WO2020216210A1 - 抗EpCAM抗体及其应用 - Google Patents

抗EpCAM抗体及其应用 Download PDF

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WO2020216210A1
WO2020216210A1 PCT/CN2020/085838 CN2020085838W WO2020216210A1 WO 2020216210 A1 WO2020216210 A1 WO 2020216210A1 CN 2020085838 W CN2020085838 W CN 2020085838W WO 2020216210 A1 WO2020216210 A1 WO 2020216210A1
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seq
cancer
variable region
chain variable
light chain
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PCT/CN2020/085838
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English (en)
French (fr)
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杨阳
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN202080021834.4A priority Critical patent/CN113597432B/zh
Publication of WO2020216210A1 publication Critical patent/WO2020216210A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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

Definitions

  • the present disclosure relates to the field of antibody drugs. Specifically, the present disclosure relates to anti-EpCAM antibodies and their applications.
  • EpCAM (CD326) is a type I transmembrane glycoprotein that acts as a specific cell adhesion molecule for epithelial cells. It also involves other processes, including cell migration, proliferation, and differentiation. EpCAM is one of the first tumor-associated antigens identified by monoclonal antibody technology. It is widely expressed on the surface of epithelial tissues in the form of multimers, and mediates the function of calcium-independent homotypic adhesion between cells, which can be classified as adhesion. Molecular family. EpCAM also has other characteristics of the adhesion molecule family, and participates in various processes including cell-matrix interaction, migration, cell differentiation, morphology, cell cycle regulation, signal transduction, and metabolism.
  • EpCAM is overexpressed in a variety of tumors of epithelial origin, suggesting that it is closely related to tumors.
  • Pathological studies have found that EpCAM is expressed to varying degrees in adenocarcinomas, including colorectal cancer, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, hepatocellular carcinoma and retinoblastoma.
  • a number of studies have confirmed that the expression of EpCAM is related to the proliferation, cycle distribution and metastasis of breast and colon cancer cells.
  • Monospecific anti-EpCAM monoclonal antibodies such as monoclonal antibody 17-1A (glaxowellcome, Centocor), are approved for adjuvant treatment of colorectal cancer.
  • the present disclosure provides an anti-EpCAM antibody with good affinity and suitable for diagnosis and treatment.
  • the present disclosure provides an anti-EpCAM antibody.
  • the anti-EpCAM antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region contains the same sequence as that shown in SEQ ID NO: 6. LCDR1, LCDR2 and LCDR3 with the same sequence of the light chain variable region shown; or
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region includes the same sequence as that shown in SEQ ID NO: 10.
  • the LCDR1, LCDR2 and LCDR3 of the same sequence of the light chain variable region are shown.
  • the anti-EpCAM antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the light chain variable region includes SEQ ID respectively.
  • NO: 14 SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2, and LCDR3; or
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the light chain variable region includes SEQ ID NO: 17, HCDR2, and HCDR3, respectively.
  • the anti-EpCAM antibody as described above, wherein the anti-EpCAM antibody is a human antibody is a human antibody.
  • the anti-EpCAM antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, and the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 9 99% identity
  • the light chain variable region has at least 90%, 92%, 94%, 95%, 96%, 97%, and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 10. 98%, 99% identity.
  • the anti-EpCAM antibody as described above, comprises a heavy chain variable region and a light chain variable region, wherein:
  • (v-1) The heavy chain variable region whose amino acid sequence is at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 5
  • the amino acid sequence of the variable region of the light chain has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 6;
  • the heavy chain variable region whose amino acid sequence is at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 9
  • the amino acid sequence of the variable region of the light chain has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 10.
  • the anti-EpCAM antibody as previously described, wherein:
  • the CDR of the variable region of the heavy chain and the CDR of the variable region of the light chain are defined using Kabat numbering rules.
  • the anti-EpCAM antibody as described above, wherein the antibody further comprises an antibody constant region; preferably, the heavy chain constant region of the antibody constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and Conventional variants thereof, the light chain constant region of the antibody constant region is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof; most preferably, the antibody comprises: and the antibody having SEQ ID NO: 23
  • the heavy chain has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identity to the heavy chain, and has at least 90% of the light chain shown in SEQ ID NO: 24 , 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity light chain; or
  • the light chain represented by NO: 26 has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the light chain; or
  • the light chain represented by NO: 28 has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the light chain.
  • the antibody competitively binds to human EpCAM with the aforementioned anti-EpCAM antibody or antigen-binding fragment thereof.
  • the anti-EpCAM antibody is a full-length antibody or an antigen-binding fragment, and the antigen-binding fragment is selected from Fab, Fab', F(ab')2, scFv, dsFv , Ds-scFv, dimer, minibody, bifunctional antibody, bispecific antibody fragment, multimer, and any combination thereof.
  • Another aspect of the present disclosure also provides a nucleic acid molecule encoding the anti-EpCAM antibody as described above.
  • Another aspect of the present disclosure also provides an expression vector comprising the nucleic acid molecule as described above.
  • Another aspect of the present disclosure also provides a host cell, which comprises the aforementioned nucleic acid molecule or the aforementioned expression vector, preferably the cell is a bacterial cell, a fungal cell, an insect animal cell or a mammalian cell.
  • an immunoconjugate which is an immunoconjugate formed by coupling an anti-EpCAM antibody and an effector molecule as described above; the effector molecule is selected from radioisotopes, anti-tumor Agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.
  • the present disclosure provides a method for preparing EpCAM antibody as described above.
  • the present disclosure provides a method for preparing the EpCAM immunoconjugate as described above.
  • the present disclosure provides a pharmaceutical composition containing a therapeutically effective amount of the anti-EpCAM antibody according to the aforementioned, or according to the aforementioned nucleic acid molecule, or the aforementioned immunoconjugate And one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the present disclosure provides a method for immunodetection or determination of EpCAM, the method comprising the step of using the anti-EpCAM antibody or immunoconjugate as described above.
  • the method is performed in vivo or in vitro.
  • the step includes, under conditions that allow the formation of a complex between the anti-EpCAM antibody or immunoconjugate as described above, and EpCAM, combining the test sample with the anti-EpCAM antibody or immunoconjugate as described above. ⁇ contact.
  • the present disclosure provides the use of the aforementioned anti-EpCAM antibody or immunoconjugate in the preparation of reagents for immunological detection of human EpCAM.
  • the present disclosure provides an anti-EpCAM antibody or immunoconjugate as described above for immunodetection or determination of EpCAM.
  • the present disclosure provides a kit comprising an anti-EpCAM antibody or immunoconjugate according to the aforementioned.
  • the present disclosure provides a method for diagnosing EpCAM-related diseases, the method comprising using an anti-EpCAM antibody as described above, an immunoconjugate as described above, or a reagent as described above Box of steps.
  • the method is performed in vivo or in vitro.
  • the step includes, under conditions that allow the formation of a complex between the anti-EpCAM antibody or immunoconjugate as described above, and EpCAM, combining the test sample with the anti-EpCAM antibody or immunoconjugate as described above. ⁇ contact.
