Single domain antibodies that specifically bind to CLL1 protein and uses thereof

Abstract

The present invention provides a single-domain antibody that specifically binds to the CLL1 protein and uses thereof. The amino acid sequence of the single-domain antibody is shown in SEQ ID No. 1. The single-domain antibody has high affinity and can specifically target CLL1-positive cells. It effectively promotes IFNγ secretion from NK cells, activates the effector function of NK cells, significantly induces NK cell cytotoxicity, and has a strong ability to specifically kill CLL1-positive cells. The single-domain antibody can be used to produce drugs for the prevention or treatment of CLL1-targeted diseases, as well as to produce CLL1-targeting CAR cells or CLL1 protein detection kits. Single-domain antibody drugs have a stable structure, small molecule size, are easy to recombinantly express, and are inexpensive to produce. They can be used alone or as a drug delivery system for delivering related drugs.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, and specifically to single domain antibodies that specifically bind to CLL1 protein and uses thereof. [Background technology]

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous group of malignant diseases characterized by the clonal proliferation of myeloid progenitor cells in the hematopoietic system. These progenitor cells undergo malignant transformation at various stages of normal myeloid cell differentiation and development. For a long time, anthracycline and cytarabine chemotherapy has been used to treat mild cases, while hematopoietic stem cell transplantation has been used to treat intermediate- and high-risk patients. Due to the heterogeneity of AML, treatment of AML patients remains challenging. In recent years, researchers have discovered that immunotherapy targeting CLL1 is effective in treating AML. C-type lectin-like molecule 1 (CLL1), also known as C-type lectin domain family 12 member A (CLEC12A), is a type II transmembrane glycoprotein that plays an important role in immune regulation as an inhibitory receptor. CLL1 is present in peripheral blood, bone marrow myeloid cells, and most AML cells, and many AML cells express CD34 + CD38 - Normal CD34, but expressed on stem cells + CD38 - The unique expression pattern of CLL1 makes it a potential target for the treatment and diagnosis of AML, as it is not expressed in stem cells. Furthermore, CLL1 is also expressed in cells of myelodysplastic syndromes (MDS) and chronic myeloid leukemia (CML).

[0003] Antibodies are globulins that specifically bind to antigens and have a variety of immune functions. They are synthesized and secreted after B cells are stimulated by antigens and differentiate and proliferate into plasma cells. Because of their ability to specifically bind to antigens, antibodies are of great significance in disease diagnosis and immunoprophylactic treatment. Antibody drugs, which consist of antibody substances (including all antibody molecules and antibody fragments with therapeutic functions), are one of the important means of targeted therapy and are currently one of the most promising and hot fields in the biopharmaceutical industry. However, their large molecular weight and strong antigenicity have hindered their widespread use in the pharmaceutical field.

[0004] Heavy-chain antibodies (HcAbs) are a type of naturally occurring antibody unique to camelids and cartilaginous fish. Their antibody domains naturally lack light chains and consist of only two heavy chains. The antigen-recognition function of heavy-chain antibodies is primarily determined by the variable regions of the heavy chains (VHHs). Because antigen recognition is possible solely through VHHs, they are also called single-domain antibodies (sdAbs). Single-domain antibodies lack the light chains and contain only the heavy-chain variable regions. Because of their small molecular weight, they are also called nanobodies (Nb). With a molecular weight of only approximately 13–15 kDa, a diameter of approximately 2.5 nm, and a length of 4 nm, single-domain antibodies are the smallest antibody fragments yet possessing antigen-binding function. Their antigen-binding ability and stability are comparable to or even superior to that of intact antibodies. Compared to conventional antibodies, single-domain antibodies possess many unique properties, including superior stability, targeting specific antigen epitopes, flexible component combinations, and low production costs. Traditional methods for obtaining single-domain antibodies involve multiple steps, including multiple immunizations of camelids, B lymphocyte isolation, VHH domain amplification, and display library construction and screening. With the development of synthetic biology, it has become possible to construct high-quality, randomized, and large-scale single-domain antibody libraries based on total synthesis. Currently, VHH single-domain antibodies are widely used in miniaturized antibody research, drug development, and disease diagnosis and treatment due to their stable structure, small molecular weight, good solubility, resistance to various adverse environments, good formulation stability, easy recombinant expression, and ease of humanization. Research and development of single-domain antibodies holds great promise for pharmaceutical applications and clinical diagnostics, and antibody miniaturization is of great significance for the development of antibody drugs. Summary of the Invention

[0005] One object of the present invention is to provide single domain antibodies that target the CLL1 antigen and their use in tumor targeted therapy.

