TWI741216B - Monoclonal antibody or antigen-binding fragment for specifically inhibiting or alleviate the binding of ptx3 and ptx3 receptor and use of the same - Google Patents

Abstract

The present invention relates to a monoclonal antibody or antigen-binding fragmen a use of the same. The monoclonal antibody or antigen-binding fragment can specifically inhibit or alleviate the binding of PTX3 and PTX3 receptor, thereby being applied on a kit and a method of detecting PTX3.

Description

Specifically inhibit or slow down the binding of PTX3 and PTX3 receptors Combined monoclonal antibody or its antigen-binding fragment and its use

The present invention relates to an antibody and its use, in particular to a monoclonal antibody or antigen-binding fragment thereof that specifically inhibits or slows the binding of the specific sequence of the C-terminal of PTX3 to the PTX3 receptor, and its use in detection reagents.

At present, cancer cells are known to stimulate the microenvironment surrounding the tumor to produce various inflammatory factors, white blood cells, hyperproliferation of blood vessels, and proteases. The chronic inflammatory response of cancer is also related to the growth, metastasis and invasion of cancer cells. However, its formation reasons and detailed mechanisms , There are still many unclear points.

In addition to tumor microenvironment related to inflammation, other studies have also pointed out that tumor microenvironment is also closely related to tumor metastasis (metastasis) and chemotherapy resistance (chemoresistance). The tumor microenvironment is composed of a variety of stromal cells and other different types of cells. It not only protects tumors, but also allows tumor cells to escape and resist immune cells, resulting in drug resistance of tumor cells.

After the fibroblasts and macrophages in the stromal tissues around the tumor are activated by CEBPD, they induce the production of secreted factor-pentraxin-related protein 3 (PTX3), which has the activity of promoting angiogenesis , And can increase the ability of nasopharyngeal carcinoma cells to migrate and invade tissues (or invasion). In addition, past studies have also confirmed that the activation of CEBPD in the tissue cells surrounding the cancer may also promote cancer metastasis, and even promote the production of drug-resistant cancer cells during chemotherapy. These drug-resistant cancer cells will grow faster and metastasize more easily.

In view of this, it is urgent to develop an antibody that specifically binds to PTX3 to detect the content of PTX3 in biological samples.

Therefore, one aspect of the present invention is to provide a monoclonal antibody or antigen-binding fragment thereof, which specifically binds to the C-terminal specific sequence of one or more pentraxin-related proteins (PTX3).

Another aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region sequence and a light chain variable region sequence of a specific sequence.

Another aspect of the present invention is to provide a kit for detecting PTX3, comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof.

Another aspect of the present invention is to provide an in vitro test The method of PTX3 uses the above kit to detect PTX3.

According to the above aspect of the present invention, a monoclonal antibody or an antigen-binding fragment thereof is provided. In one embodiment, the aforementioned monoclonal antibody or antigen-binding fragment thereof can specifically bind to the non-denatured amino acid sequences listed in SEQ ID NO:1 to SEQ ID NO:11.

According to another aspect of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence may be, for example, SEQ ID NO: 18 , SEQ ID NO: 19 and/or SEQ ID NO: 20 listed amino acid sequence, the light chain variable region sequence can be, for example, SEQ ID NO: 21, SEQ ID NO: 22 and/or SEQ ID NO: 23 List the amino acid sequence.

In some embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof can be encoded by the nucleic acid sequence listed in SEQ ID NO: 24, SEQ ID NO: 25 and/or SEQ ID NO: 26, for example. The amino acid sequence, the light chain variable region sequence may be, for example, the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 27, SEQ ID NO: 28 and/or SEQ ID NO: 29.

In other embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof can be, for example, the amino acid sequence listed in SEQ ID NO: 30, and the light chain variable region sequence can be, for example, SEQ ID NO: 31 The listed amino acid sequence.

In other embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof can be, for example, the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 32, and the light chain variable region sequence can be, for example, SEQ ID The amino acid sequence encoded by the nucleic acid sequence listed in NO: 33.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof. In one example, the aforementioned monoclonal antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof may be, for example, a single-chain variable fragment (scFv) or a single-chain variable fragment dimer [(scFv) ₂ ] , Single-chain variable region fragment trimer [(scFv) ₃ ], variable fragment (Fv), Fab fragment, Fab' fragment, F(ab') ₂ fragment or any combination of the above.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is modified through conjugation or conjugation, glycation, tag attachment, or any combination of the foregoing.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or its antigen-binding fragment is an antibody-drug conjugate (ADC) or its antigen-binding fragment.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or its antigen-binding fragment is a bispecific monoclonal antibody (BsAb) or its antigen-binding fragment.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or antigen-binding fragment thereof.

