WO2023142297A1 - Muc1 binding molecule and application thereof - Google Patents

Muc1 binding molecule and application thereof Download PDF

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WO2023142297A1
WO2023142297A1 PCT/CN2022/091544 CN2022091544W WO2023142297A1 WO 2023142297 A1 WO2023142297 A1 WO 2023142297A1 CN 2022091544 W CN2022091544 W CN 2022091544W WO 2023142297 A1 WO2023142297 A1 WO 2023142297A1
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mucl
antibody
seq
binding molecule
binding
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PCT/CN2022/091544
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French (fr)
Chinese (zh)
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蔡祥海
朱伟民
孙艳
钱其军
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浙江纳米抗体技术中心有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4725—Mucins, e.g. human intestinal mucin

Definitions

  • the present invention relates to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to a MUCl binding molecule and its application.
  • the MUC1 binding molecule of the invention can bind the extracellular region of MUC1 with high specificity, has high affinity and biological activity, low immunogenicity, stable structure and good druggability.
  • MUC1 is a mucin that is expressed in a variety of epithelial cells and highly expressed in various solid tumors and blood tumor cells. MUC1 is one of the most potentially valuable targets in the field of tumor therapy. Due to the differences in MUC1 oxygen glycosylation modification and breakage, the structure of MUC1 on the surface of tumor cells and normal cells is quite different. These differences determine that MUC1 will be a specific target for solid tumors.
  • Nanobodies have natural advantages such as easy expression, high stability, and high affinity. According to the advantages of nanobodies and the biological mechanism of MUC1, the development of MUC1 nanobodies has very broad application prospects.
  • the purpose of the present invention is to provide MUCl binding molecules and applications thereof.
  • the first aspect of the present invention provides a MUC1 binding molecule, comprising an anti-MUC1 single domain antibody, the CDR of the single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprising the sequence shown in SEQ ID NO:2, and CDR3 comprising the sequence shown in SEQ ID NO:3.
  • SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein X 1 is P, F, L, A or R , X2 is H, T, S or F, X3 is L, F, S, G, T or A, X4 is E, R, D, L or N, X5 is L, R, Y, N , D, Q, I or E, X 6 is Y, H, L, E or I, X 7 is A, Y, D, M, E or T, X 8 is Y, E or none, X 9 is Y or none, X 10 is N or none.
  • CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-13.
  • SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is S, N or R, X 2 is E, T, G, R or W, X3 is S, N, R, I, F or Y, X4 is G, D or N, X5 is D, G, S, A, E, T or none, X6 is D, T, S, R, P, L, W or none, X7 is T, I or none.
  • CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 14-24.
  • SEQ ID NO: 3 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • X 1 is A, T or N
  • X2 is A or T
  • X3 is C
  • X4 is T
  • X5 is F, Y, T, I, A, N or P
  • X 6 is Y, P, R, G, L or A
  • X 7 is G, S, F, R, L or H
  • X 8 is S, Q, I, T, W, K or Y
  • X 9 is T, L, F, P, S or none
  • X 10 is F, S, G, C, T or none
  • X 11 is R, Y, T, L, S, C or none
  • X 12 is E , D, C, F, Y or none
  • X 13 is
  • the CDR3 comprises the sequence shown in any one of SEQ ID NOs: 25-35.
  • the CDR1 of the single domain antibody comprises the sequence shown in any of SEQ ID NO:4-13
  • the CDR2 comprises the sequence shown in any of SEQ ID NO:14-24
  • CDR3 comprises the sequence shown in any one of SEQ ID NO: 25-35.
  • the single domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of the following group a1 to group a11:
  • the FR1 of the single domain antibody VHH can be selected from FR1 of any VHH of SEQ ID NO:36-46
  • the FR2 of VHH can be selected from any of SEQ ID NO:36-46 FR2 of VHH
  • FR3 of VHH can be selected from FR3 of any VHH of SEQ ID NO:36-46
  • FR4 of VHH can be selected from FR4 of any VHH of SEQ ID NO:36-46.
  • the FR region of the single domain antibody is selected from the FR region of any VHH of SEQ ID NO: 36-46.
  • the single domain antibody VHH is as shown in any one of SEQ ID NO: 36-46.
  • the MUCl binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain comprising one, two or more anti-MUCl single domain antibodies described herein. Antibodies or antigen-binding fragments thereof, antibodies or antigen-binding fragments thereof.
  • the multivalent single domain antibody or multispecific single domain antibody is connected to multiple single domain antibodies through a linker.
  • the linker consists of 1-15 amino acids selected from G and S.
  • the antigen-binding fragment of the heavy chain antibody is a single chain heavy chain antibody.
  • the heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the heavy chain antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is that of a camelid heavy chain antibody, comprising CH2 and CH3.
  • the CH2 and CH3 are the CH2 and CH3 of a human IgG Fc, such as the CH2 and CH3 of IgGl or IgG4.
  • the heavy chain constant region is CH2 and CH3 of IgG4, and its amino acid sequence is shown in SEQ ID NO:47.
  • the heavy chain constant region is that of a cartilaginous fish heavy chain antibody comprising CH1, CH2, CH3, CH4 and CH5.
  • said antibody is an antibody comprising said anti-MUCl single domain antibody as a heavy chain variable domain.
  • the antibody further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.
  • the antigen-binding fragment of an antibody is selected from Fab, F(ab')2, Fv, scFv.
  • the binding molecule of any embodiment of the invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.
  • the invention also provides polynucleotides selected from:
  • the fragment is a primer.
  • the present invention also provides a nucleic acid construct comprising the polynucleotide described herein.
  • the nucleic acid construct is a recombinant vector or an expression vector.
  • the invention also provides a phage comprising a MUCl binding molecule according to any of the embodiments herein.
  • said MUCl-binding molecule is displayed on the surface of said phage.
  • the present invention also provides a host cell selected from:
  • the present invention also provides a method for producing a MUCl-binding molecule, comprising: producing a MUCl-binding molecule (such as a monovalent or multivalent single-domain antibody, a multispecific single-domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof) suitable for producing a MUCl-binding molecule.
  • a MUCl-binding molecule such as a monovalent or multivalent single-domain antibody, a multispecific single-domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof
  • the host cells described herein are grown under conditions, and the MUCl-binding molecule is optionally purified from the culture.
  • the present invention also provides a pharmaceutical composition, comprising the MUCl binding molecule, polynucleotide, nucleic acid construct, phage or host cell described in any embodiment herein, and pharmaceutically acceptable adjuvants.
  • the pharmaceutical composition is used to treat cancer.
  • the cancer is a MUCl-associated cancer.
  • the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present invention also provides the use of the MUCl-binding molecule according to any embodiment herein in the preparation of a medicament for preventing or treating cancer.
  • the cancer is a MUCl-associated cancer.
  • the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present invention also provides a method for treating or preventing cancer, the method comprising administering to a patient in need a therapeutically effective amount of the MUCl-binding molecule described in any embodiment of the present invention, or containing the MUCl described in any embodiment of the present invention Pharmaceutical composition of binding molecules.
  • the cancer is a MUCl-associated cancer.
  • the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present invention also provides a kit for detecting MUCl, which is used for evaluating drug treatment effects or diagnosing cancer, and the kit includes MUCl binding molecules, polynucleotides, nucleic acid constructs, phages, and hosts described in any embodiment herein cell.
  • the kit further includes reagents for detecting binding of MUCl to a single domain antibody, antibody, or antigen-binding fragment thereof.
  • the bound reagent is detected, for example, by enzyme-linked immunoassay.
  • the detection binding reagent is a detectable label, such as biotin, that can be attached to the MUCl binding molecule.
  • the detectable label is attached to the MUCl binding molecule or present separately in a kit.
  • the present invention also provides a non-diagnostic method for detecting the presence of MUCl in a sample, the method comprising: incubating the sample with the MUCl binding molecule according to any of the embodiments herein, and detecting MUCl with a single domain antibody, antibody or antigen thereof The binding of the binding fragments to determine the presence of MUCl in the sample.
  • the detection is an enzyme-linked immunoreaction method detection.
  • the present invention also provides the use of the MUCl-binding molecule according to any embodiment herein in the preparation of a kit for detecting MUCl in a sample, evaluating the effect of drug treatment or diagnosing cancer.
  • Figure 1 shows the titer detection results of alpaca antiserum against MUC1 protein.
  • Figure 2 shows the results of ELISA detection of candidate antibodies and MUC1 protein.
  • Figure 3 is the result of the affinity test between the candidate antibody and MUC1 protein.
  • Fig. 4 is the detection result of the binding of the candidate antibody to the MB468 tumor cell line.
  • MUC1-binding molecules comprising anti-MUCl single domain antibodies.
  • the MUC1-binding molecule of the invention can bind MUC1 with high specificity, has high affinity and biological activity, low immunogenicity, stable structure and good druggability.
  • the single domain antibody of the present invention is easy to produce.
  • MUC1-binding molecules are proteins that specifically bind to MUC1, including but not limited to: antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies (nanobodies), minibodies (minibodies), affibodies, receptors Target binding domains of antibodies, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.
  • antibody includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), Diabodies and single chain molecules, as well as antibody fragments, especially antigen binding fragments, eg, Fab, F(ab')2 and Fv).
  • immunoglobulin Ig
  • antibody antibodies are used interchangeably.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H).
  • IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called the J chain, which contains 10 antigen-binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with the J chain Polymerization forms multivalent assemblies.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the heavy chain isotype.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each ⁇ and ⁇ chain) and four (CH1, CH1, CH2, CH3 and CH4) constant domain (CH) and the hinge region (Hinge) between the CH1 domain and the CH2 domain.
  • Each light chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH, while CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains.
  • the paired VH and VL together form an antigen binding site.
  • Light chains from any vertebrate species can, based on their constant domain amino acid sequence, be assigned to one of two distinct types called kappa and lambda.
  • immunoglobulins can be assigned to different classes, or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • a “heavy chain antibody” as described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibodies, the heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), and only contain 2 heavy chains consisting of the variable region (VHH) and other constant regions. The variable region passes through a similar hinge region The structure is linked to the constant region. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous heavy chain antibody contains 1 variable region and 5 Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc.
  • single domain antibody As used herein, the terms “single domain antibody”, “anti-MUCl single domain antibody”, “heavy chain variable region domain of a heavy chain antibody”, “VHH”, “Nanobody” are used interchangeably and refer to specific A single domain antibody that recognizes and binds to MUC1.
  • Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs.
  • the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3.
  • Single domain antibodies are the smallest functional antigen-binding fragments. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • a binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificities is a multispecific single domain antibody.
  • a multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies through a linker.
  • the linker usually consists of 1-15 amino acids selected from G and S.
  • heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to the similarity in the structures of the two, the following structural descriptions for antibodies are also applicable to heavy chain antibodies except for the light chain.
  • variable region or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • the variable domains of the heavy and light chains can be referred to as “VH” and “VL”, respectively. These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable refers to the fact that certain segments of the variable domains vary widely among antibody sequences.
  • the variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across all amino acids spanned by a variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 (heavy Chain antibodies may be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3 of the light chain variable region.
  • HVRs hypervariable regions
  • CDR1, CDR2, CDR3 heavy Chain antibodies may be abbreviated as CDR1, CDR2, CDR3
  • LCDR1, LCDR2 and LCDR3 of the light chain variable region.
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a ⁇ -sheet conformation connected by the formation of loops and in some cases forming ⁇ -sheet structures Part of three HVR connections.
  • the HVRs in each chain are held together in close proximity by the FR regions and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site.
  • the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4
  • the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • the constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the involvement of antibodies in antibody-dependent cell-mediated cytotoxicity.
  • Fc region fragment crystallizable region
  • Fc domain Fc domain refers to the C-terminal region of an antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including those located in the immune system. Binding to Fc receptors on various cells (eg, effector cells), or to the first component (Clq) of the classical complement system.
  • the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE is present in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4).
  • the human IgG heavy chain Fc region is generally defined as the sequence stretch from the amino acid residue at positions C226 or P230 of the heavy chain to the carboxy-terminus, where this numbering is according to the EU index, as in Same in Kabat.
  • the Fc region can be a native sequence Fc or a variant Fc.
  • an “antibody fragment” comprises a portion of an intact antibody, preferably the antigen-binding and/or variable region of an intact antibody.
  • Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; Or any fragment that should be able to increase the half-life by incorporation into liposomes. Digestion of antibodies with papain yields two identical antigen-binding fragments, termed "Fab" fragments, and one residual "Fc” fragment, which has the ability to readily crystallize.
  • the Fab fragment consists of the complete light chain and the variable domains of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody yields a larger F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region.
  • F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of the two heavy chains held together by disulfide bonds.
  • the effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.
  • Fv is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) protrude from the fold of these two domains, contributing the amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower avidity than the full binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv”
  • sFv is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen-binding structure.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation, ), the individual antibodies constituting the population were identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures without contamination by other immunoglobulins.
  • monoclonal indicates that the antibody has acquired characteristics from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and the use of antibodies that have part or all of the human immunoglobulin loci or encode human immunoglobulin loci.
  • Monoclonal antibodies also include herein "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remaining portions are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity.
  • “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • “humanized antibody” generally refers to a non-human antibody in which the variable domain framework regions have been exchanged with sequences found in human antibodies.
  • the entire antibody (except for the CDRs) is encoded by or is identical to such an antibody (except for the CDRs) by a polynucleotide of human origin.
  • CDRs some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the ⁇ -sheet framework of human antibody variable regions to produce antibodies whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems.
  • antibodies, single domain antibodies, heavy chain antibodies and the like all include humanized variants of each of these antibodies.
  • human antibody refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or has been produced using any of the techniques disclosed herein for the production of human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
  • the present invention also provides a single domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof that binds to the same epitope of MUCl as any anti-MUCl single domain antibody of the present invention, that is, capable of binding to any single domain of the present invention.
  • Antibodies Cross-competing single domain antibodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof for binding to MUCl.
  • the CDR1 of the single domain antibody includes the sequence shown in SEQ ID NO: 1, and SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein, X 1 is P, F, L, A or R, X 2 is H, T, S or F, X 3 is L, F, S, G, T or A, X 4 is E, R, D, L or N , X 5 is L, R, Y, N, D, Q, I or E, X 6 is Y, H, L, E or I, X 7 is A, Y, D, M, E or T, X 8 Y, E or nothing, X9 is Y or nothing, X10 is N or nothing.
  • CDR1 comprises the sequence set forth in any one of SEQ ID NO: 4-13.
  • the CDR2 of the single domain antibody includes the sequence shown in SEQ ID NO: 2, and SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is S, N or R, X2 is E, T, G, R or W, X3 is S, N, R, I, F or Y, X4 is G, D or N, X5 is D, G, S, A, E, T or none, X6 is D, T, S, R, P, L, W or none, X7 is T, I or none.