[0006] To achieve the above object, the present invention provides a single domain antibody designated CLL1-VHH-1. This single domain antibody is composed of a heavy chain variable region (VHH), which may include a complementarity determining region (CDR1) having the amino acid sequence of positions 26 to 35 of SEQ ID NO. 1, a complementarity determining region (CDR2) having the amino acid sequence of positions 50 to 59 of SEQ ID NO. 1, and a complementarity determining region (CDR3) having the amino acid sequence of positions 99 to 118 of SEQ ID NO. 1.

[0007] The single domain antibody may be a single domain antibody that specifically binds to the CLL1 protein (an anti-CLL1 single domain antibody).

[0008] Furthermore, the amino acid sequence of the heavy chain variable region may be SEQ ID No. 1, or an amino acid sequence having at least 80% identity and the same function as SEQ ID No. 1.

[0009] Single domain antibodies (anti-CLL1 single domain antibodies) have only the variable region (VHH) of a heavy chain antibody and are composed of the following in order: framework region FR1, complementarity determining region CDR1, framework region FR2, complementarity determining region CDR2, framework region FR3, complementarity determining region CDR3, and framework region FR4.

[0010] The amino acid sequence of the single domain antibody CLL1-VHH-1 may be SEQ ID NO. 1. In SEQ ID NO. 1, positions 1 to 25 represent the framework region FR1, positions 26 to 35 represent the complementarity-determining region CDR1, positions 36 to 49 represent the framework region FR2, positions 50 to 59 represent the complementarity-determining region CDR2, positions 60 to 98 represent the framework region FR3, positions 99 to 118 represent the complementarity-determining region CDR3, and positions 119 to 129 represent the framework region FR4. The nucleotide sequence of a nucleic acid molecule encoding the single domain antibody CLL1-VHH-1 (CLL1-VHH-1 gene) is shown in SEQ ID NO. 2.

[0011] In this regard, positions 1 to 75 of SEQ ID NO. 2 are the coding sequence of framework region FR1, positions 76 to 105 are the coding sequence of complementarity-determining region CDR1, positions 106 to 147 are the coding sequence of framework region FR2, positions 148 to 177 are the coding sequence of complementarity-determining region CDR2, positions 178 to 294 are the coding sequence of framework region FR3, positions 295 to 354 are the coding sequence of complementarity-determining region CDR3, and positions 355 to 387 are the coding sequence of framework region FR4.

[0012] The sequences of the complementarity determining regions are defined according to the Kabat numbering system.

[0013] Other variants of the single domain antibody sequences of the present invention with improved affinity and / or potency can be obtained using methods known in the art and are within the scope of the present invention. For example, antibodies with improved affinity can be obtained by amino acid substitution. Alternatively, codon optimization of the nucleotide sequence can improve the translation efficiency in an expression system for producing the antibody. Furthermore, polynucleotides containing sequences in which antibody specificity has been optimized by applying directed evolution techniques to any nucleic acid sequence of the present invention are also within the scope of the present invention.

[0014] In some embodiments, substitutions, insertions, or deletions may be made in one or more complementarity-determining regions or framework regions of the single domain antibodies of the present invention, as long as they do not substantially reduce the antigen-binding ability of the antibody. For example, conservative modifications (e.g., conservative substitutions; as is well known to those skilled in the art, conservative substitutions of amino acids do not alter the properties and function of a protein) may be made in the complementarity-determining regions and / or framework regions that do not substantially reduce binding affinity. For example, such modifications may be made outside of the antigen-contacting residues in the complementarity-determining regions.

[0015] The present invention further provides a biomaterial related to the single domain antibody CLL1-VHH-1, which may be any one of C1) to C4) below: C1) a nucleic acid molecule encoding the heavy chain variable region of the single domain antibody CLL1-VHH-1; C2) an expression cassette comprising the nucleic acid molecule according to C1); C3) A recombinant vector comprising the nucleic acid molecule according to C1), or a recombinant vector comprising the expression cassette according to C2). C4) A recombinant host cell comprising a nucleic acid molecule according to C1), or a recombinant host cell comprising an expression cassette according to C2), or a recombinant host cell comprising a recombinant vector according to C3).

