Anti-interleukin-6 antibody, reagent and kit for detecting interleukin-6

Abstract

The present invention discloses an anti-interleukin-6 antibody or a reagent and a kit for detecting interleukin-6, relating to the field of antibodies. The anti-interleukin-6 antibody disclosed by the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. This antibody provides an important raw material source for the detection of interleukin-6 and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and more particularly, to an anti-interleukin-6 antibody, a reagent and a kit for detecting interleukin-6. Background Art

[0002] Interleukin 6 (IL-6), also known as leukocyte interleukin 6, is a pleiotropic cytokine with a wide range of functions and belongs to the interleukin family. It can be produced by fibroblasts, monocytes / macrophages, T lymphocytes, B lymphocytes, epithelial cells, keratinocytes, and various tumor cells. IL-6 has functions such as regulating the growth and differentiation of various cells and immune responses, and is involved in various acute and chronic inflammatory disease processes including bacterial infections, neonatal sepsis, respiratory failure, systemic lupus erythematosus, enteritis, cardiovascular diseases, rheumatoid arthritis, etc., and plays a role in, for example, acute inflammatory responses, autoimmune diseases, and tumor formation. Abnormal and excessive secretion of IL-6 may lead to severe inflammatory responses, including systemic inflammatory response syndrome and cytokine release syndrome.

[0003] Clinically, during acute inflammatory response processes such as trauma, surgery, and infection, IL-6 will rapidly increase. The combined use of IL-6 and procalcitonin (PCT) can more efficiently distinguish between bacterial and viral infections, and thus can be used to guide the rational use of antibiotics and reduce the abuse of antibiotics. In addition, IL-6 can be used for early identification of sepsis and monitoring of treatment effects, monitoring of postoperative infections, auxiliary diagnosis of neonatal sepsis, avoiding missed diagnosis of focal bacterial infections such as community-acquired pneumonia (CAP) by PCT, and reducing missed diagnoses. Therefore, detecting IL-6 is of great significance for early diagnosis, prognosis judgment, guiding drug use, and studying the pathogenesis.

[0004] Currently, the main detection methods for IL-6 are colloidal gold immunochromatography and fluorescence immunochromatography, both of which are immunological detection methods based on the specific reaction between antibodies and antigens, and at the same time use labeling substances (such as colloidal gold, fluorescence) to amplify and display the detected signal. Similar immunological detection methods also include biochemical immunoturbidimetry, radioimmunoassay, chemiluminescence assay, etc. The above immunological detection methods all require antibodies against IL-6.

[0005] Therefore, there is a strong demand in the art for anti-interleukin-6 antibodies with good performance. Summary of the Invention

[0006] The present application provides an antibody or its antigen-binding fragment, which provides an important raw material source for the detection of interleukin-6 and has good activity or affinity.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, which comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequence SEQ ID NO: 17 or 18 and three complementary determining regions of the light chain variable region having the amino acid sequence SEQ ID NO: 19.

[0008] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, which comprises the following complementary determining regions:

[0009] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0010] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0011] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0012] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0013] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; and

[0014] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0015] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is any one of SEQ ID NO: 17 and 18; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19.

[0016] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, which comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is any one of SEQ ID NO: 20 and 21; and the amino acid sequence of the light chain is as shown in SEQ ID NO: 22.

[0017] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above-mentioned antibody or an antigen-binding fragment thereof.

[0018] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or kit, which comprises the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0019] To achieve the above object, according to the seventh aspect of the present invention, there is provided a method for detecting interleukin-6, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, or reagent or kit with interleukin-6 in a test sample under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0020] To achieve the above object, according to the eighth aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment.

[0021] To achieve the above object, according to the ninth aspect of the present invention, there is provided a vector comprising the above-mentioned nucleic acid.

[0022] To achieve the above object, according to the tenth aspect of the present invention, there are provided cells comprising the above-mentioned nucleic acid, vector, or expressing the above-mentioned antibody or its antigen-binding fragment.

[0023] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above-mentioned antibody or its antigen-binding fragment, the method comprising culturing the above-mentioned cells.

