WO2024124637A1 - 抗cll1单域抗体及其应用 - Google Patents

抗cll1单域抗体及其应用 Download PDF

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WO2024124637A1
WO2024124637A1 PCT/CN2022/143892 CN2022143892W WO2024124637A1 WO 2024124637 A1 WO2024124637 A1 WO 2024124637A1 CN 2022143892 W CN2022143892 W CN 2022143892W WO 2024124637 A1 WO2024124637 A1 WO 2024124637A1
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cll1
domain antibody
target
protein
antibody
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PCT/CN2022/143892
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French (fr)
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杨鑫
朱建高
杨文君
俞建东
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浙江康佰裕生物科技有限公司
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Publication of WO2024124637A1 publication Critical patent/WO2024124637A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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

Definitions

  • the present invention belongs to the field of biomedicine, and specifically relates to an anti-CLL1 single domain antibody and an application thereof.
  • AML Acute myeloid leukemia
  • CLL1 immune targeted therapy targeting CLL1 is effective in treating AML.
  • CLL1 C-type lectin-like molecule 1
  • CLEC12A C-type lectin domain family 12 member A
  • CLL1 is a type II transmembrane glycoprotein that plays an important role in immune regulation as an inhibitory receptor.
  • CLL1 is present in myeloid cells in peripheral blood and bone marrow and in most AML cells. It is also expressed on most AML CD34 + CD38 - stem cells, but not on normal CD34 + CD38 - stem cells.
  • CLL1 has become a potential target for AML treatment and diagnosis due to its special expression pattern.
  • CLL1 is also expressed on myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML) cells.
  • MDS myelodysplastic syndrome
  • CML chronic myeloid leukemia
  • Antibodies are effector immune molecules produced by B cells proliferating and differentiating into plasma cells after being specifically stimulated by B cell antigen epitopes. They mediate humoral immunity and are a type of immunoglobulin (Ig) that can specifically bind to antigens. Ig specifically recognizes and binds to the corresponding antigen epitope through the antigen binding groove composed of its V region CDR in a lock-key complementary relationship. The specific binding ability of antibodies to antigens makes them of great significance in disease diagnosis and immune prevention and treatment.
  • Antibody drugs composed of antibody substances (including whole antibody molecules and antibody fragments with therapeutic functions) are one of the important means of targeted therapy and have become the most promising and application-valuable hot area in the current biopharmaceutical industry.
  • Heavy chain antibody is a naturally occurring antibody type. It is a unique antibody type possessed by camelids or cartilaginous fish. Its antibody domain naturally lacks light chains and is composed of only two heavy chains.
  • the antigen recognition function of heavy chain antibodies is mainly determined by the variable region (VHH) of heavy chain antibodies. VHH alone can recognize antigens, so it is also called single-domain antibody (sdAb).
  • Single-domain antibodies lack antibody light chains and only have heavy chain variable regions. Because of their small molecular weight, they are also called nanobodies (Nanobody, Nb). The molecular weight of single-domain antibodies is only about 13-15KDa, with a diameter of about 2.5nm and a length of 4nm.
  • single-domain antibodies also have many unique properties, such as good stability, the ability to reach specific antigen epitopes, arbitrary combination of building blocks, and low production costs.
  • the conventional method of obtaining single-domain antibodies consists of many steps such as multiple immunization of camelids, isolation of B lymphocytes, amplification of VHH regions, construction and screening of display libraries. With the development of synthetic biology, it has become possible to construct a high-quality, randomized, large-capacity single-domain antibody library based on total synthesis.
  • VHH single-domain antibodies have been widely used in miniaturized genetic engineering antibody research, new drug development, and disease diagnosis and treatment due to their advantages such as stable structure, small molecule, good solubility, tolerance to various adverse environments, good formulation stability, easy recombinant expression, and easy humanization.
  • the research and development of single-domain antibodies has very broad prospects and important significance in the fields of drug application and clinical diagnosis.
  • One of the purposes of the present invention is to provide a single domain antibody targeting CLL1 antigen and its application in tumor targeted therapy.
  • the present invention first provides a single domain antibody (anti-CLL1 single domain antibody), named CLL1-VHH-16, which is composed of a heavy chain variable region (VHH), and the heavy chain variable region may include a complementary determining region CDR1 with amino acid sequences of positions 26-35 of SEQ ID No.1, a complementary determining region CDR2 with amino acid sequences of positions 50-59 of SEQ ID No.1, and a complementary determining region CDR3 with amino acid sequences of positions 99-118 of SEQ ID No.1.
  • the single domain antibody (anti-CLL1 single domain antibody) may be a single domain antibody that specifically binds to a CLL1 protein.
  • amino acid sequence of the heavy chain variable region may be SEQ ID No.1 or an amino acid sequence that has at least 80% identity with SEQ ID No.1 and has the same function.
  • the single domain antibody (anti-CLL1 single domain antibody) has only the variable region (VHH) of the heavy chain antibody, which is composed of the framework region FR1, the complementary determining region CDR1, the framework region FR2, the complementary determining region CDR2, the framework region FR3, the complementary determining region CDR3 and the framework region FR4 in sequence.
  • VHH variable region
  • the amino acid sequence of the single domain antibody CLL1-VHH-16 may be SEQ ID No. 1.
  • positions 1-25 of SEQ ID No. 1 are framework region FR1
  • positions 26-35 are complementary determining region CDR1
  • positions 36-49 are framework region FR2
  • positions 50-59 are complementary determining region CDR2
  • positions 60-98 are framework region FR3
  • positions 99-118 are complementary determining region CDR3
  • positions 119-129 are framework region FR4.
  • the nucleotide sequence of the nucleic acid molecule encoding the single domain antibody CLL1-VHH-16 (CLL1-VHH-16 gene) is shown in SEQ ID No. 2.
  • sequences of the complementarity determining regions are defined according to the Kabat numbering system.
  • variants of the single domain antibody sequences of the present invention with improved affinity and/or titer can be obtained by using methods known in the art and are included in the scope of protection of the present invention.
  • amino acid substitutions can be used to obtain antibodies with further improved affinity.
  • codon optimization of nucleotide sequences can also be used to improve translation efficiency in expression systems for producing antibodies.
  • polynucleotides containing sequences that optimize antibody specificity by applying directed evolution methods to any nucleic acid sequence of the present invention also fall within the scope of the present invention.
  • substitutions, insertions or deletions may occur in one or more complementary determining regions or framework regions of the single domain antibodies of the present invention, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen.
  • conservative changes e.g., conservative substitutions, as is well known to those skilled in the art, conservative substitutions of amino acids do not change the properties and functions of proteins
  • conservative changes may be made to the complementary determining regions and/or framework regions, which do not substantially reduce binding affinity.
  • such changes may be outside the antigen contact residues in the complementary determining regions.
  • the present invention also provides a biological material related to the single-domain antibody CLL1-VHH-16, and the biological material may be any one of the following C1) to C4):
  • C1 a nucleic acid molecule encoding the heavy chain variable region of the single domain antibody CLL1-VHH-16;
  • C2 an expression cassette containing the nucleic acid molecule described in C1);
  • C4 a recombinant host cell containing the nucleic acid molecule described in C1), or a recombinant host cell containing the expression cassette described in C2), or a recombinant host cell containing the recombinant vector described in C3).
  • the recombinant host cell described in C4) can express the single-domain antibody CLL1-VHH-16.
  • nucleic acid molecule may be any of the following:
  • the nucleotide sequence or coding sequence is a DNA molecule of SEQ ID No. 2;
  • D2 A DNA molecule that has 75% or more identity with the nucleotide sequence defined in D1) and has the same function.
  • the DNA molecule shown in SEQ ID No.2 encodes the single domain antibody CLL1-VHH-16 with the amino acid sequence of SEQ ID No.1.
  • the aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
  • the 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the 90% or more identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the greater than 95% identity may be at least 95%, 96%, 97%, 98% or 99% identity.
  • identity refers to the identity of an amino acid sequence or a nucleotide sequence.
  • the identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of the amino acid sequence, the value of identity (%) can then be obtained.
  • the vector described herein refers to a vector capable of carrying exogenous DNA or target gene into host cells for amplification and expression.
  • the vector may be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC), etc.), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus), etc.).
  • the vector is pComb3XSS plasmid and/or pcDNA3.1 vector.
  • the host cell (also referred to as recipient cell) described herein refers to any type of cell that can be used to introduce a vector.
  • the host cell can be a eukaryotic cell (e.g., a plant cell, an animal cell, a fungus, or an algae), or can be a prokaryotic cell (e.g., a bacterium or a protozoan).
  • the host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and/or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell.
  • the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but are not limited thereto;
  • the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cell), fish cell (e.g., grass carp, carp, rainbow trout or cat
  • CHO cell Chinese hamster
  • the fungus may be yeast, and the yeast may be from the genus Saccharomyces (such as Saccharomyces cerevisiae), the genus Kluyveromyces (such as Kluyveromyces lactis), the genus Pichia (such as Pichia pastoris), the genus Schizosaccharomyces (such as Schizosaccharomyces pombe), the genus Hansenula (such as Hansenula polymorpha), etc., but not limited thereto.
  • Saccharomyces such as Saccharomyces cerevisiae
  • the genus Kluyveromyces such as Kluyveromyces lactis
  • the genus Pichia such as Pichia pastoris
  • Schizosaccharomyces such as Schizosaccharomyces pombe
  • Hansenula such as Hansenula polymorpha
  • the fungus may also be from the genus Fusarium (Fusarium sp.), the genus Rhizoctonia (Rhizoctonia sp.), the genus Verticillium (Verticillium sp.), the genus Penicillium (Penicillium sp.), the genus Aspergillus (Aspergillus sp.), the genus Cephalosporium (Cephalosporium sp.), etc., but not limited thereto.
  • the algae may be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but not limited thereto.
  • the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto.
  • the bacteria may be Escherichia coli, Bacillus subtilis or Bacillus pumilus.
  • the host cell is Escherichia coli TG1 and/or HEK293 cells.
  • the recombinant vector described herein refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro, and can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with replication, integration, amplification and/or expression capabilities in the recipient cell.
  • the recombinant vector is pcDNA3.1-CLL1-VHH-16-hFc.
  • the recombinant vector pcDNA3.1-CLL1-VHH-16-hFc is designed to facilitate purification.
  • a human IgG1Fc gene (hFc gene, nucleotide sequence is SEQ ID No. 3) is added to the 3' end of the coding gene of the single-domain antibody CLL1-VHH-16 (SEQ ID No. 2) to obtain the CLL1-VHH-16-hFc fusion gene, and then a BamHI restriction site is added before the 5' start codon and an EcoRI restriction site is added after the 3' stop codon of the fusion gene.
  • the fusion gene is cloned between the BamHI and EcoRI recognition sites of the eukaryotic expression vector pcDNA3.1 to obtain the recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-16-hFc expressing the single-domain antibody CLL1-VHH-16 with a C-terminal fusion hFc gene.
  • the recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-16-hFc contains the coding gene of the single domain antibody CLL1-VHH-16 shown in SEQ ID No.2.
