞MB231(图7C)、肺癌细胞A549(图8C)、鼻咽癌细胞HONE1(图9C)及神经胶母细胞瘤细胞株U87MG(图10C)的细胞球团数柱形图，其中图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图7C、图8C、图9C及图10C结果可知，相较于控制组抗体IgG1k，实施例一的PTX3单株抗体可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549、鼻咽癌细胞HONE1及神经胶母细胞瘤细胞株U87MG的细胞球团数，且此项差异具有统计显著性。
4.评估实施例一的PTX3单株抗体与市售PTX3单株抗体对癌细胞活性的影响
此实施例是利用与实施例二相同的方式，评估实施例一的PTX3单株抗体与市售PTX3单株抗体(型号：ab90806；abcam plc.，U.K.)对于癌细胞活动之抑制效果，其结果如图11A至图14C所示。
请参阅图11A、图12A及图13A，其是分别示出利用本发明实施例一的PTX3单株抗体或与市售PTX3单株抗体抑制乳癌细胞MB231(图11A)、肺癌细胞A549(图12A)及鼻 咽癌细胞HONE1(图13A)的移行细胞数柱形图，其中图号「＊」代表具有统计显著性(P＜0.05)的数据，图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图11A、图12A及图13A的结果可知，相较于控制组抗体IgG1k，实施例一的PTX3单株抗体与市售PTX3单株抗体(ab90806)可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549及鼻咽癌细胞HONE1的移行细胞数，但实施例一的PTX3单株抗体抑制或减缓癌细胞移行的效果，明显高于市售PTX3单株抗体(ab90806)，且具有统计显著性。
请参阅图11B、图12B及图13B，其是分别示出利用本发明实施例一的PTX3单株抗体或与市售PTX3单株抗体抑制乳癌细胞MB231(图11B)、肺癌细胞A549(图12B)及鼻咽癌细胞HONE1(图13B)的侵袭细胞数柱形图，其中图号「＊」代表具有统计显著性(P＜0.05)的数据，图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图11B、图12B及图13B的结果可知，相较于控制组抗体IgG1k，实施例一的PTX3单株抗体与市售PTX3单株抗体(ab90806)可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549及鼻咽癌细胞HONE1的侵袭细胞数，但实施例一的PTX3单株抗体抑制或减缓癌细胞侵袭的效果，明显高于市售PTX3单株抗体(ab90806)，且具有统计显著性。
请参阅图14A、图14B及图14C，其是分别示出利用本发明实施例一的PTX3单株抗体或与市售PTX3单株抗体抑制乳癌细胞MB231(图14A)、肺癌细胞A549(图14B)及鼻咽癌细胞HONE1(图14C)的细胞球团数柱形图，其中图号「＊＊」代表具有统计显著性(P＜0.01)的数据，图号「＊＊＊」代表具有统计显著性(P＜0.001)的数据。
由图14A、图14B及图14C的结果可知，相较于控制组抗体IgG1k，实施例一的PTX3单株抗体与市售PTX3单株抗体(ab90806)可显著抑制或减缓乳癌细胞MB231、肺癌细胞A549及鼻咽癌细胞HONE1的细胞球团数，但实施例一的PTX3单株抗体抑制或减缓细胞球团数的效果，仍明显高于市售PTX3单株抗体(ab90806)，且具有统计显著性。
实施例六、评估PTX3单株抗体在活体内对肿瘤的生长及转移的影响
1.评估实施例一的PTX3单株抗体在活体内抑制原位异种移植的乳癌肿瘤的生长及转移的效果
此实施例是将上述人类癌细胞株原位注射至免疫缺陷小鼠的乳腺脂肪垫中，待形成肿瘤后，再施用实施例一的PTX3单株抗体，借此评估实施例一的PTX3单株抗体抑制或减 缓肿瘤的效果。
首先，将乳癌细胞MB231-Luc2〔MB231为人类乳癌细胞，不表现雌激素受体(estrogen receptor；ER)α及ERβ；Luc2为表现荧光素酶的基因〕原位接种到NOD-SCID小鼠(购自于乐斯科生物科技股份有限公司)的乳腺脂肪垫中。待肿瘤的平均体积达到80mm 3后，对实验小鼠施予实施例一的PTX3抗体(8mg/kg体重)或控制组抗体(IgG1k，8mg/kg体重)，每周施予一次，其结果如图15A至图15B所示。图15B是在接种乳癌细胞MB231-Luc2后第11周活体成像结果，其中活体成像是利用市售活体动物荧光成像(in vivo bioluminescent images)系统〔例如：非侵入式3D活体分子影像系统(IVIS system)，PerkinElmer〕观察肿瘤的大小，发光图像处代表小鼠体内具有乳癌细胞MB231-Luc2形成的肿瘤。然后，牺牲所有小鼠，测量其体内肿瘤大小，并利用下式(I)计算肿瘤体积：
V＝(w×l 2)×0.52       (I)
在式(I)中，l代表肿瘤长度，w代表肿瘤宽度。
请参阅图15A至图15B，其是分别示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠原位异种移植的乳癌细胞MDA-MB231的肿瘤体积(图15A)及肿瘤转移(图15B)的结果。图15A的数据是将每一时间点及每一样品的六重复实验数据，取其正负平均标准偏差而获得，而图号「＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.05)。
由图15A及图15B的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可显著抑制或减缓原位异种移植的乳癌细胞MB231的肿瘤体积及肿瘤转移，且此项差异具有统计显著性。
2.评估实施例一的PTX3单株抗体在活体内抑制原位同种移植之乳癌肿瘤的生长及转移的效果
此实施例是将上述小鼠癌细胞株原位注射至免疫系统正常小鼠的乳腺脂肪垫中，待形成肿瘤并建立小鼠三阴性乳癌(tri-negative breast cancer；TNBC)模式后，再施用实施例一的PTX3单株抗体，借此评估实施例一的PTX3单株抗体抑制肿瘤的效果。
