An antibody against inhibin b and uses thereof

Abstract

The application discloses an anti-inhibin B antibody and application thereof, and relates to the field of antibodies. The anti-inhibin B antibody disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of inhibin B, and has good performance.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antibody against inhibin B and its application. Background Technology

[0002] Inhibin B (INHB) is a heterogeneous glycoprotein derived from germ cells, composed of BB and α subunits linked together. Its predicted molecular weight is approximately 28 kDa, and it belongs to the superfamily of transforming growth factor-β (TGF-β). In males, Inhibin B is primarily produced by testicular Sertoli cells, while in females it is mainly produced by ovarian cells. High levels of INHB in the body regulate the pituitary gland and inhibit FSH release through negative feedback; it also affects paracrine function of the gonads by regulating estradiol (E2) levels. INHB levels are closely related to male spermatogenesis, testicular volume, and total sperm count, as well as female ovarian reserve and infertility caused by ovarian factors. Its levels decrease with age, directly reflecting the gonadal reserve in both sexes.

[0003] Clinically, inhibin B is commonly used for the detection and diagnosis of various spermatogenesis disorders, ovarian reserve function, and other gonadal functions. In men, inhibin B is a direct product of the vas deferens and reflects the function of the entire testicular tissue. It is often used to diagnose diseases such as impaired spermatogenesis, cryptorchidism, azoospermia, primary testicular failure, hypogonadotropic hypofunction, Kallmann syndrome, Klinefelter syndrome, precocious puberty, and complete or selective loss of thalamic or pituitary function. In women, inhibin B is an important indicator of ovarian reserve, directly involved in the regulation of follicle formation and development. It is of great significance in the diagnosis and treatment of diseases such as decreased ovarian reserve, premature ovarian failure, polycystic ovary syndrome, and endometriosis.

[0004] Currently, the detection methods for inhibin B are mainly immunological methods, such as enzyme-linked immunosorbent assay (ELISA). The principle is to 1) bind the antigen or antibody to the surface of a solid-phase carrier while maintaining its immunological activity, and 2) connect the antigen or antibody to an enzyme to form an enzyme-labeled antigen or antibody. This enzyme-labeled antigen or antibody retains both its immunological activity and the activity of the enzyme. The test sample (containing the antibody or antigen to be measured) and the enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid-phase carrier in different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing. After adding the substrate for the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. The amount of product is directly related to the amount of the test substance in the sample; therefore, qualitative or quantitative analysis can be performed based on the intensity of the color reaction. Similar immunological detection methods include radioimmunoassay, fluorescence immunochromatography, and chemiluminescence immunoassay.

[0005] The aforementioned immunological detection methods all require antibodies targeting inhibin B. Therefore, there is a strong demand in the field for antibodies against inhibin B with good performance. Summary of the Invention

[0006] This application provides an antibody against inhibin B, which provides an important source of raw materials for the detection of inhibin B and has good performance.

[0007] To achieve the above objectives, according to one aspect of the present invention, an antibody against inhibin B is provided, the antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:17 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in SEQ ID NO:19.

[0008] To achieve the above objective, according to a second aspect of the present invention, an antibody against inhibin B is provided, the antibody comprising the following complementarity-determining region:

[0009] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0010] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0011] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0012] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0013] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5;

[0014] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0015] To achieve the above objectives, according to a third aspect of the present invention, an antibody against inhibin B is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in SEQ ID NO:17; and the amino acid sequence of the aforementioned light chain variable region is shown in SEQ ID NO:19.

[0016] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against inhibin B is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18; and the amino acid sequence of the light chain is shown in SEQ ID NO:20.

[0017] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0018] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody or antibody conjugate described above.

[0019] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody and antibody-drug conjugate in the preparation of a product for detecting inhibin B is provided.

[0020] To achieve the above objectives, the present invention also provides a nucleic acid, a vector, a cell, and a method for preparing the above-mentioned antibody. Detailed Implementation

[0021] In a first aspect, embodiments of the present invention provide an antibody against inhibin B, the aforementioned antibody comprising three complementary determinant regions having a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:17 and three complementary determinant regions having a light chain variable region having an amino acid sequence as shown in SEQ ID NO:19.

[0022] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments, as long as they exhibit the desired biological activity.

[0023] Antigen-binding fragments typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the aforementioned antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0024] Antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0025] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of antibodies.

