WO2019056991A1 - 单克隆抗体或其抗原结合片段及其用途 - Google Patents

单克隆抗体或其抗原结合片段及其用途 Download PDF

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Publication number
WO2019056991A1
WO2019056991A1 PCT/CN2018/105733 CN2018105733W WO2019056991A1 WO 2019056991 A1 WO2019056991 A1 WO 2019056991A1 CN 2018105733 W CN2018105733 W CN 2018105733W WO 2019056991 A1 WO2019056991 A1 WO 2019056991A1
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Prior art keywords
antigen
monoclonal antibody
binding fragment
seq
amino acid
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PCT/CN2018/105733
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English (en)
French (fr)
Inventor
李尹甄
王育民
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臻崴生物科技有限公司
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Priority to TW107132851A priority Critical patent/TWI786186B/zh
Priority to PCT/CN2018/106144 priority patent/WO2019057024A1/zh
Publication of WO2019056991A1 publication Critical patent/WO2019056991A1/zh
Priority to TW108133090A priority patent/TWI754171B/zh
Priority to MX2021003032A priority patent/MX2021003032A/es
Priority to EP19861063.6A priority patent/EP3862363A4/en
Priority to CA3112678A priority patent/CA3112678A1/en
Priority to CN202311402782.7A priority patent/CN117417443A/zh
Priority to CN201980058876.2A priority patent/CN112739714B/zh
Priority to US17/274,960 priority patent/US20220119507A1/en
Priority to AU2019337248A priority patent/AU2019337248A1/en
Priority to JP2021538885A priority patent/JP2022500503A/ja
Priority to BR112021004586-4A priority patent/BR112021004586A2/pt
Priority to SG11202102514QA priority patent/SG11202102514QA/en
Priority to PCT/CN2019/105824 priority patent/WO2020052675A1/zh
Priority to KR1020217010919A priority patent/KR20210062036A/ko
Priority to PH12021550529A priority patent/PH12021550529A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present invention relates to an antibody and its use, 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 is applied to a detection reagent the use of.
  • 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.
  • the cause and details of the formation are detailed. There are still many unclear aspects of the mechanism.
  • the tumor microenvironment 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 metastasize and invade tissue (or invasion).
  • PTX3 factor-like protein-like protein 3
  • 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.
  • PTX3 pentraxin-related proteins
  • Another object of the present invention is to provide a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence of a specific sequence and a light chain variable region sequence.
  • a monoclonal antibody or an antigen-binding fragment thereof is proposed.
  • the monoclonal antibody or antigen-binding fragment thereof described above can specifically bind to the non-denatured amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 11.
  • 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 SEQ ID NO: 18, for example
  • the heavy chain variable region sequence is SEQ ID NO: 18, for example
  • the light chain variable region sequence can be, for example, set forth in SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: Amino acid sequence.
  • the heavy chain variable region sequence of the above 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.
  • the heavy chain variable region sequence of the above 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, set forth in SEQ ID NO:31 Amino acid sequence.
  • the heavy chain variable region sequence of the above 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
  • the light chain variable region sequence can be, for example, SEQ ID. NO: The amino acid sequence encoded by the nucleic acid sequence listed in 33.
  • 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 is a humanized antibody or an antigen-binding fragment thereof.
  • 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.
  • the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation or binding, glycosylation, tag attachment or any combination thereof.
  • the monoclonal antibody or antigen-binding fragment thereof is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof.
  • ADC antibody-drug conjugate
  • the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody (BsAb) or an antigen-binding fragment thereof.
  • BsAb bispecific monoclonal antibody
  • the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or an antigen-binding fragment thereof.
  • the non-denatured amino acid sequence may include, but is not limited to, the amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 or any combination thereof.
  • the non-denatured amino acid sequences described above can include, but are not limited to, the amino acid sequences set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 or any combination thereof.
  • the monoclonal antibody or antigen-binding fragment thereof is of the IgG class, the IgM class or the IgA class.
  • the monoclonal antibody or antigen-binding fragment thereof described above is of the IgG class and has an IgGl, IgG2, IgG3 or IgG4 isotype.
  • the monoclonal antibody or antigen-binding fragment thereof is an inert antibody or an antagonist antibody.
  • the monoclonal antibody or antigen binding fragment thereof specifically inhibits or slows the binding of the PTX3 receptor to one or more PTX3.
  • the monoclonal antibodies or antigen-binding fragments thereof thereof inhibit or slow the activity of one or more PTX3.
  • the monoclonal antibodies or antigen-binding fragments thereof 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 foregoing.
  • a kit for detecting PTX3 comprising the monoclonal antibody or antigen-binding fragment thereof according to any of the above, wherein the monoclonal antibody or antigen-binding fragment thereof specifically binds to the non-denatured amino acid sequence
  • the non-denatured amino acid sequence can include, but is not limited to, the amino acid sequences set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 11.
  • a method for detecting PTX3 in vitro which detects PTX3 using the above kit for detecting PTX3, wherein the sensitivity of analysis of the monoclonal antibody or antigen-binding fragment thereof contained in the kit for detecting PTX3 can be, for example, Not less than 0.244 ng/mL.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention is a kit for detecting PTX3 by specifically inhibiting or slowing the binding of PTX3 to the PTX3 receptor by using a specific PTX3 monoclonal antibody or an antigen-binding fragment thereof.
  • FIG. 1 is a graph showing the affinity of a PTX3 monoclonal antibody to a recombinant protein of PTX3 according to an embodiment of the present invention.
  • 2 and 3 are epitope mapping maps showing the binding of PTX3 monoclonal antibodies to 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 monoclonal antibody to the binding of the CD44 receptor to the CD44 receptor according to an embodiment of the present invention.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof and use thereof for specifically inhibiting pentraxin-related protein (PTX3) by using a monoclonal antibody or an antigen-binding fragment thereof.
  • PTX3 pentraxin-related protein
  • the combination with the PTX3 receptor can be applied to kits and methods for detecting PTX3.
  • the monoclonal antibody or antigen-binding fragment thereof referred to herein may comprise a specific sequence of a heavy chain variable region sequence and a light chain variable region sequence, which specifically inhibits or slows the C-terminal specific sequence of the PTX3 and PTX3 receptors. Combination of.
  • the monoclonal antibody or antigen-binding fragment thereof 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, listed in NO:1 to SEQ ID NO:17.
