WO2019057024A1

WO2019057024A1 - Medical composition comprising monoclonal antibody or antigen binding fragment thereof and use thereof

Info

Publication number: WO2019057024A1
Application number: PCT/CN2018/106144
Authority: WO
Inventors: 王育民; 萧郁韦; 纪智瑛; 杜军毅; 郑朝峻
Original assignee: 王育民
Priority date: 2017-09-19
Filing date: 2018-09-18
Publication date: 2019-03-28
Also published as: TW201915024A; WO2019056991A1; TWI741216B; TW201915016A; TWI786186B

Abstract

Disclosed are a medical composition comprising a monoclonal antibody or an antigen binding fragment thereof and the use thereof. The monoclonal antibody or antigen binding fragment thereof can be used as an active ingredient, specifically inhibiting or slowing down the binding of PTX3 and a PTX3 receptor, and further inhibiting or slowing down diseases or symptoms associated with the binding of PTX3 and the PTX3 receptor.

Description

Medicinal composition containing monoclonal antibody or antigen-binding fragment thereof and use thereof

Technical field

The present invention relates to a pharmaceutical composition and use thereof, and more particularly to a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof which specifically inhibits or slows the binding of PTX3 to a PTX3 receptor, and uses thereof.

Background technique

It is known that cancer cells stimulate the microenvironment around the tumor to produce various inflammatory factors, white blood cells, hypervascular hyperplasia and proteases. The chronic inflammatory response of cancer is also related to the growth, metastasis and invasion of cancer cells. However, the cause and details of the formation are detailed. There are still many unclear aspects of the mechanism.

In addition to the inflammatory response, the tumor microenvironment also pointed out that the tumor microenvironment is closely related to metastasis and chemoresistance. The tumor microenvironment is composed of a variety of stromal cells and other different types of cells, which not only protects the tumor, but also allows the tumor cells to escape and resist immune cells, resulting in resistance of the tumor cells.

Fibroblasts and macrophages in the stromal tissue surrounding the tumor are activated by CEBPD, which induces the secretion of factor-like protein-like protein 3 (PTX3), which has an activity of promoting angiogenesis. And can increase the ability of nasopharyngeal carcinoma cells to migrate and invade tissue (or invasion). In addition, past studies have also confirmed that CEBPD is activated in peripheral tissues of cancer, which may also promote cancer metastasis, and even promote the development of drug-resistant cancer cells during chemotherapy. These drug-resistant cancer cells grow faster and are easier to metastasize.

Although there are some small molecule anticancer drugs on the market, such as cis-diammine dichloroplatinum (II); CDDP; trade name Cisplatin, paclitaxel (trade name Taxol) and 5-fluorouracil (5-FU), but recent studies have found that the above small molecule anticancer drugs not only activate CEBPD expression in cancer cells, but also activate CEBPD in macrophages and fibroblasts. On the contrary, it promotes cancer drug resistance and rapid metastasis, resulting in poor treatment of cancer.

In view of this, there is an urgent need to develop a pharmaceutical composition to overcome the problems of conventional cancer drug resistance and rapid metastasis of tumor cells.

Summary of the invention

Accordingly, it is an aspect of the present invention to provide a pharmaceutical composition comprising an effective amount of a monoclonal antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, and the above monoclonal antibody or antigen-binding thereof Fragments are used as active ingredients.

Yet another aspect of the present invention is to provide a monoclonal antibody or antigen-binding fragment thereof for use in the preparation of a pharmaceutical composition for specifically inhibiting or slowing the binding of PTX3 to a PTX3 receptor, wherein the monoclonal antibody or antigen-binding thereof Fragments are active ingredients, and monoclonal antibodies or antigen-binding fragments thereof have an effective amount to inhibit or slow down the disease or condition associated with PTX3 and PTX3 receptor binding.

A further aspect of the present invention provides a method for inhibiting or slowing the activity of a tumor cell in vitro comprising administering to the tumor cell an effective amount of the above pharmaceutical composition, thereby inhibiting or slowing down the activity of the tumor cell.

According to the above aspect of the present invention, there is provided a pharmaceutical composition comprising an effective amount of a monoclonal antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, and the above monoclonal antibody or antigen-binding fragment thereof As an active ingredient.