[0026] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0027] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an 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 those in Table 1, are also within the scope of this disclosure.

[0028] Table 1: CDR Definition 1

[0029]

[0030]

[0031] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0032] 2As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0033] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0034] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0035] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0036] According to embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0037] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0038] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0039] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0040] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0041] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0042] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0043] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may be selected from Table 2.

[0044] Table 2 shows the exemplary antibody Anti-INHB 12F11RMb1 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19).

[0045] Table 2

[0046]

[0047] Secondly, embodiments of the present invention provide an antibody against inhibin B, wherein the antibody includes the following complementarity-determining region:

[0048] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1.

[0049] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2.

[0050] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3.

[0051] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4.

[0052] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5.

[0053] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0054] According to an embodiment of the present invention, the above-mentioned HCDRs and LCDRs are defined by the Kabat system.

[0055] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0056] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: 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 combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In an optional embodiment, the antibody described in the first or second aspect further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0058] In an optional embodiment, the HFR1 described above includes / such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0059] The HFR2 mentioned above includes / such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0060] The HFR3 mentioned above includes / such as SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0061] The HFR4 mentioned above includes / such as SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it;

[0062] The LFR1 mentioned above includes / such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it;

[0063] The LFR2 mentioned above includes / such as SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it;

[0064] The LFR3 mentioned above includes / such as SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; and

[0065] The LFR4 mentioned above includes / such as SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

[0066] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the anti-inhibin B antibody 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 frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0067] In an optional implementation, the above-mentioned antibody has a KD ≤ 10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds to inhibin B with its affinity.

[0068] In an optional embodiment, the above-mentioned antibody has a KD ≤ 3.17 × 10⁻⁶. -7 M binds to inhibin B with its affinity.

[0069] There are many methods for determining antibody affinity (KD), which can be categorized into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods based on their detection principles. Common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biomembrane optical interferometry (BLI); and common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA).

[0070] In an optional implementation, KD is measured using a kinetic detection method; alternatively, surface plasmon resonance, for example, by using a method such as The system's biosensor system.

[0071] In an optional embodiment, the antibody described above comprises a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in SEQ ID NO:17 and the amino acid sequence of the aforementioned light chain variable region is shown in SEQ ID NO:19.

[0072] Thirdly, embodiments of the present invention provide an antibody against inhibin B, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the aforementioned light chain variable region is shown in SEQ ID NO:19.

[0073] In optional embodiments, the antibodies described in the first, second, and third aspects above further include a constant region.

[0074] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0075] In an optional embodiment, the aforementioned heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0076] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0077] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0078] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0079] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0080] In an optional implementation, the species source of the aforementioned constant region is mice.

[0081] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.org Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0082] In an optional embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO:15, and the light chain constant region sequence (CL) is shown in SEQ ID NO:16.

[0083] It should be noted that, in other embodiments, the above-mentioned 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-mentioned constant region (SEQ ID NO: 15 or 16).

[0084] In an optional embodiment, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0085] In an optional embodiment, the antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18 and the amino acid sequence of the light chain is shown in SEQ ID NO:20.

[0086] Fourthly, the present invention provides an antibody against inhibin B, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the aforementioned heavy chain is shown in SEQ ID NO:18 and the amino acid sequence of the aforementioned light chain is shown in SEQ ID NO:20.

[0087] Fifthly, the present invention provides an antibody conjugate, wherein the aforementioned antibody conjugate includes the antibody described above.

[0088] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody.

[0089] In an optional embodiment, the antibody conjugate may further include a marker or purification tag conjugated to the antibody.

[0090] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0092] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0093] In optional embodiments, the fluorescent dyes mentioned above include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0094] In optional embodiments, the enzymes mentioned above include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0095] In optional embodiments, the aforementioned radioactive isotopes 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.

[0096] In optional embodiments, the chemiluminescent reagents mentioned above include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0097] In optional embodiments, the above-mentioned nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0098] In optional embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0099] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an optional embodiment, the colloidal metal is colloidal gold.

[0101] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated with the antibody.

[0102] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0103] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

[0104] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antibody conjugate described above.

[0105] As previously described, the antibodies in some embodiments or examples of this invention can effectively bind to inhibin B. Therefore, reagents or kits containing the aforementioned inhibin B antibodies can effectively perform qualitative or quantitative detection of inhibin B. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of inhibin B and its antibodies, such as immunoblotting and immunoprecipitation. As previously described, the antibodies in some embodiments or examples of this invention have higher binding activity or affinity for inhibin B; therefore, reagents or kits containing the aforementioned antibodies have higher detection sensitivity or specificity.