According to an embodiment of the present invention, the aforementioned non-denatured amino acid sequence It may include, but is not limited to, the amino acid sequence listed in any one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 or any combination of the foregoing. In other embodiments, the aforementioned non-denatured amino acid sequence may include, but is not limited to, the amino acid sequence listed in any one of SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 or any combination of the foregoing .

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class, the IgM class, or the IgA class. In another embodiment, the aforementioned monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is an inert antibody or an antagonist antibody.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the binding of the PTX3 receptor to one or more PTX3. In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof inhibits or slows the activity of one or more PTX3. In other examples, the aforementioned monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the interaction of the PTX3 receptor with one or more PTX3, inhibits or slows the transmission of PTX3, or any combination of the foregoing.

According to another aspect of the present invention, a kit for detecting PTX3 is provided, comprising a monoclonal antibody or antigen-binding fragment thereof as described above, wherein the monoclonal antibody or antigen-binding fragment thereof can specifically bind to non-denatured amine groups Acid sequence, and this non-denatured amino acid sequence may include, but is not limited to, the amino acid sequence listed in any one of SEQ ID NO:1 to SEQ ID NO:11.

According to another aspect of the present invention, an in vitro test is provided The method of PTX3 uses the above-mentioned PTX3 detection kit to detect PTX3, wherein the analysis sensitivity of the monoclonal antibody or antigen-binding fragment thereof contained in the PTX3 detection kit can be, for example, not less than 0.244ng/mL.

Application of the monoclonal antibody or its antigen-binding fragment of the present invention, which uses specific PTX3 monoclonal antibody or its antigen-binding fragment to specifically inhibit or slow down the binding of PTX3 and PTX3 receptor, which can be applied to the kit and detection of PTX3 PTX3 content method.

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [FIG. 1] A PTX3 monoclonal antibody pair according to an embodiment of the present invention Affinity graph of PTX3 recombinant protein.

[Fig. 2] and [Fig. 3] are the epitope mapping maps of the binding of PTX3 monoclonal antibody to different fragments of PTX3 recombinant protein according to an embodiment of the present invention.

[Figure 4] is a graph showing the competitive inhibition of the PTX3 monoclonal antibody blocking the binding of the PTX3 recombinant protein to the CD44 receptor according to an embodiment of the present invention.

Based on the foregoing, the present invention provides a monoclonal antibody or its antigen-binding fragment and its use, which utilizes the monoclonal antibody or its antigen-binding fragment to specifically inhibit pentraxin-related protein (pentraxin-related protein). The combination of protein; PTX3) and PTX3 receptor can be applied to kits and methods for detecting PTX3.

The monoclonal antibody or antigen-binding fragment thereof referred to herein may include a heavy chain variable region sequence and a light chain variable region sequence of a specific sequence to specifically inhibit or slow down the C-terminal specific sequence of PTX3 and PTX3 receptor. Combine.

In one embodiment, the aforementioned monoclonal antibody or antigen-binding fragment thereof can specifically bind to the C-terminal amino acid sequence of human PTX3, and the sequence range is not limited, such as those shown in NO:1 to SEQ ID NO:17. The non-denatured amino acid sequences listed are preferably combined with the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11, and the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11 are preferably combined. The non-denatured amino acid sequences listed in SEQ ID NO: 5 and 11 are preferred, and the non-denatured amino acid sequences listed in SEQ ID NO: 2 to SEQ ID NO: 4 are more preferably combined specifically. In the above embodiments, the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11 correspond to the 200th amino acid to the 236th amino acid sequence of the non-denatured amino acid sequence of the human PTX3 recombinant protein. An amino acid. In another example, the aforementioned non-denatured amino acid sequence listed in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 corresponds to the 200th non-denatured amino acid sequence of the human PTX3 recombinant protein. Amino acid to the 220th amino acid. In another example, the aforementioned non-denatured amino acid sequence listed in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 corresponds to the 203rd of the non-denatured amino acid sequence of the human PTX3 recombinant protein. Amino acids to the 217th amino acid.