According to another aspect of the present invention, a monoclonal antibody or antigen-binding fragment thereof is provided for the preparation of a medicament for specifically inhibiting or slowing the binding of a pentraxin-related protein (PTX3) to a PTX3 receptor The use of the composition. In one embodiment, the monoclonal antibody or antigen-binding fragment thereof is an active ingredient, and the monoclonal antibody or antigen-binding fragment thereof has an effective amount to inhibit or slow down the disease or condition associated with PTX3 and PTX3 receptor binding.

According to an embodiment of the present invention, the pharmaceutical composition is for inhibiting or slowing down a disease or a symptom associated with binding of PTX3 to a PTX3 receptor, wherein the disease or symptom includes lung cancer, breast cancer, nasopharyngeal Cancer (nasopharyngeal carcinoma; NPC) and glioblastoma multiforme (GBM).

According to still another aspect of the present invention, a method for inhibiting or slowing down the activity of a tumor cell in vitro comprises administering an effective dose of the above pharmaceutical composition to a tumor cell, thereby inhibiting or slowing down the activity of the tumor cell.

According to an embodiment of the invention, the activity comprises proliferation, cancer stemness, migration, invasiveness, metastasis or drug resistance.

A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention, which utilizes a specific PTX3 monoclonal antibody or an antigen-binding fragment thereof as an active ingredient, specifically inhibits or slows down the binding of PTX3 to the PTX3 receptor, This in turn inhibits or slows down the disease or condition associated with PTX3 binding to the PTX3 receptor.

DRAWINGS

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

[Fig. 1A] to [Fig. 1C] are diagrams showing that the PTX3 monoclonal antibody inhibits the number of transitional cells of the breast cancer cell line MDA-MB231 (Fig. 1A), the number of invading cells (Fig. 1B), and cell pellets according to an embodiment of the present invention. The result of the number (Figure 1C).

2A to 2C are diagrams showing the number of transitional cells (Fig. 2A), the number of invading cells (Fig. 2B), and the number of cell pellets of the lung cancer cell line A549 inhibited by the PTX3 monoclonal antibody according to an embodiment of the present invention (Fig. 2A). The result of Figure 2C).

[Fig. 3A] to [Fig. 3C] are diagrams showing that the PTX3 monoclonal antibody inhibits the number of transitional cells of the nasopharyngeal carcinoma cell line HONE1 (Fig. 3A), the number of invading cells (Fig. 3B), and cell pellets according to an embodiment of the present invention. The result of the number (Figure 3C).

[Fig. 4A] to [Fig. 4C] are diagrams showing the number of transition cells of the glioblastoma cell line U87MG inhibited by the PTX3 monoclonal antibody (Fig. 4A), the number of invading cells (Fig. 4B), and cells according to an embodiment of the present invention. The result of the number of pellets (Fig. 4C).

[Fig. 5] is a graph showing the results of binding of a PTX3 monoclonal antibody or a commercially available antibody to different PTX3 recombinant proteins according to an embodiment of the present invention.

[Fig. 6A] to [Fig. 6B] are diagrams showing the number of transitional cells (Fig. 6A) and the number of invading cells (Fig. 6B) of the PTX3 monoclonal antibody or the commercially available antibody inhibiting the breast cancer cell line MDA-MB231 according to an embodiment of the present invention. the result of.

[Fig. 7A] to [Fig. 7B] are diagrams showing the results of the inhibition of the number of transitional cells (Fig. 7A) and the number of invading cells (Fig. 7B) of the PTX3 monoclonal antibody or the commercially available antibody inhibiting the lung cancer cell line A549 according to an embodiment of the present invention. .

[Fig. 8A] to [Fig. 8B] are diagrams showing the number of transitional cells (Fig. 8A) and the number of invading cells (Fig. 8B) of the PTX3 monoclonal antibody or the commercially available antibody inhibiting the nasopharyngeal carcinoma cell line HONE1 according to an embodiment of the present invention. the result of.

[Fig. 9A] to [Fig. 9C] are diagrams showing a PTX3 monoclonal antibody or a commercially available antibody-inhibiting breast cancer cell line MDA-MB231 (Fig. 9A), a lung cancer cell line A549 (Fig. 9B), and a nasopharynx according to an embodiment of the present invention. The result of the number of cell pellets in the cancer cell line HONE1 (Fig. 9C).

[Fig. 10A] to [Fig. 10B] are diagrams showing that the PTX3 monoclonal antibody or the control group antibody inhibits the tumor volume of mouse xenografted breast cancer cell line MDA-MB231 (Fig. 10A) and tumor metastasis according to an embodiment of the present invention. (Fig. 10B) results.