[0106] In a seventh aspect, the present invention provides a method for detecting inhibin B, comprising: a) contacting the antibody, antibody conjugate, reagent or kit described above with inhibin B in a sample to be tested under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the aforementioned immune complex, the presence of the aforementioned complex indicating the presence of the aforementioned antigen in the test sample.

[0107] In an optional embodiment, the immune complex further includes a second antibody, which binds to the antibody.

[0108] In an optional embodiment, the immune complex further includes a second antibody that binds to inhibin B.

[0109] Eighthly, the present invention provides the use of the above-mentioned anti-inhibin B antibody and antibody conjugate in the preparation of products for detecting inhibin B.

[0110] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0111] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0112] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0113] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0114] In a twelfth aspect, the present invention provides a method for preparing an anti-inhibin B antibody, comprising: culturing cells as described above.

[0115] In a thirteenth aspect, the present invention provides the use of the above-described antibody, antibody-drug conjugate, or the above-described reagent or kit in detecting inhibin B or indicating inhibin B-related diseases.

[0116] In a fourteenth aspect, the present invention provides a method for indicative of inhibin B-related disease in a subject, comprising:

[0117] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody, antibody conjugate, or the aforementioned reagent or kit is contacted with inhibin B in a sample from the subject to form an immune complex; and

[0118] b) Detect the presence of the aforementioned immune complexes, the presence of which indicates the presence or status of inhibin B-related disease in the subject.

[0119] In an optional embodiment, the immune complex further includes a second antibody, which binds to the antibody.

[0120] In an optional embodiment, the immune complex further includes a second antibody that binds to inhibin B.

[0121] In an optional embodiment, the inhibin B-related diseases described in aspect thirteen or fourteen are selected from gonadal dysfunction diseases such as spermatogenesis disorders and ovarian reserve dysfunction.

[0122] In an optional embodiment, the inhibin B-related diseases described in aspect thirteen or fourteen are selected from decreased ovarian reserve, premature ovarian failure, polycystic ovary syndrome, endometriosis, spermatogenesis disorders, cryptorchidism, azoospermia, primary testicular failure, hypogonadotropic hypofunction, Kallmann syndrome, Klinefelter syndrome, precocious puberty, and complete or selective loss of function of the thalamus or pituitary gland.

[0123] Based on the amino acid sequence of the anti-inhibin B antibody disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-inhibin B antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing any of the antibodies described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-inhibin B antibody of this invention, it falls within the protection scope of this invention.

[0124] 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0126] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0127] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0128] Example 1: Antibody Discovery of Monoclonal Antibodies

[0129] 1. Animal immunization

[0130] Purified inhibin B antigen (from Phypeng Biotechnology) was used as an immunogen for immunizing mice. Specifically, 6-8 week old female BALB / c mice were selected and immunized four times, with each immunization occurring one week apart. The immunogen was 100 μg per mouse. For the first immunization, inhibin B antigen was mixed with an equal volume of Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple sites on the back. For the subsequent three immunizations, inhibin B antigen was mixed with an equal volume of Freund's incomplete adjuvant (Sigma) and injected intraperitoneally. Seven days after the third immunization, blood was collected from the orbital sinus of the mice, serum was separated, and the antibody titer of the immunized mice was detected using an indirect ELISA method. Mice with suitable serum antibody titers were selected for cell fusion experiments. Three days before the cell fusion experiment, a booster immunization was performed by intraperitoneal injection of inhibin B antigen without adjuvant (100 μg per mouse).

[0131] 2. Preparation of hybridoma cell lines

[0132] On the third day after booster immunization of mice, spleens were removed under sterile conditions. Mouse tumor cells and immune spleen cells were mixed at a ratio of 1:5, fused, and cultured. The culture medium was changed twice on the sixth day. On the seventh day after fusion, cell supernatant was collected for ELISA detection, and positive pools were selected for subcloning. On the sixth day of subcloning, cell supernatant was collected for ELISA detection, positive clone wells were marked, and positive single clone wells were selected under a microscope. Based on the OD value and growth status of the single clones detected by ELISA, one dominant hybridoma cell line was finally selected and named Anti-INHB 12F11.

