WO2022116853A1 - 抗fshr抗体及其抗体-药物偶联物的制备和应用 - Google Patents

抗fshr抗体及其抗体-药物偶联物的制备和应用 Download PDF

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WO2022116853A1
WO2022116853A1 PCT/CN2021/131515 CN2021131515W WO2022116853A1 WO 2022116853 A1 WO2022116853 A1 WO 2022116853A1 CN 2021131515 W CN2021131515 W CN 2021131515W WO 2022116853 A1 WO2022116853 A1 WO 2022116853A1
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antibody
amino acid
seq
antigen
cancer
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PCT/CN2021/131515
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French (fr)
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余宁辉
江旭亮
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康威(广州)生物科技有限公司
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Priority to US18/255,459 priority Critical patent/US20240043548A1/en
Priority to EP21899887.0A priority patent/EP4257606A1/en
Priority to CA3201124A priority patent/CA3201124A1/en
Priority to JP2023557475A priority patent/JP2023552664A/ja
Priority to AU2021390722A priority patent/AU2021390722A1/en
Priority to KR1020237022197A priority patent/KR20230117390A/ko
Publication of WO2022116853A1 publication Critical patent/WO2022116853A1/zh

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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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Definitions

  • the invention belongs to the field of biotechnology, and relates to the preparation and application of an anti-FSHR antibody and an antibody-drug conjugate thereof.
  • the human follicle stimulating hormone receptor is an 80 kD G protein-coupled membrane receptor.
  • the FSHR extracellular domain contains a 9-leucine-rich repeat (LRR) domain, an extracellular hinge loop, a 7-helical transmembrane domain, and a cytoplasmic domain.
  • LRR domain is responsible for the high-affinity binding of FSH
  • the hinge loop is responsible for receptor activation
  • the transmembrane and cytoplasmic domains are responsible for receptor signaling [1-4] .
  • Human FSHR is mainly expressed in ovarian granulosa cells and testicular Sertoli cells [5] .
  • the natural protein ligand of FSHR has been used clinically to treat male and female infertility.
  • FSH recombinant follicle-stimulating hormone
  • Several small molecule allosteric modulators of FSHR have also been discovered [6-9] .
  • FSHR has recently been detected to be expressed in some extragonadal normal and tumor tissues.
  • FSHR is transcribed in human female reproductive tract and placenta [10] , osteoclasts [11] , adipocytes [12] , chondrocytes [13] , benign prostatic hyperplasia and prostate cancer [13] , and ovarian cancer [14] .
  • FSH receptors are also expressed by endothelial cells in tissue samples from patients with various tumors [15] located in the prostate, breast, colon, pancreas, bladder, kidney, lung, liver, stomach, testis, and ovary.
  • anti-FSHR humanized antibodies should in principle be applicable to multiple tumor types.
  • FSHR-targeting antibody drugs on the market for cancer treatment.
  • ADCs antibody-drug conjugates
  • the present invention provides an antibody or antigen-binding fragment thereof comprising a variable region that specifically binds to the human follicle stimulating hormone receptor FSHR, comprising one, two, three, Four, five or six are selected from CDRs having the amino acid sequences shown in: SEQ ID NOs: 6, 7, 8, 10, 11 and 12.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region and/or a heavy chain variable region, and the antibody or antigen-binding fragment thereof comprises one, two one or three selected from the group consisting of light chain CDRs having the amino acid sequences shown in: SEQ ID NOs: 6, 7 and 8; and/or,
  • the antibody or antigen-binding fragment thereof comprises one, two or three heavy chain CDRs selected from the group consisting of the amino acid sequences shown below: SEQ ID NOs: 10, 11 and 12.
  • the antibody or antigen-binding fragment thereof of any of the foregoing comprises a light chain CDR having the amino acid sequence shown below: SEQ ID NOs: 6, 7, and 8 and heavy chain CDRs having the amino acid sequences shown below: SEQ ID NOs: 10, 11 and 12.
  • the antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, human antibody, humanized antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence with at least 90% identity with the amino acid sequence shown in SEQ ID NO:5, or with SEQ ID NO:
  • the amino acid sequence shown in 5 is compared to the amino acid sequence with one or more conservative amino acid substitutions; and/or,
  • the heavy chain variable region comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence with at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 9.
  • the amino acid sequences shown are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the antibody or antigen-binding fragment thereof is of the mouse IgG isotype selected from the group consisting of IgGl subtype, IgG2A subtype, IgG2B subtype, IgG2C subtype, or IgG3 subtype, which can be performed by conventional methods Humanize.
  • the antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain
  • the light chain comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence with at least 90% identity with the amino acid sequence shown in SEQ ID NO:3, or with the amino acid sequence shown in SEQ ID NO:3 compared to the amino acid sequence with one or more conservative amino acid substitutions; and/or,
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence with at least 90% identity to the amino acid sequence shown in SEQ ID NO:4, or the amino acid sequence shown in SEQ ID NO:4 Sequences are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the present invention also provides variants of any of the above-described antibodies or antigen-binding fragments thereof comprising no more than 10 amino acid substitutions, deletions and/or additions, such as one, two or three conserved amino acids Substitutions, deletions and/or additions, such as amino acid substitutions, deletions and/or additions in the FR regions, or at least one substitution, deletion and/or addition are in the CDR regions.
  • the amino acid substitutions, deletions and/or additions are accomplished by nucleotide mutagenesis by a method selected from the group consisting of oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, degenerate oligonucleotides Acid-primed PCR, error-prone PCR, DNA shuffling, and use of bacterial mutators.
  • the present invention also provides biological materials related to any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants, which are B1) or B2) as follows:
  • B2 An expression cassette, recombinant vector, recombinant cell or recombinant microorganism comprising the nucleic acid molecule of B1).
  • the nucleic acid molecule in B1) can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.
  • nucleotide sequences of the present invention can readily mutate the nucleotide sequences of the present invention using known methods such as directed evolution and point mutation.
  • Those artificially modified nucleotides with a certain identity to the nucleotide sequence of the present invention, as long as they encode the antibody or antigen-binding fragment or variant thereof and have the antibody or antigen-binding fragment or variant thereof All functions are derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention.
  • identity refers to sequence similarity to a nucleic acid sequence. “Identity” can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the expression cassette described in B2) containing the nucleic acid molecule described in B1) refers to the DNA capable of expressing the antibody or its antigen-binding fragment or variant in a host cell. Not only a promoter that initiates transcription of the gene encoding the antibody or antigen-binding fragment or variant thereof may be included, but also a terminator that terminates transcription of the gene encoding the antibody or antigen-binding fragment or variant thereof. Further, the expression cassette may also include an enhancer sequence;
  • a recombinant vector containing a nucleic acid molecule encoding the antibody or its antigen-binding fragment or variant can be constructed using an existing expression vector, such as an existing expression vector such as a pcDNA3.1 vector, and the light chain expression vector can be pcDNA3.1_CAN001 LC.
  • the chain expression vector can be pcDNA3.1_CAN001 HC;
  • the vector may be a plasmid, cosmid, phage or viral vector;
  • the recombinant microorganism can specifically be bacteria, algae and fungi, wherein the bacteria can be Escherichia coli;
  • the recombinant cells are non-propagating material.
  • the nucleic acid molecule comprises the nucleic acid fragment shown in 1) or 2) or 3) below:
  • nucleic acid fragment that hybridizes with the nucleic acid fragment defined in 1) and encodes any of the above-described antibodies or antigen-binding fragments thereof or variants thereof;
  • nucleic acid fragment that is more than 90% identical to the DNA molecule defined in 1) or 2) and encodes any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants.
  • the present invention also provides a method for preparing any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants, the method comprising:
  • the antibody or antigen-binding fragment or variant thereof is produced by culturing a recombinant cell containing the nucleic acid molecule in a culture medium under conditions capable of expressing the nucleic acid molecule described in B1) above.
  • the light chain expression vector pcDNA3.1_CAN001 LC and the heavy chain expression vector pcDNA3.1_CAN001 HC are transfected into HEK293T cells together, the transfected HEK293T cells are cultured, and the supernatant is collected to obtain the antibody or its antigen binding Fragments or variants, preferably, vectors pcDNA3.1_CAN001 HC and pcDNA3.1_CAN001 LC (mass ratio 1:2) encoding CAN001 heavy and light chains with a 25kD linear PEI polymer (polyscience) in a vector:polymer mass ratio HEK293T cells were co-transfected at a ratio of 1:3.
  • the antibody or its antigen-binding fragment or variant can be further purified by protein-A affinity chromatography (Genescript).
  • the present invention also provides an antibody-drug conjugate obtained by conjugating any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants with a cytotoxic drug through a linker.
  • the antibody-drug conjugate allows the antibody or antigen-binding fragment or variant thereof to target the human follicle-stimulating hormone receptor FSHR bound to the surface of the target cell, and causes the antibody or antigen-binding fragment or variant thereof to and The drug is endocytosed into the target cell together.
  • drug refers to cytotoxic drugs, examples of which include chemotherapeutic agents and toxins.
  • drug may be independently selected from: (a) camptothecin derivatives and metabolites (eg, SN-38).
  • the linker is selected from maleimidoacetyl (mc) or an analog thereof.
  • the antibody-drug conjugate has an average drug-to-antibody ratio DAR in the range of 1-30, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and an average drug-to-antibody ratio DAR between any two of the above values, eg, a mean drug-to-antibody ratio DAR of 5-6.
  • the present invention also provides a method for preparing any of the antibody-drug conjugates described above, comprising: linking the linker to the drug, and coupling the linker-drug moiety to the antibody , purify the antibody-drug conjugate.
  • the cytotoxic drug is MC-MMAF (Maleimidocaproyl monomethylauristatin F, maleimidocaproyl-monomethylauristatin F), which is linked
  • the antibody-drug conjugate is obtained by attaching to the S atom of the cysteine of any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants.
  • any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants are reduced under the action of TCEP (tris(2-carboxyethyl)phosphine) to expose the S atom of cysteine, and then the drug
  • TCEP tris(2-carboxyethyl)phosphine
  • the linker MC-MMAF was attached to this sulfur atom for coupling, and the sample was purified by illustra NAP-10 column (purchased from GE Healthcare).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants or any of the above-mentioned antibody-drug conjugates and a pharmaceutically acceptable accepted vector.