  • the CDR2 comprises the sequence set forth in any one of SEQ ID NO: 14-24.
  • the CDR3 of the single domain antibody includes the sequence shown in SEQ ID NO: 3, and SEQ ID NO: 3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , wherein X 1 is A, T or N, X 2 is A or T, X 3 is C, I, E, G or V, X 4 is T, G, R, V or D, X 5 is F, Y, T, I, A, N or P, X 6 is Y, P, R, G, L or A, X 7 is G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 is T, L, F, P, S or none, X 10 is F, S, G, C , T or none, X 11 is R, Y
  • the single domain antibody contains CDR1, CDR2 and CDR3 shown in any one of the SEQ ID NOs of group a1 to group a11 in Table 1:
  • the FR1 of the single domain antibody VHH can be selected from FR1 of any VHH of SEQ ID NO:36-46
  • the FR2 of VHH can be selected from any of SEQ ID NO:36-46 FR2 of VHH
  • FR3 of VHH can be selected from FR3 of any VHH of SEQ ID NO:36-46
  • FR4 of VHH can be selected from FR4 of any VHH of SEQ ID NO:36-46.
  • the FR region of the VHH of the single domain antibody of the present invention is selected from the FR region of any VHH of SEQ ID NO: 36-46.
  • the CDRs of such antibodies are selected from any one of the aforementioned group a1 to group a11.
  • the single domain antibody VHH is as shown in any one of SEQ ID NO: 36-46.
  • the MUCl binding molecules described herein may be monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof comprising one, two or more anti-MUCl single domain antibodies described herein, Antibodies or antigen-binding fragments thereof.
  • a MUCl binding molecule herein comprises an anti-MUCl single domain antibody and an immunoglobulin Fc region.
  • Fc regions useful in the present invention may be from different subtypes of immunoglobulins, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM.
  • the immunoglobulin Fc region is IgG4 Fc, the amino acid sequence of which is shown in SEQ ID NO:47.
  • a MUCl binding molecule described herein is a heavy chain antibody.
  • the heavy chain antibody also comprises a heavy chain constant region, such as that of a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the heavy chain constant region is as shown in SEQ ID NO:47.
  • the present invention also includes said antibody derivatives and analogs.
  • “Derivatives” and “analogues” refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present invention.
  • Derivatives or analogs of the present invention may be (i) polypeptides having substituent groups in one or more amino acid residues, or (ii) mature polypeptides in combination with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol A polypeptide formed by fusion of diol), or (iii) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify this polypeptide, or with a 6His tag formed fusion protein).
  • Such derivatives and analogs are within the purview of those skilled in the art from the teachings herein.
  • those skilled in the art can change one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) of the sequence of the present invention.
  • Multiple) amino acids to obtain variants of the antibody or functional fragment sequence thereof.
  • These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • conservative substitutions with amino acids with similar or similar properties usually do not change the function of the protein.
  • Amino acids with similar properties are substituted eg in the FR and/or CDR regions of the variable region.
  • Amino acid residues that may be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the protection scope of the present invention.
  • Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and those capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the protein encoded by DNA, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention are included in the immunoglobulin.
  • sequence of the variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to its source sequence.
  • Sequence identity according to the invention can be measured using sequence analysis software. For example the computer program BLAST, especially BLASTP or TBLASTN, using default parameters.
  • the present invention also includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein .
  • the antibody of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art.
  • the heavy chain antibody of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art.
  • antibodies or heavy chain antibodies of the invention may be expressed in other cell lines.
  • Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used will depend on the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , Encapsulation of polynucleotides in liposomes and microinjection of DNA directly into nuclei, etc.
  • Mammalian cell lines useful as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO ) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial MUCl binding properties.
  • ATCC American Type Culture Collection
  • the present invention also provides polynucleotides encoding the above antibodies or fragments thereof.
  • polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and respective CDRs.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • the present invention also relates to polynucleotides that hybridize to the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. Vectors generally contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing and the complete intron sequence of the acceptor splice site, sequence encoding the leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, polylinker for insertion of nucleic acid encoding the antibody to be expressed zone and optional marker elements.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the polypeptide in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • nanobody library By constructing a nanobody library, the inventors discovered, expressed and purified multiple nanobodies that can bind to the MUCl protein. All aspects of the antibodies described herein are useful in the manufacture of a medicament for the prevention or treatment of the various conditions and diseases described herein, especially those conditions or conditions associated with MUCl expressing cells.
  • the condition and disease is cancer, including but not limited to: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, Cancer of the cervix, head and neck, fallopian tubes, multiple myeloma, bile ducts, gallbladder, esophagus, prostate, or glioblastoma.
  • cancer including but not limited to: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, Cancer of the cervix, head and neck, fallopian tubes, multiple myeloma, bile ducts, gallbladder, esophagus, prostate, or glioblastoma.
  • compositions herein contain the binding molecules described herein, together with pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants.
  • excipients include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • a pharmaceutical composition may contain ingredients for improving, maintaining or retaining, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of a substance. These substances are known from the prior art. The optimum pharmaceutical composition will be determined by the intended route of administration, mode of delivery and desired dosage.
  • compositions for in vivo administration are generally presented as sterile preparations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, this method can be used for sterilization either before or after lyophilization and reconstitution.
  • Pharmaceutical compositions of the invention may be selected for parenteral delivery.
  • Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliary agents.
  • Parenteral compositions are usually presented in containers with sterile access ports, eg, intravenous solution strips or vials with a hypodermic needle-punctureable stopper.
  • compositions may be selected for inhalation or delivery through the alimentary tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating various other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • kits for producing single dosage administration units may each contain a first container with a dry protein and a second container with an aqueous formulation.
  • kits containing single and multi-lumen prefilled syringes eg, liquid syringes and lyophilized syringes are provided.
  • the present invention also provides a method for treating a patient, especially a MUCl-associated disease in a patient, by administering a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
  • a patient especially a MUCl-associated disease in a patient
  • a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
  • the terms "patient”, “subject”, “individual”, “subject” are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog , cats, rabbits, etc.), and most preferably humans.
  • Treatment refers to the subject's use of the treatment regimens described herein to achieve at least one positive therapeutic effect (e.g., a decrease in cancer cell number, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in tumor metastasis or tumor growth. speed reduction).
  • Therapeutic regimens that effectively treat a patient can vary depending on factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.
  • the therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the invention to be employed will depend, for example, on the extent and goal of the treatment. Those skilled in the art will appreciate that appropriate dosage levels for therapy will depend in part on the molecule being delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. conditions) vary. In certain embodiments, the clinician can titrate the dose and vary the route of administration to achieve optimal therapeutic effect. For example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the binding molecule in the formulation used.
  • the clinician typically administers the composition until a dosage is reached to achieve the desired effect.
  • the composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
  • the route of administration of the pharmaceutical composition is according to known methods, such as oral, injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Either by a sustained release system or by an implanted device.
  • binding molecules of the invention are useful in assays, such as binding assays, to detect and/or quantify MUCl expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in studies to further investigate the role of MUCl in disease.
  • the method for detecting MUC1 is generally as follows: obtain a cell and/or tissue sample; detect the level of MUC1 in the sample.
  • the MUCl-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with MUCl.
  • the present invention provides detection of the presence of MUCl in a sample using classical immunohistological methods known to those skilled in the art. Detection of MUCl can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of MUCl include ELISA, FACS, RIA, and the like.
  • binding molecules such as single domain antibodies are typically labeled with a detectable labeling group.
  • Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (e.g., FITC, Rhodane luminescent, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups Or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (Computed X-ray tomography) contrast agent.
  • MRI
  • Another aspect of the invention provides methods for detecting the presence of a test molecule that competes with an antibody of the invention for binding to MUCl.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of MUCl in the presence or absence of a test molecule. An increase in the amount of free antibody (ie, antibody that does not bind MUCl) will indicate that the test molecule is able to compete with the antibody for MUCl binding.
  • the antibody is labeled with a labeling group.
  • the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
  • the present invention also provides a detection kit for detecting the level of MUCl, which includes an antibody that recognizes the MUCl protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers, detection labeling, detection substrates, etc.
  • the test kit may be an in vitro diagnostic device.
  • Embodiment 1 alpaca immunization
  • the MUC1 protein sequence queried on NCBI it was fused with the human IgG Fc fragment sequence, and Suzhou Jinweizhi Company was entrusted to synthesize and construct the eukaryotic expression vector of pCDNA3.4 (Thermo) plasmid, and the synthesized plasmid was expressed by EXpiCHOTM (Thermo Fisher) System expression. After expression, a 5mL Protein A prepacked column (GE) was used for one-step affinity purification. The purified sample was replaced into PBS buffer. The purity was identified by SDS-PAGE electrophoresis gel and HPLC, and the activity was identified by ELISA. Aliquots were stored in a -80°C refrigerator for subsequent immunization.
  • the amount of the first immunization antigen (MUC1-hFc) was 1000 ⁇ g, mixed with the adjuvant (GERBU FAMA), and the back of the alpaca was selected for subcutaneous injection at four points, and the amount of each point was 1 mL.
  • the second immunization was performed at intervals of 3 weeks.
  • the 2nd to 9th immunizations the amount of immunizing antigen is 500 ⁇ g, and four subcutaneous injections are selected on the back of the alpaca for immunization, the amount of immunization at each point is 1 mL, and the interval between each immunization is one week.
  • RNAiso reagent After 5 times of immunization, 50 mL of camel peripheral blood lymphocytes were extracted and total RNA was extracted. The extraction of RNA was carried out according to the instruction manual of RNAiso reagent from TAKARA company.
  • RNA was used as a template, oligo dT was used as a primer, and the first strand of cDNA was synthesized according to the reverse transcriptase instructions of TAKARA Company.
  • variable region fragments of heavy chain antibodies was obtained by nested PCR. Amplify variable region fragments of heavy chain antibodies by nested PCR:
  • Upstream primer GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 48)
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 49)
  • Upstream primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 50)
  • Centrifuge the culture resuspend the pellet with 200mL of 2 ⁇ YT (containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin), cultivate overnight at 37°C, 250r/min, centrifuge at 8000rpm to get the supernatant, add 5 ⁇ PEG/NaCl solution, put it on ice for 60min, centrifuge at 8000rpm for 30min, resuspend the pellet in 5mL of PBS, and obtain the anti-MUC1 single domain antibody (VHH) immune library, take 10 ⁇ L to measure the titer, and put the rest at -80°C Save for later.
  • VHH anti-MUC1 single domain antibody
  • ELISA binding detection uses anti-c-myc Antibody HRP (Bethyl) as the secondary antibody to block Biotinylated Human B7-1/CD80 Protein
  • Fc molecule (Acrobiosystem) binding detection uses HRP-labeled streptavidin (Thermo) as the secondary antibody .
  • HRP-labeled streptavidin Thermo
  • the nanobody was constructed on the pCDNA3.4-IgG4 vector, constructed in the form of VHH-IgG4, and then expressed by the EXpiCHOTM (Thermo Fisher) expression system. After one week of expression, the supernatant was collected for Protein A (GE) purification. Then use Nanodrop to detect protein quantity, and HPLC to detect protein purity. The purity and yield of the obtained protein met the needs of subsequent experiments.
  • the affinity of the antibody to human MUCl-His antigen was determined by enzyme-linked immunosorbent assay (ELISA).
  • the MUC1 antigen was diluted to 2 ⁇ g/mL with coating solution, 100 ⁇ L/well was added to a 96-well strip, and incubated at 37°C for 2 hours. Wash 3 times. After washing, add 100 ⁇ L of 3% BSA blocking solution to each well, and incubate at 37°C for 2 hours. Wash 3 times. Add 100 ⁇ L/well of MUC1 antibody, the initial concentration of antibody dilution is 4 ⁇ g/mL, 4-fold dilution in 8 gradients, and incubate at 37°C for 1 hour. Wash each plate 3 times.
  • the binding kinetics and affinity of antibodies to human MUCl-His antigen were determined using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the purified antibody was passed through the sensor chip with pre-immobilized protein A, the antibody was captured by protein A, and then 5 different concentrations of MUC1.his protein were used as the mobile phase, and the binding time and dissociation time were 30min and 60min, respectively.
  • Association rates (ka), dissociation rates (kd) and equilibrium constants (KD) were analyzed using Biacore Evaluation Software 2.0 (GE).
  • BMK1 is an IgG4 antibody in the form of scFv (VH-VL) prepared according to the antibody sequences SEQ ID NO:73 and SEQ ID NO:75 in the US 2016/0340442A1 patent of GENUS oncology company, and BMK2 is based on the sequence in the antibody US20200024361A1 patent of Peptron Company IgG4 antibodies in the form of scFv (VH-VL) prepared by SEQ ID NO: 24 and SEQ ID NO: 25.
  • the amino acid sequence of each candidate antibody is shown in Table 4, and the CDR sequence of each candidate antibody is shown in Table 5.
  • the protein level affinity test results are shown in Table 2 below, and the binding kinetic curve is shown in Figure 3.
  • Isotype is an isotype control (negative control, the sequence is derived from SEQ ID NO: 2 of patent CN 106146653A).
  • the results of the candidate antibodies are shown in Figure 4. It can be seen from Figure 4 that the candidate antibodies have high, medium and low binding activities to the tumor cell MB468.
  • NBL502-1-C4, NBL502-EFP-B02, NBL502-EFP-B03, NBL502-1D2-2, NBL502-AI-201 have higher binding activity to tumor cell MB468, and the positive rate is greater than 90%.
  • the binding activity of NBL502-1-A2, NBL502-AI-120, NBL502-AI-121, NBL502-AI-181, NBL502-A12-6-LST-D5-2 to tumor cell MB468 is relatively weak, and the positive rate is 45 %-90%.
  • the binding activity of NBL502-AI212 to tumor cell MB468 was the weakest.
  • the test results are shown in Table 3, and the results show that among all the 34 tested tissues, the binding level of the candidate antibody to the tissue is not higher than that of the positive controls SM3 and BMK2.
  • the MUC1 candidate antibody has no obvious strong binding to normal tissues, and the results in various key tissues such as lung, kidney, pancreas, stomach, and brain are negative or have no obvious binding. These data indicate that the candidate antibody has good tissue safety.

Abstract

The present invention relates to an MUC1 binding molecule and the application thereof. Specifically provided is an MUC1 binding molecule. The MUC1 binding molecule contains an anti-MUC1 single-domain antibody, and complementarity-determining regions (CDR) of the single-domain antibody include a CDR1, a CDR2 and a CDR3, wherein the CDR1 comprises a sequence as shown in SEQ ID NO: 1, the CDR2 comprises a sequence as shown in SEQ ID NO: 2, and the CDR3 comprises a sequence as shown in SEQ ID NO: 3. The MUC1 binding molecule can be bound to MUC1 in a high-specificity manner, and exhibits high affinity and a high biological activity.

