Pharmaceutical compositions containing monoclonal antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The present invention provides a PTX3 monoclonal antibody or an antigen-binding fragment thereof and uses thereof. The monoclonal antibody or the antigen-binding fragment thereof specifically inhibits or delays the binding of PTX3 to a PTX3 receptor, and is applicable to a PTX3 detection kit and detection method therefor, and a pharmaceutical composition for inhibiting or delaying diseases or symptoms associated with the binding of PTX3 to a PTX3 receptor and uses thereof. [Selected Figure] Figure 2

Description

[Technical field]

[0001] The present invention relates to an antibody and uses thereof, and in particular to a monoclonal antibody or an antigen-binding fragment thereof that specifically inhibits or delays binding between a specific sequence at the C-terminus of PTX3 and a PTX3 receptor, as well as a detection reagent using the same, and a pharmaceutical composition that specifically inhibits or delays a disease or symptom associated with the binding between PTX3 and a PTX3 receptor, and uses thereof. [Background technology]

[0002] Currently, it is known that cancer cells stimulate the microenvironment surrounding the tumor to produce various inflammatory factors, white blood cells, vascular hyperproliferation, proteases, etc., and that chronic inflammatory responses in cancer are also related to the growth, metastasis, and invasion of cancer cells. However, there are still many unknowns regarding the causes of its formation and detailed mechanisms.

[0003] In addition to being related to inflammatory responses, other studies have also pointed out that the tumor microenvironment is closely related to tumor metastasis and chemotherapy resistance. The tumor microenvironment is composed of a variety of stromal cells and other different types of cells, and not only protects tumors but also allows tumor cells to escape and resist immune cells, thereby resulting in drug resistance of tumor cells.

[0004] When fibroblasts and macrophages in the stromal tissue surrounding the tumor are activated by CEBPD, they induce the production of the secreted factor pentraxin-related protein 3 (PTX3). PTX3 has angiogenesis-promoting activity and can improve the migration and tissue invasion (also called invasiveness) ability of nasopharyngeal carcinoma cells. Previous studies have also demonstrated that activation of CEBPD in cells in the tissue surrounding the cancer can promote cancer metastasis and further promote the production of drug-resistant cancer cells during chemotherapy, which grow faster and metastasize more easily.

[0005] Currently, small molecule anticancer drugs such as cis-diamminedichloroplatinum (II; CDDP; trade name: Cisplatin), paclitaxel (trade name: Taxol), and 5-fluorouracil (5-FU) are commercially available. However, recent research has found that the above small molecule anticancer drugs can activate the expression of CEBPD not only in cancer cells, but also in macrophages and fibroblasts, which in turn causes drug resistance in cancer cells and rapid metastasis, resulting in poor cancer treatment effects.

[0006] The diseases or conditions associated with the binding of PTX3 to the PTX3 receptor also relate to fibrotic diseases and / or fibrotic conditions, in addition to the above-mentioned cancers.

[0007] Therefore, in order to overcome problems such as poor therapeutic effects of existing drugs against cancer and fibrosis, it is urgent to develop an antibody that specifically binds to PTX3 to detect the content of PTX3 in biological samples. Summary of the Invention

[0008] Therefore, one embodiment of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to a specific sequence at the C-terminus of one or more pentraxin-related proteins (PTX3).

[0009] Another embodiment of the present invention is to provide a monoclonal antibody or antigen-binding fragment thereof that comprises a heavy chain variable region sequence and a light chain variable region sequence of a particular sequence.

[0010] Another embodiment of the present invention is to provide a detection set for PTX3 comprising the above-mentioned monoclonal antibody or an antigen-binding fragment thereof.

[0011] A further embodiment of the present invention is to provide an in vitro method for detecting PTX3 by using the above set.

[0012] Another embodiment of the present invention provides a pharmaceutical composition comprising an effective dose of a monoclonal antibody or an antigen-binding fragment thereof and a pharma- ceutical acceptable carrier, and containing the monoclonal antibody or the antigen-binding fragment thereof as an active ingredient.

[0013] 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 delays the binding of PTX3 to a PTX3 receptor, wherein the monoclonal antibody or the antigen-binding fragment thereof is an active ingredient and has an effective dosage for inhibiting or delaying a disease or symptom associated with the binding of PTX3 to a PTX3 receptor.

[0014] A further embodiment of the present invention is to provide a method for inhibiting or slowing the activity of tumor cells in vitro, comprising administering to tumor cells an effective dosage of the above-described pharmaceutical composition to inhibit or slow the activity of tumor cells.

[0015] Another embodiment of the present invention provides a method for inhibiting or delaying a fibrotic disease and / or a fibrotic condition in vitro comprising administering an effective dose of the above-described pharmaceutical composition to an organ affected by the fibrotic disease and / or the fibrotic condition to inhibit or delay the fibrotic disease and / or the fibrotic condition in the organ.

[0016] According to the above embodiment of the present invention, there is provided a monoclonal antibody or antigen-binding fragment thereof, in one example, the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to a non-denatured amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:11.

[0017] In one embodiment, the non-modified amino acid sequence may include, but is not limited to, any of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:5 and SEQ ID NO:11, or any combination thereof. In another embodiment, the non-modified amino acid sequence may include, but is not limited to, any of the amino acid sequences set forth in SEQ ID NO:2 to SEQ ID NO:4 and SEQ ID NO:11, or any combination thereof.

[0018] According to another embodiment of the invention, there is provided a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence and a light chain variable region sequence, where the heavy chain variable region sequence may be, for example, an amino acid sequence set forth in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and / or SEQ ID NO:21, and the light chain variable region sequence may be, for example, an amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and / or SEQ ID NO:25.

[0019] In some embodiments, the heavy chain variable region sequence of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may be, for example, an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and / or SEQ ID NO:29, and the light chain variable region sequence may be, for example, an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and / or SEQ ID NO:33.

[0020] In other examples, the heavy chain variable region sequence of the monoclonal antibody or antigen-binding fragment thereof may be, for example, the amino acid sequence set forth in SEQ ID NOs:34 or 35, and the light chain variable region sequence may be, for example, the amino acid sequence set forth in SEQ ID NOs:36 or 37.

[0021] In other examples, the heavy chain variable region sequence of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may be, for example, the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NOs:38 or 39, and the light chain variable region sequence may be, for example, the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NOs:40 or 41.

[0022] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof. In one example, the monoclonal antibody or antigen-binding fragment thereof is a mouse-derived antibody, a human / mouse chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0023] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof may be, for example, a single-chain variable region fragment (scFv), a single-chain variable region fragment dimer (scFv)2, a single-chain variable region fragment trimer (scFv)3, a variable region fragment (Fv), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a nanobody, or any combination thereof.

[0024] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation, conjugation, glycosylation, tag attachment, or any combination thereof.

[0025] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is an antibody-drug conjugate (ADC) or antigen-binding fragment thereof.

[0026] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody (BsAb) or antigen-binding fragment thereof.

[0027] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or antigen-binding fragment thereof.

[0028] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is of the IgG, IgM, IgA, IgD or IgE type, in another embodiment, the monoclonal antibody or antigen-binding fragment thereof is of the IgG type and has an IgG1, IgG2, IgG3 or IgG4 isotype.

[0029] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof belongs to the class of inactive or antagonistic antibodies.

[0030] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or delays the recognition of the binding of the PTX3 receptor to a specific sequence at the C-terminus of one or more types of PTX3. In some examples, the monoclonal antibody or antigen-binding fragment thereof inhibits or delays the activity of one or more types of PTX3. In another example, the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or delays the interaction of the PTX3 receptor with one or more types of PTX3, signal transduction by PTX3, or any combination thereof.

[0031] According to another embodiment of the present invention, there is provided a detection set for PTX3 comprising any one of the above monoclonal antibodies or antigen-binding fragments thereof, wherein the monoclonal antibody or antigen-binding fragment thereof can specifically bind to a non-denatured amino acid sequence, and the non-denatured amino acid sequence may include, but is not limited to, any of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:11.

[0032] According to yet another embodiment of the present invention, there is provided an in vitro PTX3 detection method for detecting PTX3 using the above-mentioned PTX3 detection set, wherein the analytical sensitivity of the monoclonal antibody or the antigen-binding fragment thereof included in the PTX3 detection set may be, for example, 0.0016 pM or more.

[0033] According to another embodiment of the present invention, there is provided a pharmaceutical composition comprising an effective dose of a monoclonal antibody or an antigen-binding fragment thereof and a pharma- ceutical acceptable carrier, and containing the monoclonal antibody or the antigen-binding fragment thereof as an active ingredient.

[0034] According to one embodiment of the present invention, the pharmaceutical composition may further comprise an active drug ingredient.

[0035] According to another embodiment of the present invention, there is provided a use of a monoclonal antibody or an antigen-binding fragment thereof for preparing a pharmaceutical composition for specifically inhibiting or delaying binding of pentraxin-related protein (PTX3) to a PTX3 receptor. In one embodiment, the monoclonal antibody or an antigen-binding fragment thereof is an active ingredient and has an effective dosage for inhibiting or delaying a disease or condition associated with the binding of PTX3 to a PTX3 receptor.

[0036] According to one embodiment of the present invention, the pharmaceutical composition is used to inhibit or delay a disease or condition associated with the binding of PTX3 to a PTX3 receptor, where the disease or condition may include epithelial cell carcinoma, adenocarcinoma, glioblastoma multiforme (GBM) and fibrosis. In some examples, the epithelial cell carcinoma may include lung cancer, breast cancer and nasopharyngeal carcinoma (NPC). The adenocarcinoma may be, for example, colon cancer.

[0037] According to one embodiment of the present invention, organs affected by the above fibrotic diseases or conditions may include, but are not limited to, lungs, liver, kidneys and skin.

[0038] According to one embodiment of the present invention, the pharmaceutical composition may be administered by subcutaneous (sc) injection, intramuscular injection, intravenous injection, intraperitoneal (ip) injection, orthotopic injection, oral administration or oronasal inhalation.

[0039] According to another embodiment of the present invention, there is provided a method for inhibiting or slowing the activity of tumor cells in vitro, comprising the step of administering to the tumor cells an effective dose of the pharmaceutical composition described above, thereby inhibiting or slowing the activity of the tumor cells.

[0040] According to another embodiment of the present invention, there is provided a method for inhibiting or slowing the activity of tumor cells in vitro, comprising the step of administering to the tumor cells an effective dose of the pharmaceutical composition described above, thereby inhibiting or slowing the activity of the tumor cells.

[0041] According to another embodiment of the present invention, there is provided a method for inhibiting or delaying a fibrotic disease and / or a fibrotic condition in vitro, comprising the step of administering to an organ affected by a fibrotic disease and / or a fibrotic condition an effective dose of the pharmaceutical composition described above, thereby inhibiting or delaying the fibrotic disease and / or a fibrotic condition in said organ.

[0042] The application of the monoclonal antibody or its antigen-binding fragment according to the present invention specifically inhibits or delays the binding of PTX3 to a PTX3 receptor using a specific PTX3 monoclonal antibody or its antigen-binding fragment, and can be applied not only to an in vitro PTX3 detection set and an in vitro method for detecting the PTX3 content, but also to a pharmaceutical composition and its use that inhibits or delays diseases or symptoms associated with the recognition of PTX3 receptor by PTX3. [Brief description of the drawings]