In one embodiment, the aforementioned 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 may, for example, have SEQ ID NO: 18 and SEQ ID NO: 19 and/or the amino acid sequence listed in SEQ ID NO: 20, the light chain variable region sequence may be, for example, the amino acid sequence listed in SEQ ID NO: 21, SEQ ID NO: 22 and/or SEQ ID NO: 23 sequence.

In other embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof may have a nucleic acid sequence code as listed in SEQ ID NO: 24, SEQ ID NO: 25 and/or SEQ ID NO: 26 The amino acid sequence of the light chain variable region may have the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 27, SEQ ID NO: 28 and/or SEQ ID NO: 29.

In other embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof may have an amino acid sequence as listed in SEQ ID NO: 30, and the light chain variable region sequence may have as SEQ ID NO: 30 ID NO: 31 listed amino acid sequence.

In other embodiments, the heavy chain variable region sequence of the aforementioned monoclonal antibody or antigen-binding fragment thereof may have an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 32, and the light chain variable region sequence may It has the amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO:33.

In some embodiments, the types of monoclonal antibodies or antigen-binding fragments thereof are not limited, and may be, for example, chimeric antibodies or antigen-binding fragments thereof. In other examples, the aforementioned monoclonal antibody or antigen-binding fragment thereof may be, for example, a humanized antibody or antigen-binding fragment thereof.

In some embodiments, the structure of the aforementioned monoclonal antibody or antigen-binding fragment thereof is not limited. Under the premise of considering the stability of the antibody structure, it can be a complete antibody structure or a simplified antibody structure, such as a single-chain variable region fragment. (single-chain variable fragment; scFv), single-chain variable region fragment dimer [(scFv) ₂ ], single-chain variable region fragment trimer [(scFv) ₃ ], variable region fragment (variable fragment; Fv), Fab fragments, Fab' fragments, F(ab') ₂ fragments or any combination of the above, thereby simplifying the manufacturing process of recombinant antibodies. The aforementioned monoclonal antibodies or antigen-binding fragments thereof can be carried out using fusion tumor cells or recombinant gene expression, etc., which are well known to those with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here.

In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof can be further modified through conjugation or binding, saccharification, tag attachment, or any combination of the foregoing, depending on actual needs. For example, the aforementioned monoclonal antibody or antigen-binding fragment thereof can further form an antibody-drug conjugate (ADC) or antigen-binding fragment thereof with the drug.

In some embodiments, the aforementioned 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.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is modified through conjugation or conjugation, glycation, tag attachment, or any combination of the foregoing.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or its antigen The binding fragment is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or its antigen-binding fragment is a bispecific monoclonal antibody (BsAb) or its antigen-binding fragment.

According to an embodiment of the present invention, the aforementioned monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or antigen-binding fragment thereof.

In one embodiment, the aforementioned monoclonal antibody or antigen-binding fragment thereof may belong to the IgG class, the IgM class, or the IgA class. In a specific embodiment, the aforementioned monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype. In one example, the aforementioned monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype, for example, an IgG1k isotype. In some specific examples, the aforementioned monoclonal antibodies or antigen-binding fragments thereof are inert antibodies or antagonist antibodies. In some specific examples, the aforementioned monoclonal antibodies or antigen-binding fragments thereof can specifically inhibit or slow down the activity of one or more PTX3. In other specific examples, the aforementioned monoclonal antibodies or antigen-binding fragments thereof can specifically inhibit or slow down the interaction between the PTX3 receptor and one or more PTX3, inhibit or slow down the transmission of PTX3 messages, or any combination of the above.

In application, the aforementioned monoclonal antibodies or antigen-binding fragments thereof can be used in the kits and methods for detecting PTX3, by specifically binding to the non-denatured amino acid sequences listed in SEQ ID NO:1 to SEQ ID NO:11, Improve the analytical sensitivity of PTX3 in biological samples. The form of the biological sample referred to in the present invention is not limited, and may include, but is not limited to, for example, cells, tissues, blood, Urine, lymphatic fluid, tissue fluid, body fluid, etc. The above-mentioned detection kit for PTX3 can be applied to various conventional detection components/equipment, such as flow cytometer, enzyme-linked immunosorbent assay (ELISA) detection reagent kit, biochip, etc.; or applied to conventional detection kits. Know the detection methods such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining and Western bloting analysis Wait. In a specific example, the analytical sensitivity (analytical sensitivity) of the aforementioned monoclonal antibody or its antigen-binding fragment can also be referred to as the lower limit of detection (LLOD), which is generally not less than 0.244ng/mL .