Description

MUC1结合分子及其应用MUC1 binding molecule and its application 技术领域technical field
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种MUC1结合分子及其应用。本发明的MUC1结合分子能够高特异性地结合MUC1胞外区，具有较高的亲和力和生物活性，以及低的免疫原性，结构稳定，成药性良好。The present invention relates to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to a MUCl binding molecule and its application. The MUC1 binding molecule of the invention can bind the extracellular region of MUC1 with high specificity, has high affinity and biological activity, low immunogenicity, stable structure and good druggability.
背景技术Background technique
MUC1是一种粘蛋白，在多种上皮细胞表达，在各种实体瘤和血液瘤细胞中高表达，MUC1是肿瘤治疗领域的最具潜在价值的靶点之一。由于MUC1氧糖基化修饰的差异以及会发生断裂，MUC1在肿瘤细胞与正常细胞表面的结构有着较大的差异，这些差异决定了MUC1将会是一种针对实体瘤的特异性靶点。MUC1 is a mucin that is expressed in a variety of epithelial cells and highly expressed in various solid tumors and blood tumor cells. MUC1 is one of the most potentially valuable targets in the field of tumor therapy. Due to the differences in MUC1 oxygen glycosylation modification and breakage, the structure of MUC1 on the surface of tumor cells and normal cells is quite different. These differences determine that MUC1 will be a specific target for solid tumors.
纳米抗体具有易表达、稳定性高、亲和力高等天然优势，根据纳米抗体自身优势和MUC1生物学机制，开发MUC1纳米抗体具有非常广阔的应用前景。Nanobodies have natural advantages such as easy expression, high stability, and high affinity. According to the advantages of nanobodies and the biological mechanism of MUC1, the development of MUC1 nanobodies has very broad application prospects.
发明内容Contents of the invention
本发明的目的在于提供MUC1结合分子及其应用。The purpose of the present invention is to provide MUCl binding molecules and applications thereof.
本发明第一方面提供一种MUC1结合分子，包含抗MUC1单域抗体，所述单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1所示的序列、CDR2包括SEQ ID NO:2所示的序列、和CDR3包括SEQ ID NO:3所示的序列。The first aspect of the present invention provides a MUC1 binding molecule, comprising an anti-MUC1 single domain antibody, the CDR of the single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprising the sequence shown in SEQ ID NO:2, and CDR3 comprising the sequence shown in SEQ ID NO:3.
在一个或多个实施方案中，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7X 8X 9X 10，其中，X 1为P、F、L、A或R，X 2为H、T、S或F，X 3为L、F、S、G、T或A，X 4为E、R、D、L或N，X 5为L、R、Y、N、D、Q、I或E，X 6为Y、H、L、E或I，X 7为A、Y、D、M、E或T，X 8为Y、E或无，X 9为Y或无，X 10为N或无。 In one or more embodiments, SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein X 1 is P, F, L, A or R , X2 is H, T, S or F, X3 is L, F, S, G, T or A, X4 is E, R, D, L or N, X5 is L, R, Y, N , D, Q, I or E, X 6 is Y, H, L, E or I, X 7 is A, Y, D, M, E or T, X 8 is Y, E or none, X 9 is Y or none, X 10 is N or none.
在一个或多个实施方案中，CDR1包含SEQ ID NO:4-13中任一所示的序列。In one or more embodiments, CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-13.
在一个或多个实施方案中，SEQ ID NO:2是IX 1X 2X 3X 4X 5X 6X 7，其中，X 1为S、N或R，X 2为E、T、G、R或W，X 3为S、N、R、I、F或Y，X 4为G、D或N，X 5为D、G、S、A、E、T或无，X 6为D、T、S、R、P、L、W或无，X 7为T、I或无。 In one or more embodiments, SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is S, N or R, X 2 is E, T, G, R or W, X3 is S, N, R, I, F or Y, X4 is G, D or N, X5 is D, G, S, A, E, T or none, X6 is D, T, S, R, P, L, W or none, X7 is T, I or none.
在一个或多个实施方案中，CDR2包含SEQ ID NO:14-24中任一所示的序列。In one or more embodiments, CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 14-24.
在一个或多个实施方案中，SEQ ID NO:3是In one or more embodiments, SEQ ID NO: 3 is
X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20，其中，X 1为A、T或 N，X 2为A或T，X 3为C、I、E、G或V，X 4为T、G、R、V或D，X 5为F、Y、T、I、A、N或P，X 6为Y、P、R、G、L或A，X 7为G、S、F、R、L或H，X 8为S、Q、I、T、W、K或Y，X 9为T、L、F、P、S或无，X 10为F、S、G、C、T或无，X 11为R、Y、T、L、S、C或无，X 12为E、D、C、F、Y或无，X 13为M、Y、V、L或无，X 14为G、P、H、A、T或无，X 15为Y、S、Q、R、W、V或无，X 16为N、R、T、M、P或无，X 17为F、E、A、Q或无，X 18为A、G、Y或无，X 19为S、D、R或无，X 20为Y或无。 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , wherein, X 1 is A, T or N, X2 is A or T, X3 is C, I, E, G or V, X4 is T, G, R, V or D, X5 is F, Y, T, I, A, N or P, X 6 is Y, P, R, G, L or A, X 7 is G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 is T, L, F, P, S or none, X 10 is F, S, G, C, T or none, X 11 is R, Y, T, L, S, C or none, X 12 is E , D, C, F, Y or none, X 13 is M, Y, V, L or none, X 14 is G, P, H, A, T or none, X 15 is Y, S, Q, R, W, V or nothing, X 16 is N, R, T, M, P or nothing, X 17 is F, E, A, Q or nothing, X 18 is A, G, Y or nothing, X 19 is S, D, R or none, X 20 is Y or none.
在一个或多个实施方案中，CDR3包含SEQ ID NO:25-35中任一所示的序列。In one or more embodiments, the CDR3 comprises the sequence shown in any one of SEQ ID NOs: 25-35.
在一个或多个实施方案中，所述单域抗体的CDR1包含SEQ ID NO:4-13中任一所示的序列，CDR2包含SEQ ID NO:14-24中任一所示的序列，和CDR3包含SEQ ID NO:25-35中任一所示的序列。In one or more embodiments, the CDR1 of the single domain antibody comprises the sequence shown in any of SEQ ID NO:4-13, and the CDR2 comprises the sequence shown in any of SEQ ID NO:14-24, and CDR3 comprises the sequence shown in any one of SEQ ID NO: 25-35.
在一个或多个实施方案中，所述单域抗体含有以下组a1到组a11中任一组的SEQ ID NO所示的CDR1、CDR2和CDR3：In one or more embodiments, the single domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of the following group a1 to group a11:
组Group CDR1CDR1 CDR2CDR2 CDR3CDR3
a1a1 44 1414 2525
a2a2 55 1515 2626
a3a3 66 1616 2727
a4a4 77 1717 2828
a5a5 88 1818 2929
a6a6 99 1919 3030
a7a7 1010 2020 3131
a8a8 1010 21twenty one 3232
a9a9 1111 22twenty two 3333
a10a10 1212 23twenty three 3434
a11a11 1313 24twenty four 3535
在一个或多个实施方案中，所述单域抗体VHH的FR1可选自SEQ ID NO:36-46的任一VHH的FR1，VHH的FR2可选自SEQ ID NO:36-46的任一VHH的FR2，VHH的FR3可选自SEQ ID NO:36-46的任一VHH的FR3，VHH的FR4可选自SEQ ID NO:36-46的任一VHH的FR4。In one or more embodiments, the FR1 of the single domain antibody VHH can be selected from FR1 of any VHH of SEQ ID NO:36-46, and the FR2 of VHH can be selected from any of SEQ ID NO:36-46 FR2 of VHH, FR3 of VHH can be selected from FR3 of any VHH of SEQ ID NO:36-46, and FR4 of VHH can be selected from FR4 of any VHH of SEQ ID NO:36-46.
在一个或多个实施方案中，所述单域抗体的FR区为选自SEQ ID NO:36-46的任一VHH的FR区。In one or more embodiments, the FR region of the single domain antibody is selected from the FR region of any VHH of SEQ ID NO: 36-46.
在一个或多个实施方案中，所述单域抗体VHH如SEQ ID NO:36-46中任一所示。In one or more embodiments, the single domain antibody VHH is as shown in any one of SEQ ID NO: 36-46.
在一个或多个实施方案中，所述MUC1结合分子是包含一条、两条或多条本文所述的抗MUC1单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。In one or more embodiments, the MUCl binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain comprising one, two or more anti-MUCl single domain antibodies described herein. Antibodies or antigen-binding fragments thereof, antibodies or antigen-binding fragments thereof.
在一个或多个实施方案中，所述多价单域抗体或多特异性单域抗体通过连接子连接多个单域抗体。所述连接子由选自G和S的1-15个氨基酸组成。In one or more embodiments, the multivalent single domain antibody or multispecific single domain antibody is connected to multiple single domain antibodies through a linker. The linker consists of 1-15 amino acids selected from G and S.
在一个或多个实施方案中，所述重链抗体的抗原结合片段是单链重链抗体。In one or more embodiments, the antigen-binding fragment of the heavy chain antibody is a single chain heavy chain antibody.
在一个或多个实施方案中，所述重链抗体是骆驼重链抗体或软骨鱼重链抗体。In one or more embodiments, the heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
在一个或多个实施方案中，所述重链抗体还包含重链恒定区。In one or more embodiments, the heavy chain antibody further comprises a heavy chain constant region.
在一个或多个实施方案中，所述重链恒定区是骆驼重链抗体的恒定区，包含CH2和CH3。在一个或多个实施方案中，所述CH2和CH3是人IgG Fc的CH2和CH3，例如IgG1或IgG4的CH2和CH3。优选地，所述重链恒定区为IgG4的CH2和CH3，其氨基酸序列如SEQ ID NO:47所示。In one or more embodiments, the heavy chain constant region is that of a camelid heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are the CH2 and CH3 of a human IgG Fc, such as the CH2 and CH3 of IgGl or IgG4. Preferably, the heavy chain constant region is CH2 and CH3 of IgG4, and its amino acid sequence is shown in SEQ ID NO:47.
在一个或多个实施方案中，所述重链恒定区是软骨鱼重链抗体的恒定区，包含CH1、CH2、CH3、CH4和CH5。In one or more embodiments, the heavy chain constant region is that of a cartilaginous fish heavy chain antibody comprising CH1, CH2, CH3, CH4 and CH5.
在一个或多个实施方案中，所述抗体是包含所述抗MUC1单域抗体作为重链可变结构域的抗体。In one or more embodiments, said antibody is an antibody comprising said anti-MUCl single domain antibody as a heavy chain variable domain.
在一个或多个实施方案中，所述抗体还包含轻链可变结构域、重链恒定域和轻链恒定域。In one or more embodiments, the antibody further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.
在一个或多个实施方案中，抗体的抗原结合片段选自Fab、F(ab’)2、Fv、scFv。In one or more embodiments, the antigen-binding fragment of an antibody is selected from Fab, F(ab')2, Fv, scFv.
在一个或多个实施方案中，本发明任一实施方案所述的结合分子为嵌合抗体或完全人抗体；优选为完全人抗体。In one or more embodiments, the binding molecule of any embodiment of the invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.
本发明还提供多核苷酸，选自：The invention also provides polynucleotides selected from:
(1)本文任一实施方案所述单域抗体或本文所述抗体或其抗原结合片段的编码序列；(1) the coding sequence of the single domain antibody described in any embodiment herein or the antibody or antigen-binding fragment thereof described herein;
(2)(1)的互补序列；(2) the complementary sequence of (1);
(3)(1)或(2)中任一序列的5-50bp的片段。(3) A 5-50 bp fragment of any sequence in (1) or (2).
在一个或多个实施方案中，所述片段是引物。In one or more embodiments, the fragment is a primer.
本发明还提供一种核酸构建物，包含本文所述的多核苷酸。The present invention also provides a nucleic acid construct comprising the polynucleotide described herein.
在一个或多个实施方案中，所述核酸构建物是重组载体或表达载体。In one or more embodiments, the nucleic acid construct is a recombinant vector or an expression vector.
本发明还提供包含本文任一实施方案所述MUC1结合分子的噬菌体。The invention also provides a phage comprising a MUCl binding molecule according to any of the embodiments herein.
在一个或多个实施方案中，所述MUC1结合分子展示于所述噬菌体表面。In one or more embodiments, said MUCl-binding molecule is displayed on the surface of said phage.
本发明还提供一种宿主细胞，选自：The present invention also provides a host cell selected from:
(1)表达本文任一实施方案所述MUC1结合分子；(1) expressing the MUCl binding molecule described in any embodiment herein;
(2)包含本文所述的多核苷酸；和/或(2) comprising a polynucleotide described herein; and/or
(3)包含本文所述的核酸构建物。(3) comprising a nucleic acid construct as described herein.
本发明还提供一种产生MUC1结合分子的方法，包括：在适合产生MUC1结合分子(例如单价或多价单域抗体、多特异性单域抗体、重链抗体、抗体或其抗原结合片段)的条件下培养本文所述的宿主细胞，和任选的从培养物中纯化所述MUC1结合分子。The present invention also provides a method for producing a MUCl-binding molecule, comprising: producing a MUCl-binding molecule (such as a monovalent or multivalent single-domain antibody, a multispecific single-domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof) suitable for producing a MUCl-binding molecule. The host cells described herein are grown under conditions, and the MUCl-binding molecule is optionally purified from the culture.
本发明还提供一种药物组合物，包含本文任一实施方案所述MUC1结合分子、多核苷酸、核酸构建物、噬菌体或宿主细胞，和药学上可接受的辅料。The present invention also provides a pharmaceutical composition, comprising the MUCl binding molecule, polynucleotide, nucleic acid construct, phage or host cell described in any embodiment herein, and pharmaceutically acceptable adjuvants.
在一个或多个实施方案中，所述药物组合物用于治疗癌症。In one or more embodiments, the pharmaceutical composition is used to treat cancer.
在一个或多个实施方案中，所述癌症是MUC1相关癌症。优选地，所述癌症选自：乳腺癌、肾癌、卵巢癌、胃癌、胰腺癌、肺癌、结肠癌、骨肉瘤、腺癌、膀胱癌、大肠癌、***、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、***癌或胶质母细胞瘤。In one or more embodiments, the cancer is a MUCl-associated cancer. Preferably, the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
本发明还提供本文任一实施方案所述MUC1结合分子在制备用于预防或治疗癌症的药物中的用途。The present invention also provides the use of the MUCl-binding molecule according to any embodiment herein in the preparation of a medicament for preventing or treating cancer.