首先，将1×10 6乳癌细胞4T1(4T1为小鼠乳癌细胞株，转染含有Luc2基因的载体，表现ERβ但不表现ERα，
Figure PCTCN2019105824-appb-000001
CRL-2539 TM)作为原位同种移植物，原位接种到野生型BALB/c雌性小鼠(6～8周龄，购自于乐斯科生物科技股份有限公司)的乳腺脂肪垫中。待肿瘤的平均体积达到50mm 3后，对实验小鼠施予实施例一的PTX3抗体(10mg/kg体重)或控 制组抗体(IgG1k，10mg/kg体重)，每周施予一次。利用活体动物荧光成像系统观察肿瘤的大小与转移。然后，牺牲所有小鼠，测量其体内肿瘤大小，并利用上式(I)计算肿瘤体积。
请参阅图16A至图16B，其是分别示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠原位原位同种移植的乳癌细胞4T1的肿瘤体积(图16A)及肿瘤转移(图16B)的结果。图16B是在接种乳癌细胞4T1后第5周活体成像结果，其中发光图像处代表小鼠体内具有乳癌细胞4T1形成的肿瘤，且图16A的数据是将每一时间点及每一样品的六重复实验数据，取其正负平均标准偏差而获得，而图号「＊＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.01)。
由图16A及图16B的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可显著抑制或减缓原位同种移植的乳癌细胞4T1的肿瘤体积及肿瘤转移，且此项差异具有统计显著性。
3.评估实施例一的PTX3单株抗体并用抗癌药物在活体内抑制原位同种移植之乳癌肿瘤的生长的效果(I)
此实施例是利用与实施例六第2点相同的方式进行评估，不同的处在于，此实施例是将上述小鼠乳癌细胞株4T1原位接种至BALB/C雌性小鼠(6～8周龄，购自于乐斯科生物科技股份有限公司)的乳腺脂肪垫中。待肿瘤的平均体积达到50mm 3后，施用实施例一PTX3单株抗体〔2.5mg/kg体重(也称为mpk)、5.0mg/kg体重或10.0mg/kg体重的PTX3Ab，臻崴生技〕、控制组抗体(10mg/kg体重的IgG1k，型号10101，伟乔生医)或并用紫杉醇(Taxol或称Paclitaxel，30.0mg/kg体重)，每周一次以腹腔注射的方式投予小鼠，共投予六周。第六周老鼠牺牲后，利用活体动物荧光成像系统观察肿瘤的大小与转移。
六周后，利用市售活体动物荧光成像(in vivo bioluminescent images)系统〔例如：非侵入式3D活体分子影像系统(IVIS system)，PerkinElmer〕观察肿瘤的大小，其结果如图17A所示，其中发光图像处代表小鼠体内具有乳癌细胞4T1形成的肿瘤。由上述系统测量小鼠体内肿瘤大小及转移，并根据上式(I)计算肿瘤体积，其结果如图17B至图17C所示。
请参阅图17A至图17C，其是分别示出根据本发明一实施例的PTX3单株抗体在并用或未并用紫杉醇的情形下，抑制小鼠原位同种移植的乳癌细胞4T1的肿瘤体积与转移的影像图(图17A)及肿瘤体积变化折线图(图17B至图17C)。图17B至图17C的数据是将每一时间点及每一样品的六重复实验数据，取其正负平均标准偏差而获得，其中图号「＊＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.01)。
由图17A至图17C的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体或紫杉醇可抑制或减缓原位同种移植的乳癌细胞4T1的肿瘤体积及转移，如图17A及图17B所示。然而，小鼠并用PTX3单株抗体与紫杉醇后，可提升抑制或减缓原位同种移植的乳癌细胞4T1的肿瘤体积及转移，且二者并用的效果远超过单独处理效果的综效，如图17A及图17C所示。以上差异皆具有统计显著性。
4.评估实施例一的PTX3单株抗体并用抗癌药物在活体内抑制原位同种移植之乳癌肿瘤的生长的效果(II)
此实施例是按照图18A的实验流程示意图，利用与实施例六第3点相同的方式进行评估，其结果如图18B至图18D所示。
请参阅图18B至图18D，其是分别示出根据本发明另一实施例的PTX3单株抗体在并用或未并用紫杉醇的情形下，小鼠原位同种移植的乳癌细胞4T1的肿瘤体积与转移的影像图(图18B)、肿瘤体积折线图(图18C)及小鼠存活率(图18D)。图18C至图18D的数据是将每一时间点及每一样品的六重复实验数据，取其正负平均标准偏差而获得，其中图号「＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.05)，而图号「＊＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.01)。
由图18B至图18D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体或紫杉醇可抑制或减缓原位同种移植的乳癌细胞4T1的肿瘤体积及转移，如图18B及图18C所示。然而，小鼠并用PTX3单株抗体与紫杉醇后，可提升抑制或减缓原位同种移植的乳癌细胞4T1的肿瘤体积，并提升小鼠的存活率达80％以上，且二者并用的效果远超过单独处理效果的综效，如图18D所示。以上差异皆具有统计显著性。
5.评估实施例一的PTX3单株抗体在活体内抑制同种异体移植之大肠结肠癌肿瘤的生长的效果
此实施例是按照图19A的实验流程示意图，利用与实施例六第3点相同的方式进行评估，不同的处在于，此实施例是将上述小鼠大肠癌细胞株MC38皮下(s.c.)注射至C57BL/6J雄性小鼠(6～8周龄，购自于乐斯科生物科技股份有限公司)。待MC38细胞注射第7天后，施用实施例一PTX3单株抗体〔10.0mg/kg体重的PTX3Ab，臻崴生技〕或控制组抗体(10mg/kg体重的IgG1k，型号10101，伟乔生医)，每周一次以腹腔(i.p.)注射的方式投予小鼠，共投予三重复。每周利用活体动物荧光成像系统观察肿瘤的大小。第23天后，牺牲所有 小鼠，测量其体内肿瘤大小，并利用上式(I)计算肿瘤体积。