  • the present disclosure provides an anti-EpCAM antibody as described above, or a nucleic acid molecule as described above, or an immunoconjugate as described above or a pharmaceutical composition as described above in preparation for Use in a medicine for treating cancer or tumor, wherein the cancer or tumor is preferably EpCAM-positive cancer or malignant tumor, more preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, and more Glioblastoma, neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, liver cells Tumor, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma Tumor, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histiocytosis, and lymphoplasmacytic lymphoma;
  • the lung cancer is selected from: non-small cell lung cancer and small cell Lung cancer;
  • the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.
  • the present disclosure provides a method of treating diseases associated with EpCAM, the method comprising administering to a subject a therapeutically effective amount of an anti-EpCAM antibody as described above, or a nucleic acid molecule as described above , Or the aforementioned immunoconjugate or the aforementioned pharmaceutical composition, wherein the disease is preferably cancer or tumor; more preferably EpCAM-positive cancer or malignant tumor, more preferably selected from: head and neck scales Shape cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, Thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, liver and gallbladder cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorec
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, Mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from: non-small cell lung cancer and small cell lung cancer,
  • the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg or 1-1000 mg of the aforementioned anti-EpCAM antibody or the aforementioned immunocon
  • the present disclosure provides an anti-EpCAM antibody as described above, or a nucleic acid molecule as described above, or an immunoconjugate as described above, or an anti-EpCAM antibody as described above for the treatment of diseases associated with EpCAM.
  • the pharmaceutical composition wherein the disease is preferably cancer or tumor; more preferably EpCAM-positive cancer or malignant tumor, more preferably selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neurogel Glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, Liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, Cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma,
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, Mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histiocytes, and lymphoplasmacytic lymphoma, said lung cancer being selected from: non-small cell lung cancer and small cell lung cancer,
  • the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.
  • the cancer is stomach cancer, esophageal cancer, lung cancer, pancreatic cancer.
  • the aforementioned antibodies and immunoconjugates can play a therapeutic role in the aforementioned cancers with high, medium, and low expression of EpCAM.
  • EpCAM antibodies and immunoconjugates provided by the present disclosure have good affinity with cell surface antigens, good endocytosis efficiency and tumor suppression efficiency, as well as tumor cell positioning and display effects, and have a wider drug application window and good The safety is suitable for clinical diagnosis and treatment.
  • Figures 1A to 1C binding experiments of EpCAM antigens of different species and three antibodies.
  • Figure 1A is the binding experiment of antibody and human EpCAM
  • Figure 1B is the binding experiment of antibody and mouse EpCAM
  • Figure 1C is the binding experiment of antibody and monkey EpCAM.
  • Figure 2 The binding experiment of the three antibodies and human EpCAM at the cellular level.
  • Figure 2A is the cell-level binding experiment of h1905;
  • Figure 2B is the cell-level binding experiment of h1906;
  • Figure 2C is the cell-level binding experiment of h1907.
  • EpCAM (CD326) (Epithelial Cell Adhesion Molecule) is a type I transmembrane glycoprotein. As a specific cell adhesion molecule for epithelial cells, it also involves other processes, including cell migration, proliferation, and differentiation. EpCAM is one of the first tumor-associated antigens identified using monoclonal antibody technology. It is widely expressed on the surface of epithelial tissues in the form of multimers and mediates the function of calcium-independent cell-to-cell homotype adhesion, which can be classified as adhesion. Molecular family. EpCAM also has other characteristics of the adhesion molecule family, and participates in various processes including cell-matrix interaction, migration, cell differentiation, morphology, cell cycle regulation, signal transduction, and metabolism.
  • EpCAM is overexpressed in a variety of tumors of epithelial origin, suggesting that it is closely related to tumors.
  • Pathological studies have shown that EpCAM is expressed to varying degrees in adenocarcinomas, including colorectal cancer, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, hepatocellular carcinoma and retinoblastoma.
  • Tumor EpCAM positive rate Tumor EpCAM positive rate Ovarian cancer 88-100% Breast cancer 90% Stomach cancer 98% Endometrial cancer 91-96% Colon cancer 99% Lung cancer 87% Pancreatic cancer 96% Prostate cancer 98%
  • immunoconjugates are provided.
  • the immunoconjugate disclosed herein may be an antibody attached to an effector molecule, where such an antibody comprises a heavy chain and a light chain.
  • the antibody can be an antibody fragment, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, minibody, bifunctional antibody, bispecific antibody fragment, Multimer, and any combination thereof.
  • the effector molecule may be a radioisotope, an antitumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal Ions, and any combination thereof.
  • antibodies or antibody fragments described in the present disclosure can be coupled to effector molecules by any means.
  • antibodies or antibody fragments can be chemically or recombinantly attached to the toxin.
  • the chemical methods for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates.
  • the method used to couple the antibody or antibody fragment and the toxin must be able to link the antibody and toxin without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.
  • cytotoxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Including toxins, chemotherapy drugs and other compounds that can be used to kill tumor cells.
  • toxin refers to any substance that can have a harmful effect on the growth or proliferation of cells, which can be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as ixati Kang, maytansinoid and its derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria Toxins, exotoxins, ricin A chain, abrin A chain, modeccin, ⁇ -toxin (sarcin), Aleutitesfordii toxic protein, carnation (dianthin) ) Toxin, Phytolaca Americaa Toxin (PAPI, PAPII and PAP-S), Momordica Charantia Inhibitor, Jatropha curcin, Crotin, Soapweed ( sapaonaria officinalis)
  • chemotherapeutic drugs are chemical compounds that can be used to treat tumors. This definition also includes antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They can be hormones themselves.
  • chemotherapeutic drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN TM ); alkyl sulfonates such as busulfan, improsulfan, and pippo Piposulfan; aziridine such as benaodopa, carboquone, metrodopa and uredopa; aziridine and methylamelamine include Altretamine, triethylenemelamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil , Chlorophosphamide, cholophosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride; L-phenylalanine mustard (melphalan), new Nitrogen mustard (novembichin), cholesterol phenylacetate mustard, prednimustine (prednimustine), trifosf
  • anti-hormonal agents that can regulate or inhibit the effect of hormones on tumors.
  • anti-estrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxy tamoxifen, trioxifene (trioxifene), keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgenic agents such as flutamide, nilutamide ( nilutamide), bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the antibody and toxin are both proteins and can be coupled using techniques well known in the art.
  • cross-linking agents are generally selected based on reactive functional groups available or inserted on the antibody or toxin.
  • a photoactivatable crosslinker can be used. In some cases, it may be necessary to include a spacer between the antibody and the toxin.
  • Crosslinking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipamide and bis(diazo benzidine), and heterobifunctional agents: metamaleimide Benzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.
  • Crosslinking agents that can be used to couple effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine hydrazide) and TMPPH (S-(2-thiopyridyl) ) Mercapto-propionyl hydrazide).
  • TPCH and TMPPH react on the carbohydrate portion of glycoproteins that have previously been oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide portion of the crosslinker and the aldehyde produced by periodate.
  • Heterobifunctional crosslinking agent GMBS N-( ⁇ -maleimidobutyryloxy)-succinimide
  • SMCC succinimidyl 4-(N-maleimido- (Methyl) cyclohexane) reacts with primary amine, thereby introducing maleimide groups onto the component. This maleimide group can then react with the sulfhydryl group on another component that can be introduced by the crosslinking agent, thereby forming a stable thioether bond between the components.
