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WO2025026358A1 - Muc1结合分子和包含其的嵌合抗原受体 - Google Patents

Muc1结合分子和包含其的嵌合抗原受体 Download PDF

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Publication number
WO2025026358A1
WO2025026358A1 PCT/CN2024/108846 CN2024108846W WO2025026358A1 WO 2025026358 A1 WO2025026358 A1 WO 2025026358A1 CN 2024108846 W CN2024108846 W CN 2024108846W WO 2025026358 A1 WO2025026358 A1 WO 2025026358A1
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Prior art keywords
muc1
antibody
binding molecule
cells
seq
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PCT/CN2024/108846
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English (en)
French (fr)
Inventor
李加国
许文峰
王信浩
朱伟民
孙艳
钱其军
Original Assignee
浙江纳米抗体技术中心有限公司
上海乘黄纳米抗体科技有限公司
上海细胞治疗集团药物技术有限公司
上海细胞治疗集团股份有限公司
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Priority claimed from CN202310959241.8A external-priority patent/CN119431600A/zh
Priority claimed from CN202310958766.XA external-priority patent/CN119431584A/zh
Application filed by 浙江纳米抗体技术中心有限公司, 上海乘黄纳米抗体科技有限公司, 上海细胞治疗集团药物技术有限公司, 上海细胞治疗集团股份有限公司 filed Critical 浙江纳米抗体技术中心有限公司
Publication of WO2025026358A1 publication Critical patent/WO2025026358A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to a MUC1 binding molecule and a chimeric antigen receptor comprising the same.
  • CAR Chimeric antigen receptor
  • T cell therapy made from the patient's own immune cells, has brought a turnaround for some types of aggressive leukemia and other blood cancers.
  • This therapy uses genetic engineering technology to splice the variable region gene sequence of antibodies that can recognize tumor antigen molecules with the intracellular region sequence of T lymphocyte immune receptors, and then introduces these fusion gene sequences into the T lymphocyte genome through a lentiviral vector system or a transposon transposase system.
  • T cells can express CAR, recognize and attack specific antigens, and enhance their ability to kill tumor cells.
  • CAR-T cells can even cure people whose cancer has relapsed after multiple other treatments.
  • CAR-T cells are not yet able to achieve the effect of long-term survival for most people.
  • CAR-T therapy has achieved some success in treating blood cancers, there is still a long way to go in the treatment of solid tumors (such as pancreatic cancer, lung cancer or colorectal cancer).
  • MUC1 is a mucin expressed in a variety of epithelial cells and highly expressed in various solid tumors and hematological tumor cells. MUC1 is one of the most potential targets in the field of tumor treatment. Due to the differences in MUC1 oxygen glycosylation modification and the occurrence of 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. Tumor-associated MUC1 is different from MUC1 expressed by normal cells.
  • Nanobodies have natural advantages such as easy expression, high stability, and high affinity. Based on the advantages of nanoantibodies themselves and the biological mechanism of MUC1, the development of MUC1 nanoantibodies has very broad application prospects.
  • An object of the present disclosure is to provide MUCl binding molecules and chimeric antigen receptors comprising the same.
  • the first aspect of the present disclosure provides a MUC1 binding molecule, comprising an anti-MUC1 single domain antibody, wherein the complementarity determining region (CDR) of the single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises a sequence shown in any one of SEQ ID NOs: 1-10, CDR2 comprises a sequence shown in any one of SEQ ID NOs: 11-21, and CDR3 comprises a sequence shown in any one of SEQ ID NOs: 22-32.
  • CDR complementarity determining region
  • the single domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of Group a1 to Group a11 in Table 1 below:
  • the single domain antibody VHH is as shown in any one of SEQ ID NO: 33-43.
  • the present disclosure also performs mutations on the basis of the MUC1 single domain antibody (referred to as the wild-type single domain antibody) in the first aspect to obtain MUC1 single domain antibodies with different affinities.
  • the second aspect of the present disclosure also provides a MUC1 binding molecule, comprising a MUC1 single domain antibody, wherein the complementary determining region CDR of the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, and the MUC1 single domain antibody has one or more of the following characteristics (1)-(3):
  • the CDR1 contains one or more of the following mutations compared to SEQ ID NO: 2: T3Q, R5D, R5H, R5K, R5E, R5Y, R6E, R6H, R6K, R6D, R6Y, preferably contain one or more mutations selected from the following group: T3Q, R5D, R6E, R5H ⁇ R6H, R5K ⁇ R6K, R5D ⁇ R6D, R5E ⁇ R6E, R5Y ⁇ R6Y,
  • the CDR2 contains one or more of the following mutations compared to SEQ ID NO: 12: I1G, T3Q, T3W, F4D, D6A, D6G, D7S, T8Q, preferably one or more mutations selected from the following group: I1G, T3W, D6G, F4D ⁇ D6A, T3Q ⁇ T8Q, D6G ⁇ D7S,
  • the CDR3 contains one or more of the following mutations compared with SEQ ID NO: 23: T1Q, I3G, Y6H, Q8E, Q8Y, L9G, L9Y, L9S, S10N, D12Q, preferably contains one or more mutations selected from the following group: T1Q, Q8E, Q8Y, L9G, I3G ⁇ L9G, L9Y, L9S, 6H ⁇ S10N ⁇ D12Q.
  • the MUCl single domain antibody comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 contain mutations shown in any one of Groups A1 to A17 in Table 2 below:
  • the MUCl single domain antibody has one or more of the following characteristics:
  • the CDR1 is as shown in any one of SEQ ID Nos: 2, 49-56,
  • the CDR3 is shown in any one of SEQ ID NOs: 23, 63-70.
  • the MUC1 single domain antibody contains CDR1, CDR2 and CDR3 shown in any group of SEQ ID NOs from Group B1 to Group B18 in Table 3 below:
  • the amino acid sequence of the MUCl single domain antibody is as shown in any one of SEQ ID NOs: 71-87, preferably as shown in SEQ ID NOs: 71, 72, 78 or 83.
  • the MUCl binding molecule may comprise one, two or more anti-MUCl single domain antibodies described herein.
  • the MUCl binding molecule may be a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen binding fragment thereof.
  • the multivalent binding molecule or multispecific binding molecule connects multiple anti-MUC1 single domain antibodies or antigen-binding fragments thereof via a linker, wherein the linker consists of 1-15 amino acids selected from G and S.
  • the single domain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the single domain antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is a constant region of a human or camel heavy chain antibody, comprising CH2 and CH3.
  • the CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG4.
  • the heavy chain constant region is as shown in SEQ ID NO: 44.
  • the heavy chain constant region is a constant region of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4 and CH5.
  • the MUCl binding molecule described in any embodiment of the present disclosure is a chimeric antibody or a fully human antibody; preferably a fully human antibody.
  • the binding molecule is an antibody comprising the anti-MUCl single domain antibody as a heavy chain variable domain.
  • the binding molecule further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.
  • the antigen-binding fragment of the antibody is selected from Fab, F(ab')2, Fv, scFv.
  • the third aspect of the present disclosure also provides a fusion protein comprising the anti-MUC1 binding molecule according to any embodiment of the present disclosure and other polypeptides.
  • the additional polypeptide is located at the N-terminus and/or C-terminus of the binding molecule.
  • the other polypeptide is selected from a polypeptide that localizes the binding molecule to a different organelle, a tag for purification or a tag for immune response, a transmembrane protein or its transmembrane region, a chimeric antigen receptor component.
  • the fusion protein is a chimeric antigen receptor, whose antigen binding domain comprises the MUC1 binding molecule; preferably, the chimeric antigen receptor comprises: an optional signal peptide sequence, a MUC1 binding molecule containing a MUC1 single domain antibody, a hinge region, a transmembrane region, and an intracellular region.
  • the present disclosure also provides a nucleic acid molecule comprising a sequence selected from the following: (1) a coding sequence of a MUC1 binding molecule or fusion protein according to any embodiment of the present disclosure;
  • the coding sequence is DNA or RNA.
  • the present disclosure also provides a nucleic acid construct comprising the nucleic acid molecule described herein.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector or an integration vector.
  • the expression vector is a constitutive expression vector.
  • the constitutive expression vector is a transposable vector.
  • the nucleic acid construct is mRNA.
  • the mRNA comprises in the 5′-3′ direction: a 5′ cap structure, a 5′ UTR, an open reading frame (ORF), a 3′ UTR and Poly (A), wherein the open reading frame contains the sequence of the nucleic acid molecule.
  • the present disclosure also provides a host cell selected from:
  • the host cell is a T cell, a TIL cell, or a NK cell
  • the fusion protein is a chimeric antigen receptor
  • the cell contains: a coding sequence of the chimeric antigen receptor described in any embodiment of the present disclosure.
  • the cell contains mRNA encoding the chimeric antigen receptor.
  • the cell contains DNA encoding the chimeric antigen receptor, and preferably, the DNA of the chimeric antigen receptor is integrated into the genome of the cell.
  • the cells express and/or contain coding sequences for a therapeutic agent, which is an antibody (eg, a single chain antibody, a single domain antibody, a bispecific antibody) or a cytokine.
  • a therapeutic agent which is an antibody (eg, a single chain antibody, a single domain antibody, a bispecific antibody) or a cytokine.
  • the present disclosure also provides a method for producing a MUC1 binding molecule, comprising: culturing the host cells described herein under conditions suitable for producing a MUC1 binding molecule (e.g., a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, an antibody or an antigen-binding fragment thereof), and optionally purifying the MUC1 binding molecule from the culture.
  • a MUC1 binding molecule e.g., 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 present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the MUCl binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is used to treat cancer.
  • the cancer is a MUC1-related cancer.
  • the cancer is selected from the group consisting of 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, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present disclosure also provides use of the MUCl binding molecule described in any embodiment herein in the preparation of a medicament for preventing or treating cancer.
  • the cancer is a MUC1-related cancer.
  • the cancer is selected from the group consisting of 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, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present disclosure also provides a method for treating or preventing cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the MUCl binding molecule described in any embodiment of the present disclosure, or a pharmaceutical composition containing the MUCl binding molecule described in any embodiment of the present disclosure.
  • the cancer is a MUC1-related cancer.
  • the cancer is selected from the group consisting of 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, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.
  • the present disclosure also provides a kit for detecting MUC1, which is used for evaluating the effect of drug treatment or diagnosing cancer, wherein the kit comprises the MUC1 binding molecule, nucleic acid construct, bacteriophage, and host cell described in any embodiment of the present invention.
  • the kit further comprises a reagent for detecting the binding of MUCl to a single domain antibody, an antibody or an antigen-binding fragment thereof, for example, a reagent for detecting the binding by enzyme-linked immunosorbent assay.
  • the detection binding agent is a detectable label that can be linked to the MUC1 binding molecule, such as biotin.
  • the detectable label is linked to the MUC1 binding molecule or exists separately in the kit.
  • the present disclosure also provides a non-diagnostic method for detecting the presence of MUC1 in a sample, the method comprising: incubating the sample with a MUC1 binding molecule as described in any embodiment of the present invention, and detecting the presence of MUC1 binding to a single domain.
  • the antibody, antibody or antigen-binding fragment thereof is used to determine the presence of MUC1 in the sample.
  • the detection is an enzyme-linked immunosorbent assay.
  • the present disclosure also provides use of the MUCl binding molecule described in any embodiment herein in preparing a kit for detecting MUCl in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
  • FIG1 shows the titer test result of alpaca antiserum against MUC1 protein.
  • FIG. 2 shows the ELISA test results of the candidate antibodies and MUC1 protein.
  • FIG3 shows the affinity test results of the candidate antibodies and the MUC1 protein.
  • FIG. 4 shows the binding test results of the candidate antibodies and the MB468 tumor cell line.
  • Figure 5-6 shows the ELISA test results of candidate mutant antibodies and MUC1 protein.
  • FIG. 7 shows the binding test results of the candidate mutant antibodies and the MB468 tumor cell line.
  • FIG8 shows the binding test results of candidate mutant antibodies and H226 tumor cell lines.
  • FIG. 9 shows the binding test results of the candidate mutant antibodies and the RPMI8226 tumor cell line.
  • FIG. 10 shows the binding test results of candidate mutant antibodies and SKOV3 tumor cell lines.
  • Figure 11 shows the proliferation multiples of CAR-T cells.
  • FIG. 12 shows the cell viability during CAR-T cell proliferation.
  • FIG. 13 shows the CAR positivity rate of CAR-T cells on day 9.
  • Figure 14 shows the ratio of CD4 and CD8 of CAR-T cells on the 9th day.
  • FIG. 15 shows the time required for each group of CAR-T to kill 50% of MUC1 highly expressing tumor cells MB468 under different effector-target ratios.
  • FIG. 16 shows the 48-hour tumor lysis rate of each group of CAR-T cells for HCT116 tumor cells with extremely low MUC1 expression.
  • FIG. 17 shows the CAR positivity rate of the two donors after each round of co-culture.
  • FIG18 shows the theoretical proliferation multiples of the CAR-positive T cells of the two donors at the end of the fifth round compared to the number of CAR-positive cells initially inoculated.
  • Figure 19 shows the IFN- ⁇ secretion of CAR-T in each group in the first three rounds of the co-culture experiment.
  • Figure 20 shows the cell killing results of MUC1 CAR-T from two donors prepared by the JL system.
  • Figure 21 shows the cell viability and positive rate of CAR-T prepared by LNP delivery of mRNA.
  • Figure 22 shows the 24h transfection positivity rate of CAR-T and MOCKT cells prepared by LNP delivery of mRNA and electroporation.
  • Figure 23 shows the killing results of MAD-MB468 by different effector targets of CAR-T cells prepared by LNP and electroporation.
  • FIG. 24 shows the killing results of SKOV3 by different effector targets of CAR-T cells prepared by LNP and electroporation.
  • the inventors have conducted extensive and in-depth research and a large number of screenings and have discovered a class of MUC1 binding molecules comprising anti-MUC1 single domain antibodies.
  • the MUC1 binding molecules of the present disclosure can bind to MUC1 with high specificity, have high affinity and biological activity, as well as low immunogenicity, stable structure, and good drugability.
  • the present disclosure also performs mutations on the basis of the MUC1 single domain antibody to obtain a MUC1 single domain antibody with medium affinity to reduce nonspecific binding and reduce risks.
  • the present disclosure also provides chimeric antigen receptors and cells comprising the MUC1 single domain antibody.
  • MUC1 binding molecule is a protein that specifically binds to MUC1, including but not limited to antibodies, heavy chain antibodies, nanobodies or antigen-binding fragments thereof.
  • antibody includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv.
  • antibody and “immunoglobulin” are used interchangeably.
  • Antibodies contain a basic 4-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H).
  • 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, CH2, CH3 and CH4 for ⁇ and ⁇ isotypes) constant domains (CH) and a hinge region (Hinge) between the CH1 domain and the CH2 domain.
  • Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. Pairs of VH and VL together form an antigen binding site.
  • Light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • the gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, for example humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.
  • the "heavy chain antibody” described in this article is an antibody derived from camelids or chondrichthyes.
  • heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions through a hinge region-like structure.
  • 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 chondrichthyes heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5).
  • the heavy chain antibody can have the CH2 and CH3 of human IgG Fc.
  • single domain antibody As used herein, the terms “single domain antibody”, “anti-MUC1 single domain antibody”, “heavy chain variable region domain of heavy chain antibody”, “VHH”, and “nanoantibody” are used interchangeably and refer to single domain antibodies that specifically recognize and bind to MUC1.
  • a single domain antibody is a variable region of a heavy chain antibody.
  • a single domain antibody contains three CDRs and four FRs.
  • the single domain antibody of the present disclosure has a CDR1 shown in any one of SEQ ID NOs: 1-10, a CDR2 shown in any one of SEQ ID NOs: 11-21, and a CDR3 shown in any one of SEQ ID NOs: 22-32.
  • the single domain antibody of the present disclosure has a FR1 of any VHH of SEQ ID NOs: 33-43, a FR2 of any VHH of SEQ ID NOs: 33-43, a FR3 of any VHH of SEQ ID NOs: 33-43, and a FR4 of any VHH of SEQ ID NOs: 33-43.