[0024] To achieve the above object, according to the twelfth aspect of the present invention, there is provided the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of a product for detecting interleukin-6. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 Results of reducing SDS-PAGE for Anti-IL-6 12D9Rmb1~Anti-IL-6 12D9 Rmb2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] In a first aspect, embodiments of the present invention provide an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequence SEQ ID NO: 17 or 18 and three complementary determining regions of the light chain variable region having the amino acid sequence SEQ ID NO: 19.

[0028] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody or the antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody or the antigen-binding fragment thereof described in the first aspect.

[0029] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17; LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 19.

[0030] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0031] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or an antigen-binding fragment thereto to its recognized antigen or epitope. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0032] In the present invention, the heavy chain complementary determining regions are represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are represented by LCDR, which includes LCDR1, LCDR2, and LCDR3.

[0033] The method for defining CDRs is well-known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a part of the CDR region defined by Kabat, although they may be shortened or extended according to the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0034] Table 1: CDR Definitions 1

[0035]

[0036]

[0037] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly correspond this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0039] 3If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0040] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0041] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0042] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0045] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0047] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0048] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0049] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0050]

[0051] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0052] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising the following complementarity-determining regions:

[0053] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same;

[0054] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same;

[0055] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same;

[0056] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same;

[0057] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same; and

[0058] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0059] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, and refers to the regions other than the CDRs in the variable regions of the heavy chain and the light chain of the antibody; wherein, the heavy-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0060] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0061] In an alternative embodiment, the antibody or its antigen-binding fragment according to the first aspect or the second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0062] The HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity therewith;

[0063] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity therewith;

[0064] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith;

[0065] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith;

[0066] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity therewith;

[0067] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity therewith;

[0068] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity therewith;

[0069] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity therewith.

[0070] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13, or 14).

[0071] In an alternative embodiment, the HFR1 comprises the amino acid sequence as shown in SEQ ID NO: 7 or 23.

[0072] In an alternative embodiment, the antibody or its antigen-binding fragment binds interleukin-6 with an affinity of KD < 4.97×10 -9 M.

[0073] In an alternative embodiment, the antibody or its antigen-binding fragment binds interleukin-6 with an affinity of KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M, KD ≤ 10 -12 M, or KD ≤ 10 -13 M.

[0074] In an alternative embodiment, the antibody or its antigen-binding fragment binds interleukin-6 with an affinity of KD ≤ 3.06×10 -10 M.

[0075] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0076] In an alternative embodiment, the KD is measured by a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as the system.

[0077] In a third aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 17 and 18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19.

[0078] In an alternative embodiment, the antibody or its antigen-binding fragment further comprises a constant region.

[0079] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0080] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0081] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0082] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0083] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant regions.

[0084] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cattle, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, gamecock, or human.

[0085] In an alternative embodiment, the species origin of the constant region is mouse.

[0086] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0087] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above constant regions (SEQ ID NO: 15 or 16).

[0088] In an alternative embodiment, the antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

[0089] The antigen-binding fragments of the above antibodies generally have the same binding specificity as their source antibodies. Those skilled in the art can easily understand from the content recorded in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of chemically reducing and cleaving disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0090] The antigen-binding fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using an automated peptide synthesizer, such as those sold by Applied BioSystems, etc.

[0091] Fourth aspect, the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 20 and 21, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 22.

[0092] Fifth aspect, the present invention provides an antibody conjugate, which comprises the above-mentioned antibody or an antigen-binding fragment thereof.

[0093] In an alternative embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or an antigen-binding fragment thereof.

[0094] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody or an antigen-binding fragment thereof.

[0095] In an alternative embodiment, the above-mentioned label refers to a class of substances having characteristics such as luminescence, chromogenicity, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through this characteristic.

[0096] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0097] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0098] In an alternative embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0099] In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0100] In alternative embodiments, the radioisotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0101] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, rosamines and their derivatives, and peroxyoxalates and their derivatives.

[0102] In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0103] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0104] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0105] In alternative embodiments, the colloidal metal is colloidal gold.