  • a cross-linking agent can be used to introduce a long spacer between the components, such as 3-(2-pyridyldithio)propionic acid n -Succinimidyl ester (SPDP). Therefore, there are many suitable cross-linking agents that can be used and each is selected depending on its effect on the optimal immunoconjugate yield.
  • SPDP 3-(2-pyridyldithio)propionic acid n -Succinimidyl ester
  • antibody refers to immunoglobulin.
  • a full-length antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • antibodies of the present disclosure also include antigen-binding fragments capable of binding antigen.
  • the antibodies of the present disclosure include human antibodies.
  • murine antibody in the present disclosure is a monoclonal antibody against human EpCAM prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with EpCAM antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-EpCAM antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine ⁇ , ⁇ chain or a variant thereof, or further comprise murine IgG1, The heavy chain constant region of IgG2, IgG3, or variants thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the EpCAM chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the EpCAM chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising the human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations (E.g. L234A and/or L235A mutation, and/or S228P mutation) IgG1, IgG2 or IgG4 variants.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the variable region framework of human antibodies, that is, different types of human germline antibodies The antibody produced in the framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody carrying a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast to undergo affinity maturation mutations to CDRs.
  • human antibody and “human antibody” are used interchangeably and refer to one or more variable and constant regions derived from human immunoglobulin sequences.
  • One of the preferred methods is that all variable regions and constant regions are derived from human immunoglobulin sequences, that is, “fully human antibodies” or “fully human antibodies”.
  • These antibodies can be prepared in a variety of ways, including phage display technology to isolate B cells from human PBMC, spleen, and lymph node tissues to construct a library of natural single-chain phage human antibodies, or to immunize transgenic mice that can express human antibody light and heavy chains , Screen the antibodies obtained.
  • the human antibodies of the present disclosure also include antibodies that are obtained by mutation of one or more amino acids on the basis of human antibodies and still bind to the target antigen.
  • the grafting of the CDR may result in the weakened affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody.
  • the amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that are different from the germline can be considered related.
  • the sequence can be compared with the consensus sequence of a subclass or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of hypermutation of somatic cells and thus play an important role in binding.
  • the "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to the heavy chain constant region that has been disclosed in the prior art and does not change the structure and function of the antibody variable region. Or variants of the constant region of the light chain. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions. The specific substitutions are YTE known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to an antibody in a substantially intact form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to antibodies whose light and heavy chains contain constant regions.
  • antibody of the present disclosure includes “full-length antibodies” and antigen-binding fragments thereof.
  • the full-length antibody of the present disclosure includes a full-length antibody formed by connecting a light chain variable region and a light chain constant region and a heavy chain variable region and a heavy chain constant region.
  • a full-length antibody formed by connecting a light chain variable region and a light chain constant region and a heavy chain variable region and a heavy chain constant region.
  • antigen-binding fragment or “functional fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, EpCAM). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • EpCAM an antigen
  • binding fragments included in the term "antigen-binding fragment” include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, including through hinge region The bivalent fragment of the two Fab fragments connected by the disulfide bond, (iii) the Fd fragment composed of VH and CH1 domains; (iv) the Fv fragment composed of the VH and VL domains of one arm of an antibody; ( v) dsFv, a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; (vi) diabodies, bispecific antibodies and multispecific antibodies containing fragments such as scFv, dsFv, and Fab.
  • single chain Fv single chain Fv
  • scFv single chain Fv
  • Such single chain antibodies are also included in the term "antigen-binding fragment" of antibodies.
  • the antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulin.
  • the antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment that has a molecular weight of about 50,000 and has antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein the H chain is N-terminal About half of the side and the entire L chain are joined together by disulfide bonds.
  • F(ab')2 is obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen binding activity and contains two Fab regions connected at the hinge position. Antibody fragments.
  • Fab' is an antibody fragment obtained by cleaving the disulfide bond of the hinge region of F(ab')2 and having a molecular weight of about 50,000 and having antigen-binding activity.
  • the Fab' of the present disclosure can be produced by treating the F(ab')2 of the present disclosure that specifically recognizes EpCAM and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • a suitable prior art linker consists of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • Diabodies are antibody fragments in which scFv or Fab are dimerized, and are antibody fragments with bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • Bispecific antibodies and multispecific antibodies refer to antibodies that can bind to two or more antigens or epitopes at the same time, including scFv or Fab fragments that can bind to EpCAM.
  • the diabody of the present disclosure can be produced by the following steps: Obtain the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human EpCAM and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the scFv encoding cDNA. DNA such that the amino acid sequence length of the peptide linker is 8 residues or less, insert the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introduce the expression vector into a prokaryotic or eukaryotic organism Medium to express diabodies.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7, 697 (1994)) based on the prediction of the three-dimensional structure of the antibody.
  • the full-length antibody or antigen-binding fragment of the present disclosure can be produced by the following steps: obtaining the cDNA encoding the antibody of the present disclosure that specifically recognizes human EpCAM and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs DNA encoding dsFv , Inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or eukaryotic organism to express dsFv.
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one, two, three or one mutation on the basis of the original protein or polypeptide. Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR in the variable domain of the heavy chain (VH) The amino acid residue numbers are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acids in VL
  • the residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDR is defined by the amino acid residues 26-35 (HCDR1) in human VH. ), 50-65 (HCDR2) and 95-102 (HCDR3) and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • HCDR1 amino acid residues 26-35
  • HCDR2 amino acid residues 26-65
  • HCDR3 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align. Unless otherwise specified, the antibody variable regions and CDR sequences involved in the examples of this disclosure are applicable to "Kabat "Numbering rules.
  • epitopes refers to a site on an antigen where an immunoglobulin or antibody specifically binds (for example, a specific site on an EpCAM molecule).
  • Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10-8M, for example, about less than 10-9M, 10-10M, 10-11M, 10-12M or less.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibody of the present disclosure binds to EpCAM with a dissociation equilibrium constant (KD) of less than about 10-7M, for example, less than about 10-8M or 10-9M.
  • KD dissociation equilibrium constant
  • the affinity of the antibody to the cell surface antigen in the present disclosure uses the FACS method Determine the KD value.
  • the term “competition” in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be detected for example, an antibody or immunologically functional fragment
  • prevents or inhibits for example, reduces
  • a reference antigen-binding protein for example, a ligand or a reference antibody
  • a common antigen for example, EpCAM antigen or Its fragment
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J.Immunol.137:3614-3619
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); solid-phase direct labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec.
  • the assay involves the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess.
  • the antigen binding proteins identified by the competition assay include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the Examples herein.
  • the competing antigen binding protein is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule can be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • amino acid sequence identity refers to aligning amino acid sequences and introducing gaps when necessary to achieve the maximum percentage of sequence identity, and does not consider any conservative substitutions as part of sequence identity, in the first sequence and in the second sequence The percentage of amino acid residues that are identical.
  • the alignment can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequence being compared.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of the host cell after being introduced into the host cell, thereby The host genome replicates together (e.g., non-episomal mammalian vectors).
  • mice can be immunized with human EpCAM or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • Human FR germline sequence can be obtained from ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the family Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae).
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line), 293 cells and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human EpCAM. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224 (4th edition)).
  • substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in the table "Exemplary Amino Acid Conservative Substitutions" below.
  • Effective amount refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the effectiveness of other agents required to treat the disorder
  • the dosage enhances the efficacy of another agent, and/or delays the progression of the patient’s target antigen-related disorder of the present disclosure.
  • Exogenous refers to substances produced outside organisms, cells, or humans according to circumstances.
  • Endogenous refers to substances produced in cells, organisms, or human bodies according to circumstances.
  • PCR polymerase chain reaction
  • oligonucleotide primers it is necessary to obtain sequence information from the end or outside of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified.
  • the 5'terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA.
  • PCR used herein is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acid.
  • isolated refers to a purified state, and in this case means that the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers or salts unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredients and thus the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • Suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffer, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes agents for treating diseases related to target antigen (for example, EpCAM) positive cells, the agents comprising the anti-EpCAM antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • target antigen for example, EpCAM
  • the diseases related to EpCAM in the present disclosure are not limited, as long as it is a disease related to EpCAM.
  • the therapeutic response induced by the molecules of the present disclosure can be achieved by binding to human EpCAM and then inhibiting the binding of EpCAM to its ligand, or killing Tumor cells expressing EpCAM. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of the present disclosure are very useful for people who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, Colorectal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or determination of target antigens (for example, EpCAM), reagents for immunodetection or determination of target antigens (for example, EpCAM), and cells for immunodetection or determination of target antigens (for example, EpCAM)
  • target antigens for example, EpCAM
  • reagents for immunodetection or determination of target antigens for example, EpCAM
  • cells for immunodetection or determination of target antigens for example, EpCAM
  • the method and the diagnostic agent for diagnosing diseases related to target antigen (for example, EpCAM) positive cells which comprises the disclosed specific recognition target antigen (for example, human EpCAM) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure
  • the antibody or antibody fragment as the active ingredient.
  • the method for detecting or measuring the amount of the target antigen may be any known method.
  • it includes immunodetection or assay methods.
  • the immunoassay or measurement method is a method of detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, western blotting, physicochemical method Wait.
  • the above-mentioned diseases related to EpCAM-positive cells can be diagnosed by detecting or measuring EpCAM-expressing cells using the antibodies or antibody fragments of the present disclosure.
  • the living body sample used to detect or measure the target antigen eg EpCAM
  • the target antigen eg EpCAM
  • cells expressing the target antigen such as tissue cells, blood, plasma , Serum, pancreatic juice, urine, stool, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • the reagents used to perform the antigen-antibody reaction include buffers, salts and the like.
  • the reagents used for detection include reagents commonly used in immunological detection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label.
  • EpCAM human EpCAM shown in SEQ ID NO: 1 as the template of the EpCAM of the present disclosure
  • amino acid sequences of the antigens and detection proteins involved in the present disclosure are designed.
  • the following EpCAM antigens without special instructions refer to human EpCAM.
  • EpCAM Human EpCAM full-length protein: EpCAM (NP_002345.2, SEQ ID NO: 1):
  • Human EpCAM antigen for screening and detection (huEpCAM, SEQ ID NO: 3), the sequence is as follows:
  • Murine EpCAM antigen for screening and detection (mEpCAM, SEQ ID NO: 4), the sequence is as follows:
  • CynoEpCAM antigen for screening and detection (cynoEpCAM, SEQ ID NO: 29)
  • PBMC PBMC
  • spleen lymph node tissue
  • RNA extract RNA to construct a natural single-stranded phage antibody library (capacity 3.2 ⁇ 10 10 ).
  • the constructed natural single-chain phage antibody library is packaged to form phage particles, it is panned by the liquid phase method, the phage is combined with the biotinylated EpCAM in the liquid phase, and then separated by streptavidin magnetic beads.
  • biotinylated human EpCAM was used for panning, and 500 monoclonal colonies were picked and packaged into phage single-chain antibody for phage ELISA test.