  • the VHH obtained by recombining the CDR and FR is also within the scope of the present disclosure.
  • Single-domain antibodies are the smallest functional antigen-binding fragments. Usually, antibodies that naturally lack the light chain and heavy chain constant region 1 (CH1) are first obtained, and then 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
  • nanoantibody refers to an immune molecule based on VHH containing CDR or CDR combination described herein. It can be a heavy chain antibody as described above, or a multivalent or multispecific molecule containing multiple VHHs, or a recombinant molecule obtained by recombining VHH and antibody Fc (e.g., CH2 and CH3 or CH2, CH3 and CH4).
  • a binding molecule comprising two or more single domain antibodies of the same specificity is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificity is a multispecific single domain antibody.
  • the multispecificity can be directed against MUC1 and another antigen, or against two different epitopes of MUC1.
  • a multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies via a linker.
  • the linker is usually composed of 1-15 amino acids selected from G and S. amino acid composition.
  • heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies. Due to the similarity between the two structures, the following structural description of antibodies is also applicable to heavy chain antibodies in addition to light chains.
  • variable region refers to the amino terminal domain of the heavy or light chain of an antibody.
  • variable domains of the heavy and light chains may be referred to as "VH” and “VL”, respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable refers to the situation that certain segments in the variable domain differ widely in the antibody sequence.
  • the variable domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen.
  • the more highly conserved part of the variable domain is called the framework region (FR).
  • FR framework region
  • 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 domain does not directly participate in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of antibodies in antibody-dependent cell-mediated cytotoxicity.
  • variable region annotation schemes for antibodies including: Chothia, Kabat, IMGT and Contact.
  • the IMGT annotation scheme is used exemplarily herein.
  • Fc region fragment crystallizable region
  • Fc domain Fc domain
  • Fc refers to the C-terminal region of an antibody heavy chain that mediates the binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system.
  • the Fc region is composed of two identical protein fragments from the CH2 domain and the CH3 domain of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the human IgG heavy chain Fc region is generally defined as a sequence segment from an amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, wherein the numbering is according to the EU index, as in Kabat.
  • the Fc region may be a native sequence Fc or a variant Fc.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen binding region 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'), F(ab')2, Fd, and Fv fragments, disulfide-linked Fv; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes.
  • Antigen binding fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies, and expression by host cells containing antigen binding fragments.
  • Fv is the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. From the folding of these two domains protrude six hypervariable loops (3 loops each in the heavy and light chains) that contribute the amino acid residues for antigen binding. And give the antibody antigen binding specificity.
  • a single variable domain or half an Fv containing only three HVRs specific for an antigen
  • Single-chain Fv also abbreviated as “sFv” or “scFv”
  • sFv is an antibody fragment comprising the VH and VL domains of an antibody connected into a single polypeptide chain.
  • the sFv polypeptide also comprises a polypeptide linker between the VH and VL domains so that the sFv forms a desired antigen binding structure.
  • the term "monoclonal antibody” refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts, the individual antibodies constituting the group are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Compared with 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.
  • monoclonal antibodies are synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates that the antibody is obtained from a substantially homogeneous antibody group and should not be interpreted as requiring the production of antibodies by any particular method.
  • the monoclonal antibodies used according to the present disclosure can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having a partial or entire human immunoglobulin locus or genes encoding human immunoglobulin sequences, and single cell sequencing methods.
  • Monoclonal antibodies herein also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • a “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains at least a sequence derived from a non-human immunoglobulin. Therefore, a “humanized antibody” generally refers to a non-human antibody in which the variable domain framework region is exchanged with a sequence found in a human antibody. Typically in a humanized antibody, the entire antibody (except for the CDR) is encoded by a nucleic acid molecule of human origin or is identical to such an antibody (except for the CDR).
  • the CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are transplanted into the ⁇ -folded skeleton of the variable region of a human antibody to produce an antibody, the specificity of which is determined by the transplanted CDRs.
  • Methods for producing such antibodies are well known in the art, for example, using mice with a genetically engineered immune system.
  • antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of each of the antibodies.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody expressly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. In some embodiments, the present disclosure also provides for use with the present disclosure.
  • a single domain antibody, heavy chain antibody, antibody or antigen-binding fragment thereof that binds to the same epitope of MUC1 as any anti-MUC1 single domain antibody of the present disclosure that is, a single domain antibody, heavy chain antibody, antibody or antigen-binding fragment thereof that can cross-compete with any single domain antibody of the present disclosure for binding to MUC1.
  • CDR1 of a single domain antibody comprises a sequence shown in any one of SEQ ID NOs: 1-10;
  • CDR2 of a single domain antibody comprises a sequence shown in any one of SEQ ID NOs: 11-21;
  • CDR3 of a single domain antibody comprises a sequence shown in any one of SEQ ID NOs: 22-32.
  • the single domain antibody contains CDR1, CDR2 and CDR3 shown in any group of SEQ ID NOs from Group a1 to Group a11 in Table 1:
  • the FR1 of the single-domain antibody VHH can be selected from the FR1 of any VHH of SEQ ID NO: 33-43
  • the FR2 of VHH can be selected from the FR2 of any VHH of SEQ ID NO: 33-43
  • the FR3 of VHH can be selected from the FR3 of any VHH of SEQ ID NO: 33-43
  • the FR4 of VHH can be selected from the FR4 of any VHH of SEQ ID NO: 33-43.
  • the FR region of the single-domain antibody VHH of the present disclosure is the FR region of any VHH selected from SEQ ID NO: 33-43.
  • the CDR of such an antibody is selected from any group from group a1 to group a11 in Table 1.
  • the single-domain antibody VHH is as shown in any one of SEQ ID NO: 33-43.
  • the present disclosure also performs mutations on the above-mentioned MUC1 single domain antibody (referred to as wild-type single domain antibody) to obtain a MUC1 single domain antibody with desired affinity.
  • the present disclosure also provides a MUC1 binding molecule comprising a mutated anti-MUC1 single domain antibody.
  • a mutation is performed on a wild-type single-domain antibody having CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 23, and the mutation is located in the CDR region of the wild-type single-domain antibody, and may have one or more mutations.
  • one or more of the following mutations may be performed: T3Q, R5D, R5H, R5K, R5E, R5Y, R6E, R6H, R6K, R6D, R6Y, preferably containing one or more mutations selected from the following group: T3Q, R5D, R6E, R5H ⁇ R6H, R5K ⁇ R6K, R5D ⁇ R6D, R5E ⁇ R6E, R5Y ⁇ R6Y.
  • one or more of the following mutations may be performed: I1G, T3Q, T3W, F4D, D6A, D6G, D7S, T8Q, preferably containing one or more mutations selected from the following group: I1G, T3W, D6G, F4D ⁇ D6A, T3Q ⁇ T8Q, D6G ⁇ D7S.
  • one or more of the following mutations may be performed: T1Q, I3G, Y6H, Q8E, Q8Y, L9G, L9Y, L9S, S10N, D12Q, preferably containing one or more mutations selected from the following group: T1Q, Q8E, Q8Y, L9G, I3G ⁇ L9G, L9Y, L9S, 6H ⁇ S10N ⁇ D12Q.
  • the mutated MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 comprise mutations as shown in any one of Groups A1 to A17 in Table 2 below.
  • the mutated anti-MUC1 single domain antibody has reduced affinity for MUC1 compared to a wild-type single domain antibody having CDR1 as shown in SEQ ID NO: 2, CDR2 as shown in SEQ ID NO: 12 and CDR3 as shown in SEQ ID NO: 23.
  • the anti-MUC1 single domain antibody binds to MUC1 with a KD of about 1 ⁇ 10 -8 to 5 ⁇ 10 -7 M, preferably 1.5 ⁇ 10 -8 to 2 ⁇ 10 -7 M.
  • the KD is determined by surface plasmon resonance (SPR).
  • the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3 as shown in any one of Groups B1 to B17 in Table 3 below.
  • the FR region of the MUC1 single domain antibody can be the same as the FR region of the wild-type single domain antibody, or humanized and optimized.
  • the amino acid sequence of the MUC1 single domain antibody is as shown in any one of SEQ ID NOs: 71-87, preferably as shown in SEQ ID NOs: 71, 72, 78 or 83.
  • the MUC1 binding molecule described herein may comprise one, two or more anti-MUC1 single domain antibodies described herein.
  • the MUC1 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.
  • the MUC1 binding molecules herein comprise an anti-MUC1 single domain antibody and an immunoglobulin Fc region.
  • the Fc region useful in the present disclosure may be from immunoglobulins of different subtypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.
  • the immunoglobulin Fc region is IgG4 Fc, the amino acid sequence of which is shown in SEQ ID NO: 44.
  • the MUCl binding molecules described herein are heavy chain antibodies.
  • the heavy chain antibodies further comprise a heavy chain constant region, such as a constant region of a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the disclosure also includes the antibody derivatives and analogs.
  • “Derivatives” and “analogs” refer to polypeptides that substantially maintain the same biological function or activity of the antibodies of the disclosure.
  • the derivatives or analogs of the disclosure can be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by the fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by the fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify this polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these derivatives and analogs belong to the scope known to those skilled in the art.
  • those skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present disclosure.
  • variants of the antibody or its functional fragment sequence include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • amino acids with similar properties are substituted in the FR and/or CDR regions of the variable region.
  • Amino acid residues that can 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 generally does not change the function of the protein. They are all considered to be included in the scope of protection of the present disclosure.
  • Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize to DNA encoding the antibodies of the present disclosure under high or low stringency conditions, and polypeptides or proteins obtained using antisera against antibodies of the present disclosure.
  • the sequence of the variant described in the present disclosure can have at least 95%, 96%, 97%, 98% or 99% identity with its source sequence.
  • the sequence identity described in the present disclosure can be measured using sequence analysis software.
  • the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used.
  • the present disclosure also includes 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 antibodies of the present disclosure can be prepared by conventional methods in the art, such as hybridoma technology well known in the art.
  • the heavy chain antibodies of the present disclosure can be prepared by conventional methods in the art, such as phage display technology well known in the art.
  • the antibodies or heavy chain antibodies of the present disclosure can be expressed in other cell lines.
  • Suitable mammalian host cells can be transformed with sequences encoding antibodies of the present disclosure. Transformation can be carried out by any known method, for example, including packaging nucleic acid molecules in viruses (or viral vectors) and transducing host cells with viruses (or vectors). The transformation procedure used depends on the host to be transformed.
  • Methods for introducing heterologous nucleic acid molecules into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of nucleic acid molecules in liposomes, and direct microinjection of DNA into the nucleus, etc.
  • Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, a variety of 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 (e.g., HepG2), etc. Particularly preferred cell lines are identified by determining which cell lines have high expression levels and produce proteins with Antibodies with basic MUC1 binding properties were selected.
  • ATCC American Type Culture Collection
  • the present disclosure also includes fusion proteins containing the MUC1 binding molecules described herein and other polypeptides.
  • the other polypeptides are located at the N-terminus and/or C-terminus of the binding molecule.
  • the other polypeptides are selected from polypeptides that localize the binding molecule to different organelles, tags for purification or tags for immune response, transmembrane proteins or their transmembrane regions, chimeric antigen receptor components (extracellular domains, hinge regions, transmembrane regions, signal transduction domains, costimulatory domains, etc.).
  • the fusion protein is a chimeric antigen receptor, whose antigen binding domain comprises the MUC1 binding molecule.
  • the chimeric antigen receptor comprises: an optional signal peptide sequence, a MUC1 binding molecule containing a MUC1 single domain antibody, a hinge region, a transmembrane region, and an intracellular region.
  • the signal peptide includes a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide,
  • the hinge region includes a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region or an IgG4 Fc CH2CH3 hinge region,
  • the transmembrane region includes a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region or a DAP10 transmembrane region,
  • the intracellular region includes an intracellular co-stimulatory domain and/or an intracellular signaling domain.
  • the intracellular co-stimulatory domain includes the intracellular domain of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulator (ICOS) or DNAX activation protein 10,
  • the intracellular signaling domain includes a CD3 ⁇ intracellular signaling domain or a Fc ⁇ RI ⁇ intracellular signaling domain.
  • the present disclosure also provides nucleic acid molecules encoding the above-mentioned MUC1 binding molecules or the fusion proteins.
  • Nucleic acid molecules encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and each CDR are provided herein.
  • the nucleic acid molecules of the present disclosure can be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • RNA can be mRNA that expresses the binding molecule in vivo and/or in vitro.
  • the present disclosure also relates to nucleic acid molecules that hybridize to the above-mentioned nucleic acid molecule sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present disclosure particularly relates to nucleic acid molecules that can hybridize to the nucleic acid molecules described in the present disclosure under stringent conditions.
  • stringent conditions refers to nucleic acid molecules that can hybridize to the nucleic acid molecules described in the present disclosure under stringent conditions.
  • hybridization and elution at lower ionic strength and higher temperature such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.
  • the polypeptide encoded by the hybridizable nucleic acid molecule has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present disclosure or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the host cell after proliferation by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present disclosure include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present disclosure (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present disclosure by chemical synthesis.
  • the present disclosure also relates to nucleic acid constructs containing nucleic acid molecule sequences described herein and one or more regulatory sequences operably linked to these sequences, such as regulatory sequences suitable for expressing DNA or RNA as binding molecules in vivo or in vitro.
  • the regulatory sequence can be a suitable promoter sequence.
  • the promoter sequence is usually operably connected to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence showing transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from the gene encoding the extracellular or intracellular polypeptide homologous or heterologous to the host cell.
  • An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • the promoter sequence is a strong constitutive promoter sequence that can drive any nucleic acid molecule sequence operably connected thereto to express at a high level.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • an inducible promoter provides a molecular switch that can open the expression of a nucleic acid molecule sequence operably connected to an inducible promoter when the deadline is expressed, and close the expression when expression is undesirable.