[Fig. 11A] to [Fig. 11B] are diagrams showing inhibition of tumor volume (Fig. 11A) and tumor metastasis of mouse orthotopically transplanted breast cancer cell line 4T1 by a PTX3 monoclonal antibody or an isotype control antibody according to an embodiment of the present invention (Fig. 11A) 11B) results.

Detailed ways

As described above, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof, and a use thereof, which comprises a monoclonal antibody or an antigen-binding fragment thereof as an active ingredient, which can specifically inhibit or slow down positive five The binding of a pentraxin-related protein (PTX3) receptor to PTX3, thereby inhibiting or slowing down the disease or condition associated with PTX3 binding to the PTX3 receptor.

The monoclonal antibody or antigen-binding fragment thereof referred to herein may comprise a specific sequence of a heavy chain variable region sequence and a light chain variable region sequence, which specifically blocks the C-terminal specific sequence of the PTX3 and PTX3 receptors. Combine. 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 heavy chain variable region sequence can have, for example, the sequence identification number SEQ ID NO The amino acid sequence listed in 1 and the light chain variable region sequence may, for example, be the amino acid sequence set forth in SEQ ID NO: 2.

In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof thereof specifically inhibits or slows the binding of PTX3 to the PTX3 receptor, thereby specifically inhibiting or slowing the interaction of the PTX3 receptor with one or more PTX3 , suppress or slow down PTX3 information transmission, etc.

The above monoclonal antibody or antigen-binding fragment thereof can be added as an active ingredient to a pharmaceutical composition. In one embodiment, the aforementioned pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means a carrier, diluent, adjuvant, and/or vehicle that is not itself an active ingredient but is used to deliver the active ingredient to the individual, or Addition to the above composition to improve the handling or storage properties of the composition, or to allow or facilitate the dosage unit of the composition to form an excipient or any substance suitable for pharmaceutical compositions and convenient for administration. The aforementioned pharmaceutically acceptable carrier should not destroy the pharmacological activity of the active ingredient and should be non-toxic when delivering a sufficient therapeutic amount of the active ingredient.

The foregoing pharmaceutically acceptable carriers are well known to those of ordinary skill in the art of making pharmaceutical compositions and include, but are not limited to, buffers, diluents, disintegrants, binders, adhesives, humectants. A polymer, a lubricant, a slip agent, a substance added to mask or counteract a bad taste or odor, a dye, a fragrance, and a substance added to improve the appearance of the composition. Specific examples of the aforementioned pharmaceutically acceptable carrier may include, but are not limited to, citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, oxidation. Sodium and calcium salts of magnesium, phosphoric acid and sulfuric acid, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose Substances (such as cellulose esters of alkanoic acids and cellulose alkyl esters), low melting waxes, cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (eg serum albumin), ethylenediaminetetraacetic acid (EDTA) ), dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, glycerol or powder, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol and polyethylene glycol) and other pharmaceutically acceptable Accepted substance.

The diseases or symptoms referred to herein as inhibiting or slowing down the binding of PTX3 to the PTX3 receptor may include epithelial cell carcinoma and glioblastoma multiforme (GBM), wherein epithelial cell carcinoma may include, for example, lung cancer, breast cancer, and Nasopharyngeal cancer.

In use, the above monoclonal antibody or antigen-binding fragment thereof can be used to administer an effective amount of the above monoclonal antibody or antigen-binding fragment thereof or the above-described pharmaceutical composition to tumor cells, thereby inhibiting or slowing down the activity of tumor cells. The "effective dose" as referred to in the present invention means that 2 mg to 10 mg of the PTX3 monoclonal antibody or antigen-binding fragment thereof is administered per kilogram of body weight, and the dose is administered once a week. In another embodiment, an effective dose of the aforementioned PTX3 monoclonal antibody or antigen-binding fragment thereof may be, for example, 5 mg/kg body weight to 10 mg/kg body weight, and preferably 6 mg/kg body weight to 9 mg/kg body weight. It is stated herein that if the effective dose of the PTX3 monoclonal antibody is less than 2 mg/kg body weight, it is not possible to effectively reduce or inhibit or slow the binding of PTX3 to the PTX3 receptor within a preset time.

The activity of the tumor cells referred to herein is not limited, but may be, for example, proliferation, cancer stemness, migration, invasion, metastasis, tumor volume or drug resistance. (drug resistance).