  • primer PCR was designed to construct the VH/VK/VL gene fragment of each single-chain antibody sequence.
  • the heavy and light chain variable regions of h1905, h1906 and h1907 were obtained.
  • CDR sequences in the light and heavy chains of each antibody are shown in Table 1.
  • the antibody variable region and the constant region gene (CH1-FC/CL) fragment were homologously recombined to construct a complete antibody VH-CH1-FC/VL-CL.
  • h1906 heavy chain (SEQ ID NO: 25)
  • Plasmids expressing antibody light chain and heavy chain were transfected into HEK293E cells at a ratio of 1.5:1. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. Elute the target protein with pH3.0-pH3.5 acidic eluent, and neutralize with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample is properly concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE) to remove aggregates, collect monomer peaks, and divide them for use.
  • PBS-balanced gel chromatography Superdex200 GE
  • test methods are used to verify the performance and beneficial effects of the antibody of the present invention.
  • Test Example 1 ELISA binding experiment
  • the ELISA experiment was used to detect the binding properties of epithelial cell adhesion molecule antibodies and epithelial cell adhesion molecule proteins.
  • PBST buffer pH 7.4PBS containing 0.05% Tween-20
  • sample diluent pH7.4PBS containing 1% skimmed milk
  • HRP-labeled goat anti-human secondary antibody Jackson Immuno Research, 109-035-003
  • Test case 2 FACS-based combination test
  • the cells MCF-7 (ATCC) and A431 (ATCC) expressing epithelial cell adhesion molecules were prepared into 1 ⁇ with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)) cell suspension 10 6 / ml in, 100 ⁇ l / well of 96-well round bottom plate (Corning, 3795) in. After centrifugation to remove the supernatant, add 50 ⁇ l/well of different concentrations of epithelial cell adhesion molecule antibodies diluted with FACS buffer, and incubate for 1 hour in a refrigerator at 4°C in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)

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Abstract

提供了一种抗EpCAM抗体及其抗原结合片段，以及其作为抗癌药物的用途，尤其在制备用于治疗EpCAM阳性疾病或病症的药物中的用途。

Description

抗EpCAM抗体及其应用 技术领域
本公开涉及抗体药物领域。具体地本公开涉及抗EpCAM抗体及其应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
EpCAM(CD326)是I型跨膜糖蛋白，作为上皮细胞的特异性细胞粘附分子。它还涉及到其他一些过程，包括细胞迁移，增殖，分化等。EpCAM是最早应用单克隆抗体技术鉴定出的肿瘤相关抗原之一，它以多聚体的形式广泛表达于上皮组织表面，介导钙非依赖性细胞间同型黏附功能，据此可归入粘附分子家族。EpCAM还具备粘附分子家族的其他特性，参与包括细胞与基质的相互作用、迁移、细胞分化、形态、细胞周期调节、信号传导、代谢等多种过程。同时，EpCAM在多种上皮来源的肿瘤中呈过表达，提示其与肿瘤密切相关。病理研究发现，EpCAM不同程度的表达于腺癌中，包括结直肠癌、胃腺癌、乳腺癌、卵巢癌、肺腺癌、前列腺癌、胰腺癌以及肝细胞癌和视网膜母细胞瘤。多项研究已证实EpCAM的表达与乳腺癌和结肠癌细胞的增殖、周期分布、转移有关。单特异性抗EpCAM单克隆抗体(MAB)，例如单抗17-1A(glaxowellcome，Centocor)，被批准用于结直肠癌的辅助治疗。本公开提供了一种亲和力好，并适用于诊断和治疗的抗EpCAM抗体。
发明内容
本公开提供一种抗EpCAM抗体。
在一些实施方案中，如前所述抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
i)所述重链可变区包含与如SEQ ID NO：5所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3；或
ii)所述重链可变区包含与如SEQ ID NO：9所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：10所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前所述抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
iii)所述重链可变区包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
iv)所述重链可变区包含分别如SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前所述抗EpCAM抗体，其中所述抗EpCAM抗体是人抗体。
在一些实施方案中，如前所述抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
(v)所述重链可变区与具有SEQ ID NO：5中所示氨基酸序列的重链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性，和所述轻链可变区与具有SEQ ID NO：6中所示氨基酸序列的轻链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性；
(vi)所述重链可变区与具有SEQ ID NO：7所示氨基酸序列的重链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性，和所述轻链可变区与具有SEQ ID NO：8所示氨基酸序列的轻链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性；或
(vii)所述重链可变区与具有SEQ ID NO：9所示氨基酸序列的重链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性，和所述轻链可变区与具有SEQ ID NO：10所示氨基酸序列的轻链可变区有至少90％、92％、94％、95％、96％、97％、98％、99％同一性。
在一些实施方案中，如前所述抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
(v-1)所述重链可变区，其氨基酸序列与SEQ ID NO：5具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：6具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性；
(vi-1)所述重链可变区，其氨基酸序列与SEQ ID NO：7具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：8具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性；或
(vii-1)所述重链可变区，其氨基酸序列与SEQ ID NO：9具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：10具有至少90％、92％、94％、95％、96％、97％、98％、99％的序列同一性。在一些实施方案中，如前所述抗EpCAM抗体，其中：
(viii)所述重链可变区序列如SEQ ID NO：5所示，和所述轻链可变区序列如SEQ ID NO：6所示；
(ix)所述重链可变区序列如SEQ ID NO：7所示，和所述轻链可变区序列如 SEQ ID NO：8所示；或
(x)所述重链可变区序列如SEQ ID NO：9所示，和所述轻链可变区序列如SEQ ID NO：10所示。
在一些实施方案中，如前所述抗EpCAM抗体，所述重链可变区的CDR和轻链可变区的CDR是采用Kabat编号规则定义的。
在一些实施方案中，如前所述抗EpCAM抗体，其中所述抗体进一步包含抗体恒定区；优选地，所述抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；最优选地，所述抗体包含：与具有SEQ ID NO：23所示的重链具有至少90％、92％、94％、95％、96％、97％、98％、99％同一性的重链，和与具有SEQ ID NO：24所示的轻链有至少90％、92％、94％、95％、96％、97％、98％、99％序列同一性的轻链；或
与具有SEQ ID NO：25所示的重链具有至少90％、92％、94％、95％、96％、97％、98％、99％同一性的重链，和/或与具有SEQ ID NO：26所示的轻链有至少90％、92％、94％、95％、96％、97％、98％、99％序列同一性的轻链；或
与具有SEQ ID NO：27所示的重链具有至少90％、92％、94％、95％、96％、97％、98％、99％同一性的重链，和/或与具有SEQ ID NO：28所示的轻链有至少90％、92％、94％、95％、96％、97％、98％、99％序列同一性的轻链。
在一些实施方案中，如前所述抗EpCAM抗体，其中所述抗EpCAM抗体包含：
(d)如SEQ ID NO：23所示的重链和如SEQ ID NO：24所示的轻链；