  • the regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present disclosure.
  • the regulatory sequence may also be a suitable leader sequence, which is a sequence that is recognized by the host cell to terminate transcription. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present disclosure.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector.
  • a vector such as a cloning vector, an expression vector, and an integration vector.
  • the expression of the nucleic acid molecule sequence of the present disclosure is achieved by operably connecting the nucleic acid molecule sequence of the present disclosure to the expression vector.
  • Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences that are used for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences.
  • the type of vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus and cosmid, which can be changed according to the host cell to be introduced.
  • Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses.
  • the vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector.
  • the nucleic acid construct further comprises a coding sequence of a transposase, which is used to transpose the coding sequence of the chimeric antigen receptor, for example, to integrate the coding sequence or expression cassette of the transposed chimeric antigen receptor into the genome.
  • the coding sequence of the chimeric antigen receptor is located between the recognition sequences of the transposase.
  • the nucleic acid construct comprises an expression cassette for CAR.
  • the nucleic acid construct further comprises an expression cassette for a transposase.
  • the one or two expression cassettes are contained in one or more vectors.
  • the nucleic acid construct comprises: a vector comprising (a) an expression cassette for CAR and optionally (b) an expression cassette for a transposase; or two vectors, each comprising an expression cassette for CAR and an expression cassette for a transposase.
  • the nucleic acid construct is mRNA, which can be directly transduced into cells for transient expression.
  • the mRNA comprises in the 5'-3' direction: a 5' cap structure, a 5' UTR, an open reading frame (ORF), a 3' UTR and Poly (A), wherein the open reading frame contains the sequence of the nucleic acid molecule.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.
  • the host cell suitable for introducing the nucleic acid construct described herein can 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 include: 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.
  • mammalian cells include immune cells, preferably immune effector cells.
  • Immuno effector cells are immune cells that can perform immune effector functions, including: T cells, NK cells, TIL cells, peripheral blood mononuclear cells (PBMC), neutrophils, eosinophils, hematopoietic stem cells.
  • T cells suitable for the present disclosure can be various types of T cells from various sources.
  • nucleic acids or vectors into mammalian cells
  • the vectors can be transferred into cells by physical, chemical or biological methods.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art.
  • the host is a eukaryotic organism
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the transduced or transfected immune effector cells are propagated in vitro after the introduction of nucleic acids or vectors.
  • the fusion protein is a chimeric antigen receptor (CAR), whose antigen binding domain comprises the MUC1 binding molecule, and cells expressing the chimeric antigen receptor can be produced by the following methods: (1) contacting the cells with an activator for activation; (2) contacting the cells with a nucleic acid molecule encoding the CAR, the nucleic acid molecule encoding the CAR being on a vector to introduce the nucleic acid molecule into the cells; (3) harvesting the cells.
  • Cells expressing the chimeric antigen receptor can be prepared in vitro, using a viral vector or a non-viral vector; or cells expressing the chimeric antigen receptor can be produced directly in the patient's body, using a viral vector.
  • the nucleic acid molecule encoding CAR is DNA
  • the vector is a plasmid vector.
  • the vector is a plasmid vector containing a transposon
  • the transposon contains a nucleic acid molecule encoding CAR
  • the step (2) cell is also contacted with a transposase or a nucleic acid molecule encoding a transposase.
  • the transposon and the transposase belong to the same transposon system, and the transposon system is selected from: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helaizer transposon system, ZB transposon system, BZ transposon system, Intruder transposon system, SPINON transposon system, TcBuster transposon system, Passer transposon system, JL transposon system, Yabusame-1 transposon Transposon system, Uribo2 transposon system, PiggyBac (PB) transposon system, SleepingBeauty (SB) transposon system, and various variants or derivatives of the above transposon systems.
  • Tol1 transposon system Tol2 transposon system
  • Frog Prince transposon system Minos transposon system
  • Hsmar1 transposon system Helaizer transposon system
  • ZB transposon system
  • the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.
  • the JL transposon system is the JL transposon system described in any embodiment of CN202310081106.8, and the entire contents of this application are incorporated herein by reference.
  • the nucleic acid molecule encoding the transposase is DNA or RNA.
  • the cell is contacted with a transposase or a nucleic acid molecule encoding a transposase, and cell transduction is performed by electroporation.
  • the introduction is electroporated.
  • the step (2) includes: contacting the cell with a DNA vector comprising a JL transposon and an mRNA encoding a JL transposase, wherein the JL transposon includes a CAR gene expression cassette and terminal inverted repeats located on both sides of the CAR gene expression cassette.
  • the CAR gene expression cassette is loaded on a viral vector that infects T cells, and the viral vector is injected into the patient to directly produce CAR-T in vivo.
  • the nucleic acid molecule encoding CAR is RNA, such as mRNA, saRNA, and the carrier is LNP, LPX, VLP, inorganic nanoparticles or exosomes.
  • the mRNA or saRNA encoding CAR can be transduced into cells by electroporation, or mRNA or saRNA can be transduced into cells by carriers such as LNP, thereby obtaining a cell that transiently expresses CAR.
  • the cell may express a therapeutic agent and/or a coding sequence containing a therapeutic agent, and the step (2) cell is also contacted with a nucleic acid molecule of a therapeutic agent to introduce the nucleic acid molecule of the therapeutic agent into the cell.
  • the therapeutic agent is an antibody (e.g., a single-chain antibody, a single-domain antibody, a bispecific antibody) or a cytokine.
  • the therapeutic agent is an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is an antibody or fragment thereof targeting any one or more of PD-1, LAG-3, TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, CTLA-4, BTLA and LAIR1.
  • the therapeutic agent is an antibody targeting PD-1, preferably a single-domain antibody targeting PD-1.
  • the sequence of the single-domain antibody targeting PD-1 is the single-domain antibody targeting PD-1 described in any embodiment of patent CN202011582908.X, and this application incorporates its entire contents into this article by reference.
  • the therapeutic agent is an antibody targeting CTLA-4, preferably a single-domain antibody targeting CTLA-4.
  • the sequence of the single-domain antibody targeting CTLA-4 is the single-domain antibody targeting CTLA-4 described in any embodiment of patent CN202111152925.4, and this application incorporates its entire contents into this article by reference.
  • the therapeutic agent is a bispecific antibody containing a first functional region targeting PD-1, and a second functional region targeting CTLA4.
  • the bispecific antibody is patent CN The bispecific antibody described in any embodiment of CN202310338674.1 is incorporated herein by reference in its entirety.
  • the obtained transformant can be cultivated by conventional methods to express the polypeptide encoded by the gene of the present disclosure.
  • the culture medium used in the cultivation can be selected from various conventional culture media. Cultivate under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, induce the selected promoter with a suitable method (such as temperature conversion or chemical induction), and cultivate the cells for a period of time.
  • the polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • nanobody library By constructing a nanobody library, the inventors discovered and expressed and purified multiple nanobodies that can bind to the MUC1 protein. All aspects of the antibodies described herein can be used to prepare drugs for preventing or treating various conditions and diseases described herein, especially diseases or conditions associated with cells expressing MUC1.
  • the conditions and diseases are cancers, 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, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer or glioblastoma.
  • cancers 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, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer or glioblastoma.
  • the pharmaceutical compositions herein contain binding molecules as described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Excipients are preferably non-toxic to the recipient at the dose and concentration used. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are known in the prior art.
  • the optimal pharmaceutical composition can be determined based on the expected route of administration, delivery method, and required dosage.
  • compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization and rehydration.
  • the pharmaceutical compositions of the present disclosure can be selected for parenteral delivery.
  • Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, with physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution strip or vial with a stopper pierceable by a hypodermic needle.
  • compositions may be selected for inhalation or delivery through the digestive 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 antibodies in sustained or controlled release delivery formulations. Techniques for preparing a variety of other sustained or controlled delivery modes (such as liposome carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art.
  • the present disclosure also provides a kit for producing a single-dose administration unit.
  • the kit of the present disclosure can each contain a first container with a dried protein and a second container with an aqueous formulation.
  • a kit containing a single-chamber and multi-chamber prefilled syringe e.g., a liquid syringe and a lyophilized syringe is provided.
  • the present disclosure also provides methods for treating a patient (particularly a patient's MUC1-related disease) by administering a binding molecule or a pharmaceutical composition thereof as described in any embodiment of the present disclosure.
  • a patient particularly a patient's MUC1-related disease
  • the terms "patient,” “subject,” “individual,” and “object” are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human.
  • Treatment refers to the use of a treatment regimen described herein in a subject to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor volume, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth).
  • a treatment regimen that is effective in treating a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to stimulate an anti-cancer response in the subject.
  • the therapeutically effective amount of the pharmaceutical composition containing the binding molecules of the present disclosure that will be adopted will depend on, for example, the degree of treatment and the target. It will be appreciated by those skilled in the art that the appropriate dosage level for the treatment of will depend in part on the size (body weight, body surface or organ size) and/or condition (age and general health status) of the delivered molecule, indication, route of administration and the patient and change. In certain embodiments, the clinician can titrate the dosage and change the route of administration to obtain the best therapeutic effect. For example, about 10 micrograms/kg body weight-about 50 milligrams/kg body weight every day.
  • the frequency of administration will depend on the pharmacokinetic parameters of the binding molecule in the formulation used.
  • the clinician typically administers the composition until a dose is achieved to achieve the desired effect.
  • the composition can therefore 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 via an implant device or catheter.
  • the administration route of the pharmaceutical composition is according to known methods, such as oral, intravenous, intraperitoneal, intracerebral Intracerebral (intracerebrospinal), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional routes; via sustained-release systems or via implanted devices.
  • known methods such as oral, intravenous, intraperitoneal, intracerebral Intracerebral (intracerebrospinal), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional routes; via sustained-release systems or via implanted devices.
  • binding molecules of the present disclosure can be used in assays, such as binding assays, to detect and/or quantify MUC1 expressed in tissues or cells due to their high affinity for MUC1. Binding molecules such as single domain antibodies can be used in studies to further study the role of MUC1 in disease.
  • the method of detecting MUC1 is generally as follows: obtaining a cell and/or tissue sample; detecting the level of MUC1 in the sample.
  • the MUC1 binding molecules of the present disclosure can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with MUC1.
  • the present disclosure provides for detecting the presence of MUC1 in a sample using classical immunohistological methods known to those skilled in the art. Detection of MUC1 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of MUC1 include ELISA, FACS, RIA, etc.
  • 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), fluorescent groups (e.g., FITC, rhodamine, 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 (computer tomography) contrast agents.
  • MRI magnetic resonance imaging
  • CT computer to
  • Another aspect of the disclosure provides a method for detecting the presence of a test molecule that competes with an antibody of the disclosure for binding to MUC1.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing a certain amount of MUC1 in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody that is not bound to MUC1) would indicate that the test molecule is able to compete with the antibody for binding to MUC1.
  • 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 the antibody.
  • the present disclosure also provides a detection kit for detecting MUC1 levels, which includes an antibody that recognizes MUC1 protein, a lysis medium for dissolving a sample, and common reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.
  • the detection kit can be an in vitro diagnostic device.
  • the MUC1 protein sequence queried on NCBI it was fused with the human IgGFc fragment sequence and commissioned to Suzhou GeneWeizhi Co., Ltd. to synthesize and construct a eukaryotic expression vector of pCDNA3.4 (Thermo) plasmid.
  • the synthesized plasmid was expressed using the EXpiCHOTM (Thermo Fisher) expression system.
  • EXpiCHOTM Thermo Fisher
  • GE Protein A pre-packed column
  • the purified sample was replaced into PBS buffer, and the purity was identified by SDS-PAGE electrophoresis gel and HPLC. After the activity was identified by ELISA, it was aliquoted and frozen in a -80 degree refrigerator for subsequent immunization.
  • the first immunization antigen (MUC1-hFc) was 1000 ⁇ g, mixed with adjuvant (GERBU FAMA), and injected subcutaneously at four points on the back of the alpaca.
  • the immunization volume at each point was 1mL.
  • the second immunization was performed 3 weeks apart. From the 2nd to the 9th immunization: the immunization antigen was 500 ⁇ g, injected subcutaneously at four points on the back of the alpaca.
  • the immunization volume at each point was 1mL, and the interval between each immunization was one week.
  • MUC1-His antigen was coated overnight at 4°C, blocked and washed, and the gradient diluted serum was added to the ELISA plate for incubation, and then incubated with anti-llama IgG HRP (Abcam) antibody. After washing, TMB colorimetric solution was added for color development, and the reaction was terminated with 2M HCl. The absorbance at OD450 nanometers was then detected with an enzyme marker.
  • Figure 1 After 5 immunizations, the alpaca titer reached a high level (>72,9000).
  • RNA extraction was performed according to the instructions of TAKARA RNAiso reagent.
  • RNA as template and oligo dT as primer, synthesize the first chain of cDNA according to the instructions of TAKARA reverse transcriptase.
  • Upstream primer GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 45)
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 46)
  • the fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified, annealed at 55°C for 30 cycles; a DNA fragment of about 600 bp was recovered and used as a template for the second round of PCR.
  • Upstream primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 47)
  • the fragments between the heavy chain antibody FR1 region and the long and short hinge regions were amplified, annealed at 55°C for 30 cycles, and the target fragment was recovered.
  • the results showed that the size of the fragment was about 500 bp, that is, the electrophoresis band of the single domain antibody gene was about 500 bp.
  • the phagemid pME207 and the PCR amplification product were double-digested with Sfi I and Not I (NEB), respectively. After recovery and quantification, the two fragments were ligated with T4 DNA ligase (TaKaRa) at a molar ratio of 1:3 at 16°C overnight.
  • T4 DNA ligase T4 DNA ligase