[0106] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0107] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0108] In alternative embodiments, the solid-phase carriers include, but are not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0109] In a sixth aspect, the present invention provides a reagent or a kit, which comprises the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0110] As described above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to interleukin-6. Therefore, reagents or kits containing the interleukin-6 antibodies or antigen-binding fragments thereof can effectively qualitatively or quantitatively detect interleukin-6. Applying the reagents or kits provided by the present invention, for example, can be used in detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of interleukin-6 and its antibodies. As described above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity with interleukin-6. Therefore, the reagents or kits containing the antibodies or antigen-binding fragments thereof have higher detection sensitivity or specificity.

[0111] In a seventh aspect, the present invention provides a method for detecting interleukin-6, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with interleukin-6 in a test sample to form an immune complex under conditions sufficient for an antibody / antigen binding reaction; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0112] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or its antigen-binding fragment.

[0113] In an alternative embodiment, the immune complex further comprises a second antibody that binds to interleukin-6.

[0114] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0115] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0116] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0117] In an eleventh aspect, the present invention provides a method for preparing an antibody or its antigen-binding fragment, which comprises: culturing the cells as described above.

[0118] In a twelfth aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting interleukin-6.

[0119] Based on the amino acid sequences of the antibodies or their antigen-binding fragments disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare such antibodies or their antigen-binding fragments. For example, the antibodies or their antigen-binding fragments can be isolated and purified from the culture products of recombinant cells capable of recombinantly expressing the antibodies or their antigen-binding fragments described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the antibodies or their antigen-binding fragments of the present invention, they all fall within the protection scope of the present invention.

[0120] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained by purchasing in the market.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0122] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0123] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0124] Example 1 Preparation of Anti-IL-6 12D9 Monoclonal Antibody

[0125] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-IL-6 12D9 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0126] (1) Preparation of antibody genes

[0127] mRNA was extracted from the hybridoma cell line secreting the Anti-IL-6 12D9 monoclonal antibody, and DNA products were obtained by RT-PCR. After adding A to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, 4 clones of the Heavy Chain and Light Chain genes were respectively taken for cloning and sent to a gene sequencing company for sequencing.

[0128] (2) Sequence analysis of the variable region genes of the Anti-IL-6 12D9 antibody

[0129] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 321bp, and there was a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 372bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0130] (3) Construction of recombinant antibody expression plasmids

[0131] pcDNA TM 3.4 The vector pcDNA3.4 was the recombinant antibody eukaryotic expression vector constructed. This expression vector had introduced multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI, and was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and 0.70kb Light Chain gene fragments and 1.40kb Heavy Chain gene fragments were amplified by PCR.

[0132] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0133] 2. Recombinant antibody production

[0134] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system until the cell density reaches 3 - 5×10 6 cells / ml. Select the antibody concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place it in a 35°C constant temperature incubator for culture at a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0135] The obtained antibody was named Anti-IL-6 12D9Rmb1, and Anti-IL-6 12D9Rmb1 was mutated to obtain a mutant antibody named Anti-IL-6 12D9Rmb2

[0136] The amino acid sequences of the heavy chain and light chain of the Anti-IL-6 12D9Rmb1 antibody are shown in SEQ ID NO:20 and 22 respectively;

[0137] The amino acid sequences of the heavy chain and light chain of the Anti-IL-6 12D9Rmb2 antibody are shown in SEQ ID NO:21 and 22 respectively.

[0138] Example 2 Performance detection of the antibody

[0139] 1. Affinity analysis

[0140] Dilute the purified antibody in advance, and at the same time perform gradient dilution on the interleukin-6 antigen (from Fapon Biotech); use the CM5 chip that has been pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0141] Table 2 Affinity data

[0142] Sample Name KD ka kd Control 4.97E-09 3.65E+05 1.81E-03 Anti-IL-6 12D9Rmb1 2.96E-10 1.71E+06 5.06E-04 Anti-IL-6 12D9Rmb2 3.06E-10 2.22E+06 6.79E-04 。

[0143] 2. Activity identification

[0144] Dilute the interleukin-6 antigen (from Fapon Biotech) with the coating solution (main component NaHCO3) to 1 μg / ml, 100 μL per well, and incubate overnight at 4°C; the next day, wash twice with the washing solution (main components Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the termination solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0145] Note: Solution A (main components citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components citric acid + EDTA·2Na + TMB + concentrated HCl); Termination solution (EDTA·2Na + concentrated H2SO4)