(e)如SEQ ID NO：25所示的重链和如SEQ ID NO：26所示的轻链；或
(f)如SEQ ID NO：27所示的重链和如SEQ ID NO：28所示的轻链。
在一些实施方案中，如前所述抗EpCAM抗体，所述抗体与前述的抗EpCAM抗体或其抗原结合片段竞争性结合人EpCAM。
在一些实施方案中，如前所述抗EpCAM抗体，所述抗EpCAM抗体是全长抗体或抗原结合片段，所述抗原结合片段选自Fab、Fab'、F(ab')2、scFv、dsFv、ds-scFv、二聚体、微型抗体、双功能抗体、双特异性抗体片段、多聚体，以及其任何组合。
本公开的另一方面还提供一种核酸分子，其编码如前所述的抗EpCAM抗体。
本公开的另一方面还提供一种表达载体，其包含如前所述的核酸分子。
本公开的另一方面还提供一种宿主细胞，其包含如前所述的核酸分子或如前所述的表达载体，优选所述细胞为细菌细胞、真菌细胞、昆虫动物细胞或哺乳动物细胞。
本公开的另一方面还提供一种免疫偶联物，其为由根据如前所述的抗EpCAM抗体与效应分子偶联形成的免疫偶联物；所述效应分子选自放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
在一些实施方案中，本公开提供一种如前所述的EpCAM抗体的制备方法。
在一些实施方案中，本公开提供一种如前所述的EpCAM免疫偶联物的制备方法。
在一些实施方案中，本公开提供一种药物组合物，其含有治疗有效量的根据如前所述的抗EpCAM抗体，或根据如前所述的核酸分子，或如前所述的免疫偶联物，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
在一些实施方案中，本公开提供一种用于免疫检测或测定EpCAM的方法，所述方法包括使用如前所述的抗EpCAM抗体或免疫偶联物的步骤。示例性的，所述方法是在体内或体外进行的。示例性的，所述步骤包括在允许如前所述的抗EpCAM抗体或免疫偶联物和EpCAM之间形成复合物的条件下，将待测样品与如前所述的抗EpCAM抗体或免疫偶联物接触。
在一些实施方案中，本公开提供如前所述的抗EpCAM抗体或免疫偶联物在制备免疫检测人EpCAM的试剂中的用途。
在一些实施方案中，本公开提供一种用于免疫检测或测定EpCAM的如前所述的抗EpCAM抗体或免疫偶联物。
在一些实施方案中，本公开提供一种试剂盒，其包含根据如前所述的抗EpCAM抗体或免疫偶联物。
在一些实施方案中，本公开提供一种诊断与EpCAM相关的疾病的方法，所述方法包括使用如前所述的抗EpCAM抗体、如前所述的免疫偶联物或如前所述的试剂盒的步骤。示例性的，所述方法是在体内或体外进行的。示例性的，所述步骤包括在允许如前所述的抗EpCAM抗体或免疫偶联物和EpCAM之间形成复合物的条件下，将待测样品与如前所述的抗EpCAM抗体或免疫偶联物接触。
在一些实施方案中，本公开提供如前所述的抗EpCAM抗体，或如前所述的核酸分子，或如前所述的免疫偶联物或如前所述的药物组合物在制备用于治疗癌症或肿瘤的药物中的用途，其中所述癌症或肿瘤优选为EpCAM阳性癌症或恶性肿瘤，更优选为头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
在一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞 性淋巴瘤；所述肺癌选自：非小细胞肺癌和小细胞肺癌；所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，本公开提供一种治疗与EpCAM相关的疾病的方法，所述方法包括向受试者施用治疗有效量的如前所述的抗EpCAM抗体，或如前所述的核酸分子，或如前所述的免疫偶联物或如前所述的药物组合物，其中所述疾病优选为癌症或肿瘤；更优选为EpCAM阳性癌症或恶性肿瘤，更优选选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌。更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg或1-1000mg如前所述的抗EpCAM抗体或如前所述的免疫偶联物。
在一些实施方案中，本公开提供一种用于治疗与EpCAM相关的疾病如前所述的抗EpCAM抗体，或如前所述的核酸分子，或如前所述的免疫偶联物或如前所述的药物组合物，其中所述疾病优选为癌症或肿瘤；更优选的EpCAM阳性癌症或恶性肿瘤，更优选选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌。更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺 癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，所述癌症为胃癌、食道癌、肺癌、胰腺癌。
在一些实施方案中，如前所述抗体、免疫偶联物可以在EpCAM高表达、中表达、低表达的如前所述癌症中发挥治疗作用。
本公开提供的EpCAM抗体及免疫偶联物具有与细胞表面抗原良好的亲和力，良好的细胞内吞效率和肿瘤抑制效率，以及肿瘤细胞的定位和显示作用，并且具有更宽的药物应用窗口和良好的安全性，适于临床诊断和治疗的药物应用。
附图说明
图1A至图1C：不同物种EpCAM抗原与三种抗体的结合实验。其中图1A是抗体与人EpCAM的结合实验；图1B是抗体与小鼠EpCAM结合实验；图1C是抗体与猴EpCAM结合实验。
图2：三种抗体与人EpCAM在细胞水平的结合实验。图2A是h1905的细胞水平结合实验；图2B是h1906的细胞水平结合实验；图2C是h1907的细胞水平结合实验。
发明详述
术语(定义)
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
EpCAM(CD326)(上皮细胞粘附分子)，是I型跨膜糖蛋白，作为上皮细胞的特异性细胞粘附分子，它还涉及到其他一些过程，包括细胞迁移，增殖，分化等。EpCAM是最早应用单克隆抗体技术鉴定出的肿瘤相关抗原之一，它以多聚体的形式广泛表达于上皮组织表面，介导钙非依赖性细胞间同型黏附功能,据此可归入粘附分子家族。EpCAM还具备粘附分子家族的其他特性，参与包括细胞与基质的相互作用、迁移、细胞分化、形态、细胞周期调节、信号传导、代谢等多种过程。同时，EpCAM在多种上皮来源的肿瘤中呈过表达，提示其与肿瘤密切相关。病理研究显示，EpCAM不同程度的表达于腺癌中，包括结直肠癌、胃腺癌、乳腺癌、卵巢癌、肺腺癌、前列腺癌、胰腺癌以及肝细胞癌和视网膜母细胞瘤。
EpCAM广泛的肿瘤分布：
肿瘤 EpCAM阳性率 肿瘤 EpCAM阳性率
卵巢癌 88-100％ 乳腺癌 90％
胃癌 98％ 子宫内膜癌 91-96％
结肠癌 99％ 肺癌 87％
胰腺癌 96％ 前列腺癌 98％
免疫偶联物
在一些实施方案中，提供了免疫偶联物。在一些实施方案中，本文所公开的免疫偶联物可以是附接至效应分子的抗体，其中这种抗体包含重链和轻链。在一些实施方案中，抗体可以是抗体片段，诸如Fab、Fab'、F(ab')2、scFv、dsFv、ds-scFv、二聚体、微型抗体、双功能抗体、双特异性抗体片段、多聚体，以及其任何组合。
在本文所描述的实施方案中，效应分子可以是放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
本公开所描述的抗体或抗体片段可以通过任何方式偶联至效应分子。举例来说，抗体或抗体片段可以通过化学或重组方式附接至毒素。制备融合物或偶联物的化学方式在本领域中是已知的并且可以用于制备免疫偶联物。用于偶联抗体或抗体片段和毒素的方法必须能够连接抗体与毒素而不会干扰抗体或抗体片段结合至标靶分子的能力。
术语“细胞毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。包括毒素、化疗药物等能用于肿瘤细胞杀伤的化合物。
术语“毒素”是指能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“化疗药物”是可用于治疗肿瘤的化学化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXAN TM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)， 卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和methylamelamine包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin,洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin,嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine),依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone),丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如frolinic acid；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；elfomithine；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
Figure PCTCN2020085838-appb-000001
雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺(spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；哌溴烷坑(pipobroman)；gacytosine；阿拉伯糖苷("Ara-C")；环磷酰胺； 三胺硫磷(thiotepa)；紫杉烷，如紫杉醇(
Figure PCTCN2020085838-appb-000002
Bristol-Myers Squibb Oncology,Princeton,NJ)和docetaxel(
Figure PCTCN2020085838-appb-000003
Rhone-Poulenc Rorer,Antony,France)；苯丁酸氮芥；吉西他滨(gemcitabine)；6-硫代鸟嘌呤；巯基嘌呤；氨甲蝶呤；铂类似物如顺铂和卡铂；长春花碱；铂；依托泊甙(etoposide)(VP-16)；异环磷航胶；丝裂霉素C；米托蒽醌；长春新碱；长春瑞宾(vinorelbine)；新霉酰胺(navelbine)；novantrone；替尼泊甙(teniposide)；柔红霉素；氨基蝶呤；xeloda；伊拜磷酸盐(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；维甲酸esperamicins；capecitabine；以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene),keoxifene，LY117018,onapristone，和托瑞米芬(Fareston)；和抗雄激素制剂如氟他氨(flutamide)，尼鲁米特(nilutamide)，bicalutamide，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)；和上述任何物质的可药用盐，酸或衍生物。
在一个实施方案中，抗体和毒素都是蛋白质并且可以使用本领域中熟知的技术偶联。存在本领域中所公开的数百种交联剂，其可以偶联两种蛋白质。交联剂一般基于在抗体或毒素上可用或插入的反应官能团来选择。另外，如果不存在反应基团，那么可以使用光可活化交联剂。在某些情况下，可能需要在抗体与毒素之间包括间隔子。本领域中已知的交联剂包括同型双功能剂：戊二醛、二甲基己二亚酰胺化物和双(重氮基联苯胺)，以及异型双功能剂：间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺和磺基-间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺。