  • the ligation product was dissolved in 100 ⁇ L sterile water and electroporated to transform E. coli TG1 ten times. Take 100 ⁇ L of the bacterial solution after electroporation and culture, dilute it in multiples, spread it on the ampicillin LB culture plate, calculate the storage capacity, and spread the rest on the ampicillin 2 ⁇ YT culture plate, and culture it upside down at 37°C for 13 to 16 hours. Use 10 mL of 2 ⁇ YT culture medium to scrape the bacterial moss on the culture plate, add glycerol at a final concentration of 25%, divide it into aliquots, and store it at -80°C for later use. The storage capacity is 4.3 ⁇ 10 9. In order to detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached more than 90%.
  • MUC1 protein was coated on the ELISA plate at 5 ⁇ g/mL, 100 ⁇ L per well, and incubated at 4°C overnight. A negative control was set up at the same time.
  • 200 ⁇ L of 3% BSA was added to each of the five wells and blocked at room temperature for 2 hours. After 2 hours, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). After washing the plate, 100 ⁇ L of phage (2-3 ⁇ 10 11 tfu immune camel single domain antibody phage display gene library) pre-blocked with 5% skim milk was added to each negative screen hole, and the plate was allowed to act at room temperature for 1.5 hours.
  • PBST PBS containing 0.05% Tween 20
  • the supernatant after negative screening was then transferred to the target antigen-coated well and allowed to act at room temperature for 1.5 hours.
  • the plate was washed 12 times with PBST (PBS containing 0.05% Tween 20) to wash away the unbound phage.
  • PBST PBS containing 0.05% Tween 20
  • the phage that specifically binds to MUC1 was dissociated with Glycine (SIGMA), and the eluted phage was neutralized with Tris (Invitrogen, 1M, PH 8.0) and infected with TG1 in the logarithmic phase. After propagation and amplification, the next round of "adsorption-elution" was carried out.
  • TG1 The supernatant of TG1 expression was used for ELISA binding detection and blocking activity detection.
  • the secondary antibody used in ELISA binding detection was anti-c-myc Antibody HRP (Bethyl), and the secondary antibody used in blocking Biotinylated Human B7-1/CD80 Protein and Fc molecule (Acrobiosystem) binding detection was HRP-labeled streptavidin (Thermo).
  • HRP-labeled streptavidin Thermo
  • the primary antibody used was biotin-anti-his antibody (GenScript), and the fluorescent antibody used was PE Streptavidin (Biolegend).
  • the single domain antibody was constructed into the pCDNA3.4-IgG4 vector and constructed into the VHH-IgG4 format, and then expressed by the EXpiCHO TM (Thermo Fisher) expression system. After one week of expression, the supernatant was collected for Protein A (GE) purification. The protein quality was then detected by Nanodrop and the protein purity was detected by HPLC. The purity and yield of the obtained protein met the needs of subsequent experiments.
  • ELISA enzyme-linked immunosorbent assay
  • the binding kinetics and affinity of antibodies to human MUC1-His antigen were determined using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the purified antibodies were passed through a sensor chip pre-fixed with protein A, and the antibodies were captured by protein A. Then, 5 different concentrations of MUC1.his protein were used as the mobile phase, and the binding and dissociation times were 30 min and 60 min, respectively.
  • the association rate (ka), dissociation rate (kd), and equilibrium constant (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 2016/0340442 A1 patent of GENUS oncology
  • BMK2 is an IgG4 antibody in the form of scFv (VH-VL) prepared according to the sequences SEQ ID NO: 24 and SEQ ID NO: 25 in the US20200024361A1 patent of Peptron.
  • the results of protein level affinity detection are shown in Table 4 below, and the binding kinetic curves are shown in Figure 3.
  • MB468 tumor cells expressing MUC1 protein were plated in a 96-well plate, with 3 ⁇ 10 5 cells per well, and the MB468 cells were incubated with the candidate antibodies in gradient dilutions at 2-8°C. After half an hour, the cells were washed and incubated with the detection antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008), and then the cells were used. CytoFLEX flow cytometer detection. Isotype is the 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.
  • the candidate antibodies have high, medium and low binding activities to tumor cells MB468.
  • NBL502-1-C4, NBL502-EFP-B02, NBL502-EFP-B03, NBL502-1D2-2, and NBL502-AI-201 have higher binding activities with tumor cells MB468, and the positive rates are all greater than 90%.
  • the binding activities of NBL502-1-A2, NBL502-AI-120, NBL502-AI-121, NBL502-AI-181, and NBL502-A12-6-LST-D5-2 with tumor cells MB468 are relatively weak, and the positive rates are between 45% and 90%.
  • the binding activity of NBL502-AI212 to tumor cells MB468 was the weakest.
  • the test results are shown in Table 5, which show that: in all 34 tissues tested, the level of tissue binding of the candidate antibody is no 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 are negative or no obvious binding in a variety of key tissues such as lungs, kidneys, pancreas, stomach, brain, etc. These data indicate that the candidate antibody has good tissue safety.
  • the full-length nucleic acid sequence was synthesized according to the sequence of the MUC1 single domain antibody.
  • the sequence of the MUC1 single domain antibody is shown in Table 6.
  • Antibodies other than NBL502-1-C4 are mutants of NBL502-1-C4.
  • ELISA enzyme-linked immunosorbent assay
  • the binding kinetics and affinity of antibodies to human MUC1-His antigen were determined using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the purified antibodies were passed through a sensor chip pre-fixed with protein A, and the antibodies were captured by protein A. Then, 5 different concentrations of MUC1.his protein were used as the mobile phase, and the binding time and dissociation time were 30 min and 60 min, respectively.
  • the binding rate (ka), dissociation rate (kd), and equilibrium constant (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 sequence SEQ ID NO: 73 in the US 2016/0340442 A1 patent of GENUS oncology.
  • the results of protein level affinity detection are shown in Table 7 below.
  • Tumor cells MB468, H226, RPMI8226 and SKOV3 expressing MUC1 protein were plated in 96-well plates, with 3 ⁇ 10 5 cells per well, and then the gradient diluted candidate antibodies were incubated with MB468 cells at 2-8°C; after half an hour, the detection antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008) was added for incubation after washing, and then detected using CytoFLEX flow cytometer.
  • Isotype is the isotype control (negative control, the sequence is derived from SEQ ID NO: 1 of patent CN 106146653A). The results of the candidate antibodies are shown in Figures 7-10, and the candidate antibodies have different binding activities with different tumor cells.
  • test results are shown in Tables 8-10, and the results show that the mutated MUC1 binding molecule has no staining on normal tissues, but shows good staining on tumor tissues, while 1C4 also stains on normal tissues, which may cause damage to normal tissues.
  • the chimeric antigen receptor structure involved in Example 9 is: CD8 signal peptide (SEQ ID NO: 88), antigen binding domain, CD8 hinge region (SEQ ID NO: 89), CD8 transmembrane domain (SEQ ID NO: 90), costimulatory domain 4-1BB (SEQ ID NO: 91), activation domain CD3 ⁇ (SEQ ID NO: 92).
  • the CAR-T number and the sequence of the antigen binding domain are shown in Table 11.
  • 1C4 contains the MUC1 single domain antibody NBL502-1-C4 provided by the present invention as an antigen binding domain, and the antigen binding domains of 555, 1C4-z7, and 1C4-z25 are mutants of NBL502-1-C4.
  • the chimeric antigen receptor BMK6.3 is derived from CN109715670A, and the sequence of the antigen binding domain of BMK6.3 is shown in SEQ ID NO: 93.
  • MUC1 CAR and BMK3 CAR derived from single-domain antibodies of different sequences were constructed and the in vitro performance of CAR-T cells was verified, including proliferation ability, CAR positivity rate, cell phenotype, and ability to kill target cells in vitro.
  • the full-length nucleic acid sequence and primers were synthesized, a large number of target fragments were obtained by PCR, the PC23S vector was double-digested (EcoR1 and Sal1) to obtain the vector fragment, and the target fragment and the vector fragment were connected by DNA ligase to obtain the plasmid.
  • the constructed vector was sequenced, and the plasmid was extracted in large quantities after the sequence was correct.
  • the PC23S vector sequence is shown in SEQ ID NO: 94.
  • Mock-T and MUC1 CAR-T were prepared by electroporation.
  • AIM-V medium containing 2% FBS
  • AIM-V medium containing 2% FBS
  • PBMCs inoculate into culture flasks at 5E6/mL, and place in an incubator (37°C; 5% CO 2 ) overnight.
  • the PBMCs were transferred to a 6-well plate containing 2mL of AIM-V (containing 2% FBS and 100IU/mL IL-2) culture medium (need to be preheated to 37°C in advance), placed in an incubator (37°C; 5% C02) and cultured for 24h-48h, and then the culture medium was supplemented to 4mL.
  • AIM-V containing 2% FBS and 100IU/mL IL-2
  • the first subculture was performed on the 3rd day after electroporation, the second subculture was performed on the 6th day, and the CAR-T cell preparation was completed on the 9th day.
  • PBMCs during the subculture process were cultured in AIM-V culture medium containing 2% FBS and 100IU/ml IL-2, and the subculture cell density was 5 ⁇ 10 5 /ml.
  • flow cytometry was used to detect the cell surface CAR positivity rate and other surface markers.
  • the purpose of this experiment is to evaluate the quality of the prepared mock-T, BMK CAR-T cells, and MUC1 CAR-T cells by proliferation and positive rate.
  • the passaging density was 5 ⁇ 10 5 cells/mL, and the cells were passaged and counted on Day 3, 6, and 9; the IL-2 concentration was 100 U/mL. See the experimental operation method for details.
  • the CAR positive rate of cells during proliferation was detected by flow cytometry. See the experimental procedure for details.
  • the phenotype of the CART cells we prepared was evaluated by detecting the CD3/CD4/CD8 and TCM/TEM of MUC1 CART using flow cytometry.
  • FIGs 11-14 The proliferation, viability, positive rate and phenotype of CAR-T cells are shown in Figures 11-14. As shown in Figures 11 and 12, the total proliferation times of T cells in each group at D9 were about 15-20 times that of D0, and the cell viability was maintained above 90%. As shown in Figure 13, the CAR positive rate of D9 was quite different among the groups, and the positive rates of 555 and 1C4 were higher.
  • Figure 14 shows the proportion of CD4 and CD8 in the preparation of D9 CAR-T. The proportion of CD8+T cells in each group was significantly higher than that of CD4+T cells.
  • the killing effect of CAR-T cells on tumor cells was detected using the xCELLigence RTCA device (Roche Applied Science, Canada), and the detection process was carried out according to the device operating instructions.
  • 10,000 target cells (MB468, HCT116) were resuspended in 50ul culture medium and inoculated into the microplate of the RTCA device.
  • the tumor cells were cultured on the RTCA device for about 24 hours.
  • the cell index (CI, indicating cell growth) reached about 1.5
  • effector cells CAR-T cells
  • effector cells effector cells
  • target cells were resuspended in 50ul culture medium according to different effector-target ratios (effector cells: target cells), mixed with tumor cells, and continued to be cultured on the RTCA device for 48h-96h.
  • the cell killing curve was recorded by the RTCA device, and the data was analyzed using RTCA Pro 2.3.0 software.
  • Figure 15 shows the time required for each group of CAR-T to kill 50% of MB468 MUC1-high-expressing tumor cells under different effector-target ratios, i.e., the KT50 value.
  • Each group of CAR-T can achieve KT50 within 24 hours under an effector-target ratio of 1:4. As the effector-target ratio decreases, the killing ability of each group also decreases accordingly, and the difference between the groups is not significant.
  • Figure 16 shows the 48h tumor lysis rate of each group of CAR T for HCT116 tumor cells with extremely low MUC1 expression. The results show that each group of CAR T has no killing ability for tumor cells with low MUC1 expression, proving that the CAR-T in this patent has low non-target specific killing and high safety.
  • MB468 tumor cells were inoculated into six-well plates one day in advance (600,000 tumor cells per well). After overnight incubation at 37°C, the tumor cells were treated with mitomycin C (25ug/mL) for 4h to inhibit their growth. The tumor cells were washed 5 times with PBS. CAR-positive T cells were inoculated at a 1:1 effector-target ratio, and one round was performed every 5 days. After one round, the number of cells was counted using a cell counter, and the CAR positivity rate was detected using a flow cytometer. The number of CAR-positive cells was calculated, and a new round of tumor co-culture experiments was started at a 1:1 effector-target ratio. This process was repeated until the end of the fifth round.
  • Figure 17 shows the CAR positivity rates of the two donors after each round of co-culture.
  • the CAR positivity rates of each group gradually increased in the first two rounds of co-culture, and began to show large differences after the third round. 555 and 1C4 were still able to maintain a high level.
  • Figure 18 shows the theoretical proliferation multiples of CAR-positive T cells from two donors compared to the number of CAR-positive cells inoculated at the beginning at the end of the fifth round. For both donors, 555 and 1C4 had the highest proliferation multiples.
  • Figure 19 shows the IFN- ⁇ secretion of CAR T in each group in the first three rounds of the co-culture experiment. Both donors showed that in the second round of the experiment, the release of cytokines from each group of CART was the highest, and began to decline in the third round, which was consistent with the results of the change in CAR positivity rate.
  • the JL transposon system was used to prepare MUC1 CAR-T that secretes PD1/CTLA4 bispecific antibodies.
  • the PB enzyme mRNA was replaced with JL enzyme mRNA, the dosage was 10 ⁇ g, 3 ⁇ g of MUC1 CAR plasmid was added, and 1 ⁇ g of PD1/CTLA4 bispecific antibody plasmid was added, wherein the plasmids all used the Tai plasmid framework.
  • the sequence of JL enzyme is shown in SEQ ID NO: 95
  • the sequence of Tai plasmid empty frame is shown in SEQ ID NO: 96
  • the sequence of PD1/CTLA4 bispecific antibody is shown in SEQ ID NO: 97.
  • the prepared CAR-T was co-cultured with cancer cells MB468, as well as normal bronchial, gastric and kidney cells, with an effector-target ratio of 1:4, and cell killing was measured at 43h and 72h.
  • the cell killing results of the two donors are shown in Figure 20.
  • the MUC1 CAR-T prepared by the JL transposon system can significantly kill MB468 cancer cells, but has a lower killing effect on normal cells.
  • the ionizable lipid compound B-C14 was dissolved in anhydrous ethanol with DOPE, cholesterol and DMG-PEG2000 (all purchased from Synobond) at a molar ratio of 30:15:53.125:1.875 to prepare an ethanol lipid solution (total lipid concentration: 10.78 mg/mL).
  • DOPE dimethylcellulose
  • DMG-PEG2000 dimethylcellulose
  • the structure and specific synthesis method of B-C14 can be found in Chinese patent CN116456967A.
  • the ethanol lipid solution and the mRNA aqueous solution were mixed in a volume ratio of 1:3 by a microfluidic device (Microflow S, Mingtai Medical Equipment (Shanghai) Co., Ltd.), with a total flow rate of 24 mL/min.
  • the resulting lipid nanoparticle solution was diluted 6 times with a DPBS solution containing 10% sucrose and filtered with a 0.2 ⁇ m sterile filter to obtain an LNP preparation (lipid nanoparticle) encapsulating MUC1 CAR mRNA.
  • the sample was cooled at -80 °C Keep frozen for later use.
  • Activated T cells were collected, resuspended with OPTI-MEM medium, and counted using a life counter and Trypan Blue Stain staining.
  • Activated T cells were inoculated into a 24-well plate at a cell volume of 5E5 cells/300 ⁇ L/well, and then apolipoprotein ApoE4 was added at a dose of 0.5 ⁇ g/5E5 cells/well to increase the transfection efficiency of T cells.
  • Lipid nanoparticles were then added at a dose of 0.2 ⁇ g/5 ⁇ 10 5 cells/200 ⁇ L/well, and the plate was gently shaken to ensure sufficient mixing, and then the plate was placed in a 37°C, 5% CO 2 incubator for 24 hours to obtain CAR-T cells.
  • the transfected CAR-T cells were collected by centrifugation for 24 hours, resuspended in OPTI-MEM medium, and inoculated into 24-well plates at a cell volume of 5E5 cells/500 ⁇ L/well.
  • the well plates were placed in a 37°C, 5% CO 2 incubator for different periods of time to characterize the CAR-T cells, with blank T cells as the control group.
  • the cell viability of the cell experimental group was greater than 90%, and the delivery of mRNA by LNP was non-toxic to the cells, with a positive rate of greater than 80%.
  • mRNA was transfected into cells to prepare CAR-T, the transfection dose of LNP was 0.5 ⁇ g/5E5 cells, the transfection time was 24h, recorded as "LNP-500ng”; electroporation was used, and the same dose of CAR mRNA was used, recorded as "electroporation”.
  • MOCKT used eGFP mRNA, and also used LNP and electroporation, respectively, using the same dose of mRNA, as a control, recorded as LNP-MOCKT, electroporation-MOCKT.
  • target cells SKOV3 and MDA-MB468 were co-cultured with CAR-T cells or MOCKT cells at an effector-target ratio of 2:1, 1:2, 1:4, or 1:8 for 24h, and the killing effect of CART cells was analyzed according to the value of the cell index.
  • the 24h positive rate of transfection prepared by each group of cells is shown in Figure 22. There is no significant difference in the positive rate of CAR-T cells prepared by LNP and electroporation.
  • the killing of MAD-MB468 and SKOV3 by different effector-target ratios are shown in Figures 23-24, respectively.
  • the killing effect of CAR-T cells prepared by LNP and electroporation is not much different. Under the same effector-target ratio, CART cells have a better killing effect on MDA-MB468 with high expression of MUC1, and the killing efficiency of MDA-MB468 reaches 90% under the condition of effector-target ratio of 1:2.

