TW201915016A - Monoclonal antibody or antigen-binding fragment 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

 Monoclonal antibody or antigen-binding fragment thereof and use thereof  

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

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, its formation and detailed mechanism There are still many unclear points.

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.

In view of this, there is an urgent need to develop a specific antibody that binds to PTX3 to detect PTX3 content in biological samples.

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

Another aspect of the invention provides a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence of a particular sequence and a light chain variable region sequence.

A further aspect of the invention provides a kit for detecting PTX3 comprising the above-described monoclonal antibody or antigen-binding fragment thereof.

A further aspect of the invention provides a method of detecting PTX3 in vitro, which utilizes 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 proposed. In one embodiment, the above-described monoclonal antibodies or antigen-binding fragments thereof can specifically bind to the non-denaturing amino acid sequences set forth 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 antigen-binding fragment thereof comprising a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence is, for example, SEQ ID NO: 18. The amino acid sequence set forth in SEQ ID NO: 19 and/or SEQ ID NO: 20, the light chain variable region sequence can be, for example, SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 23. Column of amino acid sequence.

In some embodiments, the heavy chain variable region sequence of the above-described monoclonal antibody or antigen-binding fragment thereof can be encoded, for example, by the nucleic acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25, and/or SEQ ID NO: The amino acid sequence, light chain variable region sequence can be, for example, the amino acid sequence encoded by the nucleic acid sequence set forth 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 above-described monoclonal antibody or antigen-binding fragment thereof can be, for example, the amino acid sequence set forth in SEQ ID NO: 30, and the light chain variable region sequence can be, for example, SEQ ID NO: 31. The amino acid sequence listed.

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

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

According to an embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof may be, for example, a single-chain variable fragment (scFV) or a single-chain variable region 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 thereof.

According to an embodiment of the invention, the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation or binding, saccharification, tag attachment or any combination thereof.

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

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

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

According to an embodiment of the present invention, the non-denaturing amino acid sequence may include, but is not limited to, the amino acid sequence listed by any one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 or any of the above combination. In other embodiments, the non-denaturing amino acid sequence described above can include, but is not limited to, the amino acid sequence set forth by any one of SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 or any combination thereof .

According to an embodiment of the invention, the monoclonal antibody or antigen-binding fragment thereof is of the IgG class, the IgM class or the IgA class. In another embodiment, the monoclonal antibody or antigen-binding fragment thereof described above is of the IgG class and has an IgGl, IgG2, IgG3 or IgG4 isotype.

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

According to an embodiment of the invention, the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the binding of the PTX3 receptor to one or more PTX3. In some illustrations, the monoclonal antibodies or antigen-binding fragments thereof described above inhibit or slow the activity of one or more PTX3. In other embodiments, the monoclonal antibodies or antigen-binding fragments thereof specifically inhibit or slow the interaction of the PTX3 receptor with one or more PTX3, inhibit or slow PTX3 signaling, or any combination of the above.

According to still another aspect of the present invention, a kit for detecting PTX3 comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of the above, wherein the monoclonal antibody or antigen-binding fragment thereof can specifically bind to a non-denatured amino group The acid sequence, and the non-denaturing amino acid sequence can include, but is not limited to, the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11.

According to still another aspect of the present invention, a method for detecting PTX3 in vitro is provided, which uses the above-mentioned kit for detecting PTX3 to detect PTX3, wherein the assay sensitivity of the monoclonal antibody or antigen-binding fragment thereof contained in the kit of PTX3 is detected. It can be, for example, not less than 0.244 ng/mL.

Application of the monoclonal antibody or antigen-binding fragment thereof of the present invention, which specifically inhibits or slows the binding of PTX3 to the PTX3 receptor by using a specific PTX3 monoclonal antibody or an antigen-binding fragment thereof, and can be applied to the detection and detection of the PTX3 set and detection. Method of PTX3 content.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Affinity curve of PTX3 recombinant protein.

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

Fig. 4 is a graph showing the competitive inhibition of binding of the PTX3 recombinant protein to the CD44 receptor in accordance with an embodiment of the present invention.

As described above, the present invention provides a monoclonal antibody or antigen-binding fragment thereof and use thereof, which specifically inhibits pentraxin-related protein (PTX3) and PTX3 by using a single antibody or an antigen-binding fragment thereof. The combination of receptors can be applied to the kit and method for detecting PTX3.