  • the present invention also provides the use of any of the above-mentioned antibodies or antigen-binding fragments thereof or the above-mentioned variants or any of the above-mentioned antibody-drug conjugates in the preparation of any of the following products :
  • the cancer is prostate cancer, breast cancer, colon cancer, pancreatic cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, testicular cancer, and ovarian cancer.
  • the present invention also provides a kit comprising any of the above-mentioned antibodies or antigen-binding fragments or variants thereof or any of the above-mentioned antibody-drug conjugates.
  • the present invention also provides a method of inhibiting the growth of cancer cells, comprising contacting the cells with the antibody-drug conjugate of any one of the above.
  • the cancer cells are prostate cancer cells, breast cancer cells, colon cancer cells, pancreatic cancer cells, bladder cancer cells, kidney cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, testicular cancer cells, and ovarian cancer cells cell.
  • the present invention also provides a method of treating a cancer patient, comprising administering to the patient a therapeutically effective amount of any of the antibody-drug conjugates described above.
  • the cancer is prostate cancer, breast cancer, colon cancer, pancreatic cancer, bladder cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, testicular cancer, and ovarian cancer.
  • any of the methods described above further comprises administering to the patient an additional therapeutic agent.
  • Suitable therapeutic agents include existing drugs and/or surgical therapies for specific applications (eg, cancer).
  • the antibody-drug conjugate is used in combination with one or more other chemotherapeutic or antineoplastic agents.
  • the other therapeutic agent is radiation therapy.
  • the therapeutic agent is a cytotoxic agent.
  • the present invention also provides a method of diagnosing a subject suspected of having cancer, the method comprising administering to the subject any of the above-described antibodies or antigen-binding fragments or variants thereof linked to a detectable label , and detect the distribution of the labeled antibody or antigen-binding fragment or variant thereof in the subject.
  • the antibodies or antigen-binding fragments or variants thereof of the present invention or antibody-drug conjugates of the present invention have various uses, for example, the antibody-drug conjugates of the present invention can be used as therapeutic agents, the Antibodies or antigen-binding fragments or variants thereof are used as reagents in diagnostic kits or as diagnostic tools.
  • the anti-FSHR antibody CAN001 provided by the present invention can be specifically combined with FSHR, and the CAN001 antibody-drug conjugate CAN001-MC-MMAF obtained on the basis has a significant killing effect on cancer cells.
  • the anti-FSHR antibody and its antibody-drug conjugate provided by the invention have broad application prospects in the detection, screening and treatment of cancer.
  • Figure 1 is a map of the pMSG ⁇ 3_FSHR plasmid.
  • Figure 2 shows that the supernatant containing CAN001 antibody specifically stains FSHR-expressing cells, but does not stain GFP-expressing cells.
  • Figure 3 shows the best CAN001 supernatant for screening.
  • Figure 4 shows the IHC staining of human placenta by CAN001.
  • Figure 5 shows IHC staining of ovarian cancer tissue and adjacent tissue by CAN001.
  • Figure 6 is a hydrophobic interaction chromatography (HIC) of CAN001-MC-MMAF.
  • Figure 7 is a size exclusion chromatography (SEC) of CAN001-MC-MMAF.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques are well described in the existing literature, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Third Edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates: the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, 3rd Edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Volume 304 , Chromatin (P.M. Wassarman and A.P. Wolfe, eds.), Academic Press, San Diego, 1999 and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.), Humana Press, Tortova, 1999 et al.
  • antibody refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, ie, molecules comprising an antigen-binding site that specifically binds (immunoreactively reacts with) an antigen.
  • Specifically binds or “immunoreacts” or “targets” means that an antibody reacts with one or more epitopes of the target antigen and does not react with other polypeptides, or with very low affinity (Kd> 10-6 g/ ml) combined with other polypeptides.
  • Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab' and F(ab')2 fragments, Fv and Fab expression libraries.
  • the basic antibody building block is known to include a tetramer.
  • Each tetramer (or “holoantibody”) consists of two identical pairs of polypeptide chains, each pair having one "light” and one "heavy” chain.
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector functions.
  • antibody molecules obtained from humans relate to any of the species IgG, IgM, IgA, IgE and IgD, which differ from each other due to the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgGl, IgG2, and others.
  • the light chain can be a kappa chain or a lambda chain.
  • mAb refers to a population of antibody molecules that contain only one molecular species of antibody molecules consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of monoclonal antibodies are the same in all molecules of the population. mAbs contain antigen binding sites capable of immunoreacting with specific epitopes of the antigen.
  • the term "specific binding” refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for the immunoglobulin.
  • the strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd represents a larger affinity.
  • Kd dissociation constant
  • the immunobinding properties of selected polypeptides can be quantified using methods well known in the art.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • Each LCVR and HCVR consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of the light chain are referred to as "LCDR1, LCDR2 and LCDR3", and the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2 and HCDR3".
  • the CDRs contain most of the residues that form specific interactions with the antigen.
  • the numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions in the present invention conform to Kabat's rules.
  • an “isolated” antibody is one that has been separated and/or recovered from components of its natural environment. Contaminant components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody is purified to the following extent: (1) the antibody is greater than 95% by weight, such as greater than 99%, as determined by the Lowry method; (2) by using spin-cup sequencing The instrument can obtain at least 15 residues of the N-terminal or internal amino acid sequence; or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions, using Coomassie blue or silver staining.
  • Isolated antibodies include antibodies in situ within recombinant cells.
  • isolated antibodies will be prepared by at least one or more purification steps.
  • the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any two of these values range between (including the endpoint) or any value therein.
  • sequence identity refers to the fact that two polynucleotide or amino acid sequences are identical (ie, identical on a nucleotide-to-nucleotide or residue-to-residue basis) over a comparison window.
  • percent sequence identity is calculated by comparing two optimally aligned sequences in a comparison window, determining the occurrence of identical nucleic acid bases (eg A, T, C, G or U) in both sequences or The number of positions of amino acid residues to obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to obtain the percent sequence identity.
  • amino acid sequence of antibodies or immunoglobulin molecules are encompassed by the present disclosure, provided that the identity of the amino acid sequence remains at least 75%, such as at least 80%, 90%, 95% and again such as 99%.
  • the changes are conservative amino acid substitutions.
  • Conservative amino acid substitutions are those that take place within a family of amino acids that are related in their side chains.
  • amino acids encoded by genes are roughly classified into the following categories: (1) acidic amino acids are aspartate and glutamate; (2) basic amino acids are lysine, arginine and histidine; (3) non-polar amino acids The sexual amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) the uncharged polar amino acids are Glycine, Asparagine, Glutamine, Cysteine, Serine, Threonine, Tyrosine.
  • amino acids include (i) serine and threonine of the aliphatic-hydroxy family; (ii) asparagine and glutamine of the amide-containing family; (iii) alanine, valine, leucine and isoleucine; and (iv) phenylalanine, tryptophan and tyrosine of the aromatic family.
  • the conservative amino acid substitution group is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, Glutamate-aspartate, and asparagine-glutamine.
  • pharmaceutically acceptable refers to a component that is suitable for human and/or animal use without undue adverse side effects (eg, toxicity, irritation and allergic reactions) commensurate with a reasonable benefit/risk ratio.
  • carrier refers to a therapeutically useful diluent, adjuvant, excipient or carrier with which the active ingredient is used.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • the carrier when the pharmaceutical composition is administered intravenously, can be water. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference.
  • compositions will contain a clinically effective dose of the antibody or antibody fragment, together with a suitable carrier, to provide a form of administration suitable for the patient.
  • the formulation should be suitable for the mode of administration.
  • the formulation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • gene is used to refer to a coding unit for a functional protein, polypeptide or peptide.
  • this functional term includes genomic sequences, cDNA sequences or fragments or combinations thereof, and gene products, including those that may have been artificially altered. Purified genes, nucleic acids, proteins and the like, when identified and separated from at least one contaminating nucleic acid or protein with which they are normally associated, are used to identify these entities.
  • sequence as used herein is used to refer to nucleotides or amino acids, whether natural or artificial, eg, modified nucleic acids or amino acids.
  • the amino acid substitutions have the effect of (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering binding affinity for protein complex formation, ( 4) alter binding affinity, and (5) confer or improve other physicochemical or functional properties of such analogs.
  • Analogs can include various muteins with sequences that differ from the sequences of naturally occurring peptides. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domains that form intermolecular contacts).
  • Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence (eg, the substituted amino acids should not tend to disrupt the helical structure present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence).
  • Examples of artificially identified secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (edited by C. Branden and J. Tooze) , Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).
  • Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction in immune serum. Additionally, specific antigens or epitopes targeted by immunoglobulins can be immobilized on columns to purify immunospecific antibodies by immunoaffinity chromatography. For the purification of immunoglobulins, refer to the article by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28) .
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, stabilizers, buffers, dispersion media, coatings, bacteriostatic agents, isotonic agents and absorption delaying agents compatible with pharmaceutical administration agent, etc. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Choices of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • administer and “treat” in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to combining an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, or biological fluid. Person, cell, tissue, organ or biological fluid contact.
  • administering and “treatment” can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, eg, by agents, diagnostic agents, binding compositions, or by other cells.
  • inhibiting includes delaying the development of symptoms associated with a disease and/or reducing the severity of those symptoms that will or are expected to develop in the disease.
  • the term also includes alleviation of existing symptoms, prevention of additional symptoms, and alleviation or prevention of underlying causes of such symptoms.
  • the term indicates that a beneficial outcome has been conferred on a vertebrate subject suffering from a disease, such as a human.
  • therapeutically effective amount refers to an antibody-drug conjugate that, when administered alone or in combination with an additional therapeutic agent, is effective to prevent or slow the treatment of a cell, tissue, or subject. Amount of disease or condition.
  • a therapeutically effective dose further refers to an amount of the antibody-drug conjugate sufficient to cause alleviation of symptoms, such as treating, curing, preventing or alleviating a related medical condition, or increasing the rate of treatment, cure rate, Prevention rate or mitigation rate.
  • the effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • the therapeutically effective amount refers to that ingredient alone.
  • a therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether it is administered in combination, consecutively, or simultaneously.
  • a therapeutically effective amount will generally reduce symptoms by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.
  • Monoclonal antibodies directed against FSHR can be prepared according to knowledge and skill in the art.