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Abstract

提供了MUC1结合分子，包括MUC1单域抗体，特异性地结合MUC1胞外区。还涉及抗MUC1单域抗体嵌合抗原受体及其应用，具体提供一种嵌合抗原受体，其抗原结合域包括含抗MUC1单域抗体的MUC1结合分子。含有所述嵌合抗原受体的免疫细胞具有较高的细胞杀伤效果。

Description

MUC1结合分子和包含其的嵌合抗原受体
本申请要求2024年8月1日提交的中国专利申请第CN202310958766.X号、以及2024年8月1日提交的中国专利申请第CN202310959241.8号的优先权，其内容通过引用全文纳入本文。
技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及MUC1结合分子和包含其的嵌合抗原受体。
背景技术
在过去的十年中，癌症个性化免疫反应的工程设计从理论变成了现实。利用患者自身免疫细胞制成的嵌合抗原受体(CAR，Chimeric antigen receptor)T细胞疗法，已经为某些类型的侵袭性白血病和其他血液癌症带来了转机。该疗法通过基因工程技术将能够识别肿瘤抗原分子的抗体可变区基因序列与T淋巴细胞免疫受体的胞内区序列拼接在一起，然后将这些融合基因序列通过慢病毒载体系统或转座子转座酶系统等，引入到T淋巴细胞基因组内。这样，T细胞就能表达CAR，识别和攻击特定抗原，增强自身杀伤肿瘤细胞的能力。在某些情况下，CAR-T细胞甚至能够治愈经过多种其他治疗后癌症复发的人。但是，CAR-T细胞还不能达到让使大多数人长期生存的效果。此外，虽然CAR-T疗法治疗血液癌症方面已经取得了一定的成功，但是在实体肿瘤(如胰腺癌、肺癌或结肠直肠癌)治疗方面，仍然有很长的路要探索。
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。肿瘤相关MUC1与正常细胞表达的MUC1不同，由于Core 2转移酶的丢失，以及唾液酸转移酶的异常激活，导致肿瘤表达的MUC1大多是低糖基化的或者糖基链被截短的，这使得一些新的抗体结合表位被暴露出来。针对这种肿瘤特异性的抗原表位设计的CAR-T往往被认为具有更少的脱靶效应。
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。
发明内容
本公开内容的目的在于提供MUC1结合分子和包含其的嵌合抗原受体。
本公开内容第一方面提供一种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包含SEQ ID NO：1-10中任一所示的序列，CDR2包含SEQ ID NO：11-21中任一所示的序列，和CDR3包含SEQ ID NO：22-32中任一所示的序列。
在一些实施方案中，所述单域抗体含有以下表1中组a1到组a11中任一组的SEQ ID NO所示的CDR1、CDR2和CDR3：
表1
在一些实施方案中，所述单域抗体VHH如SEQ ID NO：33-43中任一所示。
本公开内容还在第一方面MUC1单域抗体(称为野生型单域抗体)的基础上，进行了突变，以获得不同亲和力的MUC1单域抗体。本公开内容第二方面还提供了一种MUC1结合分子，包含MUC1单域抗体，所述MUC1单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，所述MUC1单域抗体具有如下特征(1)-(3)中的一种或多种：
(1)所述CDR1与SEQ ID NO：2相比含有以下一个或多个突变：T3Q、R5D、 R5H、R5K、R5E、R5Y、R6E、R6H、R6K、R6D、R6Y，优选含有选自以下组中的一个或多个突变：T3Q、R5D、R6E、R5H\R6H、R5K\R6K、R5D\R6D、R5E\R6E、R5Y\R6Y，
(2)所述CDR2与SEQ ID NO：12相比含有以下一个或多个突变：I1G、T3Q、T3W、F4D、D6A、D6G、D7S、T8Q，优选含有选自以下组中的一个或多个突变：I1G、T3W、D6G、F4D\D6A、T3Q\T8Q、D6G\D7S，
(3)所述CDR3与SEQ ID NO：23相比含有以下一个或多个突变：T1Q、I3G、Y6H、Q8E、Q8Y、L9G、L9Y、L9S、S10N、D12Q，优选含有选自以下组中的一个或多个突变：T1Q、Q8E、Q8Y、L9G、I3G\L9G、L9Y、L9S、6H\S10N\D12Q。
在一些实施方案中，所述MUC1单域抗体包含CDR1、CDR2和CDR3，所述CDR1、CDR2、CDR3含有以下表2中组A1到A17中任一组所示的突变：
表2