The above pharmaceutical composition can be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, orthotopic injection, oral administration, oral and nasal inhalation, etc., thereby specifically inhibiting Endogenous PTX3 activity, which in turn inhibits or slows the activity of cancer cells. Specifically, it has been confirmed by in vitro cell experiments that the monoclonal antibody or antigen-binding fragment thereof of the present invention and the pharmaceutical composition containing the same can inhibit or slow down the cancer cells after being used for a predetermined period of time, for example, from 4 weeks to 11 weeks. active.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Movement and retouching.

Example 1 Preparation of PTX3 Monoclonal Antibody

In this example, a PTX3 monoclonal antibody that specifically recognizes the C-terminal amino acid sequence of the PTX3 recombinant protein is prepared by a conventional fusion tumor method or recombinant protein expression method.

Briefly, the recombinant PTX3 protein of the non-denatured amino acid sequence set forth in SEQ ID NO: 3 was used as an immunogen, and Balb/C mice or PTX3 were injected intraperitoneally (ip) at a dose of 50 μg per mouse. The knockout (PTX3knockout) mice were intraperitoneally, and after 2 weeks, the mice were boosted with a dose of 50 μg per mouse, twice a week for four times. Next, the activated splenocytes are fused with melanoma cells to produce a fusion tumor cell line.

The culture supernatant of the fusion tumor cell strain obtained above was collected to purify the PTX3 monoclonal antibody via a commercially available column, and then the complementation decision of the heavy chain variable region and the light chain variable region was analyzed by Taiwan Weiqiao Biomedical Co., Ltd. The amino acid sequence of the complementarity-determining region (CDR) and the corresponding nucleic acid sequence, the amino acid sequence of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region having The amino acid sequence set forth in SEQ ID NO: 2.

Further, the above PTX3 monoclonal antibody was analyzed by a commercially available monoclonal antibody typing kit, and it was confirmed that the antibody was classified into IgG1k.

Example 2: Assessing the effect of PTX3 monoclonal antibody on cancer cell activity

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

1. Assess the effect of PTX3 monoclonal antibody on cancer cell migration

In the migration experiment, the above cancer cells were seeded at a cell density of 1 × 10 ⁵ cells/well in an upper layer (bottom pore diameter of 8 μm) of a 24-well Boyden cell transitioner (8 μm), and cultured for 3 hours. Next, replace the upper cell culture medium with a serum-free cell culture medium, and add 2.5 μg/mL of PTX3 recombinant protein to the lower serum-free cell culture medium (as listed in SEQ ID NO: 4). Denatured amino acid sequence), 2 μg/mL of PTX3 monoclonal antibody or 2 μg/mL of control group antibody (IgG1k).

After 16 hours of culture, the cells on the inner side of the upper layer were scraped with a cotton swab and transferred to the cells outside the bottom of the upper layer using 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole DAPI; Invitrogen) was stained, and the number of cells moving to the outer side of the upper layer was calculated under a field of magnification of 200 times the magnification of the fluorescence microscope. The results are shown in FIGS. 1A, 2A, 3A, and 4A.

Please refer to FIG. 1A, FIG. 2A, FIG. 3A and FIG. 4A, which respectively illustrate the inhibition of breast cancer cell line MB231 ( FIG. 1A ), lung cancer cell A549 ( FIG. 2A ) and nasopharyngeal carcinoma by using PTX3 monoclonal antibody of Example 1 of the present invention. Straight bar graph of the number of transition cells in cells HONE1 (Fig. 3A) and glioblastoma cell line U87MG (Fig. 4A). The data of the above embodiments are obtained by comparing the three repeated experimental data of each time point and each sample with the mean standard deviation, and all the values are analyzed by one way ANOVA. . The figure number "**" of the above embodiment represents data having statistical significance (P < 0.01), and the figure number "***" represents data having statistical significance (P < 0.001).

From the results of FIG. 1A, FIG. 2A, FIG. 3A and FIG. 4A, the PTX3 monoclonal antibody significantly inhibited or slowed down breast cancer cell line MB231, lung cancer cell line A549, nasopharyngeal carcinoma cell line HONE1 and gliacin compared to the control group antibody IgG1k. The number of transitional cells of the cell tumor cell line U87MG, and this difference was statistically significant.