The monoclonal antibodies or antigen-binding fragments thereof referred to herein may comprise a heavy chain variable region sequence of a specific sequence and a light chain variable region sequence, which specifically inhibit or slow down the C-terminal specific sequence of the PTX3 and PTX3 receptors. Combine.

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 thereof is not limited, and can be, for example, NO:1 to SEQ ID NO:17. The non-denaturing amino acid sequence of the column is preferably specifically bound to the non-denaturing amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 11, and specifically binds as SEQ ID NO: 1 to The non-denaturing amino acid sequences set forth in SEQ ID NOS: 5 and 11 are preferred, and more preferably, the non-denatured amino acid sequence as set forth in SEQ ID NO: 2 to SEQ ID NO: 4 is more preferably combined. In the above embodiments, the non-denaturing amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 11 corresponds to the 200th amino acid of the non-denaturing amino acid sequence of the human PTX3 recombinant protein to the 236th Amino acid. In another illustration, the aforementioned non-denaturing amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 corresponds to the 200th of the non-denaturing amino acid sequence of the human PTX3 recombinant protein. Amino acid to the 220th amino acid. In yet another illustration, the non-denaturing amino acid sequence set forth in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 above corresponds to the 203 of the non-denaturing amino acid sequence of the human PTX3 recombinant protein. Amino acid to the 217th amino acid.

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, SEQ ID NO: 18, SEQ ID NO: 19 and/or the amino acid sequence set forth in SEQ ID NO: 20, the light chain variable region sequence may be, for example, the amino acid listed in SEQ ID NO: 21, SEQ ID NO: 22 and/or SEQ ID NO: sequence.

In other embodiments, the heavy chain variable region sequence of the above-described monoclonal antibody or antigen-binding fragment thereof can have the nucleic acid sequence encoding as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and/or SEQ ID NO: The amino acid sequence may, and the light chain variable region sequence may have an amino acid sequence as encoded by the nucleic acid sequence set forth 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 above-described monoclonal antibody or antigen-binding fragment thereof may have an amino acid sequence as set forth in SEQ ID NO: 30, and the light chain variable region sequence may have SEQ. ID NO: The amino acid sequence listed in 31.

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

In some embodiments, the type of monoclonal antibody or antigen-binding fragment thereof is not limited and may be, for example, a chimeric antibody or antigen-binding fragment thereof. In other embodiments, the monoclonal antibody or antigen-binding fragment thereof can be, for example, a humanized antibody or an antigen-binding fragment thereof.

In some embodiments, the above-described monoclonal antibody or antigen-binding fragment thereof has a structure that is independent of the structure of the antibody, and may be an intact 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 fragment, Fab' fragment, F(ab') ₂ fragment or any combination of the above, thereby simplifying the process of recombinant antibody. The above-mentioned monoclonal antibodies or antigen-binding fragments thereof can be carried out by means of fusion tumor cells or recombinant gene expression, etc., which are well known to those skilled in the art, and will not be described herein.

In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof described above may be further modified via conjugation or binding, saccharification, tag attachment, or any combination thereof, as desired. For example, the above-described monoclonal antibody or antigen-binding fragment thereof can further form an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof with the drug.

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

According to an embodiment of the invention, the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation or binding, saccharification, tag attachment or any combination thereof.

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

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

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

In one embodiment, the monoclonal antibody or antigen-binding fragment thereof described above may belong to the IgG class, the IgM class, or the IgA class. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof is of the IgG class and has an IgGl, IgG2, IgG3 or IgG4 isotype. In an exemplary embodiment, the monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype, for example, has an IgG1k isotype. In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof is an inert antibody or an antagonist antibody. In some specific embodiments, the monoclonal antibodies or antigen-binding fragments thereof described above specifically inhibit or slow the activity of one or more PTX3. In other specific embodiments, the monoclonal antibodies or antigen-binding fragments thereof described above specifically inhibit or slow the interaction of the PTX3 receptor with one or more PTX3s, inhibit or slow PTX3 signaling, or any combination of the above.