[0133] Example 2: Preparation of Monoclonal Antibodies

[0134] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen.

[0135] 1. Antibody sequence acquisition

[0136] (1) Antibody gene preparation

[0137] mRNA was extracted from the Anti-INHB 12F11 hybridoma cell line, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after being subjected to an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After the cells grew, four clones of the heavy chain and four clones of the light chain were collected and sent to a gene sequencing company for sequencing.

[0138] (2) Sequence analysis of antibody variable region genes

[0139] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 333 bp, with a 57 bp leader peptide sequence preceding it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 348 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0140] (3) Construction of recombinant antibody expression plasmid

[0141] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.71kb Light Chain gene fragment and the 1.38kb Heavy Chain gene fragment were amplified by PCR.

[0142] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0143] 2. Recombinant antibody production

[0144] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Diluents for plasmid DNA and transfection reagent were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. Cells were then incubated at 35°C (120 rpm) with 8% CO2 for 13 days. The samples were collected by centrifugation. The supernatant was purified using a protein A affinity chromatography column to obtain purified antibodies.

[0145] The resulting antibody was named Anti-INHB 12F11Rmb1. The heavy chain (H) and light chain (L) sequences of antibody Anti-INHB 12F11Rmb1 are shown in the table below:

[0146] Table 3: Antibody Sequences

[0147] Anti-INHB 12F11Rmb1 SEQ ID NO:18 SEQ ID NO:20

[0148] Example 2: Antibody Performance Detection

[0149] 1. Activity identification

[0150] Dilute the inhibin B recombinant antigen (from Feipeng Biotechnology) with coating buffer (main component NaHCO3) to 2.5ug / ml, 100ul per well, and incubate overnight at 4℃. The next day, wash twice with washing buffer (main component Na2HPO4 + NaCl) and blot dry. Add blocking buffer (20% BSA + 80% PBS), 120ul per well, incubate at 37℃ for 1 hour, and blot dry. Add diluted purified antibody and control antibody, 100ul / well, incubate at 37℃ for 30 minutes. Wash 5 times with washing buffer and blot dry. Add goat anti-mouse IgG-HRP, 100ul per well, incubate at 37℃ for 30 minutes. Wash 5 times with washing buffer and blot dry. Add chromogenic solution A (50ul / well) and chromogenic solution B (50ul / well), incubate for 10 minutes. Add stop solution, 50ul / well. Read the OD value at 450nm (reference 630nm) on the microplate reader.

[0151] Notes: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0152] Table 4: Activity Data

[0153] Comparison 0.989 0.502 0.218 0.087 0.024 0.014 Anti-INHB 12F11Rmb1 1.208 0.614 0.478 0.237 0.173 0.081

[0154] 2. Application of antibodies in chemiluminescence platforms

[0155] The antibody Anti-INHB 12F11Rmb1 from the above embodiments was used as the labeling antibody, labeled with acridinium ester; another five antibodies, INHB-1, INHB-2, INHB-3, INHB-4, and INHB-5 (from Phytobio), were used as coating antibodies, coated with magnetic beads. The performance of the above antibodies was tested on a chemiluminescence platform using a double-antibody sandwich method.

[0156] Instrument used: Shine I2910, Reaction mode:

[0157] 1) Add 50 μl of inhibin antigen quality control or 50 μL of sample processing solution to 50 μl of magnetic bead-labeled antibody to form a magnetic bead antibody-antigen complex, and react at 37°C for 20 min.

[0158] 2) Add 50 μl of acridine ester-labeled antibody Anti-INHB 12F11Rmb1 to form a magnetic bead antibody-antigen-acridine antibody complex. React at 37°C for 20 min and wash 3 times with PBST.

[0159] 3) Add 100 μl of pre-excitation solution and 100 μl of excitation solution, and read the instrument values. The specific performance evaluation results are shown in Table 5 below.

[0160] The results showed that Anti-INHB 12F11Rmb1 had good binding activity when paired with different INHB antibodies to detect inhibin B, even at low antigen concentrations, and Anti-INHB 12F11Rmb1 had high sensitivity.

[0161] Table 5: Performance data of the chemiluminescence platform 2

[0162]

[0163] 3. Stability assessment

[0164] The above-mentioned antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the above-mentioned antibodies were stable. Table 6 below shows the OD results of enzyme immunoassay for antibody Anti-INHB 12F11Rmb1 after 21 days of testing.