在一些实施方案中，所述MUC1单域抗体具有如下特征中的一种或多种：
(1)所述CDR1如SEQ ID NO：2、49-56任一项所示，
(2)所述CDR2如SEQ ID NO：12、57-62任一项所示，
(3)所述CDR3如SEQ ID NO：23、63-70任一项所示。
在一些实施方案中，所述MUC1单域抗体含有以下表3中组B1到组B18中任一组SEQ ID NO所示的CDR1、CDR2和CDR3：
表3
在一些实施方案中，所述MUC1单域抗体的氨基酸序列如SEQ ID NO：71-87任一所示，优选如SEQ ID NO：71、72、78或83所示。
在一些实施方案中，所述MUC1结合分子可以包含一条、两条或多条本文所述的抗MUC1单域抗体。所述MUC1结合分子可以是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。
在一些实施方案中，所述多价结合分子或多特异性结合分子通过连接子连接多个抗MUC1单域抗体或其抗原结合片段。所述连接子由选自G和S的1-15个氨基酸组成。
在一些实施方案中，所述单域抗体是骆驼重链抗体或软骨鱼重链抗体。
在一些实施方案中，所述单域抗体还包含重链恒定区。
在一些实施方案中，所述重链恒定区是人或骆驼重链抗体的恒定区，包含CH2和CH3。在一些实施方案中，所述CH2和CH3是人IgG Fc的CH2和CH3，例如IgG4的CH2和CH3。优选地，所述重链恒定区如SEQ ID NO：44所示。在一些实施方案中，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。
在一些实施方案中，本公开内容任一实施方案所述的MUC1结合分子为嵌合抗体或完全人抗体；优选为完全人抗体。
在一些实施方案中，所述结合分子是包含所述抗MUC1单域抗体作为重链可变结构域的抗体。
在一些实施方案中，所述结合分子还包含轻链可变结构域、重链恒定域和轻链恒定域。
在一些实施方案中，抗体的抗原结合片段选自Fab、F(ab’)2、Fv、scFv。
本公开内容第三方面还提供含有本公开内容任一实施方案所述抗MUC1结合分子和其它多肽的融合蛋白。
在一些实施方式中，其它多肽位于所述结合分子的N端和/或C端。
在一些实施方式中，其它多肽选自将结合分子定位到不同细胞器的多肽、用于纯化的标签或者用于免疫反应的标签、跨膜蛋白或其跨膜区、嵌合抗原受体组件。
在一些实施方案中，所述融合蛋白为嵌合抗原受体，其抗原结合域包含所述MUC1结合分子；优选地，所述嵌合抗原受体包含：任选的信号肽序列、含MUC1单域抗体的MUC1结合分子、铰链区、跨膜区、胞内区。
本公开内容还提供一种核酸分子，包含选自以下的序列：(1)本公开内容任一实施方案所述MUC1结合分子或融合蛋白的编码序列；
(2)(1)的互补序列。
在一些实施方案中，所述编码序列是DNA或RNA。
本公开内容还提供一种核酸构建物，包含本文所述的核酸分子。
在一个或多个实施方案中，核酸构建物是载体，例如克隆载体、表达载体或整合载体。在一个或多个实施方案中，所述表达载体是组成型表达载体。优选地，所述组成型表达载体是转座载体。
在一些实施方案中，核酸构建物为mRNA。在一些实施方案中，所述mRNA在5′-3′方向上包含：5′帽结构、5′UTR、开放阅读框(ORF)、3′UTR和Poly(A)，所述开放阅读框含有所述核酸分子的序列。
本公开内容还提供一种宿主细胞，选自：
(1)表达本文任一实施方案所述MUC1结合分子或所述融合蛋白；
(2)包含本文所述的核酸分子；和/或
(3)包含本文所述的核酸构建物。
在一些实施方案中，所述宿主细胞为T细胞、TIL细胞或NK细胞，所述融合蛋白为嵌合抗原受体。
在一些实施方案中，所述细胞含有：本公开内容任一实施方案中所述嵌合抗原受体的编码序列。
在一些实施方案中，所述细胞含有编码所述嵌合抗原受体的mRNA。
在一些实施方案中，所述细胞含有编码所述嵌合抗原受体的DNA，优选地，所述嵌合抗原受体的DNA整合到细胞的基因组中。
在一些实施方案中，所述细胞表达治疗剂和/或含有治疗剂的编码序列，所述治疗剂是抗体(例如单链抗体、单域抗体、双特异性抗体)或细胞因子。
本公开内容还提供一种产生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。
本公开内容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、核酸分子、核酸构建物或宿主细胞，和药学上可接受的辅料。
在一些实施方案中，所述药物组合物用于治疗癌症。
在一些实施方案中，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、宫颈癌、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、前列腺癌或胶质母细胞瘤。
本公开内容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。
在一些实施方案中，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、宫颈癌、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、前列腺癌或胶质母细胞瘤。
本公开内容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公开内容任一实施方案所述的MUC1结合分子，或含有本公开内容任一实施方案所述的MUC1结合分子的药物组合物。
在一些实施方案中，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、宫颈癌、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、前列腺癌或胶质母细胞瘤。
本公开内容还提供一种检测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、、核酸构建物、噬菌体、宿主细胞。
在一些实施方案中，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。
在一些实施方案中，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。
本公开内容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。
本公开内容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。
附图说明
图1为针对MUC1蛋白的羊驼抗血清效价检测结果。
图2为候选抗体与MUC1蛋白的ELISA检测结果。
图3为候选抗体与MUC1蛋白的亲和力检测结果。
图4为候选抗体与MB468肿瘤细胞株的结合检测结果。
图5-6为候选突变抗体与MUC1蛋白的ELISA检测结果。
图7为候选突变抗体与MB468肿瘤细胞株的结合检测结果。
图8为候选突变抗体与H226肿瘤细胞株的结合检测结果。
图9为候选突变抗体与RPMI8226肿瘤细胞株的结合检测结果。
图10为候选突变抗体与SKOV3肿瘤细胞株的结合检测结果。
图11为CAR-T细胞增殖倍数。
图12为CAR-T细胞增殖过程中的细胞活率。
图13为CAR-T细胞在第9天的CAR阳性率。
图14为CAR-T细胞在第9天CD4与CD8的占比。
图15为不同效靶比条件下，各组CAR-T杀伤50％MUC1高表达肿瘤细胞MB468所需的时间。
图16为各组CAR-T对于MUC1极低表达的肿瘤细胞HCT116的48h肿瘤裂解率。
图17为每一轮共培后两个donor的CAR阳性率。
图18为第五轮结束，两个donor的CAR阳性T细胞相较于最开始接种的CAR阳性细胞数的理论增殖倍数。
图19为共培养实验前三轮各组CAR-T的IFN-γ分泌量。
图20为JL系统制备的两个donor的MUC1 CAR-T对细胞的杀伤结果。
图21为LNP递送mRNA制备CAR-T的细胞活率和阳性率。
图22为采用LNP递送mRNA和电转制备CAR-T和MOCKT细胞的24h转染阳性率。
图23为LNP和电转方式制备的CAR-T细胞不同效靶比对MAD-MB468的杀伤结果。
图24为LNP和电转方式制备的CAR-T细胞不同效靶比对SKOV3的杀伤结果。
具体实施方式
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。本公开内容的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。本公开内容还在MUC1单域抗体的基础上进行突变，获得中等亲和力的MUC1单域抗体，以减少非特异性的结合，降低风险。本公开内容还提供了包含所述MUC1单域抗体的嵌合抗原受体和细胞。
抗体
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、重链抗体、纳米抗体或它们的抗原结合片段。
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，“抗体”与“免疫球蛋白”可互换使用。
传统的“抗体”含有基本的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。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)。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。
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)和其它恒定区组成重链，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)。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。
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，本公开内容的单域抗体具有SEQ ID NO：1-10任一所示的CDR1、SEQ ID NO：11-21任一所示的CDR2、和SEQ ID NO：22-32任一所示的CDR3。优选地，本公开内容的单域抗体具有SEQ ID NO：33-43的任一VHH的FR1、SEQ ID NO：33-43的任一VHH的FR2、SEQ ID NO：33-43的任一VHH的FR3、SEQ ID NO：33-43的任一VHH的FR4。所述CDR和FR重组获得的VHH也在本公开内容的范围内。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。
本文中，“纳米抗体”是指含有本文所述CDR或CDR组合的基于VHH的免疫分子。其可以是如上所述的重链抗体，还可以是含有多条VHH的多价或多特异性分子，也可以是将VHH和抗体Fc(例如CH2和CH3或CH2、CH3和CH4)重组获得的重组分子。
包含两条或多条相同特异性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。多特异性可以是针对MUC1和另一种抗原，也可以是针对MUC1的两种不同表位。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。
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。
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。
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。可变结构域中更为高度保守的部分称为构架区(FR)。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。抗体的有多种可变区标注方案，包括：Chothia、Kabat、IMGT和Contact。本文示例性使用IMGT标注方案。
“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，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体系统的第一组分(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。
“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’)、F(ab’)2、Fd、和Fv片段、二硫键连接的Fv；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。抗原结合片段可以通过多种技术制备，包括但不限于将完整的抗体蛋白水解消化，以及由包含抗原结合片段的宿主细胞表达产生。
“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。
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，将根据本公开内容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。
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。
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)由人来源的核酸分子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系统的小鼠而产生。本公开内容中，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。
“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。在一些实施方案中，本公开内容还提供与本公开内容 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，即能够与本公开内容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。
本公开内容中，单域抗体的CDR1包括SEQ ID NO：1-10中任一所示的序列；单域抗体的CDR2包括SEQ ID NO：11-21中任一所示的序列；单域抗体的CDR3包括SEQ ID NO：22-32中任一所示的序列。
在一些实施方案中，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：
在一些实施方案中，所述单域抗体VHH的FR1可选自SEQ ID NO：33-43的任一VHH的FR1，VHH的FR2可选自SEQ ID NO：33-43的任一VHH的FR2，VHH的FR3可选自SEQ ID NO：33-43的任一VHH的FR3，VHH的FR4可选自SEQ ID NO：33-43的任一VHH的FR4。
在优选的实施方案中，本公开内容单域抗体VHH的FR区为选自SEQ ID NO：33-43的任一VHH的FR区。进一步优选地，这类抗体的CDR选自表1中组a1到组a11中的任一组。在一些实施方案中，所述单域抗体VHH如SEQ ID NO：33-43中任一所示。
本公开内容还在上述MUC1单域抗体(称为野生型单域抗体)的基础上，进行了突变，以获得所需亲和力的MUC1单域抗体。本公开内容还提供了一种一种MUC1结合分子，包含突变的抗MUC1单域抗体。
优选地，在具有SEQ ID NO：2所示的CDR1、SEQ ID NO：12所示的CDR2和SEQ ID NO：23所示的CDR3野生型单域抗体进行突变，所述突变位于野生型单域抗体的CDR区，可以具有一个或多个突变。在SEQ ID NO：2所述的CDR1区上，可以进行以下一个或多个突变：T3Q、R5D、R5H、R5K、R5E、R5Y、R6E、R6H、R6K、R6D、R6Y，优选含有选自以下组中的一个或多个突变：T3Q、R5D、R6E、R5H\R6H、R5K\R6K、R5D\R6D、R5E\R6E、R5Y\R6Y。在SEQ ID NO：12所述的CDR2区上，可以进行以下一个或多个突变：I1G、T3Q、T3W、F4D、D6A、D6G、D7S、T8Q，优选含有选自以下组中的一个或多个突变：I1G、T3W、D6G、F4D\D6A、T3Q\T8Q、D6G\D7S。在SEQ ID NO：23所述的CDR3区上，可以进行以下一个或多个突变：T1Q、I3G、Y6H、Q8E、Q8Y、L9G、L9Y、L9S、S10N、D12Q，优选含有选自以下组中的一个或多个突变：T1Q、Q8E、Q8Y、L9G、I3G\L9G、L9Y、L9S、 6H\S10N\D12Q。
突变的MUC1单域抗体包含CDR1、CDR2和CDR3，所述CDR1、CDR2、CDR3含有以下表2中组A1到A17中任一组所示的突变。在一些实施方案中，所述突变的抗MUC1单域抗体与具有SEQ ID NO：2所示的CDR1、SEQ ID NO：12所示的CDR2和SEQ ID NO：23所示的CDR3的野生型单域抗体相比，与MUC1的亲和力降低。所述抗MUC1单域抗体以约1×10-8～5×10-7M的KD结合MUC1，优选1.5×10-8～2×10-7M。所述KD是通过表面等离子共振技术(SPR)测定的。在一些实施方案中，所述MUC1单域抗体含有以下表3中组B1到组B17中任一组SEQ ID NO所示的CDR1、CDR2和CDR3。
所述MUC1单域抗体的FR区可以与野生型单域抗体的FR区相同，或者进行人源化优化。在一些实施方案中，所述MUC1单域抗体的氨基酸序列如SEQ ID NO：71-87任一所示，优选如SEQ ID NO：71、72、78或83所示。
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。所述MUC1结合分子为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。
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。可用于本公开内容的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IgG4 Fc，其氨基酸序列如SEQ ID NO：44所示。
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。
本公开内容还包括所述抗体衍生物和类似物。“衍生物”和“类似物”是指基本上保持本公开内容抗体相同的生物学功能或活性的多肽。本公开内容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。
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公开内容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、插入和/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公开内容保护的范围内。
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公开内容抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本公开内容抗体的抗血清获得的多肽或蛋白。
在一些实施方案中，本公开内容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。本公开内容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。本公开内容还包括那些具有带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。
可采用本领域常规的方法制备本公开内容的抗体，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公开内容的重链抗体，如本领域熟知的噬菌体展示技术。或者，本公开内容的抗体或重链抗体可在其他细胞系中表达。可用编码本公开内容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，例如包括将核酸分子包装在病毒(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。用于将异源核酸分子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、将核酸分子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。
融合蛋白
本公开内容还包括含有本文所述MUC1结合分子和其它多肽的融合蛋白。在一些实施方式中，其它多肽位于所述结合分子的N端和/或C端。在一些实施方式中，其它多肽选自将结合分子定位到不同细胞器的多肽、用于纯化的标签或者用于免疫反应的标签、跨膜蛋白或其跨膜区、嵌合抗原受体组件(胞外结构域、铰链区、跨膜区、信号转导结构域、共刺激结构域等)。在一些实施方案中，所述融合蛋白为嵌合抗原受体，其抗原结合域包含所述MUC1结合分子。所述嵌合抗原受体包含：任选的信号肽序列、含MUC1单域抗体的MUC1结合分子、铰链区、跨膜区、胞内区。
所述嵌合抗原受体还具有选自以下的一项或多项特征：
所述信号肽包括CD8信号肽、CD28信号肽、CD4信号肽或轻链信号肽，
所述铰链区包括CD8铰链区、IgD铰链区、IgG1 Fc CH2CH3铰链区或IgG4 Fc CH2CH3铰链区，
所述跨膜区包括CD28跨膜区、CD8跨膜区、CD3ζ跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区或DAP10跨膜区，
所述胞内区包括胞内共刺激域和/或胞内信号域，
所述胞内共刺激域包括CD28、CD134/OX40、CD137/4-1BB、淋巴细胞特异性蛋白酪氨酸激酶、诱导性T细胞共刺激因子(ICOS)或DNAX激活蛋白10的胞内结构域，
所述胞内信号域包括CD3ζ胞内信号域或FcεRIγ胞内信号域。
核酸分子和核酸构建物
本公开内容还提供了编码上述MUC1结合分子或所述融合蛋白的核酸分子。本文提供编码重链可变区、轻链可变区、重链、轻链以及各CDR的核酸分子。本公开内容的核酸分子可以是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。RNA可以是在体内和/或体外表达结合分子的mRNA。
本公开内容还涉及与上述核酸分子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％相同性的核酸分子。本公开内容特别涉及在严谨条件下与本公开内容所述核酸分子可杂交的核酸分子。在本公开内容中，“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，可杂交的核酸分子编码的多肽与成熟多肽有相同的生物学功能和活性。
本公开内容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。
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。本公开内容所涉及的生物分子(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公开内容蛋白(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公开内容蛋白序列中。
本公开内容也涉及核酸构建物，该核酸构建物含有本文所述的核酸分子序列，以及与这些序列操作性连接的一个或多个调控序列，例如适合在体内或体外将DNA或RNA表达为结合分子的调控序列。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何核酸分子序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达时打开可操作地连接诱导型启动子的核酸分子序列的表达，而在当表达是不期望的时关闭表达。
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本公开内容。调控序列也可以是合适的前导序列，对宿 主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本公开内容。
在一些实施方案中，所述核酸构建物是载体，例如克隆载体、表达载体和整合载体。通常通过可操作地连接本公开内容的核酸分子序列至表达载体，实现本公开内容核酸分子序列的表达。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。整合载体含有将靶序列整合到细胞基因组上的组件。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。
此外，载体的类型不受限制，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒，可根据待导入的宿主细胞而改变。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001，Molecular Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory，New York)和其它病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。
为了评估多肽或其部分的表达，被引入细胞的载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。
在一些实施方案中，所述核酸构建物还包含转座酶的编码序列，所述转座酶用于转座嵌合抗原受体的编码序列，例如将转座嵌合抗原受体的编码序列或表达框整合到基因组中。嵌合抗原受体的编码序列位于所述转座酶的识别序列之间。
在一些实施方案中，所述核酸构建物包含CAR的表达框。所述核酸构建物还包含转座酶的表达框。所述一种或两种表达框包含在一个或多个载体中。例如，所述核酸构建物包含：一种载体，其包含(a)CAR的表达框和任选的(b)转座酶的表达框；或两种载体，分别包含CAR的表达框和转座酶的表达框。
在一些实施方案中，核酸构建物为mRNA，可直接将mRNA转导入细胞，用于瞬时表达。在一些实施方案中，所述mRNA在5′-3′方向上包含：5′帽结构、5′UTR、开放阅读框(ORF)、3′UTR和Poly(A)，所述开放阅读框含有所述核酸分子的序列。
细胞
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。
适用于导入本文所述核酸构建物的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。哺乳动物细胞的示例包括免疫细胞，优选免疫效应细胞。“免疫效应细胞”是可执行免疫效应功能的免疫细胞，包括：T细胞、NK细胞、TIL细胞、外周血单个核细胞(PBMC)、中性粒细胞、嗜酸性粒细胞、造血干细胞。适用于本公开内容的T细胞可以是各种来源的各种类型的T细胞。
将核酸或载体引入哺乳动物细胞的方法是本领域已知的，所述载体可以通过物理、化学或生物方法转入细胞。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CaCl2法处理，所用的步骤在本领域众所周知。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。在一些实施方案中，转导的或转染的免疫效应细胞在引入核酸或载体之后离体繁殖。
所述融合蛋白为嵌合抗原受体(CAR)，其抗原结合域包含所述MUC1结合分子，表达嵌合抗原受体的细胞可以采用如下方法：(1)使细胞与激活剂接触进行激活；(2)将细胞与编码CAR的核酸分子接触，所述编码CAR的核酸分子在载体上，以将所述核酸分子引入细胞；(3)收获细胞。可以在体外制备表达嵌合抗原受体的细胞，通过病毒载体或者非病毒载体；也可以在患者体内直接产生表达嵌合抗原受体的细胞，通过病毒载体实现。
在一些实施方案中，所述编码CAR的核酸分子为DNA，所述载体为质粒载体。在一些实施方案中，所述载体为含有转座子的质粒载体，所述转座子包含编码CAR的核酸分子，所述步骤(2)细胞还与转座酶或编码转座酶的核酸分子接触。所述转座子和转座酶属于同一个转座子系统，所述转座子系统选自：Tol1转座子系统、Tol2转座子系统、Frog Prince转座子系统、Minos转座子系统、Hsmar1转座子系统、Helraiser转座子系统、ZB转座子系统、BZ转座子系统、Intruder转座子系统、SPINON转座子系统、TcBuster转座子系统、Passer转座子系统、JL转座子系统、Yabusame-1转 座子系统、Uribo2转座子系统、PiggyBac(PB)转座子系统、SleepingBeauty(SB)转座子系统，以及上述转座子系统的各种变体或衍生物。在一些实施方案中，所述转座子系统是PB转座子系统、BZ转座子系统或JL转座子系统。所述JL转座子系统为CN202310081106.8任一实施方案所述的JL转座子系统，本申请将其全部内容以引用的方式纳入本文。
在一些实施方案中，所述编码转座酶的核酸分子为DNA或RNA。在一些实施方案中，细胞与转座酶或编码转座酶的核酸分子接触，通过电转进行细胞转导。在一些实施方案中，所述引入采用电穿孔。所述步骤(2)包括：将细胞与包含JL转座子的DNA载体和编码JL转座酶的mRNA接触，所述JL转座子包括CAR基因表达框和位于CAR基因表达框两侧的末端反向重复序列。在一些实施方案中，将CAR基因表达框负载在感染T细胞的病毒载体上，将病毒载体注射到患者体内，以在体内直接产生CAR-T。
在一些实施方案中，所述编码CAR的核酸分子为RNA，例如mRNA、saRNA，所述载体为LNP、LPX、VLP、无机纳米粒或外泌体。可以将编码CAR的mRNA或saRNA通过电穿孔转导入细胞，或者mRNA或saRNA通过LNP等载体转导入细胞，从而获得一种瞬时表达CAR的细胞。
在一些实施方案中，所述细胞可以表达治疗剂和/或含有治疗剂的编码序列，步骤(2)细胞还与治疗剂的核酸分子接触，以将所述治疗剂的核酸分子引入细胞。在一些实施方案中，所述治疗剂是抗体(例如单链抗体、单域抗体、双特异性抗体)或细胞因子。在一些实施方案中，所述治疗剂为免疫检查点抑制剂。所述免疫检查点抑制剂为靶向PD-1、LAG-3、TIM3、B7-H1、CD160、P1H、2B4、CEACAM(例如CEACAM-1、CEACAM-3和/或CEACAM-5)、TIGIT、CTLA-4、BTLA和LAIR1中任一个或多个的抗体或其片段。在一些实施方案中，所述治疗剂为靶向PD-1的抗体，优选靶向PD-1的单域抗体。所述靶向PD-1的单域抗体的序列为专利CN202011582908.X任一实施方案所述的靶向PD-1的单域抗体，本申请将其全部内容以引用的方式纳入本文。在一些实施方案中，所述治疗剂为靶向CTLA-4的抗体，优选靶向CTLA-4的单域抗体。所述靶向CTLA-4的单域抗体的序列为专利CN202111152925.4任一实施方案所述的靶向CTLA-4的单域抗体，本申请将其全部内容以引用的方式纳入本文。在一些实施方案中，所述治疗剂为双特异性抗体，含有靶向PD-1的第一功能区，和靶向CTLA4的第二功能区。在一些实施方案中，所述双特异性抗体为专利CN CN202310338674.1任一实施方案所述的双特异性抗体，本申请将其全部内容以引用的方式纳入本文。
获得的转化子可以用常规方法培养，表达本公开内容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。
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。
治疗用途和药物组合物
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、宫颈癌、头颈癌、输卵管癌、多发性骨髓瘤、胆管癌、胆囊癌、食管癌、前列腺癌或胶质母细胞瘤。
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。
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。可选择本公开内容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。
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)。本公开内容还提供用于产生单剂量施用单位的试剂盒。本公开内容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。在本公开内容的某些实施方案中，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。
本公开内容也提供通过施用本公开内容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。
将采用的含有本公开内容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。
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。
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；通过持续释放系统或通过植入装置。
诊断、检测和试剂盒
本公开内容的结合分子因其与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。
本公开内容的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。本公开内容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。
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公开内容。
本公开内容的另一方面提供检测与本公开内容的抗体竞争结合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。
本公开内容还提供了用于检测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。
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。
实施例
实施例1羊驼免疫
1.1免疫原制备：
根据NCBI上查询MUC1蛋白序列，与人IgGFc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)表达系统表达，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。
1.2羊驼免疫：
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。
1.3免疫血清效价检测：
1.3.1蛋白水平效价检测
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)。
实施例2针对MUC1的单域抗体免疫文库的构建及筛选
(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。
(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。
(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：
第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGGC(SEQ ID NO：45)
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO：46)
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。
第二轮PCR：
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO：47)
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO：48)
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，即单域抗体基因电泳带约为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T4 DNA连接酶(TaKaRa)连接两个片段，在16℃，过夜连接。
(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4.3×109。为检测文库的插入率，随机选取48个克隆做菌落PCR，结果显示插入率已达到90％以上。
(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。
(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(2～3×1011tfu免疫骆驼单域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表达单域抗体，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)。
(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个单域抗体作为候选抗体。
实施例3候选抗体表达纯化
将单域抗体构建到pCDNA3.4-IgG4载体上，构建成VHH-IgG4形式，然后经EXpiCHOTM(Thermo Fisher)表达系统表达，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。
实施例4候选抗体表征
(1)蛋白水平ELISA检测：
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100μ L 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。
(2)蛋白水平亲和力检测：
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 2016/0340442 A1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。蛋白水平亲和力检测结果如下表4所示，结合动力学曲线见图3。
表4.不同抗体亲和力检测结果
(3)细胞水平结合活性检测
将表达MUC1蛋白的肿瘤细胞MB468铺于96孔板中，每孔3×105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。
实施例5：VHH抗体特异性验证
1)组织交叉反应
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。
检测结果见表5，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。
表5. 34种人体组织交叉反应试验结果汇总