  • the nucleotide sequence of human IgG1Fc gene (696bp) is shown in SEQ ID No.3, and the amino acid sequence it encodes is shown in SEQ ID No.4.
  • the recombinant host cell described herein refers to a recombinant host cell whose function has changed by manipulating and modifying the genes of the target host cell.
  • a recombinant host cell obtained by introducing an exogenous target gene or a recombinant vector into a target host cell or a recombinant host cell obtained by directly editing the endogenous genes of the target host cell.
  • the recombinant host cell can be understood to refer not only to a specific recombinant host cell, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and/or changes, the offspring may not necessarily be completely identical to the original parent cell, but is still included in the scope of the recombinant host cell.
  • the recombinant host cell is a recombinant host cell obtained by introducing the recombinant vector pcDNA3.1-CLL1-VHH-16-hFc into HEK293 cells.
  • the present invention also provides a pharmaceutical composition, which may contain the single-domain antibody CLL1-VHH-16.
  • the pharmaceutical composition may further contain a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.
  • the pharmaceutical composition has at least one of the following uses:
  • M3 is used to mediate drug-specific recognition of tumors expressing CLL1 antigen.
  • the present invention also provides a reagent or a kit, which may contain the single-domain antibody CLL1-VHH-16, and the reagent or the kit has at least one of the following uses:
  • F3 is used for in vivo imaging of CLL1 protein.
  • the kit may be a chemiluminescent immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, or a fluorescent immunoassay kit, but is not limited thereto.
  • the present invention also provides any of the following uses of the single-domain antibody CLL1-VHH-16 and/or the biomaterial:
  • E8 is used in the preparation of CAR cells targeting CLL1.
  • the CLL1 target-related disease may be a CLL1-positive cancer (such as leukemia).
  • the CLL1-positive cancer may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), but is not limited thereto.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • CML chronic myeloid leukemia
  • the product described in E6) that mediates drug-specific recognition of tumors expressing CLL1 antigen may be a drug delivery system that specifically targets CLL1 protein, and the drug delivery system contains the single-domain antibody CLL1-VHH-16.
  • the drug delivery system can be a liposome drug delivery system, a polymer micelle drug delivery system, a polymer disc drug delivery system or a nanoparticle drug delivery system.
  • the drug-carrying system can be used for targeted transport and/or site-specific release of drugs.
  • the CLL1-targeting CAR cell expresses a chimeric antigen receptor (CAR), and the chimeric antigen receptor may contain the single-domain antibody CLL1-VHH-16.
  • CAR chimeric antigen receptor
  • the antigen recognition domain of the chimeric antigen receptor may be the single domain antibody CLL1-VHH-16.
  • the CAR cells include but are not limited to CAR-T cells, CAR-NK cells, CAR-macrophages (CAR-M cells), CAR-iPSCs or CAR-PSCs.
  • the detection of CLL1 protein described herein may be detecting whether the sample to be tested contains CLL1 protein and/or detecting the content of CLL1 protein in the sample to be tested.
  • the sample to be tested may be a cell or tissue sample.
  • the products for detecting CLL1 protein include products for detecting antigen-antibody binding using enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination assay or immunoturbidimetry.
  • the product described herein may be a reagent, a test kit, a chip or a test paper.
  • the present invention also provides a method for preparing the single-domain antibody CLL1-VHH-16, which may include: constructing a recombinant expression vector containing a nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody CLL1-VHH-16; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single-domain antibody; culturing the recombinant cell, and obtaining the single-domain antibody by separation and purification.
  • nucleic acid molecule encoding the heavy chain variable region of the single-domain antibody CLL1-VHH-16 may be any of the following:
  • the nucleotide sequence or coding sequence is a DNA molecule of SEQ ID No. 2;
  • D2 A DNA molecule that has 75% or more identity with the nucleotide sequence defined in D1) and has the same function.
  • the host cell may be a HEK293 cell.
  • the introduction can be carried out by transforming the vector carrying the single domain antibody gene of the present invention into the host cell through any known transfection method such as calcium phosphate co-precipitation method, liposome-mediated method, electroporation method or viral vector method.
  • the present invention also provides a method for diagnosing or assisting in the diagnosis of a CLL1 target-related disease, which may include: isolating a sample from a subject, then using the single-domain antibody CLL1-VHH-16 to detect the content of CLL1 protein in the sample, and diagnosing or assisting in the diagnosis of the CLL1 target-related disease based on the content of the CLL1 protein (compared with the content of the CLL1 protein in the disease diagnosis standard).
  • the present invention also provides a method for screening CLL1 target-related diseases, which may include: isolating a sample from a subject, then using the single-domain antibody CLL1-VHH-16 to detect the content of CLL1 protein in the sample, and screening for CLL1 target-related diseases based on the content of CLL1 protein (compared with the content of CLL1 protein in a disease screening standard).
  • the present invention also provides a method for preventing or treating a CLL1 target-related disease, which may include administering the single-domain antibody CLL1-VHH-16 or the pharmaceutical composition described herein to a subject suffering from a CLL1 target-related disease.
  • the CLL1 target-related disease may be a CLL1 target-related tumor.
  • the CLL1 target-related tumor may be a CLL1-positive cancer.
  • the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.
  • the present invention also provides a method for detecting CLL1 protein in vitro, which may include using the single domain antibody CLL1-VHH-16 or the kit described herein to detect CLL1 protein.
  • the present invention also provides an antibody-drug conjugate, comprising an antibody portion and a conjugated portion, wherein the antibody portion may comprise the single-domain antibody CLL1-VHH-16.
  • the conjugated moiety may include a detectable label, a chemical drug or a cytotoxin.
  • the antibody moiety and the conjugated moiety may be covalently linked directly or through a linker.
  • the detectable label may be selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent agent such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives
  • a fluorescent dye such as fluorescein or fluorescent protein
  • the chemical drug can be a drug that can kill tumor cells.
  • the cytotoxin may be a microtubulin inhibitor (such as maytansine, auristatin), a DNA synthesis inhibitor (such as calicheamicin) or an RNA synthesis inhibitor (such as amatoxin).
  • a microtubulin inhibitor such as maytansine, auristatin
  • a DNA synthesis inhibitor such as calicheamicin
  • RNA synthesis inhibitor such as amatoxin
  • the purpose of the applications and methods described in the present invention may be disease diagnosis, disease prognosis and/or disease treatment, or non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and/or disease treatment, or non-disease diagnosis, non-disease prognosis and/or non-disease treatment.
  • CLL1 C-type lectin-like molecule 1 (C-type lectin-like molecule 1), also known as C-type lectin domain family 12 member A (CLEC12A). It is a type II transmembrane glycoprotein that plays an important role in immune regulation as an inhibitory receptor.
  • CLL1 is present in myeloid cells in peripheral blood and bone marrow and in most acute myeloid leukemia (AML) cells. It is also expressed on most AML CD34 + CD38 -stem cells, but not on normal CD34 + CD38 -stem cells.
  • AML acute myeloid leukemia
  • CLL1 has become a potential target for AML treatment and diagnosis due to its special expression pattern.
  • CLL1 is also expressed on myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML) cells.
  • MDS myelodysplastic syndrome
  • CML chronic myeloid leukemia
  • the present invention first uses CLL1 recombinant protein to immunize alpacas to obtain single-domain antibody genes, and constructs a single-domain antibody expression library, and then screens high-affinity anti-CLL1 single-domain antibodies (CLL1-VHH-16) from the single-domain antibody expression library through phage display screening technology. After sequencing to obtain the antibody gene sequence, CLL1-VHH-16 is prepared by genetic engineering methods. The obtained antibodies are further subjected to functional experiments such as affinity and specificity. The results show that the single-domain antibody of the present invention has high affinity, can specifically target CLL1 positive cells (cells expressing CLL1 protein) well, and can be successfully applied to the detection of CLL1 expression in AML patient bone marrow cells.
  • the single-domain antibody of the present invention can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system, and can be made into specific antibody drugs for clinical prevention and treatment of CLL1 target-related diseases (such as acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia), or CLL1 protein detection kits, etc.
  • CLL1 target-related diseases such as acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia
  • CLL1 protein detection kits etc.
  • the single-domain antibody drug of the present invention has a stable structure, small molecule, easy recombinant expression, and low production cost. It can be used alone or as a drug delivery system to carry related drugs. It has very broad prospects and important significance in the fields of drug application and clinical diagnosis.
  • Figure 1 is the agarose gel electrophoresis result of total RNA extracted from alpaca PBMC.
  • Figure 2 is the result of agarose gel electrophoresis of bacterial library colony PCR.
  • Figure 3 shows the results of detecting CLL1 expression on different tumor cell lines using FITC-labeled CLL1-VHH-16 antibody, where Isotype represents CLL1-VHH-16-hFc antibody not labeled with FITC, and CLL1 represents FITC-CLL1-VHH-16-hFc antibody.
  • FIG. 4 shows the results of using FITC-labeled CLL1-VHH-16 antibody to detect the expression of CLL1 on bone marrow cells of AML patients.
  • CLL1VHH-FITC represents FITC-labeled CLL1-VHH-16 antibody, i.e., FITC-CLL1-VHH-16-hFc antibody in Example 2;
  • CLL1-APC represents APC-labeled anti-human CLL1 specific antibody (Cat. No. 143405) of Biolegend.
  • the pComb3XSS plasmid in the following examples is a product of Beijing Zhuangmeng International Biogene Technology Co., Ltd., with the product number ZK129.
  • helper phage M13K07 in the following examples is a product of Chengdu Renyu Biotechnology Co., Ltd., item number A001-1.
  • the K562 cells (CLL1-negative cells) in the following examples are products of Nanjing Kebai Biotechnology Co., Ltd., with the product number CBP60529; the MM1.S cells (CLL1-negative cells) are products of Beijing Biobo Biotechnology Co., Ltd., with the product number bio-129535; and the U937 cells (CLL1-positive cells) are products of Beijing Biobo Biotechnology Co., Ltd., with the product number bio-73180.
  • the bone marrow cells of AML patients were obtained from Zhejiang Provincial People's Hospital.
  • the method for preparing CLL1-positive cells K562-CLL1 is to insert CLL1 between the BamHI and EcoRI sites of pcDNA3.1, and transfect K562 cells with the prepared recombinant vector to obtain CLL1-positive cells K562-CLL1.
  • the vector pcDNA3.1 in the following examples is a product of Changsha Abiwei Biotechnology Co., Ltd., product number: HG-VPI0001.
  • CLL1 recombinant protein was used to immunize alpacas to obtain single domain antibody genes, and a single domain antibody expression library was constructed. Then, high-affinity anti-CLL1 single domain antibodies were screened from the single domain antibody expression library through phage display screening technology.
  • the CLL1 recombinant protein used as antigen is as follows:
  • CLL1 immune antigen CLL1-hIgG1Fc (B918601) protein is a product of Shanghai Baiying Biotechnology Co., Ltd.
  • CLL1 screening antigen CLL1-6 ⁇ His (B918602) protein is a product of Shanghai Baiying Biotechnology Co., Ltd.