[0165] Table 6: Stability Data

[0166]

[0167]

[0168] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0169] The partial amino acid sequences involved in this application are shown in Table 7:

[0170] Table 7: Amino Acid Sequence List

[0171]

[0172]

Claims

1. An anti-inhibin B antibody, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody are, in order: HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO:17, and LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO:

19. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against inhibin B, characterized in that, The antibodies include the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: HCDR1, whose amino acid sequence is shown in SEQ ID NO:1; HCDR2, the amino acid sequence of which is shown in SEQ ID NO:2; HCDR3, the amino acid sequence of which is shown in SEQ ID NO:3; LCDR1, whose amino acid sequence is shown in SEQ ID NO:4; LCDR2, whose amino acid sequence is shown in SEQ ID NO:5; LCDR3, whose amino acid sequence is shown in SEQ ID NO:6; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

3. The antibody according to claim 1 or 2, characterized in that, The antibody further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; the amino acid sequence of HFR1 is at least 80% identical to that of SEQ ID NO:7; the amino acid sequence of HFR2 is at least 80% identical to that of SEQ ID NO:8; the amino acid sequence of HFR3 is at least 80% identical to that of SEQ ID NO:9; the amino acid sequence of HFR4 is at least 80% identical to that of SEQ ID NO:10; the amino acid sequence of LFR1 is at least 80% identical to that of SEQ ID NO:11; the amino acid sequence of LFR2 is at least 80% identical to that of SEQ ID NO:12; the amino acid sequence of LFR3 is at least 80% identical to that of SEQ ID NO:13; and the amino acid sequence of LFR4 is at least 80% identical to that of SEQ ID NO:

14.

4. The antibody according to claim 3, characterized in that, The amino acid sequence of HFR1 is shown in SEQ ID NO:7; the amino acid sequence of HFR2 is shown in SEQ ID NO:8; the amino acid sequence of HFR3 is shown in SEQ ID NO:9; the amino acid sequence of HFR4 is shown in SEQ ID NO:10; the amino acid sequence of LFR1 is shown in SEQ ID NO:11; the amino acid sequence of LFR2 is shown in SEQ ID NO:12; the amino acid sequence of LFR3 is shown in SEQ ID NO:13; and the amino acid sequence of LFR4 is shown in SEQ ID NO:

14.

5. An anti-inhibin B 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 shown in SEQ ID NO:17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

19.

6. The antibody according to claim 1 or 2, characterized in that, The antibody also contains a constant region.

7. The antibody according to claim 6, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

8. The antibody according to claim 7, characterized in that, The heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, and IgD.

9. The antibody according to claim 7, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

10. The antibody according to claim 6, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

11. The antibody according to claim 6, characterized in that, The species source of the constant region is mice.

12. The antibody according to claim 7, characterized in that, The heavy chain constant region sequence is an amino acid sequence with at least 80% identity to SEQ ID NO:15, and the light chain constant region sequence is an amino acid sequence with at least 80% identity to SEQ ID NO:

16.

13. The antibody according to claim 12, characterized in that, The heavy chain constant region sequence is shown in SEQ ID NO:15, and the light chain constant region sequence is shown in SEQ ID NO:

16.

14. The antibody according to any one of claims 1, 2, 4 to 5, characterized in that, The antibody comprises any one of F(ab')2, Fab', Fab, Fv, and scFv.

15. An anti-inhibin B antibody, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:18; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

16. An antibody conjugate, characterized in that, The antibody-drug conjugate consists of the antibody as described in any one of claims 1 to 15 and biotin conjugated to the antibody.

17. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody as described in any one of claims 1 to 15 and a label, purification tag, or solid-phase carrier conjugated to the antibody.

18. The antibody conjugate according to claim 17, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

19. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 15 or the antibody conjugate as described in any one of claims 16 to 18.

20. Use of the antibody according to any one of claims 1 to 15, or the antibody conjugate according to any one of claims 16 to 18, in the preparation of a product for detecting inhibin B.

21. A nucleic acid, characterized in that, It encodes the antibody as described in any one of claims 1 to 15.

22. A carrier, characterized in that, It contains the nucleic acid as described in claim 21.

23. A cell characterized in that, It contains the nucleic acid as described in claim 21 or the vector as described in claim 22.

24. A method for preparing the antibody according to any one of claims 1 to 15, characterized in that, It includes: Culture the cells as described in claim 23.

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