请参阅图19B，其是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠植入大肠癌细胞株MC38的肿瘤体积的结果。图19B的数据是将每一时间点及每一样品的四重复实验，取其正负平均标准偏差而获得，其中图号「＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.05)，图号「＊＊＊」则代表相较于控制组抗体(IgG1k)具有统计显著性(p＜0.001)。
由图19B的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可抑制或减缓大肠癌细胞株MC38的肿瘤体积，且此差异具有统计显著性。
6.评估实施例一的PTX3单株抗体在活体内抑制异种移植的神经胶母细胞瘤的生长的效果
此实施例是利用与实施例六第3点相同的方式进行评估，不同的处在于，此实施例是将上述人类神经胶母细胞瘤(glioblastoma multiforme；GBM)细胞株U87MG皮下(s.c.)注射至NOD-SCID雄性小鼠(6～8周龄，购自于乐斯科生物科技股份有限公司)。待U87MG细胞注射第20天后，施用实施例一PTX3单株抗体〔10.0mg/kg体重的PTX3Ab，臻崴生技〕或控制组抗体(10mg/kg体重的IgG1k，型号10101，伟乔生医)，每周一次以腹腔(i.p.)注射的方式投予小鼠，共投予四周。20天后，测量肿瘤大小，并利用上式(I)计算肿瘤体积。
请参阅图20A及图20B，其是示出根据本发明一实施例的PTX3单株抗体或控制组抗体抑制小鼠植入人类神经胶母细胞瘤细胞株U87MG的肿瘤体积折线图(图20A)及小鼠存活率(图20B)。图20A的数据是将每一时间点及每一样品的三或四重复实验数据，取其正负平均标准偏差而获得，其中图号「p＝0.0008」则代表相较于控制组抗体(IgG1k)具有统计显著性。
由图20A及图20B的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可抑制或减缓异种移植的神经胶母细胞瘤细胞U87MG的肿瘤体积(图20A)，且可提升小鼠的存活率达75％(图20B)。以上差异皆具有统计显著性。
实施例七、评估PTX3单株抗体在活体内减缓或逆转纤维化的效果
1.评估实施例一的PTX3单株抗体在活体内逆转急性肝纤维化的肝坏死的效果
此实施例是按照图21A的实验流程示意图进行，此乃根据义守大学义大医院动物中心的指南设计。首先，对C57BL/6J雄性小鼠(8周龄，购自于乐斯科生物科技股份有限公司) 肌肉注射1mL/kg体重的四氯甲烷(CCl 4与橄榄油以1：1的体积比混合)。四氯甲烷引起的急性肝纤维化会在12小时内导致肝细胞凋亡(apoptosis)及坏死(necrosis)。接着，在注射四氯甲烷后的第4个小时及第28小时，以腹腔(i.p.)注射的方式对小鼠施用实施例一PTX3单株抗体〔10mg/kg体重的PTX3Ab，臻崴生技〕或控制组抗体(10mg/kg体重的IgG1k，型号10101，伟乔生医)。第2天牺牲所有小鼠，观察肝组织切片变化、肝坏死区域所占比例及肝脏重/体重比，其结果如图21B至图21D所示。
请参阅图21B至图21D，其是分别示出根据本发明一实施例的PTX3单株抗体在急性肝纤维化小鼠体内以苏木精-伊红(hematoxylin and eosin，H&E)染色的左肝叶组织切片(图21B，放大倍率：20倍，观察肝细胞凋亡的情况)、肝坏死区域比例(图21C)及肝脏重/体重比(图21D)。图21C是利用市售影像分析软件ImageJ(W.S.Rasband,NIH,Bethesda,Maryland,USA)的自动检测阈值的功能，测量总扫描区域中，肝坏死区域所占的百分比。
由图21B至图21D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可减缓四氯甲烷引起急性肝纤维化的细胞凋亡(图21B)及坏死(图21C)，且可减缓小鼠因急性肝纤维化而增加肝脏重量的程度(图21D)。以上差异皆具有统计显著性。
2.评估实施例一的PTX3单株抗体在活体内逆转慢性肝纤维化的肝坏死的效果
此实施例是按照图22A的实验流程示意图进行，其是利用与实施例七第1点相同方式设计。不同的处在于，此实施例是对C57BL/6J雄性小鼠(8周龄，购自于乐斯科生物科技股份有限公司)肌肉注射1mL/kg体重的四氯甲烷(CCl 4与橄榄油以1：1的体积比混合)，一周注射2次，共八周。接着，在注射四氯甲烷后的第3周至第8周，以腹腔(i.p.)注射的方式对小鼠施用实施例一PTX3单株抗体〔10mg/kg体重的PTX3Ab，臻崴生技〕或控制组抗体(10mg/kg体重的IgG1k，型号10101，伟乔生医)，每周一次。第8周结束，牺牲所有小鼠，观察肝组织切片变化、肝坏死区域所占比例及肝脏重/体重比，其结果如图22B至图22D所示。
请参阅图22B至图22D，其是分别示出根据本发明一实施例的PTX3单株抗体在慢性肝纤维化小鼠体内以天狼星红(Picro-Sirius Red)染色的左肝叶组织切片(图22B，放大倍率：20倍，观察肝纤维化的情况)、肝纤维化区域比例(图22C)及肝脏重/体重比(图22D)。图22C是利用市售影像分析软件ImageJ(W.S.Rasband,NIH,Bethesda,Maryland,USA)的自动检测阈值的功能，测量总扫描区域中，肝坏死区域所占的百分比。
由图22B至图22D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株 抗体可减缓四氯甲烷引起的慢性肝纤维化(图22B)及区域比例(图22C)，且可减缓小鼠因慢性肝纤维化而增加肝脏重量的程度(图22D)。以上差异皆具有统计显著性。
4.评估实施例一的PTX3单株抗体对肾纤维母细胞的纤维化相关蛋白表现的影响
此实施例是利用大鼠肾纤维母细胞株(NRK49F，寄存编号：BCRC 60084，
Figure PCTCN2019105824-appb-000002
CRL-1570 TM)，通过下述试验评估实施例一的PTX3单株抗体对肾纤维化的影响。
首先，将肾纤维母细胞株NRK49F细胞培养在杜贝可氏改良伊格氏培养基(Dulbecco's Modified Eagle Medium，DMEM)(12800-082,Gibco)﹝含10％FBS、100μg/mL的链霉素及100U/mL的青霉素﹞中。