[0016] Here, the expression cassette described in C2), the recombinant vector described in C3) and the recombinant host cell described in C4) are capable of expressing the single domain antibody CLL1-VHH-1.

[0017] In the biomaterial, the nucleic acid molecule may be any one of the following: D1) a DNA molecule whose nucleotide sequence or coding sequence is 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.

[0018] The DNA molecule shown in SEQ ID No. 2 encodes the single domain antibody CLL1-VHH-1 having the amino acid sequence of SEQ ID No. 1.

[0019] 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0020] 80% or greater 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. 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. 95% or greater identity may be at least 95%, 96%, 97%, 98% or 99% identity.

[0021] As used herein, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. Amino acid sequence identity can be measured using an online homology search site, such as the BLAST page on the NCBI homepage. For example, in the advanced BLAST 2.1, the program blastp is used, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as the matrix, and the gap existence cost, per residue gap cost, and lambda ratio are set to 11, 1, and 0.85 (default values), respectively. The percent identity can be calculated by searching for amino acid sequence identity.

[0022] As used herein, a vector refers to a vector capable of introducing, amplifying, and expressing foreign DNA or a target gene in a host cell. The vector may be a cloning vector or an expression vector, including, but not limited to, a plasmid, a phage (e.g., λ phage or M13 filamentous phage), a cosmid, an artificial chromosome (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC), Ti plasmid artificial chromosome (TAC)), or a viral vector (e.g., baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, or herpes virus (e.g., herpes simplex virus)). In one or more embodiments of the present invention, the vector is a pComb3XSS plasmid and / or a pcDNA3.1 vector.

[0023] As used herein, a host cell (also referred to as a recipient cell) refers to any type of cell that can be used to introduce a vector. Host cells can be eukaryotic (e.g., plant cells, animal cells, fungi, algae) or prokaryotic (e.g., bacteria, protozoa). Host cells include not only the particular recipient cell but also its progeny. While progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or deliberate mutations and / or modifications, they are still considered to be within the scope of host cells. Suitable host cells are known in the art. Here, plant cells can be, but are not limited to, Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc. The animal cells may be, but are not limited to, mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse mammary carcinoma cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells or Japanese giant salamander (Andrias davidianus) cells), fish cells (e.g., grass carp, common carp, rainbow trout, or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc. The fungus may be a yeast, including, but not limited to, Saccharomyces (e.g., Saccharomyces cerevisiae), Kluyveromyces (e.g., Kluyveromyces lactis), Pichia (e.g., Pichia pastoris), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), and Hansenula (e.g., Hansenula polymorpha).Examples of fungi include, but are not limited to, Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp. Examples of algae include, but are not limited to, Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp. Bacteria include, but are not limited to, Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. For example, the bacterium may be Escherichia coli, Bacillus subtilis, or Bacillus pumilus. In one or more embodiments of the present invention, the host cell is E. coli TG1 and / or HEK293 cells.

[0024] As used herein, a recombinant vector refers to a recombinant DNA molecule constructed in vitro by ligating an exogenous target gene to a vector, and can be constructed by any suitable method, so long as the constructed recombinant vector delivers the exogenous target gene to a recipient cell and confers on the exogenous target gene the ability to replicate, integrate, amplify, and / or express in the recipient cell. In one or more embodiments of the present invention, the recombinant vector is pcDNA3.1-CLL1-VHH-1-hFc.

[0025] To facilitate purification, the recombinant vector pcDNA3.1-CLL1-VHH-1-hFc was prepared by adding the human IgG1Fc gene (hFc gene, nucleotide sequence SEQ ID No. 3) to the 3' end of the gene encoding the single-domain antibody CLL1-VHH-1 (SEQ ID No. 2) to obtain the CLL1-VHH-1-hFc fusion gene. A BamHI site was then added before the initiation codon at the 5' end of the fusion gene and an EcoRI site was added after the termination codon at the 3' end. The fusion gene was then cloned between the BamHI and EcoRI sites of the eukaryotic expression vector pcDNA3.1 to obtain the recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-1-hFc, which expresses the single-domain antibody CLL1-VHH-1 fused to hFc (SEQ ID No. 4) at its C-terminus. The recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-1-hFc contains the gene encoding the single domain antibody CLL1-VHH-1 (SEQ ID No. 2).