  • Alpacas (2-3 years old, weighing about 100 catties) were immunized with CLL1 immune antigen CLL1-hIgG1Fc for a total of four times, with 0.5 mg CLL1-hIgG1Fc injected subcutaneously each time, and the interval between two immunizations was 2 weeks. Before the next immunization, 5 ml of blood was drawn, and the plasma was separated to test the antibody titer after the previous immunization. The titer of the four immunizations after four immunizations was tested one week after the injection.
  • the titer of antibodies in serum was detected by indirect ELISA method. The steps are as follows:
  • the immune antigen CLL1-hIgG1Fc was diluted to 2ug/ml with PBS and coated on the ELISA plate at 100 ⁇ L/well at 4°C overnight; the control group was coated with 3% BSA;
  • the results of serum titer test are shown in Table 1, and the ones in bold are positive wells. After four immunizations, the serum titer in the alpaca reached 10 5 , that is, the antibody level reached 10 5 .
  • the antibody level in the alpaca reached 10 5 .
  • the Ag coating was the immune antigen CLL1-hIgG1Fc coating.
  • PBSTB was the blocking solution.
  • PBMC peripheral blood mononuclear cells
  • alpaca peripheral blood mononuclear cells (abbreviated as alpaca PBMC cells) were separated by density gradient centrifugation and dissolved in Trizol for storage. Total RNA of alpaca PBMC cells was extracted and the total RNA was reverse transcribed into cDNA using Takara reverse transcription kit. The specific steps are as follows:
  • RNA sample was divided into two portions, one using the Oligo dT Primer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer.
  • the total RNA was reverse transcribed into cDNA according to the operating procedures in the kit manual, and a total of 31.7 ⁇ g of cDNA was obtained.
  • the VHH target fragment was amplified by conventional PCR using 8 ng of cDNA as a template, and a total of 83 ⁇ g was obtained.
  • pComb3XSS as the phage plasmid vector, and use restriction endonucleases Spe I and Sac I to digest the pComb3XSS vector and the PCR product of VHH (i.e., the VHH target fragment), respectively, and incubate at 37°C for 4 hours;
  • helper phage M13K07 at a ratio of 1:20 for the number of bacteria and phage, and incubate at 37°C and 250 rpm for 30 min;
  • the phage library was subjected to three rounds of affinity screening using the screening antigen (CLL1-6 ⁇ His), including titer detection, amplification of phage eluates, and purification of phages.
  • CLL1-6 ⁇ His the screening antigen
  • V is the volume of phage added (in ⁇ L)
  • T library is the phage titer
  • T is the phage titer (unit: pfu/mL)
  • D is the dilution multiple
  • N is the number of single colonies in the plate with the corresponding dilution multiple.
  • V is the volume of the transferred bacterial solution (unit: ⁇ L)
  • OD 600 is the OD 600 of the constructed eluate bacterial library
  • V is the volume of helper phage added (unit: mL)
  • T helper-phage is the helper phage titer
  • OD 600 is the OD 600 value of the bacterial solution
  • the method is the same as step 3.1.2 for initial phage titer detection.
  • the screening method is the same as the first round screening method in step 3.1.1.
  • the phage sub-library obtained by the first round of screening amplification and purification (the first round of screening phage sub-library) is used as the input phage library for the second round of screening to obtain the phage eluate for the second round of screening.
  • the detection method is the same as step 3.1.2 for initial phage titer detection.
  • the method is the same as steps 3.1.3 and 3.1.4 for amplification and purification of the first round of phage eluate to obtain the second round of screening phage sub-library.
  • the method is the same as step 3.1.5 for titer detection of the first round of screening of phage sub-library.
  • the screening method is the same as the first round screening method in step 3.1.1.
  • the phage sub-library obtained by the second round of screening amplification and purification (second round screening phage sub-library) is used as the input phage library for the third round of screening to obtain the phage eluate for the third round of screening.
  • the detection method is the same as step 3.1.2 for initial phage titer detection.
  • the method is the same as step 3.1.3 for amplifying the first round of phage eluate to obtain the third round of amplification of phage eluate.
  • *1 enrichment degree is the screening titer divided by the input titer.
  • *2 difference multiple is the screening titer divided by the control titer.
  • V is the volume of helper phage added (unit: mL)
  • T helper-phage is the helper phage titer
  • OD 450 > 0.5 was determined as a positive result, and the obtained positive clone was the phage containing the single domain antibody that could bind to the CLL1 protein.
  • clones initially identified as positive were subjected to secondary ELISA verification using the same method as step 4.1.
  • the positive monoclonal antibody in step 4.3 was selected and sequenced to obtain the antibody gene sequence, and an anti-CLL1 single domain antibody (CLL1-VHH) was prepared using a genetic engineering method.
  • CLL1-VHH-16 One of the antibodies was named CLL1-VHH-16.
  • VHH variable region of the heavy chain antibody, which is composed of the framework region FR1, the complementary determining region CDR1, the framework region FR2, the complementary determining region CDR2, the framework region FR3, the complementary determining region CDR3 and the framework region FR4.
  • VHH variable region
  • the amino acid sequence of the single domain antibody CLL1-VHH-16 is SEQ ID No. 1. Positions 1-25 of SEQ ID No. 1 are framework region FR1, positions 26-35 are complementarity determining region CDR1, positions 36-49 are framework region FR2, positions 50-59 are complementarity determining region CDR2, positions 60-98 are framework region FR3, positions 99-118 are complementarity determining region CDR3, and positions 119-129 are framework region FR4.
  • the nucleotide sequence of the nucleic acid molecule encoding the single domain antibody CLL1-VHH-16 (CLL1-VHH-16 gene) is shown in SEQ ID No. 2.
  • Amino acid sequence of the single domain antibody CLL1-VHH-16 The three underlined parts are CDR1, CDR2 and CDR3 respectively.
  • sequences of the complementarity determining regions are defined according to the Kabat numbering system.
  • the above-mentioned single domain antibody is a single domain antibody that specifically binds to the CLL1 protein.
  • the eukaryotic expression vector pcDNA3.1 was used to express the anti-CLL1 single domain antibody (CLL1-VHH-16).
  • a human IgG1Fc gene (hFc gene, nucleotide sequence is SEQ ID No.3, and its encoded amino acid sequence is SEQ ID No.4) was added to the 3' end of the coding gene of the single-domain antibody CLL1-VHH-16 (SEQ ID No.2) to obtain the CLL1-VHH-16-hFc fusion gene. Then, a BamHI restriction site was added before the 5' start codon and an EcoRI restriction site was added after the 3' stop codon of the fusion gene.
  • the fusion gene was cloned between the BamHI and EcoRI recognition sites of the eukaryotic expression vector pcDNA3.1 to obtain the recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-16-hFc expressing the single-domain antibody CLL1-VHH-16 with a C-terminal fusion hFc gene.
  • the eukaryotic expression vector pcDNA3.1-CLL1-VHH-16-hFc obtained in step 5.2.1 was transfected into HEK293 cells by conventional electroporation for expression.
  • Protein A has the activity of binding to the Fc fragment of IgG, so the anti-CLL1 single domain antibody expressed by HEK293 cells was purified using Protein A column (conventional purification steps).
  • Antibody to be tested the anti-CLL1 single domain antibody (CLL1-VHH-16-hFc) fused with the hFc gene prepared in step 5.2.3.
  • K562-CLL1 and U937 cells Resuspend CLL1-positive cells K562-CLL1 and U937 cells to a density of 2 ⁇ 10 6 cells/mL, take 100 ⁇ L and place it in a 96-well V-bottom plate, centrifuge and discard the supernatant and collect the cells (K562 cells are used as CLL1-negative cell controls);
  • the affinity is determined by calculating the equilibrium dissociation constant (KD) of the binding of CLL1 VHH antibody to target cells according to the formula.
  • KD equilibrium dissociation constant
  • MFI-Con represents the relative mean fluorescence intensity, which is the value of the mean fluorescence intensity of the experimental group minus the mean fluorescence intensity of the background;
  • CLL1-scfv IgG Fc represents the amount of antibody used (unit: ng).
  • the standard curve was drawn with the reciprocal of the antibody usage as the horizontal axis and the reciprocal of the relative mean fluorescence intensity as the vertical axis to obtain the linear regression equation.
  • the intersection of the standard curve and the vertical axis is 1/Fmax
  • KD is the slope of the line multiplied by Fmax.
  • FITC-labeled CLL1-VHH-16 antibody (FITC-CLL1-VHH-16-hFc) was used to detect the expression of CLL1 on different tumor cells.
  • CLL1-negative cell lines control group: K562 cells and MM1.S cells; CLL1-positive cell lines (experimental group): U937 cells and CLL1-overexpressing cell line K562-CLL1 were resuscitated at 37°C and added to cell culture flasks, cultured at 37°C, 5% CO2 , and used for detection of anti-CLL1 single domain antibody (FITC-CLL1-VHH-16-hFc) after two passages.
  • control group K562 cells and MM1.S cells
  • CLL1-positive cell lines experimental group: U937 cells and CLL1-overexpressing cell line K562-CLL1 were resuscitated at 37°C and added to cell culture flasks, cultured at 37°C, 5% CO2 , and used for detection of anti-CLL1 single domain antibody (FITC-CLL1-VHH-16-hFc) after two passages.
  • FITC-labeled CLL1-VHH-16 antibody (FITC-CLL1-VHH-16-hFc) was used to detect bone marrow cells from three patients with acute myeloid leukemia (AML) and compared with the APC-labeled anti-human CLL1 specific antibody (Cat. No. 143405) from Biolegend.
  • AML acute myeloid leukemia
  • APC-labeled anti-human CLL1 specific antibody Cat. No. 143405
  • CLL1-APC Biolegend's APC-labeled anti-human CLL1 specific antibody
  • the anti-CLL1 single domain antibody (CLL1-VHH-16) of the present invention has high affinity, can specifically target CLL1 positive cells (cells expressing CLL1 protein) well, and can be successfully applied to the detection of CLL1 expression in bone marrow cells of AML patients.
  • the single domain antibody of the present invention can be made into a specific antibody drug for clinical prevention and treatment of CLL1 target-related diseases (such as acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia), and can also be used to prepare CAR cells targeting CLL1, or for preparing a detection kit for CLL1 protein.
  • the single domain antibody drug of the present invention has a stable structure, small molecules, easy recombinant expression, and low production cost. It can be used alone or as a drug delivery system to carry related drugs, and has very broad prospects and important significance in the fields of drug application and clinical diagnosis.