  • the non-denatured amino acid sequence preferably binds specifically to the non-denatured amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 11, and specifically binds as SEQ ID NO: 1 to SEQ ID NO
  • the non-denatured amino acid sequences listed in 5 and 11 are preferred, and it is more preferred to specifically bind the non-denatured amino acid sequences set forth in SEQ ID NO: 2 to SEQ ID NO: 4.
  • the non-denatured amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 11 are from the 200th amino acid to the 236th amino acid corresponding to the non-denatured amino acid sequence of the human PTX3 recombinant protein.
  • the non-denatured amino acid sequences set forth in the aforementioned SEQ ID NO: 1 to SEQ ID NO: 5 and SEQ ID NO: 11 are the 200th amino acid corresponding to the non-denatured amino acid sequence of the human PTX3 recombinant protein. 220 amino acids.
  • the non-denatured amino acid sequence set forth in the aforementioned SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 11 is the 203th amino acid corresponding to the non-denatured amino acid sequence of the human PTX3 recombinant protein. 217 amino acids.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence and a light chain variable region sequence
  • 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, for example, be the amino acid sequence set forth in SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 23.
  • the heavy chain variable region sequence of the above 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, and the light chain variable region sequence may have 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.
  • the heavy chain variable region sequence of the above monoclonal antibody or antigen-binding fragment thereof can have the amino acid sequence set forth in SEQ ID NO: 30, and the light chain variable region sequence can have SEQ ID NO :31 The amino acid sequence listed.
  • the heavy chain variable region sequence of the above monoclonal antibody or antigen-binding fragment thereof may have the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 32
  • the light chain variable region sequence may have The amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:33.
  • the monoclonal antibody or antigen-binding fragment thereof is of a different type, such as a chimeric antibody or antigen-binding fragment thereof.
  • the monoclonal antibody or antigen-binding fragment thereof can be, for example, a humanized antibody or an antigen-binding fragment thereof.
  • the 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.
  • a single-chain variable region fragment such as single-chain variable region fragment.
  • single-chain variable fragment such as 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 monoclonal antibody or antigen-binding fragment 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.
  • 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.
  • the above monoclonal antibody or antigen-binding fragment thereof can further form an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof with the drug.
  • ADC antibody-drug conjugate
  • the 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.
  • BsAb bispecific monoclonal antibody
  • a trifunctional monoclonal antibody or an antigen-binding fragment thereof.
  • the monoclonal antibody or antigen-binding fragment thereof is modified by conjugation or binding, saccharification, tag attachment or any combination thereof.
  • the monoclonal antibody or antigen-binding fragment thereof is an antibody-drug conjugate (ADC) or an antigen-binding fragment thereof.
  • ADC antibody-drug conjugate
  • the monoclonal antibody or antigen-binding fragment thereof is a bispecific monoclonal antibody (BsAb) or an antigen-binding fragment thereof.
  • BsAb bispecific monoclonal antibody
  • the monoclonal antibody or antigen-binding fragment thereof is a trifunctional monoclonal antibody or an antigen-binding fragment thereof.
  • the monoclonal antibody or antigen-binding fragment thereof described above may belong to the IgG class, the IgM class, or the IgA class.
  • the monoclonal antibody or antigen-binding fragment thereof is of the IgG class and has an IgGl, IgG2, IgG3 or IgG4 isotype.
  • the monoclonal antibody or antigen-binding fragment thereof has an IgG1 isotype, for example, has an IgG1k isotype.
  • the monoclonal antibody or antigen-binding fragment thereof is an inert antibody or an antagonist antibody.
  • 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 PTX3, inhibit or slow PTX3 signaling, or any combination of the above.
  • the above monoclonal antibody or antigen-binding fragment thereof can be used in a kit and method for detecting PTX3, and the detection is enhanced by specific binding to the non-denatured amino acid sequences listed in SEQ ID NO: 1 to SEQ ID NO: 11.
  • Analytical sensitivity of PTX3 in biological samples is not limited and may include, but is not limited to, cells, tissues, blood, urine, lymph, tissue fluid, body fluid, and the like.
  • the above kit for detecting PTX3 can be applied to various components/devices for conventional detection, such as flow cytometry, enzyme linked immunosorbent assay (ELISA) reagent kit, biochip, etc.; or Conventional detection methods such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining, and Western blotting analysis )Wait.
  • ELISA enzyme linked immunosorbent assay
  • Conventional detection methods such as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, immunohistochemical staining, and Western blotting analysis
  • the analytical sensitivity of the monoclonal antibody or antigen-binding fragment thereof may also be referred to as a lower limit of detection (LLOD), and generally not lower than 0.244 ng/mL. .
  • a PTX3 monoclonal antibody that specifically recognizes the C-terminal amino acid sequence of the PTX3 recombinant protein is prepared by a conventional fusion tumor method or recombinant protein expression method.
  • the recombinant PTX3 protein of the non-denatured amino acid sequence set forth in SEQ ID NO: 13 was used as an immunogen, and Balb/C mice or PTX3 were injected intraperitoneally (ip) at a dose of 50 ⁇ g per mouse.
  • the knockout (PTX3knockout) mice were intraperitoneally, and after 2 weeks, the mice were boosted at a dose of 50 ⁇ g per mouse, twice a week for four times.
  • the activated splenocytes are fused with melanoma cells to produce a fusion tumor cell line.
  • a fusion tumor cell strain having a high binding affinity to a recombinant protein of the non-denatured amino acid sequence set forth in SEQ ID NO: 11 is selected, and the obtained PTX3 monoclonal antibody produced by the fusion tumor cell strain is obtained.
  • the non-denatured amino acid sequences set forth in the sequence identification numbers (SEQ ID NO:) 1 to SEQ ID NO: 11 can be specifically combined.
  • the culture supernatant of the fusion tumor cell strain obtained above was collected to purify the PTX3 monoclonal antibody via a commercially available column, and then the complementation decision of the heavy chain variable region and the light chain variable region was analyzed by Taiwan Weiqiao Biomedical Co., Ltd.
  • the sequence-encoded amino acid sequence, the amino acid sequence of the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 31 or the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:33.
  • the above PTX3 monoclonal antibody was analyzed by a commercially available monoclonal antibody typing kit, and it was confirmed that the antibody was classified into IgG1k.
  • This example evaluates the affinity of the PTX3 recombinant protein to the PTX3 monoclonal antibody of Example 1 using a commercially available ELISA kit.