实施例6：MUC1单域抗体突变体表达纯化
根据MUC1单域抗体的序列合成全长核酸序列，MUC1单域抗体的序列如表6所示，NBL502-1-C4之外的抗体均为NBL502-1-C4的突变体。
表6：MUC1单域抗体氨基酸序列

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，将单域抗体构建到pCDNA3.4-IgG4载体上，构建成VHH-IgG4形式，然后经EXpiCHOTM(Thermo Fisher)表达系统表达，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。
实施例7，候选突变体抗体表征
(4)蛋白水平ELISA检测：
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5-6所示，突变后的MUC1抗体与MUC1抗原具有结合活性。
(5)蛋白水平亲和力检测：
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 2016/0340442 A1专利中的抗体序列SEQ ID NO：73制备的scFv(VH-VL)形式的IgG4抗体。蛋白水平亲和力检测结果如下表7所示。
表7.不同抗体亲和力检测结果
(6)细胞水平结合活性检测
将表达MUC1蛋白的肿瘤细胞MB468、H226、RPMI8226和SKOV3铺于96孔板中，每孔3×105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：1)。候选抗体的结果如图7-10所示，候选抗体与不同的肿瘤细胞结合活性不同。
实施例8，VHH抗体免疫组化验证
选取正常组织和9种肿瘤组织做冰冻切片，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BMK1，阴性对照为IgG4同型对照。
检测结果见表8-10，结果表明：突变后的MUC1结合分子在正常组织上无染色，而在肿瘤组织上表现出良好的染色，而1C4在正常组织上也有染色，可能会造成正常组织的损伤。
表8.候选抗体正常组织免疫组化试验结果