When applied, the above-described monoclonal antibodies or antigen-binding fragments thereof can be used to detect the set and method of PTX3, by specifically binding to the non-denaturing amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 11, Improve the analytical sensitivity of PTX3 in biological samples. The biological sample referred to herein is not limited in form, and may include, but is not limited to, cells, tissues, blood, urine, lymph, tissue fluid, body fluid, and the like. The above-mentioned kit for detecting PTX3 can be applied to various components/devices for conventional detection, such as flow cytometry, enzyme linked immunosorbent assay (ELISA) test kit, biochip, etc.; Known methods for detection such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining, and Western blotting analysis Wait. In a specific example, the analytical sensitivity of the above-mentioned monoclonal antibody or antigen-binding fragment thereof may also be referred to as lower limit of detection (LLOD), and generally not lower than 0.244 ng/mL. .

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. Retouching.

Example 1. Preparation of PTX3 monoclonal antibody

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

Briefly, the PTX3 recombinant protein of the non-denaturing amino acid sequence set forth in SEQ ID NO: 13 was used as an immunogen, and Balb/C mice were injected intraperitoneally (ip) at a dose of 50 μg per mouse. Or PTX3 knockout (PTX3 knockout) mice were intraperitoneally, and after 2 weeks, the mice were boosted with 50 μg of each mouse, once every two weeks for four times. Next, the activated splenocytes are fused with melanoma cells to produce a fusion tumor cell line.

From the above-mentioned fusion tumor cell strain, a fusion tumor cell strain having a high binding affinity to a recombinant protein of the non-denaturing amino acid sequence set forth in SEQ ID NO: 11 is selected, wherein the obtained fusion tumor cell strain produces a PTX3 single The strain antibodies can specifically bind to the non-denaturing amino acid sequence as set forth in the sequence identification numbers (SEQ ID NO:) 1 to SEQ ID NO: 11.

After collecting the culture supernatant of the fusion tumor cell strain obtained above and purifying the PTX3 monoclonal antibody via a commercially available column, the Taiwan Weiqiao Biomedical Co., Ltd. analyzed the complementary determination of the heavy chain variable region and the light chain variable region. Amino acid sequence of the complementarity-determining region (CDR) and 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: 30 or as SEQ ID NO The amino acid sequence encoded by the nucleic acid sequence set forth in 32, the amino acid sequence of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 31 or the nucleic acid sequence set forth in SEQ ID NO: 33 The encoded amino acid sequence.

Further, the 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 affinity of PTX3 monoclonal antibodies

This example uses a commercially available ELISA kit to assess the affinity of the PTX3 recombinant protein to the PTX3 monoclonal antibody of Example 1.

First, 5 μg/mL of PTX3 recombinant protein (such as the non-denaturing amino acid sequence set forth in SEQ ID NO: 14) or bovine serum albumin (BSA; as a control group) was coated in 96-well cell culture. The wells (model: 9018, Corning Costar) were reacted at 4 ° C until overnight. Next, a blocking solution [phosphate buffer solution (PBS) containing 3% skim milk powder] was added to the well, and a blocking reaction was carried out at room temperature (4 ° C to 40 ° C) for 1 hour. After removing the blocking solution, the wells were rinsed with PBS, and the primary antibody was added to the primary antibody for 1 hour at room temperature (4 ° C to 40 ° C), wherein the primary antibody was serially diluted with the PTX3 monoclonal antibody of Example 1. It is 2.44 × 10 ^-4 μg / mL to 1.00 μg / mL). Then, the unbound PTX3 monoclonal antibody of each well was washed away with PBS, and the secondary antibody was added to react at room temperature (4 ° C to 40 ° C). Thereafter, each well was added with tetramethyl benzidine (TMB) for a certain period of time, and then reacted with 0.1 M sulfuric acid (H ₂ SO ₄ ) for 10 minutes to terminate the reaction, wherein the secondary antibody was combined with horseradish peroxidation. Anti-mouse horse peroxidase (HRP) anti-mouse IgG (IgG-HRP). Next, the absorbance at 450 nm was read using a commercially available enzyme immunoassay analyzer (ELISA reader), and the results are shown in Fig. 1. Each value is three repetitions. The reaction time of the above secondary antibody is carried out in accordance with the manufacturer's manual, which should be well known to those of ordinary skill in the art to which the present invention pertains, and therefore will not be further described.

1 is a graph showing the affinity curves of PTX3 monoclonal antibodies against PTX3 recombinant protein according to an embodiment of the present invention, wherein the curve indicated by the figure: indicates the affinity curve of the PTX3 monoclonal antibody against the PTX3 recombinant protein. The numbered curve indicates the affinity curve of BSA for PTX3 recombinant protein.