Several embodiments are used below to illustrate the application of the present invention, but they are not used to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.

Example 1. Preparation of PTX3 monoclonal antibody

This example uses the conventional fusion tumor method or recombinant protein expression method to prepare a PTX3 monoclonal antibody that specifically recognizes the C-terminal amino acid sequence of the PTX3 recombinant protein.

In short, the PTX3 recombinant protein of the non-denatured amino acid sequence listed in SEQ ID NO: 13 was used as the immunogen, and 50 μg per mouse was injected intraperitoneally (ip) into Balb/C mice or PTX3 knockout mice were immunized in the abdominal cavity with a dose of 50 μg per mouse two weeks later, once every two weeks, for a total of four times. Then, the activated spleen cells are fused with the melanoma cells to produce a fusion tumor cell line.

From the above fusion tumor cell lines, a fusion tumor cell line with higher binding affinity to the recombinant protein of the non-denatured amino acid sequence listed in SEQ ID NO: 11 was screened, and the PTX3 monomer produced by the obtained fusion tumor cell line was selected. Strain antibodies can specifically bind to the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11.

After collecting the culture supernatant of the fusion tumor cell line obtained above and purifying the PTX3 monoclonal antibody through a commercially available column, it was entrusted to Taiwan Weiqiao Biomedical Co., Ltd. to analyze the complementarity determination of the variable region of the heavy chain and the variable region of the light chain. Region (complementarity-determining region; CDR) amino acid sequence and corresponding nucleic acid sequence, the amino acid sequence of its heavy chain variable region has the amino acid sequence listed in SEQ ID NO: 30 or as SEQ ID NO : The amino acid sequence encoded by the nucleic acid sequence listed in 32, the amino acid sequence of the light chain variable region has the amino acid sequence listed in SEQ ID NO: 31 or the nucleic acid sequence listed in SEQ ID NO: 33 Coded amino acid sequence.

In addition, the aforementioned PTX3 monoclonal antibody was analyzed by a commercially available monoclonal antibody typing kit, and it was confirmed that its antibody typing was IgG1k.

Example 2: Evaluation of the affinity of PTX3 monoclonal antibodies

In this example, a commercially available ELISA kit was used to evaluate the affinity of the PTX3 recombinant protein and the PTX3 monoclonal antibody of Example 1.

First, 5μg/mL of PTX3 recombinant protein (such as the non-denatured amino acid sequence listed in SEQ ID NO: 14) or bovine serum albumin (BSA; as a control group) was spread on 96-well cell culture In each well of the plate (model: 9018, Corning Costar), react at 4°C until overnight. Then, the barrier solution (phosphate buffered solution (PBS) containing 3% skimmed milk powder) was added to the well, and the barrier reaction was carried out at room temperature (4°C to 40°C) for 1 hour. After removing the barrier solution, rinse each well with PBS, and then add the primary antibody to react at room temperature (4°C to 40°C) for 1 hour. The primary antibody is the serially diluted PTX3 monoclonal antibody of Example 1 (concentration It is 2.44×10 ^-4 μg/mL to 1.00 μg/mL). Then, the unbound PTX3 monoclonal antibody in each well was washed with PBS, and the secondary antibody was added to react at room temperature (4°C to 40°C). After that, tetramethyl benzidine (TMB) was added to each well to react for a period of time, and 0.1M sulfuric acid (H ₂ SO ₄ ) was added to react for 10 minutes to stop the reaction. The secondary antibody was conjugated horseradish peroxide. Hydrogenase (anti-mouse horse peroxidase; HRP) anti-mouse IgG (IgG-HRP). Next, a commercially available enzyme immunoassay (ELISA reader) was used to read the absorbance at 450 nm, and the results are shown in Figure 1. Each value is repeated in three. The reaction time of the above-mentioned secondary antibody is carried out with reference to the manufacturer's operating manual, which should be well known to anyone with ordinary knowledge in the technical field to which the present invention belongs, so it will not be repeated.