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Abstract

涉及抗CLL1单域抗体及其应用。具体涉及氨基酸序列为 SEQ ID No.1 的单域抗体。单域抗体亲和力高，能够特异性靶向 CLL1 阳性细胞，可应用于AML 患者骨髓细胞中CLL1 表达的检测。单域抗体可制成临床上用于预防和治疗CLL1靶点相关疾病(如急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病等)的特异性抗体药物，也可用于制备靶向 CLL1 的 CAR 细胞，或用于制备 CLL1蛋白的检测试剂盒等。单域抗体药物结构稳定、分子小、易于重组表达、生产成本低，可单独使用或作为载药***携带相关药物，在药物应用和临床诊断等领域具有十分广阔的前景和重要的意义。

Description

抗CLL1单域抗体及其应用 技术领域
本发明属于生物医药领域，具体涉及抗CLL1单域抗体及其应用。
背景技术
急性髓细胞白血病(Acute Myeloid Leukemia，AML)是一种髓系造血干/祖细胞恶性疾病，长期以来，一直采用蒽环类和阿糖胞苷化疗来治疗轻度患者，使用造血干细胞移植来治疗中、高危患者。急性髓细胞白血病的异质性导致目前对于AML患者的治疗仍是一种挑战。近年来，研究人员发现，以CLL1为靶点的免疫靶向疗法治疗AML的效果显著。C型凝集素样分子1(C-type lectin-like molecule 1，CLL1)也称为C型凝集素结构域家族12成员A(C-type lectin domain family 12 member A，CLEC12A)，是一种Ⅱ型跨膜糖蛋白，作为抑制性受体在免疫调节中发挥重要作用。CLL1存在于外周血和骨髓的髓系细胞以及大部分AML细胞中，同时表达于多数AML的CD34 +CD38 -干细胞上，正常人的CD34 +CD38 -干细胞则不表达，CLL1因其特殊的表达模式成为AML治疗和诊断的潜在靶点。此外，CLL1还在骨髓增生异常综合征(Myelodysplastic Syndromes，MDS)和慢性髓细胞白血病(Chronic Myeloid Leukemia，CML，慢性粒细胞白血病)细胞上表达。
抗体(Antibody，Ab)是B细胞被B细胞抗原表位特异性刺激后增殖分化为浆细胞所产生的效应免疫分子，介导体液免疫，是一类能与抗原特异性结合的免疫球蛋白(Immunoglobulin，Ig)。Ig通过其V区CDR所组成的抗原结合槽与相应抗原表位以锁-匙(lock-key)互补关系进行特异性识别与结合。抗体同抗原的特异性结合能力使其在疾病诊断和免疫防治中具有重要意义。由抗体物质(包括具有治疗功能的全抗体分子和抗体片段)组成的抗体药物是靶向治疗的重要手段之一，已经成为目前生物医药产业最有前景和应用价值的热点领域。
重链抗体(Heavy chain antibody，HcAb)是天然存在的一种抗体类型，是骆驼科动物或软骨鱼类所具有的独特抗体类型，其抗体结构域天然缺失轻链，只由两条重链组成。重链抗体的抗原识别功能主要由重链抗体的可变区(VHH)决定。单独的VHH即可以识别抗原，因此其又被称为单域抗体(Single-domain antibody，sdAb)。单域抗体缺失抗体轻链，只有重链可变区，因其分子量小，也被称为纳米抗体(Nanobody，Nb)。单域抗体的分子量只有约13-15KDa，直径约为2.5nm，长4nm，是迄今为止最小的具有抗原结合功能的抗体片段，其与抗原结合的能力及其稳定性同完全抗体基本一致或具有更高的特异性抗原亲和力。与传统抗体相比，单域抗体还具有许多独特的性质，比如稳定性好、可抵达特殊抗原表位、可砌块模式任意组合、生产成本低等。获得单域抗体的常规方法由多次免疫骆驼科动物、B淋巴细胞分离、VHH区扩增、展示文库构建和 筛选等众多步骤构成。随着合成生物学的发展，构建基于全合成的高质量随机化大容量单域抗体库成为可能。目前，VHH单域抗体因其结构稳定、分子小、溶解性好、耐受各类不良环境、制剂稳定性好、易于重组表达、易于人源化等优点，已经被广泛使用在小型化基因工程抗体研究、新药开发以及疾病的诊断治疗上。单域抗体的研究和开发在药物应用和临床诊断等领域具有十分广阔的前景和重要的意义。
发明公开
本发明的目的之一是提供一种靶向CLL1抗原的单域抗体及其在肿瘤靶向治疗中的应用。
为实现上述目的，本发明首先提供了单域抗体(抗CLL1单域抗体)，名称为CLL1-VHH-16，所述单域抗体由重链可变区(VHH)组成，所述重链可变区可包含氨基酸序列分别是SEQ ID No.1的第26-35位的互补决定区CDR1、SEQ ID No.1的第50-59位的互补决定区CDR2和SEQ ID No.1的第99-118位的互补决定区CDR3。
所述单域抗体(抗CLL1单域抗体)可为特异性结合CLL1蛋白的单域抗体。
进一步地，所述重链可变区的氨基酸序列可为SEQ ID No.1或与SEQ ID No.1具有至少80％的同一性且具有相同的功能的氨基酸序列。
所述单域抗体(抗CLL1单域抗体)只有重链抗体的可变区(VHH)，依次由框架区FR1、互补决定区CDR1、框架区FR2、互补决定区CDR2、框架区FR3、互补决定区CDR3和框架区FR4组成。
所述单域抗体CLL1-VHH-16的氨基酸序列可为SEQ ID No.1。其中，SEQ ID No.1的第1-25位为框架区FR1，第26-35位为互补决定区CDR1，第36-49位为框架区FR2，第50-59位为互补决定区CDR2，第60-98位为框架区FR3，第99-118位为互补决定区CDR3，第119-129位为框架区FR4。编码单域抗体CLL1-VHH-16的核酸分子(CLL1-VHH-16基因)的核苷酸序列如SEQ ID No.2所示。
所述互补决定区的序列根据Kabat编号***定义。
本发明单域抗体序列具有改善的亲和力和/或效价的其他变体可通过采用在本领域已知的方法来获得，并包括在本发明的保护范围内。例如，氨基酸置换可用于获得具有进一步改善的亲合力的抗体。或者，核苷酸序列的密码子优化也可用来改善用于产生抗体的表达***中的翻译效率。此外，包含通过对本发明的任何核酸序列应用定向进化法而优化抗体特异性的序列的多核苷酸也属于本发明的范围内。
在某些实施方案中，可以在本发明所述单域抗体的一个或多个互补决定区或框架区内发生替代、***或删除，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对互补决定区和/或框架区做出保守变化(例如，保守替代，本领域技术人员所熟知，氨基酸的保守性取代不改变蛋白质的性质和功能)，其不实质性降低结合亲和力。例如，此类变化可以在互补决定区中的抗原接触 残基以外。
本发明还提供了与所述单域抗体CLL1-VHH-16相关的生物材料，所述生物材料可为下述C1)至C4)中的任一种：
C1)编码所述单域抗体CLL1-VHH-16重链可变区的核酸分子；
C2)含有C1)所述核酸分子的表达盒；
C3)含有C1)所述核酸分子的重组载体、或含有C2)所述表达盒的重组载体；
C4)含有C1)所述核酸分子的重组宿主细胞、或含有C2)所述表达盒的重组宿主细胞、或含有C3)所述重组载体的重组宿主细胞。
其中，C4)所述的重组宿主细胞可表达所述单域抗体CLL1-VHH-16。
上述生物材料中，所述核酸分子可为下述任一种：
D1)核苷酸序列或编码序列是SEQ ID No.2的DNA分子；
D2)与D1)限定的核苷酸序列具有75％或75％以上同一性，且具有相同功能的DNA分子。
SEQ ID No.2所示的DNA分子编码氨基酸序列为SEQ ID No.1的单域抗体CLL1-VHH-16。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述85％以上的同一性可为至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述90％以上的同一性可为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。所述95％以上的同一性可为至少95％、96％、97％、98％或99％的同一性。
本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索以对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
本文所述载体是指能够把外源DNA或目的基因运载进入宿主细胞进行扩增和表达的载体，所述载体可以是克隆载体也可以是表达载体，包括但不限于：质粒、噬菌体(如λ噬菌体或M13丝状噬菌体等)、黏粒(即柯斯质粒)、人工染色体(如酵母人工染色体(YAC)、细菌人工染色体(BAC)、P1人工染色体(PAC)或Ti质粒人工染色体(TAC)等)、病毒载体(如杆状病毒载体、逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒或疱疹病毒(如单纯疱疹病毒) 等)。在本发明的一个或多个实施方案中，载体为pComb3XSS质粒和/或pcDNA3.1载体。
本文所述宿主细胞(也称为受体细胞)是指可用于导入载体的任何类型的细胞。宿主细胞可以是真核细胞(例如植物细胞、动物细胞、真菌、或藻类)，或者可以是原核细胞(例如细菌或原生动物)。所述宿主细胞可理解为不仅是指特定的受体细胞，而且也指这种细胞的后代，且由于天然的、偶然的或有意的突变和/或改变，该子代可以不必与原始的亲代细胞完全一致，但仍包括在宿主细胞的范围中。合适的宿主细胞为本领域已知的，其中：所述植物细胞可为拟南芥(Arabidopsis thaliana)、烟草(Nicotiana tabacum)、玉米(Zea mays)、水稻(Oryza sativa)、小麦(Triticum aestivum)等植物细胞但不限于此；所述动物细胞可为哺乳动物细胞(例如中国仓鼠卵巢细胞(CHO细胞)、非洲绿猴肾细胞(Vero细胞)、幼仓鼠肾细胞(BHK细胞)、小鼠乳腺癌细胞(C127细胞)、人胚胎肾细胞(HEK293细胞)、人HeLa细胞、成纤维细胞、骨髓细胞系、T细胞或NK细胞等)、禽类细胞(例如鸡或鸭细胞)、两栖类细胞(例如非洲爪蟾(Xenopus laevis)细胞或大鲵(Andrias davidianus)细胞)、鱼类细胞(例如草鱼、鲤鱼、虹鳟鱼或鲶鱼细胞)、昆虫细胞(例如Sf21细胞或Sf-9细胞)等但不限于此。所述真菌可为酵母，所述酵母可来自酵母属(如酿酒酵母Saccharomyces cerevisiae)、克鲁维酵母属(如乳酸克鲁维酵母Kluyveromyces lactis)、毕赤酵母属(如巴斯德毕赤酵母Pichia pastoris)、裂殖酵母属(如非洲粟酒裂殖酵母Schizosaccharomyces pombe)、汉逊酵母属(如多态汉逊酵母Hansenula polymorpha)等但不限于此。所述真菌还可来自镰刀菌属(Fusarium sp.)、丝核菌属(Rhizoctonia sp.)、轮枝菌属(Verticillium sp.)、青霉属(Penicillium sp.)、曲霉属(Aspergillus sp.)、头孢霉(Cephalosporium sp.)等但不限于此。所述藻类可来自墨角藻属(Fucus sp.)、曲壳藻属(Achnanthes sp.)、茧形藻属(Amphiprora sp.)、双眉藻属(Amphora sp.)、纤维藻属(Ankistrodesmus sp.)、星胞藻属(Asteromonas sp.)、黄金色藻属(Boekelovia sp.)等但不限于此。细菌可来自埃希氏菌属(Escherichia sp.)、欧文氏菌属(Erwinia sp.)、土壤杆菌属(Agrobacterium sp.)、黄杆菌属(Flavobacterium sp.)、产碱菌属(Alcaligenes sp.)、假单胞菌属(Pseudomonas sp.)、芽胞杆菌属(Bacillus sp.)等但不限于此。例如所述细菌可为大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)或短小芽孢杆菌(Bacillus pumilus)。在本发明的一个或多个实施方案中，所述宿主细胞为大肠杆菌TG1和/或HEK293细胞。
