WO2023109803A1 - 抗抑制素抗体及其应用 - Google Patents

抗抑制素抗体及其应用 Download PDF

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WO2023109803A1
WO2023109803A1 PCT/CN2022/138630 CN2022138630W WO2023109803A1 WO 2023109803 A1 WO2023109803 A1 WO 2023109803A1 CN 2022138630 W CN2022138630 W CN 2022138630W WO 2023109803 A1 WO2023109803 A1 WO 2023109803A1
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antibody
variant
nucleic acid
seq
cell
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PCT/CN2022/138630
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English (en)
French (fr)
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张永
郑兴昌
王爱珂
金佳敏
杨佳乐
孙迪
陈宇
杨军帅
陈勇
陈璐
周思超
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宁波三生生物科技股份有限公司
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Priority to EP22906548.7A priority Critical patent/EP4339206A1/en
Priority to AU2022416227A priority patent/AU2022416227A1/en
Publication of WO2023109803A1 publication Critical patent/WO2023109803A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present disclosure belongs to the technical field of animal reproduction. Specifically, the present disclosure relates to an anti-inhibin antibody and its use in assisting animal reproduction.
  • Inhibin is a water-soluble protein hormone secreted by animal gonads and belongs to the transforming growth factor ⁇ superfamily (TGF- ⁇ ), because it has a strong effect on the production and secretion of pituitary follicle-stimulating hormone (FSH). named for its inhibitory effect.
  • Inhibin exists in the form of ⁇ heterodimers, and the ⁇ subunits are divided into five types: ⁇ A, ⁇ B, ⁇ C, ⁇ D, and ⁇ E. At present, inhibin A ( ⁇ A) and inhibin B ( ⁇ B) are mainly studied.
  • inhibin A is mainly secreted by dominant follicles and corpus luteum
  • inhibin B is secreted by small and medium antral follicles.
  • neutralizing inhibin through active or passive immunization can promote the production and secretion of endogenous FSH, which can stimulate follicle development and increase the number of ovulation in female animals, indicating that both methods have the potential to regulate the reproductive performance of livestock. It is of great value to livestock production and economic animal reproduction.
  • inhibition of FSH secretion can significantly increase the number of ovulations after immunization, but inhibin is an influencing factor for fertilized egg implantation, and active immunization induces long-term antibody neutralization of inhibin, which has a negative impact on conception rate and may lead to ovulation The number of cases increased but the conception rate decreased.
  • AIS inhibin antiserum
  • AIS has also shown a clinical effect better than that of pregnant horse serum gonadotropin (PMSG) in the timing insemination procedure of pigs. , reducing the rate of ovarian cysts and improving the hormone secretion of gilts are better than PMSG. It can be seen that if AIS is used instead of PMSG during superovulation or timed insemination, the reproductive performance of female animals will be further improved.
  • PMSG pregnant horse serum gonadotropin
  • AIS is a polyclonal antibody with complex components and large batch-to-batch differences, which makes it difficult to meet the basic principle of drug quality control.
  • it is necessary to repeatedly inject immunogens into animals each time it is prepared which takes a long time to prepare and finally extracts
  • the blood of immunized animals may lead to abnormal death of animals in the middle, which is not in line with animal ethics.
  • inhibin active immunization has a negative impact on the conception rate
  • the components of AIS used in passive immunization are complex and difficult to make medicines.
  • the field of animal reproduction, especially economic animals, requires monoclonal antibodies with specific components that can specifically neutralize inhibin.
  • the present disclosure relates to an anti-inhibin antibody or an antigen-binding portion thereof, which is used to immunoneutralize inhibin, promote endogenous FSH production and secretion, stimulate follicular development and increase ovulation number in female animals.
  • the present disclosure provides a method for preparing anti-inhibin monoclonal antibody hybridoma cell lines 4D826 and 1E57, the method comprising:
  • the present disclosure provides an anti-inhibin antibody or an antigen-binding portion thereof selected from 4D8 and 1E5.
  • the present disclosure provides an anti-inhibin antibody or an antigen-binding portion thereof, which has a heavy chain CDR as shown in the amino acid sequence of SEQ ID NO.1, 2, and 3, and the amino acid sequence of SEQ ID NO.4, 5, or 6 the light chain CDRs shown; and an anti-inhibin antibody or an antigen-binding portion thereof, which has the heavy chain CDRs shown in the amino acid sequence SEQ ID NO.11, 12, 13, and the amino acid sequence SEQ ID NO.14 , 15, 16 the light chain CDRs shown.
  • the present disclosure provides an anti-inhibin antibody or an antigen-binding portion thereof, which has the heavy chain variable region of the 4D8 monoclonal antibody shown in the amino acid sequence of SEQ ID NO.7, and the amino acid sequence of SEQ ID NO.9.
  • the present disclosure provides a nucleic acid encoding the above-mentioned anti-inhibin antibody or its antigen-binding portion, such as the nucleic acid sequence encoding the heavy chain variable region of the 4D8 monoclonal antibody shown in SEQ ID NO.8, and the nucleic acid sequence shown in SEQ ID NO.10
  • the nucleic acid sequence encoding the light chain variable region of the 4D8 monoclonal antibody; and the nucleic acid sequence encoding the heavy chain variable region of the 1E5 monoclonal antibody as shown in SEQ ID NO.18, and the nucleic acid sequence shown in SEQ ID NO.20 Nucleic acid sequence encoding the light chain variable region of the 1E5 monoclonal antibody.
  • the present disclosure provides an expression vector, which contains the above-mentioned isolated nucleic acid sequence and an expression control sequence operably linked to the sequence.
  • the present disclosure provides an antibody expression system constructed by transfecting the above construct into host cells.
  • the present disclosure provides a method for recombinant preparation of the above-mentioned anti-inhibin antibody, comprising the following steps: cultivating an expression system containing the above-mentioned antibody under conditions suitable for expressing the above-mentioned antibody, thereby expressing the above-mentioned antibody, and purifying and isolating the above-mentioned antibody.
  • the host cells used therein are all prior art and can be obtained directly through commercial channels.
  • the culture medium used in the culture is also a variety of conventional culture medium. Those skilled in the art can select suitable culture medium based on experience to optimize the culture conditions.
  • an appropriate method can be selected to induce and start the expression of monoclonal antibodies, and continue to culture the cells.
  • the recombinant monoclonal antibody can be expressed in the cell or on the cell membrane, of course, it may also be secreted outside the cell, and then the recombinant protein can be separated and purified by physical, chemical and other characteristics.
  • these techniques are well known in the art by those skilled in the art.
  • the above methods include but are not limited to: protein denaturation, centrifugation, ultrafiltration, chromatography, HPLC and other single techniques or a combination of several techniques.
  • the target monoclonal antibody gene sequence obtained from the monoclonal hybridoma cell line can reproduce the activity of the monoclonal antibody after constructing an expression vector and CHO recombinant expression, and obtain a recombinant anti-inhibin monoclonal antibody.
  • the present disclosure provides a use of the above-mentioned anti-inhibin antibody in the preparation of drugs for promoting animal reproduction, estrus synchronization, conception and birth, embryo transfer, improving ovulation quality, promoting livestock reproduction, and inducing estrus in female animals;
  • the above-mentioned animals include rats , pigs, cattle, sheep.
  • Figure 1 shows the effect of recombinant monoclonal antibody 4D8 on improving the ovulation quality of ICR mice.
  • Figure 2 shows the effect of recombinant monoclonal antibody 1E5 on improving the ovulation quality of ICR mice.
  • Figure 3 shows the effect of ascites monoclonal antibody 4D8 on improving the ovulation quality of ICR mice.
  • Figure 4 shows the effect of ascites monoclonal antibody 1E5 on improving the ovulation quality of ICR mice.
  • Figure 5 shows the effect of PMSG on improving the ovulation quality of ICR mice.
  • Figure 6 shows the effect of goat polyclonal antibodies on improving the ovulation quality of ICR mice.
  • antibodies eg, monoclonal antibodies
  • antigen-binding portions thereof that specifically bind inhibin.
  • anti-inhibin monoclonal antibodies that specifically bind inhibin, wherein the anti-inhibin antibodies include variants of the parent antibody.
  • antibodies that specifically bind inhibin eg, mammalian inhibin.
  • anti-inhibin antibodies comprising a modification in one or more amino acid residues (e.g., 5-13 amino acid substitutions in the framework region of the heavy chain variable region), versus those without the modification. It retains an affinity for the antigen compared to the parental antibody.
  • inhibin refers to any inhibin molecule known to those skilled in the art.
  • the aforementioned inhibins can be derived from mammals, for example, inhibins can be derived from mice, pigs, cattle, and sheep.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where integers are required, the term means rounding up or down to the nearest whole number within plus or minus 10% of a given value or range.
  • the phrase "substantially identical" is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains with 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequences the term is understood to mean exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of a reference nucleic acid sequence. %, 99% or higher sequence identity of nucleic acid sequences.
  • sequence identity or “identity” has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleic acid or polypeptide, or along a region of the molecule. Although there are many methods of measuring the identity between two polynucleic acids or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • substitution-type variant is one in which at least one amino acid residue has been removed from the native sequence and a different amino acid has been inserted in its same position.
  • the substitution can be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions may be at consecutive positions.
  • an amino acid may be substituted by multiple residues, where such variants include both substitutions and insertions.
  • An “insertional” variant is one in which one or more amino acids are inserted immediately adjacent to an amino acid at a specified position in a native sequence. Immediately adjacent to an amino acid means attached to the ⁇ -carboxyl or ⁇ -amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids have been removed from the native amino acid sequence. Typically, deletion variants have one or two amino acids deleted from a specific region of the molecule.
  • variable domains of antibodies refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target.
  • variability is not evenly distributed throughout the variable domains of antibodies.
  • the variability is concentrated in three segments called complementarity determining regions (CDRs; namely CDR1, CDR2 and CDR3) or hypervariable regions, which are located within the variable domains of the light and heavy chains.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of native heavy and light chains consists of four FR regions, predominantly in a ⁇ -sheet configuration, connected by three CDRs that form a loop that connects the ⁇ -sheet structures and In some cases a partial ⁇ -sheet structure is formed.
  • the CDRs of each chain are usually connected adjacently by FR regions and contribute to the formation of the antibody target binding site (epitope or determinant) by means of CDRs from other chains (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)).
  • numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • a CDR may have the ability to specifically bind a cognate epitope.