[0043] [Figure 1] FIG. 2 is a curve diagram showing the affinity of a PTX3 monoclonal antibody according to one embodiment of the present invention to a PTX3 recombinant protein. [Diagram 2] FIG. 11 is a curve diagram showing the affinity of a PTX3 monoclonal antibody according to another embodiment of the present invention to a PTX3 recombinant protein. [Diagram 3] FIG. 2 shows epitope mapping of binding between a PTX3 monoclonal antibody according to one embodiment of the present invention and different fragments of the PTX3 recombinant protein. [Figure 4] FIG. 2 shows epitope mapping of binding between a PTX3 monoclonal antibody according to one embodiment of the present invention and different fragments of the PTX3 recombinant protein. [Diagram 5] FIG. 1 shows the results of binding between a PTX3 monoclonal antibody according to an embodiment of the present invention or a commercially available antibody and a heterologous PTX3 recombinant protein. [Figure 6] FIG. 2 is a diagram showing competitive inhibition of the binding between PTX3 recombinant protein and PTX3 receptor by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 7A] FIG. 13 is a diagram showing the results of the number of migratory cells of the breast cancer cell line MDA-MB231 suppressed by the PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 7B] FIG. 13 is a diagram showing the results of the number of invasive cells of breast cancer cell line MDA-MB231 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 7C] FIG. 13 is a diagram showing the results of the number of cell aggregates of breast cancer cell line MDA-MB231 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 8A] FIG. 1 is a diagram showing the results of the number of migratory cells of the lung cancer cell line A549 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 8B] FIG. 1 is a diagram showing the results of the number of invasive cells of the lung cancer cell line A549 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 8C]FIG. 1 is a diagram showing the results of the number of cell aggregates of the lung cancer cell line A549 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 9A] FIG. 13 is a diagram showing the results of the number of migratory cells of the nasopharyngeal carcinoma cell line HONE1 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 9B] FIG. 13 is a diagram showing the results of the number of invasive cells of the nasopharyngeal carcinoma cell line HONE1 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 9C] FIG. 13 is a diagram showing the results of the number of cell aggregates of the nasopharyngeal carcinoma cell line HONE1 suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 10A] FIG. 13 is a diagram showing the results of the number of migratory cells of the glioblastoma cell line U87MG inhibited by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 10B] FIG. 13 is a diagram showing the results of the number of invasive cells of the glioblastoma cell line U87MG suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 10C] FIG. 13 is a diagram showing the results of the number of cell clusters of the glioblastoma cell line U87MG suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 11A] FIG. 1 shows the results of the number of migratory cells of breast cancer cell line MDA-MB231 inhibited by an example of the PTX3 monoclonal antibody of the present invention or a commercially available antibody. [Figure 11B] FIG. 13 is a diagram showing the results of the number of invasive cells of breast cancer cell line MDA-MB231 inhibited by an example of the PTX3 monoclonal antibody of the present invention or a commercially available antibody. [Figure 12A] FIG. 1 shows the results of the number of migratory cells of the lung cancer cell line A549 inhibited by a PTX3 monoclonal antibody according to an embodiment of the present invention or a commercially available antibody. [Figure 12B] FIG. 1 shows the results of the number of invasive cells of the lung cancer cell line A549 inhibited by an example of the PTX3 monoclonal antibody of the present invention or a commercially available antibody. [Figure 13A]FIG. 1 shows the results of the number of migratory cells of the nasopharyngeal carcinoma cell line HONE1 inhibited by the PTX3 monoclonal antibody according to one embodiment of the present invention or a commercially available antibody. [Figure 13B] FIG. 13 is a diagram showing the results of the number of invasive cells of the nasopharyngeal carcinoma cell line HONE1 inhibited by an example of the PTX3 monoclonal antibody of the present invention or a commercially available antibody. [Figure 14A] FIG. 13 is a diagram showing the results of the number of cell aggregates of breast cancer cell line MDA-MB231 inhibited by an example of the PTX3 monoclonal antibody of the present invention or a commercially available antibody. [Figure 14B] FIG. 1 shows the results of the number of cell aggregates of the lung cancer cell line A549 inhibited by a PTX3 monoclonal antibody according to an embodiment of the present invention or a commercially available antibody. [Figure 14C] FIG. 1 shows the results of the number of cell aggregates of the nasopharyngeal carcinoma cell line HONE1 suppressed by a PTX3 monoclonal antibody according to an embodiment of the present invention or a commercially available antibody. [Figure 15A] FIG. 13 is a diagram showing the results of tumor volume of orthotopic xenografted breast cancer cells MDA-MB231 in mice suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody. [Figure 15B] FIG. 13 is a diagram showing the results of tumor metastasis of orthotopic xenografted breast cancer cells MDA-MB231 in mice, which was inhibited by a PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody. [Figure 16A] FIG. 13 is a diagram showing the results of tumor volume of orthotopic 4T1 breast cancer cells in mice suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention or an isotype control antibody. [Figure 16B] FIG. 1 shows the results of tumor metastasis of orthotopic 4T1 breast cancer cells in mice suppressed by a PTX3 monoclonal antibody according to one embodiment of the present invention or an isotype control antibody. [Figure 17A] 1 is an image showing the tumor volume of orthotopic xenografted breast cancer cells 4T1 in mice suppressed when a PTX3 monoclonal antibody according to an embodiment of the present invention is used in combination with or without paclitaxel. [Figure 17B] 1 is a line graph showing the change in tumor volume of orthotopic xenografted breast cancer cells 4T1 in mice suppressed when a PTX3 monoclonal antibody according to an embodiment of the present invention is used in combination with or without paclitaxel. [Figure 17C] 1 is a line graph showing the change in tumor volume of orthotopic xenografted breast cancer cells 4T1 in mice suppressed when a PTX3 monoclonal antibody according to an embodiment of the present invention is used in combination with or without paclitaxel. [Figure 18A] FIG. 1 is a schematic diagram showing an experimental flow for evaluating mouse orthotopic breast cancer cell 4T1 when a PTX3 monoclonal antibody according to one embodiment of the present invention is used in combination with or without paclitaxel. [Figure 18B] 13 is an image showing the tumor volume and metastasis of orthotopic mouse breast cancer cell 4T1 when a PTX3 monoclonal antibody according to another embodiment of the present invention is used in combination with or without paclitaxel. [Figure 18C] 13 is a line graph showing the tumor volume of mouse orthotopic breast cancer cell 4T1 when a PTX3 monoclonal antibody according to another embodiment of the present invention is used in combination with or without paclitaxel. [Figure 18D] FIG. 11 is a graph showing the survival rate of mice with orthotopic transplantation of breast cancer cells 4T1 in mice when a PTX3 monoclonal antibody according to another embodiment of the present invention is used in combination with or without paclitaxel. [Figure 19A] FIG. 1 is a schematic diagram showing an experimental flow of colon cancer cell line MC38 transplanted into mice and inhibited by a PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody. [Figure 19B] 1 is a line graph showing the tumor volume of colon cancer cell line MC38 transplanted into mice, which was inhibited by an example of the PTX3 monoclonal antibody of the present invention or a control antibody. [Figure 20A]1 is a line graph showing the tumor volume of human glioblastoma cell line U87MG transplanted into mice, which was inhibited by an example of the PTX3 monoclonal antibody of the present invention or a control antibody. [Figure 20B] FIG. 13 is a graph showing the survival rate of mice in which human glioblastoma cell line U87MG was transplanted into mice and inhibited by a PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody. [Figure 21A] FIG. 1 is a schematic diagram showing an experimental flow for evaluating the ameliorative effect of a PTX3 monoclonal antibody according to one embodiment of the present invention on acute hepatic fibrosis in mice. [Figure 21B] FIG. 2 is a diagram showing a tissue section of the left lobe of the liver stained with hematoxylin and eosin (H&E) in a mouse with acute hepatic fibrosis caused by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 21C] FIG. 2 is a graph showing the proportion of necrotic areas in the liver of mice with acute hepatic fibrosis treated with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 21D] FIG. 2 is a graph showing liver weight / body weight ratio in mice with acute hepatic fibrosis induced by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 22A] FIG. 1 is a schematic diagram showing an experimental flow for evaluating the ameliorative effect of a PTX3 monoclonal antibody according to one embodiment of the present invention on chronic hepatic fibrosis in mice. [Figure 22B] FIG. 2 is a diagram showing a tissue section of the left lobe of the liver stained with Picro-Sirius Red in a mouse with chronic liver fibrosis caused by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 22C] FIG. 2 is a graph showing the proportion of hepatic fibrosis areas in mice with chronic hepatic fibrosis treated with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 22D] FIG. 2 is a graph showing liver weight / body weight ratio in mice with chronic hepatic fibrosis induced by a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 23A]1 shows the results of Western blotting analysis showing the expression of fibrosis-related proteins in renal fibroblasts by the PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 23B] 1 is a cell staining image of a renal fibroblast cell nodule using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 23C] FIG. 2 is a histogram showing the results of absorbance measurement of renal fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 24A] FIG. 2 is a schematic diagram showing an experimental flow of renal fibrosis in UUO mice using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 24B] 1 is a stained image of a kidney tissue section of a UUO mouse using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 24C] FIG. 2 is a schematic diagram showing an experimental flow of renal fibrosis in UUO mice using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 24D] 1 is a stained image of a kidney tissue section of a UUO mouse using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 25A] 1 shows the results of Western blotting analysis showing the expression of fibrosis-related proteins in pulmonary fibroblasts by the PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 25B] FIG. 1 is a histogram of the number of transitional cells of pulmonary fibroblasts treated with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 25C] 1 is a cell staining image of a pulmonary fibroblast cell nodule using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 25D] FIG. 2 is a histogram showing the results of absorbance measurement of pulmonary fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 26A] FIG. 26A is a schematic diagram showing an experimental flow of BLM-induced pulmonary fibrosis in mice using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 26B]FIG. 2 is a curve diagram showing changes in body weight in mice with BLM-induced pulmonary fibrosis treated with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 26C] 1 shows images of the appearance and tissue sections of the lungs of a mouse with pulmonary fibrosis induced by BLM using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 26D] 1 shows images of the appearance and tissue sections of the lungs of a mouse with pulmonary fibrosis induced by BLM using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 27A] FIG. 1 is a schematic diagram showing an experimental flow of BLM-induced pulmonary fibrosis in mice using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 27B] FIG. 2 is a curve diagram showing changes in body weight in mice with BLM-induced pulmonary fibrosis treated with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 27C] 1 shows images of the appearance and tissue sections of the lungs of a mouse with pulmonary fibrosis induced by BLM using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 27D] 1 shows images of the appearance and tissue sections of the lungs of a mouse with pulmonary fibrosis induced by BLM using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 28A] 1 shows the results of Western blotting analysis showing the expression of fibrosis-related proteins in fetal fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 28B] 1 is a cell staining image showing cell nodules of embryonic fibroblasts stained with a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 28C] FIG. 2 is a histogram showing the results of absorbance measurement of embryonic fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention. [Figure 29A] 1 is a Western blotting analysis result showing the expression of fibrosis-related proteins in liver fibroblasts treated with different PTX3 monoclonal antibodies according to an embodiment of the present invention. [Figure 29B]1 is a Western blotting analysis result showing the expression of fibrosis-related proteins in liver fibroblasts treated with different PTX3 monoclonal antibodies according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent, the following detailed description will be given with reference to the accompanying drawings.

[0045] As described above, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically inhibits the binding of pentraxin-related protein (PTX3) to a PTX3 receptor by the monoclonal antibody or an antigen-binding fragment thereof, and use of the same. The present invention is applicable to a detection set and a detection method for PTX3, and a pharmaceutical composition for suppressing or delaying a disease or symptom associated with the binding of PTX3 to a PTX3 receptor, and use of the same.

[0046] The monoclonal antibody or antigen-binding fragment thereof referred to in the present invention may contain specific sequences of heavy chain variable region sequences and light chain variable region sequences in order to specifically inhibit or delay the binding of PTX3 to a specific sequence at the C-terminus of the PTX3 receptor.

[0047] In other words, in one embodiment, the 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, and examples thereof include non-modified amino acid sequences described in SEQ ID NO:1 to SEQ ID NO:17, but it is preferable that the monoclonal antibody or antigen-binding fragment thereof specifically binds to non-modified amino acid sequences described in SEQ ID NO:1 to SEQ ID NO:11, more preferably non-modified amino acid sequences described in SEQ ID NO:1 to SEQ ID NO:5 and 11, and even more preferably non-modified amino acid sequences described in SEQ ID NO:2 to SEQ ID NO:4. In the above embodiment, the non-modified amino acid sequence described in SEQ ID NO:1 to SEQ ID NO:11 corresponds to the amino acids 200 to 236 of the non-modified amino acid sequence of human PTX3 recombinant protein. In another example, the non-denatured amino acid sequences described in SEQ ID NO:1 to SEQ ID NO:5 and SEQ ID NO:11 correspond to the amino acids 200 to 220 of the non-denatured amino acid sequence of human PTX3 recombinant protein. In another example, the non-denatured amino acid sequences described in SEQ ID NO:2 to SEQ ID NO:4 and SEQ ID NO:11 correspond to the amino acids 203 to 217 of the non-denatured amino acid sequence of human PTX3 recombinant protein.

[0048] In one embodiment, the 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, for example, the amino acid sequence set forth in SEQ ID NO:18. The amino acid sequence of CDR2 of the heavy chain variable region may be RIDPANX1X2TKYDPX3FQG, wherein X1 represents G or D, X2 represents D or N, and X3 represents K or M, and the specific amino acid sequences are set forth in SEQ ID NOs:19 or 20. The sequence of CDR3 of the heavy chain variable region has, for example, the amino acid sequence set forth in SEQ ID NO:21. The sequence of CDR1 of the light chain variable region has, for example, the amino acid sequence set forth in SEQ ID NO:22. The sequence of CDR2 of the light chain variable region has, for example, the amino acid sequence set forth in SEQ ID NO:23. The amino acid sequence of CDR3 of the light chain variable region may be HQX4QRSPLT. Here, X4 represents F or Y, and the specific amino acid sequence is set forth in SEQ ID NOs:24 or 25.

[0049] In other examples, the heavy chain variable region sequence of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may have an amino acid sequence encoded by a nucleic acid sequence set forth in, for example, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and / or SEQ ID NO:29, while the light chain variable region sequence may have an amino acid sequence encoded by a nucleic acid sequence set forth in, for example, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and / or SEQ ID NO:33.

[0050] In other examples, the heavy chain variable region sequence of the above monoclonal antibodies or antigen-binding fragments thereof may have, for example, the amino acid sequence set forth in SEQ ID NOs:34 or 35, while the light chain variable region sequence may have the amino acid sequence set forth in SEQ ID NOs:36 or 37.

[0051] In other examples, the heavy chain variable region sequence of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof may have, for example, an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NOs:38 or 39, while the light chain variable region sequence may have, for example, an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NOs:40 or 41.