本文所述重组载体是指将外源目的基因与载体在体外连接构建而成的重组DNA分子，可以任何合适的方式构建，只要构建的重组载体可携带外源目的基因进入受体细胞、并为外源目的基因提供在受体细胞的复制、整合、扩增和/或表达能力即可。在本发明的一个或多个实施方案中，所述重组载体为pcDNA3.1-CLL1-VHH-16-hFc。
所述重组载体pcDNA3.1-CLL1-VHH-16-hFc是为了便于纯化，在单域抗体CLL1-VHH-16的编码基因(SEQ ID No.2)3'末端添加人源IgG1Fc基因(hFc基因，核苷酸序列为SEQ ID No.3)，得到CLL1-VHH-16-hFc融合基因，然后分别在该融合基因的5’端起始密码子前加入BamHI酶切位点，3’端终止密码子后加入EcoRI酶切位点，将其克隆至真核表达载体pcDNA3.1的BamHI和EcoRI识别位点之间，得到表达C端融合hFc基因的单域抗体CLL1-VHH-16的重组真核表达载体pcDNA3.1-CLL1-VHH-16-hFc。所述重组真核表达载体pcDNA3.1-CLL1-VHH-16-hFc含有SEQ ID No.2所示的单域抗体CLL1-VHH-16的编码基因。
人源IgG1Fc基因核苷酸序列(696bp)如SEQ ID No.3所示，其编码的氨基酸序列如SEQ ID No.4所示。
本文所述重组宿主细胞是指对目的宿主细胞的基因进行操作和修饰，从而得到功能发生变化的重组宿主细胞。如将外源目的基因或重组载体导入目的宿主细胞后得到的重组宿主细胞，或直接对目的宿主细胞的内源基因进行基因编辑后得到的重组宿主细胞。所述重组宿主细胞可理解为不仅是指特定的重组宿主细胞，而且也指这种细胞的后代，且由于天然的、偶然的或有意的突变和/或改变，该子代可以不必与原始的亲代细胞完全一致，但仍包括在重组宿主细胞的范围中。在本发明的一个或多个实施方案中，所述重组宿主细胞是将所述重组载体pcDNA3.1-CLL1-VHH-16-hFc导入HEK293细胞得到的重组宿主细胞。
本发明还提供了药物组合物，所述药物组合物可含有所述单域抗体CLL1-VHH-16。
进一步地，所述药物组合物还可含有药学上可接受的载体。
所述药学上可接受的载体可为稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂。
所述药物组合物具有下述至少一种用途：
M1)用于预防或***；
M2)用于预防或治疗CLL1靶点相关疾病；
M3)用于介导药物特异性地识别表达CLL1抗原的肿瘤。
本发明还提供了试剂或试剂盒，所述试剂或试剂盒可含有所述单域抗体CLL1-VHH-16，所述试剂或试剂盒具有下述至少一种用途：
F1)检测CLL1蛋白；
F2)筛查、诊断或辅助诊断CLL1靶点相关疾病；
F3)用于CLL1蛋白的体内显像。
所述试剂盒可为化学发光免疫试剂盒、酶联免疫检测试剂盒、胶体金免疫试剂盒、或荧光免疫试剂盒，但不限于此。
本发明还提供了所述单域抗体CLL1-VHH-16和/或所述生物材料的下述任一种应用：
E1)在制备用于预防或***的药物中的应用，或在预防或***中 的应用；
E2)在制备用于预防或治疗CLL1靶点相关疾病的药物中的应用，或在预防或治疗CLL1靶点相关疾病中的应用；
E3)在制备用于筛查、诊断或辅助诊断CLL1靶点相关疾病的产品中的应用，或在筛查、诊断或辅助诊断CLL1靶点相关疾病中的应用；
E4)在检测CLL1蛋白或制备用于检测CLL1蛋白的产品中的应用；
E5)在制备用于结合CLL1蛋白的产品中的应用；
E6)在介导药物特异性地识别表达CLL1抗原的肿瘤或制备介导药物特异性地识别表达CLL1抗原的肿瘤的产品中的应用；
E7)在CLL1蛋白的体内显像或制备用于CLL1蛋白的体内显像的产品中的应用；
E8)在制备靶向CLL1的CAR细胞中的应用。
本文中，所述CLL1靶点相关疾病可为CLL1阳性癌症(如白血病)。
本文中，所述CLL1阳性癌症可为急性髓细胞白血病(Acute Myeloid Leukemia，AML)、骨髓增生异常综合征(Myelodysplastic Syndromes，MDS)、慢性髓细胞白血病(Chronic Myeloid Leukemia，CML，慢性粒细胞白血病)但不限于此。
E6)中所述介导药物特异性地识别表达CLL1抗原的肿瘤的产品可为特异性靶向CLL1蛋白的载药***，所述载药***含有所述单域抗体CLL1-VHH-16。
所述载药***可为脂质体递药***、聚合物胶束递药***、聚合物圆盘递药***或纳米粒递药***。
所述载药***可用于药物的靶向运输和/或定点释放。
E8)中所述靶向CLL1的CAR细胞表达嵌合抗原受体(CAR)，所述嵌合抗原受体可含有所述单域抗体CLL1-VHH-16。
进一步地，所述嵌合抗原受体的抗原识别域可为所述单域抗体CLL1-VHH-16。
进一步地，所述CAR细胞包括但不限于CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞(CAR-M细胞)、CAR-iPSC或CAR-PSC。
本文所述检测CLL1蛋白可为检测待测样品中是否含有CLL1蛋白和/或检测待测样品中CLL1蛋白的含量。
所述待测样品可为细胞或组织样品。
所述用于检测CLL1蛋白的产品包括利用酶联免疫吸附法、免疫荧光检测法、放射免疫法、发光免疫测定法、胶体金免疫层析法、凝集法或免疫比浊法等检测抗原抗体结合的产品。
本文所述产品可为试剂、试剂盒、芯片或试纸。
本发明还提供了所述单域抗体CLL1-VHH-16的制备方法，所述方法可包括：构建含有编码所述单域抗体CLL1-VHH-16重链可变区的核酸分子的重组表达载 体；将所述重组表达载体导入宿主细胞，获得表达所述单域抗体的重组细胞；培养所述重组细胞，经分离纯化获得所述单域抗体。
进一步地，所述编码所述单域抗体CLL1-VHH-16重链可变区的核酸分子可为下述任一种：
D1)核苷酸序列或编码序列是SEQ ID No.2的DNA分子；
D2)与D1)限定的核苷酸序列具有75％或75％以上同一性，且具有相同功能的DNA分子。
进一步地，所述宿主细胞可为HEK293细胞。
进一步地，所述导入可为通过磷酸钙共沉淀法、脂质体介导法、电穿孔法或病毒载体法等任何已知的转染方法将携带本发明单域抗体基因的载体转化宿主细胞。
本发明还提供了诊断或辅助诊断CLL1靶点相关疾病的方法，所述方法可包括：从受试者分离获得样品，然后利用所述单域抗体CLL1-VHH-16检测所述样品中CLL1蛋白的含量，根据所述CLL1蛋白的含量(与疾病诊断标准的CLL1蛋白的含量比较)进行CLL1靶点相关疾病的诊断或辅助诊断。
本发明还提供了筛查CLL1靶点相关疾病的方法，所述方法可包括：从受试者分离获得样品，然后利用所述单域抗体CLL1-VHH-16检测所述样品中CLL1蛋白的含量，根据所述CLL1蛋白的含量(与疾病筛查标准的CLL1蛋白的含量比较)进行CLL1靶点相关疾病的筛查。
本发明还提供了预防或治疗CLL1靶点相关疾病的方法，所述方法可包括向患有CLL1靶点相关疾病的受试者施用所述单域抗体CLL1-VHH-16或本文所述药物组合物。
上述方法中，所述CLL1靶点相关疾病可为CLL1靶点相关肿瘤。
上述方法中，所述CLL1靶点相关肿瘤可为CLL1阳性癌症。
上述方法中，所述CLL1阳性癌症可为急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病。
本发明还提供了体外检测CLL1蛋白的方法，所述方法可包括利用所述单域抗体CLL1-VHH-16或本文所述试剂盒检测CLL1蛋白。
本发明还提供了抗体药物偶联物，包括抗体部分和偶联部分，所述抗体部分可包含所述单域抗体CLL1-VHH-16。
进一步地，所述偶联部分可包括可检测的标记、化学药物或细胞毒素。
所述抗体部分和偶联部分可直接或通过接头共价连接。
所述可检测的标记可选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
所述化学药物可为可杀伤肿瘤细胞的药物。
所述细胞毒素可为微管蛋白抑制剂(如美登素、阿里他汀)、DNA合成抑制 剂(如卡奇霉素)或RNA合成抑制剂(如鹅膏毒素)。
本发明所述应用和方法的目的可以是疾病诊断目的、疾病预后目的和/或疾病治疗目的，它们的目的也可以是非疾病诊断目的、非疾病预后目的和非疾病治疗目的；它们的直接目的可以是获取疾病诊断结果、疾病预后结果和/或疾病治疗结果的中间结果的信息，它们的直接目的可以是非疾病诊断目的、非疾病预后目的和/或非疾病治疗目的。
本文所述CLL1为C型凝集素样分子1(C-type lectin-like molecule 1)也称为C型凝集素结构域家族12成员A(C-type lectin domain family 12 member A，CLEC12A)，是一种Ⅱ型跨膜糖蛋白，作为抑制性受体在免疫调节中发挥重要作用。CLL1存在于外周血和骨髓的髓系细胞以及大部分急性髓细胞白血病(Acute Myeloid Leukemia，AML)细胞中，同时表达于多数AML的CD34 +CD38 -干细胞上，正常人的CD34 +CD38 -干细胞则不表达，CLL1因其特殊的表达模式成为AML治疗和诊断的潜在靶点。CLL1还在骨髓增生异常综合征(Myelodysplastic Syndromes，MDS)和慢性髓性白血病(Chronic Myeloid Leukemia，CML)细胞上表达。