[0026] The nucleotide sequence (696 bp) of the human IgG1Fc gene is shown in SEQ ID No. 3, and the amino acid sequence encoded by the human IgG1Fc gene is shown in SEQ ID No. 4.

[0027] As used herein, a recombinant host cell refers to a recombinant host cell whose function has been altered by genetic manipulation or modification of a target host cell. Examples include recombinant host cells obtained by introducing an exogenous target gene or recombinant vector into a target host cell, or recombinant host cells obtained by directly editing an endogenous gene in a target host cell. Recombinant host cells encompass not only the specific recombinant host cell but also its progeny. While progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or intentional mutations and / or modifications, they are still considered to be within the scope of recombinant host cells. In one or more embodiments of the present invention, the recombinant host cell is a recombinant host cell obtained by introducing the recombinant vector pcDNA3.1-CLL1-VHH-1-hFc into HEK293 cells.

[0028] The present invention further provides any one of the following uses of the single domain antibody CLL1-VHH-1 or a biological material: E1) Use in the manufacture of a medicament for the prevention or treatment of tumors (including tumors expressing the CLL1 antigen), or use in the prevention or treatment of tumors; E2) Use in the manufacture of a medicament for the prevention or treatment of a CLL1 target-related disease, or use in the prevention or treatment of a CLL1 target-related disease; E3) Use in the manufacture of a product for screening, diagnosing or aiding in diagnosis of a CLL1 target-related disease, or use in screening, diagnosing or aiding in diagnosis of a CLL1 target-related disease; E4) Use in the detection of CLL1 protein or in the manufacture of a product for the detection of CLL1 protein; E5) Use in the manufacture of a product for binding to CLL1 protein; E6) Use in the drug-mediated specific recognition of tumors expressing the CLL1 antigen or in the manufacture of a product for the drug-mediated specific recognition of tumors expressing the CLL1 antigen; E7) Use in in vivo imaging of CLL1 protein or in the manufacture of a product for in vivo imaging of CLL1 protein; E8) Use in the production of CAR cells targeting CLL1.

[0029] As used herein, a CLL1 target-associated disease may be a CLL1-positive cancer (such as leukemia).

[0030] As used herein, CLL1-positive cancer may be, but is not limited to, acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myeloid leukemia (CML).

[0031] E6) The product for drug-mediated specific recognition of tumors expressing the CLL1 antigen may be a drug delivery system that specifically targets the CLL1 protein, which drug delivery system comprises the single domain antibody CLL1-VHH-1.

[0032] Additionally, the drug delivery system further comprises a therapeutically active drug (therapeutic agent) or diagnostic agent bound or linked to the single domain antibody CLL1-VHH-1.

[0033] Additionally, therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-tumor agents, anti-viral agents, autoimmune disease agents, and anti-infective agents.

[0034] Anti-tumor agents may include chemical drugs or cytotoxins.

[0035] The cytotoxin may be a microtubule inhibitor (such as maytansine or auristatin), a DNA synthesis inhibitor (such as a calicheamicin), or an RNA synthesis inhibitor (such as an amatoxin).

[0036] Additionally, the therapeutic agent may be an agent for treating a CLL1 target-related disease.

[0037] Additionally, the therapeutic agent may be one or more of a monoclonal antibody or binding fragment thereof, a protein, a peptide, an RNA molecule, a DNA molecule, an siRNA molecule, an RNAi molecule, an ssRNA molecule, a growth factor, an enzyme inhibitor, or a binding protein.

[0038] Additionally, the diagnostic agent may be one or more selected from an imaging agent, a contrast agent, a fluorescent label, a radioactive label, a magnetic resonance imaging label, and a spin label.

[0039] The imaging agent may be one or more selected from radionuclides, biotin, fluorophores, antibodies, horseradish peroxidase, alkaline phosphatase, nanoparticles, quantum dots, nanodroplets capable of detecting anti-cancer drugs, liposomal drugs, and cytokines.

[0040] The drug delivery system may be a liposomal drug delivery system, a polymeric micelle drug delivery system, a polymeric disc drug delivery system, or a nanoparticle drug delivery system.