可以用于使效应分子偶合至抗体片段的交联剂包括例如TPCH(S-(2-硫代吡啶基)-L-半胱氨酸酰肼)和TPMPH(S-(2-硫代吡啶基)巯基-丙酰肼)。TPCH和TPMPH在先前已经通过温和高碘酸盐处理而氧化的糖蛋白的碳水化合物部分反应，由此在交联剂的酰肼部分与高碘酸盐产生的醛之间形成腙键。异型双功能交联剂GMBS(N-(γ-马来酰亚胺基丁酰氧基)-琥珀酰亚胺)和SMCC(琥珀酰亚胺基4-(N-马来酰亚胺基-甲基)环己烷)与伯胺反应，由此将马来酰亚胺基引入组分上。这个马来酰亚胺基随后可以与另一种组分上的可以由交联剂引入的硫氢基反应，由此在组分之间形成稳定的硫醚键。如果组分之间的位阻干扰任一种组分的活性，那么可以使用交联剂，将长的间隔臂引入组分之间，诸如3-(2-吡啶基二硫基)丙酸n-琥珀酰亚胺酯(SPDP)。因此，存在许多适合的交联剂，其可以被使用并且各自取决于其对最佳免疫偶联物产量的影响来选择。
本公开所述的“抗体”指免疫球蛋白，全长抗体是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为 μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开“抗体”除包括全长抗体外，还包括能结合抗原的抗原结合片段。
本公开的抗体包括人抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人EpCAM的单克隆抗体。制备时用EpCAM抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源抗EpCAM抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的EpCAM嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的EpCAM嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区 框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
术语“人抗体”与“人源抗体”可以互换使用，是指有一或多个可变区和恒定区来源于人免疫球蛋白序列。其中一个优选的方式是所有的可变区和恒定区均来自于人免疫球蛋白序列，即“完全人源抗体”或“全人抗体”。这些抗体可以通过多种方式制备获得，包括通过噬菌体展示技术，从人PBMC、脾脏、淋巴结组织分离B细胞，构建天然单链噬菌体人抗体库，或者通过免疫可表达人抗体轻重链的转基因小鼠，筛选获得的抗体。本公开的人抗体还包括在人抗体基础上经过一个或更多个氨基酸的突变获得的仍结合目标抗原的抗体。
由于抗原的接触残基，CDR的移植可由于与抗原接触的构架残基而导致产生的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以可能是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚类共有序列或具有高相似性百分数的动物抗体序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
本公开中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指轻链和重链包含恒定区的抗体。本公开“抗体”包含“全长抗体”及其抗原结合片段。
在一些实施方案中，本公开的全长抗体包括由轻链可变区与轻链恒定区连接和重链可变区与重链恒定区连接后所形成的全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区和重链恒定区。
术语“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，EpCAM)的能力的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含通过铰链区上的二硫键连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组 成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50，000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100，000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50，000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别EpCAM并与胞外区的氨基酸序列或其三维结构结合的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
双抗体是其中scFv或Fab被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
双特异性抗体和多特异性抗体是指能同时结合两个或多个抗原或抗原决定簇 的抗体，其中包含能结合EpCAM的scFv或Fab片段。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人EpCAM并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering，7，697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的全长抗体或抗原结合片段可以通过以下步骤来生产：获得本公开的特异性识别人EpCAM并与胞外区的氨基酸序列或其三维结构结合的抗体的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基 24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。除非另有说明，本公开实施例涉及的抗体可变区和CDR序列均适用“Kabat”编号规则。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，EpCAM分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-8M，例如大约小于10-9M、10-10M、10-11M、10-12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10-7M，例如小于大约10-8M或10-9M的解离平衡常数(KD)结合EpCAM，例如，在本公开中抗体与细胞表面抗原的亲和力采用FACS法测定KD值。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如EpCAM抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白， 所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人EpCAM或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细 胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人EpCAM特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”，在其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”，在其应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224 页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来 说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本公开包括用于治疗与目标抗原(例如EpCAM)阳性细胞相关的疾病的药剂，所述药剂包含本公开的抗EpCAM抗体或其抗原结合片段作为活性成分。
本公开中与EpCAM相关的疾病没有限制，只要它是与EpCAM相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类EpCAM，然后阻遏EpCAM与其配体的结合，或杀伤过表达EpCAM的肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本公开涉及用于免疫检测或测定目标抗原(例如EpCAM)的方法、用于免疫检测或测定目标抗原(例如EpCAM)的试剂、用于免疫检测或测定表达目标抗原(例如EpCAM)的细胞的方法和用于诊断与目标抗原(例如EpCAM)阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别目标抗原(例如人 EpCAM)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定目标抗原(例如EpCAM)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与EpCAM阳性细胞相关的疾病可以通过用本公开的抗体或抗体片段检测或测定表达EpCAM的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原(例如EpCAM)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如EpCAM)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本公开一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本发明，但是以下描述优选的方法和材料。通过说明书和权利要求书，本发明的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本发明所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本发明的优选实施方式。这些实施例不应以任何方式理解为限制本发明的范围，本发明的范围由权利要求书限定。
具体实施方式
实施例
实施例1.EpCAM抗原及检测用蛋白的制备
EpCAM抗原设计
以SEQ ID NO：1所示人EpCAM作为本公开EpCAM的模板，设计本公开涉 及的抗原及检测用蛋白的氨基酸序列。以下EpCAM抗原未特殊说明的均指人EpCAM。
人EpCAM全长蛋白：EpCAM(NP_002345.2，SEQ ID NO：1)：
Figure PCTCN2020085838-appb-000004
鼠EpCAM全长蛋白氨基酸序列(NP_032558.2，SEQ ID NO：2)
Figure PCTCN2020085838-appb-000005
筛选及检测用人EpCAM抗原(huEpCAM，SEQ ID NO：3)，序列如下：
Figure PCTCN2020085838-appb-000006
注释：斜体部分为His-tag标记。
筛选及检测用鼠EpCAM抗原(mEpCAM，SEQ ID NO：4)，序列如下：
Figure PCTCN2020085838-appb-000007
筛选及检测用食蟹猴EpCAM抗原(cynoEpCAM，SEQ ID NO：29)
Figure PCTCN2020085838-appb-000008
Figure PCTCN2020085838-appb-000009
实施例2.特异性结合人EpCAM的阳性序列的筛选