  • PTX3 recombinant protein such as the non-denatured amino acid sequence set forth in SEQ ID NO: 14
  • BSA bovine serum albumin
  • 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.
  • the wells were rinsed with PBS, and the primary antibody was added to the reaction at room temperature (4 ° C to 40 ° C) for 1 hour, wherein the primary antibody was the serially diluted PTX3 monoclonal antibody of Example 1 (concentration was 2.44 x 10 -4 ⁇ g/mL to 1.00 ⁇ g/mL). Then, each well of the unbound PTX3 monoclonal antibody was washed away with PBS, and a secondary antibody was added thereto to react at room temperature (4 ° C to 40 ° C).
  • 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.
  • TMB tetramethyl benzidine
  • HRP horse peroxidase
  • IgG-HRP anti-mouse IgG
  • ELISA reader enzyme-linked immunosorbent analyzer
  • FIG. 1 is a graph showing the affinity of the PTX3 monoclonal antibody to the PTX3 recombinant protein according to an embodiment of the present invention, wherein the curve indicated by the figure ⁇ represents the affinity curve of the PTX3 monoclonal antibody to the PTX3 recombinant protein. ⁇ The indicated curve represents the affinity curve of BSA for PTX3 recombinant protein.
  • the PTX3 monoclonal antibody specifically binds to the 200th amino acid to the 359th amino acid of the PTX3 recombinant protein (such as the non-denatured amino acid sequence set forth in SEQ ID NO: 13) or the 200th amino acid to The 236th amino acid (such as the non-denatured 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 uses a commercially available ELISA kit to assess the epitope mapping region of the PTX3 monoclonal antibody binding to PTX3.
  • This example uses the same manner as in Example 1 to evaluate the smaller binding region of the PTX3 monoclonal antibody to PTX3, except that this example is a 200 ⁇ g/mL recombinant protein of PTX3 (eg SEQ ID NOs: 12).
  • the non-denatured amino acid sequences listed in 16, 17 were dissolved in a 0.1 M aqueous solution of sodium hydrogencarbonate, pH 8.3) or BSA (as a control group) and coated in wells of a 96-well cell culture dish, and reacted 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.
  • 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 the unbound 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.
  • a secondary antibody anti-mouse IgG-HRP, diluted 1:5000
  • 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, and the results are shown in FIG. Each value is three repetitions.
  • RI37 represents a PTX3 recombinant protein fragment as 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
  • the figure number "***" represents the control group (ie, BSA) Group) was statistically significant (p ⁇ 0.001).
  • PTX3 monoclonal antibody has higher affinity for the PTX3 recombinant protein fragment as listed in SEQ ID NO: 12 than the other fragments, and is statistically significant.
  • FIG. 3 shows an epitope mapping map of a PTX3 monoclonal antibody binding to different fragments of the PTX3 recombinant protein according to an embodiment of the present invention, wherein the horizontal axis represents the PTX3 as set forth in SEQ ID NOs: 1-10, respectively. Recombinant protein fragment.
  • 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: 2-4 is much higher than the other fragments, wherein SEQ ID NOs:
  • the PTX3 recombinant protein fragment listed in 2 to 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 the epitope mapping region of the PTX3 monoclonal antibody which binds to PTX3 of Example 1 is located.
  • 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.
  • Example 1 demonstrates that the PTX3 monoclonal antibody of Example 1 can competitively bind to the binding region of PTX3 to the PTX3 receptor (e.g., CD44) or its vicinity, which can improve the analytical sensitivity of detecting PTX3.
  • the PTX3 monoclonal antibody of Example 1 can competitively bind to the binding region of PTX3 to the PTX3 receptor (e.g., CD44) or its vicinity, which can improve the analytical sensitivity of detecting PTX3.
  • the competitive binding assay used in this example is similar to Example 1, except that this example is 10 ⁇ g/mL of CD44 N-terminal recombinant protein (CD-44 N-terminal 1 to 220).
  • CD44 N-terminal recombinant protein CD-44 N-terminal 1 to 220.
  • the sequence of the amino acid residue, dissolved in PBS, pH 7.2; Sino Biological Inc., Beijing, China) was plated in the wells of a 96-well cell culture dish and reacted overnight at 4 °C.
  • 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.
  • the PPT3 recombinant protein such as the non-denaturing amino acid sequence set forth in SEQ ID NO: 14, HRP-PTX3, concentration 5 ⁇ g/mL
  • HRP concentration 5 ⁇ g/mL
  • the cloned antibody concentration of 1 ⁇ g/mL or 2 ⁇ g/mL was pre-reacted at room temperature (4 ° C to 40 ° C) for 1 hour to form a pre-reactant.
  • 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.
  • FIG. 4 is a competitive inhibition diagram of the PTX3 monoclonal antibody blocking the binding of the PTX3 recombinant protein to the CD44 receptor, wherein the vertical axis represents the competitive inhibition rate (%), below the horizontal axis, according to an embodiment of the present invention.
  • the figure number "+” or “-” indicates that the binding reaction is carried out with or without the addition of specific components.
  • the first straight line on the left side of Figure 4 represents the control group (ie, the group of PTX3 recombinant protein without the PTX3 monoclonal antibody).
  • the figure number "* * *” represents statistical significance (p ⁇ 0.001) compared to the control group.
  • 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 the PTX3 monoclonal antibody was reacted with the PTX3 recombinant protein before reacting with the CD44 receptor.
  • the resulting competitive inhibition rate (%) showed a dose-dependent relationship with the concentration of the PTX3 monoclonal antibody, and was statistically significant, and the PTX3 monoclonal antibody representing Example 1 did compete with PTX3 for the binding region of CD44 or its adjacent region. And its inhibition mechanism is competitive inhibition.
  • 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 a kit and method for detecting PTX3 for detecting the PTX3 content in a biological sample in vitro.
  • a kit and method for detecting PTX3 for detecting the PTX3 content in a biological sample in vitro Regarding the applicable biological samples, methods, kits, components/equipments suitable for detecting PTX3, etc., as described above, no further explanation is given.
  • the present invention is exemplified by a specific sequence of a PTX3 monoclonal antibody, a specific analysis mode or a specific evaluation mode, and the monoclonal antibody or antigen-binding fragment thereof of the present invention and the use thereof, 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 the monoclonal antibodies or antigen-binding fragments thereof of the present invention and uses thereof may be used in other analytical modes or other, without departing from the spirit and scope of the present invention.