Please refer to Figure 1, which shows the affinity curve of the PTX3 monoclonal antibody to the PTX3 recombinant protein according to an embodiment of the present invention, wherein the curve marked with the figure number ● represents the affinity curve of the PTX3 monoclonal antibody to the PTX3 recombinant protein. The curve marked with number ■ represents the effect of BSA on PTX3 recombinant protein Affinity curve.

It can be seen from the results in Fig. 1 that even after ^4-6- fold dilution (antibody concentration equivalent to 0.244ng/mL), the PTX3 monoclonal antibody still maintains a good affinity for the PTX3 recombinant protein and can be applied to the PTX3 detection kit.

In addition, in other experiments, the PTX3 monoclonal antibody can specifically bind to the 200th amino acid to the 359th amino acid of the PTX3 recombinant protein (such as the non-denatured amino acid sequence listed in SEQ ID NO: 13) or The 200th amino acid to the 236th amino acid (such as the non-denatured amino acid sequence listed in SEQ ID NO: 12) (not shown in the figure).

Example Three: Evaluate the epitope localization region of PTX3 monoclonal antibody binding to PTX3

In this example, a commercially available ELISA kit was used to evaluate the epitope mapping region of PTX3 monoclonal antibody binding to PTX3.

This example uses the same method as the first example to evaluate the smaller binding area of the PTX3 monoclonal antibody to PTX3, but the difference is that this example uses 200μg/mL of PTX3 recombinant protein (such as SEQ ID NOs: The non-denatured amino acid sequences listed in 12, 16, and 17, dissolved in a 0.1M sodium bicarbonate aqueous solution, pH 8.3) or BSA (as a control group), are coated in the wells of a 96-well cell culture dish, and reacted at 4°C To overnight. Then, the barrier solution (PBS containing 1% BSA) was added to the well, and the barrier reaction was carried out at room temperature (4°C to 40°C) for 1 hour. After removing the barrier solution, the wells were rinsed with PBS, and then the PTX3 monoclonal antibody (concentration of 125ng/mL) of Example 1 was added to react at room temperature (4°C to 40°C) for 2 hours. Then, use PBS to wash out the holes in each well The bound PTX3 monoclonal antibody was added to the secondary antibody (anti-mouse IgG-HRP, diluted 1:5000) and reacted at room temperature (4°C to 40°C) for 1 hour. Then, after adding TMB to each well to react for a period of time, 0.1M sulfuric acid was added to react for 10 minutes to terminate the reaction, and a commercially available enzyme immunoassay instrument was used to read the absorbance at 450 nm. The result is shown in Figure 2. Each value is repeated in three.

Please refer to Figure 2, which shows an epitope mapping map of the binding of a PTX3 monoclonal antibody to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention, where RI37 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 12 , KT44 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 16, GI40 represents the PTX3 recombinant protein fragment as listed in SEQ ID NO: 17, and the figure number "**" represents the comparison with the control group (ie BSA group) was statistically significant ( p <0.001).

It can be seen from the results in Figure 2 that the affinity of the PTX3 monoclonal antibody to the PTX3 recombinant protein fragment as listed in SEQ ID NO: 12 is higher than that of other fragments and is statistically significant.

Please refer to FIG. 3, which shows the epitope mapping map of the binding of PTX3 monoclonal antibody to different fragments of PTX3 recombinant protein according to an embodiment of the present invention, where the horizontal axis represents the ones listed in SEQ ID NOs: 1 to 10, respectively. PTX3 recombinant protein fragment.

It can be seen from the results in Figure 3 that the affinity of the PTX3 monoclonal antibody to the PTX3 recombinant protein fragments listed in SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 4 is much higher than that of other fragments. Among them, SEQ ID NOs: The PTX3 recombinant protein fragments listed in 2~4 correspond to PTX3 No. 203 to 217 The amino acid, such as the amino acid sequence listed in SEQ ID NO: 11, represents the epitope mapping region where the PTX3 monoclonal antibody of Example 1 binds to PTX3 and is located as listed in SEQ ID NOs: 2~4 or as listed in SEQ ID NOs: 2~4. ID NO: Within the range of the amino acid sequence listed in 11.