[0052] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is not limited in type and may be, for example, a chimeric antibody or antigen-binding fragment thereof. In other examples, the monoclonal antibody or antigen-binding fragment thereof may be, for example, a mouse-derived antibody, a human / mouse chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0053] In some embodiments, the structure of the monoclonal antibody or antigen-binding fragment thereof is not limited, and may be a complete antibody structure or a simplified antibody structure, taking into account the stability of the complementarity-determining region (CDR) structure, such as a single-chain variable fragment (scFv), a single-chain variable fragment dimer (scFv2), a single-chain variable fragment trimer (scFv3), a variable fragment (Fv), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a nanobody (also called a nanobody, a single domain antibody (sdAb), or a heavy-chain antibody), or any combination thereof, thereby simplifying the process of recombinant antibodies. The monoclonal antibody or antigen-binding fragment thereof may be prepared by tumor cell fusion or recombinant gene expression, which are well known to those skilled in the art and will not be described in detail here.

[0054] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof may be further modified by conjugation, binding, glycosylation, tag attachment, or any combination thereof according to actual needs. For example, the monoclonal antibody or antigen-binding fragment thereof may further form an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof with a drug. In another example, the monoclonal antibody or antigen-binding fragment thereof may be bound to a specific signal peptide to enter a specific site, such as the blood-brain barrier (BBB).

[0055] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof may be, for example, a bispecific monoclonal antibody (BsAb), a trifunctional monoclonal antibody, or an antigen-binding fragment thereof.

[0056] According to one embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation, binding, glycosylation, tag attachment, or any combination thereof.

[0057] According to one embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof is an antibody-drug conjugate (ADC) or antigen-binding fragment thereof.

[0058] According to one embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody (BsAb) or antigen-binding fragment thereof.

[0059] According to one embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or antigen-binding fragment thereof.

[0060] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof can belong to the IgG type, IgM type, IgA type, IgD type, or IgE type. In one specific embodiment, the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG type and has an IgG1, IgG2, IgG3, or IgG4 isotype. In one example, the monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype, for example, an IgG1k isotype. In some specific examples, the monoclonal antibody or antigen-binding fragment thereof belongs to an inactive antibody or an antagonist antibody. In some specific examples, the monoclonal antibody or antigen-binding fragment thereof can specifically inhibit or delay the activity of one or more types of PTX3. In other specific examples, the monoclonal antibody or antigen-binding fragment thereof can specifically inhibit or delay the interaction of a PTX3 receptor with one or more types of PTX3, signal transduction by PTX3, or any combination thereof.

[0061] In application, the above-mentioned monoclonal antibody or its antigen-binding fragment can be applied to a detection set and method for PTX3, and improves the analytical sensitivity of detecting PTX3 in a biological sample by specifically binding to the non-denatured amino acid sequence described in SEQ ID NO:1 to SEQ ID NO:11. The form of the biological sample referred to in the present invention may include, but is not limited to, cells, tissues, blood, urine, lymph, tissue fluid, body fluid, etc. The above-mentioned detection set for PTX3 can be applied to various existing detection components / devices such as a flow cytometer, an enzyme-linked immunosorbent assay (ELISA) detection reagent set, a biochip, etc., or can be applied to existing detection methods such as a direct ELISA, an indirect ELISA, a sandwich ELISA, a competitive ELISA, an immunohistochemical staining, and a Western blotting analysis. In one specific example, the analytical sensitivity of the monoclonal antibody or antigen-binding fragment thereof, which can also be referred to as the lower limit of detection (LLOD), is generally 0.0016 pM or more.

[0062] The monoclonal antibody or antigen-binding fragment thereof may be added to a pharmaceutical composition as an active ingredient. In one embodiment, the pharmaceutical composition may optionally include a pharma- ceutically acceptable carrier. In the present invention, the term "pharma-ceutically acceptable carrier" refers to a carrier, diluent, adjuvant and / or vehicle for delivering an active ingredient to an individual, which is not an active ingredient itself, or an additive added to the above composition to improve the processing or storage properties of the composition, or an excipient or any substance for permitting or assisting the formation of a dosage unit of the composition suitable for a pharmaceutical composition and easy administration. The pharma-ceutically acceptable carrier must not destroy the pharmacological activity of the active ingredient and must be non-toxic when delivering a sufficient therapeutic dose of the active ingredient.

[0063] The pharma- ceutically acceptable carriers applied are well known to those of ordinary skill in the art of preparing pharmaceutical compositions and include, but are not limited to, buffers, diluents, disintegrants, adhesives, sticking agents, wetting agents, polymers, lubricants, slip agents, substances added to mask or remove unpleasant tastes and odors, dyes, fragrances and substances added to improve the appearance of the composition. Specific examples of the pharma- ceutically acceptable carrier may include, but are not limited to, citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose-based substances (e.g., cellulose esters of alkanoic acids, cellulose alkyl esters), low melting point waxes, cacao butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethylsulfoxide (DMSO), sodium chloride or other salts, liposomes, glycerin or powder, polymers (e.g., polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, etc.), and other pharma- ceutically acceptable substances.

[0064] The suppression or delay of the disease or symptom associated with the binding of PTX3 to the PTX3 receptor in the present invention may include, but is not limited to, epithelial cell carcinoma, glioblastoma multiforme (GBM), adenocarcinoma, and fibrosis. Examples of the epithelial cell carcinoma include lung cancer, breast cancer, and nasopharyngeal carcinoma. Examples of the adenocarcinoma include colon cancer. Organs affected by the fibrotic disease or symptom may include, but are not limited to, the lung, liver (e.g., acute hepatic fibrosis, chronic hepatic fibrosis), kidney, skin, etc.

[0065] In application, the monoclonal antibody or antigen-binding fragment thereof can be used to suppress or delay a disease or symptom associated with the binding of PTX3 to a PTX3 receptor by administering an effective dose of the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition to a target cell or a subject. Taking a mouse as an example, the "effective dose" in the present invention refers to administering 2 mg to 10 mg of the PTX3 monoclonal antibody or antigen-binding fragment thereof per kilogram of body weight once a week. In another example, the effective dose of the PTX3 monoclonal antibody or antigen-binding fragment thereof is, for example, preferably 5 mg / kg body weight to 10 mg / kg body weight, more preferably 6 mg / kg body weight to 9 mg / kg body weight. When applied to other subjects, it can be converted to an appropriate effective dose according to the bioequivalence. Note that if the effective dose of the PTX3 monoclonal antibody is less than 2 mg / kg body weight, it cannot effectively reduce, suppress, or delay the binding of PTX3 to a PTX3 receptor within a given time period.

[0066] With respect to tumors, the above-mentioned monoclonal antibodies or antigen-binding fragments thereof can inhibit or delay tumor cell activities such as proliferation, cancer stemness, migration, invasion, metastasis, tumor volume, or drug resistance.

[0067] Fibrosis, as used herein, is defined as the formation of excess fibrous tissue, e.g., fibrous connective tissue, in organs or tissues, or the formation of fibrous tissue during inflammation, repair, or reaction. The fibrous tissue may be reactive, benign, or pathological. Physiologically, fibrosis is used to describe pathological conditions of excess deposition of fibrous tissue and the process of deposition of connective tissue during wound healing. Examples include scars formed from wounds, fibromas formed from a single type of cell, and pathological conditions of excess deposition of fibrous tissue. For organs affected by fibrotic diseases or conditions, the monoclonal antibodies or antigen-binding fragments thereof can inhibit or delay diseases or conditions associated with fibrosis, e.g., acute liver fibrosis, chronic liver fibrosis.

[0068] The above pharmaceutical composition can be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, orthotopic injection, oral administration, oral nasal inhalation, etc. to specifically suppress endogenous PTX3 activity and further suppress or delay cancer cell activity. Specifically, in vitro cell experiments have demonstrated that the monoclonal antibody or its antigen-binding fragment of the present invention and the pharmaceutical composition comprising the same can be used for a certain period of time, for example, 4 to 11 weeks, to suppress or delay cancer cell activity.

[0069] The following examples are provided to illustrate the application of the present invention, but are not intended to limit the scope of the present invention. Those skilled in the art can make various changes and modifications without departing from the concept and scope of the present invention.

[0070] Example 1. Preparation of PTX3 monoclonal antibody In this example, a PTX3 monoclonal antibody that specifically recognizes the C-terminal amino acid sequence of PTX3 recombinant protein was prepared by a conventional tumor fusion method or a recombinant protein expression method.

[0071] Briefly, the PTX3 recombinant protein with the non-denatured amino acid sequence described in SEQ ID NO:13 was used as an immunogen, and was injected intraperitoneally (ip) into the abdominal cavity of Balb / C mice at a dose of 50 μg per mouse, and after 2 weeks, the mice were boosted with 50 μg per mouse once every 2 weeks for a total of 4 times. The activated splenocytes were then fused with bone marrow cancer cells, and then fusion tumor cell lines were produced.

[0072] From the above fusion tumor cell lines, fusion tumor cell lines having high binding affinity to the recombinant protein of the non-denatured amino acid sequence described in SEQ ID NO:11 are screened, and the PTX3 monoclonal antibody produced from the obtained fusion tumor cell line can specifically bind to the non-denatured amino acid sequence described in the sequence identification numbers SEQ ID NO:1 to SEQ ID NO:11.

[0073] The culture supernatant of the fusion tumor cell line obtained above was collected, and the PTX3 monoclonal antibody (Zhenwei Biotechnology Co., Ltd.) was purified using a commercially available column, and then the amino acid sequences of the complementarity-determining regions (CDRs) of the heavy chain variable region and the light chain variable region and the corresponding nucleic acid sequences were analyzed by Weiqiao Biomedical Co., Ltd. The sequence of the heavy chain variable region may have, for example, the amino acid sequence described in SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:21, and the sequence of the light chain variable region may have, for example, the amino acid sequence described in SEQ ID NO:22, SEQ ID NO:23, and / or SEQ ID NO:24. The amino acid sequence of the heavy chain variable region may have, for example, the amino acid sequence described in SEQ ID NO:34 or the amino acid sequence encoded by the nucleic acid sequence described in SEQ ID NO:38. The amino acid sequence of the light chain variable region has, for example, the amino acid sequence set forth in SEQ ID Ns:36 or the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:40.

[0074] Furthermore, when the above-mentioned PTX3 monoclonal antibody was analyzed using a commercially available monoclonal antibody isotyping kit, the isotype of the antibody was confirmed to be IgG1k.

[0075] Example 2. Evaluation of affinity of PTX3 monoclonal antibody In this example, the affinity between the PTX3 recombinant protein and the PTX3 monoclonal antibody of Example 1 was evaluated using a conventional ELISA kit.

[0076] First, 5 μg / mL of PTX3 recombinant protein (e.g., the non-denatured amino acid sequence described in SEQ ID NO: 14) or bovine serum albumin (BSA; as a control group) was applied to each well of a 96-well cell culture plate (product code: 9018, Corning Costar) and reacted overnight at 4°C. Thereafter, a blocking solution (phosphate buffer solution (PBS) containing 3% skim milk powder) was placed in the well and a blocking reaction was performed for 1 hour at room temperature (4°C to 40°C). After removing the blocking solution, each well was wet washed with PBS, and a primary antibody was added and reacted for 1 hour at room temperature (4°C to 40°C). Here, the primary antibody was the PTX3 monoclonal antibody of Example 1 (concentration 2.44 × 10 -4The concentration of the secondary antibody was 1.0 μg / mL to 1.00 μg / mL. After that, the wells were washed with PBS to remove unbound PTX3 monoclonal antibody, and the secondary antibody was added and reacted at room temperature (4°C to 40°C). Then, tetramethyl benzidine (TMB) was added to each well and reacted for a certain period of time, and then 0.1 M sulfuric acid (H2SO4) was added and reacted for 10 minutes to stop the reaction. Here, the secondary antibody was anti-mouse IgG (IgG-HRP) conjugated with horseradish peroxidase (anti-mouse horse peroxidase; HRP). Next, the absorbance at 450 nm was read using a commercially available ELISA reader, and the results are shown in Figure 1. Each value was repeated three times. The reaction time of the secondary antibody was performed according to the manufacturer's manual and is well known to those skilled in the art, so a detailed explanation will be omitted here.

[0077] Referring to Figure 1, there is shown a curve graph showing the affinity of a PTX3 monoclonal antibody according to one embodiment of the present invention for PTX3 recombinant protein, in which the curve marked with the symbol "●" represents the affinity curve of the PTX3 monoclonal antibody for PTX3 recombinant protein, and the curve marked with the symbol "■" represents the affinity curve of BSA for PTX3 recombinant protein.

[0078] As can be seen from the results in Figure 1, 4 6 After high dilution (corresponding to an antibody concentration of 0.244 ng / mL or 0.0016 pM), the PTX3 monoclonal antibody still retains good affinity for the PTX3 recombinant protein.