  • the evaluation method is carried out.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention and its use have the advantage that the specific PTX3 monoclonal antibody or antigen-binding fragment thereof specifically inhibits or slows the binding of PTX3 to the PTX3 receptor. It can be applied to kits and methods for detecting PTX3.

Abstract

一种单克隆抗体或其抗原结合片段及其用途。前述单克隆抗体或其抗原结合片段专一性抑制或减缓PTX3与PTX3受体之结合，可应用于检测PTX3的试剂盒及方法。

Description

单克隆抗体或其抗原结合片段及其用途 【技术领域】
本发明是有关于一种抗体及其用途，特别是有关于一种专一性抑制或减缓PTX3的C端特定序列与PTX3受体结合的单克隆抗体或其抗原结合片段及其应用于检测试剂的用途。
【背景技术】
目前已知癌细胞会刺激肿瘤周边微环境产生各种发炎因子、白血球、血管过度增生及蛋白酶等，而癌症的慢性发炎反应也与癌细胞的生长、转移与侵袭有关，然而其形成原因及详细机制，仍有诸多未明之处。
肿瘤微环境除了与发炎反应有关之外，其他研究也指出，肿瘤微环境与肿瘤转移(metastasis)及化疗抗药性(chemoresistance)亦息息相关。肿瘤微环境是由多种的基质细胞(stromal cells)及其他不同型态的细胞所构成，不仅可保护肿瘤，使肿瘤细胞得以逃脱和抵抗免疫细胞，而造成肿瘤细胞的抗药性。
在肿瘤周边基质组织中的纤维母细胞及巨噬细胞受到CEBPD活化后，会诱导产生分泌型因子-正五聚蛋白相关蛋白3(pentraxin-related protein 3；PTX3)，其具有促进血管新生的活性，且可增加鼻咽癌细胞的转移及侵入组织(或称侵袭)的能力。另外，过去研究亦证实癌周边组织细胞中CEBPD受到活化，亦可能促使癌转移，甚至促使在化疗过程中产生抗药性癌细胞，这些抗药性癌细胞会生长的更快并且更容易转移。
有鉴于此，亟需发展一种专一性结合PTX3的抗体，以检测生物样本中的PTX3含量。
【发明内容】
因此，本发明的一个目的是提供一种单克隆抗体或其抗原结合片段，其为专一性结合一种或多种正五聚蛋 白相关蛋白(pentraxin-related protein；PTX3)的C端特定序列。
本发明的另一个目的是提供一种单克隆抗体或其抗原结合片段，其包含特定序列的重链可变区序列以及轻链可变区序列。
本发明的又一目的是提供一种检测PTX3的试剂盒，包含上述的单克隆抗体或其抗原结合片段。
本发明的再一目的是提供一种体外检测PTX3的方法，其利用上述试剂盒检测PTX3。
根据本发明的上述目的，提出一种单克隆抗体或其抗原结合片段。在一实施例中，上述单克隆抗体或其抗原结合片段可专一性结合如SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列。
根据本发明的另一目的，提供一种单克隆抗体或其抗原结合片段，其包含重链可变区序列以及轻链可变区序列，其中重链可变区序列可例如SEQ ID NO：18、SEQ ID NO：19及/或SEQ ID NO：20所列的氨基酸序列，轻链可变区序列可例如SEQ ID NO：21、SEQ ID NO：22及/或SEQ ID NO：23所列的氨基酸序列。
在一些实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NO：24、SEQ ID NO：25及/或SEQ ID NO：26所列的核酸序列编码的氨基酸序列，轻链可变区序列可例如SEQ ID NO：27、SEQ ID NO：28及/或SEQ ID NO：29所列的核酸序列编码的氨基酸序列。
在其他实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NO：30所列的氨基酸序列，轻链可变区序列可例如SEQ ID NO：31所列的氨基酸序列。
在另一些实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可例如SEQ ID NO：32所列的核酸序列编码的氨基酸序列，轻链可变区序列可例如SEQ ID NO：33所列的核酸序列编码的氨基酸序列。
依据本发明一实施例，上述单克隆抗体或其抗 原结合片段为嵌合抗体或其抗原结合片段。在一例示中，上述单克隆抗体或其抗原结合片段为人源化抗体或其抗原结合片段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段可例如为单链可变区片段(single-chain variable fragment；scFv)、单链可变区片段二聚体﹝(scFv) 2﹞、单链可变区片段三聚体﹝(scFv) 3﹞、可变区片段(variable fragment；Fv)、Fab片段、Fab'片段、F(ab') 2片段或上述之任意组合。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为通过复合(conjugation)或结合、糖基化、标签附接(tag attachment)或上述任意组合予以修饰。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为双功能单克隆抗体(bispecific monoclonal antibody；BsAb)或其抗原结合片段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为三功能单克隆抗体(trifunctional monoclonal antibody)或其抗原结合片段。
依据本发明一实施例，上述非变性氨基酸序列可包含但不限于由SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11任一所列的氨基酸序列或上述任意组合。在其他实施例中，上述非变性氨基酸序列可包含但不限于由SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11任一所列的氨基酸序列或上述任意组合。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段属于IgG类、IgM类别或IgA类别。在另一实施例中，上述单克隆抗体或其抗原结合片段属于IgG类且具有IgG1、IgG2、IgG3或IgG4同型。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段属于惰性抗体或拮抗剂抗体。
依据本发明一实施例，上述单克隆抗体或其抗 原结合片段为专一性抑制或减缓PTX3受体与一种或多种PTX3的结合。在一些例示中，上述单克隆抗体或其抗原结合片段抑制或减缓一种或多种PTX3的活性。在另一些例示中，上述单克隆抗体或其抗原结合片段专一性抑制或减缓PTX3受体与一或多种PTX3的相互作用、抑制或减缓PTX3信号传递或上述之任意组合。
根据本发明的又一目的，提出一种检测PTX3的试剂盒，包含如上述任一的单克隆抗体或其抗原结合片段，其中单克隆抗体或其抗原结合片段可专一性结合非变性氨基酸序列，且此非变性氨基酸序列可包括但不限于如SEQ ID NO：1至SEQ ID NO：11任一所列的氨基酸序列。