NRK49F细胞利用0.4μg/mL IgG1k(型号10101，伟乔生医)、0.4μg/mL PTX3抗体(臻崴生技)处理后，再以200ng/mL PTX3处理6小时。接着，利用改良式放射免疫沉淀分析(modified radioimmunoprecipitation assay，RIPA)缓冲液〔modified RIPA buffer，含有50mM Tris-HCl(pH 7.4),150mM NaCl,1mM EDTA、1％NP-40、0.25％脱氧胆酸钠(sodium deoxycholate)、1mM二硫苏糖醇(dithiothreitol，DTT)、1mM苯甲基磺酰氟(phenylmethylsulfonyl fluoride，PMSF)、抑肽酶(aprotinin，1mg/ml)以及亮肽素(leupeptin，1mg/ml)〕〕溶解NRK49F细胞。之后，利用特定的抗体进行西方墨点法(western blotting)，以检测α-微管蛋白(α-tubulin，型号T6199，Sigma)、纤维连接蛋白(Fibronectin，型号15613-1-AP，ProteinTech)的表现，并以α-微管蛋白的表现量作为注入控制(loading control)组，其结果如图23A所示。
另外，将NRK49F细胞种入24孔细胞培养盘，利用0.4μg/mL IgG1k(型号10101，伟乔生医)、PTX3抗体(臻崴生技)或200ng/mL PTX3处理24小时。接着，NRK49F细胞利用甲醇固定于-20℃至隔夜。隔天，利用天狼星红溶液(Picro-Sirius Red Solution，型号ab246832，Abcam)在室温下染色20分钟后，利用醋酸润洗二次。利用光学显微镜在放大倍率200倍的视野下，判定细胞的结节数。然后，利用0.1N NaOH溶解这些细胞，利用市售ELISA读取设备测量于490nm的吸光值，其结果如图23B及图23C所示。
请参阅图23A至图23C，其是示出根据本发明一实施例的PTX3单株抗体对小鼠肾纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图23A)、细胞结节的细胞染色影像(图23B)及其柱形图(图23C)。图号「＊＊＊」则代表相较于控制组具有统计显著性(p＜0.001)。
由图23A至图23C的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株 抗体可降低肾纤维母细胞的纤维化相关蛋白表现(图23A)、减少细胞结节数(图23B，图23C)，且此差别具有统计显著差异性。
5.评估实施例一的PTX3单株抗体在单侧输尿管阻塞(unilateral ureteral obstruction，UUO)动物模式中减缓肾纤维化的效果
此实施例是分别按照图24A及图24C的实验流程示意图进行评估。首先，取六至八周龄C57BL/6J品系的雄性小鼠(购自于乐斯科生物科技股份有限公司)。接着，由小鼠左侧切口进行单侧输尿管阻塞(unilateral ureteral obstruction，UUO)手术。取出左肾露出输尿管(ureter)，并利用丝线(4-O Silk)缝合输尿管。之后，利用手术缝合器缝合伤口。接下来，术后第0天及第7天(前处理，如图24A及图24B)或术后第7天(后处理，如图24C及图24D)，小鼠利用10mg/kg IgG1k(型号10101，伟乔生医)、10mg/kg PTX3抗体(臻崴生技)处理，在术后第14天将小鼠安乐死。将半颗肾脏以福尔马林固定，并包埋于石蜡中。组织切片以苏木紫-伊红(Haematoxylin Eosin，HE)或天狼星红溶液(Picro-Sirius Red Solution，型号ab246832，Abcam)染色后，覆盖上玻片后封片，以20倍的放大倍率观察影像，其结果如图24B及图24D所示，图中比例尺代表100μm。
请参阅图24B及图24D，其是分别示出根据本发明一实施例的PTX3单株抗体对UUO小鼠的肾脏组织切片染色影像。
由图24B及图24D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体确实可减缓UUO动物的肾纤维化。
6.评估实施例一的PTX3单株抗体对PTX3处理的肺纤维母细胞的纤维化相关蛋白表现及移行的影响
此实施例是利用人类肺纤维母细胞株(HFL1，寄存编号：BCRC 60299，
Figure PCTCN2019105824-appb-000003
CCL-153 TM)，通过下述试验评估实施例一的PTX3单株抗体对肺纤维化的影响。
首先，将HFL1细胞培养在汉氏F-12K(F12K)培养基〔凯恩(Kaighn)改良〕(21127-022,Gibco)﹝含10％FBS、100μg/mL的链霉素及100U/mL的青霉素﹞中。
HFL1细胞利用0.4μg/mL IgG1k(型号10101，伟乔生医)、0.4μg/mL PTX3抗体(臻崴生技)处理后，再以200ng/mL PTX3处理6小时。接着，利用改良式RIPA缓冲液溶解HFL1细胞。之后，利用特定的抗体进行西方墨点法(western blotting)，以检测α-微管蛋白(α-tubulin，型号T6199，Sigma)、纤维连接蛋白(Fibronectin，型号15613-1-AP，ProteinTech)、 胶原蛋白第1型(Collagen I，型号14695-1-AP，ProteinTech)以及α-平滑肌动蛋白(α-smooth muscle sctin，α-SMA，型号GTX 100904，GeneTex)的表现，并以α-微管蛋白的表现量作为注入控制(loading control)组，其结果如图25A所示。
另外，将HFL1细胞种入24孔细胞培养盘〔内含孔径8-μm的插入式培养皿(353097,BD Biosciences)〕，下层培养孔含或不含0.4μg/mL IgG1k(型号10101，伟乔生医)、PTX3抗体(臻崴生技)或200ng/mL PTX3处理。经培养16小时后，利用棉棒刮下插入式培养皿内的细胞。贴附在插入式培养皿的聚碳酸酯薄膜下表面的总细胞数移行到插入式培养皿底部外的细胞，则利用DAPI染色，利用荧光显微镜在放大倍率200倍的视野下，判定细胞数，其结果如图25B所示。