[0079] The above ELISA kit also demonstrated that the PTX3 monoclonal antibody of one embodiment of the present invention has high affinity for the PTX3 recombinant protein. First, 10 μg / mL of PTX3 recombinant protein (for example, the non-denatured amino acid sequence described in SEQ ID NO:14, dissolved in PBS at pH 7.2) was applied to each well of a 96-well cell culture plate (the product code is the same as above) and reacted overnight at 4° C. Then, a blocking solution (PBS containing 3% bovine serum albumin (BSA)) was placed in the well, and a blocking reaction was performed at room temperature (4° C. to 40° C.) for 1 hour. After removing the blocking solution, each well was wet washed with PBS, and a primary antibody was added and reacted at room temperature (4° C. to 40° C.) for 1 hour. Here, the primary antibody was the PTX3 monoclonal antibody of Example 1 serially diluted (concentration 0.01 ng / mL to 1000 ng / mL). Then, each well was wet washed several times with PBST to remove unbound PTX3 monoclonal antibody in each well, and a secondary antibody was added and reacted at room temperature (4°C to 40°C). Here, the secondary antibody was anti-mouse IgG bound to HRP (IgG-HRP). Then, TMB was added to each well and reacted for a certain period of time, and then 0.1M sulfuric acid (H2SO4) was added and reacted for 10 minutes to stop the reaction. Next, the absorbance at 450 nm was read using a commercially available ELISA reader, and the results are shown in Figure 2. Each value was repeated four times. The reaction time of the secondary antibody described above is well known to those skilled in the art, so a detailed explanation will be omitted here.

[0080] FIG. 2 is a curve showing the affinity of a PTX3 monoclonal antibody according to another embodiment of the present invention to a PTX3 recombinant protein.

[0081] As can be seen from the results in FIG. 2, the dissociation constant (KD) of the PTX3 monoclonal antibody to the PTX3 recombinant protein (i.e., antigen) is 85 pM, and the PTX3 monoclonal antibody has high affinity to the PTX3 recombinant protein, so that it can be applied to the detection set of PTX3.

[0082] In other experiments, the PTX3 monoclonal antibody can specifically bind to amino acids 200 to 359 of the PTX3 recombinant protein (e.g., the non-denatured amino acid sequence set forth in SEQ ID NO:13; not shown) or amino acids 200 to 236 (e.g., the non-denatured amino acid sequence set forth in SEQ ID NO:12; as shown in FIG. 3).

[0083] Example 3. Evaluation of the binding region between PTX3 monoclonal antibody and PTX3 1. Evaluation of epitope mapping region of binding between PTX3 monoclonal antibody of Example 1 and PTX3 In this example, epitope mapping regions of binding between PTX3 monoclonal antibodies and PTX3 were evaluated using a conventional ELISA kit.

[0084] In this example, 200 μg / mL of PTX3 recombinant protein (e.g., non-denatured amino acid sequence described in SEQ ID NOs: 12, 16, 17, dissolved in 0.1 M sodium bicarbonate aqueous solution, pH 8.3) or BSA (as a control group) was applied to the wells of a 96-well cell culture plate and reacted overnight at 4 ° C., but the small binding region of the PTX3 monoclonal antibody to PTX3 was evaluated in the same manner as in Example 1. Next, a blocking solution (PBS containing 1% BSA) was placed in the wells, and a blocking reaction was performed at room temperature (4 ° C. to 40 ° C.) for 1 hour. After removing the blocking solution, each well was wet washed with PBS, and the PTX3 monoclonal antibody of Example 1 (concentration 125 ng / mL) was added and reacted at room temperature (4 ° C. to 40 ° C.) for 2 hours. Then, the wells were washed with PBS to remove unbound PTX3 monoclonal antibody, and a secondary antibody (anti-mouse IgG-HRP, diluted 1:5000) was added and reacted at room temperature (4°C to 40°C) for 1 hour. Then, TMB was added to each well and reacted for a certain period of time, and then 0.1M sulfuric acid was added and reacted for 10 minutes to stop the reaction. The absorbance at 450 nm was read using a commercially available ELISA reader, and the results are shown in Figure 3. Each value was repeated three times.

[0085] 3, which shows epitope mapping of binding between PTX3 monoclonal antibody according to one embodiment of the present invention and different fragments of PTX3 recombinant protein, in which RI37 represents the PTX3 recombinant protein fragment described in SEQ ID NO:12, KT44 represents the PTX3 recombinant protein fragment described in SEQ ID NO:16, GI40 represents the PTX3 recombinant protein fragment described in SEQ ID NO:17, and the symbol "***" indicates statistical significance (p<0.001) compared to the control group (i.e., BSA group).

[0086] As can be seen from the results in FIG. 3, the affinity of the PTX3 monoclonal antibody to the PTX3 recombinant protein fragment set forth in SEQ ID NO:12 is higher than that of the other fragments and is statistically significant.

[0087] 4, which shows epitope mapping of binding between the PTX3 monoclonal antibody according to one embodiment of the present invention and different fragments of PTX3 recombinant protein, in which the horizontal axis represents the PTX3 recombinant protein fragments described in SEQ ID NOs: 1 to 10, respectively.

[0088] As can be seen from the results in Fig. 4, the affinity of the PTX3 monoclonal antibody to the PTX3 recombinant protein fragment described in SEQ ID NOs:1-5 or SEQ ID NOs:2-4 is much higher than that of other fragments. Here, the PTX3 recombinant protein fragment described in SEQ ID NOs:2-4 corresponds to the amino acids 203 to 217 of PTX3, for example, the amino acid sequence described in SEQ ID NO:11, and the epitope mapping region of the binding between the PTX3 monoclonal antibody of Example 1 and PTX3 is within the range of the amino acid sequence described in SEQ ID NOs:2-4 or SEQ ID NO:11.

[0089] 2. Evaluation of differences in epitope mapping regions of binding between the PTX3 monoclonal antibody of Example 1 and commercially available PTX3 monoclonal antibodies and PTX3 In this Example, similarly to Example 3, epitope mapping regions of binding between the PTX3 monoclonal antibody of Example 1 and a commercially available PTX3 monoclonal antibody (product code: ab90806; Abcam plc., UK) and PTX3 were evaluated, and the results are shown in Figure 5. Each value was repeated three times.

[0090] 5 shows epitope mapping of binding between the PTX3 monoclonal antibody according to one embodiment of the present invention and a commercially available PTX3 monoclonal antibody and different fragments of the PTX3 recombinant protein, where PTX3 / FL represents the PTX3 recombinant protein fragment described in SEQ ID NO:15, RI37 represents the PTX3 recombinant protein fragment described in SEQ ID NO:12, KT44 represents the PTX3 recombinant protein fragment described in SEQ ID NO:16, GI40 represents the PTX3 recombinant protein fragment described in SEQ ID NO:17, and the symbol "***" indicates statistical significance (p<0.001) compared to the control group (BSA group).

[0091] As can be seen from the results in FIG. 5, the affinity of both the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) for PTX3 / FL (SEQ ID NO: 15) is high, but the affinity of the PTX3 monoclonal antibody of Example 1 for the PTX3 recombinant protein fragment RI37 (SEQ ID NO: 12) is higher than that of the commercially available PTX3 monoclonal antibody (ab90806) and is statistically significant, indicating that there is a difference between the PTX3 monoclonal antibody and the commercially available PTX3 monoclonal antibody (ab90806) in the epitope mapping region for binding to PTX3.

[0092] Example 4. Evaluation of the effect of PTX3 monoclonal antibodies on the binding of PTX3 to the PTX3 receptor in vitro A PTX3 monoclonal antibody can specifically inhibit or delay the opportunity for PTX3 to bind to the PTX3 receptor by competitively binding to the binding region of the PTX3 receptor or a region adjacent thereto. In this example, CD44 is used as an example of a PTX3 receptor, and the effect of a PTX3 monoclonal antibody to competitively inhibit the binding of PTX3 to the PTX3 receptor was evaluated by a competitive binding test.

[0093] This example demonstrates that the PTX3 monoclonal antibody of Example 1 neutralizes PTX3, preventing it from binding to the binding domain of the PTX3 receptor (eg, CD44) or a domain nearby.

[0094] Briefly, the competitive binding assay used in this example was the same as in Example 1, except that 10 μg / mL of PTX3 receptor (e.g., CD44 N-terminal recombinant protein (the sequence of amino acid residues 1-220 of the N-terminus of CD44, dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing, China)) was applied to the wells of a 96-well cell culture plate and reacted overnight at 4° C. Then, blocking solution (PBS containing 3% nonfat powder) was placed in the wells, and a blocking reaction was performed at room temperature (4° C. to 40° C.) for 1 hour.

[0095] When a competitive binding test was performed, PTX3 recombinant protein bound to HRP (e.g., the non-denatured amino acid sequence described in SEQ ID NO:14, HRP-PTX3, concentration 5 μg / mL) was pre-reacted with different concentrations of the PTX3 monoclonal antibody of Example 1 (concentration 1 μg / mL or 2 μg / mL) at room temperature (4°C to 40°C) for 1 hour to form a pre-reaction product.

[0096] After removing the blocking solution, each well was wet washed with PBS, and the above-mentioned preliminary reaction product was added and reacted at room temperature (4°C to 40°C) for 2 hours. Next, each well was washed with PBS to remove unbound preliminary reaction product, and each well was added with TMB and reacted for a certain period of time. Then, 0.1M sulfuric acid was added and reacted for 10 minutes to stop the reaction, and the absorbance at 450 nm was read using a commercially available ELISA reader, and the results are shown in Figure 6. Each value was repeated four times.

[0097] 6 is a diagram showing competitive inhibition of the binding between PTX3 recombinant protein and PTX3 receptor by PTX3 monoclonal antibody according to one embodiment of the present invention, where the vertical axis represents the competitive inhibition rate (%), the sign "+" or "-" below the horizontal axis represents the presence or absence of addition of a specific component when performing a binding reaction, the first bar on the left side of FIG. 6 represents the control group (i.e., the group of PTX3 recombinant protein without PTX3 monoclonal antibody), and the sign "***" represents statistical significance (p<0.001) compared to the control group.

[0098] As can be seen from the results in Figure 6, the value in the first bar on the left side of Figure 6 without adding PTX3 monoclonal antibody is taken as 0% competitive inhibition rate, and when the PTX3 monoclonal antibody and PTX3 recombinant protein are pre-reacted and then reacted with the CD44 receptor, the obtained competitive inhibition rate (%) shows a dose-dependent relationship with the concentration of the PTX3 monoclonal antibody and is statistically significant, indicating that the PTX3 monoclonal antibody of Example 1 neutralizes PTX3 and prevents it from binding to the CD44 binding region or its neighboring region.

[0099] Example 5. Evaluation of the effect of PTX3 monoclonal antibody on the activity of cancer cells Breast cancer, lung cancer, nasopharyngeal carcinoma, and glioblastoma multiforme (GBM) are malignant tumors, and the cancer cells have activities such as migration, invasion, and cancer stemness. In this example, human breast cancer cell line (MDA-MB231, registration number: BCRC 60425, ATCC HTB-26; triple negative breast cancer cell line, hereinafter simply referred to as "MB231"), human lung cancer cell line A549 (registration number: BCRC 60074; ATCC CCL-185), human nasopharyngeal cancer cell line HONE1 (Int. J. Cancer. 1990 Jan 15; 45 (1): 83-9; Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 9524-9528, December 1989), human GBM cancer cell U87MG (ATCC registration number: HTB-14; BCRC registration number: 60360), etc. were used to evaluate the effect of the PTX3 monoclonal antibody of Example 1 on the activity of cancer cells by the following test.

[0100] 1.Evaluation of the effect of PTX3 monoclonal antibody on cancer cell migration When performing migration experiments, the above cancer cells were added at 1 × 10 5 The cells were seeded at a cell density of 1 cell / well into the upper layer (diameter of the bottom well is 8 μm) of a 24-well Boyden chamber and cultured for 3 hours. Next, the cell culture medium in the upper layer was replaced with a serum-free cell culture medium, and 0.2 μg / mL of PTX3 recombinant protein (e.g., the non-denatured amino acid sequence described in SEQ ID NO: 14), 0.4 μg / mL of PTX3 monoclonal antibody, or 0.4 μg / mL of a control antibody (IgG1k) was added to the serum-free cell culture medium in the lower layer.

[0101] After culturing for 16 hours, the cells on the inside of the upper layer were scraped off with a cotton swab, and the cells that had migrated to the outside bottom of the upper layer were stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). The number of cells that had migrated to the outside bottom of the upper layer was calculated under a fluorescent microscope with a magnification of 200x. The results are shown in Figures 7A, 8A, 9A, and 10A.

[0102] 7A, 8A, 9A and 10A are histograms showing the number of migratory cells in breast cancer cells MB231 (FIG. 7A), lung cancer cells A549 (FIG. 8A), nasopharyngeal carcinoma cells HONE1 (FIG. 9A) and glioblastoma cell line U87MG (FIG. 10A) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention. The data in the above examples were obtained as positive and negative mean standard deviations from the data of three repeated experiments for each time point and each sample, and all values ​​were analyzed by one-way ANOVA. The symbol "**" in the above examples represents data with statistical significance (P<0.01), and the symbol "***" represents data with statistical significance (P<0.001).