2. Assess the effect of PTX3 monoclonal antibody on cancer cell invasion

In the invasion experiment, the upper layer (bottom pore size of 8 μm) of the 24-well Bodenden cell transition chamber was previously coated with a basement membrane matrix (matrigel, purchased from BD Bioscience), and the above cancer cells were 1×. The cell density of 10 ⁵ cells/well was seeded in the upper layer of a 24-well Borden cell transferer and cultured for 3 hours. Next, replace the upper cell culture medium with a serum-free cell culture medium, and add 2.5 μg/mL of PTX3 recombinant protein to the lower serum-free cell culture medium (as listed in SEQ ID NO: 4). Denatured amino acid sequence), 2 μg/mL of PTX3 monoclonal antibody or 2 μg/mL of control group antibody (IgG1k).

After 16 hours of culture, the cells on the inner side of the upper layer were scraped with a cotton swab and transferred to the cells outside the bottom of the upper layer using 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole DAPI; Invitrogen) was stained, and the number of cells moving to the outer side of the upper layer was calculated under a field of magnification of 200 times the magnification of the fluorescence microscope, and the results are shown in FIGS. 1B, 2B, 3B, and 4B.

Please refer to FIG. 1B, FIG. 2B, FIG. 3B and FIG. 4B, which respectively illustrate the inhibition of breast cancer cell line MB231 ( FIG. 1B ), lung cancer cell A549 ( FIG. 2B ) and nasopharyngeal carcinoma by using PTX3 monoclonal antibody of Example 1 of the present invention. The number of invading cells in the cells HONE1 (Fig. 3B) and the glioblastoma cell line U87MG (Fig. 4B), wherein the figure number "**" represents statistically significant (P < 0.01) data, the figure number " ***" represents data with statistical significance (P < 0.001).

From the results of FIG. 1B, FIG. 2B, FIG. 3B and FIG. 4B, the PTX3 monoclonal antibody significantly inhibited or slowed down breast cancer cell line MB231, lung cancer cell line A549, nasopharyngeal carcinoma cell line HONE1 and gliacin compared to the control group antibody IgG1k. The number of invading cells of the cell tumor cell line U87MG, and this difference was statistically significant.

3. Assess the effect of PTX3 monoclonal antibody on cancer cell migration

The above cancer cells have cancer stemness, and the addition of the PTX3 monoclonal antibody causes the aforementioned cancer cells to form cell globules.

In the cell pellet experiment, the above cancer cells were added to a 10% fetal bovine serum containing 2.5 μg/mL of PTX3 recombinant protein, 2 μg/mL of PTX3 monoclonal antibody or 2 μg/mL of control group antibody (IgG1k). Fetal Bovine Serum; FBS) RPMI-1640 cell culture medium (containing 10% fetal bovine serum (FBS), 50-100 μg/mL streptomycin and 50-100 U/mL penicillin], and Incubation at a concentration of 5% carbon dioxide at 37 ° C to maintain humidity, which is generally known in the art to which the present invention pertains, and therefore will not be further described.

Then, the above-mentioned differently treated cancer cells were seeded at a cell density of 5 × 10 ³ cells/well in a multi-well plate with ultra-low attachment surface (Corning Inc.) to Serum-free cell culture medium DMEM/F12 (Gibco) [B27 (Invitrogen), 20 ng/mL epidermal growth factor (EGF; Abcam) and 10 ng/mL basic fibroblasts) Growth factor (basic Fibroblast Growth Factor (bFGF; Peprotech)] was co-cultured. After 2 weeks of culture, the number of cell pellets was observed by an optical microscope, and the results are shown in Fig. 1C, Fig. 2C, Fig. 3C, and Fig. 4C.

Please refer to FIG. 1C, FIG. 2C, FIG. 3C and FIG. 4C, which respectively illustrate the inhibition of breast cancer cell line MB231 (FIG. 1C), lung cancer cell A549 (FIG. 2C), nasopharyngeal carcinoma by using PTX3 monoclonal antibody of Example 1 of the present invention. The cell pellet number of the cell HONE1 (Fig. 3C) and the glioblastoma cell line U87MG (Fig. 4C), wherein the figure number "**" represents statistically significant (P < 0.01) data, the figure number "***" represents data with statistical significance (P < 0.001).

As can be seen from the results of FIG. 1C, FIG. 2C, FIG. 3C and FIG. 4C, the PTX3 monoclonal antibody of Example 1 can significantly inhibit or slow down breast cancer cell line MB231, lung cancer cell A549, nasopharyngeal carcinoma cell line HONE1 and the control group antibody IgG1k. The number of cell pellets of the glioma cell line U87MG, and this difference was statistically significant.