根据本发明的另一目的，提出一种体外检测PTX3的方法，其利用上述检测PTX3的试剂盒检测PTX3，其中检测PTX3的试剂盒所含的单克隆抗体或其抗原结合片段的分析灵敏度可例如不低于0.244ng/mL。
应用本发明的单克隆抗体或其抗原结合片段，其是利用特定的PTX3单克隆抗体或其抗原结合片段专一性抑制或减缓PTX3与PTX3受体的结合，可应用于检测PTX3的试剂盒及检测PTX3含量的方法。
【附图说明】
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，所附图式的详细说明如下：
图1为绘示根据本发明一实施例的PTX3单克隆抗体对PTX3重组蛋白的亲和力曲线图。
图2与图3为绘示根据本发明一实施例的PTX3单克隆抗体与不同片段的PTX3重组蛋白结合的表位定位图谱。
图4为绘示根据本发明一实施例的PTX3单克隆抗体阻碍PTX3重组蛋白与CD44受体的结合的竞争性抑制图。
【实施方式】
承前所述，本发明提供一种单克隆抗体或其抗原结合片段及其用途，其是利用单克隆抗体或其抗原结合片段专一性抑制正五聚蛋白相关蛋白(pentraxin-related  protein；PTX3)与PTX3受体的结合，可应用于检测PTX3的试剂盒及方法。
本发明此处所称的单克隆抗体或其抗原结合片段可包含特定序列的重链可变区序列以及轻链可变区序列，以专一性抑制或减缓PTX3与PTX3受体的C端特定序列的结合。
申言之，在一实施例中，前述单克隆抗体或其抗原结合片段可专一性结合人类PTX3的C端氨基酸序列，其序列范围不拘，可例如NO：1至SEQ ID NO：17所列的非变性氨基酸序列，然以专一性结合如SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列为佳，又以专一性结合如SEQ ID NO：1至SEQ ID NO：5以及11所列的非变性氨基酸序列为较佳，又以专一性结合如SEQ ID NO：2至SEQ ID NO：4所列的非变性氨基酸序列为更佳。在上述实施例中，SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列为对应于人类PTX3重组蛋白的非变性氨基酸序列的第200个氨基酸至第236个氨基酸。在另一个例示中，前述SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的非变性氨基酸序列为对应于人类PTX3重组蛋白的非变性氨基酸序列的第200个氨基酸至第220个氨基酸。在又一个例示中，前述SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的非变性氨基酸序列为对应于人类PTX3重组蛋白的非变性氨基酸序列的第203个氨基酸至第217个氨基酸。
在一实施例中，上述单克隆抗体或其抗原结合片段包含重链可变区序列以及轻链可变区序列，其中重链可变区序列可例如具有SEQ ID NO：18、SEQ ID NO：19及/或SEQ ID NO：20所列的氨基酸序列，轻链可变区序列可例如SEQ ID NO：21、SEQ ID NO：22及/或SEQ ID NO：23所列的氨基酸序列。
在其他实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可具有如SEQ ID NO：24、SEQ ID NO：25及/或SEQ ID NO：26所列的核酸序列编码的氨基酸序列，而轻链可变区序列可具有如SEQ ID NO：27、 SEQ ID NO：28及/或SEQ ID NO：29所列的核酸序列编码的氨基酸序列。
在其他实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可具有具有如SEQ ID NO：30所列的氨基酸序列，而轻链可变区序列可具有如SEQ ID NO：31所列的氨基酸序列。
在其他实施例中，上述单克隆抗体或其抗原结合片段的重链可变区序列可具有如SEQ ID NO：32所列的核酸序列编码的氨基酸序列，而轻链可变区序列可具有如SEQ ID NO：33所列的核酸序列编码的氨基酸序列。
在一些实施例中，单克隆抗体或其抗原结合片段的种类不拘，可例如嵌合抗体或其抗原结合片段。在其他例示中，上述单克隆抗体或其抗原结合片段可例如为人源化抗体或其抗原结合片段。
在一些实施例中，上述单克隆抗体或其抗原结合片段的结构不拘，在兼顾抗体结构稳定性的前提下，可为完整的抗体结构，或是简化的抗体结构，例如单链可变区片段(single-chain variable fragment；scFv)、单链可变区片段二聚体﹝(scFv) 2﹞、单链可变区片段三聚体﹝(scFv) 3﹞、可变区片段(variable fragment；Fv)、Fab片段、Fab'片段、F(ab') 2片段或上述的任意组合，藉此简化重组抗体的制程。上述单克隆抗体或其抗原结合片段可利用融合瘤细胞或重组基因表达等方式进行，此为本发明所属技术领域中具有通常知识者所熟知，此处不再赘述。
在一些实施例中，上述单克隆抗体或其抗原结合片段可视实际需求，进一步经由复合(conjugation)或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。举例而言，上述单克隆抗体或其抗原结合片段可与药物进一步形成抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。
在一些实施例中，上述单克隆抗体或其抗原结合片段可例如为双功能单克隆抗体(bispecific monoclonal antibody；BsAb)、三功能单克隆抗体(trifunctional monoclonal antibody)或其抗原结合片 段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为通过复合(conjugation)或结合、糖化、标签附接(tag attachment)或上述任意组合予以修饰。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为抗体药物复合体(antibody-drug conjugate；ADC)或其抗原结合片段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为双功能单克隆抗体(bispecific monoclonal antibody；BsAb)或其抗原结合片段。
依据本发明一实施例，上述单克隆抗体或其抗原结合片段为三功能单克隆抗体(trifunctional monoclonal antibody)或其抗原结合片段。
在一实施例中，上述单克隆抗体或其抗原结合片段可属于IgG类、IgM类别或IgA类别。在一具体实施例中，上述单克隆抗体或其抗原结合片段属于IgG类且具有IgG1、IgG2、IgG3或IgG4同型。在一例示中，上述单克隆抗体或其抗原结合片段具有IgG1同型，例如具有IgG1k同型。在一些具体例示中，上述单克隆抗体或其抗原结合片段属于惰性抗体或拮抗剂抗体。在一些具体例示中，上述单克隆抗体或其抗原结合片段可专一性抑制或减缓一或多种PTX3的活性。在另一些具体例示中，上述单克隆抗体或其抗原结合片段可专一性抑制或减缓PTX3受体与一或多种PTX3的相互作用、抑制或减缓PTX3信号传递或上述的任意组合。