[0103] As can be seen from the results in Figures 7A, 8A, 9A and 10A, compared with the control antibody IgG1k, the PTX3 monoclonal antibody can significantly inhibit or delay the number of migratory cells in breast cancer cells MB231, lung cancer cells A549, nasopharyngeal carcinoma cells HONE1 and glioblastoma cell line U87MG, and this difference is statistically significant.

[0104] 2.Evaluation of the effect of PTX3 monoclonal antibody on cancer cell invasion When performing the invasion experiment, the bottom surface of the upper layer (bottom well diameter 8 μm) of a 24-well Boyden chamber was coated with basement membrane matrix (Matrigel, purchased from BD Bioscience) in advance, and the above cancer cells were inoculated at 1 × 10 5The cells were seeded in the upper layer of a 24-well Boyden chamber at a cell density of 10 cells / well and cultured for 3 hours. Then, the upper cell culture medium was replaced with a serum-free cell culture medium, and 0.2 μg / mL of PTX3 recombinant protein (e.g., the non-denatured amino acid sequence shown in SEQ ID NO:4), 0.4 μg / mL of PTX3 monoclonal antibody, or 0.4 μg / mL of a control antibody (IgG1k) was added to the serum-free cell culture medium in the lower layer.

[0105] After culturing for 16 hours, the cells on the inside of the upper layer were scraped off with a cotton swab, and the cells that had migrated to the outside bottom of the upper layer were stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). The number of cells that had migrated to the outside bottom of the upper layer was calculated under a fluorescent microscope with a magnification of 200x. The results are shown in Figures 7B, 8B, 9B, and 10B.

[0106] 7B, 8B, 9B and 10B are histograms showing the number of invasive cells of breast cancer cells MB231 (FIG. 7B), lung cancer cells A549 (FIG. 8B), nasopharyngeal carcinoma cells HONE1 (FIG. 9B) and glioblastoma cell line U87MG (FIG. 10B) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention, where the symbol "**" indicates statistically significant data (P<0.01) and the symbol "***" indicates statistically significant data (P<0.001).

[0107] As can be seen from the results in Figures 7B, 8B, 9B and 10B, compared with the control antibody IgG1k, the PTX3 monoclonal antibody can significantly inhibit or delay the number of invasive cells in breast cancer cells MB231, lung cancer cells A549, nasopharyngeal carcinoma cells HONE1 and glioblastoma cell line U87MG, and this difference is statistically significant.

[0108] 3. Evaluation of the effect of PTX3 monoclonal antibody on cancer cell migration The above cancer cells have cancer stemness, and the addition of PTX3 recombinant protein enables the cancer cells to form cell masses (spheres).

[0109] When a cell mass experiment was performed, the above cancer cells were added to RPMI-1640 cell culture medium containing 10% fetal bovine serum (FBS) [containing 10% fetal bovine serum (FBS), 50-100 μg / mL streptomycin, and 50-100 U / mL penicillin] containing 0.2 μg / mL PTX3 recombinant protein, 0.4 μg / mL PTX3 monoclonal antibody, or 0.4 μg / mL control antibody (IgG1k), and cultured at 37° C. in 5% carbon dioxide with humidity maintained. This is known to those skilled in the art, so detailed explanation will be omitted.

[0110] Then, 5 × 10 cancer cells that had been treated with the different methods were 3 The cells were seeded at a cell density of 10 cells / well in a multi-well plate with ultra-low attachment surface (Corning Inc.) and co-cultured in serum-free cell culture medium DMEM / F12 (Gibco) [containing B27 (Invitrogen), 20 ng / mL epidermal growth factor (EGF; Abcam), and 10 ng / mL basic fibroblast growth factor (bFGF; Peprotech)]. After 2 weeks of culture, the number of cell clusters was observed under an optical microscope, and the results are shown in Figures 7C, 8C, 9C, and 10C.

[0111] 7C, 8C, 9C and 10C are histograms showing the number of cell clusters of breast cancer cells MB231 (FIG. 7C), lung cancer cells A549 (FIG. 8C), nasopharyngeal carcinoma cells HONE1 (FIG. 9C) and glioblastoma cell line U87MG (FIG. 10C) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention, respectively. Here, the symbol "**" represents statistically significant data (P<0.01), and the symbol "***" represents statistically significant data (P<0.001).

[0112] As can be seen from the results in Figures 7C, 8C, 9C and 10C, the PTX3 monoclonal antibody of Example 1 can significantly inhibit or delay the number of cell masses in breast cancer cells MB231, lung cancer cells A549, nasopharyngeal cancer cells HONE1 and glioblastoma cell line U87MG compared with the control antibody IgG1k, and this difference is statistically significant.

[0113] 4. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 and a commercially available PTX3 monoclonal antibody on the activity of cancer cells In this example, similar to Example 2, the inhibitory effects of the PTX3 monoclonal antibody of Example 1 and a commercially available PTX3 monoclonal antibody (product code: ab90806; abcam plc., UK) on cancer cell activity were evaluated, and the results are shown in Figures 11A to 14C.

[0114] 11A, 12A and 13A are histograms showing the number of migration cells of breast cancer cells MB231 (FIG. 11A), lung cancer cells A549 (FIG. 12A) and nasopharyngeal cancer cells HONE1 (FIG. 13A) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention and a commercially available PTX3 monoclonal antibody, respectively. Here, the symbol "*" represents statistically significant data (P<0.05), the symbol "**" represents statistically significant data (P<0.01), and the symbol "***" represents statistically significant data (P<0.001).

[0115] As can be seen from the results in Figures 11A, 12A, and 13A, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or delay the number of migrating cells in breast cancer cells MB231, lung cancer cells A549, and nasopharyngeal cancer cells HONE1 compared to the control group antibody IgG1k, but the inhibitory or delaying effect of the PTX3 monoclonal antibody of Example 1 on the number of migrating cancer cells is significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806) and is statistically significant.

[0116] 11B, 12B and 13B are histograms showing the number of invasive cells of breast cancer cells MB231 (FIG. 11B), lung cancer cells A549 (FIG. 12B) and nasopharyngeal cancer cells HONE1 (FIG. 13B) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention and a commercially available PTX3 monoclonal antibody, respectively. Here, the symbol "*" represents statistically significant data (P<0.05), the symbol "**" represents statistically significant data (P<0.01), and the symbol "***" represents statistically significant data (P<0.001).

[0117] As can be seen from the results in Figures 11B, 12B, and 13B, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or delay the number of invasive cells in breast cancer cells MB231, lung cancer cells A549, and nasopharyngeal cancer cells HONE1 compared to the control group antibody IgG1k, but the inhibitory or delaying effect of the PTX3 monoclonal antibody of Example 1 on the number of invasive cancer cells is significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806) and is statistically significant.

[0118] 14A, 14B and 14C are histograms showing the number of cell masses of breast cancer cells MB231 (FIG. 14A), lung cancer cells A549 (FIG. 14B) and nasopharyngeal cancer cells HONE1 (FIG. 14C) inhibited by the PTX3 monoclonal antibody of Example 1 of the present invention and a commercially available PTX3 monoclonal antibody, respectively. Here, the symbol "**" represents statistically significant data (P<0.01), and the symbol "***" represents statistically significant data (P<0.001).

[0119] As can be seen from the results in Figures 14A, 14B, and 14C, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) can significantly inhibit or delay the number of cell clumps in breast cancer cells MB231, lung cancer cells A549, and nasopharyngeal cancer cells HONE1 compared to the control group antibody IgG1k, but the inhibitory or delaying effect on the number of cell clumps by the PTX3 monoclonal antibody of Example 1 is still significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806) and is statistically significant.

[0120] Example 6. Evaluation of the effect of PTX3 monoclonal antibody on tumor growth and metastasis in vivo 1. Evaluation of the inhibitory effect of the PTX3 monoclonal antibody of Example 1 on the growth and metastasis of orthotopic xenografted breast cancer tumors in vivo In this example, the above-mentioned human cancer cell line was orthotopically injected into the mammary fat pad of an immunodeficient mouse to form a tumor, and then the PTX3 monoclonal antibody of Example 1 was administered to evaluate the tumor suppressing or delaying effect of the PTX3 monoclonal antibody of Example 1 on the tumor.

[0121] First, breast cancer cells MB231-Luc2 (MB231 is a human breast cancer cell line that does not express estrogen receptor (ER) α and ERβ; Luc2 is a gene expressing luciferase) were orthotopically inoculated into the mammary fat pad of NOD-SCID mice (purchased from Lucco Biotechnology Co., Ltd.). The average tumor volume was 80 mm 3After reaching the age of 100, the experimental mice were administered the PTX3 antibody of Example 1 (8 mg / kg body weight) or the antibody of the control group (IgG1k, 8 mg / kg body weight) once a week, and the results are shown in Figures 15A to 15B. Figure 15B shows the in vivo imaging results 11 weeks after inoculation with breast cancer cells MB231-Luc2, where the in vivo imaging was performed by observing the size of the tumor using a commercially available in vivo fluorescent imaging (in vivo bioluminescent images) system (e.g., non-invasive 3D biomolecular imaging system (IVIS system), PerkinElmer), and the luminescent image area represents the tumor formed from breast cancer cells MB231-Luc2 in the mouse body. Then, all the mice were sacrificed, and the size of the tumor in the mouse body was measured, and the tumor volume was calculated according to the following formula (I).

[0122] V = (w × l 2 )×0.52 (I) In formula (I), l represents the length of the tumor and w represents the width of the tumor.

[0123] 15A-15B are diagrams showing the results of tumor volume (FIG. 15A) and tumor metastasis (FIG. 15B) of orthotopic xenografted breast cancer cells MDA-MB231 in mice, which were inhibited by a PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody. The data in FIG. 15A was obtained as the positive and negative mean standard deviation from data from six repeated experiments for each time point and each sample, and the symbol "*" indicates statistical significance (p<0.05) against the control antibody (IgG1k).

[0124] As can be seen from the results in Figures 15A and 15B, the PTX3 monoclonal antibody of Example 1 can significantly inhibit or delay the tumor volume and metastasis of orthotopic xenografted breast cancer cells MB231 compared with the control antibody (IgG1k), and this difference is statistically significant.

[0125] 2. Evaluation of the inhibitory effect of the PTX3 monoclonal antibody of Example 1 on the growth and metastasis of orthotopic breast cancer tumors in vivo In this example, the above-mentioned mouse cancer cell line was orthotopically injected into the mammary fat pad of a mouse with a normal immune system to form a tumor. A mouse triple-negative breast cancer (TNBC) model was then established, and the PTX3 monoclonal antibody of Example 1 was then administered to evaluate the tumor-suppressing effect of the PTX3 monoclonal antibody of Example 1 on the tumor.

[0126] First, 1×10 6 Breast cancer cell 4T1 (4T1 is a mouse breast cancer cell line, transfected with a vector containing the Luc2 gene, expressing ERβ but not ERα, ATCC® CRL-2539) TM ) were orthotopically inoculated into the mammary fat pads of wild-type BALB / c female mice (6-8 weeks old, purchased from Lexa Biotechnology Co., Ltd.). The average tumor volume was 50 mm 3 After reaching the target tumor volume, the experimental mice were administered the PTX3 antibody of Example 1 (10 mg / kg body weight) or the control antibody (IgG1k, 10 mg / kg body weight) once a week. The tumor size and metastasis were observed using an in vivo fluorescence imaging system. Then, all the mice were sacrificed, and the tumor size in the mouse body was measured, and the tumor volume was calculated according to the above formula (I).

[0127] 16A-16B are diagrams showing the results of suppression of tumor volume (FIG. 16A) and metastasis (FIG. 16B) of orthotopic breast cancer cell 4T1 in mice suppressed by PTX3 monoclonal antibody according to one embodiment of the present invention or antibody of the control group. FIG. 16B shows the in vivo imaging results 5 weeks after inoculation of breast cancer cell 4T1, in which the luminescent image area shows that the mouse has a tumor formed from breast cancer cell 4T1 in the mouse body, and the data in FIG. 16A is obtained as the positive and negative average standard deviation from the data of six repeated experiments for each time point and each sample, and the symbol "**" indicates statistical significance (p<0.01) against the antibody of the control group (IgG1k).

[0128] As can be seen from the results in Figures 16A and 16B, the PTX3 monoclonal antibody of Example 1 can significantly inhibit or delay the tumor volume and metastasis of orthotopic 4T1 breast cancer cells compared with the control antibody (IgG1k), and this difference is statistically significant.

[0129] 3. Evaluation of the inhibitory effect of the combination of the PTX3 monoclonal antibody of Example 1 and an anticancer drug on the growth of orthotopic breast cancer tumors in vivo (I) This example was evaluated in the same manner as in point 2 of Example 6, except that the above-mentioned mouse breast cancer cell line 4T1 was orthotopically inoculated into the mammary fat pad of BALB / C female mice (6-8 weeks old, purchased from Lexa Biotechnology Co., Ltd.). 3 After reaching 100 mg / kg body weight, the PTX3 monoclonal antibody of Example 1 (PTX3Ab at 2.5 mg / kg body weight (also called mpk), 5.0 mg / kg body weight or 10.0 mg / kg body weight, Zhenwei Biotechnology Co., Ltd.), the control antibody (IgG1k at 10 mg / kg body weight, product code 10101, Weiqiao Biomedical Co., Ltd.) or combined with paclitaxel (also called Taxol or Paclitaxel, 30.0 mg / kg body weight) was administered to the mice by intraperitoneal injection once a week for a total of 6 weeks. After the 6th week, the mice were sacrificed, and the tumor size and metastasis were observed by in vivo fluorescence imaging system.