本发明首先利用CLL1重组蛋白免疫羊驼得到单域抗体基因，并构建单域抗体表达文库，然后通过噬菌体展示筛选技术从单域抗体表达文库中筛选得到高亲和力的抗CLL1单域抗体(CLL1-VHH-16)。测序得到抗体基因序列后，采用基因工程的方法制备CLL1-VHH-16。进一步对获得的抗体进行了亲和力、特异性等功能实验。结果表明，本发明的单域抗体亲和力高，能够很好地特异性靶向CLL1阳性细胞(表达CLL1蛋白的细胞)，并可以成功应用于AML患者骨髓细胞中CLL1的表达的检测。本发明的单域抗体可以在原核细胞、酵母细胞、真核细胞及任何重组***中表达和生产，可制成临床上用于预防和治疗CLL1靶点相关疾病(如急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病)的特异性抗体药物，或CLL1蛋白的检测试剂盒等。本发明单域抗体药物结构稳定、分子小、易于重组表达、生产成本低，可单独使用或作为载药***携带相关药物，在药物应用和临床诊断等领域具有十分广阔的前景和重要的意义。
附图说明
图1为羊驼PBMC提取总RNA的琼脂糖凝胶电泳结果。
图2为细菌文库菌落PCR琼脂糖凝胶电泳结果。
图3为使用FITC标记的CLL1-VHH-16抗体检测不同肿瘤细胞株上的CLL1表达结果。其中Isotype表示未用FITC标记的CLL1-VHH-16-hFc抗体，CLL1表示FITC-CLL1-VHH-16-hFc抗体。
图4为使用FITC标记的CLL1-VHH-16抗体检测AML患者骨髓细胞上CLL1的表达结果。图4中CLL1VHH-FITC表示FITC标记的CLL1-VHH-16抗体，即实施例2中的FITC-CLL1-VHH-16-hFc抗体；CLL1-APC表示Biolegend公司APC标记的抗人CLL1特异性抗体(货号143405)。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的pComb3XSS质粒为北京庄盟国际生物基因科技有限公司产品，货号ZK129。
下述实施例中的辅助噬菌体M13K07为成都仁域生物技术有限公司产品，货号A001-1。
下述实施例中的K562细胞(CLL1阴性细胞)为南京科佰生物科技有限公司产品，货号CBP60529；MM1.S细胞(CLL1阴性细胞)为北京百欧博伟生物技术有限公司产品，货号bio-129535；U937细胞(CLL1阳性细胞)为北京百欧博伟生物技术有限公司产品，货号bio-73180。
下述实施例中AML患者骨髓细胞来源于浙江省人民医院。
下述实施例中CLL1阳性细胞K562-CLL1的制备方法为将CLL1***pcDNA3.1的BamHI和EcoRI位点之间，制备的重组载体转染K562细胞，得到CLL1阳性细胞K562-CLL1。
下述实施例中的载体pcDNA3.1为长沙艾碧维生物科技有限公司产品，货号：HG-VPI0001。
实施例1、抗CLL1单域抗体的制备
本实施例利用CLL1重组蛋白免疫羊驼得到单域抗体基因，并构建单域抗体表达文库，然后通过噬菌体展示筛选技术从单域抗体表达文库中筛选得到高亲和力的抗CLL1单域抗体。
1、动物免疫
1.1、抗原的获得
作为抗原的CLL1重组蛋白如下所示：
CLL1免疫抗原：CLL1-hIgG1Fc(B918601)蛋白为上海百英生物科技有限公司产品。
CLL1筛选抗原：CLL1-6×His(B918602)蛋白为上海百英生物科技有限公司产品。
1.2、免疫羊驼
使用CLL1免疫抗原CLL1-hIgG1Fc免疫羊驼(2-3岁，体重100斤左右)共四次，每次皮下注射0.5mg CLL1-hIgG1Fc，两次免疫间隔2周。下一次免疫前，抽取5ml血液，分离血浆后检测上一次免疫后的抗体效价。免疫四次后的四免 效价在注射一周后进行检测。
通过间接ELISA法来检测血清中抗体的效价，步骤如下所示：
(1)免疫抗原CLL1-hIgG1Fc用PBS稀释到2ug/ml，包被酶标板，100μL/孔，4℃过夜；对照组用3％BSA包被；
(2)弃抗原溶液，用200μL PBST溶液(含0.05％Tween20的1×PBS溶液)冲洗酶标板3次，每次5min；
(3)用200μL封闭液PBSTB(含2％BSA的PBST)封闭酶标板，室温封闭1.5h；
(4)弃封闭液，用200μL PBST溶液冲洗酶标板3次，每次5min；
(5)用封闭液按照1:10、1:100、1:1000、1:10000、1:100000、1:1000000、1:10000000对待检效价的血清进行梯度稀释，100μL/孔加入酶标板，室温孵育2h；
(6)弃血清，用200μL PBST溶液冲洗酶标板4次，每次5min；
(7)向每个孔中加入1:10000封闭液稀释的山羊抗美洲驼抗体(Goat Anti-Llama IgG H&L(HRP)，Abcam，ab112786)，100μL/孔，室温孵育1h；
(8)弃检测抗体，用200μL PBST溶液冲洗酶标板4次，每次5min；
(9)向每孔加入100μL TMB底物溶液，显色2-3min；
(10)向每孔加入100μL反应终止液；
(11)30min内使用酶标仪测定450nm处吸光值。阳性孔判定标准：免疫血清样品的OD 450读值大于未免血清的OD 450读值，且读值大于0.5。
1.3、免疫结果
血清效价检测结果如表1所示，加粗字体的为阳性孔。经过四次免疫后，羊驼体内的血清效价达到10 5，即抗体水平达到10 5。
表1、血清效价检测结果
Figure PCTCN2022143892-appb-000001
如表1所示：经过第四次免疫后，羊驼体内抗体水平达到10 5。Ag包被为免疫抗原CLL1-hIgG1Fc包被。PBSTB为封闭液。
2、噬菌体文库构建
免疫成功之后采集羊驼外周血单个核细胞(PBMC)用于构建噬菌体文库：即抗CLL1 VHH噬菌体抗体库。
2.1、RNA提取和反转录
采用密度梯度离心方法分离羊驼外周血单个核细胞(简称羊驼PBMC细胞)， 溶于Trizol中保存。提取羊驼PBMC细胞总RNA，使用Takara反转录试剂盒对得到的总RNA进行反转录成cDNA。具体步骤如下：
(1)将用Trizol保存的羊驼PBMC细胞转移至1.5mL离心管中，加入1/5体积的氯仿混匀，室温静置5min；
(2)4℃、12000g离心15min；
(3)上清液转移到新的离心管中，加入等体积的异丙醇，室温静置10min；
(4)4℃、12000g离心10min；
(5)用1mL 75％乙醇清洗沉淀，7500g离心5min去除乙醇，干燥；
(6)沉淀溶解于适量无RNA酶的水中即为提取得到的总RNA，共获得181.2ug总RNA(图1)；
(7)使用Takara反转录试剂盒对总RNA进行反转录：上述总RNA样品分为两份，一份使用试剂盒内的Oligo dT Primer作为引物，另一份用试剂盒内的Random 6-mers作为引物，按照试剂盒说明书操作步骤将总RNA反转录成cDNA，共获得31.7μg cDNA。
2.2、CLL1抗体的VHH目的片段PCR
以8ng cDNA为模板常规PCR扩增VHH目的片段，共获得83μg。
2.3、酶切和连接
1、使用pComb3XSS作为噬菌体质粒载体，用限制性内切酶Spe I、Sac I分别酶切pComb3XSS载体和VHH的PCR产物(即VHH目的片段)，37℃孵育4h；
2、使用DNA回收纯化试剂盒对酶切后的pComb3XSS载体和VHH的PCR产物进行纯化，4℃保存。
3、双酶切后的pComb3XSS载体和VHH目的片段进行连接、回收，共获得3.1μg连接产物。
2.4、细菌文库构建
(1)取100ng连接产物同50μL TG1感受态细胞进行电转化；
(2)37℃复苏1h，取100μL转化产物进行10个梯度稀释后涂板，37℃过夜培养；
(3)根据稀释倍数和单菌落数计算所有电转反应所获得的转化菌落数目，即细菌文库的库容量为2.6×10 8cfu；
(4)从梯度稀释板中随机挑选若干个单克隆进行菌落PCR，琼脂糖凝胶电泳结果显示(图2)，挑选的单克隆均为阳性克隆，阳性率为100％；
(5)收集剩下所有菌液(即转化产物的各梯度稀释液)均匀涂布到20个15cm培养板(2×YT培养基，含100μg/mL Amp、2％琼脂糖)中，37℃倒置培养过夜；
(6)过夜培养的平板菌落使用2×YT液体培养基刮下，置于50mL离心管，测量其OD 600＝120，添加终浓度20％甘油于-80℃保存，即得到细菌文库。
(7)上述单克隆阳性菌落的测序序列翻译成蛋白质序列，序列多样性比对显示均为独立序列，多样性良好，细菌文库多样性符合要求。
2.5、噬箘体文库制备
(1)接种细菌文库到100mL 2×YT液体培养基(含100μg/mL Amp)中，37℃，250rpm培养至OD 600为0.5-0.55；
(2)按照细菌个数:噬箘体数＝1:20的比例加入辅助噬菌体M13K07，37℃、250rpm孵育30min；
(3)加入终浓度为50μg/mL的Kan，30℃、250rpm培养过夜；
(4)将过夜培养的菌液于4℃、4000rpm离心20min，上清液转移到新的50mL离心管中；
(5)加入1/4体积4℃预冷的20％PEG/2.5M NaCl溶液，混合均匀，冰上静置孵育至少30min；
(6)4℃、4000rpm离心20min，弃上清，倒置2min；
(7)加入1mL PBS重悬，4℃、12000rpm离心10min；
(8)转移上清至新的离心管中，再次加入1/4体积4℃的预冷20％PEG/2.5M NaCl溶液，混合均匀成白色悬液，冰上孵育至少10min；
(9)4℃、12000rpm离心10min，弃上清，用1mL PBS重悬，12000rpm离心10min，转移上清至新的离心管，-80℃保存，即为纯化后的噬菌体文库，其效价为1×10 13pfu/mL。
3、亲和筛选
利用筛选抗原(CLL1-6×His)对噬菌体文库进行3轮亲和筛选，包含效价检测、噬菌体洗脱液的扩增与噬菌体的纯化。
3.1、第一轮筛选
3.1.1、第一轮筛选方法
(1)用筛选抗原(CLL1-6×His)包被免疫管(50μg/管，包被液为2mL/管的PBS)，4℃缓慢旋转过夜，同时平行包被BSA(50μg/管，包被液为2mL/管的PBS)作对照；
(2)弃除过夜包被免疫管中的液体，加入2mL PBS缓冲液于室温清洗3次，每次旋转5min；
(3)加入2mL 3％脱脂奶粉，室温旋转封闭2h；
(4)弃除封闭后免疫管中的液体，加入2mL PBST缓冲液于室温清洗3次，每次旋转5min；
(5)弃除免疫管中的清洗液，按照下列公式加入噬菌体文库作为第一轮筛选的输入噬菌体文库，加入PBS缓冲液至2mL，室温旋转孵育1h。
Figure PCTCN2022143892-appb-000002
其中，V为加入的噬菌体体积(单位μL)，T library为噬菌体效价。
(6)弃除免疫管内液体，加入2mL PBST(1×PBS加0.1％Tween20，下同)缓冲液，于室温清洗免疫管20次，每次旋转5min；
(7)弃除免疫管内液体，加入1mL 0.25mg/mL的Trypsin溶液，室温旋 转洗脱30min；
(8)加入10μL 10％AEBSF(丝氨酸蛋白酶抑制剂)终止洗脱，将免疫管中的溶液转移至新的1.5mL离心管中，即为第一轮筛选的噬菌体洗脱液(简称第一轮噬菌体洗脱液)。
3.1.2、噬菌体初始效价检测
(1)-80℃冰箱保存的TG1菌株在2×YT固体培养基上进行单菌落划线，37℃培养过夜；
(2)从单菌落平板上挑取一个单菌落到5mL 2×YT培养基中，37℃培养过夜；