  • an "antibody fragment” or “antigen-binding portion” of an antibody refers to any portion of a full-length antibody that is less than full-length but includes at least a portion of the variable region (e.g., one or more CDRs and and/or one or more antibody combining sites), and thus retain binding specificity and at least part of the specific binding ability of the full-length antibody.
  • an antigen-binding portion refers to an antibody fragment comprising an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as derivatives produced synthetically, eg, recombinantly.
  • Antibodies include antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragment may comprise multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen binding moieties include any antibody fragment which, when inserted into an antibody framework (eg, by substituting corresponding regions), results in an antibody that immunospecifically binds (ie exhibits a Ka of at least or at least about 107-108 M-1) an antigen.
  • a "functional fragment” or “analog of an anti-inhibin antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • a functional fragment generally has the same meaning as an "antibody fragment” and, with respect to an antibody, may refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2 and so on.
  • the "Fv” fragment consists of a dimer of the variable domains of one heavy chain and one light chain in a non-covalent association (VH-VL dimer).
  • VH-VL dimer non-covalent association
  • the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer, as is the case with intact antibodies.
  • the above six CDRs collectively confer the target binding specificity of the intact antibody.
  • bispecific antibody refers to an antibody and/or antigen-binding molecule capable of specifically binding to two different antigenic determinants, usually, a bispecific antibody and/or antigen-binding molecule Contains two antigen-binding sites, each of which is specific for a different antigenic determinant.
  • bispecific antibodies and/or antigen binding molecules are capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the population of monoclonal antibodies is identical to the other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies in vitro from clonal populations of host cells by transforming the host cells with a plasmid carrying an artificial sequence of nucleic acid encoding the antibody.
  • hybridomas refers to a cell or cell line (typically a myeloma or lymphoma cell) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and sustain production of specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, eg, Harlow & Lane, 1988).
  • hybridodoma or “hybridoma cell” is referred to, it also includes subclones and progeny cells of the hybridoma.
  • a full-length antibody is one that has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region.
  • Antibodies such as those produced naturally by antibody-secreting B cells, as well as antibodies produced synthetically with the same domain.
  • Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or Antigen-binding subsequences of antibodies), containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are derived from a non-human species having the desired specificity, affinity and capacity ( Donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDRs complementarity determining regions
  • the humanized antibody mentioned in the present disclosure also covers the antibody containing 1 or 2 two amino acid mutations in the CDR.
  • CDR refers to complementarity-determining regions, and it is known that each heavy and light chain of an antibody molecule has 3 CDRs. CDRs are also called hypervariable regions, and are present in the variable regions of each heavy and light chain of an antibody, having very high variability sites in the primary structure of the CDRs.
  • the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually comprise chemically active surface types of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • telomere binding portion As used herein, “specifically binds” or “immunospecifically binds” with respect to an antibody or antigen-binding portion thereof is used interchangeably herein and refers to the passage of an antibody or antigen-binding portion between the antibody-binding site of the antibody and the antigen. The ability of non-covalent interactions to form one or more non-covalent bonds with the same antigen.
  • the above-mentioned antigens may be isolated antigens or present in tumor cells.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1 ⁇ 10 7 M ⁇ 1 or 1 ⁇ 10 8 M ⁇ 1 or greater (or 1 ⁇ 10 ⁇ 7 M or 1 ⁇ 10 -8 M or lower dissociation constant (Kd)) binds to the above-mentioned antigens.
  • Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst.
  • ITC isothermal titration calorimetry
  • kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies. Instruments and methods for real-time detection and monitoring of association rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem.Soc.Trans.27 :335).
  • nucleic acid and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleic acids or nucleic acid derivatives, including deoxyribonucleic acid (DNA ) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules as well as RNA molecules.
  • a nucleic acid molecule can be single- or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that exist in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding a provided antibody or antigen binding portion.
  • operably linked with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleic acid.
  • Expression levels of a polypeptide can be assessed by any method known in the art, including, for example, methods for determining the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell for receiving, maintaining, replicating and amplifying a vector. Host cells can also be used to express polypeptides encoded by vectors. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • Host cells can be prokaryotic cells, such as Escherichia coli; or lower eukaryotic cells, such as yeast cells-Pichia/Saccharomyces cerevisiae or filamentous fungi; or higher eukaryotic cells, such as mammalian cells CHO/293T, various COS cells, HeLa cells, HEK cells such as HEK 293 cells, murine myeloma (NSO) cells, baby hamster kidney (BHK) cells, etc.
  • prokaryotic cells such as Escherichia coli
  • lower eukaryotic cells such as yeast cells-Pichia/Saccharomyces cerevisiae or filamentous fungi
  • higher eukaryotic cells such as mammalian cells CHO/293T, various COS cells, HeLa cells, HEK cells such as HEK 293 cells, murine myeloma (NSO) cells, baby hamster kidney (BHK) cells, etc.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • Reference to vectors includes those into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to vectors also includes those vectors comprising a nucleic acid encoding a polypeptide.
  • Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplifying nucleic acids or expressing/displaying polypeptides encoded by nucleic acids. Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome. Also contemplated are vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
  • vector also includes "viral vector” or “viral vector”.
  • viral vectors are engineered viruses that are operably linked to foreign genes to transfer (either as vehicles or shuttles) foreign genes into cells.
  • expression vector includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA.
  • expression vectors are well known to those skilled in the art and include expression vectors replicable in eukaryotic cells and/or prokaryotic cells as well as expression vectors that remain episomal or integrate into the host cell genome.
  • the expression vector used herein refers to bacterial plasmid, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known to those skilled in the art.
  • treating an individual suffering from a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged following treatment.
  • treatment includes prophylaxis, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease.
  • Treatment also includes any pharmaceutical use of any provided antibody, or antigen-binding portion thereof, and compositions provided herein.
  • the present disclosure provides an anti-inhibin antibody or an antigen-binding portion thereof comprising a heavy chain CDR selected from the amino acid sequence of SEQ ID NO. 1, 2, 3, 11, 12, 13, or any variant thereof , and the light chain CDRs selected from the amino acid sequence SEQ ID NO.4, 5, 6, 14, 15, 16 or any variant thereof.
  • An antibody or antigen-binding portion thereof comprising a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.1, 11 or any variant thereof, selected from the amino acid sequence of SEQ ID NO.2, 12 or any variant thereof
  • the heavy chain CDR2 of the body is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.3, 13 or any variant thereof; and the light chain CDR1 selected from the amino acid sequence of SEQ ID NO.4, 14 or any variant thereof, selected from From the light chain CDR2 of amino acid sequence SEQ ID NO.5, 15 or any variant thereof, from the light chain CDR3 of amino acid sequence SEQ ID NO.6, 16 or any variant thereof.
  • the antibody or antigen-binding portion thereof comprising a combination of CDRs of a heavy chain and a light chain selected from:
  • Heavy chain CDR1, CDR2 and CDR3 comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.1, 2 and 3, or any variant thereof, respectively, and SEQ ID NO.4, 5 and 6 respectively
  • heavy chain CDR1, CDR2 and CDR3 comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO.11, 12 and 13 respectively or any variant thereof, and comprising SEQ ID NO.14, 15 and 16 respectively Light chain CDR1 , CDR2 and CDR3 sequences or light chain CDR1 , CDR2 and CDR3 of any variant thereof.
  • the above-mentioned antibody or antigen-binding portion thereof comprising a heavy chain variable region selected from the amino acid sequence of SEQ ID NO.7, 17 or any variant thereof, and selected from the amino acid sequence of SEQ ID NO.9, 19 or the light chain variable region of any variant thereof.
  • the above-mentioned antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO.7 or any variant thereof, and a light chain of the amino acid sequence of SEQ ID NO.9 or any variant thereof. chain variable region.
  • the above-mentioned antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence of SEQ ID NO.17 or any variant thereof, and a light chain of the amino acid sequence of SEQ ID NO.19 or any variant thereof. chain variable region.
  • the disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen-binding portion thereof of any preceding aspect.
  • the disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof or bispecific or multispecific molecule according to any of the preceding aspects.
  • the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NO.8, 18 or any variant thereof, and an antibody selected from SEQ ID NO.10, 20 or any variant thereof Antibody light chain variable region nucleic acid sequence.
  • said nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO.8 or any variant thereof, and the light chain variable region of nucleotide sequence SEQ ID NO.10 or any variant thereof nucleic acid sequence.
  • the nucleic acid comprises a heavy chain variable region nucleic acid sequence of nucleotide sequence SEQ ID NO.18 or any variant thereof, and an antibody light chain variable region of nucleotide sequence SEQ ID NO.20 or any variant thereof region nucleic acid sequence.
  • the present disclosure provides an anti-inhibin antibody or antigen-binding portion thereof that has at least greater than 60%, 65%, 70%, 75%, 80%, 85% %, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the present disclosure provides nucleic acid molecules encoding antibodies or antigen-binding portions thereof according to any of the preceding aspects, or having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99% or higher sequence identity nucleic acid molecules.
  • the present disclosure provides a vector comprising a nucleic acid according to any of the preceding aspects.
  • the present disclosure provides a cell comprising a vector according to any of the preceding aspects.
  • the present disclosure provides an expression system constructed by introducing an expression vector comprising the aforementioned nucleic acid into a host cell.
  • the expression vector comprises any one selected from bacterial plasmids, yeast plasmids, plant cell viruses, and mammalian cell viruses (such as adenovirus or retrovirus).
  • the expression vector is pcDNA3.1(+).
  • the host cell is a prokaryotic host cell or a eukaryotic host cell.
  • the prokaryotic host cell is a bacterial cell.
  • the aforementioned bacterial cells are Escherichia coli.
  • the eukaryotic host cell is selected from any of fungi, plants, insects, and mammals.
  • the fungal eukaryotic host cell is selected from any one of yeast (such as Pichia pastoris and Saccharomyces cerevisiae) and filamentous fungi.
  • the mammalian eukaryotic host cell is selected from Chinese hamster ovary (CHO) cells, murine myeloma (NSO) cells, baby hamster kidney (BHK) cells, and human embryonic kidney (HEK) cells (such as HEK293 cells) any of the.
  • CHO Chinese hamster ovary
  • NSO murine myeloma
  • BHK baby hamster kidney
  • HEK human embryonic kidney
  • the host cell is a Chinese Hamster Ovary (CHO) cell.