表9. 9种人体肿瘤组织免疫组化试验结果

表10.免疫组化试验结果

实施例9：MUC1 CAR-T细胞质粒构建
实施例9涉及的嵌合抗原受体结构为：CD8信号肽(SEQ ID NO：88)、抗原结合域、CD8铰链区(SEQ ID NO：89)、CD8跨膜域(SEQ ID NO：90)、共刺激域4-1BB(SEQ ID NO：91)、激活域CD3ζ(SEQ ID NO：92)。CAR-T编号和抗原结合域的序列如表11所示。其中1C4含有本发明提供的MUC1单域抗体NBL502-1-C4作为抗原结合域，555、1C4-z7、1C4-z25的抗原结合域为NBL502-1-C4的突变体，嵌合抗原受体BMK6.3来源于CN109715670A，BMK6.3的抗原结合域的序列如SEQ ID NO：93所示。
表11：实施例9涉及的抗原结合域序列
通过使用不同序列的单域抗体来源的MUC1 CAR与BMK3 CAR构建质粒并通过电转的方式将基因转入健康人来源的PBMC中构建MUC1 CAR-T细胞，验证CAR-T细胞在体外的表现，包括增殖能力、CAR阳性率、细胞表型、体外对靶细胞的杀伤能力等指标。
实验仪器：普通PCR仪(耶拿，TRIO)、电泳仪(Bio-Rad，1645050)、电泳槽(Bio-Rad，1704483)、台式微量离心机(Thermo，Pico17)、金属浴加热模块(Thermo， DCD)、电热恒温水槽(上海一恒，DK-8D)、自动凝胶成像仪(天能Tanon，2500)
实验试剂
实验方法
根据MUC1单域抗体和BMK6.3的序列合成全长核酸序列和引物，通过PCR获得大量目的片段，对PC23S载体进行双酶切(EcoR1和Sal1)获得载体片段，将目的片段和载体片段通过DNA连接酶连接获取质粒。将构建的载体进行测序，序列正确后大量提取该质粒。PC23S载体序列如SEQ ID NO：94所示。
实施例10，制备靶向MUC1 CAR-T细胞
通过电转的方法制备Mock-T和MUC1 CAR-T。
实验仪器：电转仪(Lonza4D)、自动化细胞计数器(CellomterK2)、显微镜(江南XD-202)、生物安全柜(Thermo Fisher Scientific)、冷藏柜(青岛海尔)、二氧化碳培养箱(Thermo)、台式低速离心机(湘仪)、Pico17台式微量离心机(Thermo)。
使用AIM-V培养基(含2％FBS)复苏PBMCs，按照5E6/mL接种进培养瓶中，放置于培养箱(37℃；5％CO2)中过夜。使用5ug/mL anti-CD3以及anti-CD28在4℃条件过夜包被6孔板或者培养瓶。将放置过夜的PBMCs接种到经过包被的6孔板或者培养瓶中，刺激48h。收集细胞并采用Lonza 4D电转仪进行电转，电转过程根据电转仪说明书操作步骤进行优化。用100uL电转液重悬5×106到7×106PBMC细胞， 将6ug上述制备质粒加入到细胞悬液中，同时电转体系中加入20ug PB酶mRNA(PB酶的氨基酸序列如SEQ ID NO：99所示)和200IU Rnase inhibitor，用EO-115程序进行电转。电转后室温静置10min。之后，将PBMCs转移到含2mL AIM-V(含有2％FBS以及100IU/mL IL-2)培养液的6孔板中(需要提前预热至37℃)，放置到培养箱(37℃；5％C02)中培养24h-48h，再将培养液补充至4mL。电转后的第3天进行第一次传代，第6天进行第二次传代，第9天CAR-T细胞制备完成。传代过程中的PBMCs培养于含2％FBS和100IU/ml IL-2的AIM-V培养液中，传代细胞密度为5×105/ml。CAR-T细胞制备过程及制备完成后，利用流式细胞仪检测细胞表面CAR阳性率及其它表面标志物。
实施例11，MUC1 CAR-T细胞体外增殖、阳性率和细胞表型检测
本实验的目的旨在通过增殖情况和阳性率来评价所制备mock-T、BMK CAR-T细胞、MUC1 CAR-T细胞的质量。
实验仪器：自动化细胞计数器(Cellomter K2)、显微镜(江南XD-202)、生物安全柜(Thermo Fisher Scientific)、冷藏柜(青岛海尔)、二氧化碳培养箱(Thermo)、台式低速离心机(湘仪)、流式细胞仪(Beckman Coulter CytoFLEX S Flow Cytometer)Pico17台式微量离心机(Thermo)。
实验试剂
供体细胞信息
实验方法
1)CAR-T细胞培养和传代
通过细胞计数和等密度传代的方法，计算各组细胞的增殖倍数并绘制细胞增殖曲线。传代密度为5×105个/mL，在Day3、6、9传代并计数；IL-2浓度为100U/mL。详见实验操作方法。
2)MUC1 CAR-T阳性细胞比例检测
通过流式细胞术的方法检测细胞在增殖过程中的CAR阳性率。详见实验操作方法。
3)MUC1 CAR-T细胞表型检测
通过流式细胞仪检测MUC1 CART的CD3/CD4/CD8、TCM/TEM来评价我们制备的CART细胞的表型。
实验仪器：自动化细胞计数器(Invitrogen Countess II FL Cellomter K2)、显微镜(江南XD-202)、Pico17台式微量离心机(Thermo)、流式细胞仪(Beckman Coulter CytoFLEX S Flow Cytometer)
实验试剂
实验结果
CAR-T细胞的增殖、活率、阳性率与表型如图11-14所示。如图11、图12所示，各组T细胞在制备D9总增殖倍数相较于D0约增殖15-20倍，细胞活率能维持在90％以上。如图13所示，制备D9的CAR阳性率各组之间差异较大，555与1C4阳性率较高。图14显示制备D9 CAR-T的CD4与CD8的占比，各组CD8+T细胞要明显高于CD4+T细胞的比例。
实施例12，MUC1 CAR-T细胞的杀伤实验
CAR-T细胞对肿瘤细胞的杀伤作用利用xCELLigence RTCA设备(Roche Applied Science，Canada)进行检测，检测过程根据设备操作说明书进行。将10000个靶细胞(MB468、HCT116)重悬于50ul培养液，接种到RTCA设备配套的微孔板中。将肿瘤细胞在RTCA设备上培养约24h，待细胞指数(cell index,CI，指示细胞生长情况)达到1.5左右，按照不同的效靶比(效应细胞：靶细胞)，将不同数量的效应细胞(CAR-T细胞)重悬于50ul培养液中，与肿瘤细胞混合，继续在RTCA设备上培养48h-96h。细胞杀伤曲线由RTCA设备记录，数据用RTCA Pro 2.3.0软件进行分析。
图15显示的是不同效靶比条件下，各组CAR-T杀伤50％MB468这种MUC1高表达肿瘤细胞所需的时间，即KT50值。各组CAR-T在1：4的效靶比情况下均能做到24h以内达到KT50。随着效靶比的下降，各组杀伤能力也相应的下降，各组间的差异不显著。
图16是各组CAR T对于MUC1极低表达的肿瘤细胞HCT116的48h肿瘤裂解率，结果表明各组CAR T对于MUC1表达低的肿瘤细胞并无杀伤能力，证明本专利中的CAR-T非靶点特异性杀伤低，安全性高。
实施例13，MUC1 CAR-T细胞的连续增殖实验
将MB468肿瘤细胞提前一天接种到六孔板中(每孔600000个肿瘤细胞)，37℃培养箱过夜后，使用丝裂霉素C处理(25ug/mL)肿瘤细胞4h，抑制其生长。PBS清洗肿瘤细胞5遍。按照1：1的效靶比接种CAR阳性T细胞，每5天为1轮，1轮结束后使用细胞计数仪统计细胞数，并使用流式细胞仪检测CAR阳性率，计算得到CAR阳性细胞数，再按照1：1的效靶比开启新一轮的肿瘤共培养实验。如此往复至第5轮结束。同时在每一轮共培养实验开始24h后，即CAR-T接种进肿瘤细胞板24h后，收集部分上清液，使用Cytokine Beads Assay检测试剂盒对每组CAR-T细胞因子释放水平进行检测。
图17显示了每一轮共培后两个donor的CAR阳性率，前两轮共培结果各组CAR阳性率逐步上升，第三轮之后开始出现较大差异，555与1C4仍能维持较高水平。
图18展示了第五轮结束，两个donor的CAR阳性T细胞相较于最开始接种的CAR阳性细胞数的理论增殖倍数。两个donor均为555与1C4的增殖倍数最高。
图19展示了共培养实验前三轮各组CAR T的IFN-γ分泌量，两donor均显示第二轮实验，各组CART细胞因子释放量最高，第三轮开始下降，与CAR阳性率变化结果较为一致。
实施例14，JL转座子系统制备分泌双抗的MUC1 CAR-T细胞
按照实施例10的步骤，采用JL转座子系统制备分泌PD1/CTLA4双特异性抗体的MUC1 CAR-T。其中PB酶mRNA替换成JL酶mRNA，用量为10μg，加入MUC1 CAR的质粒3μg，并加入PD1/CTLA4双特异性抗体的质粒1μg，其中质粒均采用太质粒框架。JL酶的序列如SEQ ID NO：95所示，太质粒空载框架的序列如SEQ ID NO：96所示，PD1/CTLA4双特异性抗体的序列如SEQ ID NO：97所示。将制备的CAR-T与癌细胞MB468、以及支气管、胃和肾正常细胞共培养，效靶比为1：4，在43h和72h测定细胞杀伤。两个donor的细胞杀伤结果如图20所示。从图中可以看出，JL转座子系统制备的MUC1 CAR-T能够对MB468癌细胞产生明显的杀伤，但对正常细胞的杀伤较低。
实施例15，嵌合抗原受体的mRNA制备MUC1 CAR-T细胞
(1)LNP负载mRNA
将可电离脂质化合物B-C14分别与DOPE、胆固醇和DMG-PEG2000(均购自赛诺帮格)以30：15：53.125：1.875的摩尔比溶于无水乙醇制备为乙醇脂质溶液(总脂质浓度为：10.78mg/mL)，B-C14的结构和具体合成方法参见中国专利CN116456967A。用10mM柠檬酸盐缓冲液(pH＝4.0)稀释MUC1 CAR mRNA(其CAR结构同实施例9中的555CAR结构，mRNA序列如SEQ ID NO：98所示)，得到mRNA水溶液(0.2mg/mL)。通过微流控装置(Microflow S，铭汰医药设备(上海)有限公司)以1：3的体积比混合乙醇脂质溶液和mRNA水溶液，总流速24mL/min，所得的脂质纳米颗粒溶液用含有10％蔗糖的DPBS溶液稀释6倍，并用0.2μm无菌过滤器过滤，得到包封MUC1 CAR mRNA的LNP制剂(脂质纳米颗粒)。样品-80℃冷 冻保存待用。
(2)CAR-T制备
收集活化的T细胞，用OPTI-MEM培养基重悬后，选用life计数仪，Trypan Blue Stain染色进行细胞计数，按照5E5 cells/300μL/孔的细胞量将活化的T细胞接种至24孔板，随后按照0.5μg/5E5 cells/孔的剂量加入载脂蛋白ApoE4，以增加T细胞的转染效率，再按照0.2μg/5×105cells/200μL/孔的剂量分别加入脂质纳米颗粒，然后轻轻摇晃孔板，保证充分混匀后，将孔板置于37℃，5％CO2培养箱培养24h，即获得CAR-T细胞。
(3)CAR-T细胞的培养
离心收集转染24h的CAR-T细胞，用OPTI-MEM培养基重悬后，按照5E5cells/500μL/孔的细胞量将转染后的T细胞接种至24孔板。将孔板置于37℃，5％CO2培养箱培养不同时间，对CAR-T细胞进行表征，以空白T细胞作为Control组。如图21所示，细胞实验组的细胞活率大于90％，用LNP递送mRNA对细胞无毒性，阳性率均大于80％。
(4)杀伤癌细胞
将mRNA转染至细胞制备CAR-T，LNP的转染剂量为0.5μg/5E5 cells，转染时间为24h，记为“LNP-500ng”；采用电转的方式，使用相同剂量的CAR mRNA，记为“电转”。MOCKT采用eGFP mRNA，也分别采用LNP和电转的方式，使用相同剂量的mRNA，作为对照，分别记为LNP-MOCKT、电转-MOCKT。采用实施例12类似的方法，将靶细胞SKOV3和MDA-MB468与CAR-T细胞或者MOCKT细胞，按照2：1、1：2、1：4、或1：8的效靶比共培养24h，根据细胞指数的数值来分析CART细胞的杀伤效果。
各组细胞制备的转染24h阳性率如图22所示，LNP和电转方式制备的CAR-T细胞阳性率没有明显差异。不同效靶比对MAD-MB468、SKOV3的杀伤分别如图23-24所示。LNP和电转方式制备的CAR-T细胞杀伤效果差异不大，相同的效靶比条件下，CART细胞对高表达MUC1的MDA-MB468有更好的杀伤效果，其中效靶比1：2的条件下MDA-MB468的杀伤效率达到90％。

Claims (15)

  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；
    所述CDR1包含：SEQ ID NO：2所示的序列，或与SEQ ID NO：2相比含有以下一个或多个突变的序列：T3Q、R5D、R5H、R5K、R5E、R5Y、R6E、R6H、R6K、R6D、R6Y；
    所述CDR2包含：SEQ ID NO：12所示的序列，或与SEQ ID NO：12相比含有以下一个或多个突变的序列：I1G、T3Q、T3W、F4D、D6A、D6G、D7S、T8Q；
    所述CDR3包含：SEQ ID NO：23所示的序列，或与SEQ ID NO：23相比含有以下一个或多个突变的序列：T1Q、I3G、Y6H、Q8E、Q8Y、L9G、L9Y、L9S、S10N、D12Q。
  2. 如权利要求1所述的MUC1结合分子，其特征在于，所述CDR1的序列如SEQ ID NO：2所示，所述CDR2如SEQ ID NO：12所示，所述CDR3如SEQ ID NO：23所示；
    或所述CDR1、CDR2和CDR3含有以下表2中组A1到A17中任一组所示的突变：
    表2

  3. 如权利要求1或2任一项所述的MUC1结合分子，其特征在于，所述MUC1单域抗体含有以下表3中组B1到组B18中任一组SEQ ID NO所示的CDR1、CDR2和CDR3：
    表3

    优选地，所述MUC1单域抗体的氨基酸序列如SEQ ID NO：34、71-87任一所示，更优选如SEQ ID NO：34、71、72、78或83所示。
  4. 如权利要求1-3任一项所述的结合分子，其特征在于，所述MUC1结合分子包含一条、两条或多条本文所述的抗MUC1单域抗体；和/或，所述MUC1结合分子为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。
  5. 含有权利要求1-4中任一项所述MUC1结合分子和其它多肽的融合蛋白，
    优选地，所述其它多肽位于所述结合分子的N端和/或C端，所述其它多肽选自将结合分子定位到不同细胞器的多肽、用于纯化的标签或者用于免疫反应的标签、跨膜蛋白或其跨膜区、嵌合抗原受体组件；
    更优选地，所述融合蛋白为嵌合抗原受体，其抗原结合域包含所述MUC1结合分子。
  6. 一种核酸分子，其特征在于，所述核酸分子包含选自以下的序列：
    (1)权利要求1-4中任一项所述MUC1结合分子或权利要求5所述融合蛋白的编码序列；
    (2)(1)的互补序列。
  7. 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6所述的核酸分子；优选地，所述核酸构建物是克隆载体、表达载体、整合载体或mRNA。
  8. 一种宿主细胞，其特征在于：
    (1)表达权利要求1-4中任一项所述MUC1结合分子或权利要求5所述融合蛋白；
    (2)包含权利要求6所述的核酸分子；和/或
    (3)包含权利要求7所述的核酸构建物；
    优选地，所述宿主细胞是免疫效应细胞，更优选T细胞、TIL细胞或NK细胞。
  9. 如权利要求8所述的宿主细胞，其特征在于，所述融合蛋白为嵌合抗原受体，所述宿主细胞表达嵌合抗原受体，和/或含有所述嵌合抗原受体的编码序列；
    优选地，所述嵌合抗原受体的编码序列为DNA或mRNA；
    更优选地，所述宿主细胞还表达治疗剂，和/或含有治疗剂的编码序列。
  10. 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8或9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。
  11. 一种药物组合物，包含权利要求1-4中任一项所述的MUC1结合分子、权利要求5所述的融合蛋白、权利要求6所述的核酸分子、权利要求7所述的核酸构建物、或权利要求8或9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。
  12. 权利要求1-4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。
  13. 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1-4中任一项所述的MUC1结合分子、权利要求5所述的融合蛋白、权利要求6所述的核酸分子、权利要求7所述的核酸构建物、或权利要求8或9所述的宿主细胞；
    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；
    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。
  14. 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1-4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。
  15. 权利要求1-4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。
PCT/CN2024/108846 2023-08-01 2024-07-31 Muc1结合分子和包含其的嵌合抗原受体 WO2025026358A1 (zh)

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