  • Monoclonal antibody DNA (such as light chain DNA and heavy chain DNA) can be readily synthesized by conventional methods, the DNA can be placed in expression vectors (such as constructed to obtain light chain expression vectors and heavy chain expression vectors), and then These vectors are transfected into host cells such as HEK293T cells, CHO cells, NSO cells, or myeloma cells that do not otherwise produce immunoglobulins to effect synthesis of monoclonal antibodies in recombinant host cells. Recombinant production of antibodies is described in more detail below.
  • Monoclonal antibodies to FSHR can be conjugated to a drug linker (eg, MC-MMAF), usually at the S atom of the cysteine of the antibody.
  • a drug linker eg, MC-MMAF
  • the antibodies or antigen-binding fragments or antibody-drug conjugates of the present invention that specifically bind to FSHR can be used to specifically recognize cells, tissues, and the like that express FSHR.
  • the present invention also provides a method of treating a disease.
  • the method comprises administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate of the present invention.
  • the disease is cancer.
  • the antibody-drug conjugates of the present invention can be used to treat cancer (ie, inhibit the growth or survival of tumor cells).
  • cancers whose growth can be inhibited with the antibody-drug conjugates of the invention include, typically, prostate, breast, colon, pancreatic, bladder, kidney, lung, liver, stomach, testicular cancers and ovarian cancer.
  • Antibodies of the invention and antibody-drug conjugates thereof can be used in diagnostic assays, eg, to detect the expression of FSHR in specific cells, tissues or serum.
  • antibodies are typically labeled (directly or indirectly) with a detectable moiety.
  • Numerous labels are available, which generally fall into the following categories: biotin, fluorescent dyes, radionucleotides, enzymes, iodine, and biosynthetic labels.
  • Antibodies of the invention can also be used in in vivo diagnostic assays. Antibodies are typically labeled with radionuclides (eg,111In, 99Tc , 4C , 125I , 3H , 32P , 35S , or18F ) to allow localization of the antigen by immunoscintigraphy or positron imaging or antibody-expressing cells.
  • radionuclides eg,111In, 99Tc , 4C , 125I , 3H , 32P , 35S , or18F
  • compositions comprise an effective dose of the antibody-drug conjugate and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a substance approved by a regulatory agency of the government or listed in other generally recognized pharmacopeias for use in animals, particularly in humans.
  • a “pharmaceutically acceptable carrier” will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.
  • Example 1 Mouse anti-human FSHR monoclonal antibody CAN001
  • Synthesize codon-optimized DNA fragments (shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively) encoding the light chain and heavy chain of the anti-human FSHR monoclonal antibody by Genescript, and verify the synthesized DNA fragments by sequencing nucleotide sequence.
  • SEQ ID NO: 1 Light chain DNA sequence
  • the DNA sequence of the light chain variable region is the 64th to the 405th position of SEQ ID NO: 1
  • the DNA sequence of the light chain CDR1 is the 130th to the 168th position
  • the DNA sequence of the light chain CDR2 is the 214th to the 168th position.
  • the DNA sequence of the light chain CDR3 is from position 331 to position 357.
  • SEQ ID NO: 2 Heavy chain DNA sequence
  • the DNA sequence of the heavy chain variable region is the 64th to the 441st position of SEQ ID NO:2, the DNA sequence of the heavy chain CDR1 is the 154th to the 168th position, and the DNA sequence of the heavy chain CDR2 is the 211th to the 16th position. 261, the DNA sequence of the heavy chain CDR3 is from position 358 to position 378.
  • the light chain DNA sequence was cloned between the Hind III site and the EcoR I site of pcDNA3.1 (purchased from Thermo Fisher) by the In-Fusion cloning method to construct the light chain expression vector pcDNA3.1_CAN001 LC.
  • the heavy chain DNA sequence was cloned between the Hind III site and the EcoR I site of pcDNA3.1 by the In-Fusion cloning method to construct the heavy chain expression vector pcDNA3.1_CAN001 HC.
  • SEQ ID NO:3 Light chain amino acid sequence
  • the amino acid sequence of the light chain variable region is the 22nd to the 135th position of SEQ ID NO:3 (SEQ ID NO:5), and the amino acid sequence of the light chain CDR1 is the 44th to the 56th position.
  • CSSSSSVSSSYLY SEQ ID NO: 6
  • the amino acid sequence of the light chain CDR2 is from position 72 to position 78
  • STSNLAS SEQ ID NO: 7
  • the amino acid sequence of the light chain CDR3 is from position 111 to position 119 (HQWSSYPPT (SEQ ID NO: 8)).
  • SEQ ID NO:4 Heavy chain amino acid sequence
  • the amino acid sequence of the heavy chain variable region is the 22nd to the 147th position of SEQ ID NO:4 (SEQ ID NO:9), and the amino acid sequence of the heavy chain CDR1 is the 52nd to the 56th position.
  • SYWMH SEQ ID NO: 10
  • the amino acid sequence of heavy chain CDR2 is from position 71 to position 87
  • the amino acid sequence of heavy chain CDR3 is from position 120 to position 126 (LSVGFAY (SEQ ID NO: 12)).
  • Extract the RNA of OVCAR3 cell line (purchased from ATCC company, product catalog number is htb-161) and reverse-transcribe it into cDNA, and use cDNA as template to carry out PCR amplification with FSHR-F and FSHR-R as primers to obtain the fragment containing the FSHR gene .
  • FSHR-F 5'-gtttCCACAACCatggccctgctcctgg-3' (SEQ ID NO: 13);
  • FSHR-R 5'-ggttgattaactcgagttagttttgggctaaatgacttagagggacaag-3' (SEQ ID NO: 14).
  • the fragment containing the FSHR gene was cloned into the cloning vector pJET2.1 (purchased from Thermo Fisher, product catalog number SO501), and the pJET2.1-FSHR plasmid was constructed. After sequencing and verification, the insert was subcloned between the Nco I and Xho I sites of pMSG ⁇ 3, and the pMSG ⁇ 3_FSHR plasmid was constructed.
  • the pMSG ⁇ 3_FSHR plasmid map is shown in Figure 1.
  • HEK293T cells were cultured in serum-free medium and treated with vectors pcDNA3.1_CAN001 HC and pcDNA3.1_CAN001 LC (mass ratio 1:2) encoding CAN001 heavy and light chains with 25 kD linear PEI polymer (polyscience ) according to the ratio of carrier:polymer mass ratio of 1:3 to transfect HEK293T cells. After transfection, HEK293T cells were cultured for 72 hours, and the culture supernatant was collected, which was the supernatant containing CAN001 antibody.
  • HEK293T cells were transfected with pMSG ⁇ 3_FSHR plasmid to obtain HEK293T cells expressing FSHR as the experimental group.
  • HEK293T cells were transfected with pmaxGFP (Lonza) to obtain HEK293T cells expressing GFP protein as a negative control group.
  • the cells of the experimental group and the negative control group were incubated with the supernatant containing the CAN001 antibody obtained in Example 3, the cells coated with the CAN001 antibody were washed and incubated with APC-conjugated goat anti-mouse IgG secondary antibody .
  • Flow cytometry was performed using BD calibur and analyzed using Cellquest software. The results, shown in Figure 2, show that FSHR-expressing cells were specifically stained with the CAN001 antibody-containing supernatant, but not GFP-expressing cells.
  • a mixture of plasmids pcDNA3.1_CAN001 HC and pcDNA3.1_CAN001 LC in different ratios from left to right in Figure 3, the mass ratio of pcDNA3.1_CAN001 HC and pcDNA3.1_CAN001 LC was 1, respectively).
  • :2, 1:1 and 1:3 and 25kD linear PEI polymer were transfected into HEK293T cells according to the ratio of carrier:polymer mass ratio of 1:3.
  • the supernatant was collected and used to stain HEK293T cells expressing FSHR and HEK293T cells expressing GFP protein as a negative control, as described above.
  • Example 5 IHC staining of human placenta and tumor tissue with CAN001 antibody
  • PBS buffer 1000 ml distilled water, 8.0 g NaCl, 0.2 g KCl, 1.149 g Na 2 HPO 4 , 0.2 g NaH 2 PO 4 .
  • Liquid A 29.41g trisodium citrate-2H 2 O + 1000ml distilled water
  • Liquid B 21g citric acid + 1000ml distilled water
  • Hematoxylin chromogenic solution prefferved in the dark, oxidized when exposed to light: the proportioning is according to the instructions on the hematoxylin kit (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd., product catalog number is CTS1097), or it can be prepared according to the pre-experiment
  • the usual ratio is as follows: 1ml hematoxylin stock solution + 4ml distilled water.
  • GTVisionIII anti-mouse/rabbit universal immunohistochemical detection kit was purchased from Gene Technology (Shanghai) Co., Ltd., catalog number GK500705, A solution (secondary antibody working solution): HRP-labeled polymer (anti-mouse, rabbit); Solution B: DAB buffer dilution; Solution C: DAB stock solution. DAB chromogenic solution preparation: 1ml B solution + 20 ⁇ l C solution. Color Result: Brown. The kit does not contain avidin, streptavidin, and is not interfered by endogenous biotin.
  • paraffin-embedded sections of human placenta or ovarian cancer tissue were baked in a 70°C incubator for about 40 minutes. The sections were then soaked in xylene for 15 minutes, followed by gradient hydration in absolute ethanol, 95% ethanol, and 75% ethanol for 5 minutes each.
  • the sections were soaked in 3% hydrogen peroxide for about 15 minutes to remove tissue endogenous catalase, and then washed with PBS buffer three times for 5 minutes each.
  • Figure 4 shows the IHC staining of human placenta with CAN001 antibody.
  • a PBS solution (14.7 ⁇ L, 2.5 mM, pH 7) containing TCEP (tris(2-carboxyethyl) phosphine) was added to the above CAN001 antibody solution, and the resulting solution was vortexed gently and incubated at 37°C. 2 hours.
  • Example 7 Cell killing assay of CAN001 antibody-drug conjugate
  • This example tested the effect of CAN001 antibody-drug conjugates against cancer cell lines by CellTiter-Glo cell viability assay using 96-well plates.
  • Human ovarian cancer cell lines OVCAR-3, A2780, 3AO, all purchased from ATCC.
  • the cells were seeded in a 96-well cell culture plate, with 1000 cells per well, and the total amount of culture medium in each well was controlled to be 200 ⁇ L. After the cells adhered, the cells were treated with corresponding drugs, and the drugs were withdrawn at different time points (0, 24h, 48h, 72h, 96h), and MTS detection solution (MTS reagent 10 ⁇ L+190 ⁇ L complete medium) was added (MTS cell proliferation).