  • the method for introducing the expression vector into the host cell is selected from transfection, transformation or infection.
  • the expression vector is introduced into the host cell by transfection.
  • transfection methods include: electroporation transfection, calcium phosphate transfection, liposome transfection, protoplast fusion transfection, microinjection, electroporation, gene gun method, cationic polymerization Infection with biological and viral vectors.
  • the present disclosure provides a composition comprising the antibody or antigen-binding portion thereof, bispecific or multispecific molecule, nucleic acid, vector and cell of any of the preceding aspects.
  • the present disclosure provides antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acids, vectors, cells, and compositions of any of the preceding aspects in preparation for promoting animal reproduction, estrus synchronization, and conception. It is used in drugs for offspring, embryo transplantation, improving ovulation quality, promoting livestock reproduction, and inducing estrus in female animals.
  • the aforementioned animals include mice, pigs, cattle, and sheep.
  • the present disclosure provides methods for promoting animal reproduction, estrus synchronization, fertilization, embryo transfer, improving ovulation quality, promoting livestock reproduction, or inducing estrus in dams, the method comprising administering the aforementioned antibodies or antigen-binding combinations thereof to the animal. part.
  • the administered dose is 0.1-1000 ⁇ g/kg.
  • the administration is by intraperitoneal, intramuscular, or subcutaneous injection.
  • the antibody or antigen-binding portion thereof and the bispecific or multispecific molecule are administered at a dose of 0.1-1000 ⁇ g/kg, administered intraperitoneally, intramuscularly or subcutaneously.
  • the antibodies herein, and derivatives, fragments, analogs and homologues thereof, can be incorporated into pharmaceutical compositions suitable for administration.
  • compositions suitable for administration suitable for administration.
  • the principles and considerations involved in the preparation of such compositions, as well as guidance for selecting components, are well known in the art.
  • compositions generally comprise an antibody and a pharmaceutically acceptable carrier.
  • antibody fragments minimal inhibitory fragments that specifically bind to the binding domain of the target protein may be preferred.
  • peptide molecules can be designed that retain the ability to bind target protein sequences.
  • Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference work in the field, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles, such as fixed oils, may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Their use in compositions is envisioned except for any conventional media or reagents with which the antibodies are incompatible.
  • a pharmaceutical composition of the above embodiments is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, the maintenance of the desired particle size in the case of dispersions, and the use of surfactants.
  • Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation are vacuum-drying and freeze-drying to obtain a powder containing the active ingredient plus any additional desired ingredient from a sterile-filtered solution of these ingredients as previously described .
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant such as a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to permeate are used in the formulation.
  • penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the antibody or antibodies may be formulated into ointments, ointments, gels, or creams as generally known in the art.
  • the compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • antibodies are prepared with carriers that will protect them from rapid elimination from the body, such as slow/controlled release formulations, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Dosage unit form refers to physically separable units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of a drug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • one or more antibodies one or more antibodies.
  • the specifications for dosage unit forms of the above embodiments are dictated by and directly dependent on the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulation of such antibodies for treatment of individuals.
  • the pharmaceutical composition can be presented in a container, pack, or dispenser together with instructions for administration.
  • compositions described herein may also contain more than one antibody, preferably those with complementary activities that do not adversely affect each other, depending on the particular condition to be treated.
  • a composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytotoxic agent such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytokine such as cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • growth inhibitory agent such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts effective for the intended purpose. For example, they may be present in combination in the kit or in use.
  • one or more antibodies may be administered in combination therapy, i.e., with other agents, such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases) combined.
  • agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases
  • the term "in conjunction" herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably detectable at the site of treatment at an effective concentration when administration of the second compound is initiated.
  • “combination” can also be a kit comprising both an antibody of the present disclosure and another therapeutic agent.
  • combination therapy can comprise one or more antibodies described herein and one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , enzyme inhibitors, and/or cytotoxins or cytostatic agents, as described in more detail below) are co-formulated and/or co-administered.
  • additional therapeutic agents e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , enzyme inhibitors, and/or cytotoxins or cytostatic agents, as described in more detail below.
  • mice Three 6-8-week-old female BALB/c mice were selected, all of which were of SPF grade, and were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.
  • the immunological reagent was Freund's adjuvant, which was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • the immunization scheme is shown in Table 1:
  • mice were killed by pulling their necks, soaked in alcohol for 5-10 minutes, took out the inverted mice, placed them on the dissecting table, fixed the limbs, scrubbed the abdomen with 75% alcohol again, and cut the abdominal skin to expose the peritoneum under aseptic conditions. Wipe and disinfect with 75% alcohol;
  • the cells cultured in the T75 culture flask (good growth, in the logarithmic phase) were lightly patted and washed once with PBS, then suspended with 10ml of PBS, and counted for later use.
  • mice after the macrophages were extracted the peritoneum was cut open by aseptic operation, the spleen was taken out, placed in incomplete medium, crushed by pressing to obtain a large number of cells, centrifuged at 1000rpm for 5min, and washed with PBS for 2 -3 times, finally suspend with 10ml PBS, take an appropriate amount for counting, and set aside.
  • HAT medium Add 5ml of HAT medium, gently blow and aspirate the cell pellet to suspend and mix well, then add macrophages and add HAT medium to 60ml;
  • the HAT medium was replaced with HT medium.
  • OVA ovalbumin
  • the cells in the six-well plate underwent two rounds of subcloning screening, and a total of 6 subclones were obtained, among which the cell culture supernatants of 4D826 and 1E57 had the highest titers, and the titer detection results are shown in Table 2.
  • the antibody subtypes secreted by the two hybridoma cells 4D826 and 1E57 were identified, and the result was: the immunoglobulin heavy chain secreted by the two cell lines All subtypes were IgG1, and all light chain subtypes were Kappa.
  • mice were intraperitoneally injected with 0.5 ml of paraffin oil (purchased from Sigma).
  • paraffin oil purchased from Sigma.
  • the vigorously growing hybridoma cells 4D826 and 1E57 were centrifuged to discard the medium, resuspended with PBS or incomplete medium, adjusted the cell concentration to 2 ⁇ 10 7 cells/ml, and injected 0.1ml of the cell suspension into the peritoneal cavity of the mouse.
  • the abdominal cavity of the mice can be seen to be obviously enlarged.
  • the ascites is collected, and the ascites can be collected directly by killing.
  • Example 2 Cloning to obtain the variable region coding sequence of anti-inhibin monoclonal antibody and vector construction
  • the two monoclonal cell lines 4D826 and 1E57 with the titer of the above cell culture supernatant reaching 1:25600 were sent for sequencing.
  • amino acid sequence of the heavy chain variable region of the 4D8 monoclonal antibody is shown in SEQ ID NO.7
  • its nucleic acid sequence is shown in SEQ ID NO.8
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2, and 3, respectively shown.
  • amino acid sequence of the light chain variable region containing 4D8 monoclonal antibody is shown in SEQ ID NO.9
  • its nucleic acid sequence is shown in SEQ ID NO.10
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.4, 5, 6.
  • amino acid sequence of the heavy chain variable region of the 1E5 monoclonal antibody is shown in SEQ ID NO.17
  • its nucleic acid sequence is shown in SEQ ID NO.18
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.11, 12, and 13, respectively shown.
  • amino acid sequence of the light chain variable region of the 1E5 monoclonal antibody is shown in SEQ ID NO.19
  • its nucleic acid sequence is shown in SEQ ID NO.20
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.14, 15, and 16, respectively shown.
  • amino acid sequences of the signal peptides of the heavy chain and the light chain are SEQ ID NO.21; the amino acid sequences of the constant regions of the heavy chain and the light chain are SEQ ID NO.22 and SEQ ID NO.23, respectively.
  • the vector pcDNA3.1(+) was subjected to double digestion reaction with the target genes of 4D8 and 1E5 respectively using restriction enzymes Nhe I and Xho I, and the system is as follows in Table 3:
  • the prepared reaction solution was put into a 1.5ml centrifuge tube, sealed and bathed in water at 37°C for 6 hours, and the digested product was taken for agarose gel electrophoresis experiment.
  • the target DNA band in the agarose gel after electrophoresis was recovered according to the instructions.
  • T4 DNA ligase to ligate the target fragments of the heavy chain and light chain with the digested pcDNA3.1(+) vector, and see the instructions for the reaction system.
  • the antibody was purified by Protein G affinity chromatography.
  • the samples to be purified included the ascites fluid prepared in Example 1, the transient cell culture supernatant prepared in Example 2, and the goat polyantiserum prepared in Example 3. Specific steps are as follows:
  • the inhibin content in the human body is about 2.5 ⁇ g/L, and its content will increase to more than 5 ⁇ g/L when the follicles are well developed. On this basis, it is estimated that the inhibin content in the body is 5-10 ⁇ g according to the weight of a mouse of 25 g.
  • concentration gradients were set up in the antibody group, which were 0.002 ⁇ g, 0.02 ⁇ g, 0.2 ⁇ g, and 20 ⁇ g per mouse, namely 0.1 ⁇ g/kg, 1 ⁇ g/kg, 10 ⁇ g/kg, and 1000 ⁇ g/kg. kg.
  • the dosage was set at 10 IU/mouse.
  • 5-week-old female ICR mice were selected and randomly divided into 22 groups, with 5 ICR mice in each group, specifically: four groups of recombinant monoclonal antibody 4D8, four groups of recombinant monoclonal antibody 1E5, four groups of ascites monoclonal antibody 4D8, and ascites monoclonal antibody 1E5
  • Four groups sheep polyclonal antibody four groups and standard product PMSG; normal saline was used as negative control.
  • the above-mentioned dose of antibody and 10 IU PMSG were injected intraperitoneally, and 10 IU HCG was also injected intraperitoneally 48 hours later. Oocytes were dissected and counted at 9:00 a.m. on Thursday.
  • Embodiment 6 Effects of different administration methods on ovulation in mice
  • 5-week-old female ICR mice were selected and randomly divided into 17 groups, with 5 ICR mice in each group, specifically: 1 ⁇ g/kg recombinant monoclonal antibody 4D8 three groups, 1 ⁇ g/kg recombinant monoclonal antibody 1E5 three groups, 1 ⁇ g/kg ascites monoclonal antibody Three groups of anti-4D8, three groups of 1 ⁇ g/kg ascites monoclonal antibody 1E5, three groups of 1 ⁇ g/kg goat polyclonal antibody and 10IU PMSG; normal saline was used as negative control. At 5:00 p.m. on Monday, 0.02 ⁇ g antibody and 10 IU PMSG were injected intraperitoneally, subcutaneously, and intramuscularly.