From the results of FIG. 1, after up to ^46-fold dilution (antibody concentration corresponding to 0.244ng / mL), PTX3 PTX3 monoclonal antibodies to remain good recombinant protein affinity can be applied to detect PTX3 kit.

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

Example 3: Assessing the epitope localization region of PTX3 monoclonal antibody binding to PTX3

This example utilizes a commercially available ELISA kit to assess the epitope mapping region of PTX3 monoclonal antibody binding to PTX3.

This example evaluates the smaller binding region of PTX3 monoclonal antibody to PTX3 in the same manner as in Example 1, except that this example is a recombinant protein of PTX3 at 200 μg/mL (eg SEQ ID NOs: The non-denaturing amino acid sequence listed in 12, 16, and 17 was dissolved in a 0.1 M sodium bicarbonate solution, pH 8.3) or BSA (as a control group) in a well of a 96-well cell culture dish at 4 ° C. Until overnight. Next, a blocking solution [PBS containing 1% BSA] was added to the well, and a blocking reaction was carried out at room temperature (4 ° C to 40 ° C) for 1 hour. After the blocking solution was removed, each well was rinsed with PBS, and then the PTX3 monoclonal antibody of Example 1 (concentration: 125 ng/mL) was added to carry out a reaction at room temperature (4 to 40 ° C) for 2 hours. Then, each well of unconjugated PTX3 monoclonal antibody was washed away with PBS, and a secondary antibody (anti-mouse IgG-HRP, diluted 1:5000) was added thereto to react at room temperature (4 ° C to 40 ° C) for 1 hour. Thereafter, each well was added to the TMB reaction for a while, and 0.1 M sulfuric acid was added for 10 minutes to terminate the reaction, and the absorbance at 450 nm was read using a commercially available enzyme immunoassay. The results are shown in Fig. 2. Each value is three repetitions.

2 is a map showing the epitope mapping of PTX3 monoclonal antibodies and different fragments of PTX3 recombinant protein according to an embodiment of the present invention, wherein RI37 represents the PTX3 recombinant protein fragment set forth in SEQ ID NO: 12. KT37 represents the PTX3 recombinant protein fragment set forth in SEQ ID NO: 16, GI40 represents the PTX3 recombinant protein fragment set forth in SEQ ID NO: 17, and the figure number "***" represents the control group (ie, The BSA group) was statistically significant ( p < 0.001).

From the results of Fig. 2, it was revealed that the PTX3 monoclonal antibody had higher affinity for the PTX3 recombinant protein fragment as listed in SEQ ID NO: 12 than the other fragments, and was statistically significant.

Please refer to FIG. 3 , which illustrates an epitope mapping map of PTX3 monoclonal antibody binding to different PTX3 recombinant proteins according to an embodiment of the present invention, wherein the horizontal axis represents SEQ ID NOS: 1 to 10, respectively. PTX3 recombinant protein fragment.

From the results of FIG. 3, the affinity of the PTX3 monoclonal antibody for the PTX3 recombinant protein fragment as set forth in SEQ ID NOs: 1-5 or SEQ ID NOs: 2 to 4 is much higher than the other fragments, wherein SEQ ID NOs: The PTX3 recombinant protein fragment listed in 2~4 corresponds to the 203 to 217 amino acids of PTX3, such as the amino acid sequence set forth in SEQ ID NO: 11, and represents the binding of PTX3 monoclonal antibody of Example 1 to PTX3. The epitope localization region is located within the range of the amino acid sequence set forth in SEQ ID NOs: 2 to 4 or as set forth in SEQ ID NO: 11.

Example 4: Assessing the effect of PTX3 monoclonal antibody on the binding of PTX3 to CD44 in vitro

The PTX3 monoclonal antibody competitively binds to the PTX3 receptor binding region of PTX3 or its adjacent region, thereby specifically inhibiting or slowing the chance of binding of PTX3 to the PTX3 receptor. This example uses CD44 as an example to evaluate the effect of PTX3 monoclonal antibody competitively inhibiting the binding of PTX3 to the PTX3 receptor using a competitive binding assay.