4. To evaluate the difference in the epitope localization region of the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody binding to PTX3.

In this example, the epitope localization region of the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (model: ab90806; abcam plc., UK) for PTX3 binding was evaluated in the same manner as in Example 3. The result is shown in Figure 5. Each value is three repetitions.

5 is a map showing the epitope binding of a PTX3 monoclonal antibody and a commercially available PTX3 monoclonal antibody to different PTX3 recombinant proteins, wherein PTX3/FL represents SEQ ID NO according to an embodiment of the present invention. : a PTX3 recombinant protein fragment listed in 4, RI37 represents a PTX3 recombinant protein fragment as set forth in SEQ ID NO: 5, KT37 represents a PTX3 recombinant protein fragment as set forth in SEQ ID NO: 6, and GI40 represents SEQ ID NO: 7 The PTX3 recombinant protein fragment listed, while the figure number "***" represents statistical significance (p < 0.001) compared to the control group (ie, the BSA group).

As can be seen from the results of FIG. 5, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) have higher affinity for PTX3/FL (SEQ ID NO: 4), but the PTX3 monoclonal of Example 1. The antibody PTX3 monoclonal antibody has higher affinity for the PTX3 recombinant protein fragment of RI37 (SEQ ID NO: 5) than the commercially available PTX3 monoclonal antibody (ab90806), and is statistically significant, representing the PTX3 monoclonal antibody and the commercially available PTX3 monoclonal The antibody (ab90806) does have a difference in the epitope localization region for PTX3 binding.

5. To evaluate the effect of the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody on the activity of cancer cells.

In this example, the inhibitory effect of the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (model: ab90806; abcam plc., UK) on cancer cell activity was evaluated in the same manner as in Example 2. As shown in Figures 6A to 9C.

Please refer to FIG. 6A, FIG. 7A and FIG. 8A, which respectively illustrate the inhibition of breast cancer cell line MB231 ( FIG. 6A ) and lung cancer cell A549 by using the PTX3 monoclonal antibody of the first embodiment of the present invention or with the commercially available PTX3 monoclonal antibody ( FIG. 7A ). And the nasopharyngeal carcinoma cell line HONE1 (Fig. 8A), the transition cell number bar graph, wherein the figure number "**" represents statistically significant (P < 0.01) data, and the figure number "***" represents statistically significant Sexual (P < 0.001) data.

From the results of FIG. 6A, FIG. 7A and FIG. 8A, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) significantly inhibited or slowed down breast cancer cell line MB231 and lung cancer cells compared to the control group antibody IgG1k. The number of transitional cells of A549 and nasopharyngeal carcinoma cell line HONE1, but the effect of PTX3 monoclonal antibody of Example 1 on inhibiting or slowing the migration of cancer cells was significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and was statistically significant.

Please refer to FIG. 6B, FIG. 7B and FIG. 8B, which respectively illustrate the inhibition of breast cancer cell line MB231 ( FIG. 7B ) and lung cancer cell A549 by using the PTX3 monoclonal antibody of the first embodiment of the present invention or with the commercially available PTX3 monoclonal antibody ( FIG. 8B ). And the nasopharyngeal carcinoma cell line HONE1 (Fig. 9B), the number of invading cells is a bar graph, wherein the figure number "**" represents statistically significant (P < 0.01) data, and the figure number "***" represents statistically significant Sexual (P < 0.001) data.

From the results of FIG. 6B, FIG. 7B and FIG. 8B, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) significantly inhibited or slowed down breast cancer cell line MB231 and lung cancer cells compared to the control group antibody IgG1k. The number of invading cells of A549 and nasopharyngeal carcinoma cell line HONE1, but the effect of the PTX3 monoclonal antibody of Example 1 on inhibiting or slowing the invasion of cancer cells was significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and was statistically significant.

Please refer to FIG. 9A, FIG. 9B and FIG. 9C, which respectively illustrate the inhibition of breast cancer cell line MB231 ( FIG. 9A ) and lung cancer cell A549 by using the PTX3 monoclonal antibody of Example 1 of the present invention or with a commercially available PTX3 monoclonal antibody ( FIG. 9B ). And the cell pellet number of the nasopharyngeal carcinoma cell line HONE1 (Fig. 9C), wherein the figure number "**" represents statistically significant (P<0.01) data, and the figure number "***" represents statistical Significant (P < 0.001) data.