  • Kit colorimetric method is a product of Ai Meijie
  • put the 96-well cell culture plate in a CO 2 cell constant temperature incubator for about 2 hours in the dark then take out the cell culture plate and place it on the microplate reader, The absorbance at 492 nm was determined for the different groups of cells.
  • Table 1 shows that CAN001 antibody-drug conjugate CAN001-MC-MMAF has significantly improved killing effect on ovarian cancer cells compared with MMAF.

Abstract

提供了抗FSHR抗体及其抗体-药物偶联物的制备和应用。具体提供一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含可变区，其特异性结合人卵泡刺激素受体FSHR，包含一个、两个、三个、四个、五个或六个选自具有以下所示的氨基酸序列的CDR：SEQ ID NO：6、7、5、10、11和12。提供的抗FSHR抗体及其抗体-药物偶联物在癌症的检测、筛查、预防和治疗中具有广阔的应用前景。

Description

抗FSHR抗体及其抗体-药物偶联物的制备和应用 技术领域
本发明属于生物技术领域，涉及抗FSHR抗体及其抗体-药物偶联物的制备和应用。
背景技术
人卵泡刺激素受体(FSHR)是80kD G蛋白偶联的膜受体。FSHR胞外域包含一个9-富亮氨酸重复序列(LRR)域，一个胞外铰链环，一个7-螺旋跨膜域和胞质域。LRR域负责FSH的高亲和力结合，铰链环负责受体激活，跨膜域和胞质域负责受体信号传导 [1-4]。
人FSHR主要在卵巢颗粒细胞和睾丸支持细胞中表达 [5]。FSHR的天然蛋白配体重组卵泡刺激素(FSH)已在临床上用于治疗男性和女性不育症。还发现了几种FSHR小分子变构调节剂 [6-9]。
最近已经检测到FSHR在一些性腺外正常组织和肿瘤组织中表达。FSHR在人类女性生殖道和胎盘 [10]、破骨细胞 [11]、脂肪细胞 [12]、软骨细胞 [13]、良性前列腺增生和前列腺癌 [13]以及卵巢癌 [14]中转录。FSH受体也通过内皮细胞在患有多种肿瘤的患者的组织样本中表达 [15]，该肿瘤位于前列腺、乳腺、结肠、胰腺、膀胱、肾、肺、肝、胃、睾丸和卵巢。
由于肿瘤周围血管、前列腺癌和卵巢癌中共同的FSHR标记，抗FSHR人源化抗体原则上应适用于多种肿瘤类型。当前，市场上没有批准的针对FSHR的抗体药物用于癌症治疗。显然需要发明可以有效治疗各种癌症的新型抗FSHR抗体。因此亟需研发新型的抗FSHR单克隆抗体及其抗体-药物偶联物(ADC)，用于癌症的检测、筛查和治疗，使其具有更低的毒副作用，更佳的临床药效，这将给患者提供更多的药物选择。
参考文献：
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发明内容
在一个方面，本发明提供一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含可变区，其特异性结合人卵泡刺激素受体FSHR，包含一个、两个、三个、四个、五个或六个选自具有以下所示的氨基酸序列的CDR：SEQ ID NO:6、7、8、10、11和12。
在一些实施方案中，上述抗体或其抗原结合片段中，所述抗体或其抗原结合片段包含轻链可变区和/或重链可变区，所述抗体或其抗原结合片段包含一个、两个或三个选自具有以下所示的氨基酸序列的轻链CDR：SEQ ID NO:6、7和8；和/或，
所述抗体或其抗原结合片段包含一个、两个或三个选自具有以下所示的氨基酸序列的重链CDR：SEQ ID NO:10、11和12。
在一些实施方案中，上述任一所述的抗体或其抗原结合片段中，所述抗体或其抗原结合片段包含具有以下所示的氨基酸序列的轻链CDR：SEQ ID NO:6、7和8以及具有以下所示的氨基酸序列的重链CDR：SEQ ID NO:10、11和12。
在一些实施方案中，所述抗体或其抗原结合片段为分离的抗体或其抗原结合片段。
在一些实施方案中，所述抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人抗体、人源化抗体或其抗原结合片段。
在一些实施方案中，上述任一所述的抗体或其抗原结合片段中，所述抗体或其抗原结合片段包含轻链可变区和重链可变区；
其中，所述轻链可变区包含具有SEQ ID NO:5所示的氨基酸序列，或与SEQ ID NO:5所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或，
所述重链可变区包含具有SEQ ID NO:9所示的氨基酸序列，或与SEQ ID NO:9所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:9所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或其抗原结合片段是小鼠IgG同种型，其选自IgG1亚型、IgG2A亚型、IgG2B亚型、IgG2C亚型或者IgG3亚型，可以通过常规方法进行人源化。
在一些实施方案中，上述任一所述的抗体或其抗原结合片段中，所述抗体或其抗原结合片段包含轻链和重链；
其中，所述轻链包含具有SEQ ID NO:3所示的氨基酸序列，或与SEQ ID NO:3所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:3所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或，
所述重链包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在另一个方面，本发明还提供上述任一所述的抗体或其抗原结合片段的变体，其包含不超过10个氨基酸取代、缺失和/或添加，例如一个、两个或三个保守氨基酸取代、缺失和/或添加，例如在FR区的氨基酸取代、缺失和/或添加，或者至少一个取代、缺失和/或添加是在CDR区。
在一些实施方案中，所述氨基酸的取代、缺失和/或添加通过核苷酸突变实现，通过选自以下方法进行：寡核苷酸介导的定点突变、盒式突变、简并寡核苷酸引物PCR、易错PCR、DNA改组和使用细菌增变株。
在另一个方面，本发明还提供与上述任一所述的抗体或其抗原结合片段或上述变体相关的生物材料，为如下B1)或B2)：
B1)编码上述任一所述的抗体或其抗原结合片段或上述变体的核酸分子；
B2)含有B1)所述核酸分子的表达盒、重组载体、重组细胞或重组微生物。
在一些实施方案中，上述生物材料中，B1)所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的核苷酸序列有一定同一性的核苷酸，只要编码所述抗体或其抗原结合片段或变体并且具有所述抗体或其抗原结合片段或变体的功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与核酸序列的序列相似性。“同一性”可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
在一些实施方案中，上述生物材料中，B2)所述的含有B1)所述核酸分子的表达盒 是指能够在宿主细胞中表达所述抗体或其抗原结合片段或变体的DNA，该DNA不但可包括启动编码所述抗体或其抗原结合片段或变体的基因转录的启动子，还可包括终止编码所述抗体或其抗原结合片段或变体的基因转录的终止子。进一步，所述表达盒还可包括增强子序列；
可用现有的表达载体构建含有编码所述抗体或其抗原结合片段或变体的核酸分子的重组载体，现有的表达载体如pcDNA3.1载体，轻链表达载体可以为pcDNA3.1_CAN001 LC，重链表达载体可以为pcDNA3.1_CAN001 HC；
所述载体可为质粒、黏粒、噬菌体或病毒载体；
所述重组微生物具体可为细菌、藻和真菌，其中，细菌可为大肠杆菌；
所述重组细胞为非繁殖材料。
在一些实施方案中，上述生物材料中，所述核酸分子包含如下1)或2)或3)所示的核酸片段：
1)如下DNA分子：
SEQ ID NO:1中第130位至第168位所示的核酸片段；
SEQ ID NO:1中第214位至第234位所示的核酸片段；
SEQ ID NO:1中第331位至第357位所示的核酸片段；
SEQ ID NO:2中第154位至第168位所示的核酸片段；
SEQ ID NO:2中第211位至第261位所示的核酸片段；
SEQ ID NO:2中第358位至第378位所示的核酸片段；
SEQ ID NO:1中第64位至第405位所示的核酸片段；
SEQ ID NO:2中第64位至第441位所示的核酸片段；
SEQ ID NO:1所示的核酸片段；或
SEQ ID NO:2所示的核酸片段；
2)与1)限定的核酸片段杂交且编码上述任一所述的抗体或其抗原结合片段或上述变体的核酸片段；
3)与1)或2)限定的DNA分子具有90％以上的同一性且编码上述任一所述的抗体或其抗原结合片段或上述变体的核酸片段。
在另一个方面，本发明还提供制备上述任一所述的抗体或其抗原结合片段或上述变体的方法，所述方法包括：
在能使上述B1)所述的核酸分子表达的条件下于培养基中培养含有所述核酸分子的重组细胞，以制备所述抗体或其抗原结合片段或变体。
在一些实施方案中，将轻链表达载体pcDNA3.1_CAN001 LC和重链表达载体pcDNA3.1_CAN001 HC一起转染进HEK293T细胞，培养转染后的HEK293T细胞，收集上清得到所述抗体或其抗原结合片段或变体，优选地，编码CAN001重链和轻链的载体pcDNA3.1_CAN001 HC和pcDNA3.1_CAN001 LC(质量比1:2)与25kD线性PEI聚合物(polyscience)按照载体:聚合物的质量比为1:3的比例复合转染HEK293T细胞。
在一些实施方案中，收集上清后可以进一步纯化得到所述抗体或其抗原结合片段或变体，纯化可以采用protein-A亲和层析(Genescript)。
在另一个方面，本发明还提供一种抗体-药物偶联物，由上述任一所述的抗体或其抗原 结合片段或上述变体通过连接子与细胞毒类药物偶联得到。所述抗体-药物偶联物使得所述抗体或其抗原结合片段或变体靶向结合到靶细胞表面的人卵泡刺激素受体FSHR，并使得所述抗体或其抗原结合片段或变体和药物一起被内吞到靶细胞内部。