利用人PBMC、脾脏、淋巴结组织分离B细胞，并提取RNA，构建天然单链噬菌体抗体库(库容3.2×10 10)。将构建的天然单链噬菌体抗体文库经过包装形成噬菌体颗粒后，采用液相法进行淘筛，噬菌体与生物素化的EpCAM液相结合，再采用链霉亲和素磁珠分离。为了获得与人EpCAM结合的阳性序列，采用生物素化的人EpCAM进行淘筛，挑取500个单克隆菌落包装成噬菌体单链抗体，用于噬菌体ELISA测试。分别测试单克隆噬菌体与人EpCAM和鼠EpCAM的结合活性：ELISA板上分别包被1μg/ml人EpCAM或鼠EpCAM以及1％BSA，加入1:1封闭缓冲液稀释的噬菌体上清，最后用抗M13HRP检测；检测OD450值，筛选结合力强的分子，得到多个特异性序列。
实施例3.完整单克隆抗体的构建
噬菌体库筛选得到的多个特异性序列构建完整抗体后通过ELISA结合实验及多项实验确定其中3个性能优异的抗体，分别是h1905、h1906和h1907。对其完整单克隆抗体构建的过程如下：
根据测序得到的单链抗体序列，设计引物PCR搭建各单链抗体序列的VH/VK/VL基因片段。获得h1905、h1906和h1907的重轻链可变区。
>h1905重链可变区(SEQ ID NO：5)
Figure PCTCN2020085838-appb-000010
>h1905轻链可变区(SEQ ID NO：6)
Figure PCTCN2020085838-appb-000011
>h1906重链可变区(SEQ ID NO：7)
Figure PCTCN2020085838-appb-000012
>h1906轻链可变区(SEQ ID NO：8)
Figure PCTCN2020085838-appb-000013
>h1907重链可变区(SEQ ID NO：9)
Figure PCTCN2020085838-appb-000014
Figure PCTCN2020085838-appb-000015
>h1907轻链可变区(SEQ ID NO：10)
Figure PCTCN2020085838-appb-000016
其中各抗体轻重链中CDR序列(根据Kabat规则确定的CDR)如表1所示。
表1 各重链及轻链CDR序列
Figure PCTCN2020085838-appb-000017
抗体可变区再与恒定区基因(CH1-FC/CL)片段进行同源重组，构建完整抗体VH-CH1-FC/VL-CL。
构建的完整抗体h1905、h1906和h1907序列如下：
全人抗体h1905：
h1905重链：(SEQ ID NO：23)
Figure PCTCN2020085838-appb-000018
Figure PCTCN2020085838-appb-000019
h1905轻链(SEQ ID NO：24)
Figure PCTCN2020085838-appb-000020
全人抗体h1906
h1906重链：(SEQ ID NO：25)
Figure PCTCN2020085838-appb-000021
h1906轻链：(SEQ ID NO：26)
Figure PCTCN2020085838-appb-000022
全人抗体h1907
h1907重链：(SEQ ID NO：27)
Figure PCTCN2020085838-appb-000023
h1907轻链：(SEQ ID NO：28)
Figure PCTCN2020085838-appb-000024
实施例4.全人抗体的表达与纯化
分别表达抗体轻链和重链的质粒以1.5:1的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH 8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
以下用测试方法验证本发明抗体性能及有益效果。
测试例1：ELISA结合实验
ELISA实验被用来检测上皮细胞粘附分子抗体和上皮细胞粘附分子蛋白的结合特性。将重组上皮细胞粘附分子蛋白按100ng/孔加入96孔高结合板(Corning，3590)中，4℃过夜结合后，用PBST缓冲液(pH 7.4PBS含0.05％Tween-20)洗板3次后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入50μl/孔用样品稀释液(pH7.4PBS含1％脱脂奶)稀释的不同浓度待测抗体，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research,109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL,52-00-03)，于室温孵育10-15min，加入50μl/孔1M H 2SO 4终止反应，用MD VersaMaxTM酶标仪在450nm处读取吸收值，计算上皮细胞粘附分子抗体对上皮细胞粘附分子蛋白的结合EC50和Emax值。结果见图1A至图1C和表2。
表2 抗体与不同EpCAM的结合活性
Figure PCTCN2020085838-appb-000025
测试例2：基于FACS的结合试验
将表达上皮细胞粘附分子的细胞MCF-7(ATCC)、A431(ATCC)用FACS 缓冲液(2％胎牛血清(Gibco，10099141)pH7.4PBS(Sigma，P4417-100TAB))制备成1×10 6/ml的细胞悬液，100μl/孔加入96孔圆底板(Corning，3795)中。离心去除上清后加入50μl/孔用FACS缓冲液稀释的不同浓度待测上皮细胞粘附分子抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488山羊抗人IgG(H+L)(invitrogen,A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACSCantoII流式细胞仪上检测几何平均数荧光强度，计算上皮细胞粘附分子抗体对表达上皮细胞粘附分子的细胞的结合能力。
结果如图2A至图2C所示，结果显示三种抗体均与表达EpCAM的细胞具有良好的结合能力。

Claims (15)

  1. 一种抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
    i)所述重链可变区包含与如SEQ ID NO：5所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3；或
    ii)所述重链可变区包含与如SEQ ID NO：9所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：10所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
    iii)所述重链可变区包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
    iv)所述重链可变区包含分别如SEQ ID NO：17、SEQ ID NO：18和SEQ ID NO：19所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：20、SEQ ID NO：21和SEQ ID NO：22所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2所述的抗EpCAM抗体，其包含重链可变区和轻链可变区，其中：
    v)所述重链可变区，其氨基酸序列与SEQ ID NO：5具有至少90％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：6具有至少90％的序列同一性；
    vi)所述重链可变区，其氨基酸序列与SEQ ID NO：7具有至少90％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：8具有至少90％的序列同一性；或
    vii)所述重链可变区，其氨基酸序列与SEQ ID NO：9具有至少90％的序列同一性，和所述轻链可变区，其氨基酸序列与SEQ ID NO：10具有至少90％的序列同一性。
  4. 根据权利要求3所述的抗EpCAM抗体，其包含如下所示的重链可变区和轻链可变区：
    viii)所述重链可变区如SEQ ID NO：5所示，和所述轻链可变区如SEQ ID NO：6所示；
    ix)所述重链可变区如SEQ ID NO：7所示，和所述轻链可变区如SEQ ID NO：8所示；或
    x)所述重链可变区如SEQ ID NO：9所示，和所述轻链可变区如SEQ ID NO：10所示。
  5. 根据权利要求1至4项中任一项所述的抗EpCAM抗体，其中所述抗EpCAM抗体进一步包含重链恒定区和轻链恒定区；
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区，和所述轻链恒定区选自人κ和λ链恒定区；
    更优选地，所述抗EpCAM抗体包含：
    a)与SEQ ID NO：23具有至少90％序列同一性的重链，和与SEQ ID NO：24具有至少90％序列同一性的轻链；
    b)与SEQ ID NO：25具有至少90％同一性的重链，和与SEQ ID NO：26具有至少90％序列同一性的轻链；或
    c)与SEQ ID NO：27具有至少90％同一性的重链，和与SEQ ID NO：28具有至少90％序列同一性的轻链。
  6. 根据权利要求1至5项中任一项所述的抗EpCAM抗体，其中所述抗EpCAM抗体包含：
    d)如SEQ ID NO：23所示的重链和如SEQ ID NO：24所示的轻链；
    e)如SEQ ID NO：25所示的重链和如SEQ ID NO：26所示的轻链；或
    f)如SEQ ID NO：27所示的重链和如SEQ ID NO：28所示的轻链。
  7. 一种分离的抗EpCAM抗体，所述抗体与权利要求1至6项中任一项所述的抗EpCAM抗体竞争性结合人EpCAM。
  8. 一种核酸分子，其编码权利要求1至7项中任一项所述的抗EpCAM抗体。
  9. 一种宿主细胞，其包含如权利要求8所述的核酸分子。
  10. 一种免疫偶联物，其为由根据权利要求1至7项中任一项所述的抗EpCAM抗体与效应分子偶联形成的免疫偶联物；所述效应分子选自放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
  11. 一种药物组合物，其含有治疗有效量的根据权利要求1至7项中任一项所述的抗EpCAM抗体，或根据权利要求8所述的核酸分子，或根据权利要求10所述的免疫偶联物，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  12. 一种用于免疫检测或测定EpCAM的方法，所述方法包括使用权利要求1至7项中任一项所述的抗EpCAM抗体或权利要求10所述的免疫偶联物的步骤。
  13. 一种试剂盒，其包含根据权利要求1至7项中任一项所述的抗EpCAM抗体或权利要求10所述的免疫偶联物。
  14. 一种诊断与EpCAM相关的疾病的方法，所述方法包括使用权利要求1至7项中任一项所述的抗EpCAM抗体、权利要求10所述的免疫偶联物或权利要求13所述的试剂盒的步骤。
  15. 一种治疗与EpCAM相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至7项中任一项所述的抗EpCAM抗体，或权利要求8所述的核酸分子，或权利要求10所述的免疫偶联物或权利要求11所述的药物组合物，其中所述疾病优选为肿瘤；
    更优选地，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
    更优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；所述肺癌选自：非小细胞肺癌和小细胞肺癌；所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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