Anti-statin B antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-inhibin B antibody and applications thereof. Background Art

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

[0003] In clinical practice, inhibin B is commonly used to detect and diagnose gonadal function, including various spermatogenesis disorders and ovarian reserve. In men, INHB is a direct product of the vas deferens and reflects the function of the entire testicular tissue. It is commonly used to diagnose disorders such as spermatogenesis, cryptorchidism, azoospermia, primary testicular failure, hypogonadotropinemia, Kallmann syndrome, Klinefelter syndrome, precocious puberty, and complete or selective loss of subthalamic or pituitary function. In women, INHB is a key indicator of ovarian reserve, directly involved in the regulation of folliculogenesis and development. It is crucial for the diagnosis and treatment of conditions such as diminished ovarian reserve, premature ovarian failure, polycystic ovary syndrome, and endometriosis.

[0004] Currently, the primary detection method for inhibin B is immunological, such as enzyme-linked immunosorbent assay (ELISA). The principles are: 1) binding the antigen or antibody to a solid-phase carrier surface while maintaining its immunological activity; and 2) linking the antigen or antibody to an enzyme to form an enzyme-labeled antigen or antibody, which retains both its immunological and enzymatic activity. The specimen to be tested (the antibody or antigen to be tested) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the solid-phase carrier surface in different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing. After the addition of the enzyme reaction substrate, the substrate is catalyzed by the enzyme to produce a colored product. The amount of the product is directly related to the amount of the substance being tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color reaction. Similar immunological detection methods include radioimmunoassay, fluorescent immunochromatography, and chemiluminescence.

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

[0006] The present application provides an anti-inhibin B antibody, 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 anti-inhibin B antibody is provided, wherein the antibody comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0008] In order to achieve the above object, according to a second aspect of the present invention, an anti-inhibin B antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0009] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0010] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0011] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

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

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

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

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

[0016] To achieve the above objectives, according to the fourth aspect of the present invention, an anti-inhibin B antibody 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; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0017] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0018] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above antibody or the above antibody conjugate.

[0019] In order to achieve the above-mentioned object, according to the seventh aspect of the present invention, there is provided a use of the above-mentioned antibody or antibody conjugate in the preparation of a product for detecting inhibin B.

[0020] In order to achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. DETAILED DESCRIPTION

[0021] In a first aspect, an embodiment of the present invention provides an anti-inhibin B antibody, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

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

[0023] Antigen-binding fragments typically have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that such antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain such antigen-binding fragments.

[0024] Antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0025] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of antibodies.

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

[0027] Methods for defining CDRs are well known in the art, including the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200200201154, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by 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, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0028] Table 1: CDR Definition 1

[0029]

[0030]

[0031] 1 The numbering of all CDR definitions in Table 1 is based on 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." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0032] 2"AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0033] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0034] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0035] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0036] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[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 a Contact system.

[0042] In some optional 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, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may be selected from Table 2, respectively.

[0044] Table 2 shows an 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] In a second aspect, an embodiment of the present invention provides an anti-inhibin B antibody, wherein the antibody comprises the following complementary determining regions:

[0048] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0049] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0050] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0051] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0052] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0053] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

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

[0055] 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 region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0056] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0058] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0059] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0060] The HFR3 comprises / is as SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0062] The LFR1 comprises / is represented by SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0063] The LFR2 comprises / is as SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0064] The above LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0065] The LFR4 comprises / is represented by SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0066] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-inhibin B antibodies provided herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0067] In an optional embodiment, the above 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 inhibin B with high affinity.

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

[0069] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0070] In alternative embodiments, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example, by using a kinetic assay such as System of biosensor systems.

[0071] In an optional embodiment, the above-mentioned antibody comprises a heavy chain variable region and / or a light chain variable region, 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.

[0072] In a third aspect, an embodiment of the present invention provides an anti-inhibin B antibody 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 shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19.

[0073] In an optional embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0074] In an alternative embodiment, the above-mentioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0075] In an optional embodiment, the 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 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 embodiment, the above-mentioned IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0078] In an alternative embodiment, the above-mentioned light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0079] In an optional embodiment, the species origin of the above-mentioned constant region is cow, horse, pig, sheep, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0080] In an optional embodiment, the species origin of the above-mentioned constant region is mouse.