如本文所用，术语“药物”或“D”是指细胞毒类药物，实例包括化学治疗剂和毒素。特别地，“药物”可以独立地选自：(a)喜树碱衍生物和代谢产物(例如，SN-38)。其他包括白喉毒素、肉毒毒素、破伤风毒素、痢疾毒素、霍乱毒素、鹅膏蕈碱、O-鹅膏蕈碱、鹅膏蕈碱衍生物、吡咯并苯并二氮杂卓、吡咯并苯并二氮杂卓衍生物、河豚毒素、短裸甲藻毒素、雪卡毒素、蓖麻毒素、AM毒素、tubulysin、格尔德霉素、美登醇、卡奇霉素、道诺霉素、阿霉素、甲氨蝶呤、长春地辛、SG2285、尾海兔素、尾海兔素类似物、澳瑞他汀(例如单甲基澳瑞他汀E和单甲基澳瑞他汀F)、念珠藻素、喜树碱、根瘤菌素、根瘤菌素衍生物、CC-1065、CC-1065类似物或衍生物、倍癌霉素、烯二炔抗生素、拉霉素、埃博霉素、阿咗那非得(azonafide)、阿普立定(aplidine)、类毒素或前述任意组合；(b)厄洛替尼、硼替佐米、氟维司群、索坦、来曲唑、甲磺酸伊马替尼、PTK787/ZK 222584、奥沙利铂、5-氟尿嘧啶、亚叶酸、雷帕霉素、拉帕替尼、洛那法尼(lonafarnib)、索拉非尼、吉非替尼、AG1478、AG1571、噻替派、环磷酰胺、白消安、英丙舒凡、哌泊舒凡、苯多巴(benzodopa)、卡波醌、米得哌(meturedopa)、乌得哌(uredopa)、乙烯亚胺、六甲蜜胺、曲他胺、三亚乙基磷酰胺(trietylenephosphoramide)、riethiylenethiophosphoramide、三羟甲蜜胺(trimethylolomelamine)、布拉它辛、布拉它辛酮、amptothecin、拓扑替康、苔藓抑素、卡利他汀(callystatin)、CC-1065、阿多来新、卡折来新、比折来新、念珠藻素1、念珠藻素8、尾海兔素、倍癌霉素、KW-2189、CB1-TM1、五加素、水鬼蕉碱(pancratistatin)、匍枝珊瑚醇(sarcodictyin)、海绵抑制素、苯丁酸氮芥、萘氮芥、氯磷酰胺(cholophosphamide)、雌莫司汀、异环磷酰胺、氮芥、美法仑、新恩比兴、苯芥胆甾醇、泼尼氮芥、曲洛磷胺、乌拉莫司汀、卡莫司汀、氯脲霉素、福莫司汀、洛莫司丁、尼莫司汀、雷莫司汀、卡奇霉素、卡奇霉素γ1、卡奇霉素ω1、dynemicin、dynemicin A、氯膦酸盐、拉霉素、新制癌菌素发色团、阿克拉霉素(aclacinomysins)、放线菌素、抗霉素、重氮丝氨酸、博来霉素、放线菌素C、carabicin、洋红霉素(carninomycin)、嗜癌霉素(carzinophilin)、色霉素、放线菌素D、道诺霉素、地托比星(detorubucin)、6-重氮-5-氧代-L-正亮氨酸、阿霉素、吗啉代-阿霉素、氰基吗啉代-阿霉素、2-吡咯并阿霉素、脂质体阿霉素、脱氧阿霉素、表柔比星、依索比星、麻西罗霉素、丝裂霉素C、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、波非罗霉素(potfiromycin)、嘌呤霉素、三铁阿霉素(quelamycin)、罗多比星、streptomigrin、链脲菌素、杀结核菌素、乌苯美司、净司他丁、佐柔比星、5-氟尿嘧啶、二甲叶酸、甲氨蝶呤、蝶罗呤、曲美沙特、氟达拉滨、6-巯基嘌呤、硫咪嘌呤、硫鸟嘌呤、安西他滨、阿扎胞苷、6-氮杂尿苷、卡莫氟、阿糖孢苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷、卡普睾酮、屈他雄酮丙酸酯、环硫雄醇、美雄烷、睾内酯、氨鲁米特、米托坦、曲洛司坦、亚叶酸、醋葡醛内酯、醒磷酰胺糖苷(aldophosphamide glycoside)、氨基乙酰丙酸、恩尿嘧啶、安吖啶、阿莫司汀(bestrabucil)、比生群、edatraxate、地磷酰胺(defofamine)、秋水仙胺、亚丝醌、elfornithine、依利醋铵、乙环氧啶、硝酸镓、羟基脲、香菇多糖、洛尼达宁(lonidainine)、美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇(mopidanmol)、nitraerine、喷司他丁、蛋氨氮芥(phenamet)、吡柔比星、洛索蒽醌、2-乙基酰肼、甲基苄肼、多糖k、雷佐生、根霉菌素、西佐喃、锗螺胺、细交链孢菌酮酸 (tenuazonic acid)、三亚胺醌、2,2’,2”-三氯三乙胺、T-2毒素、verracurin A、漆斑菌素A、蛇形菌素、尿烷、长春地辛、达卡巴嗪、甘露醇氮芥、二溴甘露醇、二溴卫矛醇、胍血生、gacytosine、阿拉伯糖苷、环磷酰胺、噻替派、紫杉醇、紫杉醇的白蛋白工程纳米制剂、多西他赛、瘤可宁、吉西他滨、6-硫代鸟嘌呤、巯嘌呤、顺铂、卡铂、长春花碱、铂、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、长春瑞滨、米托蒽醌、替尼泊苷、依达曲沙(edatrexate)、道诺霉素、氨喋呤、希罗达、伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000、二氟甲基鸟氨酸、视黄酸、卡培他滨或前述任意药学上可接受的盐、溶剂化物或酸；(c)亲和配体，其中该亲和配体是底物、抑制剂、刺激剂、神经递质、放射性同位素或前述的任意组合；(d)放射性标记“P”、“S”、荧光染料、电子致密试剂、酶、生物素、链霉亲和素、地高辛、半抗原、免疫原性蛋白质、具有与靶标互补序列的核酸分子，或前述的任意组合；(e)免疫调节化合物、抗癌剂、抗病毒剂、抗细菌剂、抗真菌剂和抗寄生虫剂或前述的任意组合；(f)他莫昔芬、雷洛昔芬、屈洛昔芬、4-羟基他莫昔芬、曲沃昔芬、雷洛西芬(keoxifene)、LY117018、奥那司酮或托瑞米芬；(g)4(5)-咪唑、氨鲁米特、醋酸甲地孕酮、依西美坦、来曲唑或阿曲唑(astrozole)；(h)氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林、戈舍瑞林或曲沙他滨；(i)芳香酶抑制剂；(j)蛋白激酶抑制剂；(k)脂质激酶抑制剂；(l)反义寡核苷酸；(m)核酶；(n)疫苗；(o)抗血管生成剂。在一些实施方案中，所述毒素优选为澳瑞他汀，更优选为单甲基澳瑞他汀F(MMAF)。
在一些实施方案中，上述任一所述的抗体-药物偶联物中，所述接头选自马来酰亚胺基乙酰(mc)或其类似物。
在一些实施方案中，上述任一所述的抗体-药物偶联物中，所述抗体-药物偶联物具有1-30范围内的平均药物抗体比DAR，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30以及任意两个上述数值之间的平均药物抗体比DAR，例如5-6的平均药物抗体比DAR。
在另一个方面，本发明还提供制备上述任一所述的抗体-药物偶联物的方法，包括：将所述接头连接至所述药物，使所述接头-药物部分偶联至所述抗体，纯化所述抗体-药物缀合物。
用于制备抗体-药物偶联物的其他方法己描述于各种出版物中。例如，可在抗体中经由通过还原链间二硫键活化的半胱氨酸巯基进行化学修饰用于发生缀合反应。
在一些实施方案中，上述抗体-药物偶联物中，所述细胞毒类药物为MC-MMAF(Maleimidocaproyl monomethylauristatin F，马来酰亚胺基己酰-单甲基澳瑞他汀F)，其连接到上述任一所述的抗体或其抗原结合片段或上述变体的半胱氨酸的S原子上而得到抗体-药物偶联物。优选地，将上述任一所述的抗体或其抗原结合片段或上述变体在TCEP(三(2-羧乙基)膦)的作用下还原，暴露半胱氨酸的S原子，再将药物连接子MC-MMAF连接在该硫原子上从而实现偶联，样品通过illustra NAP-10柱(购自GE Healthcare)纯化。
在另一个方面，本发明还提供一种药物组合物，其包含上述任一所述的抗体或其抗原结合片段或上述变体或上述任一所述的抗体-药物偶联物以及药学上可接受的载体。
在另一个方面，本发明还提供上述任一所述的抗体或其抗原结合片段或上述变体或上述任一所述的抗体-药物偶联物在制备如下任一所示的产品中的应用：
(1)检测人卵泡刺激素受体表达的产品；
(2)检测、筛查、预防和/或治疗癌症的产品。
在一些实施方案中，上述应用中，所述癌症是前列腺癌、乳腺癌、结肠癌、胰腺癌、膀胱癌、肾癌、肺癌、肝癌、胃癌、睾丸癌和卵巢癌。
在另一个方面，本发明还提供一种试剂盒，其包含上述任一所述的抗体或其抗原结合片段或变体或上述任一所述的抗体-药物偶联物。
在另一个方面，本发明还提供一种抑制癌细胞生长的方法，包括将所述细胞与上述任一项所述的抗体-药物偶联物接触。
在一些实施方案中，所述癌细胞为前列腺癌细胞、乳腺癌细胞、结肠癌细胞、胰腺癌细胞、膀胱癌细胞、肾癌细胞、肺癌细胞、肝癌细胞、胃癌细胞、睾丸癌细胞和卵巢癌细胞。
在另一个方面，本发明还提供一种治疗癌症患者的方法，包括向所述患者施用治疗有效量的上述任一所述的抗体-药物偶联物。
在一些实施方案中，上述方法中，所述癌症是前列腺癌、乳腺癌、结肠癌、胰腺癌、膀胱癌、肾癌、肺癌、肝癌、胃癌、睾丸癌和卵巢癌。
在一些实施方案中，上述任一所述的方法进一步包括向所述患者施用另外的治疗剂。合适的治疗剂包括现有的用于特定应用(如癌症)的药物和/或手术疗法。例如，所述抗体-药物偶联物与一种或多种其它化疗或抗肿瘤试剂联用。或者，其它治疗剂是放射疗法。
在一些实施方案中，所述治疗剂为细胞毒性剂。
在另一个方面，本发明还提供一种诊断怀疑患有癌症的对象的方法，所述方法包括向所述对象施用连接可检测标记的上述任一所述的抗体或其抗原结合片段或变体，并检测标记的抗体或其抗原结合片段或变体在所述对象中的分布。
本领域技术人员应理解本发明的抗体或其抗原结合片段或变体或抗体-药物偶联物有多种用途，例如，本发明的抗体-药物偶联物可用作治疗剂、本发明的抗体或其抗原结合片段或变体用作诊断试剂盒中的试剂或用作诊断工具。
本发明提供的抗FSHR抗体CAN001可以与FSHR特异性结合，在其基础上得到的CAN001抗体-药物偶联物CAN001-MC-MMAF对癌细胞具有显著的杀伤作用。本发明提供的抗FSHR抗体及其抗体-药物偶联物在癌症的检测、筛查和治疗中具有广阔的应用前景。
附图说明
图1为pMSGβ3_FSHR质粒图谱。
图2为含有CAN001抗体的上清液特异性染色FSHR表达细胞，但不染色GFP表达细胞。
图3为筛选最佳CAN001上清液。
图4为CAN001对人胎盘进行IHC染色。
图5为CAN001对卵巢癌组织和邻近组织进行IHC染色。
图6为CAN001-MC-MMAF的疏水作用色谱(HIC)。
图7为CAN001-MC-MMAF的尺寸排除色谱(SEC)。
具体实施方式