  • mice 48 hours later, 10 IU HCG was also injected intraperitoneally. Oocytes were dissected and counted at 9:00 a.m. on Thursday. The results are shown in Table 6. It can be seen from the results that after administration of different samples in different ways, the ovulation effects on mice are basically the same. Therefore, in the follow-up work, the mouse experiment is still administered intraperitoneally, while other animals are appropriately adjusted according to specific operations. Preferably, pigs, cattle, and sheep are administered intramuscularly.
  • Example 7 Application of monoclonal antibody in improving ovulation quality of ICR mice
  • mice in each group 1 ⁇ g/kg recombinant monoclonal antibody 4D8, 1 ⁇ g/kg recombinant monoclonal antibody 1E5, 1 ⁇ g/kg ascites monoclonal antibody 4D8, 1 ⁇ g/kg ascites monoclonal antibody 1E5 , 10IU PMSG and 1 ⁇ g/kg goat polyclonal antibody group.
  • intraperitoneal injection of the above dose of antibody and 10IU PMSG 48 hours later, the same intraperitoneal injection of 10IU HCG, oocytes were dissected at 9:00 a.m. on Thursday, and the quality of the eggs was observed.
  • the in vivo activities of recombinant and ascites prepared monoclonal antibodies 4D8 and 1E5 were judged by comparing the conception rate and litter size of mice.
  • the disclosed product is intended to be used instead of PMSG in the field of animal reproduction, with PMSG as a standard and physiological saline as a negative control.
  • mice Seven-week-old female ICR mice were selected and randomly divided into 7 groups, 10 in each group. At 5:00 p.m. on the same day, each mouse was given 0.02 ⁇ g antibody and 10 IU PMSG intraperitoneally, and 48 hours later, 10 IU HCG was injected and bred. After 20 days, the conception rate was counted, and the litter size was counted after calving.
  • the subsequent application research is carried out with recombinant 4D8 monoclonal antibody and recombinant 1E5 monoclonal antibody.
  • Example 9 The application of different dosages of antibodies in increasing the litter size of sows
  • a total of 110 210-day-old gilts with a body weight of 85-100kg and similar signs were selected. Randomly divided into 11 groups: recombinant monoclonal antibody 4D8 (0.1 ⁇ g/kg, 1 ⁇ g/kg, 1000 ⁇ g/kg), recombinant monoclonal antibody 1E5 (0.1 ⁇ g/kg, 1 ⁇ g/kg, 1000 ⁇ g/kg), 10 IU PMSG, goat polyclonal antibody group (0.1 ⁇ g/kg, 1 ⁇ g/kg, 1000 ⁇ g/kg); physiological saline was used as negative control.
  • the above-mentioned doses of different medicines were injected intramuscularly at the back and neck of the donor pigs in each group, and 500 IU HCG was injected 80 hours later.
  • the first insemination was performed 24 hours after the administration of HCG, and the second insemination was performed at an interval of 16 hours.
  • the sows in each group were recorded in detail. Aberdeen.
  • the results are shown in Table 8: the total litter size of sows in the 1 ⁇ g/kg monoclonal antibody 4D8 group and 1E5 group (125 and 131 heads) was higher than that of the 10IU PMSG group (72 heads), 1 ⁇ g/kg and 1000 ⁇ g/kg sheep The anti-antibody group (both 66 heads) was significantly different from the PMSG group (P ⁇ 0.05).
  • the average litter size (13.9 and 13.1) of the 1 ⁇ g/kg monoclonal antibody 4D8 and 1E5 groups was also higher than that of the 10IU PMSG group (10.3), and the 1 ⁇ g/kg and 1000 ⁇ g/kg polyclonal antibody groups (9.4).
  • L means 0.1 ⁇ g/kg
  • M means 1 ⁇ g/kg
  • H means 1000 ⁇ g/kg
  • the dosage for pigs was calculated based on the theoretical dosage and the results of in vivo activity in mice, and they were randomly divided into 4 groups: 1 ⁇ g/kg recombinant monoclonal antibody 4D8 group, 1 ⁇ g/kg recombinant monoclonal antibody 1E5 group, 10IU PMSG group, 1 ⁇ g/kg sheep polyclonal antibody group; each group was further divided into primiparous sow group and multiparous sow group.
  • the donor pigs in the recombinant monoclonal antibody 4D8 group and the recombinant monoclonal antibody 1E5 group were in good estrus, and at the dosage of 1 ⁇ g/kg, the estrus rate of primiparous and multiparous sows in the recombinant monoclonal antibody group was high 95%, higher than PMSG group (85% and 80%), goat polyclonal antibody group (70% and 65%).
  • sows in each group were higher than that of normal natural estrous sows (8-14 pieces/head); similarly, at the dosage of 1 ⁇ g/kg, primiparous and multiparous in the recombinant monoclonal antibody 4D8 group
  • the average number of ovulations per head of sows was 24.8 and 25.5, respectively, and the average number of ovulations per head of primiparous and multiparous sows in recombinant monoclonal antibody 1E5 group were 24.7 and 25.2, which were higher than those of PMSG group (19.8 and 20.1 ), sheep polyclonal antibody group (18.3 and 17.9), the difference between the two groups was significant compared with the PMSG group (P ⁇ 0.05).
  • the number of available embryos (6.5 and 7.0) per head of cows in the 1 ⁇ g/kg recombinant monoclonal antibody 4D8 group and recombinant monoclonal antibody 1E5 group were higher than those in the PMSG group (4.9), 1 ⁇ g/kg and 1000 ⁇ g/kg sheep The multi-antibody group (3.5), the difference was significant (P ⁇ 0.05).
  • L means 0.1 ⁇ g/kg
  • M means 1 ⁇ g/kg
  • H means 1000 ⁇ g/kg
  • Example 11 It can be seen from Example 11 that the dosage of 1 ⁇ g/kg has achieved good results in superovulation of cows, and is more feasible in the actual application process. Therefore, it is planned to use this dosage to carry out research on improving the anestrus situation of cows .