在应用时，上述单克隆抗体或其抗原结合片段可用于检测PTX3的试剂盒及方法，通过与SEQ ID NO：1至SEQ ID NO：11所列的非变性氨基酸序列专一性结合，提高检测生物样本中PTX3的分析灵敏度。本发明此处所称的生物样本的形式不拘，可包括但不限于例如细胞、组织、血液、尿液、淋巴液、组织液、体液等。上述检测PTX3的试剂盒可应用于各种习知检测用的组件/设备，例如流式细胞仪、酵素连结免疫吸附分析(enzymelinked immunosorbent assay；ELISA)检测试剂试剂盒、生物 芯片等；或应用于习知检测用的方法例如直接ELISA(direct ELISA)、间接ELISA(indirect ELISA)、三明治ELISA(sandwich ELISA)、竞争性ELISA(competitive ELISA)、免疫组织化学染色及西方墨点分析法(Western bloting analysis)等。在一具体例中，上述单克隆抗体或其抗原结合片段的分析灵敏度(analytical sensitivity)，亦可称为检测下限值(lower limit of detection；LLOD)，一般而言不低于0.244ng/mL。
以下利用数个实施例以说明本发明之应用，然其并非用以限定本发明，本发明技术领域中具有通常知识者，在不脱离本发明之精神和范围内，当可作各种之更动与润饰。
实施例1、PTX3单克隆抗体的制备
此实施例是利用习知融合瘤法或重组蛋白表现法，制备专一性识别PTX3重组蛋白的C端氨基酸序列的PTX3单克隆抗体。
简言之，将如SEQ ID NO：13所列的非变性氨基酸序列的PTX3重组蛋白作为免疫原，以每头小鼠50μg的剂量以腹腔注射(i.p.)的方式注入Balb/C小鼠或PTX3基因剔除(PTX3knockout)小鼠腹腔内，2周后再以每头小鼠50μg的剂量加强免疫，二周一次，共四次。接着，将活化的脾细胞与黑色素瘤细胞融合后，产生融合瘤细胞株。
从上述融合瘤细胞株中，筛选出对SEQ ID NO：11所列的非变性氨基酸序列的重组蛋白的结合亲和力较高的融合瘤细胞株，其中所得的融合瘤细胞株产生的PTX3单克隆抗体可专一性结合如序列辨识编号(SEQ ID NO：)1至SEQ ID NO：11所列的非变性氨基酸序列。
收集上述所得的融合瘤细胞株的培养上清液经由市售管柱纯化出PTX3单克隆抗体后，委由台湾伟乔生医股份公司分析重链可变区及轻链可变区的互补决定区(complementarity-determining region；CDR)的氨基酸序列及对应的核酸序列，其重链可变区的氨基酸序列具有如 SEQ ID NO：30所列的氨基酸序列或如SEQ ID NO：32所列的核酸序列编码的氨基酸序列，轻链可变区的氨基酸序列具有如SEQ ID NO：31所列的氨基酸序列或如SEQ ID NO：33所列的核酸序列编码之氨基酸序列。
另外，上述PTX3单克隆抗体经市售单克隆抗体分型试剂盒分析后，确认其抗体分型为IgG1k。
实施例二、评估PTX3单克隆抗体的亲和力
此实施例利用市售ELISA试剂盒评估PTX3重组蛋白与实施例一的PTX3单克隆抗体的亲和力。
首先，将5μg/mL的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列)或牛血清白蛋白(bovine serum albumin，BSA；作为控制组)涂布在96孔细胞培养盘(型号：9018，Corning Costar)的各孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含3％脱脂奶粉之磷酸盐缓冲溶液(PBS)﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。在去除阻隔溶液后，利用PBS润洗各孔，再加入初级抗体于室温(4℃至40℃)进行反应1小时，其中初级抗体为经序列稀释的实施例一的PTX3单克隆抗体(浓度为2.44×10 -4μg/mL至1.00μg/mL)。然后，利用PBS洗去各孔未结合的PTX3单克隆抗体，并加入二级抗体于室温(4℃至40℃)反应。之后，各孔加入四甲基联苯胺(tetramethyl benzidine；TMB)反应反应一段时间后，加入0.1M硫酸(H 2SO 4)反应10分钟，以终止反应，其中二级抗体为结合辣根过氧化氢酶(anti-mouse horse peroxidase；HRP)的抗小鼠IgG(IgG-HRP)。接下来，利用市售酶联免疫分析仪(ELISA reader)读取450nm的吸光值，其结果如图1所示。每个数值为三重复。上述二级抗体的反应时间系参照制造商的操作手册进行，此应为本发明所属技术领域中任何具有通常知识者所熟知，故不另赘述。
请参阅图1，其绘示根据本发明一实施例的PTX3单克隆抗体对PTX3重组蛋白的亲和力曲线图，其中图号●标示的曲线代表PTX3单克隆抗体对PTX3重组蛋白的亲和力曲线，图号■标示的曲线代表BSA对PTX3重组蛋 白的亲和力曲线。
由图1结果可知，在高达4 6倍的稀释(抗体浓度相当于0.244ng/mL)后，PTX3单克隆抗体仍维持对PTX3重组蛋白良好的亲和力，可应用于PTX3检测试剂盒。
另外，在其他实验中，PTX3单克隆抗体可专一性结合PTX3重组蛋白的第200个氨基酸至第359个氨基酸(如SEQ ID NO：13所列的非变性氨基酸序列)或第200个氨基酸至第236个氨基酸(如SEQ ID NO：12所列的非变性氨基酸序列)(图未绘示)。
实施例三、评估PTX3单克隆抗体与PTX3结合的表位定位区域
此实施例利用市售ELISA试剂盒评估PTX3单克隆抗体与PTX3结合的表位定位(epitope mapping)区域。
此实施例利用与实施例一相同的方式，评估PTX3单克隆抗体对PTX3的较小结合区域，惟其不同之处在于，本实施例是将200μg/mL的PTX3重组蛋白(如SEQ ID NOs：12、16、17所列的非变性氨基酸序列，溶于0.1M的碳酸氢钠水溶液，pH 8.3)或BSA(作为控制组)涂布在96孔细胞培养盘的孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含1％BSA之PBS﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。在去除阻隔溶液后，利用PBS润洗各孔，再加入实施例一的PTX3单克隆抗体(浓度为125ng/mL)于室温(4℃至40℃)进行反应2小时。然后，利用PBS洗去各孔未结合的PTX3单克隆抗体，并加入二级抗体(抗小鼠IgG-HRP，以1：5000稀释)于室温(4℃至40℃)反应1小时。之后，各孔加入TMB反应反应一段时间后，加入0.1M硫酸反应10分钟以终止反应，并利用市售酵素免疫分析仪读取450nm的吸光值，其结果如图2所示。每个数值为三重复。
请参阅图2，其绘示根据本发明一实施例的PTX3单克隆抗体与不同片段的PTX3重组蛋白结合的表位定位图谱，其中RI37代表如SEQ ID NO：12所列的PTX3 重组蛋白片段，KT37代表如SEQ ID NO：16所列的PTX3重组蛋白片段，GI40代表如SEQ ID NO：17所列的PTX3重组蛋白片段，而图号「＊＊＊」则代表相较于控制组(即BSA组)具有统计显著性(p＜0.001)。
由图2的结果可知，PTX3单克隆抗体对于如SEQ ID NO：12所列的PTX3重组蛋白片段的亲和力，高于其他片段，且具有统计显著性。
请参阅图3，其绘示根据本发明一实施例的PTX3单克隆抗体与不同片段的PTX3重组蛋白结合的表位定位图谱，其中横轴分别代表如SEQ ID NO：1～10所列的PTX3重组蛋白片段。