以下通过特定的具体实施方式说明本发明，本领域技术人员可由本说明书所揭露的内容容易地了解本发明的优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。应理解，本发明的保护范围不局限于下述特定的具体实施方式；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方式，而不是为了限制本发明的保护范围。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,第二版,冷泉港实验室出版社,1989和第三版,2001；Ausubel等,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons,纽约,1987和periodic updates:the series METHODS IN ENZYMOLOGY,学术出版社,圣地亚哥；Wolffe,CHROMATIN STRUCTURE AND FUNCTION,第三版,学术出版社,圣地亚哥,1998；METHODS IN ENZYMOLOGY,第304卷,Chromatin(P.M.Wassarman和A.P.Wolffe,eds.),学术出版社,圣地亚哥,1999和METHODS IN MOLECULAR BIOLOGY,第119卷，Chromatin Protocols(P.B.Becker,ed.)，胡玛纳出版社,托托瓦，1999等。
本文所用术语“抗体”指免疫球蛋白(Ig)分子和免疫球蛋白分子的免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”或“发生免疫反应”或“针对”是指抗体与目标抗原的一个或多个抗原决定簇反应且不与其它多肽反应，或者以很低亲和力(Kd>10 -6g/ml)与其它多肽结合。抗体包括但不限于单克隆抗体、嵌合抗体、dAb(结构域抗体)、单链抗体、Fab、Fab’和F(ab’)2片段、Fv和Fab表达库。
已知基本的抗体结构单元包括一个四聚体。每个四聚体(或称为“全抗”)由两对相同的多肽链组成，每对具有一条“轻”链和一条“重”链。每条链的氨基末端部分包括约100至110个或更多个氨基酸的可变区，主要负责抗原识别。每条链的羧基端部分限定了一个恒定区，主要负责效应子功能。一般来讲，从人类获得的抗体分子涉及IgG、IgM、IgA、IgE和IgD中的任意种类，这些由于分子中存在的重链的性质而彼此不同。某些种类还具有亚类，诸如IgG1、IgG2、以及其它。此外，在人类中，轻链可为κ链或λ链。
如本文所用，术语“单克隆抗体”(mAb)是指一群这样的抗体分子：其只含有由独特的轻链基因产物和独特的重链基因产物组成的抗体分子中的一种分子种类。具体地，单克 隆抗体的互补决定区(CDR)在该群体的所有分子中是相同的。mAb含有能够与抗原的特定表位发生免疫反应的抗原结合位点。
如本文所用，术语“特异性结合”是指在免疫球蛋白分子与免疫球蛋白特异的抗原之间发生的非共价相互作用类型。免疫学结合相互作用的强度或亲和力可以以相互作用的解离常数(Kd)表示，其中较小的Kd代表较大的亲和力。所选多肽的免疫结合特性可使用本领域熟知方法进行定量。
如本文所用，术语“互补决定区”和“CDR”预期是指在抗体的HC和LC多肽的可变区内发现的不连续抗原结合位点。其他人已经描述了这些特定区域，包括Kabat等人，Ann.NY Acad.Sci.190:382-93(1971)；Kabat等人，J.Biol.Chem.252:6609-6616(1977)；Kabat等人，Sequences of Proteins of Immunological Interest,第5版,U.S.Department of Health and Human Services,NIH Publication No.91-3242(1991)；Chothia等人，J.Mol.Biol.196:901-917(1987)；MacCallum等人，J.Mol.Biol.,262:732-745(1996)；和North等人，J.Mol.Biol.,406,228-256(2011)，其中定义包括彼此比较时氨基酸残基的重叠或亚组。
CDR散布于更保守的区域，称为框架区(“FR”)。每个LCVR和HCVR由三个CDR和四个FR组成，按照以下顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的三个CDR被称为“LCDR1、LCDR2和LCDR3”，并且重链的三个CDR被称为“HCDR1、HCDR2和HCDR3”。CDR包含与抗原形成特异性的相互作用的大多数残基。本发明中的LCVR和HCVR区域内CDR氨基酸残基的编号和定位符合Kabat规则。
“分离的”抗体是从其天然环境的组分中分离和/或回收的抗体。其天然环境的污染组分是将妨碍抗体的诊断或治疗用途的物质，并且可包括酶、激素、以及其它蛋白质或非蛋白质溶质。在一些实施方案中，将所述抗体纯化到以下程度：(1)所述抗体按重量百分比计大于95％，如大于99％，如由Lowry方法所测；(2)通过使用转杯式测序仪可以获得N-末端或内部氨基酸序列的至少15个残基；或者(3)通过还原或非还原条件下的SDS-PAGE，使用考马斯蓝或银染色测出具有同质性。分离的抗体包括重组细胞内的原位抗体。通常，分离的抗体将通过至少一个或多个纯化步骤制备。在一些实施方案中，分离的抗体的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
术语“序列同一性”指的是：在比较窗口中两个多核苷酸或氨基酸序列是相同的(即基于核苷酸对核苷酸或残基对残基是相同的)。术语“序列同一性百分比”通过如下方式计算：在比较窗口中比较两个最佳比对的序列，确定在两个序列中出现相同核酸碱基(例如A、T、C、G或U)或氨基酸残基的位置的数目以得到匹配的位置的数目，将匹配的位置的数目除以比较窗口中位置的总数目(即窗口尺寸)，然后将结果乘以100以得到序列同一性百分比。
抗体或免疫球蛋白分子的氨基酸序列的微小变化都涵盖在本公开之内，条件是氨基酸序列的同一性保持至少75％、如至少80％、90％、95％并又如99％。在一些实施方案中，变化为保守氨基酸取代。保守氨基酸取代是在其侧链中相关的氨基酸家族内发生的取代。基因编码的氨基酸大致分以下类：(1)酸性氨基酸为天冬氨酸盐、谷氨酸盐；(2)碱性氨 基酸为赖氨酸、精氨酸、组氨酸；(3)非极性氨基酸为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)；以及(4)无电荷的极性氨基酸为甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。其它家族的氨基酸包括(i)脂肪族-羟基家族的丝氨酸和苏氨酸；(ii)含酰胺家族的天冬酰胺和谷氨酰胺；(iii)脂肪族家族的丙氨酸、缬氨酸、亮氨酸和异亮氨酸；以及(iv)芳族家族的苯丙氨酸、色氨酸和酪氨酸。在一些实施方案中，保守氨基酸取代组为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、谷氨酸-天冬氨酸、以及天冬酰胺-谷氨酰胺。例如，可以合理地预测用异亮氨酸或缬氨酸单独的置换亮氨酸，用谷氨酸盐置换天冬氨酸盐，用丝氨酸置换苏氨酸，或用一个结构相关的氨基酸类似的置换一个氨基酸，且对所得分子的结合或特性不会有重要影响，特别是该置换不涉及结合位点内的氨基酸。氨基酸改变是否产生功能性肽可以容易地通过测定多肽衍生物的比活性来确定。所述测定在本文进行了详细描述。抗体或免疫球蛋白分子的片段或类似物可由本领域普通技术人员容易地制备。
术语“药学上可接受”是指适合人和/或动物使用而没有与合理的益处/风险比相称的过度不良副作用(例如毒性，刺激和过敏反应)的组分。
术语“载体”是指与活性成分一起使用的用于治疗的稀释剂、佐剂、赋形剂或载体。此类药物载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。在一些实施方案中，当药物组合物静脉内给药时，载体可以是水。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗体片段，连同合适的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
术语“基因”用于指功能性蛋白质，多肽或肽的编码单元。如本领域技术人员所理解的，该功能性术语包括基因组序列，cDNA序列或其片段或组合，以及基因产物，包括可能已被人工改变的基因产物。纯化的基因，核酸，蛋白质及类似物，在从通常与其相关的至少一种污染核酸或蛋白质中识别和分离时，用于指鉴这些实体。
如本文所用的术语“序列”用于指核苷酸或氨基酸，无论是天然的还是人工的，例如修饰过的核酸或氨基酸。
在一些实施方案中，氨基酸取代具有如下效果：(1)降低对蛋白水解作用的敏感性，(2)降低对氧化作用的敏感性，(3)改变用于形成蛋白复合物的结合亲和力，(4)改变结合亲和力，和(5)赋予或改进此类类似物的其它物理化学或功能特性。类似物可包括序列不同于天然存在的肽序列的各种突变蛋白。例如，可在天然存在的序列(优选在形成分子间接触的结构域之外的多肽部分中)中进行单个或多个氨基酸取代(优选保守氨基酸取代)。保守氨基酸取代不应当显著改变亲本序列的结构特性(例如，置换的氨基酸不应当趋于破坏亲本序列中存在的螺旋结构，或破坏表征亲本序列的其它类型二级结构)。人工识别的多肽的二级和三级结构的示例描述于Proteins,Structures and Molecular Principles(Creighton编辑，W.H.Freeman and Company,New York(1984))；Introduction to Protein Structure(C.Branden和J.Tooze编辑，Garland Publishing,New York,N.Y.(1991))；和Thornton 等人Nature 354:105(1991)。
抗体可通过公知的技术纯化，例如利用蛋白A或蛋白G进行亲和层析，其主要提供了免疫血清中的IgG级分。另外，可将免疫球蛋白所靶向的特异抗原或其表位固定于柱上，以通过免疫亲和色谱来纯化免疫特异性抗体。免疫球蛋白的纯化可参考D.Wilkinson的文章(The Scientist，由The Scientist,Inc.,Philadelphia Pa.出版，第14卷，第8期(2000年4月17日)，第25-28页)。
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、稳定剂、缓冲剂、分散介质、包衣、抑菌剂、等渗剂和吸收延缓剂等。合适载体描述于最新版的Remington's Pharmaceutical Sciences中。此类载体或稀释剂可选择包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
本文所用“抑制”或“治疗”包括延缓与疾病有关的症状的发展和/或减轻所述疾病将要或预期发展的这些症状的严重程度。所述术语还包括减缓已有症状、防止另外的症状和减缓或防止这些症状的潜在原因。因此，所述术语表示业已将有益结果赋予患有疾病的脊椎动物对象，比如人。
本文所用术语“治疗有效量”或“有效量”是指当将抗体-药物偶联物单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述抗体-药物偶联物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。