[0146] Table 3 Activity data

[0147] Concentration (ng / ml) 250.00 125.00 62.50 31.25 15.63 0.00 Control 1.421 0.803 0.528 0.302 0.119 0.021 Anti-IL-6 12D9Rmb1 1.993 1.467 1.010 0.598 0.404 0.026 Anti-IL-6 12D9Rmb2 1.977 1.433 1.022 0.588 0.428 0.024

[0148] 3. Stability assessment

[0149] Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples at 7 days, 14 days, and 21 days for status observation, and perform activity detection on the 21-day samples. The results show that there is no obvious change in the protein state of the antibody after being placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. The following Table 4 shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-IL-6 12D9Rmb1 after 21 days of assessment.

[0150] Table 4 Stability Data

[0151] Sample Concentration (ng / ml) 125.00 62.50 0.00 Sample at 4°C for 21 days 1.446 1.033 0.022 Sample at -80°C for 21 days 1.422 1.025 0.023 Sample at 37°C for 21 days 1.473 1.042 0.024

[0152] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0153] Part of the amino acid sequences involved in this application are shown in Table 5 as follows:

[0154]

[0155]

Claims

1. An anti-interleukin-6 antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment has three complementarity-determining regions of any one of the heavy chain variable regions of amino acid sequences SEQ ID NO: 17 and 18 and three complementarity-determining regions of the light chain variable region of amino acid sequence SEQ ID NO: 19, and the complementarity-determining regions of the variable regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

2. An anti-interleukin-6 antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises the following complementarity-determining regions: The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5; and The amino acid sequence of LCDR3 is as shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that, The HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and 4, characterized in that, The antibody or its antigen-binding fragment binds to interleukin-6 with an affinity of KD < 4.97×10 -9 M.

6. An anti-interleukin-6 antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 17 and 18; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

19.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 6, characterized in that, The antibody or its antigen-binding fragment further comprises a constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The constant region comprises a heavy chain constant region and a light chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

10. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

11. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that, The species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose or human.

12. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that,The species origin of the constant region is mouse.

13. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity therewith; the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity therewith.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4 to 6, characterized in that, The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

15. An anti-interleukin-6 antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:20 and 21; the amino acid sequence of the light chain is as shown in SEQ ID NO:

22.

16. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 14 or the antibody according to claim 15.

17. The antibody conjugate according to claim 16, characterized in that, The antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

18. The antibody conjugate according to claim 16, characterized in that, The antibody conjugate further comprises a label conjugated to the antibody or its antigen-binding fragment.

19. The antibody conjugate according to claim 18, characterized in that, The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels.

20. The antibody conjugate according to claim 16, characterized in that, The antibody conjugate further comprises a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

21. The antibody conjugate according to claim 16, characterized in that, The solid-phase carrier is selected from microspheres, plates and membranes.

22. A reagent or kit, characterized in that, The reagent or kit comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 14 or the antibody according to claim 15 or the antibody conjugate according to any one of claims 16 to 21.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 21, or the reagent or kit according to claim 22 in the preparation of a product for detecting interleukin-6, characterized in that, Comprising: a) contacting the antibody or its antigen-binding fragment according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 21, or the reagent or kit according to claim 22 with interleukin-6 in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

24. The use according to claim 23, characterized in that, The immune complex further comprises a second antibody that binds to the antibody or its antigen-binding fragment.

25. The use according to claim 23, characterized in that, The immune complex further comprises a second antibody that binds to interleukin-6.

26. A nucleic acid, characterized in that, It encodes the antibody or its antigen-binding fragment according to any one of claims 1 to 14 or the antibody according to claim 15.

27. A vector, characterized in that, It contains the nucleic acid according to claim 26.

28. A cell, characterized in that, It contains the nucleic acid according to claim 26 or the vector according to claim 27.

29. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the method according to claim 15, characterized in that, It comprises: culturing the cell according to claim 28.

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