由图3的结果可知，PTX3单克隆抗体对于如SEQ ID NOs：1～5或如SEQ ID NOs：2～4所列的PTX3重组蛋白片段的亲和力，远高于其他片段，其中SEQ ID NOs：2～4所列的PTX3重组蛋白片段对应于PTX3第203至217个氨基酸，如SEQ ID NO：11所列之氨基酸序列，代表实施例一的PTX3单克隆抗体与PTX3结合的表位定位区域坐落于如SEQ ID NOs：2～4所列或如SEQ ID NO：11所列的氨基酸序列的范围内。
实施例四、评估PTX3单克隆抗体在体外对PTX3与CD44的结合的影响
PTX3单克隆抗体可竞争性结合至PTX3的PTX3受体结合区或其邻近区域，进而专一性抑制或减缓PTX3与PTX3受体的结合的机会。本实施例以CD44为例，利用竞争性结合试验，评估PTX3单克隆抗体竞争性抑制PTX3结合至PTX3受体的效果。
本实施例证实，实施例一的PTX3单克隆抗体可竞争性结合至PTX3与PTX3受体(例如CD44)的结合区域或其邻近区域，可提高检测PTX3的分析灵敏度。
申言之，此实施例使用的竞争性结合分析法是与实施例一相似的方式，惟不同之处在于，本实施例是将10μg/mL的CD44N端重组蛋白(CD44N端第1～220个氨基酸残基的序列，溶于PBS，pH 7.2；Sino Biological Inc.， 北京，中国)涂布在96孔细胞培养盘的孔内，4℃反应至隔夜。接着，将阻隔溶液﹝含3％脱脂奶粉之PBS﹞加入孔内，于室温(4℃至40℃)进行阻隔反应达1小时。
在进行竞争性结合试验时，将结合HRP的PTX3重组蛋白(如SEQ ID NO：14所列的非变性氨基酸序列，HRP-PTX3，浓度为5μg/mL)与不同浓度的实施例一的PTX3单克隆抗体(浓度为1μg/mL或2μg/mL)于室温(4℃至40℃)进行预反应1小时，以形成预反应物。
在去除阻隔溶液后，利用PBS润洗各孔，再加入上述预反应物，于室温(4℃至40℃)进行反应2小时。然后，利用PBS洗去各孔未结合的预反应物，于各孔加入TMB反应一段时间后，加入0.1M硫酸反应10分钟以终止反应，并利用市售酵素免疫分析仪读取450nm的吸光值，其结果如图4所示。每个数值为四重复。
请参阅图4，其绘示根据本发明一实施例的PTX3单克隆抗体阻碍PTX3重组蛋白与CD44受体的结合的竞争性抑制图，其中纵轴代表竞争性抑制率(％)，横轴下方的图号「+」或「－」代表进行结合反应时有或无添加特定成分，图4左侧第1道直条代表控制组(即未加PTX3单克隆抗体的PTX3重组蛋白的组别)，而图号「＊ ＊ ＊」则代表相较于控制组具有统计显著性(p＜0.001)。
由图4的结果可知，以图4左侧第1道未加PTX3单克隆抗体的数值作为0％之竞争性抑制率，当PTX3单克隆抗体与PTX3重组蛋白预反应后再与CD44受体反应，所得的竞争性抑制率(％)与PTX3单克隆抗体的浓度呈现剂量依存关系，且具有统计显著性，代表实施例一的PTX3单克隆抗体确实可与PTX3竞争CD44的结合区域或其邻近区域，且其抑制机制属于竞争性抑制。
补充说明的是，本发明实施例一的PTX3单克隆抗体对PTX3重组蛋白具有良好的亲和力及灵敏度，可应用于检测PTX3的试剂盒及方法，以于体外检测生物样本中的PTX3含量。关于适用的生物样本、适用于检测PTX3的方法、试剂盒、组件/设备等悉如前述，不另赘言。
综言之，本发明虽以特定序列的PTX3单克隆 抗体、特定的分析模式或特定的评估方式作为例示，说明本发明的单克隆抗体或其抗原结合片段及其用途，惟本发明所属技术领域中任何具有通常知识者可知，本发明并不限于此，在不脱离本发明之精神和范围内，本发明之单克隆抗体或其抗原结合片段及其用途，亦可使用其他的分析模式或其他的评估方式进行。
由上述实施例可知，本发明的单克隆抗体或其抗原结合片段及其用途，其优点在于利用特定的PTX3单克隆抗体或其抗原结合片段专一性抑制或减缓PTX3与PTX3受体之结合，可应用于检测PTX3之试剂盒及方法。
虽然本发明已以数个实施例揭露如上，然其并非用以限定本发明，在本发明所属技术领域中任何具有通常知识者，在不脱离本发明之精神和范围内，当可作各种更动与润饰，因此本发明的保护范围当视后附之权利要求范围所界定者为准。

Claims (34)

  1. 一种单克隆抗体或其抗原结合片段，其特征在于该单克隆抗体或其抗原结合片段专一性结合一非变性氨基酸序列，且该非变性氨基酸序列为选自序列号SEQ ID NO：1至SEQ ID NO：11所列的氨基酸序列。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为选自如SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的氨基酸序列。
  3. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为选自如SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的氨基酸序列。
  4. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：2所列的氨基酸序列。
  5. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：3所列的氨基酸序列。
  6. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：4所列的氨基酸序列。
  7. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：11所列的氨基酸序列。
  8. 一种单克隆抗体或其抗原结合片段，其特征在于包括：
    一重链可变区序列，其特征在于具有如SEQ ID NO：18、SEQ ID NO：19及/或SEQ ID NO：20所列的氨基酸序列；以及
    一轻链可变区序列，其特征在于具有如SEQ ID NO：21、SEQ ID NO：22及/或SEQ ID NO：23所列的氨基酸序列。
  9. 一种单克隆抗体或其抗原结合片段，其特征在于包括：
    一重链可变区序列，其特征在于具有如SEQ ID NO：24、SEQ ID NO：25及/或SEQ ID NO：26所列的核酸序列编码的氨基酸序列；以及
    一轻链可变区序列，其特征在于具有如SEQ ID NO：27、SEQ ID NO：28及/或SEQ ID NO：29所列的核酸序列编码的氨基酸序列。
  10. 一种单克隆抗体或其抗原结合片段，其特征在于包括：
    一重链可变区序列，其特征在于具有如SEQ ID NO：30所列的氨基酸序列；以及
    一轻链可变区序列，其特征在于具有如SEQ ID NO：31所列的氨基酸序列。
  11. 一种单克隆抗体或其抗原结合片段，包括：
    一重链可变区序列，其特征在于具有如SEQ ID NO：32所列的核酸序列编码的氨基酸序列；以及
    一轻链可变区序列，其特征在于具有如SEQ ID NO：33所列的核酸序列编码的氨基酸序列。
  12. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为嵌合抗体或其抗原结合片段。
  13. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为人源化抗体或其抗原结合片段。
  14. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该抗原结合片段为单链可变区片段、单链可变区片段二聚体、单链可变区片段三聚体、可变区片段、Fab片段、Fab'片段、F(ab') 2片段或上述的任意组合。