单克隆抗体
可根据本领域知识和技能来制备针对FSHR的单克隆抗体。
用常规方法便可容易地合成单克隆抗体的DNA(如轻链DNA和重链DNA)，所述DNA可被置于表达载体中(例如构建得到轻链表达载体和重链表达载体)，然后将这些载体转染到宿主细胞中以在重组宿主细胞中实现单克隆抗体的合成，所述宿主细胞例如有HEK293T细胞、CHO细胞、NS0细胞或不会另外产生免疫球蛋白的骨髓瘤细胞。抗体的重组制备将在下文更详细地阐述。
抗体-药物偶联物
FSHR的单克隆抗体可以与药物连接子(例如MC-MMAF)偶联，通常连接在抗体的半胱氨酸的S原子上。
本发明的抗体及其抗体-药物偶联物的治疗应用
与FSHR特异性结合的本发明抗体或抗原结合片段或抗体-药物偶联物可用于特异性识别表达FSHR的细胞、组织等。
在一些实施方案中，本发明还提供一种治疗疾病的方法。所述方法包括向有需要的患者施用本发明所述的治疗有效量的抗体-药物偶联物。在一些实施方案中，所述疾病为癌症。
癌症
本发明的抗体-药物偶联物可用于治疗癌症(即抑制肿瘤细胞的生长或存活)。在一些实施方案中，可用本发明的抗体-药物偶联物抑制其生长的癌症包括通常包括前列腺癌、乳腺癌、结肠癌、胰腺癌、膀胱癌、肾癌、肺癌、肝癌、胃癌、睾丸癌和卵巢癌。
本发明的抗体及其抗体-药物偶联物的非治疗性应用
本发明的抗体及其抗体-药物偶联物可用于诊断测定，例如用于检测FSHR在特定的细胞、组织或血清中的表达。为了诊断应用，通常用可检测的部分(直接或间接)标记抗体。可利用众多标记物，其通常分为以下类别：生物素、荧光染料、放射性核苷酸、酶、碘和生物合成标记物。
本发明抗体还可用于体内诊断测定。通常用放射性核素(例如 111In、 99Tc、 4C、 125I、 3H、 32P、 35S或 18F)标记抗体，以使可用免疫显像(immunoscintigraphy)或正电子成像术定位抗原或表达抗体的细胞。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗体-药物偶联物以及药学上可接受的载体。
在一些实施方案中，术语“药学上可接受的载体”是指由政府的监管机构批准的或其他公认的药典中列出的用于动物(特别是用于人类)的物质。此外，“药学上可接受的载体”通常将是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂。
实施例1：小鼠抗人FSHR单克隆抗体CAN001
1、由Genescript合成编码抗人FSHR单克隆抗体的轻链和重链的密码子优化的DNA片段(分别如SEQ ID NO:1和SEQ ID NO:2所示)，并测序验证合成的DNA片段的核苷酸序列。
SEQ ID NO:1：轻链DNA序列
Figure PCTCN2021131515-appb-000001
Figure PCTCN2021131515-appb-000002
轻链可变区的DNA序列为SEQ ID NO:1的第64位至第405位，轻链CDR1的DNA序列为第130位至第168位，轻链CDR2的DNA序列为第214位至第234位，轻链CDR3的DNA序列为第331位至第357位。
SEQ ID NO:2：重链DNA序列
Figure PCTCN2021131515-appb-000003
Figure PCTCN2021131515-appb-000004
重链可变区的DNA序列为SEQ ID NO:2的第64位至第441位，重链CDR1的DNA序列为第154位至第168位，重链CDR2的DNA序列为第211位至第261位，重链CDR3的DNA序列为第358位至第378位。
2、通过In-Fusion克隆方法将轻链DNA序列克隆到pcDNA3.1(购自Thermo Fisher)的Hind III位点和EcoR I位点间，构建得到轻链表达载体pcDNA3.1_CAN001 LC。
3、通过In-Fusion克隆方法将重链DNA序列克隆到pcDNA3.1的Hind III位点和EcoR I位点间，构建得到重链表达载体pcDNA3.1_CAN001 HC。
SEQ ID NO:3：轻链氨基酸序列
Figure PCTCN2021131515-appb-000005
基于Kabat编号系统编号，轻链可变区的氨基酸序列为SEQ ID NO:3的第22位至第135位(SEQ ID NO:5)，轻链CDR1的氨基酸序列为第44位至第56位(CSASSSVSSSYLY(SEQ ID NO:6))，轻链CDR2的氨基酸序列为第72位至第78位(STSNLAS(SEQ ID NO:7))，轻链CDR3的氨基酸序列为第111位至第119位(HQWSSYPPT(SEQ ID NO:8))。
SEQ ID NO:4：重链氨基酸序列
Figure PCTCN2021131515-appb-000006
基于Kabat编号系统编号，重链可变区的氨基酸序列为SEQ ID NO:4的第22位至第147位(SEQ ID NO:9)，重链CDR1的氨基酸序列为第52位至第56位(SYWMH(SEQ ID NO:10))，重链CDR2的氨基酸序列为第71位至第87位(EINPRNGRTNYNEKFKS(SEQ ID NO:11))，重链CDR3的氨基酸序列为第120位至第126位(LSVGFAY(SEQ ID NO:12))。
实施例2：人FSHR基因的克隆及表达载体的构建
提取OVCAR3细胞系(购自ATCC公司，产品目录号为htb-161)的RNA反转录为cDNA，以cDNA为模板以FSHR-F和FSHR-R为引物进行PCR扩增得到含有FSHR基因的片段。
FSHR-F：5’-gtttCCACAACCatggccctgctcctgg-3’(SEQ ID NO:13)；
FSHR-R：5’-ggttgattaactcgagttagttttgggctaaatgacttagagggacaag-3’(SEQ ID NO:14)。
将含有FSHR基因的片段克隆到克隆载体pJET2.1(购自Thermo Fisher，产品目录号为SO501)，构建得到pJET2.1-FSHR质粒。测序验证后，将插入片段亚克隆到pMSGβ3的Nco I和Xho I位点间，构建得到pMSGβ3_FSHR质粒，pMSGβ3_FSHR质粒图谱如图1所示。
实施例3：在HEK293T细胞中产生抗体CAN001
为了制备CAN001抗体，将HEK293T细胞在无血清培养基中培养，并用编码CAN001重链和轻链的载体pcDNA3.1_CAN001 HC和pcDNA3.1_CAN001 LC(质量比1:2)与25kD线性PEI聚合物(polyscience)按照载体:聚合物的质量比为1:3的比例复合转染HEK293T细胞。转染后将HEK293T细胞培养72小时，并收集培养上清液，即为含有CAN001抗体的上清液。
实施例4：CAN001抗体的流式细胞仪分析
用pMSGβ3_FSHR质粒转染HEK293T细胞,得到表达FSHR的HEK293T细胞，作为实验组。用pmaxGFP(Lonza)转染HEK293T细胞，得到表达GFP蛋白的HEK293T细胞，作为阴性对照组。将实验组和阴性对照组的细胞分别与实施例3获得的含有CAN001抗体的上清液一起孵育后，将CAN001抗体包被的细胞洗涤，并与APC偶联的山羊抗小鼠IgG二抗孵育。流式细胞仪使用BD calibur进行，并使用Cellquest软件进行分析。结果如图2所示，结果表明用含有CAN001抗体的上清液特异性染色表达FSHR的细胞，而不染色表达GFP的细胞。
为了进一步筛选最好的CAN001上清液，用不同比例的质粒pcDNA3.1_CAN001 HC和pcDNA3.1_CAN001 LC的混合物(图3中从左到右pcDNA3.1_CAN001 HC和pcDNA3.1_CAN001 LC的质量比分别为1:2、1:1和1:3)与25kD线性PEI聚合物按照载体:聚合物的质量比为1:3的比例复合转染HEK293T细胞。转染后72小时，收集上清液并用于染色表达FSHR的HEK293T细胞，表达GFP蛋白的HEK293T细胞用作阴性对照，步骤如上所述。流式细胞仪分析结果如图3所示，结果表明当pcDNA3.1_CAN001 HC与pcDNA3.1_CAN001 LC的质量比为1:2时，HEK293T细胞被染色的阳性率最高(38.3％)，而pcDNA3.1_CAN001 HC与pcDNA3.1_CAN001 LC的质量比为1:1和1:3时，HEK293T细胞被染色的阳性率分别为 30.02％和34.75％。选择pcDNA3.1_CAN001 HC与pcDNA3.1_CAN001 LC的质量比为1:2制备含有CAN001抗体的上清液。
实施例5：CAN001抗体对人胎盘和肿瘤组织进行IHC染色
实验试剂：
PBS缓冲液(pH＝7.4)：1000ml蒸馏水，8.0g NaCl，0.2g KCl，1.149g Na 2HPO 4，0.2g NaH 2PO 4。
0.01M枸橼酸缓冲液(pH6.0)：
A液：29.41g枸橼酸三钠-2H 2O+1000ml蒸馏水
B液：21g枸橼酸+1000ml蒸馏水
抗原修复工作液的配制：82ml A液+18ml B液+900ml蒸馏水
苏木素显色液(避光保存，见光被氧化)：配比根据苏木素试剂盒(购自福州迈新生物技术开发有限公司，产品目录号为CTS1097)上的说明书配比，也可以根据预实验时所得试验结果调整比例，以达到最佳染色。通常配比如下：1ml苏木素原液+4ml蒸馏水。
GTVisionⅢ抗鼠/兔通用型免疫组化检测试剂盒购自基因科技(上海)股份有限公司，产品目录号为GK500705，A液(二抗工作液)：HRP标记聚合物(抗鼠、兔)；B液：DAB缓冲稀释液；C液：DAB原液。DAB显色液配制：1ml B液+20μl C液。显色结果：棕色。该试剂盒不含亲和素、链亲和素，不受内源生物素干扰。
实验步骤：
1)脱蜡和水化
将人胎盘或卵巢癌组织石蜡包埋切片于70℃恒温箱中烘烤约40分钟。然后将切片置于二甲苯中浸泡15分钟，随后在无水乙醇、95％乙醇、75％乙醇中各浸泡5分钟进行梯度水化。
2)去除内源性过氧化物酶
3％过氧化氢中浸泡切片约15分钟，去除组织内源性过氧化氢酶，接着用PBS缓冲液洗片子3次，每次5分钟。
3)抗原修复
在沸水中加入抗原修复工作液(pH 6.0)。将切片放入修复溶液内，在最高压力下，加热约10分钟后打开盖子，待其自然冷却至室温。
4)封闭、抗体孵育
当玻片冷却到室温后，PBS缓冲液洗3次，每次5分钟，滴加10％山羊血清/PBS封闭液在标本上，湿盒内室温静置封闭1小时。后滴加目标蛋白抗体CAN001(1:200-800)在标本上，每片约50μL，置于湿盒中4℃过夜后PBS缓冲液洗3次，每次洗5分钟；滴加羊抗鼠二抗50μL(1:200)，置于湿盒中室温静置1小时，PBS缓冲液洗3次，每次洗5分钟。
5)DAB显色
每块切片组织上滴加1-2滴DAB显色液，并同时在显微镜下观察染色程度，程度适中时用PBS缓冲液终止反应，随后苏木素染细胞核复染30秒，PBS缓冲液终止反应。