  • Each donor sheep was injected intramuscularly with 300 IU of the corresponding drug (Day 10), and the suppository was withdrawn (Day 12). Observe the estrous performance of the female goats, and test the estrus with the test ram. The ewe's genital redness, mucus flow and acceptance of climbing are regarded as estrus, and the estrus rate is calculated. At the same time, the first insemination was performed 24 hours after estrus, and the second insemination was performed after an interval of 16 hours. The conception rate and double lamb rate were counted.
  • L means 0.1 ⁇ g/kg
  • M means 1 ⁇ g/kg
  • H means 1000 ⁇ g/kg

Abstract

提供了抗抑制素抗体及其应用。所述抗体能够解除抑制素对内源性促卵泡激素(FSH)分泌的抑制作用，促使内源性 FSH分泌并促进动物卵泡发育，一定程度上可替代 FSH 或孕马血清促性腺激素(PMSG)在动物繁殖生产中的使用，可以应用于促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情。

Description

抗抑制素抗体及其应用 技术领域
本公开属于动物繁殖技术领域。具体地，本公开涉及一种抗抑制素抗体及其在辅助动物繁殖中的应用。
背景技术
抑制素(Inhibin)是由动物性腺分泌的一种水溶性蛋白激素，属于转化生长因子β超家族(TGF-β)的一员，因其对垂体促卵泡激素(FSH)的产生与分泌具有强烈的抑制作用而得名。抑制素以αβ异二聚体的形式存在，其中β亚基又分为βA、βB、βC、βD和βE五种，目前研究较多的主要是抑制素A(αβA)和抑制素B(αβB)，二者均能抑制FSH的合成与分泌，抑制素A主要由优势卵泡及黄体分泌，抑制素B则由中小窦状卵泡分泌。大量研究报道，通过主动或被动免疫中和抑制素，促使内源FSH的产生和分泌，均能刺激雌性动物卵泡发育以及增加排卵数，表明这种两种方法都具有调控家畜繁殖性能的潜力，对畜牧生产和经济动物繁殖具有重要价值。
目前已有相关专利和文章报道，将抑制素α亚基的部分肽段与一些载体蛋白偶联或者直接以重组抑制素α亚基蛋白作为免疫原主动免疫雌性动物，从而解除抑制素对源性FSH分泌的抑制作用，经过免疫接种后的排卵数能够得到显著提升，但是抑制素是受精卵着床的影响因子，主动免疫诱导抑制素的长期抗体中和对受胎率有负面影响，可能导致排卵数提升但是受胎率下降的情况。
同时，抑制素的被动免疫对雌性动物繁殖性能的影响也被广泛研究。通过抑制素α亚基的部分肽段与一些载体蛋白偶联制备的抗原免疫动物制备相应的抑制素抗血清(AIS)，然后再将AIS注射雌性动物，被动免疫中和内源性抑制素，这种方法在老鼠、羊、猪、牛、马以及鱼类中显著地提升了排卵数。根据浙江省农科院潘建治等人公开的专利(CN111134084A)报道，AIS在猪的定时输精程序中也显示出了优于孕马血清促性腺激素(PMSG)的临床使用效果，在排卵数、降低卵巢囊肿率以及改善后备母猪激素分泌情况等方面均优于PMSG。由此可知，如果在超数排卵或定时输精时用AIS替代PMSG，将能进一步提升雌性动物的繁殖性能。但是，AIS是多克隆抗体，其组分复杂且批间差异大，难以满足药品质量可控这一基本原则，另外每次制备时需要反复给动物注射免疫原，制备周期长，最后还要抽取免疫动物的血液，中途可能导致动物的非正常死亡，不符合动物伦理。
在1991年公开的一篇专利(WO1991010449A1)中就提及利用抑制素中和抗体来刺激家养哺乳动物超数排卵的应用，其规定该抗体可以是多克隆抗体或者单克隆抗体。然而在其说明书中只公布了多克隆抗体的制备过程及其家养哺乳动物超排中的应用，没有公布任何与单克隆抗体制备及应用的相关信息。
因此，由于抑制素主动免疫对受胎率产生负面影响，被动免疫所用的AIS成分复杂难以成药，动物繁殖领域特别是经济动物需要成分明确且能特异性中和抑制素的单克隆抗体。
发明内容
本公开涉及抗抑制素抗体或其抗原结合部分，用于免疫中和抑制素，促使内源FSH产生和分泌，刺激雌性动物卵泡发育以及增加排卵数。
本公开提供了一种制备抗抑制素单克隆抗体杂交瘤细胞株4D826和1E57的方法，该方法包括：
(1)用预制备的猪抑制素α亚基6-25片段(多肽序列如序列表中SEQ ID NO.24所示)与载体蛋白偶联作为免疫原免疫动物；
(2)分离被免疫动物的脾细胞并在适于产生杂交瘤细胞的条件下与适当的骨髓瘤细胞融合；
(3)筛选并培养如上得到的杂交瘤细胞。
本公开提供了一种选自4D8和1E5的抗抑制素抗体或其抗原结合部分。
本公开提供了一种抗抑制素抗体或其抗原结合部分，其具有如氨基酸序列SEQ ID NO.1、2、3所示的重链CDR，和如氨基酸序列SEQ ID NO.4、5、6所示的轻链CDR；和一种抗抑制素抗体或其抗原结合部分，其具有如氨基酸序列SEQ ID NO.11、12、13所示的重链CDR，和如氨基酸序列SEQ ID NO.14、15、16所示的轻链CDR。
本公开提供了一种抗抑制素抗体或其抗原结合部分，其具有如氨基酸序列SEQ ID NO.7所示的4D8单克隆抗体重链可变区，和如氨基酸序列SEQ ID NO.9所示的4D8单克隆抗体轻链可变区；和一种抗抑制素抗体或其抗原结合部分，其具有如氨基酸序列SEQ ID NO.17所示的1E5单克隆抗体重链可变区，和如SEQ ID NO.19所示的1E5单克隆抗体轻链可变区。
本公开提供了编码上述抗抑制素抗体或其抗原结合部分的核酸，如SEQ ID NO.8所示的编码含4D8单克隆抗体重链可变区的核酸序列，以及SEQ ID NO.10所示的编码含4D8单克隆抗体轻链可变区的核酸序列；以及如SEQ ID NO.18所示的编码含1E5单克隆抗体重链可变区的核酸序列，以及SEQ ID NO.20所示的编码含1E5单克隆抗体轻链可变区的核酸序列。
本公开提供了一种表达载体，该表达载体含有上述分离的核酸序列以及与该序列操作性相连的表达调控序列。
本公开提供了一种抗体的表达系统，由上述构建体转染到宿主细胞构建而成。
本公开提供了一种提供上述抗抑制素抗体的重组制备方法，包括如下步骤：在适合表达上述抗体的条件下，培养含上述抗体的表达系统，从而表达出上述抗体，并纯化分离上述抗体。
其中所用的宿主细胞均为现有技术，可通过商业途径直接获取，培养中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，以优化培养条件。当宿主细胞生长到一定密度后，可选择合适的方式诱导启动单克隆抗体表达，并继续培养细胞。在上述方法中，重组单抗可表达在细胞内、或在细胞膜上，当然也可能分泌到细胞外，之后可通过物理、化学和其他特性将重组蛋白分离并纯化。明显的，这些技术在本领域已被本领域技术人员所熟知，上述方法包括但不限于：蛋白质的变复性、离心、超滤、层析、HPLC等单一技术或几种技术的组合使用。
其中从单克隆杂交瘤细胞株获得的目的单抗基因序列，在构建表达载体、CHO重组表达后可重现单抗的活性，获得重组抗抑制素单克隆抗体。
本公开提供了一种上述抗抑制素抗体在制备用于促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情的药物中的用途；上述动物包括鼠、猪、牛、羊。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本说明书的实施例，并与说明书一起用于解释本说明书的原理。
图1示出了重组单抗4D8对提高ICR小鼠排卵质量的效果。
图2示出了重组单抗1E5对提高ICR小鼠排卵质量的效果。
图3示出了腹水单抗4D8对提高ICR小鼠排卵质量的效果。
图4示出了腹水单抗1E5对提高ICR小鼠排卵质量的效果。
图5示出了PMSG对提高ICR小鼠排卵质量的效果。
图6示出了羊多抗对提高ICR小鼠排卵质量的效果。
具体实施方式
I.定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
本文提供的是一种特异性结合抑制素的抗体(例如，单克隆抗体)及其抗原结合部分。在具体的方面，本文提供的是特异性结合抑制素的抗抑制素单克隆抗体，其中抗抑制素抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合抑制素(例如，哺乳动物抑制素)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗抑制素抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有该修饰的亲本抗体相比，其保持与抗原的亲和力。术语“抑制素”是指本领域技术人员已知的任何抑制素分子。例如上述抑制素可以来自哺乳动物，例如抑制素可以是来自鼠、猪、牛、羊。
如本文使用的且除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.& Lipman,D.,SIAM J Applied Math 48:1073(1988))。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。该取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，该环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest,NationalInstitute of Health,Bethesda,MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人 的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合部分”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及该全长抗体的至少部分特异性结合能力。因此，抗原结合部分指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab')2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。该片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合部分包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约107-108M-1的Ka)抗原的抗体。“功能片段”或“抗抑制素抗体的类似物”是可防止或实质降低该受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低该受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab')2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定VH-VL二聚体表面上的靶结合位点，与完整抗体的情况一样。上述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow&Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所提到的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本公开所提到的人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合部分的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合部分通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。上述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x10 8M -1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合上述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul,ed.,Fundamental Immunology,2nd ed.,Raven Press,New York, pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7，229，619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，术语“多核酸”和“核酸分子”指包含至少两个连接的核酸或核酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合部分的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“操作性相连”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而该启动子调控或介导核酸的转录。