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

In other words, the competitive binding assay method used in this example is similar to that of Example 1, except that this example is a CD44 N-terminal recombinant protein of 10 μg/mL (CD44 N-terminal 1~) The sequence of 220 amino acid residues, dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing, China) was plated in wells of a 96-well cell culture dish and reacted overnight at 4 °C. Next, a blocking solution [PBS containing 3% skim milk powder] was added to the well, and a blocking reaction was carried out at room temperature (4 ° C to 40 ° C) for 1 hour.

In the competitive binding assay, the PPT3 recombinant protein that binds HRP (such as the non-denaturing amino acid sequence set forth in SEQ ID NO: 14, HRP-PTX3, concentration 5 μg/mL) and different concentrations of Example 1 A PTX3 monoclonal antibody (concentration of 1 μg/mL or 2 μg/mL) was pre-reacted at room temperature (4 ° C to 40 ° C) for 1 hour to form a pre-reactant.

After removing the blocking solution, each well was rinsed with PBS, and the above pre-reacted product was further added, and the reaction was carried out at room temperature (4 ° C to 40 ° C) for 2 hours. Then, the unbound pre-reacted materials of each well were washed away with PBS, and TMB was added to each well for a period of time, and then 0.1 M sulfuric acid was added for 10 minutes to terminate the reaction, and the absorbance at 450 nm was read using a commercially available enzyme immunoassay analyzer. The result is shown in Figure 4. Each value is four repetitions.

Please refer to FIG. 4 , which is a competitive inhibition diagram 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, wherein the vertical axis represents the competitive inhibition rate (%), and the horizontal axis The figure number "+" or "-" below indicates that the specific reaction is carried out with or without the binding reaction. The first straight line on the left side of Figure 4 represents the control group (ie, the group of PTX3 recombinant protein without PTX3 monoclonal antibody). The figure number "***" represents statistical significance ( p < 0.001) compared to the control group.

From the results of FIG. 4, the value of the PTX3 monoclonal antibody in the first lane on the left side of FIG. 4 was used as a competitive inhibition rate of 0%, and when the PTX3 monoclonal antibody was pre-reacted with the PTX3 recombinant protein, it was reacted with the CD44 receptor. The competitive inhibition rate (%) obtained showed a dose-dependent relationship with the concentration of the PTX3 monoclonal antibody, and was statistically significant, and the PTX3 monoclonal antibody of Example 1 did compete with PTX3 for the binding region of CD44 or its adjacent region. And its inhibition mechanism is competitive inhibition.

It is to be noted that the PTX3 monoclonal antibody of the first embodiment of the present invention has good affinity and sensitivity to the PTX3 recombinant protein, and can be applied to the detection of the PTX3 kit and the method for detecting the PTX3 content in the biological sample in vitro. Regarding the applicable biological samples, methods, kits, components/equipments suitable for detecting PTX3, etc., as mentioned above, no further explanation is given.

In summary, the present invention is exemplified by a specific sequence of PTX3 monoclonal antibody, a specific analysis mode or a specific evaluation mode, and the single antibody or antigen-binding fragment thereof of the present invention and its use, but the technical field of the present invention It is to be understood by those of ordinary skill in the art that the present invention is not limited thereto, and that the individual antibodies or antigen-binding fragments thereof of the present invention and their uses may also use other analytical modes or other, without departing from the spirit and scope of the present invention. The evaluation method is carried out.

It can be seen from the above examples that the monoclonal antibody or antigen-binding fragment thereof of the present invention and the use thereof have the advantages that the specific PTX3 monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the binding of PTX3 to the PTX3 receptor. The kit and method used to detect PTX3.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

<110> 臻崴Biotech Co., Ltd.

<120> Monoclonal antibody or antigen-binding fragment thereof and use thereof

<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 200 to 208 amino acids

<400> 1

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 203 to 211 amino acids

<400> 2

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 206 to 214 amino acids

<400> 3

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 209 to 217 amino acids

<400> 4

<210> 5

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 212 to 220 amino acids

<400> 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 215 to 223 amino acids

<400> 6

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 218 to 226 amino acids

<400> 7

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 221 to 229 amino acids

<400> 8

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 224 to 232 amino acids

<400> 9

<210> 10

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 227 to 236 amino acids

<400> 10

<210> 11

<211> 15

<212> PRT

<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 200 to 236 amino acids (RI37)

<400> 12

<210> 13

<211> 160

<212> PRT

<213> Artificial sequence

<220>

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

<223> Human PTX3 recombinant protein

<400> 13

<210> 14

<211> 363

<212> PRT

<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 255 to 298 amino acids (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 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 PTX3 monoclonal antibody