将HFL1细胞种入24孔细胞培养盘，利用含或不含0.4μg/mL IgG1k(型号10101，伟乔生医)、PTX3抗体(臻崴生技)或200ng/mL PTX3处理24小时。接着，HFL1细胞利用甲醇固定于-20℃至隔夜。隔天，利用天狼星红溶液(Picro-Sirius Red Solution，型号ab246832，Abcam)在室温下染色20分钟后，利用醋酸润洗二次。利用光学显微镜在放大倍率200倍的视野下，判定细胞的结节数。然后，利用0.1N NaOH溶解这些细胞，利用市售ELISA读取设备测量于490nm的吸光值，其结果如图25C及图25D所示。
请参阅图25A至图25D，其是示出根据本发明一实施例的PTX3单株抗体对肺纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图25A)、移行细胞数的柱形图(图25B)、细胞结节的细胞染色影像(图25C)及其柱形图(图25D)。图号「＊＊＊」则代表相较于控制组具有统计显著性(p＜0.001)。
由图25A至图25D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可减缓PTX3处理的肺纤维母细胞的纤维化相关蛋白表现、移行及细胞结节数。
7.评估实施例一的PTX3单株抗体对于IPF小鼠模式的博来霉素诱发的肺纤维化的影响(I)
此实施例是按照图26A的实验流程示意图进行。首先，取六至八周龄C57BL/6J品系的雄性小鼠(购自于乐斯科生物科技股份有限公司)。小鼠通过气管灌注(intratracheally instilled，I.T.)方式投予PBS或2mg/kg博来霉素(bleomycin，BLM，型号ap302，Znzo)诱发纤维化。诱发纤维化后，第14、21天以腹腔注射方式对小鼠投予10mg/kg IgG1k(型号10101，伟乔生医)或10mg/kg PTX3抗体(臻崴生技)，在第28天进行安乐死。
C57BL/6小鼠在以气管灌注方式投予博来霉素后的第7、14、21或28天，分别测量 其体重，然后以腹腔注射方式投予10mg/kg IgG1k(控制组)或投予10mg/kg PTX3抗体，其结果如图26B所示。
小鼠在安乐死后，肉眼检视以气管灌注方式投予PBS(即健康控制组)或2mg/kg博来霉素(即BLM)后第7、14、21或28天的小鼠肺部组织。左肺叶经灌流后，以三聚甲醛(paraformaldehyde)固定，并以石蜡包埋。组织切片以HE染色后，覆盖上玻片后封片，以20倍的放大倍率观察影像，其结果如图26C所示，图中比例尺代表100μm。
小鼠在安乐死后，肉眼检视以博来霉素诱导纤维化后并投予10mg/kg IgG1k或10mg/kg PTX3抗体第28天的小鼠肺部组织。左肺叶经灌流后，以三聚甲醛(paraformaldehyde)固定，并以石蜡包埋。组织切片以HE染色后，覆盖上玻片后封片，以20倍的放大倍率观察影像，其结果如图26D所示，图中比例尺代表100μm。
请参阅图26B至图26D，其是示出根据本发明一实施例的PTX3单株抗体对BLM诱发肺纤维化小鼠的体重变化曲线图(图26B)、肺部外观及组织切片影像(图26C及图26D)。
由图26B至图26D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体在诱发肺纤维化小鼠中，可逆转因肺纤维化导致的体重减轻及肺纤维化的程度。
8.评估实施例一的PTX3单株抗体对于IPF小鼠模式的博来霉素诱发的肺纤维化的影响(II)
此实施例是按照图27A的实验流程示意图进行。首先，取六至八周龄C57BL/6J品系的雄性小鼠(购自于乐斯科生物科技股份有限公司)。小鼠通过气管灌注(intratracheally instilled，I.T.)方式投予PBS或2mg/kg博来霉素(bleomycin，BLM，型号ap302，Znzo)诱发纤维化。诱发纤维化后，第21天以腹腔注射方式对小鼠投予10mg/kg IgG1k(型号10101，伟乔生医)或10mg/kg PTX3抗体(臻崴生技)，在第28天进行安乐死。
C57BL/6小鼠在以气管灌注方式投予博来霉素后的第7、14、21或28天，分别测量其体重，然后在第21天以腹腔注射方式投予10mg/kg IgG1k(控制组)或投予10mg/kg PTX3抗体，其结果如图27B所示。
小鼠在安乐死后，肉眼检视以气管灌注方式投予PBS(即健康控制组)或2mg/kg博来霉素(即BLM)后第7、14、21或28天的小鼠肺部组织。左肺叶经灌流后，以三聚甲醛(paraformaldehyde)固定，并以石蜡包埋。组织切片以HE染色后，覆盖上玻片后封片，以20倍的放大倍率观察影像，其结果如图27C所示，图中比例尺代表100μm。
小鼠在安乐死后，肉眼检视以博来霉素诱导纤维化后并投予10mg/kg IgG1k或10 mg/kg PTX3抗体第28天的小鼠肺部组织。左肺叶经灌流后，以三聚甲醛(paraformaldehyde)固定，并以石蜡包埋。组织切片以HE染色后，覆盖上玻片后封片，以20倍的放大倍率观察影像，其结果如图27D所示，图中比例尺代表100μm。
请参阅图27B至图27D，其是示出根据本发明一实施例的PTX3单株抗体对BLM诱发肺纤维化小鼠的体重变化曲线图(图27B)、肺部外观及组织切片影像(图27C及图27D)。
由图27B至图27D的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体在诱发肺纤维化小鼠中，可逆转因肺纤维化导致的体重减轻及肺纤维化的程度。
9.评估实施例一的PTX3单株抗体对NIH-3T3胚胎纤维母细胞的纤维化相关蛋白表现的影响
此实施例是利用小鼠胚胎纤维母细胞株(NIH-3T3，寄存编号：BCRC 60008，
Figure PCTCN2019105824-appb-000004
CCL-1658 TM)，通过下述试验评估实施例一的PTX3单株抗体对纤维化的影响。
首先，将NIH-3T3细胞培养在DMEM(12800-082,Gibco)﹝含10％FBS、100μg/mL的链霉素及100U/mL的青霉素﹞中。