如本文所用，“表达”指通过多核酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是原核细胞，如大肠杆菌；或是低等真核细胞，如酵母细胞-毕赤酵母/酿酒酵母或丝状真菌；或是高等真核细胞，如哺乳动物细胞CHO/293T、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞、鼠骨髓瘤(NS0)细胞、幼仓鼠肾(BHK)细胞等。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。本文所用的表达载体指本领域技术人员所熟知的细菌质粒、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示该个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合部分以及本文所提供的组合物的任何药学用途。
II.具体实施方案详述
在一方面，本公开提供了一种抗抑制素抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO.1、2、3、11、12、13或其任何变体的重链CDR，和选自氨基酸序列SEQ ID NO.4、5、6、14、15、16或其任何变体的轻链CDR。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO.1、11或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.2、12或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.3、13或其任何变体的重链CDR3；和选自氨基酸序列SEQ ID NO.4、14或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.5、15或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.6、16或其任何变体的轻链CDR3。
根据前一方面的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
(1)分别包含SEQ ID NO.1、2、3的重链CDR1、CDR2及CDR3序列或其任何变体的重链CDR1、CDR2及CDR3，和分别包含SEQ ID NO.4、5、6的轻链CDR1、CDR2及CDR3序列或其任何变体的轻链CDR1、CDR2及CDR3；
(2)分别包含SEQ ID NO.11、12、13的重链CDR1、CDR2及CDR3序列或其任何变体的重链CDR1、CDR2及CDR3，和分别包含SEQ ID NO.14、15、16的轻链CDR1、CDR2及CDR3序列或其任何变体的轻链CDR1、CDR2及CDR3。
在一些实施方案中，上述抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO.7、17或其任何变体的重链可变区，和选自氨基酸序列SEQ ID NO.9、19或其任何变体的轻链可变区。
在一些实施方案中，上述抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO.7或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.9或其任何变体的轻链可变区。
在一些实施方案中，上述抗体或其抗原结合部分，其包含氨基酸序列SEQ IN NO.17或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.19或其任何变体的轻链可变区。
在一方面，本公开提供了一种双特异性或多特异性分子，其包含前述任一方面的抗体或其抗原结合部分。
在一方面，本公开提供了编码根据前述任一方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸分子。
在一些实施方案中，所述核酸包含选自SEQ ID NO.8、18或其任何变体的抗体重链可变区核酸序列，和选自SEQ ID NO.10、20或其任何变体的抗体轻链可变区核酸序列。
在一些实施方案中，所述核酸包含核酸序列SEQ ID NO.8或其任何变体的重链可变区核酸序列，和核酸序列SEQ ID NO.10或其任何变体的轻链可变区核酸序列。
在一些实施方案中，所述核酸包含核酸序列SEQ ID NO.18或其任何变体的重链可变区核酸序列，和核酸序列SEQ ID NO.20或其任何变体的抗体轻链可变区核酸序列。
在一方面，本公开提供了抗抑制素抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
在一方面，本公开提供了编码如前述任一方面的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子。
在一方面，本公开提供了包含如前述任一方面的核酸的载体。
在一方面，本公开提供了包含如前述任一方面的载体的细胞。
在一方面，本公开提供了一种表达系统，该表达系统通过将包含前述核酸的表达载体导入宿主细胞构建而成。
在一些实施方案中，该表达载体包含选自细菌质粒、酵母质粒、植物细胞病毒和哺乳动物细胞病毒(如腺病毒或逆转录病毒)中的任一种。
在一些实施方案中，该表达载体为pcDNA3.1(+)。
在一些实施方案中，宿主细胞为原核宿主细胞或真核宿主细胞。
在一些实施方案中，原核宿主细胞为细菌细胞。
在一些实施方案中，上述细菌细胞为大肠杆菌。
在一些实施方案中，真核宿主细胞选自真菌、植物、昆虫和哺乳动物中的任一种。
在一些实施方案中，真菌真核宿主细胞选自酵母(如毕赤酵母和酿酒酵母)和丝状真菌中的任一种。
在一些实施方案中，哺乳动物真核宿主细胞选自中国仓鼠卵巢(CHO)细胞、鼠骨髓瘤(NSO)细胞、幼仓鼠肾(BHK)细胞和人胚胎肾(HEK)细胞(如HEK293细胞)中的任一种。
在一些实施方案中，宿主细胞为中国仓鼠卵巢(CHO)细胞。
根据前一方面的表达系统，该表达系统将表达载体导入宿主细胞的方法选自转染、转化或感染。
在一些实施方案中，采用转染的方法将表达载体导入宿主细胞。
在一些实施方案中，转染的方法包括：电穿孔转染法、磷酸钙转染法、脂质体转染、原生质融合转染法、显微注射法、电穿孔法、基因枪法、阳离子聚合物和病毒载体感染法。
在一方面，本公开提供了一种组合物，其包含前述任一方面的抗体或其抗原结合部分、双特异性或多特异性分子、核酸、载体和细胞。
在一方面，本公开提供了前述任一方面的抗体或其抗原结合部分、双特异性或多特异性分子、核酸、载体、细胞和组合物在制备用于促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情的药物中的用途。
在一些实施方案中，上述动物包括鼠、猪、牛、羊。
在一方面，本公开提供了用于促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖或诱导母畜发情的方法，该方法包括向动物使用前述抗体或其抗原结合部分。
在一些实施方案中，按给药剂量为0.1～1000μg/kg的剂量给药。
在一些实施方案中，经腹腔、肌肉或者皮下注射的方式给药。
在一些实施方案中，抗体或其抗原结合部分和双特异性或多特异性分子的给药剂量为0.1～1000μg/kg，经腹腔、肌肉或者皮下注射的方式给药。
可将本文抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA，90:7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适的药学上可接受的载体描述于最新版的Remington's Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将上述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的药学上可接受的载体包括生理盐水、抑菌水、Cremophor ELTM(BASF，Parsippany，N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥，该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种抗体。上述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本公开的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1：抗抑制素单克隆抗体杂交瘤细胞的制备
1.免疫原的制备
经Uniprot数据库检索发现，小鼠、大鼠、猪、牛以及羊抑制素α亚基6-25片段的同源性为100％，故选取此片段作为抗原肽以筛选能同时作用于小鼠、大鼠、猪、牛以及羊的抗抑制素单克隆抗体。将上述抗原肽片段与牛血清白蛋白(BSA)偶联，以偶联物作为免疫原，增强了抗原肽的免疫原性。
2.免疫动物
选择3只6-8周龄雌性BALB/c小鼠，上述小鼠均为SPF级别，购自浙江维通利华实验动物技术有限公司。免疫试剂为弗氏佐剂，购自上海碧云天生物技术有限公司。免疫方案如表1所示：
表1.免疫途径与周期
Figure PCTCN2022138630-appb-000001
3.制备杂交瘤细胞
3.1融合前准备
(1)巨噬细胞提取：
将免疫后的小鼠拉颈处死，酒精浸泡5-10min，取出倒立小鼠，置于解剖台上，固定四肢，再次用75％酒精擦洗腹部，在无菌条件下剪开腹部皮肤露出腹膜，并用75％酒精擦拭消毒；
用10ml注射器吸取无菌的DMEM培养基5ml，在用镊子提起腹膜后将其注入小鼠腹腔，同时从两侧用手指揉压腹部或提拉大腿，令培养基在腹腔内充分流动，5min后待液体变黄，再次用镊子提起腹膜，用注射器吸取液体，获得含有巨噬细胞的培养基，1000rpm离心5min后用PBS洗涤一次，再用10ml HAT培养基悬浮，计数备用。
(2)骨髓瘤细胞sp2/0的培养与收集
将培养在T75培养瓶中的细胞(生长良好，处于对数期)，轻拍起后用PBS洗涤一遍，再用10ml PBS将其悬浮，计数备用。
(3)脾细胞制备
对提取完巨噬细胞后的小鼠，以无菌操作剪开腹膜，取出脾脏，置于不完全培养基中，通过按压的方式将其碾碎获得大量细胞，1000rpm离心5min后用PBS洗2-3次，最后用10ml PBS悬浮，取适量计数，备用。
3.2细胞融合
取前述准备好的骨髓瘤细胞和脾细胞，按1:10的比例混合在一起，在50ml离心管中用不完全培养基洗1次，离心1000rpm 8min，弃去上清并将残留液体吸净，轻弹离心管底使细胞沉淀松动；
用吸管在60s内加预热至40℃的50％PEG(pH8.0)1ml，边加边轻轻搅拌；
用10ml吸管在90s内加20ml预热的不完全培养基，室温静置10min；
1000rpm离心6min，弃去上清；
加入5ml HAT培养基，轻轻吹吸细胞沉淀使其悬浮并混匀，然后加入巨噬细胞并补加HAT培养基至60ml；
分装96孔细胞培养板，每孔200μl，置于37℃、5％CO 2培养箱中培养；
培养7天后用HT培养基换出HAT培养基。
3.3杂交瘤细胞的单克隆化
将偶联鸡卵白蛋白(OVA)的抑制素α亚基6-25片段以10μg/ml的浓度稀释到pH9.6的碳酸氢钠溶液中，以每孔100μl的量加入到酶标板(96孔板)，2-8℃静置过夜，第二天取出洗板拍干后加5％BSA封闭，每孔200μl，37℃孵育1h；
洗板拍干后无菌操作取融合后的培养上清100μl加入到酶标板上，37℃孵育1h；
洗板后加二抗(羊抗鼠，HRP标记)，每孔100μl，37℃孵育1h；
洗板后加入显色液，每孔100μl，37℃孵育5-10min，加终止液50μl，读取460nm波长下的吸光度值。