6)封片
用封片剂滴在组织旁边，再用方形盖玻片盖上，为避免气泡产生，应先放平一侧，然后再放平另一侧，最后在盖玻片上轻压数次，封好片子晾干24小时。
CAN001抗体对人胎盘进行的IHC染色如图4所示。
CAN001抗体对卵巢癌组织和邻近组织进行的IHC染色如图5所示。
实施例6：CAN001抗体-药物偶联物的制备
用PBS缓冲液(660.0μL,pH＝7)稀释抗体溶液CAN001(1039.2μL,7.66mg/mL)，得到1699.2μL浓度为4.68mg/mL的CAN001抗体溶液。在上述CAN001抗体溶液中加入含有TCEP(三(2-羧乙基)膦)的PBS溶液(14.7μL，2.5mM，pH＝7)，将得到的溶液轻轻涡旋均匀后在37℃下孵育2小时。加入136.5μL含有3.5mM药物连接子MC-MMAF(Maleimidocaproyl monomethylauristatin F，马来酰亚胺基己酰-单甲基澳瑞他汀F)的DMSO溶液，将溶液轻轻涡旋并在25℃下保持1小时，反应得到粗样品。粗样品通过illustra NAP-10柱(购自GE Healthcare)纯化，并通过0.25μm微滤器过滤，得到最终的抗体-药物偶联物CAN001-MC-MMAF，3.1mL，2.56mg/mL，DAR(药物抗体比)＝5.95，UmAb％(未偶联抗体百分率)＝1.3％，Agg％(聚合抗体百分率)＝1.6％。
CAN001-MC-MMAF的结构式如式1所示。
CAN001-MC-MMAF的疏水作用色谱(HIC)如图6所示。
CAN001-MC-MMAF的尺寸排除色谱(SEC)如图7所示。
Figure PCTCN2021131515-appb-000007
实施例7：CAN001抗体-药物偶联物的细胞杀伤实验
本实施例使用96孔板通过CellTiter-Glo细胞活力测定法测试CAN001抗体-药物偶联物对抗癌细胞系的作用。
人卵巢癌细胞系：OVCAR-3，A2780，3AO，均购自ATCC。
将细胞接种于96孔细胞培养板中，每个孔1000个细胞，每孔培养基总量控制为200μL。待细胞贴壁后，给予相应的药物处理细胞，于不同时间点(0、24h、48h、72h、96h)撤药，并且加入MTS检测溶液(MTS试剂10μL+190μL完全培养基)(MTS细胞增殖试剂盒(比色法)为艾美捷产品)，然后将96孔细胞培养板放在CO 2细胞恒温培养箱内避光孵育大约2小时，然后取出细胞培养板，放置于酶标仪上，测定不同分组的细胞在492nm处的吸光度。
每种化合物的％抑制率和IC50将通过XLFit曲线拟合软件进行计算。结果如表1所示。
表1
Figure PCTCN2021131515-appb-000008
表1表明，与MMAF相比，CAN001抗体-药物偶联物CAN001-MC-MMAF对卵巢癌细胞 具有显著提高的杀伤作用。

Claims (24)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含可变区，其特异性结合人卵泡刺激素受体FSHR，包含一个、两个、三个、四个、五个或六个选自具有以下所示的氨基酸序列的CDR：SEQ ID NO:6、7、8、10、11和12。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于：所述抗体或其抗原结合片段包含轻链可变区和/或重链可变区，所述抗体或其抗原结合片段包含一个、两个或三个选自具有以下所示的氨基酸序列的轻链CDR：SEQ ID NO:6、7和8；和/或，
    所述抗体或其抗原结合片段包含一个、两个或三个选自具有以下所示的氨基酸序列的重链CDR：SEQ ID NO:10、11和12。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其特征在于：所述抗体或其抗原结合片段包含具有以下所示的氨基酸序列的轻链CDR：SEQ ID NO:6、7和8以及具有以下所示的氨基酸序列的重链CDR：SEQ ID NO:10、11和12。
  4. 根据权利要求1-3任一项所述的抗体或其抗原结合片段，其特征在于：所述抗体或其抗原结合片段包含轻链可变区和重链可变区；
    其中，所述轻链可变区包含具有SEQ ID NO:5所示的氨基酸序列，或与SEQ ID NO:5所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:5所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或，
    所述重链可变区包含具有SEQ ID NO:9所示的氨基酸序列，或与SEQ ID NO:9所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:9所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  5. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于：所述抗体或其抗原结合片段包含轻链和重链；
    其中，所述轻链包含具有SEQ ID NO:3所示的氨基酸序列，或与SEQ ID NO:3所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:3所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或，
    所述重链包含如SEQ ID NO:4所示的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列具有至少90％同一性的氨基酸序列，或与SEQ ID NO:4所示的氨基酸序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段的变体，其包含不超过10个氨基酸取代、缺失和/或添加。
  7. 与权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体相关的生物材料，为如下B1)或B2)：
    B1)编码权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体的核酸分子；
    B2)含有B1)所述核酸分子的表达盒、重组载体、重组细胞或重组微生物。
  8. 根据权利要求7所述的生物材料，其特征在于：B1)所述核酸分子包含如下1)或2)或3)所示的核酸片段：
    1)如下DNA分子：
    SEQ ID NO:1中第130位至第168位所示的核酸片段；
    SEQ ID NO:1中第214位至第234位所示的核酸片段；
    SEQ ID NO:1中第331位至第357位所示的核酸片段；
    SEQ ID NO:2中第154位至第168位所示的核酸片段；
    SEQ ID NO:2中第211位至第261位所示的核酸片段；
    SEQ ID NO:2中第358位至第378位所示的核酸片段；
    SEQ ID NO:1中第64位至第405位所示的核酸片段；
    SEQ ID NO:2中第64位至第441位所示的核酸片段；
    SEQ ID NO:1所示的核酸片段；或
    SEQ ID NO:2所示的核酸片段；
    2)与1)限定的核酸片段杂交且编码权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体的核酸片段；
    3)与1)或2)限定的DNA分子具有90％以上的同一性且编码权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体的核酸片段。
  9. 制备权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体的方法，所述方法包括：
    在能使权利要求7中B1)所述的核酸分子表达的条件下于培养基中培养含有所述核酸分子的重组细胞，以制备所述抗体或其抗原结合片段或变体。
  10. 一种抗体-药物偶联物，由权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体通过连接子与细胞毒类药物偶联得到。
  11. 根据权利要求10所述的抗体-药物偶联物，其特征在于：所述细胞毒类药物为化学治疗剂或毒素，所述毒素优选为澳瑞他汀，更优选为单甲基澳瑞他汀F。
  12. 根据权利要求10所述的抗体-药物偶联物，其特征在于：所述接头选自马来酰亚胺基乙酰或其类似物。
  13. 根据权利要求10-12任一项所述的抗体-药物偶联物，其特征在于：所述抗体-药物偶联物具有1-30范围内的平均药物抗体比DAR。
  14. 制备权利要求10-13任一项所述的抗体-药物偶联物的方法，包括：
    将所述接头连接至所述药物，使所述接头-药物部分偶联至所述抗体，纯化所述抗体-药物缀合物。
  15. 一种药物组合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体或权利要求10-13任一项所述的抗体-药物偶联物以及药学上可接受的载体。
  16. 权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体或权利要求10-13任一项所述的抗体-药物偶联物在制备如下任一所示的产品中的应用：
    (1)检测人卵泡刺激素受体表达的产品；
    (2)检测、筛查、预防和/或治疗癌症的产品。
  17. 根据权利要求16所述的应用，其特征在于：所述癌症是前列腺癌、乳腺癌、结肠癌、胰腺癌、膀胱癌、肾癌、肺癌、肝癌、胃癌、睾丸癌和卵巢癌。
  18. 一种抑制癌细胞生长的方法，包括将所述细胞与权利要求10-13任一项所述的抗体-药物偶联物接触。
  19. 根据权利要求18所述的方法，其特征在于：所述癌细胞为前列腺癌细胞、乳腺癌细胞、结肠癌细胞、胰腺癌细胞、膀胱癌细胞、肾癌细胞、肺癌细胞、肝癌细胞、胃癌细胞、睾丸癌细胞和卵巢癌细胞。
  20. 一种治疗癌症患者的方法，包括向所述患者施用治疗有效量的权利要求10-13任一项所述的抗体-药物偶联物。
  21. 根据权利要求20所述的方法，进一步包括向所述患者施用另外的治疗剂。
  22. 根据权利要求21所述的方法，其特征在于：所述治疗剂为细胞毒性剂。
  23. 一种诊断怀疑患有癌症的对象的方法，所述方法包括向所述对象施用连接可检测标记的权利要求1-5任一项所述的抗体或其抗原结合片段或权利要求6所述的变体，并检测标记的抗体或其抗原结合片段或变体在所述对象中的分布。
  24. 根据权利要求20-23任一项所述的方法，其特征在于：所述癌症是前列腺癌、乳腺癌、结肠癌、胰腺癌、膀胱癌、肾癌、肺癌、肝癌、胃癌、睾丸癌和卵巢癌。
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