From the results of FIG. 9A, FIG. 9B and FIG. 9C, the PTX3 monoclonal antibody of Example 1 and the commercially available PTX3 monoclonal antibody (ab90806) significantly inhibited or slowed down breast cancer cell line MB231 and lung cancer cells compared to the control group antibody IgG1k. The number of cell pellets of A549 and nasopharyngeal carcinoma cell line HONE1, but the effect of the PTX3 monoclonal antibody of Example 1 on inhibiting or slowing down the number of cell pellets was still significantly higher than that of the commercially available PTX3 monoclonal antibody (ab90806), and was statistically significant. Sex.

Example 3: Assessing the effect of PTX3 monoclonal antibody on tumor growth and metastasis in vivo

1. Evaluation of the PTX3 monoclonal antibody of Example 1 inhibiting the growth and metastasis of orthotopic xenograft tumors in vivo

In this embodiment, the human cancer cell line is injected into the mammary fat pad of the immunodeficient mouse in situ, and after the tumor is formed, the PTX3 monoclonal antibody of Example 1 is administered, thereby evaluating the PTX monoclonal of the first embodiment. Antibodies inhibit or slow the effects of tumors.

First, breast cancer cell line MB231-Luc2 [MB231 is a human breast cancer cell, does not exhibit estrogen receptor (ER) α and ERβ; Luc2 is a gene exhibiting luciferase] is inoculated in situ to NOD-SCID mice. (purchased from Leko Biotech Co., Ltd., Taiwan) in the breast fat pad. After the average volume of the tumor reached 80 mm ³ , the experimental mice were administered the PTX3 antibody (8 mg/kg body weight) of the first example or the control group antibody (IgG1k, 8 mg/kg body weight) once a week, and the results are shown in Fig. 10A. It is shown in Figure 10B. Fig. 10B is a result of in vivo imaging at week 11 after inoculation of breast cancer cell line MB231-Luc2, wherein the luminescent image represents a tumor having a formation of breast cancer cell line MB231-Luc2 in mice. Then, all mice were sacrificed, the tumor size in vivo was measured, and the tumor volume was calculated using the following formula (I):

V=(w×l ² )×0.52 (I)

In formula (I), l represents the length of the tumor and w represents the width of the tumor.

Please refer to FIG. 10A to FIG. 10B, which respectively show that the PTX3 monoclonal antibody or the control group antibody inhibits the tumor volume of mouse orthotopically transplanted breast cancer cell line MDA-MB231 ( FIG. 10A ) and tumor metastasis, respectively, according to an embodiment of the present invention. (Fig. 10B) results. The data in Fig. 10A is obtained by taking six replicate experimental data at each time point and each sample, taking the positive and negative mean standard deviations, and the figure number "*" represents statistically significant compared to the control group antibody (IgG1k). Sex (p<0.05).

From the results of FIGS. 10A and 10B, the PTX3 monoclonal antibody of Example 1 significantly inhibited or slowed down the tumor volume and tumor metastasis of the xenografted breast cancer cell line MB231 compared to the control group antibody (IgG1k), and this Item differences are statistically significant.

2. Evaluation of the PTX3 monoclonal antibody of Example 1 inhibiting the growth and metastasis of in situ allograft tumors in vivo

In this embodiment, the human cancer cell line is injected in situ into the mammary fat pad of the normal normal mouse, and after the tumor is formed, the PTX3 monoclonal antibody of Example 1 is administered, thereby evaluating the PTX of the first embodiment. Monoclonal antibodies inhibit the effects of tumors.

First, breast cancer cells 4T1-Luc2 (4T1 is a mouse breast cancer cell line, showing ERβ but not ERα) were inoculated in situ to wild-type BALB/c mice (purchased from Lesco Biotech Co., Ltd., Taiwan). The mammary fat pad. After the average volume of the tumor reached 80 mm ³ , the experimental mice were administered the PTX3 antibody (8 mg/kg body weight) or the control group antibody (IgG1k, 8 mg/kg body weight) of Example 1 once a week, and the results are shown in Fig. 11A. This is shown in Figure 11B. Fig. 11B is the result of in vivo imaging at week 11 after inoculation of breast cancer cells 4T1-Luc2, wherein the luminescent image represents a tumor having a breast cancer cell 4T1-Luc2 formation in mice. Then, all mice were sacrificed, the tumor size in vivo was measured, and the tumor volume was calculated using the above formula (I).