选取测定的吸光度值大于0.5的孔，将其内的细胞吸出转接到24孔板上(共获得38个阳性孔，阳性率偏低，一轮数据较多不展示)，37℃、5％CO 2培养箱中继续培养；
5-7天后，以同样的方式检测24孔板中培养的细胞上清，复筛确认阳性情况(数值此处未体现)，选择吸光度值最高的15个阳性孔内的细胞，将其转接到六孔板上，37℃、5％CO 2培养箱中继续培养；剩余阳性克隆则直接保种，液氮保存。
六孔板内的细胞经两轮亚克隆筛选，共获得6个亚克隆，其中4D826和1E57的细胞培养上清效价最高，其效价检测结果见表2。
表2 杂交瘤细胞株4D826和1E57的细胞培养上清效价检测
Figure PCTCN2022138630-appb-000002
Figure PCTCN2022138630-appb-000003
4.抗抑制素单克隆抗体的亚型分析
采用福因德科技(武汉)有限公司的单抗亚型鉴定试剂盒，根据说明书鉴定4D826和1E57两株杂交瘤细胞分泌的抗体亚型，结果为：2株细胞株分泌的免疫球蛋白重链亚型均为IgG1，轻链亚型均为Kappa。
5.腹水的制备
提前一周用0.5ml石蜡油(购自Sigma公司)注射小鼠腹腔。致敏一周后，将生长旺盛的杂交瘤细胞4D826和1E57分别离心弃去培养基，用PBS或者不完全培养基重悬，调整细胞浓度至2×10 7个细胞/ml，注射0.1ml细胞悬液至小鼠腹腔中。7-10天后可见小鼠腹腔明显肿大，此时采集腹水，可直接处死抽取腹水。4℃，8000rpm离心20min，去除腹水中细胞碎片与油脂，然后加入等体积甘油保存于-20℃。经间接ELISA检测，腹水的单抗效价可达到1:4000000。
实施例2：克隆获得抗抑制素单克隆抗体的可变区编码序列及载体构建
1.基因测序及序列合成
(1)单克隆细胞株4D826和1E57的重链可变区和轻链可变区的确定
将上述细胞培养上清效价达到1:25600的两株单克隆细胞株4D826和1E57送样测序。
4D8单抗的重链可变区的氨基酸序列如SEQ ID NO.7所示，其核酸序列如SEQ ID NO.8所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.1、2、3所示。
Figure PCTCN2022138630-appb-000004
核酸序列
Figure PCTCN2022138630-appb-000005
含4D8单抗的轻链可变区的氨基酸序列如SEQ ID NO.9所示，其核酸序列如SEQ ID NO.10所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.4、5、6所示。
Figure PCTCN2022138630-appb-000006
核酸序列
Figure PCTCN2022138630-appb-000007
1E5单抗的重链可变区的氨基酸序列如SEQ ID NO.17所示，其核酸序列如SEQ ID NO.18所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.11、12、13所示。
Figure PCTCN2022138630-appb-000008
核酸序列
Figure PCTCN2022138630-appb-000009
1E5单抗的轻链可变区的氨基酸序列如SEQ ID NO.19所示，其核酸序列如SEQ ID NO.20所示，其CDR1、CDR2和CDR3分别如SEQ ID NO.14、15、16所示。
Figure PCTCN2022138630-appb-000010
核酸序列
Figure PCTCN2022138630-appb-000011
(2)重组抗体信号肽及恒定序列的确定
重链和轻链的信号肽氨基酸序列为SEQ ID NO.21；重链和轻链的恒定区氨基酸序列分别为SEQ ID NO.22、SEQ ID NO.23。
将上述序列按一定要求组合后构成单抗4D8和1E5的重组抗体重链和抗体轻链全长序列。
2.重组表达载体构建
将载体pcDNA3.1(+)分别与4D8和1E5的目的基因使用限制酶Nhe I、Xho I进行双酶切反应，体系如下表3：
表3 酶切体系
成分 体积
pcDNA3.1/目的基因 10μl
Nhe I 1.5μl
Xho I 1.5μl
10X Buffer 5μl
ddH 2O 补足50μl
配好的反应液装入1.5ml离心管中，密封后37℃水浴6h，取酶切产物进行琼脂糖凝胶电泳实验。
使用天根琼脂糖凝胶DNA回收试剂盒(购自天根生化科技(北京)有限公司，货号DP209)，根据说明书对电泳后的琼脂糖凝胶中的DNA目的条带进行回收。
使用T4 DNA连接酶分别将重链和轻链的目的片段与酶切后的pcDNA3.1(+)载体连接，反应体系见说明书。
参照天根DH5a感受态细胞说明书进行连接产物转化，筛选出阳性克隆并扩大培养，用质粒提取试剂盒，按说明书提取质粒。
3.CHO细胞瞬时转染表达重组抗抑制素单克隆抗体
(1)CHO-s细胞的培养：125ml摇瓶常规培养条件下，当细胞密度生长到1×10 7个/ml时，以2×10 5个/ml的初始密度接种到新125ml摇瓶中，37℃、5％CO 2培养，2d后当密度达到5×10 5个/ml时，按转染试剂lipo2000的操作说明书开始瞬时转染；
(2)7天后收集细胞上清，5000rpm离心30min除去细胞及碎片，0.45μm过滤后4℃暂存，用于后续的抗体纯化。
实施例3：羊多抗的制备
1.抑制素抗原的制备
用常规技术合成猪抑制素α亚基6-25片段(氨基酸序列如序列表中SEQ ID NO.24所示)，通过N端与BSA偶联。
2.免疫：
第一次免疫，将2mg偶联抗原与弗氏完全佐剂1:1乳化后，颈部皮下免疫一只阉割的雄山羊，再次免疫时则改为弗氏不完全佐剂，第五次免疫一周后取小样血清进行ELISA效价检测；当效价达到要求后，加强免疫一次取全血。结果如表4所示，抗血清效价达到1:64000以上。
表4 羊多抗效价检测
Figure PCTCN2022138630-appb-000012
实施例4：抗体的纯化
采用Protein G亲和层析纯化抗体，待纯化样品包括实施例1制备的腹水、实施例2制备的瞬转细胞培养上清以及实施例3制备的羊多抗血清。具体步骤如下：
A.用3-5倍柱体积纯化水清洗Protein G亲和层析柱；
B.用3-5倍柱体积20mM磷酸钠缓冲液(pH7.0)冲洗Protein G亲和层析柱；
C.将所需纯化样品泵入层析柱；
D.用100mM柠檬酸缓冲液(pH2.7)洗脱，收集洗脱峰；并用1M Tris缓冲液(pH9.0)中和收集物；
E.10mM PB8.0缓冲液透析脱盐；
F.用SDS-PAGE电泳初步鉴定抗体，结果显示纯化后的腹水制备的单抗、经瞬时转染细胞获得的重组单抗以及羊多抗，均分别在50kD及25kD处出现目的条带，纯度均大于90％。同样的，经间接ELISA检测，纯化后的单抗在浓度为1g/L时，效价为1:1000000，而1g/L的羊多抗效价为1:16000。
实施例5：小鼠超排实验确定抗体用量
查阅文献得知(Ulcova-Gallova Z,Babcova K,Micanova Z,Bibkova K,Rumpik D.Hyperstimulation syndrome:the levels of inhibin A and B in sera and follicular fluids.Gynecol Endocrinol.2014；30(4):298-301)，人体内抑制素含量约为2.5μg/L，卵泡发育良好时其含量会升高到5μg/L以上。在此基础上按照一只小鼠体重为25g估计，体内抑制素含量为5-10μg。为了考察用量与排卵数是否存在相关性，抗体组设置四个浓度梯度，分别为每只0.002μg、0.02μg、0.2μg、20μg，即0.1μg/kg、1μg/kg、10μg/kg、1000μg/kg。参照PMSG已有成熟的用量方案，将其用量定为10IU/只小鼠。
选取5周龄雌性ICR小鼠，随机分成22组，每组5只ICR小鼠，具体为：重组单抗4D8四组、重组单抗1E5四组、腹水单抗4D8四组、腹水单抗1E5四组、羊多抗四组和标准品PMSG；以生理盐水作阴性对照。周一下午5点，经腹腔分别注射上述剂量的抗体、10IU PMSG，48小时后同样经腹腔注射10IU HCG，周四上午九点解剖取卵计数，结果如表5所示。可以看出，低剂量组(0.1μg/kg)促排效果较PMSG组差，中剂量两组(1μg/kg、10μg/kg)可使小鼠排卵明显增加，但用量再提高(高剂量组，1000μg/kg)在相同时间内并不会对排卵有促进作用；同时也可以看出，单抗组在同一用量情况下，每组样品对排卵数的影响差异不大，0.02μg/只(1μg/kg)的用量已可使排卵数较PMSG有较为明显的提高，因此后续小鼠相关实验暂以0.02μg/只的用量比对10IU的PMSG用量进行，但不排除，随着研究的深入，对用量有进行调整的可能。
表5 不同单抗及用量对小鼠排卵个数(个/只)的影响
Figure PCTCN2022138630-appb-000013
实施例6：不同给药方式对小鼠排卵的影响
小鼠超排实验一般均通过腹腔给药，但考虑到其在大动物应用上的限制性，本实验主要确定经皮下、肌肉注射两种给药方式，是否可产生同等效果。
选取5周龄雌性ICR小鼠，随机分成17组，每组5只ICR小鼠，具体为：1μg/kg重组单抗4D8三组、1μg/kg重组单抗1E5三组、1μg/kg腹水单抗4D8三组、1μg/kg腹水单抗1E5三组、1μg/kg羊多抗三组和10IU PMSG；以生理盐水作阴性对照。周一下午5点，经腹腔、皮下、肌肉分别注射0.02μg抗体，10IU PMSG，48小时后同样经腹腔注射10IU HCG，周四上午九点解剖取卵计数，结果如表6所示。从结果可以看出，不同样品分别经不同方式给药后，对小鼠的排卵效果基本一致。因此，在后续工作中，小鼠实验依旧通过腹腔方式给药，而在其他动物上根据具体操作适当调整，优选地，猪、牛、羊通过肌肉注射方式给药。
表6 不同给药方式对小鼠排卵个数(个/只)的影响
Figure PCTCN2022138630-appb-000014
实施例7：单克隆抗体在提高ICR小鼠排卵质量中的应用
选取5周龄雌性ICR小鼠，随机分成6组，每组10只：1μg/kg重组单抗4D8、1μg/kg重组单抗1E5、1μg/kg腹水单抗4D8、1μg/kg腹水单抗1E5、10IU PMSG和1μg/kg羊多抗组。周一下午5点，腹腔注射上述剂量的抗体和10IU PMSG，48小时后同样腹腔注射10IU HCG，周四上午九点解剖取卵，观察卵子质量，结果如图1-图6所示：可以看出，抗体组卵子均在M2期，透明质膜明显，且无死卵。同样的可以得出，重组单抗和腹水单抗在排卵质量的影响上相差不大。
实施例8：抗抑制素单克隆抗体对小鼠受胎率、产仔数的影响
通过比较小鼠受胎率和产仔数来判断重组和腹水制备的单克隆抗体4D8和1E5的体内活性。本公开产品旨在代替PMSG在动物繁殖领域使用，以PMSG作为标准品，生理盐水为阴性对照。
选取七周龄雌性ICR小鼠，随机分成7组，每组10只。当天下午5点，每只小鼠通过腹腔给0.02μg抗体、10IU PMSG，48h后注射10IU HCG并配种。20天后统计受胎率，产仔后统计产仔数。
结果如表7所示：在1μg/kg的抗体用量情况下，单克隆抗体组的受胎率(90％)明显高于PMSG组(80％)，也高于羊多抗组(70％)，其均产仔数平均至少13只同样也高于PMSG的11.1只和羊多抗的10只。同时可以看出，重组表达的单抗和腹水制备的单抗在受胎率和均产仔数上均表现良好，但重组单抗略优的原因可能是腹水中含有的小鼠IgG对实验造成的影响。
优选地，后续以重组4D8单抗和重组1E5单抗开展应用研究。
表7 单克隆抗体4D8和1E5对小鼠受胎、产仔的影响实验
组别 实验只数 受胎小鼠(只) 受胎率 产仔数(只) 均产仔数(只)
重组4D8 10 9 90％ 123 13.7±0.93
重组1E5 10 10 100％ 134 13.4±0.86
腹水4D8 10 9 90％ 118 13.1±1.00
腹水1E5 10 9 90％ 120 13.3±0.75
PMSG 10 8 80％ 89 11.1±0.89
羊多抗 10 7 70％ 70 10.0±0.99
生理盐水 10 4 40％ 28 7.0±1.43
实施例9：不同用量抗体在提高母猪产仔数的应用
选择110头210日龄的后备三元母猪，体重85-100kg，体征接近。随机分为11组：重组单抗4D8(0.1μg/kg、1μg/kg、1000μg/kg)、重组单抗1E5(0.1μg/kg、1μg/kg、1000μg/kg)、10IU PMSG、羊多抗组(0.1μg/kg、1μg/kg、1000μg/kg)；以生理盐水作阴性对照。分别在各组供体猪耳后颈部肌肉注射上述剂量的不同药品，80h后注射500IU HCG，HCG给药后24h第一次输精，间隔16h再次输精，详细记录各组母猪的产仔数。