(3)取50μL菌液转接到5mL 2×YT液体培养基中，37℃、250rpm培养至OD 600＝0.5-0.55，即得到TG1菌液；
(4)取10μL第一轮噬菌体洗脱液，在1.5mL离心管中进行10倍梯度稀释，即将第一轮噬菌体洗脱液取10μL至90μL PBS中，再从中取10μL至90μL PBS中，依次类推，共稀释12个梯度至10 -12；
(5)在每个稀释离心管中加入90μL TG1菌液，振荡混匀后于37℃孵育30min；
(6)从每个稀释离心管中取5μL菌液滴加到2×YT固体培养基(Amp)中，静止数分钟后于37℃倒置过夜；
(7)统计不同稀释度下平板中可以明显区分的单菌落数量，按照下面公式计算每毫升噬菌体溶液中的噬菌粒数量，即噬菌体文库效价：
T(pfu/ml)＝N×D×400
其中，T为噬菌体效价(单位pfu/mL)，D为稀释倍数，N为相应稀释倍数平板中的单菌落个数。
3.1.3、第一轮噬菌体洗脱液的扩增
(1)-80℃冰箱保存的TG1菌株在2×YT固体培养基上进行单菌落划线，37℃培养过夜；
(2)从单菌落板平上挑取一个单菌落到5mL 2×YT培养基中，37℃培养过夜；
(3)取50μL菌液转接到含5mL 2×YT液体培养基中，37℃、250rpm培养至OD 600＝0.5-0.55；
(4)加入500μL第一轮噬菌体洗脱液，37℃、250rpm继续培养30min；
(5)将全部菌液均匀涂布到3个含有100μg/mL Amp的15cm圆形培养基平板中，37℃培养过夜；
(6)培养基平板表面加入2×YT液体培养基，用涂布棒轻轻将菌落刮下并将菌液收集至15mL离心管中，即为扩增后的细菌子文库，加入终浓度为20％的甘油，同时使用分光光度计测量菌液OD 600值，获得洗脱液细菌文库OD 600值。
(7)将洗脱液细菌文库根据下面公式转接至100mL 2×YT液体培养基中(含100μg/mL Amp)，初始OD 600＝0.1：
Figure PCTCN2022143892-appb-000003
其中，V为转接菌液的体积(单位μL)，OD 600为构建的洗脱液细菌文库OD 600；
(8)37℃、250rpm培养至菌液OD 600＝0.5-0.55；
(9)按照下面公式以细菌个数:噬菌体数＝1:20加入辅助噬菌体M13K07：
Figure PCTCN2022143892-appb-000004
其中，V为加入辅助噬菌体的体积(单位mL)，T helper-phage为辅助噬菌体效价，OD 600为菌液的OD 600值；
(10)37℃、250rpm继续培养30min；
(11)加入终浓度为50μg/mL的Kan，30℃、200rpm培养过夜。
3.1.4、第一轮噬菌体纯化
(1)将过夜培养的菌液转移至新的50mL离心管中，4℃、4000rpm离心20min；
(2)将上清液转移至新的50mL离心管中，加入1/4体积4℃预冷的20％PEG/2.5M NaCl溶液，充分混匀后冰上放置至少30min；
(3)4℃、4000rpm离心20min，弃上清；
(4)加入1mL PBS重悬沉淀，转移至新的1.5mL离心管中，4℃、13000rpm离心15min；
(5)上清转移至新的1.5mL离心管中，加入1/4体积4℃预冷的20％PEG/2.5M NaCl溶液，混匀后冰上放置至少10min；
(6)4℃、13000rpm离心10min，弃上清，加入1mL PBS重悬沉淀；
(7)4℃、13000rpm离心2min，将上清液转移到新的1.5mL离心管中，即为第一轮筛选噬菌体子文库，长期于-80℃保存，短期(1-2周)可于-20℃放置保存。
3.1.5、第一轮筛选噬菌体子文库的效价检测
方法同步骤3.1.2噬菌体初始效价检测。
3.2、第二轮筛选
3.2.1、第二轮筛选方法
筛选方法同步骤3.1.1第一轮筛选方法，第一轮筛选扩增纯化得到的噬菌体子文库(第一轮筛选噬菌体子文库)作为第二轮筛选的输入噬菌体文库，得到第二轮筛选的噬菌体洗脱液。
3.2.2、第二轮噬菌体洗脱液的效价检测
检测方法同步骤3.1.2噬菌体初始效价检测。
3.2.3、第二轮噬菌体洗脱液的扩增、纯化
方法同步骤3.1.3和3.1.4第一轮噬菌体洗脱液的扩增、纯化，得到第二轮筛选噬菌体子文库。
3.2.4、第二轮筛选噬菌体子文库的效价检测
方法同步骤3.1.5第一轮筛选噬菌体子文库的效价检测。
3.3、第三轮筛选
3.3.1、第三轮筛选方法
筛选方法同步骤3.1.1第一轮筛选方法，第二轮筛选扩增纯化得到的噬菌体子文库(第二轮筛选噬菌体子文库)作为第三轮筛选的输入噬菌体文库，得到第三轮筛选的噬菌体洗脱液。
3.3.2、第三轮噬菌体洗脱液的效价检测
检测方法同步骤3.1.2噬菌体初始效价检测。
3.3.3、第三轮洗脱液的扩增
方法同步骤3.1.3第一轮噬菌体洗脱液的扩增，得到第三轮扩增的噬菌体洗脱液。
3.4、三轮筛选结果
三轮亲和筛选结果如表2所示：
表2、三轮亲和筛选结果
Figure PCTCN2022143892-appb-000005
其中，*1富集程度为筛选效价除以输入效价，数值越大表明抗体在输入库中的富集程度越高；*2相差倍数为筛选效价除以对照效价，数值越高表明阳性抗体在筛选库中的含量越高。
结果显示，经过3轮筛选后，富集程度达到1.3×10 -2倍，可以进行ELISA单克隆验证。
4、CLL1 VHH阳性单克隆挑选
4.1、CLL1 VHH阳性单克隆的ELISA检测
(1)从第三轮扩增的噬菌体洗脱液中取合适稀释度的菌液均匀涂布到含100μg/mL Amp的固体培养基平板，37℃培养过夜；
(2)随机挑取192个单克隆菌落于96孔细胞培养板(P1-P2)中，每孔加入200μL 2×YT培养基(含100μg/mL Amp)，37℃培养过夜；
(3)取5μL过夜培养的菌液转接至每孔添加了200μL 2×YT液体培养基(含100μg/mL Amp)的新96孔细胞培养板中，37℃培养5h；
(4)按照下面公式以细菌个数:噬菌体数＝1:20加入辅助噬菌体M13K07：
Figure PCTCN2022143892-appb-000006
其中，V为加入辅助噬菌体的体积(单位mL)，T helper-phage为辅助噬菌体效价；
(5)37℃孵育30min，加入终浓度为50μg/mL的Kan，30℃培养过夜；
(6)将过夜培养的菌液于4℃、4000rpm离心10min，得到上清液，4℃ 保存备用；
(7)将筛选抗原用PBS稀释到1ug/ml，包被酶标板，100μL/孔，4℃过夜；对照组用3％BSA包被；
(8)弃抗原溶液，每孔加入200μL PBS缓冲液室温清洗酶标板3次，每次10min；
(9)每孔加入200μL封闭液PBSTB(含2％BSA的PBST)封闭酶标板，室温封闭1h；
(10)弃封闭液，每孔加入200μL PBST(含0.1％Tween20的1×PBS)缓冲液，室温清洗酶标板3次，每次10min；
(11)每孔加入100μL封闭液，再加入100μL前述步骤(6)中离心后的上清液，室温孵育2h；
(12)弃掉酶标板内液体，每孔加入200μL PBST缓冲液洗涤3次，每次10min；
(13)每孔加入1:30000稀释于封闭液的M13 Bacteriophage HRP Antibody，Mouse Mab(北京义翘神州科技股份有限公司产品，货号11973-MM05T-H)，100μL/孔，室温孵育1h；
(14)弃掉ELISA板内液体，每孔加入200μL PBST缓冲液洗涤6次，每次5min；
(15)每孔加入100μL TMB单组份显色液，避光显色1-3min，每孔加入100μL 1M HCl终止，用酶标仪读取OD 450值。
OD 450＞0.5判定为阳性结果，得到的阳性克隆即为能与CLL1蛋白结合的含有单域抗体的噬菌体。
4.2、CLL1 VHH阳性单克隆的ELISA二次验证
为了排除假阳性结果，将初步认定为阳性的克隆进行ELISA二次验证，方法同步骤4.1。
4.3、CLL1 VHH阳性单克隆的测序
从ELISA二次检测板中取181个阳性克隆菌液5μL接种至1mL 2×YT培养基(含100μg/mL Amp)中，37℃、250rpm培养至OD 600＝0.8-1.0，取0.5mL菌液测序，得到不同的抗体序列。
5、抗CLL1单域抗体的制备
5.1、抗CLL1单域抗体的结构和序列
挑选步骤4.3中的阳性单克隆测序得到抗体基因序列，利用基因工程的方法制备抗CLL1单域抗体(CLL1-VHH)，将其中一个抗体命名为CLL1-VHH-16。
单域抗体只有重链抗体的可变区(VHH)，依次由框架区FR1、互补决定区CDR1、框架区FR2、互补决定区CDR2、框架区FR3、互补决定区CDR3和框架区FR4组成。具体序列信息如下：
单域抗体CLL1-VHH-16的氨基酸序列为SEQ ID No.1。SEQ ID No.1的第1-25 位为框架区FR1，第26-35位为互补决定区CDR1，第36-49位为框架区FR2，第50-59位为互补决定区CDR2，第60-98位为框架区FR3，第99-118位为互补决定区CDR3，第119-129位为框架区FR4。编码单域抗体CLL1-VHH-16的核酸分子(CLL1-VHH-16基因)的核苷酸序列如SEQ ID No.2所示。
单域抗体CLL1-VHH-16的氨基酸序列：
Figure PCTCN2022143892-appb-000007
Figure PCTCN2022143892-appb-000008
Figure PCTCN2022143892-appb-000009
三个下划线部分依次为CDR1、CDR2和CDR3。
CLL1-VHH-16基因的核苷酸序列：5’-GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCGGAAGCTATGACATGACCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGTGGGTCTCAGGAATTAATAGTGGTGGTAGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAACTATCGACCGCCTTAGTACGGTAGTAGCTGGTACGTACCAGCCGTCCGAAGAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC-3’(SEQ ID No.2)。
互补决定区的序列根据Kabat编号***定义。
上述单域抗体为特异性结合CLL1蛋白的单域抗体。
5.2、抗CLL1单域抗体的表达和纯化
采用真核表达载体pcDNA3.1来表达抗CLL1单域抗体(CLL1-VHH-16)。
5.2.1、重组载体的构建
为了便于纯化，在单域抗体CLL1-VHH-16的编码基因(SEQ ID No.2)3'末端添加人源IgG1Fc基因(hFc基因，核苷酸序列为SEQ ID No.3，其编码的氨基酸序列为SEQ ID No.4)，得到CLL1-VHH-16-hFc融合基因，然后将该融合基因的5’端起始密码子前加入BamHI酶切位点，3’端终止密码子后加入EcoRI酶切位点，将其克隆至真核表达载体pcDNA3.1的BamHI和EcoRI识别位点之间，得到表达C端融合hFc基因的单域抗体CLL1-VHH-16的重组真核表达载体pcDNA3.1-CLL1-VHH-16-hFc。