在一个或多个实施方案中，所述癌症是MUC1相关癌症。优选地，所述癌症选自：乳腺癌、肾癌、卵巢癌、胃癌、胰腺癌、肺癌、结肠癌、骨肉瘤、腺癌、膀胱癌、大肠癌、***、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、***癌或胶质母细胞瘤。In one or more embodiments, the cancer is a MUCl-associated cancer. Preferably, the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
本发明还提供一种治疗或预防癌症的方法，所述方法包括给予需要的患者治疗有效量的本发明任一实施方案所述的MUC1结合分子，或含有本发明任一实施方案所述的MUC1结合分子的药物组合物。The present invention also provides a method for treating or preventing cancer, the method comprising administering to a patient in need a therapeutically effective amount of the MUCl-binding molecule described in any embodiment of the present invention, or containing the MUCl described in any embodiment of the present invention Pharmaceutical composition of binding molecules.
在一个或多个实施方案中，所述癌症是MUC1相关癌症。优选地，所述癌症选自：乳腺癌、肾癌、卵巢癌、胃癌、胰腺癌、肺癌、结肠癌、骨肉瘤、腺癌、膀胱癌、大肠癌、***、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、***癌或胶质母细胞瘤。In one or more embodiments, the cancer is a MUCl-associated cancer. Preferably, the cancer is selected from: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, cholangiocarcinoma, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
本发明还提供一种检测MUC1的试剂盒，用于评估药物治疗效果或诊断癌症，所述的试剂盒包含本文任一实施方案所述MUC1结合分子、多核苷酸、核酸构建物、 噬菌体、宿主细胞。The present invention also provides a kit for detecting MUCl, which is used for evaluating drug treatment effects or diagnosing cancer, and the kit includes MUCl binding molecules, polynucleotides, nucleic acid constructs, phages, and hosts described in any embodiment herein cell.
在一个或多个实施方案中，所述试剂盒还包括用于检测MUC1与单域抗体、抗体或其抗原结合片段的结合的试剂。例如通过酶联免疫反应法检测所述结合的试剂。In one or more embodiments, the kit further includes reagents for detecting binding of MUCl to a single domain antibody, antibody, or antigen-binding fragment thereof. The bound reagent is detected, for example, by enzyme-linked immunoassay.
在一个或多个实施方案中，所述检测结合试剂是能与MUC1结合分子连接的可检测标记物，例如生物素。所述的可检测标记物被连接于所述MUC1结合分子或分离地存在于试剂盒中。In one or more embodiments, the detection binding reagent is a detectable label, such as biotin, that can be attached to the MUCl binding molecule. The detectable label is attached to the MUCl binding molecule or present separately in a kit.
本发明还提供一种检测样品中MUC1存在情况的非诊断性方法，所述方法包括：以本文任一实施方案所述MUC1结合分子与样品孵育，和检测MUC1与单域抗体、抗体或其抗原结合片段的结合，从而确定样品中MUC1存在情况。所述检测是酶联免疫反应法检测。The present invention also provides a non-diagnostic method for detecting the presence of MUCl in a sample, the method comprising: incubating the sample with the MUCl binding molecule according to any of the embodiments herein, and detecting MUCl with a single domain antibody, antibody or antigen thereof The binding of the binding fragments to determine the presence of MUCl in the sample. The detection is an enzyme-linked immunoreaction method detection.
本发明还提供本文任一实施方案所述MUC1结合分子在制备用于检测样品中MUC1、评估药物治疗效果或诊断癌症的试剂盒中的用途。The present invention also provides the use of the MUCl-binding molecule according to any embodiment herein in the preparation of a kit for detecting MUCl in a sample, evaluating the effect of drug treatment or diagnosing cancer.
附图说明Description of drawings
图1为针对MUC1蛋白的羊驼抗血清效价检测结果。Figure 1 shows the titer detection results of alpaca antiserum against MUC1 protein.
图2为候选抗体与MUC1蛋白的ELISA检测结果。Figure 2 shows the results of ELISA detection of candidate antibodies and MUC1 protein.
图3为候选抗体与MUC1蛋白的亲和力检测结果。Figure 3 is the result of the affinity test between the candidate antibody and MUC1 protein.
图4为候选抗体与MB468肿瘤细胞株的结合检测结果。Fig. 4 is the detection result of the binding of the candidate antibody to the MB468 tumor cell line.
具体实施方式Detailed ways
本发明人经过广泛而深入地研究，经过大量的筛选，发现一类包含抗MUC1单域抗体的MUC1结合分子。本发明的MUC1结合分子能够高特异性地结合MUC1，具有较高的亲和力和生物活性，以及低的免疫原性，结构稳定，成药性良好。本发明单域抗体生成简便。After extensive and in-depth research and extensive screening, the inventors found a class of MUCl-binding molecules comprising anti-MUCl single domain antibodies. The MUC1-binding molecule of the invention can bind MUC1 with high specificity, has high affinity and biological activity, low immunogenicity, stable structure and good druggability. The single domain antibody of the present invention is easy to produce.
抗体Antibody
本文中，“MUC1结合分子”是特异性结合MUC1的蛋白质，包括但不仅限于：抗体、抗体的抗原结合片段、重链抗体、纳米抗体(nanobody)、微型抗体(minibody)、亲和体、受体的靶结合区、细胞粘附分子、配体、酶、细胞因子、和趋化因子。Herein, "MUC1-binding molecules" are proteins that specifically bind to MUC1, including but not limited to: antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies (nanobodies), minibodies (minibodies), affibodies, receptors Target binding domains of antibodies, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.
本文中，术语“抗体”包括单克隆抗体(包括全长抗体，其具有免疫球蛋白Fc区)，具有多表位特异性的抗体组合物，多特异性抗体(例如，双特异性抗体)，双抗体和单链分子，以及抗体片段，尤其是抗原结合片段，例如，Fab，F(ab’)2和Fv)。本文 中，术语“免疫球蛋白”(Ig)和“抗体”可互换地使用。As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), Diabodies and single chain molecules, as well as antibody fragments, especially antigen binding fragments, eg, Fab, F(ab')2 and Fv). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.
基本的4链抗体单元是由两条相同的轻链(L)和两条相同的重链(H)构成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元及称作J链的另外多肽组成，包含10个抗原结合位点；而IgA抗体包含2-5个基本的4链单元，其可与J链组合聚合形成多价装配物。在IgG的情况中，4链单元通常约150,000道尔顿。每条轻链通过一个共价二硫键与重链相连，而两条重链通过一个或多个二硫键彼此相连，二硫键的数目取决于重链的同种型。每条重链和轻链还具有间隔规律的链内二硫桥。每条重链在N-末端具有可变结构域(VH)，接着是三个(对于每种α和γ链，CH1、CH2和CH3)和四个(对于μ和ε同种型，CH1、CH2、CH3和CH4)恒定结构域(CH)以及位于CH1结构域与CH2结构域之间的绞链区(Hinge)。每条轻链在N-末端具有可变结构域(VL)，接着是其另一端的恒定结构域(CL)。VL与VH排列在一起，而CL与重链的第一恒定结构域(CH1)排列在一起。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。成对的VH和VL一起形成一个抗原结合位点。关于不同类别抗体的结构和性质，参见如Basic and Clinical Immunology，第八版，Daniel P.Sties，Abba I.Terr和Tristram G.Parsolw编辑，Appleton & Lange，Norwalk，CT，1994，第71页和第6章。来自任何脊椎动物物种的轻链，根据其恒定结构域氨基酸序列，可归入两种称作κ和λ的截然不同型中的一种。根据其重链恒定结构域(CH)氨基酸序列，免疫球蛋白可归入不同的类或同种型。有五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，分别具有称作α、δ、ε、γ和μ的重链。根据CH序列和功能的相对较小差异，γ和α类可进一步分为亚类，例如人表达下列亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called the J chain, which contains 10 antigen-binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with the J chain Polymerization forms multivalent assemblies. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH1, CH2 and CH3 for each α and γ chain) and four (CH1, CH1, CH2, CH3 and CH4) constant domain (CH) and the hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at its N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH, while CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic and Clinical Immunology, Eighth Edition, edited by Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can, based on their constant domain amino acid sequence, be assigned to one of two distinct types called kappa and lambda. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
本文所述“重链抗体”是源自骆驼科生物或软骨鱼科生物的抗体。相比上述4链抗体，重链抗体缺失轻链和重链恒定区1(CH1)，仅包含2条由可变区(VHH)和其他恒定区组成的重链，可变区通过类似铰链区结构与恒定区相连。骆驼科重链抗体的每条重链包含1个可变区(VHH)和2个恒定区(CH2和CH3)，软骨鱼科重链抗体的每条重链含有1个可变区和5个恒定区(CH1-CH5)。重链抗体的抗原结合片段包括VHH和单链重链抗体。通过与人IgG Fc的恒定区融合，重链抗体可以具有人IgG Fc的CH2和CH3。A "heavy chain antibody" as described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibodies, the heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), and only contain 2 heavy chains consisting of the variable region (VHH) and other constant regions. The variable region passes through a similar hinge region The structure is linked to the constant region. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous heavy chain antibody contains 1 variable region and 5 Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc.
如本文所用，术语“单域抗体”、“抗MUC1单域抗体”、“重链抗体的重链可变区结构域”、“VHH”、“纳米抗体”可互换使用，均指特异性识别和结合于MUC1的单域抗体。单域抗体是重链抗体的可变区。通常，单域抗体含有三个CDR 和四个FR。优选地，本发明的单域抗体具有SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。单域抗体是最小的功能性抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。As used herein, the terms "single domain antibody", "anti-MUCl single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", "Nanobody" are used interchangeably and refer to specific A single domain antibody that recognizes and binds to MUC1. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Preferably, the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3. Single domain antibodies are the smallest functional antigen-binding fragments. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
包含两条或多条单域抗体的结合分子是多价单域抗体；包含两条或多条不同特异性单域抗体的结合分子是多特异性单域抗体。多价单域抗体或多特异性单域抗体通过连接子连接多个单域抗体。所述连接子通常由选自G和S的1-15个氨基酸组成。A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificities is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies through a linker. The linker usually consists of 1-15 amino acids selected from G and S.
本文中，重链抗体和抗体旨在区分抗体的不同组合方式。由于二者的结构具有相似性，下述针对抗体的结构描述除涉及轻链外也均适用于重链抗体。Herein, heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to the similarity in the structures of the two, the following structural descriptions for antibodies are also applicable to heavy chain antibodies except for the light chain.
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基末端结构域。重链和轻链的可变结构域可分别称为“VH”和“VL”。这些结构域通常是抗体的最可变的部分(相对于相同类型的其它抗体)并含有抗原结合位点。"Variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
术语“可变的”指可变结构域中的某些区段在抗体序列中差异广泛的情况。可变结构域介导抗原结合并限定特定抗体对其特定抗原的特异性。然而，变异性并非均匀分布于可变结构域跨越的全部氨基酸。相反，其集中在三个称为高变区(HVR)的区段(在轻链和重链可变结构域中均有)，即分别为重链可变区的HCDR1、HCDR2、HCDR3(重链抗体中可简称为CDR1、CDR2、CDR3)以及轻链可变区的LCDR1、LCDR2和LCDR3。可变结构域中更为高度保守的部分称为构架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(FR1、FR2、FR3和FR4)，它们大多采取β-折叠构象，通过形成环状连接且在有些情况中形成β-折叠结构一部分的三个HVR连接。每条链中的HVR通过FR区非常接近的保持在一起，并与另一条链的HVR一起促成抗体的抗原结合位点的形成。通常，轻链可变区的结构为FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4，重链可变区的结构为FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如在抗体依赖性细胞介导的细胞毒性中抗体的参与。The term "variable" refers to the fact that certain segments of the variable domains vary widely among antibody sequences. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across all amino acids spanned by a variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 (heavy Chain antibodies may be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3 of the light chain variable region. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation connected by the formation of loops and in some cases forming β-sheet structures Part of three HVR connections. The HVRs in each chain are held together in close proximity by the FR regions and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site. Generally, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as the involvement of antibodies in antibody-dependent cell-mediated cytotoxicity.
“Fc区”(可结晶片段区域)或“Fc结构域”或“Fc”是指抗体重链的C-末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫***的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体***的第一组分(C1q)的结合。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的CH2结构域和CH3结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH结构域2-4)。虽然免疫球蛋白重链的Fc区的边界可以变化，但是 人IgG重链Fc区通常定义为从重链位置C226或P230的氨基酸残基到羧基端的序列段，其中该编号是根据EU索引，如在Kabat中一样。如本文所使用的，Fc区可以是天然序列Fc或变体Fc。"Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including those located in the immune system. Binding to Fc receptors on various cells (eg, effector cells), or to the first component (Clq) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE is present in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4). Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence stretch from the amino acid residue at positions C226 or P230 of the heavy chain to the carboxy-terminus, where this numbering is according to the EU index, as in Same in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.
“抗体片段”包含完整抗体的一部分，优选完整抗体的抗原结合区和/或可变区。抗体片段优选为抗体的抗原结合片段。抗体片段的例子包括Fab、Fab’、F(ab’)2和Fv片段；双抗体；线性抗体；单链抗体分子；scFv-Fc片段；由抗体片段形成的多特异性抗体；以及通过化学修饰或通过掺入脂质体中应能够增加半衰期的任何片段。用木瓜蛋白酶消化抗体产生称作“Fab”片段的两个相同的抗原结合片段，和一个残余“Fc”片段，其具有易于结晶的能力。Fab片段由完整轻链及重链可变结构域(VH)和一条重链第一恒定结构域(CH1)组成。每个Fab片段在抗原结合方面是单价的，即其具有单个抗原结合位点。胃蛋白酶处理抗体产生一个较大F(ab’)2片段，它粗略相当于两个通过二硫键相连的Fab片段，具有不同抗原结合活性且仍能够交联抗原。Fab’片段因在CH1结构域的羧基末端增加了一些另外的残基(包括来自抗体铰链区的一个或多个半胱氨酸)而与Fab片段有所不同。F(ab’)2抗体片段最初是作为成对Fab’片段生成的，在Fab’片段之间具有铰链半胱氨酸。抗体片段的其它化学偶联也是已知的。Fc片段包含通过二硫键保持在一起的两条重链的羧基末端部分。抗体的效应子功能是由Fc区中的序列决定的，该区还是由在某些类型细胞上发现的Fc受体(FcR)所识别的区。An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and/or variable region of an intact antibody. Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; Or any fragment that should be able to increase the half-life by incorporation into liposomes. Digestion of antibodies with papain yields two identical antigen-binding fragments, termed "Fab" fragments, and one residual "Fc" fragment, which has the ability to readily crystallize. The Fab fragment consists of the complete light chain and the variable domains of the heavy chain (VH) and the first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody yields a larger F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of the two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.