Example 4: Evaluation of the effect of PTX3 monoclonal antibody on the binding of PTX3 and CD44 in vitro

The PTX3 monoclonal antibody can competitively bind to the PTX3 receptor binding region of PTX3 or its adjacent region, thereby specifically inhibiting or slowing down the chance of binding between PTX3 and PTX3 receptor. In this example, CD44 was used as an example, and the competitive binding test was used to evaluate the effect of the PTX3 monoclonal antibody on competitively inhibiting the binding of PTX3 to the PTX3 receptor.

This example demonstrates that the PTX3 monoclonal antibody of Example 1 can competitively bind to the binding region of PTX3 and PTX3 receptor (such as CD44) or its adjacent region, which can improve the analytical sensitivity of detecting PTX3.

It should be said that the competitive binding analysis method used in this example is similar to that in Example 1, except that this example uses 10 μg/mL CD44 N-terminal recombinant protein (CD44 N-terminal 1st~ A sequence of 220 amino acid residues, dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing, China) was spread in the wells of a 96-well cell culture dish, and reacted at 4°C until overnight. Then, the barrier solution (PBS containing 3% skimmed milk powder) was added to the well, and the barrier reaction was carried out at room temperature (4°C to 40°C) for 1 hour.

In the competitive binding test, the PTX3 recombinant protein that binds to HRP (such as the non-denatured amino acid sequence listed in SEQ ID NO: 14) Column, HRP-PTX3, concentration of 5μg/mL) and PTX3 monoclonal antibody of Example 1 (concentration of 1μg/mL or 2μg/mL) at different concentrations for pre-reaction at room temperature (4°C to 40°C) for 1 hour , To form a pre-reactant.

After removing the barrier solution, the wells were rinsed with PBS, and the above-mentioned pre-reactant was added, and the reaction was carried out at room temperature (4°C to 40°C) for 2 hours. Then, use PBS to wash off the unbound pre-reactants in each well. After adding TMB to each well for a period of time, add 0.1M sulfuric acid to react for 10 minutes to terminate the reaction, and use a commercially available enzyme immunoassay to read the absorbance at 450nm , And the result is shown in Figure 4. Each value is repeated in four.

Please refer to Figure 4, which is a graph showing the competitive inhibition of the PTX3 monoclonal antibody blocking the binding of the PTX3 recombinant protein to the CD44 receptor according to an embodiment of the present invention, where the vertical axis represents the competitive inhibition rate (%), and the horizontal axis The figure number "+" or "-" below represents the presence or absence of specific ingredients added during the binding reaction. The first straight bar on the left side of Figure 4 represents the control group (that is, the group of PTX3 recombinant protein without PTX3 monoclonal antibody) , And the figure number "＊＊＊" means that it is statistically significant compared to the control group ( p <0.001).

It can be seen from the results in Figure 4 that the value of the first lane on the left side of Figure 4 without PTX3 monoclonal antibody is used as the 0% competitive inhibition rate. When the PTX3 monoclonal antibody reacts with the PTX3 recombinant protein before it reacts 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. It represents that the PTX3 monoclonal antibody of Example 1 can indeed compete with PTX3 for the binding region of CD44 or its adjacent region. And its suppression mechanism belongs to competitive suppression.

It is supplemented that the PTX3 individual plant in Example 1 of the present invention The antibody has good affinity and sensitivity to the PTX3 recombinant protein, and can be applied to the kits and methods for detecting PTX3 to detect the content of PTX3 in biological samples in vitro. The applicable biological samples, methods, kits, components/equipment etc. applicable to the detection of PTX3 are as described above, and no further details will be given.

In summary, although the present invention uses PTX3 monoclonal antibodies with specific sequences, specific analysis modes or specific evaluation methods as examples to illustrate the monoclonal antibodies or antigen-binding fragments thereof of the present invention and their uses, the technical field of the present invention belongs to Anyone with ordinary knowledge in the present invention can know that the present invention is not limited to this. Without departing from the spirit and scope of the present invention, the monoclonal antibody or antigen-binding fragment thereof of the present invention and its use may also use other analysis modes or other methods. The evaluation method is carried out.