[0130] After 6 weeks, the tumor size was observed using a commercially available in vivo bioluminescent imaging system (e.g., non-invasive 3D biomolecular imaging system (IVIS system), PerkinElmer), and the results are shown in Figure 17A, in which the luminescent image area represents the tumor formed from breast cancer cell 4T1 in the mouse body. The size and metastasis of the tumor in the mouse body were measured using the above system, and the tumor volume was calculated based on the above formula (I), and the results are shown in Figures 17B to 17C.

[0131] 17A-17C are images (FIG. 17A) and line graphs (FIG. 17B-17C) showing tumor volume and metastasis of mouse orthotopic xenografted breast cancer cells 4T1 suppressed by the combination or absence of paclitaxel with a PTX3 monoclonal antibody according to one embodiment of the present invention. The data in FIG. 17B-17C are obtained as positive and negative mean standard deviations from six replicates of each time point and each sample, where the symbol "**" indicates statistical significance (p<0.01) relative to the control antibody (IgG1k).

[0132] As can be seen from the results of Figures 17A to 17C, as shown in Figures 17A and 17B, the PTX3 monoclonal antibody of Example 1 or paclitaxel can inhibit or delay the tumor volume and metastasis of orthotopic transplanted breast cancer cells 4T1, compared with the antibody of the control group (IgG1k). However, as shown in Figures 17A and 17B, when the PTX3 monoclonal antibody is used in combination with paclitaxel in mice, it can inhibit or delay the tumor volume and metastasis of orthotopic transplanted breast cancer cells 4T1, and the combined effect of the two far exceeds the comprehensive effect of single treatment. All of the above differences are statistically significant.

[0133] 4. Evaluation of the inhibitory effect of the PTX3 monoclonal antibody of Example 1 in combination with an anticancer drug on the growth of orthotopic breast cancer tumors in vivo (II) In this example, evaluation was performed in the same manner as in point 3 of Example 6, following the schematic diagram of the experimental flow in FIG. 18A, and the results are shown in FIGS. 18B to 18D.

[0134] 18B-18D are images (FIG. 18B), line graphs (18C) of tumor volume and metastasis of mouse orthotopic breast cancer cell 4T1 in the case of using PTX3 monoclonal antibody according to another embodiment of the present invention in combination with or without paclitaxel, and mouse survival rate (FIG. 18D). The data in FIG. 18C-18D are obtained as positive and negative average standard deviations from data of six repeated experiments for each time point and each sample, where the symbol "*" indicates statistical significance (p<0.05) relative to the antibody (IgG1k) of the control group, and the symbol "**" indicates statistical significance (p<0.01) relative to the antibody (IgG1k) of the control group.

[0135] As can be seen from the results of Figures 18B to 18D, as shown in Figures 18B and 18C, the PTX3 monoclonal antibody of Example 1 or paclitaxel can inhibit or delay the tumor volume and metastasis of orthotopic breast cancer cell 4T1 compared with the control antibody (IgG1k). However, as shown in Figures 18B and 18C, when the PTX3 monoclonal antibody is used in combination with paclitaxel in mice, the tumor volume of orthotopic breast cancer cell 4T1 can be inhibited or delayed, and the survival rate of the mice can be improved to 80% or more, and the combined effect of the two far exceeds the comprehensive effect of single treatment. All of the above differences were statistically significant.

[0136] 5. Evaluation of the inhibitory effect of the PTX3 monoclonal antibody of Example 1 on the growth of allogeneic colon cancer tumors in vivo This example was evaluated in the same manner as in point 3 of Example 6 according to the schematic diagram of the experimental flow in FIG. 19A, except that the above-mentioned mouse colon cancer cell line MC38 was subcutaneously (sc) injected into C57BL / 6J male mice (6-8 weeks old, purchased from Lexco Biotechnology Co., Ltd.). On the 7th day after the injection of MC38 cells, the mice were intraperitoneally (ip) injected with the PTX3 monoclonal antibody of Example 1 (10.0 mg / kg body weight PTX3Ab, Zhenwei Biotechnology Co., Ltd.) or the control antibody (10 mg / kg body weight IgG1k, product code 10101, Weiqiao Biomedical Co., Ltd.) once a week, for a total of three times. The tumor size was observed by an in vivo fluorescence imaging system every week. On the 23rd day, all the mice were sacrificed, and the tumor size in the mouse body was measured, and the tumor volume was calculated according to the above formula (I).

[0137] Referring to Figure 19B, the results of tumor volume of colon cancer cell line MC38 transplanted into mice suppressed by PTX3 monoclonal antibody according to one embodiment of the present invention or control antibody are shown. The data in Figure 19B are obtained as positive and negative mean standard deviations from data of four repeated experiments for each time point and each sample, where the symbol "*" indicates statistical significance (p<0.05) against the control antibody (IgG1k), and the symbol "***" indicates statistical significance (p<0.001) against the control antibody (IgG1k).

[0138] As can be seen from the results in FIG. 19B, the PTX3 monoclonal antibody of Example 1 can inhibit or delay the tumor volume of the colon cancer cell line MC38 compared to the control antibody (IgG1k), and this difference is statistically significant.

[0139] 6. Evaluation of the inhibitory effect of the PTX3 monoclonal antibody of Example 1 on the growth of xenografted glioblastoma in vivo This example was evaluated in the same manner as in point 3 of Example 6, except that the above human glioblastoma (GBM) cell line U87MG was subcutaneously (sc) injected into NOD-SCID male mice (6-8 weeks old, purchased from Lexco Biotechnology Co., Ltd.). On the 20th day after the injection of U87MG cells, the mice were intraperitoneally (ip) injected with the PTX3 monoclonal antibody of Example 1 (10.0 mg / kg body weight PTX3Ab, Zhenwei Biotechnology Co., Ltd.) or the control antibody (10 mg / kg body weight IgG1k, product code 10101, Weiqiao Biomedical Co., Ltd.) once a week for a total of 4 weeks. After 20 days, the tumor size was measured, and the tumor volume was calculated according to the above formula (I).

[0140] 20A and 20B are line graphs (FIG. 20A) showing tumor volumes of human glioblastoma cell line U87MG transplanted into mice inhibited by PTX3 monoclonal antibody according to one embodiment of the present invention or a control antibody, and a graph (FIG. 20B) showing the survival rate of the mice. The data in FIG. 20A was obtained as the positive and negative mean standard deviations from data from three or four repeated experiments for each time point and each sample, where the symbol "p=0.0008" indicates statistical significance relative to the control antibody (IgG1k).

[0141] As can be seen from the results of Figures 20A and 20B, the PTX3 monoclonal antibody of Example 1 can inhibit or delay the tumor volume of xenografted glioblastoma cells U87MG compared to the control antibody (IgG1k) (Figure 20A), and can improve the survival rate of mice to 75% (Figure 20B). All of the above differences were statistically significant.

[0142] Example 7. Evaluation of the effect of PTX3 monoclonal antibody in delaying or reversing fibrosis in vivo 1. Evaluation of the reversal effect of the PTX3 monoclonal antibody of Example 1 on acute liver fibrosis and hepatic necrosis in vivo This example was carried out according to the experimental flow diagram in FIG. 21A and was designed based on the guidelines of the Animal Center of E-Da Hospital, Yishou University. First, C57BL / 6J male mice (8 weeks old, purchased from Lexus Biotechnology Co., Ltd.) were intramuscularly injected with 1 mL / kg body weight of tetrachloromethane (a mixture of CCl4 and olive oil in a 1:1 volume ratio). Acute liver fibrosis caused by tetrachloromethane induced hepatocyte apoptosis and necrosis within 12 hours. Next, 4 hours and 28 hours after the injection of tetrachloromethane, the mice were administered the PTX3 monoclonal antibody of Example 1 (10 mg / kg body weight PTX3Ab, Zhenwei Biotechnology Co., Ltd.) or the control antibody (10 mg / kg body weight IgG1k, product code 10101, Weiqiao Biomedical Co., Ltd.) by intraperitoneal (ip) injection. On the second day, all the mice were sacrificed and the changes in liver tissue sections, the proportion of hepatic necrotic areas, and liver weight / body weight ratio were observed, and the results are shown in Figures 21B to 21D.

[0143] 21B to 21D are diagrams showing tissue sections of the left hepatic lobe stained with hematoxylin and eosin (H&E) in mice with acute hepatic fibrosis caused by PTX3 monoclonal antibody according to one embodiment of the present invention (FIG. 21B, magnification: 20 times, observing the state of hepatocyte apoptosis), the percentage of hepatic necrotic area (FIG. 21C), and liver weight / body weight ratio (FIG. 21D). In FIG. 21C, the percentage of hepatic necrotic area in the total scanned area was measured using the automatic threshold detection function of the commercially available image analysis software ImageJ (WS Rasband, NIH, Bethesda, Maryland, USA).

[0144] As can be seen from the results in Figures 21B to 21D, the PTX3 monoclonal antibody of Example 1 can delay cell apoptosis (Figure 21B) and necrosis (Figure 21C) in acute liver fibrosis caused by tetrachloromethane, as well as the degree of increase in liver weight caused by acute liver fibrosis in mice (Figure 21D), compared to the antibody of the control group (IgG1k). All of the above differences were statistically significant.

[0145] 2. Evaluation of the reversal effect of the PTX3 monoclonal antibody of Example 1 on hepatic necrosis caused by chronic liver fibrosis in vivo This example was carried out according to the schematic diagram of the experimental flow in FIG. 22A, and was designed similarly to point 1 of Example 7, except that C57BL / 6J male mice (8 weeks old, purchased from Lexco Biotechnology Co., Ltd.) were intramuscularly injected with 1 mL / kg body weight of tetrachloromethane (a mixture of CCl4 and olive oil in a 1:1 volume ratio) twice a week for a total of 8 weeks. Then, 3 to 8 weeks after the injection of tetrachloromethane, the mice were administered the PTX3 monoclonal antibody of Example 1 (10 mg / kg body weight PTX3Ab, Zhenwei Biotechnology Co., Ltd.) or the control antibody (10 mg / kg body weight IgG1k, product code 10101, Weiqiao Biomedical Co., Ltd.) by intraperitoneal (ip) injection once a week. At the end of the 8th week, all the mice were sacrificed, and the changes in liver tissue sections, the proportion of liver necrotic areas, and liver weight / body weight ratio were observed, and the results are shown in FIG. 22B to FIG. 22D.

[0146] 21B to 21D are diagrams showing a tissue section of the left hepatic lobe stained with Picro-Sirius Red in a mouse with chronic hepatic fibrosis caused by a PTX3 monoclonal antibody according to one embodiment of the present invention (FIG. 22B, magnification: 20 times, observing the state of hepatic fibrosis), the proportion of the hepatic fibrosis area (FIG. 22C), and the liver weight / body weight ratio (FIG. 22D). In FIG. 22C, the proportion of the hepatic necrotic area in the total scanned area was measured using the automatic threshold detection function of the commercially available image analysis software ImageJ (WS Rasband, NIH, Bethesda, Maryland, USA).

[0147] As can be seen from the results of Figures 22B to 22D, the PTX3 monoclonal antibody of Example 1 can delay the chronic liver fibrosis (Figure 22B) and the area ratio (Figure 22C) caused by tetrachloromethane, as well as the degree of increase in liver weight caused by chronic liver fibrosis in mice (Figure 22D), compared with the antibody of the control group (IgG1k). All of the above differences were statistically significant.

[0148] 4. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on the expression of fibrosis-related proteins in renal fibroblasts This example uses a rat kidney fibroblast cell line (NRK49F, registration number: BCRC 60084, ATCC® CRL-1570 TM ) was used to evaluate the effect of the PTX3 monoclonal antibody of Example 1 on renal fibrosis in the following test.

[0149] First, renal fibroblast cell line NRK49F cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (12800-082, Gibco) (containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin).

[0150] NRK49F cells were treated with 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.) and 0.4 μg / mL PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), and then further treated with 200 ng / mL PTX3 for 6 hours. Next, NRK49F cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1 mg / ml), and leupeptin (1 mg / ml). Then, western blotting was performed using specific antibodies to detect the expression of α-tubulin (product code T6199, Sigma) and fibronectin (product code 15613-1-AP, ProteinTech). The expression level of α-tubulin was used as a loading control, and the results are shown in Figure 23A.