“Fv”是含有完整抗原识别和结合位点的最小抗体片段。该片段由紧密、非共价结合的一个重链可变结构域和一个轻链可变结构域的二聚体组成。从这两个结构域的折叠中突出了六个高变环(重链和轻链各3个环)，贡献出抗原结合的氨基酸残基并赋予抗体以抗原结合特异性。然而，即使是单个可变结构域(或只包含对抗原特异的三个HVR的半个Fv)也具有识别和结合抗原的能力，尽管亲合力低于完整结合位点。“单链Fv”也可缩写为“sFv”或“scFv”，是包含抗体VH和VL结构域的连接成一条多肽链的抗体片段。优选的是，sFv多肽在VH和VL结构域之间还包含多肽接头，使得sFv形成期望的抗原结合结构。"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) protrude from the fold of these two domains, contributing the amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower avidity than the full binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen-binding structure.
本文中，术语“单克隆抗体”指从一群基本上同质的抗体中获得的抗体，即除了可能以少量存在的可能的天然出现的突变和/或翻译后修饰(例如异构化、酰胺化)之外，构成群体的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。与多克隆抗体制剂(其典型地包括针对不同决定簇(表位)的不同抗体)相比，每个单克隆抗体针对抗原上的单个决定簇。除它们的特异性外，单克隆抗体的优势在 于它们通过杂交瘤培养合成，未受到其它免疫球蛋白的污染。修饰语“单克隆”表明抗体从基本上同质的抗体群获得的特征，不应解释为要求通过任何特定方法来生产抗体。例如，将根据本发明使用的单克隆抗体可通过多种技术来生成，包括例如杂交瘤法、噬菌体展示法、重组DNA法、及用于从具有部分或整个人免疫球蛋白基因座或编码人免疫球蛋白序列的基因的动物生成人或人样抗体的技术、单细胞测序法。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation, ), the individual antibodies constituting the population were identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody has acquired characteristics from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and the use of antibodies that have part or all of the human immunoglobulin loci or encode human immunoglobulin loci. A technique for producing human or human-like antibodies from the genes of immunoglobulin sequences, and single-cell sequencing.
单克隆抗体在本文中也包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性。Monoclonal antibodies also include herein "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remaining portions are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity.
非人(例如鼠)抗体的“人源化”形式指最低限度包含衍生自非人免疫球蛋白的序列的嵌合抗体。因此，“人源化抗体”通常指可变结构域构架区与在人抗体中发现的序列交换的非人抗体。通常在人源化抗体中，整个抗体(除CDR以外)由人来源的多核苷酸编码或与这种抗体相同(除CDR以外)。CDR(其中一些或全部由源自非人生物体的核酸编码)被移植到人抗体可变区的β-折叠骨架中以产生抗体，其特异性由被移植的CDR来决定。这类抗体的产生方法本领域周知，例如使用具有基因工程免疫***的小鼠而产生。本发明中，抗体、单域抗体、重链抗体等均包括各所述抗体的经人源化的变体。"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Thus, "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions have been exchanged with sequences found in human antibodies. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by or is identical to such an antibody (except for the CDRs) by a polynucleotide of human origin. CDRs, some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the β-sheet framework of human antibody variable regions to produce antibodies whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies and the like all include humanized variants of each of these antibodies.
“人抗体”指这样的抗体，其具有与由人生成的抗体的氨基酸序列对应的氨基酸序列和/或使用本文所公开的用于生成人抗体的任何技术产生。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。人抗体可使用本领域已知的多种技术来生成，包括噬菌体展示文库。A "human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or has been produced using any of the techniques disclosed herein for the production of human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
在一些实施方案中，本发明还提供与本发明的任何抗MUC1单域抗体结合MUC1相同表位的单域抗体、重链抗体、抗体或其抗原结合片段，即能够与本发明的任何单域抗体交叉竞争与MUC1的结合的单域抗体、重链抗体、抗体或其抗原结合片段。In some embodiments, the present invention also provides a single domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof that binds to the same epitope of MUCl as any anti-MUCl single domain antibody of the present invention, that is, capable of binding to any single domain of the present invention. Antibodies Cross-competing single domain antibodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof for binding to MUCl.
本发明中，单域抗体的CDR1包括SEQ ID NO:1所示的序列，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7X 8X 9X 10，其中，X 1为P、F、L、A或R，X 2为H、T、S或F，X 3为L、F、S、G、T或A，X 4为E、R、D、L或N，X 5为L、R、Y、N、D、Q、I或E，X 6为Y、H、L、E或I，X 7为A、Y、D、M、E或T，X 8为Y、E或无，X 9为Y或无，X 10为N或无。在一个或多个实施方案中，CDR1包含SEQ ID NO:4-13中任一所示的序列。 In the present invention, the CDR1 of the single domain antibody includes the sequence shown in SEQ ID NO: 1, and SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein, X 1 is P, F, L, A or R, X 2 is H, T, S or F, X 3 is L, F, S, G, T or A, X 4 is E, R, D, L or N , X 5 is L, R, Y, N, D, Q, I or E, X 6 is Y, H, L, E or I, X 7 is A, Y, D, M, E or T, X 8 Y, E or nothing, X9 is Y or nothing, X10 is N or nothing. In one or more embodiments, CDR1 comprises the sequence set forth in any one of SEQ ID NO: 4-13.
本发明中，单域抗体的CDR2包括SEQ ID NO:2所示的序列，SEQ ID NO:2是IX 1X 2X 3X 4X 5X 6X 7，其中，X 1为S、N或R，X 2为E、T、G、R或W，X 3为S、N、R、I、F或Y，X 4为G、D或N，X 5为D、G、S、A、E、T或无，X 6为D、T、S、R、P、L、W或无，X 7为T、I或无。在一个或多个实施方案中，CDR2包含SEQ ID NO:14-24中任一所示的序列。 In the present invention, the CDR2 of the single domain antibody includes the sequence shown in SEQ ID NO: 2, and SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is S, N or R, X2 is E, T, G, R or W, X3 is S, N, R, I, F or Y, X4 is G, D or N, X5 is D, G, S, A, E, T or none, X6 is D, T, S, R, P, L, W or none, X7 is T, I or none. In one or more embodiments, the CDR2 comprises the sequence set forth in any one of SEQ ID NO: 14-24.
本发明中，单域抗体的CDR3包括SEQ ID NO:3所示的序列，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20，其中，X 1为A、T或N，X 2为A或T，X 3为C、I、E、G或V，X 4为T、G、R、V或D，X 5为F、Y、T、I、A、N或P，X 6为Y、P、R、G、L或A，X 7为G、S、F、R、L或H，X 8为S、Q、I、T、W、K或Y，X 9为T、L、F、P、S或无，X 10为F、S、G、C、T或无，X 11为R、Y、T、L、S、C或无，X 12为E、D、C、F、Y或无，X 13为M、Y、V、L或无，X 14为G、P、H、A、T或无，X 15为Y、S、Q、R、W、V或无，X 16为N、R、T、M、P或无，X 17为F、E、A、Q或无，X 18为A、G、Y或无，X 19为S、D、R或无，X 20为Y或无。在一个或多个实施方案中，CDR3包含SEQ ID NO:25-35中任一所示的序列。 In the present invention, the CDR3 of the single domain antibody includes the sequence shown in SEQ ID NO: 3, and SEQ ID NO: 3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , wherein X 1 is A, T or N, X 2 is A or T, X 3 is C, I, E, G or V, X 4 is T, G, R, V or D, X 5 is F, Y, T, I, A, N or P, X 6 is Y, P, R, G, L or A, X 7 is G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 is T, L, F, P, S or none, X 10 is F, S, G, C , T or none, X 11 is R, Y, T, L, S, C or none, X 12 is E, D, C, F, Y or none, X 13 is M, Y, V, L or none, X14 is G, P, H, A, T or nothing, X15 is Y, S, Q, R, W, V or nothing, X16 is N, R, T, M, P or nothing, X17 is F, E, A, Q or none, X18 is A, G, Y or none, X19 is S, D, R or none, X20 is Y or none. In one or more embodiments, the CDR3 comprises the sequence set forth in any one of SEQ ID NO: 25-35.
在一个或多个实施方案中，所述单域抗体含有表1中组a1到组a11中任一组SEQ ID NO所示的CDR1、CDR2和CDR3：In one or more embodiments, the single domain antibody contains CDR1, CDR2 and CDR3 shown in any one of the SEQ ID NOs of group a1 to group a11 in Table 1:
表1Table 1
组Group CDR1CDR1 CDR2CDR2 CDR3CDR3
a1a1 44 1414 2525
a2a2 55 1515 2626
a3a3 66 1616 2727
a4a4 77 1717 2828
a5a5 88 1818 2929
a6a6 99 1919 3030
a7a7 1010 2020 3131
a8a8 1010 21twenty one 3232
a9a9 1111 22twenty two 3333
a10a10 1212 23twenty three 3434
a11a11 1313 24twenty four 3535
在一个或多个实施方案中，所述单域抗体VHH的FR1可选自SEQ ID NO:36-46的任一VHH的FR1，VHH的FR2可选自SEQ ID NO:36-46的任一VHH的FR2，VHH的FR3可选自SEQ ID NO:36-46的任一VHH的FR3，VHH的FR4可选自SEQ ID NO:36-46的任一VHH的FR4。In one or more embodiments, the FR1 of the single domain antibody VHH can be selected from FR1 of any VHH of SEQ ID NO:36-46, and the FR2 of VHH can be selected from any of SEQ ID NO:36-46 FR2 of VHH, FR3 of VHH can be selected from FR3 of any VHH of SEQ ID NO:36-46, and FR4 of VHH can be selected from FR4 of any VHH of SEQ ID NO:36-46.
在优选的实施方案中，本发明单域抗体VHH的FR区为选自SEQ ID NO:36-46的任一VHH的FR区。进一步优选地，这类抗体的CDR选自前述组a1到组a11中的任一组。在一个或多个实施方案中，所述单域抗体VHH如SEQ ID NO:36-46中任一所示。In a preferred embodiment, the FR region of the VHH of the single domain antibody of the present invention is selected from the FR region of any VHH of SEQ ID NO: 36-46. Further preferably, the CDRs of such antibodies are selected from any one of the aforementioned group a1 to group a11. In one or more embodiments, the single domain antibody VHH is as shown in any one of SEQ ID NO: 36-46.
本文所述MUC1结合分子可以是包含一条、两条或多条本文所述的抗MUC1单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。The MUCl binding molecules described herein may be monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof comprising one, two or more anti-MUCl single domain antibodies described herein, Antibodies or antigen-binding fragments thereof.
在一些实施方案中，本文的MUC1结合分子包含抗MUC1单域抗体和免疫球蛋白Fc区。可用于本发明的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。在一些实施方案中，免疫球蛋白Fc区为IgG4Fc，其氨基酸序列如SEQ ID NO:47所示。In some embodiments, a MUCl binding molecule herein comprises an anti-MUCl single domain antibody and an immunoglobulin Fc region. Fc regions useful in the present invention may be from different subtypes of immunoglobulins, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM. In some embodiments, the immunoglobulin Fc region is IgG4 Fc, the amino acid sequence of which is shown in SEQ ID NO:47.
在一些实施方案中，本文所述MUC1结合分子是重链抗体。所述重链抗体还包含重链恒定区，例如骆驼重链抗体或软骨鱼重链抗体的恒定区。优选地，所述重链恒定区如SEQ ID NO:47所示。In some embodiments, a MUCl binding molecule described herein is a heavy chain antibody. The heavy chain antibody also comprises a heavy chain constant region, such as that of a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody. Preferably, the heavy chain constant region is as shown in SEQ ID NO:47.
本发明还包括所述抗体衍生物和类似物。“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的衍生物或类似物可以是(i)在一个或多个氨基酸残基中具有取代基团的多肽，或(ii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iii)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些衍生物和类似物属于本领域熟练技术人员公知的范围。The present invention also includes said antibody derivatives and analogs. "Derivatives" and "analogues" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present invention. Derivatives or analogs of the present invention may be (i) polypeptides having substituent groups in one or more amino acid residues, or (ii) mature polypeptides in combination with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol A polypeptide formed by fusion of diol), or (iii) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify this polypeptide, or with a 6His tag formed fusion protein). Such derivatives and analogs are within the purview of those skilled in the art from the teachings herein.
在不实质性影响抗体活性的前提下，本领域技术人员可以对本发明的序列改变一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段序列的变体。这些变体包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。在本领域中，用性能相近或相似的氨基酸进 行保守性取代时，通常不会改变蛋白质的功能。如在可变区的FR和/或CDR区中将具有类似性质的氨基酸进行取代。可进行保守性取代的氨基酸残基为本领域所周知。这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。它们都被视为包括在本发明保护的范围内。Under the premise of not substantially affecting the activity of the antibody, those skilled in the art can change one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) of the sequence of the present invention. Multiple) amino acids to obtain variants of the antibody or functional fragment sequence thereof. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. In this field, conservative substitutions with amino acids with similar or similar properties usually do not change the function of the protein. Amino acids with similar properties are substituted eg in the FR and/or CDR regions of the variable region. Amino acid residues that may be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the protection scope of the present invention.
本文所述抗体的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and those capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions The protein encoded by DNA, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
在一些实施方案中，本发明所述变体的序列可以与其来源序列有至少有95％、96％、97％、98％或99％的一致性。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本发明还包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。In some embodiments, the sequence of the variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to its source sequence. Sequence identity according to the invention can be measured using sequence analysis software. For example the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The present invention also includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein .
可采用本领域常规的方法制备本发明的抗体，如本领域熟知的杂交瘤技术。可采用本领域常规的方法制备本发明的重链抗体，如本领域熟知的噬菌体展示技术。或者，本发明的抗体或重链抗体可在其他细胞系中表达。可用编码本发明抗体的序列转化合适的哺乳动物宿主细胞。转化可采用任何已知的方法进行，例如包括将多核苷酸包装在病毒(或病毒载体中)并用病毒(或载体)转导宿主细胞。所用的转化程序取决于将转化的宿主。用于将异源多核苷酸引入哺乳动物细胞中的方法为本领域所熟知，包括葡聚糖介导的转染、磷酸钙沉淀、聚凝胺介导的转染、原生质体融合、电穿孔、将多核苷酸囊封在脂质体中和将DNA直接微注射至核中等。可用作用于表达的宿主的哺乳动物细胞系为本领域所熟知，包括但不限于可从美国典型培养物保藏中心(ATCC)获得的多种永生化细胞系，包括但不限于中国仓鼠卵巢(CHO)细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，HepG2)等。尤其优选的细胞系通过确定哪些细胞系具有高表达水平并产生具有基本MUC1结合特性的抗体来进行选择。The antibody of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art. The heavy chain antibody of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, antibodies or heavy chain antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used will depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , Encapsulation of polynucleotides in liposomes and microinjection of DNA directly into nuclei, etc. Mammalian cell lines useful as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO ) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial MUCl binding properties.