<400> 19

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

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

<400> 20

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

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

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> V_segment

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

<400> 24

<210> 25

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

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

<400> 26

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

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

<400> 27

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

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

<400> 28

<210> 29

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> V_segment gene

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

<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> Gene sequence of the heavy chain variable region of PTX3 monoclonal antibody

<400> 32

<210> 33

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<221> CDS

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

<400> 33

Claims (34)

 A monoclonal antibody or antigen-binding fragment thereof, characterized in that the monoclonal antibody or antigen-binding fragment thereof is specifically bound to a non-denatured amino acid sequence, and the non-denatured amino acid sequence is selected from the sequence identification number A group consisting of the amino acid sequences listed in (SEQ ID NO:) 1 to SEQ ID NO: 11.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denaturing amino acid sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: A group of amino acids consisting of a sequence of amino acids.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denaturing amino acid sequence is selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: A group of amino acids consisting of a sequence of amino acids.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denatured amino acid sequence is the amino acid sequence as set forth in SEQ ID NO: 2.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denatured amino acid sequence is the amino acid sequence as set forth in SEQ ID NO: 3.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denatured amino acid sequence is an amino acid sequence as set forth in SEQ ID NO: 4.    The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the non-denatured amino acid sequence is an amino acid sequence as set forth in SEQ ID NO: 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: 18, SEQ ID NO: 19 and/or SEQ ID NO: 20; A light chain variable region sequence having the amino acid sequence set forth in SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 23.    A monoclonal antibody or antigen-binding fragment thereof comprising: a heavy chain variable region sequence having an amino acid encoded by the nucleic acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 25 and/or SEQ ID NO: And a light chain variable region sequence having the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, and/or SEQ ID NO:29.    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 SEQ ID NO: 31 The amino acid sequence listed.    A monoclonal antibody or 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 SEQ. ID NO: The amino acid sequence encoded by the nucleic acid sequence set forth in 33.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, 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 claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof is a humanized antibody or an antigen-binding fragment thereof.   The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antigen-binding fragment is a single-chain variable fragment (scFv), single-stranded Variable region fragment dimer [(scFv) ₂ ], single chain variable region fragment trimer [(scFv) ₃ ], variable region 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 claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof is via a conjugation or binding, saccharification, label attachment (tag attachment) or any combination of the above is modified.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, 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 claims 1 to 11, 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 claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or an antigen thereof Combine the fragments.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, 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 claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof belongs to the IgG class and has an IgG1, IgG2, IgG3 or IgG4 isotype.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, 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 claims 1 to 11, 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 claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof belongs to an antagonist antibody.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows down the PTX3 receptor and one or more PTX3 The combination.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the activity of one or more PTX3.    The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows down the PTX3 receptor and one or more PTX3 Interact, suppress or slow down PTX3 messaging or any combination of the above.    A kit for detecting PTX3, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the monoclonal antibody or antigen-binding fragment thereof is specifically combined with a non- A modified amino acid sequence, and the non-denaturing amino acid sequence is selected from the group consisting of amino acid sequences as set forth in SEQ ID NO: 1 to SEQ ID NO: 11.    The kit for detecting PTX3 according to claim 27, wherein 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 A population consisting of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11.    The kit for detecting PTX3 according to claim 27, wherein 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 A group consisting of the amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11.    The kit for detecting PTX3 according to claim 27, wherein the non-denaturing amino acid sequence is the amino acid sequence as set forth in SEQ ID NO: 2.    The kit for detecting PTX3 according to claim 27, wherein the non-denaturing amino acid sequence is the amino acid sequence as set forth in SEQ ID NO: 3.    The kit for detecting PTX3 according to claim 27, wherein the non-denaturing amino acid sequence is the amino acid sequence set forth in SEQ ID NO: 4.    The kit for detecting PTX3 according to claim 27, wherein the non-denaturing amino acid sequence is the amino acid sequence as set forth in SEQ ID NO: 11.    A method for detecting PTX3 in vitro, which is capable of detecting PTX3 using a kit for detecting PTX3 according to any one of claims 27 to 33, wherein the detection of the monoclonal antibody contained in the kit of PTX3 or The sensitivity of one of the antigen-binding fragments was not less than 0.244 ng/mL.