5.2.2、抗CLL1单域抗体的表达
将步骤5.2.1中得到的真核表达载体pcDNA3.1-CLL1-VHH-16-hFc采用常规电转的方法转染至HEK293细胞中进行表达。
5.2.3、抗CLL1单域抗体的纯化
Protein A具有和IgG的Fc片段结合的活性，因此采用Protein A柱纯化HEK293细胞表达的抗CLL1单域抗体(常规纯化步骤)。
最终制备得到纯化后的融合了hFc基因的抗CLL1单域抗体(CLL1-VHH-16-hFc)。
实施例2、抗CLL1单域抗体的功能鉴定
1、抗原抗体亲和力常数测定
待测抗体：步骤5.2.3中制备的融合了hFc基因的抗CLL1单域抗体(CLL1-VHH-16-hFc)。
(1)将待测抗体CLL1-VHH-16-hFc用Pierce TM NHS-Fluorescein Antibody Labeling Kit标记获得FITC直标抗体FITC-CLL1-VHH-16-hFc，用于流式检测。
(2)将CLL1阳性细胞K562-CLL1和U937细胞重悬至密度为2×10 6个细胞/mL，取100μL分别置于96孔V底板中离心弃上清并收集细胞(K562细胞做为CLL1阴性细胞对照)；
(3)将40μL不同梯度稀释(具体分组见表3)的FITC-CLL1-VHH-16-hFc同上述细胞混合，室温避光孵育10min；
表3、CLL1-VHH-16-hFc抗体亲和力试验分组
Figure PCTCN2022143892-appb-000010
(4)200μL FACS buffer离心清洗2次，用200μL FACS buffer重悬后立即进行流式检测平均荧光强度(MFI)；
(5)根据公式计算CLL1 VHH抗体与靶细胞结合的平衡解离常数(KD)来判定亲和力的强弱。其中KD值越小，则亲和力越强。
1/(MFI-Con)＝1/Fmax+(KD/Fmax)(1/[CLL1-scfv IgG Fc])。
其中，MFI-Con代表相对平均荧光强度，系实验组平均荧光强度减去背景平均荧光强度的值；CLL1-scfv IgG Fc代表抗体使用量(单位：ng)。
以抗体使用量的倒数为横坐标、相对平均荧光强度的倒数为纵坐标绘制标准曲线，得到线性回归方程。根据线性回归方程计算，标准曲线与纵坐标的交点为1/Fmax，KD即为直线斜率乘以Fmax。
计算所得的平衡解离常数见表4：
表4、CLL1-VHH-16-hFc与K562-CLL1细胞的平衡解离常数(KD)
单域抗体 亲和常数KD(nM) 线性回归方程式 线性回归的相差系数R 2
CLL1-VHH-16 5 y＝0.004x+0.0004 0.999
2、单域抗体CLL1-VHH-16的特异性检测
使用FITC标记的CLL1-VHH-16抗体(FITC-CLL1-VHH-16-hFc)检测不同肿瘤细胞上CLL1的表达。
(1)CLL1阴性细胞株(对照组)：K562细胞和MM1.S细胞；CLL1阳性细胞株(实验组)：U937细胞和CLL1过表达细胞株K562-CLL1均于37℃复苏后加入细胞培养瓶中，于37℃、5％CO 2培养，传代2次后用于检测抗CLL1单域抗体(FITC-CLL1-VHH-16-hFc)。
(2)将各细胞浓度调整到2×10 6个/mL，200μL/孔加入96孔板中，1500rpm离心5min；
(3)用FACS buffer清洗，离心；
(4)加入20μL稀释的FITC-CLL1-VHH-16-hFc抗体(1：400)混匀，室温孵育10min；
(5)加入FACS buffer离心洗去抗体，再加入buffer重悬，用流式细胞仪进行检测。
流式结果显示：FITC-CLL1-VHH-16-hFc抗体能够特异性结合肿瘤细胞上表达的CLL1抗原分子(图3)。
3、AML患者骨髓细胞中CLL1表达的检测
使用FITC标记CLL1-VHH-16抗体(FITC-CLL1-VHH-16-hFc)检测3例急性髓细胞白血病(AML)患者的骨髓细胞，同Biolegend公司APC标记的抗人CLL1特异性抗体(货号143405)进行比较，流式分析步骤如下：
(1)冻存的3位AML患者的骨髓细胞于37℃复苏。
(2)将细胞密度调整为2×10 6个/ml，200μL/孔加入96孔板中，1500rpm离心5min；
(3)用FACS buffer清洗，离心；
(4)加入20μL稀释的FITC-CLL1-VHH-16-hFc抗体(1：400)混匀，室温孵育10min；
(5)加入FACS buffer离心洗去抗体，再加入buffer重悬，用流式细胞仪进行检测。
(6)Biolegend公司APC标记的抗人CLL1特异性抗体(CLL1-APC)作为对比，步骤同上。
结果显示：本发明的FITC-CLL1-VHH-16-hFc抗体与Biolegend公司APC标记的抗人CLL1特异性抗体两者具有相似的表达图谱(图4)，说明本发明的单域抗体CLL1-VHH-16能够特异性识别AML肿瘤细胞表达的CLL1。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明的抗CLL1单域抗体(CLL1-VHH-16)亲和力高，能够很好地特异性靶向CLL1阳性细胞(表达CLL1蛋白的细胞)，并可以成功应用于AML患者骨髓细胞中CLL1的表达的检测。本发明的单域抗体可制成临床上用于预防和治疗CLL1靶点相关疾病(如急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病)的特异性抗体药物，也可用于制备靶向CLL1的CAR细胞，或用于制备CLL1蛋白的检测试剂盒等。本发明单域抗体药物结构稳定、分子小、易于重组表达、生产成本低，可单独使用或作为载药***携带相关药物，在药物应用和临床诊断等领域具有十分广阔的前景和重要的意义。

Claims (19)

  1. 单域抗体，其特征在于，所述单域抗体由重链可变区组成，所述重链可变区包含氨基酸序列分别是SEQ ID No.1的第26-35位的互补决定区CDR1、SEQ ID No.1的第50-59位的互补决定区CDR2和SEQ ID No.1的第99-118位的互补决定区CDR3。
  2. 根据权利要求1所述的单域抗体，其特征在于，所述重链可变区的氨基酸序列为SEQ ID No.1或与SEQ ID No.1具有至少80％的同一性且具有相同的功能的氨基酸序列。
  3. 与权利要求1或2所述单域抗体相关的生物材料，其特征在于，所述生物材料为下述C1)至C4)中的任一种：
    C1)编码权利要求1或2所述单域抗体重链可变区的核酸分子；
    C2)含有C1)所述核酸分子的表达盒；
    C3)含有C1)所述核酸分子的重组载体、或含有C2)所述表达盒的重组载体；
    C4)含有C1)所述核酸分子的重组宿主细胞、或含有C2)所述表达盒的重组宿主细胞、或含有C3)所述重组载体的重组宿主细胞。
  4. 根据权利要求3所述的生物材料，其特征在于，所述核酸分子为下述任一种：
    D1)核苷酸序列或编码序列是SEQ ID No.2的DNA分子；
    D2)与D1)限定的核苷酸序列具有75％或75％以上同一性，且具有相同功能的DNA分子。
  5. 药物组合物，其特征在于，所述药物组合物含有权利要求1或2所述的单域抗体。
  6. 试剂或试剂盒，其特征在于，所述试剂或试剂盒含有权利要求1或2所述的单域抗体，所述试剂或试剂盒具有下述任一用途：
    F1)检测CLL1蛋白；
    F2)筛查、诊断或辅助诊断CLL1靶点相关疾病；
    F3)用于CLL1蛋白的体内显像。
  7. 权利要求1或2所述单域抗体和/或权利要求3或4所述生物材料的下述任一种应用：
    E1)在制备用于预防或***的药物中的应用，或在预防或***中的应用；
    E2)在制备用于预防或治疗CLL1靶点相关疾病的药物中的应用，或在预防或治疗CLL1靶点相关疾病中的应用；
    E3)在制备用于筛查、诊断或辅助诊断CLL1靶点相关疾病的产品中的应用，或在筛查、诊断或辅助诊断CLL1靶点相关疾病中的应用；
    E4)在检测CLL1蛋白或制备用于检测CLL1蛋白的产品中的应用；
    E5)在制备用于结合CLL1蛋白的产品中的应用；
    E6)在介导药物特异性地识别表达CLL1抗原的肿瘤或制备介导药物特异性地识别表达CLL1抗原的肿瘤的产品中的应用；
    E7)在CLL1蛋白的体内显像或制备用于CLL1蛋白的体内显像的产品中的应用；
    E8)在制备靶向CLL1的CAR细胞中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述CLL1靶点相关疾病为CLL1阳性癌症。
  9. 根据权利要求8所述的应用，其特征在于，所述CLL1阳性癌症为急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病。
  10. 权利要求1或2所述单域抗体的制备方法，其特征在于，所述方法包括：构建含有权利要求3或4中所述核酸分子的重组表达载体；将所述重组表达载体导入宿主细胞，获得表达所述单域抗体的重组细胞；培养所述重组细胞，经分离纯化获得所述单域抗体。
  11. 诊断或辅助诊断CLL1靶点相关疾病的方法，其特征在于，所述方法包括：从受试者分离获得样品，然后利用权利要求1或2所述的单域抗体检测所述样品中CLL1蛋白的含量，根据所述CLL1蛋白的含量进行CLL1靶点相关疾病的诊断或辅助诊断。
  12. 筛查CLL1靶点相关疾病的方法，其特征在于，所述方法包括：从受试者分离获得样品，然后利用权利要求1或2所述的单域抗体检测所述样品中CLL1蛋白的含量，根据所述CLL1蛋白的含量进行CLL1靶点相关疾病的筛查。
  13. 预防或治疗CLL1靶点相关疾病的方法，其特征在于，所述方法包括向患有CLL1靶点相关疾病的受试者施用权利要求1或2所述的单域抗体或权利要求5所述的药物组合物。
  14. 根据权利要求11-13中任一所述的方法，其特征在于，所述CLL1靶点相关疾病为CLL1靶点相关肿瘤。
  15. 根据权利要求14所述的方法，其特征在于，所述CLL1靶点相关肿瘤为CLL1阳性癌症。
  16. 根据权利要求15所述的方法，其特征在于，所述CLL1阳性癌症为急性髓细胞白血病、骨髓增生异常综合征或慢性髓细胞白血病。
  17. 体外检测CLL1蛋白的方法，其特征在于，所述方法包括利用权利要求1或2所述的单域抗体或权利要求6中所述的试剂盒检测CLL1蛋白。
  18. 抗体药物偶联物，包括抗体部分和偶联部分，其特征在于，所述抗体部分包含权利要求1或2所述的单域抗体。
  19. 根据权利要求18所述的抗体药物偶联物，其特征在于，所述偶联部分包括可检测的标记、化学药物或细胞毒素。
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