[0041] Drug delivery systems can be used for targeted delivery and / or site-specific release of drugs.

[0042] The product in E8) may be an immune cell.

[0043] Furthermore, immune cells include, but are not limited to, CAR-modified cells (CAR cells), TCR cells, TIL cells, CIK cells, CTL cells, T cells, NK cells, NKT cells, gamma-delta T cells, or macrophages.

[0044] Furthermore, the CAR-modified cells (CAR cells) contain or express a chimeric antigen receptor (CAR), which may comprise the single domain antibody CLL1-VHH-1.

[0045] Furthermore, the TCR cells, TIL cells, CIK cells, CTL cells, T cells, NK cells, NKT cells, gamma-delta T cells or macrophages contain or express the single domain antibody CLL1-VHH-1.

[0046] Furthermore, the antigen recognition domain of the chimeric antigen receptor may be the single domain antibody CLL1-VHH-1.

[0047] Furthermore, CAR cells include, but are not limited to, CAR-T cells, CAR-NK cells, CAR-macrophages (CAR-M cells), CAR-iPSCs, or CAR-PSCs.

[0048] Furthermore, TCR cells include, but are not limited to, TCR-T cells or TCR-NK cells.

[0049] The detection of CLL1 protein described herein may be detecting whether or not a test sample contains CLL1 protein and / or detecting the content of CLL1 protein in a test sample.

[0050] The test sample may be a cell or tissue sample.

[0051] Products for detecting CLL1 protein include products that detect antigen-antibody binding using enzyme immunoassay, immunofluorescence assay, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination assay, or immunoturbidimetry, etc.

[0052] The articles of manufacture described herein may be reagents, kits, chips, or test strips.

[0053] The present invention further provides a pharmaceutical composition, which may comprise the single domain antibody CLL1-VHH-1, or a chimeric antigen receptor (CAR) modified cell, wherein the chimeric antigen receptor (CAR) may comprise the single domain antibody CLL1-VHH-1.

[0054] Furthermore, the antigen recognition domain of the chimeric antigen receptor may be the single domain antibody CLL1-VHH-1.

[0055] The cell may be a T cell, an NK cell, a γδT cell, an NKT cell, a macrophage, or a stem cell.

[0056] CAR-modified cells (CAR cells) may be, but are not limited to, CAR-T cells, CAR-NK cells, CAR-macrophages (CAR-M cells), CAR-iPSCs, or CAR-PSCs.

[0057] Additionally, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0058] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.

[0059] The pharmaceutical composition has at least one of the following uses: F1) Use in the prevention or treatment of tumors, F2) Use in the prevention or treatment of CLL1 target-related diseases; F3) Use in drug-mediated specific recognition of tumors expressing the CLL1 antigen.

[0060] The present invention further provides a reagent or kit, which may comprise the single domain antibody CLL1-VHH-1 and has at least one of the following uses: G1) Use in detecting CLL1 protein; G2) Use in screening, diagnosing or assisting in diagnosis of CLL1 target-related diseases; G3) Use of CLL1 protein in in vivo imaging.

[0061] The kit may be, but is not limited to, a chemiluminescent immunoassay kit, an enzyme-linked immunoassay kit, a colloidal gold immunoassay kit, or a fluorescent immunoassay kit.

[0062] The present invention further provides a method for producing the single domain antibody CLL1-VHH-1, 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-1, introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the single domain antibody, culturing the recombinant cell, and isolating and purifying the single domain antibody.

[0063] Furthermore, the nucleic acid molecule encoding the heavy chain variable region of the single domain antibody CLL1-VHH-1 may be any one of the following: H1) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2; H2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in H1) and has the same function.

[0064] Additionally, the host cell may be a HEK293 cell.

[0065] Furthermore, the introduction may be by transforming a vector containing the single domain antibody gene of the present invention into a host cell using any known transfection method, such as calcium phosphate co-precipitation, liposome-mediated transfection, electroporation, or viral vector methods.

[0066] The present invention further provides a method for diagnosing or aiding in the diagnosis of a CLL1 target-related disease, which may include isolating a sample from a subject, detecting the content of CLL1 protein in the sample using the single domain antibody CLL1-VHH-1, and diagnosing or aiding in the diagnosis of a CLL1 target-related disease based on the content of CLL1 protein (compared to the content of CLL1 protein in a disease diagnostic criterion).