NIH-3T3细胞利用0.4μg/mL PTX3抗体(臻崴生技)处理后，利用200ng/mL PTX3处理6小时。接着，利用RIPA缓冲液溶解细胞。之后，利用特定的抗体进行西方墨点法(western blotting)，以检测α-微管蛋白(α-tubulin，型号T6199，Sigma)、胶原蛋白第1型(Collagen I，型号14695-1-AP，ProteinTech)以及α-平滑肌动蛋白(α-smooth muscle sctin，α-SMA，型号GTX 100904，GeneTex)的表现，并以α-微管蛋白的表现量作为注入控制(loading control)组，其结果如图28A所示。
另外，将NIH-3T3细胞种入24孔细胞培养盘，以含或不含0.4μg/mL IgG1k(型号10101，伟乔生医)、PTX3抗体(臻崴生技)或200ng/mL PTX3处理24小时。之后，NIH-3T3细胞利用甲醇固定于-20℃至隔夜。隔天，利用天狼星红溶液(Picro-Sirius Red Solution，型号ab246832，Abcam)在室温下染色20分钟后，利用醋酸润洗二次。利用光学显微镜在放大倍率200倍的视野下，判定细胞的结节数。然后，利用0.1N NaOH溶解这些细胞，利用市售ELISA读取设备测量于490nm的吸光值，其结果如图28B及图28C所示。
请参阅图28A至图28C，其是示出根据本发明一实施例的PTX3单株抗体对胚胎纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果(图28A)、细胞结节的细胞染色影像(图28B)及其柱形图(图28C)。图号「＊＊＊」则代表相较于控制组具有统计显著性(p＜0.001)。
由图28A至图28C的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株 抗体可减缓NIH-3T3细胞的纤维化相关蛋白表现及细胞结节数。
10.评估实施例一的PTX3单株抗体对F28肝纤维母细胞的纤维化相关蛋白表现的影响
首先，将人类癌症相关纤维母细胞/肝细胞〔cancer associated fibroblast/F28(CAF/F28)cells〕培养在DMEM(12800-082,Gibco)﹝含10％FBS、100μg/mL的链霉素及100U/mL的青霉素﹞中。
CAF/F28细胞暴露在放射剂量8格雷(Gray，Gy)后，利用0.4μg/ml PTX3抗体(臻崴生技)处理6小时。接着，利用改良式放射免疫沉淀分析(modified radioimmunoprecipitation assay，RIPA)缓冲液〔modified RIPA buffer，含有50mM Tris-HCl(pH 7.4),150mM NaCl,1mM EDTA、1％NP-40、0.25％脱氧胆酸钠(sodium deoxycholate)、1mM二硫苏糖醇(dithiothreitol，DTT)、1mM苯甲基磺酰氟(phenylmethylsulfonyl fluoride，PMSF)、抑肽酶(aprotinin，1mg/ml)以及亮肽素(leupeptin，1mg/ml)]〕溶解细胞。之后，利用特定的抗体进行西方墨点法(western blotting)，以检测α-微管蛋白(α-tubulin，型号T6199，Sigma)、纤维连接蛋白(Fibronectin，型号15613-1-AP，ProteinTech)、胶原蛋白第1型(Collagen I，型号14695-1-AP，ProteinTech)以及α-平滑肌动蛋白(α-smooth muscle sctin，α-SMA，型号GTX 100904，GeneTex)的表现，并以α-微管蛋白的表现量作为注入控制(loading control)组，其结果如图29A所示。
另外，CAF/F28细胞利用0.4μg/mL PTX3抗体(臻崴生技)处理后，利用200ng/mL PTX3处理6小时。接着，利用RIPA缓冲液溶解细胞。之后，利用特定的抗体进行西方墨点法(western blotting)，以检测α-微管蛋白(α-tubulin，型号T6199，Sigma)、纤维连接蛋白(Fibronectin，型号15613-1-AP，ProteinTech)、胶原蛋白第1型(Collagen I，型号14695-1-AP，ProteinTech)以及α-平滑肌动蛋白(α-smooth muscle sctin，α-SMA，型号GTX100904，GeneTex)的表现，并以α-微管蛋白的表现量作为注入控制(loading control)组，其结果如图29B所示。
请参阅图29A至图29B，其是示出根据本发明一实施例的PTX3单株抗体对不同处理的肝纤维母细胞的纤维化相关蛋白表现的西方墨点分析结果。
由图29A至图29B的结果可知，相较于控制组抗体(IgG1k)，实施例一的PTX3单株抗体可减缓CAF/F28肝纤维母细胞因照射放射线或PTX3引起的纤维化相关蛋白表现。
上述小鼠实验系根据成功大学实验动物照护及使用指南(Guide for Care and Use of  Laboratory Animals)进行，且相关动物的使用方案已经过实验动物照护及使用委员会(Institutional Animal Care and Use Committee，IACUC)的审查及核准。
补充说明的是，本发明实施例一的PTX3单株抗体对PTX3重组蛋白具有良好的亲和力及灵敏度，可应用于检测PTX3的套组及方法，以于体外检测生物样本中的PTX3含量。关于适用的生物样本、适用于检测PTX3的方法、套组、组件/设备等悉如前述，不另赘言。其次，本发明的含单株抗体或其抗原结合片段的医药组合物及其用途，其利用亲和力及灵敏度较高的PTX3单株抗体或其抗原结合片段作为有效成分，通过有效抑制或减缓PTX3与PTX3受体的结合，进而抑制或减缓与PTX3与PTX3受体结合相关的疾病或症状，可作为跨疾病广效药。
综言之，本发明虽以特定序列的PTX3单株抗体、特定的分析模式或特定的评估方式作为例示，说明本发明的含单株抗体或其抗原结合片段的医药组合物及其用途，而本发明所属技术领域中任何技术人员可知，本发明并不限于此，在不脱离本发明的构思和范围内，本发明的含单株抗体或其抗原结合片段的医药组合物及其用途，也可使用其它的分析模式或其它的评估方式进行。
由上述实施例可知，本发明的含单株抗体或其抗原结合片段的医药组合物及其用途，其优点在于利用特定的PTX3单株抗体或其抗原结合片段作为有效成分，专一性抑制或减缓PTX3与PTX3受体的结合，可应用于检测PTX3的套组及方法，以及抑制或减缓与PTX3与PTX3受体结合相关的疾病或症状。