核酸nucleic acid
本发明还提供了编码上述抗体或其片段的多核苷酸。本文提供编码重链可变区、轻链可变区、重链、轻链以及各CDR的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。The present invention also provides polynucleotides encoding the above antibodies or fragments thereof. Provided herein are polynucleotides encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and respective CDRs. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.
如本领域技术人员将了解，由于遗传密码的简并性，可制得极大量的核酸，它们全部编码本发明的抗体或其抗原结合片段。因此，在已鉴定特定氨基酸序列的情况下，本领域技术人员可通过以不改变编码蛋白质的氨基酸序列的方式简单地修饰一个或多个密码子的序列来制得任何数量的不同的核酸。所以，本发明还涉及与上述多核苷酸序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严谨条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严谨条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, extremely large numbers of nucleic acids can be produced, all of which encode antibodies of the invention or antigen-binding fragments thereof. Thus, having identified a particular amino acid sequence, one skilled in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the encoded protein. Therefore, the present invention also relates to polynucleotides that hybridize to the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
所以，本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的核酸构建物，例如表达载体和重组载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。载体通常含有用于质粒维系和用于克隆与表达外源性核苷酸序列的序列。所述序列(在某些实施方案中总称为“侧翼序列”)通常包括一个或多个以下核苷酸序列：启动子、一个或多个增强子序列、复制起点、转录终止序列、含有供体和受体剪接位点的完全内含子序列、编码用于多肽分泌的前导序列的序列、核糖体结合位点、聚腺苷酸化序列、用于***编码将要表达的抗体的核酸的多连接子区和可选标记元件。Therefore, the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. Vectors generally contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences (collectively referred to in certain embodiments as "flanking sequences") typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing and the complete intron sequence of the acceptor splice site, sequence encoding the leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, polylinker for insertion of nucleic acid encoding the antibody to be expressed zone and optional marker elements.
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙 门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。 Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
在上面的方法中的多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The polypeptide in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
治疗用途和药物组合物Therapeutic uses and pharmaceutical compositions
通过构建纳米抗体文库，发明人发现并表达纯化多个可以结合MUC1蛋白的纳米抗体。本文所述的抗体的所有方面都可用于制备用以预防或治疗本文所述各种病况和疾病的药物，所述病况和疾病尤其病况与表达MUC1的细胞相关的疾病或病况。在一些实施方案中，所述病况和疾病是癌症，包括但不限于：乳腺癌、肾癌、卵巢癌、胃癌、胰腺癌、肺癌、结肠癌、骨肉瘤、腺癌、膀胱癌、大肠癌、***、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、***癌或胶质母细胞瘤。By constructing a nanobody library, the inventors discovered, expressed and purified multiple nanobodies that can bind to the MUCl protein. All aspects of the antibodies described herein are useful in the manufacture of a medicament for the prevention or treatment of the various conditions and diseases described herein, especially those conditions or conditions associated with MUCl expressing cells. In some embodiments, the condition and disease is cancer, including but not limited to: breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, Cancer of the cervix, head and neck, fallopian tubes, multiple myeloma, bile ducts, gallbladder, esophagus, prostate, or glioblastoma.
本文的药物组合物含有本文所述结合分子，以及药学上可接受的辅料，包括但不限于稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂。辅料优选地在所采用的剂量和浓度下对接受者无毒。这类辅料包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的物质。这些物质为现有技术已知。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。The pharmaceutical compositions herein contain the binding molecules described herein, together with pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. The excipient is preferably nontoxic to recipients at the dosages and concentrations employed. Such excipients include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, a pharmaceutical composition may contain ingredients for improving, maintaining or retaining, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The rate of release, absorption or penetration of a substance. These substances are known from the prior art. The optimum pharmaceutical composition will be determined by the intended route of administration, mode of delivery and desired dosage.
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。可选择本发明的药物组合物用于肠胃外递送。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术内。其它药物组合物将为本领域技术人员显而易见，包括在持续或控制释放递送配制物中包含抗体的配制物。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。Pharmaceutical compositions for in vivo administration are generally presented as sterile preparations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, this method can be used for sterilization either before or after lyophilization and reconstitution. Pharmaceutical compositions of the invention may be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliary agents. Parenteral compositions are usually presented in containers with sterile access ports, eg, intravenous solution strips or vials with a hypodermic needle-punctureable stopper. Alternatively, compositions may be selected for inhalation or delivery through the alimentary tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating various other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以脱水或冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在施用前复水的形式(例如，冻干)。本发明还提供用于产生单剂量施用单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals or as dehydrated or lyophilized powders. The formulations can be stored in a ready-to-use form or reconstituted (eg, lyophilized) before administration. The invention also provides kits for producing single dosage administration units. The kits of the invention may each contain a first container with a dry protein and a second container with an aqueous formulation. In certain embodiments of the invention, kits containing single and multi-lumen prefilled syringes (eg, liquid syringes and lyophilized syringes) are provided.
本发明也提供通过施用本发明任一实施方案所述的结合分子或其药物组合物来治疗患者(尤其是患者的MUC1相关疾病)的方法。本文中，术语“患者”、“受试者”、“个体”、“对象”在本文中可互换使用，包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。“治疗”指向受试者采用本文所述治疗方案以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。有效治疗患者的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。The present invention also provides a method for treating a patient, especially a MUCl-associated disease in a patient, by administering a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof. Herein, the terms "patient", "subject", "individual", "subject" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g. rat, mouse, dog , cats, rabbits, etc.), and most preferably humans. "Treatment" refers to the subject's use of the treatment regimens described herein to achieve at least one positive therapeutic effect (e.g., a decrease in cancer cell number, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into surrounding organs, or a decrease in tumor metastasis or tumor growth. speed reduction). Therapeutic regimens that effectively treat a patient can vary depending on factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.
将采用的含有本发明结合分子的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平将部分取决于所递送的分子、适应症、施用途径和患者的大小(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方案中，临床医生可滴定剂量并改变施用途径来获得最佳的治疗效果。例如每天约10微克/千克体重-约50毫克/千克体重。The therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the invention to be employed will depend, for example, on the extent and goal of the treatment. Those skilled in the art will appreciate that appropriate dosage levels for therapy will depend in part on the molecule being delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. conditions) vary. In certain embodiments, the clinician can titrate the dose and vary the route of administration to achieve optimal therapeutic effect. For example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
给药频率将取决于所用配制物中结合分子的药物动力学参数。临床医生典型地施用组合物直到达到实现所需效果的剂量。组合物因此可作为单次剂量施用，或随时 间以作为两次或多次剂量(可含有或不含有相同量的所需分子)施用，或通过植入装置或导管以连续输液的方式施用。The frequency of dosing will depend on the pharmacokinetic parameters of the binding molecule in the formulation used. The clinician typically administers the composition until a dosage is reached to achieve the desired effect. The composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
药物组合物的施用途径是根据已知方法，例如经口、通过静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射；通过持续释放***或通过植入装置。The route of administration of the pharmaceutical composition is according to known methods, such as oral, injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Either by a sustained release system or by an implanted device.
诊断、检测和试剂盒Diagnostics, Tests and Kits
本发明的结合分子因其与MUC1的高亲合力可用于测定，例如结合测定来检测和/或定量在组织或细胞中表达的MUC1。结合分子例如单域抗体可用在进一步研究MUC1在疾病中的作用的研究中。检测MUC1的方法大致如下：获得细胞和/或组织样本；检测样本中MUC1的水平。Due to their high affinity for MUCl, the binding molecules of the invention are useful in assays, such as binding assays, to detect and/or quantify MUCl expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in studies to further investigate the role of MUCl in disease. The method for detecting MUC1 is generally as follows: obtain a cell and/or tissue sample; detect the level of MUC1 in the sample.
本发明的MUC1结合分子可用于诊断目的，用来检测、诊断或监控与MUC1相关的疾病和/或病况。本发明提供使用本领域技术人员已知的经典免疫组织学方法检测样本中MUC1的存在。可以体内或体外进行MUC1的检测。适用于检测MUC1的存在的方法实例包括ELISA、FACS、RIA等。The MUCl-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with MUCl. The present invention provides detection of the presence of MUCl in a sample using classical immunohistological methods known to those skilled in the art. Detection of MUCl can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of MUCl include ELISA, FACS, RIA, and the like.
对于诊断应用来说，通常用可检测的标记基团来标记结合分子例如单域抗体。合适的标记基团包括(但不限于)以下：放射性同位素或放射性核素(例如，3H、14C、15N、35S、90Y、99Tc、111In、125I、131I)、荧光基团(例如，FITC、罗丹明、镧系元素磷光体)、酶促基团(例如，辣根过氧化物酶、β根半乳糖苷酶、荧光素酶、碱性磷酸酶)、化学发光基团、生物素基基团或由二级报导体识别的预定多肽表位(例如，亮氨酸拉链对序列、用于二级抗体的结合位点、金属结合结构域、表位标签)、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂。用于标记蛋白质的各种方法在本领域中已知且可用来进行本发明。For diagnostic applications, binding molecules such as single domain antibodies are typically labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (e.g., FITC, Rhodane luminescent, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups Or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (Computed X-ray tomography) contrast agent. Various methods for labeling proteins are known in the art and can be used to carry out the present invention.
本发明的另一方面提供检测与本发明的抗体竞争结合MUC1的测试分子的存在的方法。一种所述测定的实例将涉及在存在或不存在测试分子的情形下检测含有一定量MUC1的溶液中的游离抗体的量。游离抗体(即，未结合MUC1的抗体)的量增加将表示测试分子能与该抗体竞争结合MUC1。在一个实施方案中，用标记基团标记抗体。或者，标记测试分子并在存在或不存在抗体的情形下监控游离测试分子的量。Another aspect of the invention provides methods for detecting the presence of a test molecule that competes with an antibody of the invention for binding to MUCl. An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of MUCl in the presence or absence of a test molecule. An increase in the amount of free antibody (ie, antibody that does not bind MUCl) will indicate that the test molecule is able to compete with the antibody for MUCl binding. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
本发明还提供了用于检测MUC1水平的检测试剂盒，该试剂盒包括识别MUC1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。The present invention also provides a detection kit for detecting the level of MUCl, which includes an antibody that recognizes the MUCl protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers, detection labeling, detection substrates, etc. The test kit may be an in vitro diagnostic device.
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的材料和方法。The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples, unless otherwise stated, are conventional materials and methods in the art.
实施例Example
实施例1，羊驼免疫Embodiment 1, alpaca immunization
1.1免疫原制备：1.1 Immunogen preparation:
根据NCBI上查询MUC1蛋白序列，与人IgG Fc片段序列进行融合，委托苏州金唯智公司进行合成构建pCDNA3.4(Thermo)质粒的真核表达载体，将合成后的质粒利用EXpiCHOTM(Thermo Fisher)表达***表达，表达后用5mL的Protein A预装柱(GE)进行一步亲和纯化，将纯化后样品置换入PBS缓冲液中，经SDS-PAGE电泳凝胶与HPLC鉴定纯度，ELISA鉴定活性后，分装冻存于-80度冰箱用于后续免疫。According to the MUC1 protein sequence queried on NCBI, it was fused with the human IgG Fc fragment sequence, and Suzhou Jinweizhi Company was entrusted to synthesize and construct the eukaryotic expression vector of pCDNA3.4 (Thermo) plasmid, and the synthesized plasmid was expressed by EXpiCHOTM (Thermo Fisher) System expression. After expression, a 5mL Protein A prepacked column (GE) was used for one-step affinity purification. The purified sample was replaced into PBS buffer. The purity was identified by SDS-PAGE electrophoresis gel and HPLC, and the activity was identified by ELISA. Aliquots were stored in a -80°C refrigerator for subsequent immunization.
1.2羊驼免疫：1.2 Alpaca immunity:
首次免疫抗原(MUC1-hFc)量为1000μg，与佐剂(GERBU FAMA)混匀，选取羊驼背部皮下四点注射免疫，每点免疫量为1mL。间隔3周进行第2次免疫。第2至9次免疫：免疫抗原量为500μg，选取羊驼背部皮下四点注射免疫，每点免疫量为1mL，每次免疫间隔时间为一周。The amount of the first immunization antigen (MUC1-hFc) was 1000 μg, mixed with the adjuvant (GERBU FAMA), and the back of the alpaca was selected for subcutaneous injection at four points, and the amount of each point was 1 mL. The second immunization was performed at intervals of 3 weeks. The 2nd to 9th immunizations: the amount of immunizing antigen is 500 μg, and four subcutaneous injections are selected on the back of the alpaca for immunization, the amount of immunization at each point is 1 mL, and the interval between each immunization is one week.
1.3免疫血清效价检测：1.3 Immune serum titer detection:
1.3.1蛋白水平效价检测1.3.1 Detection of protein level titer
4度过夜包被MUC1-His抗原，封闭洗涤后，将梯度稀释的血清加入ELISA板进行孵育后，再使用anti-llama IgG HRP(Abcam)抗体进行孵育，洗涤后加入TMB显色液显色，用2M HCl终止反应，然后用酶标仪检测OD450纳米处吸光值。实验结果如图1所示，经过5次免疫后羊驼效价达到较高水平(>72,9000)。Coat the MUC1-His antigen overnight at 4 nights. After blocking and washing, add the serially diluted serum to the ELISA plate for incubation, and then use anti-llama IgG HRP (Abcam) antibody for incubation. After washing, add TMB chromogenic solution to develop color. The reaction was terminated with 2M HCl, and then the absorbance at OD450 nm was detected with a microplate reader. The experimental results are shown in Figure 1. After 5 times of immunization, the alpaca titer reached a relatively high level (>72,9000).
实施例2，针对MUC1的纳米抗体免疫文库的构建及筛选Example 2, Construction and Screening of Nanobody Immune Library against MUC1
(1)5次免疫结束后，提取骆驼外周血淋巴细胞50mL并提取总RNA。RNA的提取参照TAKARA公司RNAiso试剂说明书进行。(1) After 5 times of immunization, 50 mL of camel peripheral blood lymphocytes were extracted and total RNA was extracted. The extraction of RNA was carried out according to the instruction manual of RNAiso reagent from TAKARA company.