[0151] In addition, NRK49F cells were seeded in 24-well cell culture plates and treated with 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.), PTX3 antibody (Zhenwei Biotechnology Co., Ltd.) or 200 ng / mL PTX3 for 24 hours. Then, NRK49F cells were fixed with methanol overnight at -20°C. The next day, they were stained with Picro-Sirius Red Solution (product code ab246832, Abcam) at room temperature for 20 minutes, and then wet washed twice with acetic acid. The number of cell nodules was determined under a 200x magnification field of an optical microscope. Then, these cells were dissolved in 0.1N NaOH and the absorbance at 490 nm was measured using a commercially available ELISA reader, and the results are shown in Figures 23B and 23C.

[0152] 23A to 23C are a Western blotting analysis result (FIG. 23A) showing the expression of fibrosis-related proteins in mouse kidney fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention, a cell staining image of a cell nodule (FIG. 23B), and a histogram thereof (FIG. 23C), respectively. The symbol "***" indicates statistical significance (p<0.001) compared to the control group.

[0153] As can be seen from the results in Figures 23A to 23C, the PTX3 monoclonal antibody of Example 1 was able to reduce the expression of fibrosis-related proteins in renal fibroblasts (Figure 23A) and decrease the number of cellular nodules (Figures 23B and 23C) compared to the control antibody (IgG1k), and the difference was statistically significant.

[0154] 5. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 in delaying renal fibrosis in an animal model of unilateral ureteral obstruction (UUO) This example was evaluated according to the schematic diagrams of the experimental flow in Figures 24A and 24C, respectively. First, 6-8 week-old male C57BL / 6J mice (purchased from LES Biotechnology Co., Ltd.) were taken. Next, unilateral ureteral obstruction (UUO) surgery was performed on the left incision of the mouse. The left kidney was removed to expose the ureter, which was then sutured with a suture (4-O Silk). The wound was then sutured with a surgical suture instrument. Then, on postoperative day 0 and day 7 (pretreatment, e.g., Figures 24A and 24B) or postoperative day 7 (posttreatment, e.g., Figures 24C and 24D), the mice were treated with 10 mg / kg IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.) and 10 mg / kg PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), and the mice were euthanized on postoperative day 14. Half of the kidney was fixed in formalin and embedded in paraffin. The tissue sections were stained with hematoxylin-eosin (HE) or Picro-Sirius Red Solution (product code ab246832, Abcam), then covered with a cover glass and sealed, and the images were observed at a magnification of 20 times. The results are shown in Figures 24B and 24D, and the scale bar in the figures represents 100 μm.

[0155] Referring to Figures 24B and 24D, there are shown images of tissue sections of kidneys of UUO mice stained with a PTX3 monoclonal antibody according to one embodiment of the present invention.

[0156] As can be seen from the results in FIG. 24B and FIG. 24D, the PTX3 monoclonal antibody of Example 1 can certainly delay renal fibrosis in UUO animals, as compared with the antibody (IgG1k) of the control group.

[0157] 6. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on the expression and migration of fibrosis-related proteins in pulmonary fibroblasts after PTX3 treatment In this example, the effect of the PTX3 monoclonal antibody of Example 1 on pulmonary fibrosis was evaluated by the following test using a human lung fibroblast cell line (HFL1, registration number: BCRC 60299, ATCC (registered trademark) CCL-153TM).

[0158] First, HFL1 cells were cultured in Ham's F-12K (F12K) medium (Kaighn modification) (21127-022, Gibco) (containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin).

[0159] HFL1 cells were treated with 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.) and 0.4 μg / mL PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), and then further treated with 200 ng / mL PTX3 for 6 hours. HFL1 cells were then lysed with improved RIPA buffer. Western blotting was then performed using specific antibodies to detect the expression of α-tubulin (α-tubulin, product code T6199, Sigma), fibronectin (Fibronectin, product code 15613-1-AP, ProteinTech), type I collagen (Collagen I, product code 14695-1-AP, ProteinTech), and α-smooth muscle actin (α-SMA, product code GTX 100904, GeneTex). The expression level of α-tubulin was used as a loading control, and the results are shown in Figure 25A.

[0160] In addition, HFL1 cells were seeded in a 24-well cell culture plate (including an insert-type culture dish with a well diameter of 8 μm (353097, BD Biosciences)), and the lower culture well was treated with or without 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.), PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), or 200 ng / mL PTX3. After 16 hours of culture, the cells in the insert-type culture dish were scraped with a cotton swab. The cells attached to the lower surface of the polycarbonate film of the insert-type culture dish that had migrated from the bottom of the insert-type culture dish were stained with DAPI, and the number of cells was determined under a fluorescent microscope with a magnification of 200 times, and the results are shown in Figure 25B.

[0161] HFL1 cells were seeded in 24-well cell culture plates and treated with or without 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.), PTX3 antibody (Zhenwei Biotechnology Co., Ltd.) or 200 ng / mL PTX3 for 24 hours. Then, HFL1 cells were fixed with methanol overnight at -20°C. The next day, they were stained with Picro-Sirius Red Solution (product code ab246832, Abcam) at room temperature for 20 minutes, and then wet washed twice with acetic acid. The number of cell nodules was determined under a 200x magnification field of an optical microscope. Then, these cells were dissolved in 0.1N NaOH and the absorbance at 490 nm was measured using a commercial ELISA reader, and the results are shown in Figures 25C and 25D.

[0162] 25A to 25D show the results of Western blotting analysis (FIG. 25A) showing the expression of fibrosis-related proteins in pulmonary fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention, a histogram of the number of migrating cells (FIG. 25B), a cell staining image of cell nodules (FIG. 25C), and a histogram of the same (FIG. 25D). The symbol "***" indicates statistical significance (p<0.001) compared to the control group.

[0163] As can be seen from the results in Figures 25A to 25D, the PTX3 monoclonal antibody of Example 1 can delay the expression, migration, and number of fibrosis-related proteins in pulmonary fibroblasts after PTX3 treatment, compared to the control antibody (IgG1k).

[0164] 7. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on bleomycin-induced pulmonary fibrosis in an IPF mouse model (I) This example was carried out according to the schematic diagram of the experimental flow in FIG. 26A. First, 6-8 week-old male C57BL / 6J mice (purchased from Lexico Biotechnology Co., Ltd.) were collected. PBS or 2 mg / kg bleomycin (BLM, product code ap302, Znzo) was administered intratracheally (IT) to the mice to induce fibrosis. After inducing fibrosis, 10 mg / kg IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.) or 10 mg / kg PTX3 antibody (Zhenwei Biotechnology Co., Ltd.) was administered intraperitoneally to the mice on the 14th and 21st days, and the mice were euthanized on the 28th day.

[0165] C57BL / 6 mice were weighed on the 7th, 14th, 21st, or 28th day after intratracheal administration of bleomycin, and then 10 mg / kg IgG1k (control group) or 10 mg / kg PTX3 antibody was administered intraperitoneally. The results are shown in Figure 26B.

[0166] After the mice were euthanized, the lung tissues of the mice were macroscopically observed on the 7th, 14th, 21st, or 28th day after intratracheal administration of PBS (i.e., healthy control group) or 2 mg / kg bleomycin (i.e., BLM). After perfusion of the left lung lobe, the tissues were fixed with paraformaldehyde and embedded in paraffin. The tissue sections were stained with HE, then covered with a cover glass and sealed, and images were observed at a magnification of 20 times. The results are shown in Figure 26C, where the scale bar represents 100 μm.

[0167] After the mice were euthanized, fibrosis was induced with bleomycin, and then 10 mg / kg IgG1k or 10 mg / kg PTX3 antibody was administered. The lung tissues of the mice were macroscopically observed on the 28th day after administration. The left lung lobe was perfused, then fixed with paraformaldehyde and embedded in paraffin. The tissue sections were stained with HE, then sealed with a cover glass, and images were observed at a magnification of 20 times. The results are shown in Figure 26D, and the scale bar in the figure represents 100 μm.

[0168] Referring to Figures 26B to 26D, there are a curve (Figure 26B) showing the weight change in mice with BLM-induced pulmonary fibrosis caused by a PTX3 monoclonal antibody according to one embodiment of the present invention, and images (Figures 26C and 26D) showing the appearance of the lungs and tissue sections.

[0169] As can be seen from the results in Figures 26B to 26D, the PTX3 monoclonal antibody of Example 1 can reverse the weight loss and the degree of pulmonary fibrosis caused by pulmonary fibrosis in mice with induced pulmonary fibrosis, compared to the control antibody (IgG1k).

[0170] 8. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on bleomycin-induced pulmonary fibrosis in an IPF mouse model (II) This example was carried out according to the schematic diagram of the experimental flow in FIG. 27A. First, 6-8 week-old male C57BL / 6J mice (purchased from Lexico Biotechnology Co., Ltd.) were collected. PBS or 2 mg / kg bleomycin (BLM, product code ap302, Znzo) was intratracheally instilled (IT) into the mice to induce fibrosis. After inducing fibrosis, on the 21st day, the mice were intraperitoneally injected with 10 mg / kg IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.) or 10 mg / kg PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), and euthanized on the 28th day.

[0171] C57BL / 6 mice were weighed on days 7, 14, 21, and 28 after intratracheal administration of bleomycin, and then on day 21, 10 mg / kg IgG1k (control group) or 10 mg / kg PTX3 antibody was administered intraperitoneally. The results are shown in Figure 27B.

[0172] After the mice were euthanized, the lung tissues of the mice were macroscopically observed on the 7th, 14th, 21st, or 28th day after intratracheal administration of PBS (i.e., healthy control group) or 2 mg / kg bleomycin (i.e., BLM). After perfusion of the left lung lobe, the tissues were fixed with paraformaldehyde and embedded in paraffin. The tissue sections were stained with HE, then covered with a cover glass and sealed, and images were observed at a magnification of 20 times. The results are shown in Figure 27C, where the scale bar represents 100 μm.

[0173] After euthanasia, the mice were induced with bleomycin, and then 10 mg / kg IgG1k or 10 mg / kg PTX3 antibody was administered. The lung tissues of the mice were macroscopically observed on the 28th day. The left lung lobe was perfused, then fixed with paraformaldehyde and embedded in paraffin. The tissue sections were stained with HE, then sealed with a cover glass, and images were observed at a magnification of 20 times. The results are shown in Figure 27D. The scale bar in the figure represents 100 μm.

[0174] Referring to Figures 27B to 27D, there is a curve showing the change in body weight of mice with BLM-induced pulmonary fibrosis caused by a PTX3 monoclonal antibody according to one embodiment of the present invention (Figure 27B), and images showing the appearance of the lungs and tissue sections (Figures 27C and 27D).

[0175] As can be seen from the results in Figures 27B to 27D, the PTX3 monoclonal antibody of Example 1 can reverse the weight loss and the degree of pulmonary fibrosis caused by pulmonary fibrosis in mice with induced pulmonary fibrosis, compared to the control antibody (IgG1k).

[0176] 9. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on the expression of fibrosis-related proteins in NIH-3T3 embryonic fibroblasts This example uses a mouse embryonic fibroblast cell line (NIH-3T3, registration number: BCRC 60008, ATCC® CCL-1658 TM ) was used to evaluate the effect of the PTX3 monoclonal antibody of Example 1 on fibrosis in the following test.

[0177] First, NIH-3T3 cells were cultured in DMEM (12800-082, Gibco) (containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin).

[0178] NIH-3T3 cells were treated with 0.4μg / mL PTX3 antibody (Zenwei Biotechnology Co., Ltd.) and then with 200ng / mL PTX3 for 6 hours. The cells were then lysed with RIPA buffer. Western blotting was then performed using specific antibodies to detect the expression of α-tubulin (α-tubulin, product code T6199, Sigma), type I collagen (Collagen I, product code 14695-1-AP, ProteinTech), and α-smooth muscle actin (α-SMA, product code GTX 100904, GeneTex), and the expression level of α-tubulin was used as a loading control group, and the results are shown in Figure 28A.

[0179] In addition, NIH-3T3 cells were seeded in 24-well cell culture plates and treated with or without 0.4 μg / mL IgG1k (product code 10101, Weiqiao Biomedical Co., Ltd.), PTX3 antibody (Zhenwei Biotechnology Co., Ltd.), or 200 ng / mL PTX3 for 24 hours. Then, NIH-3T3 cells were fixed with methanol overnight at -20°C. The next day, they were stained with Picro-Sirius Red Solution (product code ab246832, Abcam) at room temperature for 20 minutes, and then wet washed twice with acetic acid. The number of cell nodules was determined under a 200x magnification field of an optical microscope. Then, these cells were dissolved in 0.1N NaOH, and the absorbance at 490 nm was measured using a commercially available ELISA reader, and the results are shown in Figure 28B and Figure 28C.

[0180] 28A to 28C show the results of Western blotting analysis (FIG. 28A) showing the expression of fibrosis-related proteins in embryonic fibroblasts using a PTX3 monoclonal antibody according to one embodiment of the present invention, a cell staining image (FIG. 28B) showing cell nodules, and a histogram (FIG. 28C) of the same. The symbol "***" indicates statistical significance (p<0.001) compared to the control group.

[0181] As can be seen from the results in FIG. 28A to FIG. 28C, the PTX3 monoclonal antibody of Example 1 can delay the expression of fibrosis-related proteins and the number of cell nodules in NIH-3T3 cells, as compared to the control antibody (IgG1k).