[0081] In this article, the division of variable and constant region sequences refers to the IMGT division 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.. Variable regions divided by different methods may differ from the variable region C-terminus or constant region N-terminus divided by IMGT. Variable regions or constant regions divided by other methods known in the art are also within the scope of protection of the present invention.

[0082] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as 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 above-mentioned antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0085] In an optional embodiment, the above-mentioned antibody includes a heavy chain and / or a light chain, 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.

[0086] In a fourth aspect, the present invention provides an anti-inhibin B antibody 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.

[0087] In a fifth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody.

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

[0089] In an optional embodiment, the antibody conjugate further comprises a marker or purification tag coupled to the antibody.

[0090] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In an optional embodiment, the above-mentioned labels include but are not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle labels.

[0092] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0093] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

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

[0095] In an optional embodiment, the above-mentioned 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 an optional embodiment, the above-mentioned chemiluminescent reagents include but are not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0097] In an optional embodiment, 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 an optional embodiment, the colloid includes but is not limited to colloidal metal, colloidal carbon, disperse dyes, dye-labeled microspheres and latex.

[0099] In an optional embodiment, the colloidal metal includes but is 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 comprises a solid phase carrier coupled to the antibody.

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

[0103] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

[0104] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0105] As previously described, the antibodies in some embodiments or examples of the present invention are capable of effectively binding to inhibin B. Therefore, reagents or kits containing such inhibin B antibodies are capable of effectively performing qualitative or quantitative detection of inhibin B. The reagents or kits provided herein can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of inhibin B and its antibodies. As previously described, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity for inhibin B. Therefore, reagents or kits containing such 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 above-mentioned antibody, antibody conjugate, reagent or kit with inhibin B in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

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

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

[0109] In an eighth aspect, the present invention provides use of the above-mentioned anti-inhibin B antibodies and antibody conjugates in the preparation of products for detecting inhibin B.

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

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

[0112] In a tenth aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0113] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

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

[0115] In a thirteenth aspect, the present invention provides use of the above-mentioned antibody, antibody conjugate, or the above-mentioned 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 indicating an inhibin B-related disease in a subject, comprising:

[0117] a) contacting the above-mentioned antibody, antibody conjugate, or the above-mentioned reagent or kit with inhibin B in a sample from a subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and

[0118] b) detecting the presence of the aforementioned immune complex, wherein the presence of the aforementioned complex indicates the presence or status of an inhibin B-related disease in the subject.

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

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

[0121] In an optional embodiment, the inhibin B-related disease in the thirteenth aspect or the fourteenth aspect is selected from gonadal function diseases such as spermatogenesis disorders and ovarian reserve function.

[0122] In an optional embodiment, the inhibin B-related disease of the thirteenth aspect or the fourteenth aspect is selected from the group consisting of diminished ovarian reserve, premature ovarian failure, polycystic ovary syndrome, endometriosis, spermatogenic dysfunction, cryptorchidism, azoospermia, primary testicular failure, hypogonadotropin function, Kallmann syndrome, Klinefelter syndrome, precocious puberty, and complete or selective loss of subthalamic or pituitary function.

[0123] Based on the amino acid sequence of the anti-inhibin B antibody disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-inhibin B antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express any of the above-mentioned antibodies. This is easy for those skilled in the art to achieve. Based on this, no matter what technology is used to prepare the anti-inhibin B antibody of the present invention, it falls within the scope of protection of the present invention.

[0124] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled 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 preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0126] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

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

[0128] Example 1 Antibody Discovery of Monoclonal Antibodies

[0129] 1. Animal immunization

[0130] Purified inhibin B antigen (from Feipeng Bio) was used as an immunogen for immunization mice. Specifically, female BALB / c mice aged 6-8 weeks were selected and immunized 4 times, with each immunization interval of 1 week and an immunizing agent of 100 μg / mouse. In the first immunization, inhibin B antigen was mixed with equal volumes of Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple points on the back. In the second immunization, inhibin B antigen was mixed with equal volumes of Freund's incomplete adjuvant (Sigma) and injected intraperitoneally. Seven days after the third immunization, blood was collected from the mouse orbits, serum was separated, and the antibody titer of the immunized mice was detected by indirect ELISA. Mice with suitable serum antibody titers were selected for cell fusion experiments. Three days before the cell fusion experiments, inhibin B antigen without adjuvant was injected intraperitoneally to perform booster immunization (100 μg / only).