(2)以RNA为模板，oligo dT为引物，参照TAKARA公司反转录酶说明书合成cDNA第一链。(2) RNA was used as a template, oligo dT was used as a primer, and the first strand of cDNA was synthesized according to the reverse transcriptase instructions of TAKARA Company.
(3)采用PrimeSTAR高保真DNA聚合酶，经巢式PCR获得重链抗体的可变区编码基因。用巢式PCR扩增重链抗体的可变区片段：(3) Using PrimeSTAR high-fidelity DNA polymerase, the gene encoding the variable region of the heavy chain antibody was obtained by nested PCR. Amplify variable region fragments of heavy chain antibodies by nested PCR:
第一轮PCR：The first round of PCR:
上游引物：GTCCTGGCTGCTCTTCTACAAGGC(SEQ ID NO:48)Upstream primer: GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 48)
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO:49)Downstream primer: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 49)
扩增重链抗体引导肽和抗体CH2之间的片段，55℃退火，30个循环；回收约600bp的DNA片段，作为第二轮PCR的模板。Amplify the fragment between the leader peptide of the heavy chain antibody and antibody CH2, anneal at 55°C for 30 cycles; recover a DNA fragment of about 600 bp as a template for the second round of PCR.
第二轮PCR：The second round of PCR:
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO:50)Upstream primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 50)
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO:51)Downstream primer: GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 51)
扩增重链抗体FR1区和长、短铰链区之间的片段(长片段和短片段)，55℃退火，30个循环，回收目的片段，结果显示该片段的大小约为500bp，即纳米抗体基因电泳带约为500bp。Amplify the fragments (long fragments and short fragments) between the FR1 region of the heavy chain antibody and the long and short hinge regions, anneal at 55°C for 30 cycles, and recover the target fragment. The results show that the fragment is about 500bp in size, that is, the nanobody The gene electrophoresis band is about 500bp.
(4)将噬菌粒pME207和PCR扩增产物分别用Sfi I和Not I双酶切(NEB)，回收、定量后，以1∶3摩尔比，用T4DNA连接酶(TaKaRa)连接两个片段，在16℃，过夜连接。(4) Digest the phagemid pME207 and the PCR amplification product with Sfi I and Not I respectively (NEB), after recovery and quantification, connect the two fragments with T4 DNA ligase (TaKaRa) at a molar ratio of 1:3 , at 16 °C, overnight for ligation.
(5)连接产物经乙醇沉淀后，溶于100μL无菌水，分十次进行电穿孔转化大肠杆菌TG1。取100μL电击、培养后的菌液倍比稀释，涂布氨苄青霉素LB培养板，计算库容，其余部分全部涂布于氨苄青霉素2×YT培养板，37℃，倒置培养13～16h。用10mL，2×YT培养基将培养板上的菌苔刮洗后，加入终浓度25％甘油，分装，-80℃保存备用。库容的大小为4.3×10 9。为检测文库的***率，随机选取48个克隆做菌落PCR，结果显示***率已达到90％以上。 (5) After the ligation product was precipitated by ethanol, it was dissolved in 100 μL of sterile water and electroporated ten times to transform Escherichia coli TG1. Take 100 μL of the electroshocked and cultured bacteria solution and spread it on the ampicillin LB culture plate to calculate the storage capacity. The rest is spread on the ampicillin 2×YT culture plate and cultured upside down at 37°C for 13-16 hours. Scrape and wash the bacterial lawn on the culture plate with 10 mL of 2×YT medium, add glycerol with a final concentration of 25%, aliquot, and store at -80°C for later use. The size of the storage capacity is 4.3×10 9 . To test the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached over 90%.
(6)根据计算的库容量结果，接种10倍库容量的活细胞于200mL的2×YT(含2％葡萄糖，100μg/mL氨苄青霉素)，37℃，200r/min培养至OD600达0.5，按感染复数20∶1加入辅助噬菌体，37℃静置30min后，37℃，200r/min，30min。将培养物离心，用200mL的2×YT(含100μg/mL氨苄青霉素和50μg/mL卡那霉素)重悬沉淀，37℃，250r/min过夜培养后，8000rpm离心取上清，加入5×PEG/NaCl溶液，冰上放置60min，8000rpm离心30min，重悬沉淀于5mL的PBS中，即得到抗MUC1的单域抗体(VHH)免疫文库，取10μL测定滴度，其余分装于-80℃保存备用。(6) According to the calculated pool capacity results, inoculate living cells with 10 times the pool volume in 200 mL of 2×YT (containing 2% glucose, 100 μg/mL ampicillin), culture at 37°C and 200 r/min until the OD600 reaches 0.5, press The multiplicity of infection was 20:1 and the helper phage was added, and after standing at 37°C for 30min, 37°C, 200r/min, 30min. Centrifuge the culture, resuspend the pellet with 200mL of 2×YT (containing 100μg/mL ampicillin and 50μg/mL kanamycin), cultivate overnight at 37°C, 250r/min, centrifuge at 8000rpm to get the supernatant, add 5× PEG/NaCl solution, put it on ice for 60min, centrifuge at 8000rpm for 30min, resuspend the pellet in 5mL of PBS, and obtain the anti-MUC1 single domain antibody (VHH) immune library, take 10μL to measure the titer, and put the rest at -80℃ Save for later.
(7)将MUC1蛋白按5μg/mL，100μL每孔包被在酶标板上，4℃放置过夜，同 时设立负对照。第二天五个孔中分别加入200μL，3％BSA，室温封闭2小时。2小时后用PBST(PBS中含有0.05％吐温20)洗3遍。洗板后先在每个负筛孔加入100μL用5％脱脂牛奶预封闭的噬菌体(2～3×10 11tfu免疫骆驼纳米抗体噬菌展示基因库)，在室温下作用1.5小时，再将负筛后上清转移至目的抗原包被孔中，室温下作用1.5小时。用PBST(PBS中含有0.05％吐温20)洗12遍，以洗掉不结合的噬菌体。用Glycine(SIGMA)将与MUC1特异性结合的噬菌体解离下，洗脱的噬菌体经Tris(Invitrogen，1M，PH 8.0)中和后感染处于对数期的TG1，经繁殖扩增，进行下一轮“吸附-洗脱”。最后洗脱下的噬菌体浸染TG1，利用IPTG(Thermo)诱导TG1表达纳米抗体，取TG1表达的上清做ELISA结合检测与阻断活性检测。ELISA结合检测使用二抗为anti-c-myc Antibody HRP(Bethyl)，阻断Biotinylated Human B7-1/CD80 Protein,Fc分子(Acrobiosystem)结合检测使用二抗为HRP标记的链亲和素(Thermo)。同时取TG1表达的上清在过表达293T的MUC1细胞上做FACS结合检测，一抗用的是biotin-anti-his抗体(金斯瑞)，荧光抗体使用的是PE Streptavidin(Biolegend)。 (7) Coat MUC1 protein at 5 μg/mL, 100 μL per well on a microtiter plate, place at 4°C overnight, and set up a negative control at the same time. On the second day, 200 μL of 3% BSA was added to five wells respectively, and blocked for 2 hours at room temperature. After 2 hours, wash 3 times with PBST (0.05% Tween 20 in PBS). After washing the plate, add 100 μL of phage pre-blocked with 5% skimmed milk (2-3×10 11 tfu immunized camel nanobody phage display gene library) to each negative sieve hole, and let it act for 1.5 hours at room temperature. After sieving, the supernatant was transferred to the target antigen-coated well, and reacted for 1.5 hours at room temperature. Wash 12 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phages. Glycine (SIGMA) was used to dissociate the phage that specifically binds to MUC1, and the eluted phage was neutralized by Tris (Invitrogen, 1M, PH 8.0) and then infected with TG1 in the logarithmic phase. After propagation and amplification, the next step was performed. round of "adsorption-elution". Finally, the eluted phage was infected with TG1, and IPTG (Thermo) was used to induce TG1 to express nanobody, and the supernatant of TG1 expression was taken for ELISA binding detection and blocking activity detection. ELISA binding detection uses anti-c-myc Antibody HRP (Bethyl) as the secondary antibody to block Biotinylated Human B7-1/CD80 Protein, Fc molecule (Acrobiosystem) binding detection uses HRP-labeled streptavidin (Thermo) as the secondary antibody . At the same time, the supernatant of TG1 expression was taken for FACS binding detection on 293T overexpressed MUC1 cells. The primary antibody was biotin-anti-his antibody (GenScript), and the fluorescent antibody was PE Streptavidin (Biolegend).
(8)通过序列分析后，结合ELISA和FACS检测结果，选择有阻断活性的11个纳米抗体作为候选抗体。(8) After sequence analysis, combined with ELISA and FACS detection results, 11 nanobodies with blocking activity were selected as candidate antibodies.
实施例3，候选抗体表达纯化Example 3, expression and purification of candidate antibodies
将纳米抗体构建到pCDNA3.4-IgG4载体上，构建成VHH-IgG4形式，然后经EXpiCHOTM(Thermo Fisher)表达***表达，表达一周后收取上清进行Protein A(GE)纯化。然后使用Nanodrop检测蛋白质量，HPLC检测蛋白纯度。所得蛋白纯度及产量满足后续试验需要。The nanobody was constructed on the pCDNA3.4-IgG4 vector, constructed in the form of VHH-IgG4, and then expressed by the EXpiCHOTM (Thermo Fisher) expression system. After one week of expression, the supernatant was collected for Protein A (GE) purification. Then use Nanodrop to detect protein quantity, and HPLC to detect protein purity. The purity and yield of the obtained protein met the needs of subsequent experiments.
实施例4，候选抗体表征Example 4, Characterization of Candidate Antibodies
(1)蛋白水平ELISA检测：(1) Protein level ELISA detection:
使用酶联免疫吸附测定技术(ELISA)测定抗体对人MUC1-His抗原的亲和力。将MUC1抗原用包被液稀释至2μg/mL，100μL/孔加入96孔板条中，37℃孵育2小时。洗涤3次。洗涤完毕后，每个孔加入100μL 3％BSA封闭液，37℃静置孵育2小时。洗涤3次。加入100μL/孔MUC1抗体，抗体稀释起始浓度均为4μg/mL，4倍比稀释8个梯度，37℃孵育1小时。每板洗涤3次。每孔加100μL酶标二抗孵育30min。洗涤3次。每孔加入100μL TMB，显色5min。每孔加入100μL盐酸终止液终止反应，以450nm波长读板。检测结果如图2所示，MUC1抗体与MUC1 抗原具有良好的结合活性，由拟合曲线得知，EC50均小于1nM。The affinity of the antibody to human MUCl-His antigen was determined by enzyme-linked immunosorbent assay (ELISA). The MUC1 antigen was diluted to 2 μg/mL with coating solution, 100 μL/well was added to a 96-well strip, and incubated at 37°C for 2 hours. Wash 3 times. After washing, add 100 μL of 3% BSA blocking solution to each well, and incubate at 37°C for 2 hours. Wash 3 times. Add 100 μL/well of MUC1 antibody, the initial concentration of antibody dilution is 4 μg/mL, 4-fold dilution in 8 gradients, and incubate at 37°C for 1 hour. Wash each plate 3 times. Add 100 μL enzyme-labeled secondary antibody to each well and incubate for 30 min. Wash 3 times. Add 100 μL TMB to each well and develop color for 5 min. Add 100 μL hydrochloric acid stop solution to each well to stop the reaction, and read the plate at a wavelength of 450 nm. The test results are shown in Figure 2, the MUC1 antibody has good binding activity to the MUC1 antigen, and the fitted curves show that the EC50 is less than 1nM.
(2)蛋白水平亲和力检测：(2) Protein level affinity detection:
使用表面等离子共振技术(SPR)测定抗体对人MUC1-His抗原的结合动力学和亲和力。将纯化的抗体流经预先固定protein A的传感器芯片，抗体被protein A捕获，然后将5个不同浓度的MUC1.his蛋白作为流动相，结合时间和解离时间分别为30min和60min。使用Biacore Evaluation Software 2.0(GE)分析结合速率(ka)、解离速率(kd)和平衡常数(KD)。BMK1是根据GENUS oncology公司US 2016/0340442A1专利中的抗体序列SEQ ID NO:73和SEQ ID NO:75制备的scFv(VH-VL)形式的IgG4抗体，BMK2是根据Peptron公司抗体US20200024361A1专利中的序列SEQ ID NO:24和SEQ ID NO:25制备的scFv(VH-VL)形式IgG4抗体。各候选抗体的氨基酸序列如表4所示，各候选抗体的CDR序列如表5所示。蛋白水平亲和力检测结果如下表2所示，结合动力学曲线见图3。The binding kinetics and affinity of antibodies to human MUCl-His antigen were determined using surface plasmon resonance (SPR). The purified antibody was passed through the sensor chip with pre-immobilized protein A, the antibody was captured by protein A, and then 5 different concentrations of MUC1.his protein were used as the mobile phase, and the binding time and dissociation time were 30min and 60min, respectively. Association rates (ka), dissociation rates (kd) and equilibrium constants (KD) were analyzed using Biacore Evaluation Software 2.0 (GE). BMK1 is an IgG4 antibody in the form of scFv (VH-VL) prepared according to the antibody sequences SEQ ID NO:73 and SEQ ID NO:75 in the US 2016/0340442A1 patent of GENUS oncology company, and BMK2 is based on the sequence in the antibody US20200024361A1 patent of Peptron Company IgG4 antibodies in the form of scFv (VH-VL) prepared by SEQ ID NO: 24 and SEQ ID NO: 25. The amino acid sequence of each candidate antibody is shown in Table 4, and the CDR sequence of each candidate antibody is shown in Table 5. The protein level affinity test results are shown in Table 2 below, and the binding kinetic curve is shown in Figure 3.