It can be seen from the above examples that the monoclonal antibody or its antigen-binding fragment of the present invention and its use have the advantage of using a specific PTX3 monoclonal antibody or its antigen-binding fragment to specifically inhibit or slow down the binding of PTX3 to the PTX3 receptor. Sets and methods used to detect PTX3.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.

<110> Zhenwei Biotechnology Co., Ltd.

<120> Monoclonal antibody or antigen-binding fragment thereof that specifically inhibits or slows the binding of PTX3 and PTX3 receptor and its use

<130>

<150> US 62/560,202

<151> 2017-09-19

<160> 33

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 200th to 208th amino acid

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 203 to 211 amino acids

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 the 206th to 214th amino acids

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 the 209th to 217th amino acids

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 212 to 220 amino acids

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<212> PRT

<213> Artificial sequence

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<223> Human sapiens PTX3 215 to 223 amino acids

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<212> PRT

<213> Artificial sequence

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<223> Human sapiens PTX3 218th to 226th amino acid

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<212> PRT

<213> Artificial sequence

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<223> Human sapiens PTX3 221 to 229 amino acids

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 224 to 232 amino acids

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<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 227th to 236th amino acid

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<213> Artificial sequence

<220>

<223> Human sapiens PTX3 203 to 217 amino acids

<400> 11

<210> 12

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<222> (200)...(236)

<223> Human sapiens PTX3 200th to 236th amino acid (RI37)

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<210> 13

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<212> PRT

<213> Artificial sequence

<220>

<222> (200)...(359)

<223> Human PTX3 Recombinant Protein

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<213> Artificial sequence

<220>

<222> (19)...(381)

<223> Human PTX3 Recombinant Protein

<400> 14

<210> 15

<211> 381

<212> PRT

<213> Artificial sequence

<220>

<222> (1)...(381)

<223> Human PTX3 Recombinant Protein

<400> 15

<210> 16

<211> 44

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 255th to 298th amino acid (KT44)

<400> 16

<210> 17

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 320 to 359 amino acids (GI40)

<400> 17

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the heavy chain variable region of the PTX3 monoclonal antibody

<400> 18

<210> 19

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of the heavy chain variable region of the PTX3 monoclonal antibody

<400> 19

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of the heavy chain variable region of the PTX3 monoclonal antibody

<400> 20

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR1 of the light chain variable region of the PTX3 monoclonal antibody

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR2 of PTX3 monoclonal antibody light chain variable region

<400> 22

<210> 23

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

<223> CDR3 of PTX3 monoclonal antibody light chain variable region

<400> 23

<210> 24

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> Nucleic acid sequence of CDR1 of PTX3 monoclonal antibody heavy chain variable region

<400> 24

<210> 25

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> CDR2 gene of heavy chain variable region of PTX3 monoclonal antibody

<400> 25

<210> 26

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> CDR3 gene of heavy chain variable region of PTX3 monoclonal antibody

<400> 26

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> CDR1 gene of light chain variable region of PTX3 monoclonal antibody

<400> 27

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> CDR2 gene of light chain variable region of PTX3 monoclonal antibody

<400> 28

<210> 29

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

<223> CDR3 gene of light chain variable region of PTX3 monoclonal antibody

<400> 29

<210> 30

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<221> V_region

<223> PTX3 monoclonal antibody heavy chain variable region

<400> 30

<210> 31

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> V_region

<223> PTX3 monoclonal antibody light chain variable region

<400> 31

<210> 32

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<221> CDS

<223> The gene sequence of the heavy chain variable region of the PTX3 monoclonal antibody

<400> 32

<210> 33

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<221> CDS

<223> Gene sequence of light chain variable region of PTX3 monoclonal antibody

<400> 33

Claims (29)