[0182] 10. Evaluation of the effect of the PTX3 monoclonal antibody of Example 1 on the expression of fibrosis-related proteins in F28 liver fibroblasts First, human cancer associated fibroblast / hepatocytes (cancer associated fibroblast / F28 (CAF / F28) cells) were cultured in DMEM (12800-082, Gibco) (containing 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin).

[0183] CAF / F28 cells were exposed to 8 Gray (Gy) radiation and then treated with 0.4 μg / mL PTX3 antibody (Zenwei Biotechnology) for 6 hours. Then, the cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (1 mg / ml), and leupeptin (1 mg / ml). Western blotting was then performed using specific antibodies to detect the expression of α-tubulin (α-tubulin, product code T6199, Sigma), fibronectin (Fibronectin, product code 15613-1-AP, ProteinTech), type I collagen (Collagen I, product code 14695-1-AP, ProteinTech), and α-smooth muscle actin (α-SMA, product code GTX 100904, GeneTex). The expression level of α-tubulin was used as a loading control, and the results are shown in Figure 29A.

[0184] CAF / F28 cells were treated with 0.4 μg / mL PTX3 antibody (Zenwei Biotechnology Co., Ltd.) and then with 200 ng / mL PTX3 for 6 hours. The cells were then lysed with RIPA buffer. Western blotting was then performed using specific antibodies to detect the expression of α-tubulin (α-tubulin, product code T6199, Sigma), fibronectin (Fibronectin, product code 15613-1-AP, ProteinTech), type I collagen (Collagen I, product code 14695-1-AP, ProteinTech), and α-smooth muscle actin (α-SMA, product code GTX 100904, GeneTex). The expression level of α-tubulin was used as a loading control group, and the results are shown in Figure 29B.

[0185] 29A and 29B are Western blotting analysis results showing the expression of fibrosis-related proteins in liver fibroblasts treated with different PTX3 monoclonal antibodies according to one embodiment of the present invention.

[0186] As can be seen from the results in FIG. 29A to FIG. 29B, the PTX3 monoclonal antibody of Example 1 can delay the expression of fibrosis-related proteins caused by irradiation of CAF / F28 hepatic fibroblasts and PTX3, as compared to the control antibody (IgG1k).

[0187] The above mouse experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals of Cheng Kung University, and the relevant animal use protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).

[0188] In addition, the PTX3 monoclonal antibody of Example 1 of the present invention has good affinity and sensitivity to PTX3 recombinant protein, and can be applied to the detection set and method of PTX3 to detect the PTX3 content in a biological sample in vitro. The applied biological sample, the method, set, components / instruments for detecting PTX3, etc. are as described above, and needless to say. Next, the pharmaceutical composition containing the monoclonal antibody or its antigen-binding fragment of the present invention and its use can be a broad-spectrum drug for treating various diseases by effectively inhibiting or delaying the binding of PTX3 to the PTX3 receptor by using the PTX3 monoclonal antibody or its antigen-binding fragment with high affinity and sensitivity as an active ingredient, and effectively inhibiting or delaying the binding of PTX3 to the PTX3 receptor, thereby inhibiting or delaying the disease or symptoms associated with the binding of PTX3 to the PTX3 receptor.

[0189] That is, the present invention has described the pharmaceutical composition comprising the monoclonal antibody of the present invention or its antigen-binding fragment and its use by taking the PTX3 monoclonal antibody of a specific sequence, a specific analysis mode, or a specific evaluation method as an example. However, the present invention is not limited thereto, and it is obvious to those skilled in the art that the pharmaceutical composition comprising the monoclonal antibody of the present invention or its antigen-binding fragment and its use can be carried out by other analysis modes or other evaluation methods without departing from the concept and scope of the present invention.

[0190] As can be seen from the above examples, the pharmaceutical composition containing the monoclonal antibody or its antigen-binding fragment of the present invention and its use have the advantage that they specifically inhibit or delay the binding of PTX3 to the PTX3 receptor using a specific PTX3 monoclonal antibody or its antigen-binding fragment as an active ingredient, can be applied to a detection set and method for PTX3, and can inhibit or delay diseases or symptoms associated with the binding of PTX3 to the PTX3 receptor.

[0191] Although several embodiments of the present invention have been disclosed as above, they are not intended to limit the present invention, and a person skilled in the art can make various changes and modifications without departing from the concept and scope of the present invention, and therefore the scope of protection of the present invention is limited by the appended claims.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof, characterized by specifically binding to a non-denatured amino acid sequence selected from the group consisting of the amino acid sequences set forth in sequence identification numbers SEQ ID NO:2 to SEQ ID NO:4 and SEQ ID NO:

11.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

2.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

3.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

4.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

11.

6. a heavy chain variable region sequence having a CDR1 represented by the amino acid sequence SEQ ID NO: 18, a CDR2 represented by the amino acid sequence SEQ ID NO: 19 or a CDR2 represented by the amino acid sequence SEQ ID NO: 20, and a CDR3 represented by the amino acid sequence SEQ ID NO: 21; a light chain variable region sequence having a CDR1 represented by the amino acid sequence SEQ ID NO: 22, a CDR2 represented by the amino acid sequence SEQ ID NO: 23, and a CDR3 represented by the amino acid sequence SEQ ID NO: 24 or a CDR3 represented by the amino acid sequence SEQ ID NO: 25; A monoclonal antibody or an antigen-binding fragment thereof comprising:

7. a heavy chain variable region sequence having a CDR1 encoded by the nucleic acid sequence SEQ ID NO:26, a CDR2 encoded by the nucleic acid sequence SEQ ID NO:27 or a CDR2 encoded by the nucleic acid sequence SEQ ID NO:28, and a CDR3 encoded by the nucleic acid sequence SEQ ID NO:29; a light chain variable region sequence having a CDR1 encoded by the nucleic acid sequence SEQ ID NO:30, a CDR2 encoded by the nucleic acid sequence SEQ ID NO:31, and a CDR3 encoded by the nucleic acid sequence SEQ ID NO:32 or a CDR3 encoded by the nucleic acid sequence SEQ ID NO:33; A monoclonal antibody or an antigen-binding fragment thereof comprising:

8. A heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NOs: 34 or 35; A light chain variable region sequence having the amino acid sequence set forth in SEQ ID NOs: 36 or 37; A monoclonal antibody or an antigen-binding fragment thereof comprising:

9. A heavy chain variable region sequence having an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NOs: 38 or 39; A light chain variable region sequence having an amino acid sequence encoded by a nucleic acid sequence set forth in SEQ ID NOs: 40 or 41; A monoclonal antibody or an antigen-binding fragment thereof comprising:

10. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.

11. The monoclonal antibody or its antigen-binding fragment described in any one of claims 1 to 9, characterized in that the monoclonal antibody or its antigen-binding fragment is a mouse-derived antibody, a human / mouse chimeric antibody, a humanized antibody or its antigen-binding fragment.

12. The antigen-binding fragment may be a single chain variable region fragment, a single chain variable region fragment dimer (scFv) ２ ], single chain variable region fragment trimer [(scFv) ３ ], variable region fragment, Fab fragment, Fab' fragment, F(ab') ２ 10. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9, which is a fragment, a nanobody or any combination thereof.

13. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation, binding, glycosylation, tag attachment or any combination thereof.

14. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof-drug conjugate.

15. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody or antigen-binding fragment thereof.

16. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or antigen-binding fragment thereof.

17. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG, IgM, IgA, IgD or IgE type.

18. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG type and has an IgG1, IgG2, IgG3 or IgG4 isotype.

19. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype.

20. The monoclonal antibody or its antigen-binding fragment described in any one of claims 1 to 9, characterized in that the monoclonal antibody or its antigen-binding fragment is an antibody that specifically inhibits or delays the binding between PTX3 and the PTX3 receptor.

21. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof belongs to an antagonist antibody.

22. A monoclonal antibody or an antigen-binding fragment thereof described in any one of claims 1 to 9, characterized in that the monoclonal antibody or an antigen-binding fragment thereof specifically inhibits or delays binding between a pentraxin-related protein receptor and a specific sequence at the C-terminus of one or more types of PTX3.

23. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof inhibits or delays the activity of one or more types of PTX3.

24. The monoclonal antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, characterized in that the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or delays the interaction between a PTX3 receptor and one or more types of PTX3, signal transduction by PTX3, or any combination thereof.

25. A detection set for PTX3 comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, The monoclonal antibody or antigen-binding fragment thereof specifically binds to a non-denatured amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO:

11.

26. The detection set for PTX3 according to claim 25, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

2.

27. The detection set for PTX3 according to claim 25, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

3.

28. The detection set for PTX3 according to claim 25, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

4.

29. The detection set for PTX3 according to claim 25, characterized in that the non-denatured amino acid sequence is the amino acid sequence set forth in SEQ ID NO:

11.

30. A method for detecting PTX3 in vitro using the detection set for PTX3 according to any one of claims 25 to 29, comprising: A method for detecting PTX3 in vitro, wherein the analytical sensitivity of the monoclonal antibody or antigen-binding fragment thereof contained in the PTX3 detection set is 0.0016 pM or more.

31. 1. A pharmaceutical composition comprising an effective dose of a monoclonal antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier, A pharmaceutical composition comprising as an active ingredient the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

32. 32. The pharmaceutical composition of claim 31 further comprising an active pharmaceutical ingredient.

33. 32. The pharmaceutical composition of claim 31, wherein the effective dosage is from 2 mg to 10 mg per kilogram of body weight.

34. 32. The pharmaceutical composition of claim 31, wherein the effective dosage is from 5 mg / kg body weight to 10 mg / kg body weight.

35. 32. The pharmaceutical composition of claim 31, wherein the effective dosage is from 6 mg / kg body weight to 9 mg / kg body weight.

36. 1. Use of a monoclonal antibody or an antigen-binding fragment thereof for preparing a pharmaceutical composition that specifically inhibits or delays binding of pentraxin-related protein (PTX3) to a PTX3 receptor, comprising: Use of a monoclonal antibody or an antigen-binding fragment thereof, characterized in that the pharmaceutical composition is a pharmaceutical composition described in any one of claims 31 to 35, and the monoclonal antibody or an antigen-binding fragment thereof has an effective dosage for inhibiting or delaying a disease or symptom associated with the binding of PTX3 to the PTX3 receptor.

37. Use of a monoclonal antibody or its antigen-binding fragment for preparing a pharmaceutical composition that specifically inhibits or delays the binding of PTX3 to the PTX3 receptor, as described in claim 36, characterized in that the disease or condition includes epithelial cell carcinoma, adenocarcinoma, glioblastoma and fibrosis.

38. The use of a monoclonal antibody or its antigen-binding fragment for preparing a pharmaceutical composition that specifically inhibits or delays the binding of PTX3 to the PTX3 receptor, as described in claim 37, wherein the epithelial cell cancer includes lung cancer, breast cancer and nasopharyngeal carcinoma.

39. The use of a monoclonal antibody or its antigen-binding fragment for preparing a pharmaceutical composition that specifically inhibits or delays the binding of PTX3 to a PTX3 receptor, as described in claim 37, characterized in that the adenocarcinoma includes colon cancer.

40. Use of a monoclonal antibody or its antigen-binding fragment for preparing a pharmaceutical composition that specifically inhibits or delays the binding of PTX3 to the PTX3 receptor as described in claim 37, characterized in that the organ affected by the disease or condition of fibrosis is selected from the group consisting of the lungs, liver, kidneys and skin.

41. The use of a monoclonal antibody or its antigen-binding fragment for preparing a pharmaceutical composition that specifically inhibits or delays the binding of PTX3 to a PTX3 receptor, characterized in that the pharmaceutical composition is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, orthotopic injection, oral administration or oronasal inhalation.

42. A pharmaceutical composition for inhibiting or slowing tumor cell activity in vitro, comprising a pharmaceutical composition according to any one of claims 31 to 35.

43. The pharmaceutical composition for inhibiting or retarding tumor cell activity in vitro according to claim 42, characterized in that the origin of the tumor cells includes glioblastoma, epithelial cell carcinoma and adenocarcinoma.

44. The pharmaceutical composition for inhibiting or retarding the activity of tumor cells in vitro according to claim 43, characterized in that the epithelial cell cancer includes lung cancer, breast cancer and nasopharyngeal carcinoma.

45. 44. The pharmaceutical composition for inhibiting or retarding tumor cell activity in vitro according to claim 43, characterized in that the adenocarcinoma comprises colon carcinoma.

46. 43. The pharmaceutical composition for inhibiting or retarding tumor cell activity in vitro according to claim 42, characterized in that the activity comprises proliferation, cancer stemness, migration, invasion, metastasis, tumor volume or drug resistance.

47. A pharmaceutical composition for inhibiting or delaying a fibrotic disease and / or a fibrotic condition in vitro, comprising a pharmaceutical composition according to any one of claims 31 to 35.

48. The pharmaceutical composition for inhibiting or delaying a fibrotic disease and / or a fibrotic condition in vitro according to claim 47, characterized in that the organ affected by the fibrotic disease and / or the fibrotic condition is selected from the group consisting of lung, liver, kidney and skin.

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