表2.不同抗体亲和力检测结果Table 2. Affinity test results of different antibodies
Figure PCTCN2022091544-appb-000001
Figure PCTCN2022091544-appb-000001
(3)细胞水平结合活性检测(3) Detection of binding activity at the cell level
将表达MUC1蛋白的肿瘤细胞MB468铺于96孔板中，每孔3×10 5细胞，再将梯度稀释的候选抗体与MB468细胞于2～8℃孵育；半小时后，洗涤后加入检测抗体anti-human IgG PE(Jackson Immuno Research，Code：109-117-008)孵育，然后使用CytoFLEX流式细胞仪检测。Isotype为同型对照(阴性对照，序列来源于专利CN 106146653A的SEQ ID NO:2)。候选抗体的结果如图4所示，从图4可以看出，候选抗体对肿瘤细胞MB468存在高中低结合活性。其中，NBL502-1-C4，NBL502-EFP-B02，NBL502-EFP-B03，NBL502-1D2-2，NBL502-AI-201与肿瘤细胞MB468的结合活性更高，阳性率均大于90％。NBL502-1-A2，NBL502-AI-120，NBL502-AI-121，NBL502-AI-181，NBL502-A12-6-LST-D5-2与肿瘤细胞MB468的结合活性相对较弱，阳性率在45％-90％之间。NBL502-AI212与肿瘤细胞MB468的结合活性最弱。 Spread the tumor cells MB468 expressing MUC1 protein in a 96-well plate, 3×10 5 cells per well, and then incubate the MB468 cells with serially diluted candidate antibodies at 2-8°C; half an hour later, add the detection antibody anti after washing -human IgG PE (Jackson Immuno Research, Code: 109-117-008) was incubated, and then detected by CytoFLEX flow cytometry. Isotype is an isotype control (negative control, the sequence is derived from SEQ ID NO: 2 of patent CN 106146653A). The results of the candidate antibodies are shown in Figure 4. It can be seen from Figure 4 that the candidate antibodies have high, medium and low binding activities to the tumor cell MB468. Among them, NBL502-1-C4, NBL502-EFP-B02, NBL502-EFP-B03, NBL502-1D2-2, NBL502-AI-201 have higher binding activity to tumor cell MB468, and the positive rate is greater than 90%. The binding activity of NBL502-1-A2, NBL502-AI-120, NBL502-AI-121, NBL502-AI-181, NBL502-A12-6-LST-D5-2 to tumor cell MB468 is relatively weak, and the positive rate is 45 %-90%. The binding activity of NBL502-AI212 to tumor cell MB468 was the weakest.
实施例5，VHH抗体特异性验证Example 5, VHH Antibody Specific Verification
1)组织交叉反应1) Tissue cross-reaction
选取34种组织做冰冻切片，常温晾干后使用丙酮固定。使用内源性生物素阻断试剂盒(生工，E674001)的试剂A和试剂B进行封闭，将生物素标记的抗体样品孵育30min，洗涤后加入辣根过氧化物酶标记的链霉亲和素(Abcam，ab7403)孵育15min。使用DAB显色和苏木素复染，中性塑胶封片，自然风干后待镜检。2个阳性对照为分别为Anti-MUC1 antibody[SM3](来自于Abcam的ab22711)和BMK2，阴性对照为IgG4同型对照。34 tissues were selected for frozen sections, dried at room temperature and then fixed with acetone. Use reagent A and reagent B of the endogenous biotin blocking kit (Shenggong, E674001) to block, incubate the biotin-labeled antibody samples for 30 min, add horseradish peroxidase-labeled streptavidin after washing Primer (Abcam, ab7403) was incubated for 15 min. DAB was used for color development and hematoxylin counterstaining, and neutral plastic was used to seal the slides and allow them to be air-dried for microscopic examination. The two positive controls were Anti-MUC1 antibody[SM3] (ab22711 from Abcam) and BMK2, and the negative control was IgG4 isotype control.
检测结果见表3，结果表明：在所有测试的34种组织中，候选抗体与组织结合水平不高于阳性对照SM3和BMK2。MUC1候选抗体与正常组织无明显强结合，在多种关键组织如肺、肾、胰腺、胃、大脑等结果阴性或无明显结合，这些数据说明候选抗体具有很好的组织安全性。The test results are shown in Table 3, and the results show that among all the 34 tested tissues, the binding level of the candidate antibody to the tissue is not higher than that of the positive controls SM3 and BMK2. The MUC1 candidate antibody has no obvious strong binding to normal tissues, and the results in various key tissues such as lung, kidney, pancreas, stomach, and brain are negative or have no obvious binding. These data indicate that the candidate antibody has good tissue safety.
表3. 34种人体组织交叉反应试验结果汇总Table 3. Summary of 34 human tissue cross-reactivity test results
Figure PCTCN2022091544-appb-000002
Figure PCTCN2022091544-appb-000002
Figure PCTCN2022091544-appb-000003
Figure PCTCN2022091544-appb-000003
Figure PCTCN2022091544-appb-000004
Figure PCTCN2022091544-appb-000004
实施例6，序列汇总Example 6, Sequence Summary
表4.抗体序列汇总表Table 4. Antibody sequence summary table
Figure PCTCN2022091544-appb-000005
Figure PCTCN2022091544-appb-000005
Figure PCTCN2022091544-appb-000006
Figure PCTCN2022091544-appb-000006
表5.抗体CDR序列汇总表Table 5. Summary of antibody CDR sequences
Figure PCTCN2022091544-appb-000007
Figure PCTCN2022091544-appb-000007

Claims (13)

  1. 一种MUC1结合分子，包含抗MUC1单域抗体，所述单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1所示的序列、CDR2包括SEQ ID NO:2所示的序列、和CDR3包括SEQ ID NO:3所示的序列，其中，A MUC1 binding molecule, comprising an anti-MUC1 single domain antibody, the complementarity determining region CDR of said single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises SEQ ID NO:2 The sequence shown, and CDR3 include the sequence shown in SEQ ID NO: 3, wherein,
    SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7X 8X 9X 10，其中，X 1为P、F、L、A或R，X 2为H、T、S或F，X 3为L、F、S、G、T或A，X 4为E、R、D、L或N，X 5为L、R、Y、N、D、Q、I或E，X 6为Y、H、L、E或I，X 7为A、Y、D、M、E或T，X 8为Y、E或无，X 9为Y或无，X 10为N或无， SEQ ID NO:1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , wherein X 1 is P, F, L, A or R, X 2 is H, T, S Or F, X3 is L, F, S, G, T or A, X4 is E, R, D, L or N, X5 is L, R, Y, N, D, Q, I or E, X 6 is Y, H, L, E or I, X 7 is A, Y, D, M, E or T, X 8 is Y, E or none, X 9 is Y or none, X 10 is N or none ,
    SEQ ID NO:2是IX 1X 2X 3X 4X 5X 6X 7，其中，X 1为S、N或R，X 2为E、T、G、R或W，X 3为S、N、R、I、F或Y，X 4为G、D或N，X 5为D、G、S、A、E、T或无，X 6为D、T、S、R、P、L、W或无，X 7为T、I或无， SEQ ID NO:2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is S, N or R, X 2 is E, T, G, R or W, X 3 is S, N, R, I, F or Y, X 4 is G, D or N, X 5 is D, G, S, A, E, T or none, X 6 is D, T, S, R, P, L , W or nothing, X 7 is T, I or nothing,
    SEQ ID NO:3是SEQ ID NO:3 is
    X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20，其中，X 1为A、T或N，X 2为A或T，X 3为C、I、E、G或V，X 4为T、G、R、V或D，X 5为F、Y、T、I、A、N或P，X 6为Y、P、R、G、L或A，X 7为G、S、F、R、L或H，X 8为S、Q、I、T、W、K或Y，X 9为T、L、F、P、S或无，X 10为F、S、G、C、T或无，X 11为R、Y、T、L、S、C或无，X 12为E、D、C、F、Y或无，X 13为M、Y、V、L或无，X 14为G、P、H、A、T或无，X 15为Y、S、Q、R、W、V或无，X 16为N、R、T、M、P或无，X 17为F、E、A、Q或无，X 18为A、G、Y或无，X 19为S、D、R或无，X 20为Y或无。 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , wherein, X 1 is A, T or N, X2 is A or T, X3 is C, I, E, G or V, X4 is T, G, R, V or D, X5 is F, Y, T, I, A, N or P, X 6 is Y, P, R, G, L or A, X 7 is G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 is T, L, F, P, S or none, X 10 is F, S, G, C, T or none, X 11 is R, Y, T, L, S, C or none, X 12 is E , D, C, F, Y or none, X 13 is M, Y, V, L or none, X 14 is G, P, H, A, T or none, X 15 is Y, S, Q, R, W, V or nothing, X 16 is N, R, T, M, P or nothing, X 17 is F, E, A, Q or nothing, X 18 is A, G, Y or nothing, X 19 is S, D, R or none, X 20 is Y or none.
  2. 如权利要求1所述的MUC1结合分子，其特征在于，所述单域抗体的CDR1包含SEQ ID NO:4-13中任一所示的序列，CDR2包含SEQ ID NO:14-24中任一所示的序列，和CDR3包含SEQ ID NO:25-35中任一所示的序列，The MUCl binding molecule according to claim 1, wherein the CDR1 of the single domain antibody comprises the sequence shown in any of SEQ ID NO:4-13, and CDR2 comprises any of SEQ ID NO:14-24 the sequence shown, and CDR3 comprises the sequence shown in any one of SEQ ID NO:25-35,
    优选地，所述单域抗体含有以下组a1到组a11中任一组的SEQ ID NO所示的CDR1、CDR2和CDR3：Preferably, the single domain antibody contains CDR1, CDR2 and CDR3 shown in the SEQ ID NO of any one of the following group a1 to group a11:
    组Group CDR1CDR1 CDR2CDR2 CDR3CDR3 a1a1 44 1414 2525 a2a2 55 1515 2626 a3a3 66 1616 2727 a4a4 77 1717 2828
    a5 8 18 29 a6 9 19 30 a7 10 20 31 a8 10 21 32 a9 11 22 33 a10 12 23 34 a11 13 24 35
    。     a5 8 18 29 a6 9 19 30 a7 10 20 31 a8 10 twenty one 32 a9 11 twenty two 33 a10 12 twenty three 34 a11 13 twenty four 35
    .
  3. 如权利要求1或2所述的MUC1结合分子，其特征在于，MUCl binding molecule as claimed in claim 1 or 2, is characterized in that,
    所述单域抗体的FR区包含选自SEQ ID NO:36-46的任一VHH的FR区，和/或The FR region of the single domain antibody comprises a FR region selected from any VHH of SEQ ID NO: 36-46, and/or
    所述单域抗体VHH如SEQ ID NO:36-46中任一所示，或与如SEQ ID NO:36-46中任一的氨基酸序列具有至少80％、90％、95％、96％、97％、98％或99％同一性，和/或The single domain antibody VHH is as shown in any of SEQ ID NO:36-46, or has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and/or
    所述MUC1结合分子是包含一条、两条或多条所述单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。The MUCl binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof comprising one, two or more of the single-domain antibodies .
  4. 一种多核苷酸，其特征在于，所述多核苷酸选自：A polynucleotide, characterized in that, the polynucleotide is selected from:
    (1)权利要求1-3中任一项所述的MUC1结合分子的编码序列；(1) the coding sequence of the MUCl binding molecule described in any one of claims 1-3;
    (2)(1)的互补序列；(2) the complementary sequence of (1);
    (3)(1)或(2)中任一序列的5-50bp的片段。(3) A 5-50 bp fragment of any sequence in (1) or (2).
  5. 一种核酸构建物，其特征在于，所述核酸构建物包含权利要求4所述的多核苷酸；优选地，所述核酸构建物是重组载体或表达载体。A nucleic acid construct, characterized in that the nucleic acid construct comprises the polynucleotide according to claim 4; preferably, the nucleic acid construct is a recombinant vector or an expression vector.
  6. 包含权利要求1-3中任一项所述的MUC1结合分子的噬菌体；优选地，所述MUC1结合分子展示于所述噬菌体表面。A phage comprising the MUCl-binding molecule according to any one of claims 1-3; preferably, the MUCl-binding molecule is displayed on the surface of the phage.
  7. 一种宿主细胞，其特征在于，所述宿主细胞：A host cell, characterized in that the host cell:
    (1)表达权利要求1-3中任一项所述的MUC1结合分子；和/或(1) expressing the MUCl binding molecule described in any one of claims 1-3; and/or
    (2)包含权利要求4所述的多核苷酸；和/或(2) comprising the polynucleotide of claim 4; and/or
    (3)包含权利要求5所述的核酸构建物。(3) comprising the nucleic acid construct of claim 5.
  8. 一种产生MUC1结合分子的方法，包括：在适合产生MUC1结合分子的条件下培养权利要求7所述的宿主细胞，和任选的从培养物中纯化所述MUC1 结合分子。A method of producing a MUCl-binding molecule, comprising: culturing the host cell of claim 7 under conditions suitable for producing a MUCl-binding molecule, and optionally purifying the MUCl-binding molecule from the culture.
  9. 一种药物组合物，包含权利要求1-3中任一项所述的MUC1结合分子、权利要求4所述的多核苷酸、权利要求5所述的核酸构建物、权利要求6所述的噬菌体或权利要求7所述的宿主细胞，和药学上可接受的辅料；优选地，所述药物组合物用于治疗癌症。A pharmaceutical composition comprising the MUCl binding molecule according to any one of claims 1-3, the polynucleotide according to claim 4, the nucleic acid construct according to claim 5, and the phage according to claim 6 Or the host cell according to claim 7, and a pharmaceutically acceptable auxiliary material; preferably, the pharmaceutical composition is used for treating cancer.
  10. 权利要求1-3中任一项所述的MUC1结合分子在制备用于预防或治疗癌症的药物中的用途。Use of the MUCl binding molecule according to any one of claims 1-3 in the preparation of a medicament for preventing or treating cancer.
  11. 一种检测MUC1的试剂盒，用于评估药物治疗效果或诊断癌症，所述的试剂盒包含权利要求1-3中任一项所述的MUC1结合分子、权利要求4所述的多核苷酸、权利要求5所述的核酸构建物、权利要求6所述的噬菌体或权利要求7所述的宿主细胞；A kit for detecting MUCl, used for evaluating drug treatment effect or diagnosing cancer, said kit comprising the MUCl binding molecule according to any one of claims 1-3, the polynucleotide according to claim 4, The nucleic acid construct of claim 5, the phage of claim 6 or the host cell of claim 7;
    优选地，所述试剂盒还包括用于检测MUC1与单域抗体、抗体或其抗原结合片段的结合的试剂；Preferably, the kit further includes reagents for detecting the binding of MUCl to single domain antibodies, antibodies or antigen-binding fragments thereof;
    更优选地，所述试剂是通过酶联免疫反应法检测所述结合的试剂。More preferably, said reagent is a reagent for detecting said binding by enzyme-linked immunoassay.
  12. 一种检测样品中MUC1存在情况的非诊断性方法，所述方法包括：以权利要求1-3中任一项所述的MUC1结合分子与样品孵育，和检测MUC1与单域抗体、抗体或其抗原结合片段的结合，从而确定样品中MUC1存在情况。A non-diagnostic method for detecting the presence of MUCl in a sample, the method comprising: incubating the sample with the MUCl binding molecule according to any one of claims 1-3, and detecting MUCl and single domain antibody, antibody or its The binding of the antigen-binding fragment to determine the presence of MUC1 in the sample.
  13. 权利要求1-3中任一项所述的MUC1结合分子在制备用于检测样品中MUC1、评估药物治疗效果或诊断癌症的试剂盒中的用途。Use of the MUCl binding molecule according to any one of claims 1-3 in the preparation of a kit for detecting MUCl in a sample, evaluating the effect of drug treatment or diagnosing cancer.
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