[0131] 2. Preparation of hybridoma cell lines

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

[0133] Example 2 Preparation of monoclonal antibodies

[0134] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. 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 also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. 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. This product was PCR-amplified with rTaq DNA polymerase and inserted into the pMD-18T vector. The resulting cells were transformed into DH5α competent cells, and colonies were grown. The heavy and light chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

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

[0139] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and 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 them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 333 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 348 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

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

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

[0142] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0143] 2. Recombinant Antibody Production

[0144] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 cells / ml and re-dissolved in culture medium, which was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed thoroughly, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell diluent over 1 minute, mixed thoroughly, and samples were taken and counted. The viability of the cells after transfection was recorded and observed, and the cells were placed in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column to obtain purified antibodies.

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

[0146] Table 3: Antibody sequences

[0147] Antibody name Heavy chain light chain Anti-INHB 12F11Rmb1 SEQ ID NO: 18 SEQ ID NO:20

[0148] Example 2 Performance testing of antibodies

[0149] 1. Activity identification

[0150] The coating solution (main component NaHCO3) was used to dilute the inhibin B recombinant antigen (from Feipeng Bio) to 2.5ug / ml, 100ul per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution (main component Na2HPO4+NaCl) and patted dry; the blocking solution (20% BSA+80% PBS) was added, 120ul per well, incubated at 37°C for 1h, and patted dry; the diluted purified antibody and control antibody were added, 100ul / well, incubated at 37°C for 30min; the cells were washed 5 times with the washing solution and patted dry; the goat anti-mouse IgG-HRP was added, 100ul per well, incubated at 37°C for 30min; the cells were washed 5 times with the washing solution and patted dry; the colorimetric solution A (50ul / well) and the colorimetric solution B (50ul / well) were added for 10min; the stop solution was added, 50ul / well; the OD value was read at 450nm (reference 630nm) on a microplate reader.

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

[0152] Table 4: Activity data

[0153] Concentration (ng / ml) 125 62.50 31.25 15.63 7.81 0.00 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 Platform

[0155] Anti-INHB 12F11Rmb1, the antibody from the previous example, was used as the labeled antibody, labeled with an acridinium ester. Five other antibodies, INHB-1, INHB-2, INHB-3, INHB-4, and INHB-5 (from Feipeng Bio), were used as coating antibodies and coated onto magnetic beads. The performance of these antibodies was tested using a double-antibody sandwich assay on a chemiluminescence platform.

[0156] Instrument used: Yingkai Shine I2910, reaction mode:

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

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

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

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

[0161] Table 5: Chemiluminescence platform performance data2

[0162]

[0163] 3. Stability assessment

[0164] The above-mentioned antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for status observation, and the 21-day samples were tested for activity. The results showed that no obvious changes in protein status were observed in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above-mentioned antibodies are stable. Table 6 below shows the OD results of the enzyme immunoassay activity test of the antibody Anti-INHB 12F11Rmb1 for 21 days.

[0165] Table 6: Stability data

[0166]

[0167]

[0168] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0169] Some of the amino acid sequences involved in this application are shown in Table 7:

[0170] Table 7: Amino acid sequence listing

[0171]

[0172]

Claims

1. An anti-inhibin B antibody comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having the amino acid sequence shown in SEQ ID NO:

19.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems, or a combination of multiple systems.

3. An anti-inhibin B antibody, characterized in that The antibody comprises the following complementarity determining regions: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6; Optionally, the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence at least 80% identical thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. An anti-inhibin B antibody comprising a heavy chain variable region and / or 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; Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the 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; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv and scFv.

5. An anti-inhibin B antibody comprising a heavy chain and / or 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.

6. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a marker or purification tag coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or kit, characterized in that The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting inhibin B; Optionally, the use includes: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with inhibin B in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to inhibin B.

9. A nucleic acid, a vector, a cell, or a method for producing the antibody of any one of claims 1 to 5, wherein the nucleic acid encodes the antibody of any one of claims 1 to 5; the vector comprises a nucleic acid encoding the antibody of any one of claims 1 to 5; the cell comprises the nucleic acid or vector; and the method comprises the cell.

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