虽然本发明已以数个实施例公开如上，然其并非用以限定本发明，在本发明所属技术领域中任何技术人员，在不脱离本发明的构思和范围内，当可作各种的变动与修饰，因此本发明的保护范围当视后附的权利要求所界定者为准。

Claims (52)

  1. 一种单株抗体或其抗原结合片段，其特征在于，该单株抗体或其抗原结合片段是专一性结合一非变性氨基酸序列，且该非变性氨基酸序列是选自于由如序列识别编号SEQ ID NO：1至SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  2. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列是选自于由如SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  3. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列是选自于由如SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  4. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：2所列的氨基酸序列。
  5. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：3所列的氨基酸序列。
  6. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：4所列的氨基酸序列。
  7. 根据权利要求1所述的单株抗体或其抗原结合片段，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：11所列的氨基酸序列。
  8. 一种单株抗体或其抗原结合片段，其特征在于，包括：
    一重链可变区序列，具有如SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20和/或SEQ ID NO：21所列的氨基酸序列；以及
    一轻链可变区序列，具有如SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24和/或SEQ ID NO：25所列的氨基酸序列。
  9. 一种单株抗体或其抗原结合片段，其特征在于，包括：
    一重链可变区序列，具有如SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和/或SEQ ID NO：29所列的核酸序列编码的核酸序列；以及
    一轻链可变区序列，具有如SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32和/或SEQ ID NO：33所列的核酸序列编码的核酸序列。
  10. 一种单株抗体或其抗原结合片段，其特征在于，包括：
    一重链可变区序列，具有如SEQ ID NOs：34或35所列的氨基酸序列；以及
    一轻链可变区序列，具有如SEQ ID NOs：36或37所列的氨基酸序列。
  11. 一种单株抗体或其抗原结合片段，其特征在于，包括：
    一重链可变区序列，具有如SEQ ID NOs：38或39所列的核酸序列编码的核酸序列；以及
    一轻链可变区序列，具有如SEQ ID NOs：40或41所列的核酸序列编码的核酸序列。
  12. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段为嵌合抗体或其抗原结合片段。
  13. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段为鼠源抗体、人鼠嵌合抗体、人源化抗体或其抗原结合片段。
  14. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该抗原结合片段为单链可变区片段、单链可变区片段二聚体﹝(scFv) 2﹞、单链可变区片段三聚体﹝(scFv) 3﹞、可变区片段、Fab片段、Fab'片段、F(ab') 2片段、纳米抗体或上述的任意组合。
  15. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段是经由复合或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。
  16. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段为抗体药物复合体或其抗原结合片段。
  17. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段为双功能单株抗体或其抗原结合片段。
  18. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段为三功能单株抗体或其抗原结合片段。
  19. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段属于IgG类别、IgM类别、IgA类别、IgD类别或IgE类别。
  20. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段属于IgG类别且具有IgG1、IgG2、IgG3或IgG4同型。
  21. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段具有IgG1同型。
  22. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段属于惰性抗体。
  23. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段属于拮抗剂抗体。
  24. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段是专一性抑制或减缓正五聚蛋白相关蛋白受体与一或多种PTX3的一C端特定序列的结合。
  25. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段是专一性抑制或减缓一或多种PTX3的活性。