[0067] The present invention further provides a method for screening for CLL1 target-related diseases, which may include isolating a sample from a subject, detecting the content of CLL1 protein in the sample using a single domain antibody CLL1-VHH-1, and screening for CLL1 target-related diseases based on the content of CLL1 protein (compared to the content of CLL1 protein in a disease diagnostic criterion).

[0068] The present invention further provides a method for preventing or treating a CLL1 target-related disease, which may comprise administering the single domain antibody CLL1-VHH-1 or the pharmaceutical composition described herein to a subject suffering from a CLL1 target-related disease.

[0069] In the above method, the CLL1 target-associated disease may be a CLL1 target-associated tumor.

[0070] In the above method, the CLL1 target-associated tumor may be a CLL1-positive cancer.

[0071] In the above methods, the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

[0072] The present invention further provides a method for detecting CLL1 protein in vitro, which may comprise detecting CLL1 protein using the single domain antibody CLL1-VHH-1 or the reagents or kits described herein.

[0073] The present invention further provides an antibody-drug conjugate comprising an antibody portion and a binding portion. The antibody portion may comprise the single domain antibody CLL1-VHH-1.

[0074] Additionally, the binding moiety may comprise a detectable label, a chemical agent, or a cytotoxin.

[0075] The antibody moiety and the binding moiety may be covalently bonded directly or via a linker.

[0076] The detectable label may be selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0077] The chemical drug may be a drug capable of killing tumor cells.

[0078] The cytotoxin may be a microtubule inhibitor (such as maytansine or auristatin), a DNA synthesis inhibitor (such as a calicheamicin), or an RNA synthesis inhibitor (such as an amatoxin).

[0079] The purpose of the uses and methods of the present invention may be for the purpose of disease diagnosis, prognosis, and / or treatment, or may be for non-disease diagnosis, prognosis, and treatment, and the direct purpose may be to obtain information on intermediate results of disease diagnosis, prognosis, and / or treatment, or may be for the purpose of non-disease diagnosis, prognosis, and / or treatment.

[0080] CLL1, as described herein, is C-type lectin-like molecule 1 (CLEC12A), also known as C-type lectin domain family 12 member A (CLEC12A), a type II transmembrane glycoprotein that plays an important role in immunoregulation as an inhibitory receptor. CLL1 is present in peripheral blood, bone marrow myeloid cells, and most acute myeloid leukemia (AML) cells, and is expressed on the CD34 receptor of many AML cells. + CD38 - Normal CD34, but expressed on stem cells + CD38 - The unique expression pattern of CLL1 makes it a potential target for the treatment and diagnosis of AML, as it is not expressed in stem cells. Furthermore, CLL1 is also expressed in cells of myelodysplastic syndromes (MDS) and chronic myeloid leukemia (CML).

[0081] In the present invention, alpacas were immunized with CLL1 recombinant protein to obtain single-domain antibody genes, and a single-domain antibody expression library was constructed. Then, a high-affinity anti-CLL1 single-domain antibody (CLL1-VHH-1) was screened from the single-domain antibody expression library using phage display screening technology. After determining the antibody gene sequence, CLL1-VHH-1 was produced using genetic engineering techniques. Further performance experiments, such as affinity and specificity, were performed on the obtained antibody to detect antitumor activity mediated by the single-domain antibody CLL1-VHH-1, including measurement of NK cell effector function and ADCC activity. Experimental results showed that the single-domain antibody (CLL1-VHH-1) obtained by the present alpaca immunization and screening method had an affinity constant of 48.3 nM for K562-CLL1 cells, indicating high affinity for the CLL1 protein. Furthermore, the single-domain antibody CLL1-VHH-1 of the present invention exhibited excellent specificity and effectively targeted CLL1-positive cells (cells expressing the CLL1 protein, CLL1 + The antibody CLL1-VHH-1 of the present invention can more effectively promote IFNγ secretion from NK cells, activate the effector function of NK cells, and specifically target CLL1 + It enhances the efficacy of NK cells against target cells, improves antitumor activity, significantly induces NK cell cytotoxicity (ADCC), and has a strong ability to specifically kill CLL1-positive cells.