  15. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为通过复合或结合、糖基化、标签附接或上述任意组合予以修饰。
  16. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为抗体药物复合体或其抗原结合片段。
  17. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为双功能单克隆抗体或其抗原结合片段。
  18. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段为三功能单克隆抗体或其抗原结合片段。
  19. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段属于IgG类、IgM类别或IgA类别。
  20. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段属于IgG类且具有IgG1、IgG2、IgG3或IgG4同型。
  21. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段具有IgG1同型。
  22. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段属于惰性抗体。
  23. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段属于拮抗剂抗体。
  24. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段专一性抑制或减缓PTX3受体与一或多种PTX3的结合。
  25. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段专一性抑制或减缓一种或多种PTX3的活性。
  26. 根据权利要求1至11任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗 原结合片段专一性抑制或减缓PTX3受体与一或多种PTX3的相互作用、抑制或减缓PTX3信号传递或上述的任意组合。
  27. 一种检测PTX3的试剂盒，包含如权利要求1至26任一项所述的单克隆抗体或其抗原结合片段，其特征在于其中该单克隆抗体或其抗原结合片段专一性结合一非变性氨基酸序列，且该非变性氨基酸序列为选自如SEQ ID NO：1至SEQ ID NO：11所列的氨基酸序列。
  28. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该单克隆抗体或其抗原结合片段专一性结合一非变性氨基酸序列，且该非变性氨基酸序列为选自如SEQ ID NO：1至SEQ ID NO：5以及SEQ ID NO：11所列的氨基酸序列。
  29. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该单克隆抗体或其抗原结合片段专一性结合一非变性氨基酸序列，且该非变性氨基酸序列为选自如SEQ ID NO：2至SEQ ID NO：4以及SEQ ID NO：11所列的氨基酸序列。
  30. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：2所列的氨基酸序列。
  31. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：3所列的氨基酸序列。
  32. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：4所列的氨基酸序列。
  33. 根据权利要求27所述的检测PTX3的试剂盒，其特征在于其中该非变性氨基酸序列为如SEQ ID NO：11所列的氨基酸序列。
  34. 一种体外检测PTX3的方法，其特征在于利用如权利要求27至33任一项所述的检测PTX3的试剂盒检测PTX3，其中该检测PTX3的试剂盒所含的单克隆抗体或其抗原结合片段之一分析灵敏度不低于0.244ng/mL。
PCT/CN2018/105733 2017-09-19 2018-09-14 单克隆抗体或其抗原结合片段及其用途 WO2019056991A1 (zh)

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TW108133090A TWI754171B (zh) 2018-09-14 2019-09-12 含單株抗體或其抗原結合片段之醫藥組成物及其用途
KR1020217010919A KR20210062036A (ko) 2018-09-14 2019-09-13 단클론 항체 또는 그의 항원 결합 단편을 포함하는 의약 조성물 및 그의 용도
CN202311402782.7A CN117417443A (zh) 2018-09-14 2019-09-13 含单株抗体或其抗原结合片段的医药组合物及其用途
EP19861063.6A EP3862363A4 (en) 2018-09-14 2019-09-13 MEDICAL COMPOSITION WITH MONOCLONAL ANTIBODY OR ANTIBODY FAB FRAGMENT THEREOF AND USE THEREOF
CA3112678A CA3112678A1 (en) 2018-09-14 2019-09-13 Medicinal composition containing monoclonal antibody or antibody fab fragment thereof, and use thereof
MX2021003032A MX2021003032A (es) 2018-09-14 2019-09-13 Composicion medicinal que incluye un anticuerpo monoclonal o fragmento de union a antigeno y uso del mismo.
CN201980058876.2A CN112739714B (zh) 2018-09-14 2019-09-13 含单株抗体或其抗原结合片段的医药组合物及其用途
US17/274,960 US20220119507A1 (en) 2018-09-14 2019-09-13 Medicinal composition containing monoclonal antibody or antibody fab fragment thereof, and use thereof
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JP2021538885A JP2022500503A (ja) 2018-09-14 2019-09-13 モノクローナル抗体又はその抗原結合断片を含む医薬組成物及びその使用
BR112021004586-4A BR112021004586A2 (pt) 2018-09-14 2019-09-13 composição medicinal incluindo anticorpo monoclonal ou fragmento de ligação ao antígeno e seu uso
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