  26. 根据权利要求1至11中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段是专一性抑制或减缓PTX3受体与一或多种PTX3的交互作用、抑制或减缓PTX3信息传递或上述的任意组合。
  27. 一种检测PTX3的套组，包含如权利要求1至26中任一项所述的单株抗体或其抗原结合片段，其特征在于，其中该单株抗体或其抗原结合片段是专一性结合一非变性氨基酸序列，且该非变性氨基酸序列是选自于由如SEQ ID NO：1至SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  28. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该单株抗体或其抗原结合片段是专一性结合一非变性氨基酸序列，且该非变性氨基酸序列是选自于由如SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  29. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该单株抗体或其抗原结合片段是专一性结合一非变性氨基酸序列，且该非变性氨基酸序列是选自于由如SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的氨基酸序列所组成的一族群。
  30. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：2所列的氨基酸序列。
  31. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：3所列的氨基酸序列。
  32. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：4所列的氨基酸序列。
  33. 根据权利要求27所述的检测PTX3的套组，其特征在于，其中该非变性氨基酸序列为如SEQ ID NO：11所列的氨基酸序列。
  34. 一种体外检测PTX3的方法，其是利用如权利要求27至33中任一项所述的检测PTX3的套组检测PTX3，其特征在于，其中该检测PTX3的套组所含的单株抗体或其抗原结合片段的一分析灵敏度不低于0.0016pM。
  35. 一种医药组合物，包含具有一有效剂量的一单株抗体或其抗原结合片段及一医药学上可接受的载剂，其特征在于其是以如权利要求1至11中任一项所述的该单株抗体或其抗原结合片段作为一有效成分。
  36. 根据权利要求35所述的医药组合物，其特征在于，其中该医药组合物还包含一活性药物成分。
  37. 根据权利要求35所述的医药组合物，其特征在于，其中该有效剂量为每公斤体重2mg至10mg。
  38. 根据权利要求35所述的医药组合物，其特征在于，其中该有效剂量为5mg/kg体重至10mg/kg体重。
  39. 根据权利要求35所述的医药组合物，其特征在于，其中该有效剂量为6mg/kg体重至9mg/kg体重。
  40. 一种单株抗体或其抗原结合片段用于制备专一性抑制或减缓正五聚蛋白相关蛋白(PTX3)与PTX3受体结合的医药组合物的用途，其特征在于，其中该医药组合物为如权利要求34至38中任一项所述，且该单株抗体或其抗原结合片段具有一有效剂量，以抑制或减缓与该PTX3与PTX3受体结合相关的一疾病或一症状。
  41. 根据权利要求40所述的单株抗体或其抗原结合片段用于制备专一性抑制或减缓PTX3与PTX3受体结合的医药组合物的用途，其特征在于，其中该疾病或该症状包括上皮细胞癌、腺癌、神经胶母细胞瘤及纤维化。
  42. 根据权利要求41所述的单株抗体或其抗原结合片段用于制备专一性抑制或减缓PTX3与PTX3受体结合的医药组合物的用途，其特征在于，其中该上皮细胞癌包括肺癌、乳癌、鼻咽癌。
  43. 根据权利要求41所述的单株抗体或其抗原结合片段用于制备专一性抑制或减缓PTX3与PTX3受体结合的医药组合物的用途，其特征在于，其中该腺癌包括大肠癌。
  44. 根据权利要求41所述的单株抗体或其抗原结合片段用于制备专一性抑制或减缓PTX3与PTX3受体结合的医药组合物的用途，其特征在于，其中受该纤维化的该疾病或该症状影响的一器官是选自于由肺、肝、肾及皮肤所组成的一族群。
  45. 根据权利要求40所述的单株抗体或其抗原结合片段用于制备专一性抑制或减缓 PTX3与PTX3受体结合的医药组合物的用途，其特征在于，其中该医药组合物是经由皮下注射、肌肉注射、静脉注射、腹腔注射、原位注射、经口投予或口鼻吸入的方式投予。
  46. 一种用于体外抑制或减缓肿瘤细胞的活性的方法，其特征在于，包括对一肿瘤细胞投予一有效剂量的如权利要求35至39中任一项所述的医药组合物，借此抑制或减缓该肿瘤细胞的一活性。
  47. 根据权利要求46所述的用于体外抑制或减缓肿瘤细胞的活性的方法，其特征在于，其中该肿瘤细胞的一来源包括神经胶母细胞瘤、上皮细胞癌及腺癌。
  48. 根据权利要求47所述的用于体外抑制或减缓肿瘤细胞的活性的方法，其特征在于，其中该上皮细胞癌包括肺癌、乳癌及鼻咽癌。
  49. 根据权利要求47所述的用于体外抑制或减缓肿瘤细胞的活性的方法，其特征在于，其中该腺癌包括大肠癌。
  50. 根据权利要求46所述的用于体外抑制或减缓肿瘤细胞的活性的方法，其特征在于，其中该活性包含增生、癌干原细胞性、移行、侵袭、转移、肿瘤体积或抗药性。
  51. 一种用于体外抑制或减缓纤维化疾病和/或纤维化症状的方法，其特征在于，包括对受该纤维化疾病和/或该纤维化症状影响的一器官投予一有效剂量的如权利要求35至39中任一项所述的医药组合物，借此抑制或减缓该器官的该纤维化疾病和/或该纤维化症状。
  52. 根据权利要求51所述的用于体外抑制或减缓纤维化疾病和/或纤维化症状的方法，其特征在于，其中该器官是选自于由肺、肝、肾及皮肤所组成的一族群。
PCT/CN2019/105824 2018-09-14 2019-09-13 含单株抗体或其抗原结合片段的医药组合物及其用途 WO2020052675A1 (zh)

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