Please refer to FIG. 11A to FIG. 11B, which respectively show that the PTX3 monoclonal antibody or the control group antibody inhibits the tumor volume of the mouse in situ xenografted breast cancer cell 4T1 ( FIG. 11A ) and tumor metastasis ( FIG. 11A ) according to an embodiment of the present invention. The result of Figure 11B). The data in Fig. 11A is obtained by taking six replicate experimental data at each time point and each sample, taking the positive and negative mean standard deviations, and the figure number "**" means having statistics compared to the control group antibody (IgG1k). Significant (p < 0.01).

From the results of FIGS. 11A and 11B, the PTX3 monoclonal antibody of Example 1 can significantly inhibit or slow down the tumor volume and tumor metastasis of in situ allograft breast cancer cell 4T1 compared to the control group antibody (IgG1k), and This difference is statistically significant.

In summary, the present invention exemplifies a pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof of the present invention and a use thereof, using a specific sequence of a PTX3 monoclonal antibody, a specific analysis mode or a specific evaluation mode, and the use thereof. The present invention is not limited thereto, and the pharmaceutical composition containing the monoclonal antibody or antigen-binding fragment thereof of the present invention and use thereof can be used without departing from the spirit and scope of the present invention. Other analysis modes or other evaluation methods can also be used.

It can be seen from the above examples that the pharmaceutical composition containing the monoclonal antibody or the antigen-binding fragment thereof of the present invention and the use thereof have the advantages of using a specific PTX3 monoclonal antibody or an antigen-binding fragment thereof as an active ingredient, and specifically inhibiting or The binding of PTX3 to the PTX3 receptor is slowed down, thereby inhibiting or slowing the disease or condition associated with PTX3 binding to the PTX3 receptor.

While the invention has been described above in terms of several embodiments, it is not intended to limit the invention, and it is to be construed as being limited by the scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims

A pharmaceutical composition comprising an effective amount of a monoclonal antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, and the monoclonal antibody or antigen-binding fragment thereof is used as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the effective dose is from 2 mg to 10 mg/kg of body weight per kilogram of body weight.
The pharmaceutical composition according to claim 1, wherein the effective dose is from 5 mg/kg body weight to 10 mg/kg body weight.
The pharmaceutical composition according to claim 1, wherein the effective dose is from 6 mg/kg body weight to 9 mg/kg body weight.
Use of a monoclonal antibody or antigen-binding fragment thereof for the preparation of a pharmaceutical composition for specifically inhibiting or slowing the binding of a pentraxin-related protein (PTX3) to a PTX3 receptor, wherein The monoclonal antibody or antigen-binding fragment thereof is an active ingredient, and the monoclonal antibody or antigen-binding fragment thereof has an effective dose to inhibit or slow down the disease or symptom associated with the binding of the PTX3 to the PTX3 receptor.
Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 5 for the preparation of a pharmaceutical composition which specifically inhibits or slows the binding of PTX3 to the PTX3 receptor, characterized in that the disease or the symptom comprises epithelial cell carcinoma And glioblastoma multiforme (GBM).
The use of the monoclonal antibody or antigen-binding fragment thereof according to claim 5 for the preparation of a pharmaceutical composition for specifically inhibiting or slowing down the binding of PTX3 to a PTX3 receptor, characterized in that the epithelial cell carcinoma comprises lung cancer, breast cancer and Nasopharyngeal cancer.
The use of the monoclonal antibody or antigen-binding fragment thereof according to claim 5 for the preparation of a pharmaceutical composition which specifically inhibits or slows the binding of PTX3 to the PTX3 receptor, wherein the pharmaceutical composition is administered by subcutaneous injection, Intramuscular injection, intravenous injection, intraperitoneal injection, in situ injection, oral administration, oral and nasal inhalation.
A method for inhibiting or slowing the activity of a tumor cell in vitro, comprising administering an effective dose to the tumor cell, the pharmaceutical composition according to any one of claims 1 to 4, thereby inhibiting or slowing down the tumor cell Activity.
The method for inhibiting or slowing the activity of a tumor cell in vitro according to claim 9, wherein the source of the tumor cell comprises glioblastoma and epithelial cell carcinoma.
The method for inhibiting or slowing down the activity of a tumor cell in vitro according to claim 9, wherein the epithelial cell carcinoma comprises lung cancer, breast cancer, and nasopharyngeal cancer.
The method for inhibiting or slowing the activity of a tumor cell in vitro according to claim 9, wherein the activity comprises proliferation, cancer stem cell, migration, invasion, metastasis, tumor volume or drug resistance.