A monoclonal antibody or antigen-binding fragment thereof, characterized in that the monoclonal antibody or antigen-binding fragment thereof specifically binds to a non-denatured amino acid sequence, and the non-denatured amino acid sequence is selected from the sequence identification number (SEQ ID NO:) 2 to SEQ ID NO: 4 and a group of amino acid sequences listed in SEQ ID NO: 11, and the monoclonal antibody or antigen-binding fragment thereof includes: a heavy chain variable region sequence , Wherein the heavy chain variable region sequence has a complementarity-determining region (CDR) as shown in SEQ ID NO: 18, a CDR2 as shown in SEQ ID NO: 19, and a CDR2 as shown in SEQ ID NO: 20 And a light chain variable region sequence, wherein the light chain variable region sequence has the CDR1 shown in SEQ ID NO: 21, the CDR2 shown in SEQ ID NO: 22, and the CDR2 shown in SEQ ID NO: 23 CDR3 shown. The monoclonal antibody or antigen-binding fragment thereof according to item 1 of the scope of patent application, wherein the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO: 2. The monoclonal antibody or antigen-binding fragment thereof according to item 1 of the scope of patent application, wherein the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO:3. The monoclonal antibody or antigen-binding fragment thereof according to item 1 of the scope of patent application, wherein the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO:4. According to the monoclonal antibody described in item 1 of the scope of patent application Or an antigen-binding fragment thereof, wherein the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO: 11. A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region sequence having a CDR1 amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 24, and a nucleic acid sequence set forth in SEQ ID NO: 25 The encoded CDR2 amino acid sequence and the CDR3 amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 26; and a light chain variable region sequence having the nucleic acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 27 CDR1 amino acid sequence, CDR2 amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 28, and CDR3 amino acid sequence encoded by the nucleic acid sequence listed in SEQ ID NO: 29, and wherein the monoclonal antibody Or its antigen-binding fragment specifically binds to a non-denatured amino acid sequence, and the non-denatured amino acid sequence is selected from sequence identification numbers (SEQ ID NO:) 2 to SEQ ID NO: 4 and SEQ ID NO : A group of amino acid sequences listed in 11. A monoclonal antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 30; and a light chain variable region sequence having a light chain variable region sequence such as SEQ ID NO: 31 The listed amino acid sequence. A monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region sequence having an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 32; and a light chain variable region sequence having a light chain variable region sequence such as SEQ ID NO: 32 ID NO: 33 is the amino acid sequence encoded by the nucleic acid sequence listed. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the antigen-binding fragment is a single-chain variable fragment (scFv), a single-chain variable fragment (scFv), Variable region fragment dimer [(scFv) ₂ ], single chain variable region fragment trimer [(scFv) ₃ ], variable region fragment (variable fragment; Fv), Fab fragment, Fab' fragment, F( ab') ₂ fragments or any combination of the above. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is attached via conjugation or binding, saccharification, or label attachment (tag attachment) or any combination of the above to modify. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is an antibody-drug conjugate (ADC) or Its antigen-binding fragment. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody (BsAb) or Its antigen-binding fragment. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or an antigen thereof Combine fragments. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class, the IgM class or the IgA class. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class and has the isotype of IgG1, IgG2, IgG3, or IgG4. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 in the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is an inert antibody. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof is an antagonist antibody. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows down the PTX3 receptor and one or more PTX3 The combination. According to any one of items 1 to 8 of the scope of patent application The monoclonal antibody or antigen-binding fragment thereof described in item 1, wherein the monoclonal antibody or antigen-binding fragment specifically inhibits or slows down the activity of one or more PTX3. The monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 8 of the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows down the PTX3 receptor and one or more PTX3 The interaction, inhibition or slowing down of PTX3 message transmission or any combination of the above. A kit for detecting PTX3, comprising the monoclonal antibody or antigen-binding fragment thereof as described in any one of items 1 to 23 in the scope of patent application, wherein the monoclonal antibody or antigen-binding fragment specifically binds to a non- The denatured amino acid sequence, and the non-denatured amino acid sequence is selected from a group consisting of the amino acid sequences listed in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11. According to the kit for detecting PTX3 described in item 24 of the scope of patent application, the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO: 2. According to the kit for detecting PTX3 described in item 24 of the scope of patent application, the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO:3. According to the kit for detecting PTX3 described in item 24 of the scope of patent application, the non-denatured amino acid sequence is the amino acid sequence listed in SEQ ID NO:4. According to the kit for detecting PTX3 described in item 24 of the scope of patent application, the non-denatured amino acid sequence is as SEQ ID NO: The amino acid sequence listed in 11. An in vitro method for detecting PTX3, which uses the kit for detecting PTX3 as described in any one of items 24 to 28 of the scope of the patent application to detect PTX3, wherein the monoclonal antibody contained in the kit for detecting PTX3 or its The analytical sensitivity of one of the antigen-binding fragments is not less than 0.244ng/mL.

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