结果如表8所示：1μg/kg单抗4D8组和1E5组母猪总产仔数(125头和131头)高于10IU PMSG组(72头)、1μg/kg和1000μg/kg的羊多抗组(均为66头)，且与PMSG组比差异显著(P<0.05)。1μg/kg单抗4D8组和1E5组的均产仔数(13.9头和13.1头)亦高于10IU PMSG组(10.3头)、1μg/kg和1000μg/kg的羊多抗组(9.4头)。
表8重组单抗4D8/1E5、PMSG、羊多抗对后备母猪产仔数的比较
组别 实验头数 产仔母猪头数 总产仔头数 均产仔头数
单抗4D8-L 10 4 39 9.8±2.22
单抗4D8-M 10 9 125 13.9±1.52
单抗4D8-H 10 9 120 13.3±1.78
单抗1E5-L 10 5 41 8.2±2.01
单抗1E5-M 10 10 131 13.1±1.15
单抗1E5-H 10 9 118 13.1±1.43
PMSG 10 7 72 10.3±1.24
羊多抗-L 10 2 15 7.5±2.31
羊多抗-M 10 7 66 9.4±1.39
羊多抗-H 10 7 66 9.4±1.56
生理盐水 10 4 30 7.5±2.01
注：L指0.1μg/kg；M指1μg/kg；H指1000μg/kg
实施例10：抗体在促进后备和经产母猪同期发情和排卵中的应用
分别选取80头后备三元母猪，80头断奶2周后没有发情的经产母猪，体重85-100kg，品种相同，体征接近。在未进行用量优化的前提下，以理论用量和小鼠体内活性结果推算对猪的用量，随机分为4组：1μg/kg重组单抗4D8组、1μg/kg重组单抗1E5组、10IU PMSG组、1μg/kg羊多抗组；各组组内再分为初产母猪组和经产母猪组。
分别在各组供体猪耳后颈部肌肉注射上述剂量的不同药品，80h后注射500IU HCG并开始观察各组母猪的发情情况，72h后对供体猪手术采卵，计算排卵数。
现有技术中，正常自然发情母猪的排卵数通常是8-14枚/头(参见中国发明专利申请CN111134084A；King B,et al.Ovulatory and endocrine responses after active immunization of gilts against a synthetic fragment of bovine inhibin.Journal of animal science.1993；71(4):975-82；Ri-hong G,et al.A novel method to improve sow reproductive performance:Combination of pre-weaning immunization against inhibin and post-insemination hCG treatment.Journal of Integrative Agriculture.2020:0)。而如表9所示，重组单抗4D8组和重组单抗1E5组的供体猪发情均良好，在1μg/kg的用量情况下，重组单抗组初产和经产母猪发情率均高于95％，高于PMSG组(85％和80％)、羊多抗组(70％和65％)。并且各组母猪的排卵数均高于正常自然发情母猪的排卵数(8-14枚/头)；同样的，在1μg/kg的用量情况下，重组单 抗4D8组初产和经产母猪的头均排卵数分别为24.8枚和25.5枚，重组单抗1E5组初产和经产母猪的头均排卵数分别为24.7枚和25.2枚，高于PMSG组(19.8枚和20.1枚)、羊多抗组(18.3枚和17.9枚)，二者与PMSG组相比差异显著(P<0.05)。
表9 重组单抗4D8/1E5、PMSG、羊多抗对后备和经产母猪的同期发情及排卵作用
Figure PCTCN2022138630-appb-000015
实施例11：抗体在促进母牛超数排卵中的应用
自然条件下，一头母牛一次只产生一枚胚胎，这大大限制了优质牛的繁育，因此有必要开发一种一次能产多个高质量胚胎的方法。
选择110头3-6岁龄体质健康、无疾病的荷斯坦母牛，随机分为11组：重组单抗4D8(0.1μg/kg、1μg/kg、1000μg/kg)、重组单抗1E5(0.1μg/kg、1μg/kg、1000μg/kg)、10IU PMSG、羊多抗组(0.1μg/kg、1μg/kg、1000μg/kg)；以生理盐水作阴性对照。各组母牛在原有饲喂基础上加喂1kg精料，按常规的超数排卵方案经肌肉注射分别给上述剂量的相应药品，观察发情。站立发情12h后第一次输精，24h后第二次输精。于Day 16非手术冲洗采集胚胎，统计胚胎数。
结果如表10所示：1μg/kg的重组单抗4D8组和重组单抗1E5组母牛头均胚胎数(8.1枚和8.4枚)高于PMSG组(6.2枚)、1μg/kg和1000μg/kg的羊多抗组(5.1枚)，差异显著(P<0.05)。同样的，1μg/kg的重组单抗4D8组和重组单抗1E5组母牛头均可用胚胎数(6.5枚和7.0枚)均高于PMSG组(4.9枚)、1μg/kg和1000μg/kg的羊多抗组(3.5枚)，差异显著(P<0.05)。
表10 重组单抗4D8/1E5、PMSG、羊多抗对促进荷斯坦母牛超数排卵的比较
Figure PCTCN2022138630-appb-000016
注：L指0.1μg/kg；M指1μg/kg；H指1000μg/kg
实施例12：抗体在改善乏情母牛发情情况中的应用
从实施例11可以看出，1μg/kg的用量在母牛超数排卵中取得了良好结果，且在实际应用过程中可行性较高，因此计划以此用量开展改善母牛乏情情况的研究。
查情后选择40头不能正常发情的荷斯坦母牛，随机分为四组：1μg/kg重组单抗4D8、1μg/kg重组单抗1E5、10IU PMSG和1μg/kg羊多抗组。各组供体牛按同期发情程序，在固定时间点经肌肉注射分别给予上述剂量的相应药品，以接受公牛爬跨和直肠检查两种方式观察发情。
结果如表11所示，乏情母牛对药品反应敏感。1μg/kg重组单抗4D8组和重组单抗1E5组母牛的发情率为80％均高于PMSG组(60％)和羊多抗组(50％)，差异明显(P<0.05)。
表11 重组单抗4D8/1E5、PMSG、羊多抗对诱导乏情母牛发情的比较
组别 实验头数 发情头数 发情率
重组单抗4D8 10 8 80％
重组单抗1E5 10 8 80％
PMSG 10 6 60％
羊多抗 10 5 50％
实施例13：抗体在促进母羊同期发情及提高双羔率中的应用
选择110只1.5-3岁，体重30-45kg、体质健康、无疾病的母山羊，随机分为四组：重组单抗4D8(0.1μg/kg、 1μg/kg、1000μg/kg)、重组单抗1E5(0.1μg/kg、1μg/kg、1000μg/kg)、10IU PMSG、羊多抗组(0.1μg/kg、1μg/kg、1000μg/kg)；以生理盐水作阴性对照。于发情周期任一天给供体羊放置孕酮阴道栓并记为Day 0，各供体羊分别肌肉注射300IU的相应药品(Day 10)，撤栓(Day 12)。观察母山羊的发情表现，并用试情公羊进行试情。以母羊外阴发红、流黏液并接受爬跨视为发情，计算发情率。同时在发情后24h第一次输精，间隔16h后再次输精，统计受胎率和双羔率。
结果如表12所示，各组母羊发情明显，1μg/kg重组单抗4D8和重组单抗1E5组母羊发情率均达到90％以上，高于10IU PMSG组(70％)和羊多抗组(30％)，且与PMSG组比差异显著(P<0.05)；同样的，重组单抗4D8、重组单抗1E5组母羊受胎率均达到90％以上，高于PMSG组(80％)、1μg/kg和1000μg/kg的羊多抗组(30％)。从双胎结果可以看出，1μg/kg的重组单抗4D8和重组单抗1E5组的双胎率高于60％，明显高于PMSG组(43.8％)和羊多抗组(33.3％)，差异显著(P<0.05)。
表12 重组单抗4D8/1E5、PMSG、羊多抗对母山羊的发情及双羔的影响比较
组别 实验头数 发情头数 发情率 受孕头数 受胎率 双胎率
单抗4D8-L 10 4 40％ 3 30 33.3％
单抗4D8-M 10 10 90％ 9 90％ 66.7％
单抗4D8-H 10 9 90％ 8 90％ 62.5％
单抗1E5-L 10 5 50％ 4 50％ 50.0％
单抗1E5-M 10 9 90％ 10 100％ 70％
单抗1E5-H 10 9 90％ 8 80％ 62.5％
PMSG 10 7 70％ 7 70％ 43.8％
羊多抗-L 10 2 20％ 1 10％ 0％
羊多抗-M 10 4 40％ 3 30％ 33.3％
羊多抗-H 10 4 40％ 3 30％ 33.3％
生理盐水 10 2 20％ 2 20％ 0％
注：L指0.1μg/kg；M指1μg/kg；H指1000μg/kg

Claims (10)

  1. 一种抗抑制素抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO.1、2、3的重链CDR1、CDR2及CDR3序列或其任何变体的重链CDR1、CDR2及CDR3，和分别包含SEQ ID NO.4、5、6的轻链CDR1、CDR2及CDR3序列或其任何变体的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO.11、12、13的重链CDR1、CDR2及CDR3序列或其任何变体的重链CDR1、CDR2及CDR3，和分别包含SEQ ID NO.14、15、16的轻链CDR1、CDR2及CDR3序列或其任何变体的轻链CDR1、CDR2及CDR3序列；
    优选地，所述抗体或其抗原结合部分包含选自氨基酸序列SEQ ID NO.7、17或其任何变体的重链可变区，和选自氨基酸序列SEQ ID NO.9、19或其任何变体的轻链可变区。
  2. 权利要求1所述抗体或其抗原结合部分，其包含氨基酸序列SEQ ID NO.7或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.9或其任何变体的轻链可变区。
  3. 权利要求1所述抗体或其抗原结合部分，其包含氨基酸序列SEQ IN NO.17或其任何变体的重链可变区，和氨基酸序列SEQ ID NO.19或其任何变体的轻链可变区。
  4. 编码根据权利要求1-3任一项所述的抗体或其抗原结合部分的核酸；
    优选地，所述核酸包含选自SEQ ID NO.8、18或其任何变体的抗体重链可变区核酸序列，和选自SEQ ID NO.10、20或其任何变体的抗体轻链可变区核酸序列；
    更优选地，所述核酸包含核酸序列SEQ ID NO.8或其任何变体的重链可变区核酸序列，和核酸序列SEQ ID NO.10或其任何变体的轻链可变区核酸序列；
    更优选地，所述核酸包含核酸序列SEQ ID NO.18或其任何变体的重链可变区核酸序列，和核酸序列SEQ ID NO.20或其任何变体的抗体轻链可变区核酸序列。
  5. 包含权利要求4所述核酸的载体。
  6. 包含权利要求4所述的核酸或权利要求5所述的载体的细胞。
  7. 一种表达系统，所述表达系统通过将包含权利要求4所述核酸的表达载体导入宿主细胞构建而成；
    所述表达载体包含选自细菌质粒、酵母质粒、植物细胞病毒和哺乳动物细胞病毒(如腺病毒或逆转录病毒)中的任一种；更优选地，所述表达载体为pcDNA3.1(+)；
    所述宿主细胞为原核宿主细胞或真核宿主细胞；
    优选地，所述原核宿主细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌；
    优选地，所述真核宿主细胞选自真菌、植物、昆虫和哺乳动物中的任一种；
    优选地，所述真菌真核宿主细胞选自酵母(如毕赤酵母和酿酒酵母)和丝状真菌中的任一种；
    优选地，所述哺乳动物真核宿主细胞选自中国仓鼠卵巢(CHO)细胞、鼠骨髓瘤(NS0)细胞、幼仓鼠肾(BHK)细胞和人胚胎肾(HEK)细胞(如HEK293细胞)中的任一种；更优选地，所述宿主细胞为中国仓鼠卵巢(CHO)细胞。
  8. 权利要求7所述的表达系统，所述将表达载体导入宿主细胞的方法选自转染、转化或感染；
    优选地，采用转染的方法将表达载体导入宿主细胞；
    优选地，所述转染的方法包括：电穿孔转染法、磷酸钙转染法、脂质体转染、原生质融合转染法、显微注射法、电穿孔法、基因枪法、阳离子聚合物和病毒载体感染法。
  9. 一种组合物，其包含权利要求1-3任一项所述的抗体或其抗原结合部分、权利要求4所述的核酸、权利要求5所述的载体和/或权利要求6所述的细胞。
  10. 权利要求1-3任一项所述的抗体或其抗原结合部分、权利要求4所述的核酸、权利要求5所述的载体和/或权利要求6所述的细胞和/或权利要求9所述的组合物在制备用于促进动物繁殖、同期发情、受胎产仔、胚胎移植、提高排卵质量、促进家畜繁殖、诱导母畜发情的药物中的用途；
    优选地，所述动物选自鼠、猪、牛和羊。
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