[0082] The single domain antibodies of the present invention can be expressed and produced in prokaryotic cells, yeast cells, eukaryotic cells, and any recombinant system, and can be used to produce specific antibody drugs that are clinically used for the prevention and treatment of CLL1-targeted diseases (such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), as well as to produce CLL1-targeting CAR cells and CLL1 protein detection kits. The single domain antibody drugs of the present invention have a stable structure, small molecule, are easily recombinantly expressed, and are inexpensive to produce. They can be used alone or as a drug delivery system for delivering related drugs, and have very broad prospects and important significance in fields such as pharmaceutical applications and clinical diagnosis. [Brief explanation of the drawings]

[0083] [Figure 1] This shows the results of agarose gel electrophoresis of total RNA extracted from alpaca PBMCs. [Figure 2] 1 shows the results of agarose gel electrophoresis of colony PCR of a bacterial library. [Figure 3] FITC-labeled CLL1-VHH-1 antibody was used to detect CLL1 expression on various tumor cell lines, where Isotype represents non-FITC-labeled CLL1-VHH-1-hFc antibody, and CLL1 represents FITC-CLL1-VHH-1-hFc antibody. [Figure 4] 1 shows the results of measuring the effector function of NK cells mediated by CLL1-VHH-1 antibody in Example 3. Here, VHH-hFC represents CLL1-VHH-1 antibody. [Figure 5] 1 shows the results of measuring the ADCC activity of CLL1-VHH-1 antibody in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention will be described in detail below with reference to specific embodiments, but the examples shown are for the purpose of illustrating the present invention and do not limit the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, but do not limit the present invention in any way.

[0085] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and were carried out according to the techniques or conditions described in the literature in the field or in accordance with the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0086] The pComb3XSS plasmid used in the following examples is a product of Beijing Zhuangmeng International Biogene Technology Co., Ltd. (catalog number ZK129).

[0087] The helper phage M13K07 in the following examples is a product of Chengdu Renyu Biotechnology Co., Ltd. (catalog number A001-1).

[0088] In the following examples, K562 cells (CLL1-negative cells) are manufactured by Nanjing Kebai Biotechnology Co., Ltd. (catalog number CBP60529). MM1.S cells (CLL1-negative cells) are manufactured by Beijing Baiou Bowei Biotechnology Co., Ltd. (catalog number bio-129535). U937 cells (CLL1-positive cells) are manufactured by Beijing Baiou Bowei Biotechnology Co., Ltd. (catalog number bio-73180). U266 cells (CLL1-negative cells) are manufactured by Beijing Baiou Bowei Biotechnology Co., Ltd. (catalog number bio-69276). Jurkat cells (CLL1-negative cells) are manufactured by Beijing Baiou Bowei Biotechnology Co., Ltd. (catalog number bio-68218). THP-1 cells (CLL1-positive cells) are a product of Beijing Baiou Bowei Biotechnology Co., Ltd. (catalog number bio-68161).

[0089] In the following examples, primary peripheral blood mononuclear cells (PBMCs) were obtained from the venous blood of healthy volunteers and are abbreviated as primary PBMCs.

[0090] In the example below, the method for producing CLL1-positive cells K562-CLL1 involves inserting the CLL1 gene between the BamHI and EcoRI sites of pcDNA3.1, and then introducing the resulting recombinant vector into K562 cells to obtain CLL1-positive cells K562-CLL1.

[0091] The vector pcDNA3.1 used in the following examples is a product of Changsha Abiwei Biotechnology Co., Ltd. (catalog number HG-VPI0001).

[0092] Some of the sequences in the examples below are as follows: Human IgG1Fc gene nucleotide sequence (696bp): gagcccaaatctgctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccccgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID No.3) Amino acid sequence of human IgG1 Fc (232 aa): EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.4)

[0093] Example 1: Production of a single domain antibody that specifically binds to the CLL1 protein In this example, alpacas were immunized with CLL1 recombinant protein to obtain single-domain antibody genes and construct a single-domain antibody expression library. Phage display screening technology was then used to screen for single-domain antibodies (abbreviated as anti-CLL1 single-domain antibodies) that specifically bind to the CLL1 protein with high affinity from the single-domain antibody expression library.

[0094] 1. Animal immunity 1.1. Antigen acquisition The antigen, CLL1 recombinant protein, is as follows: CLL1 immunogen: CLL1-hIgG1Fc (B918601) protein (derived from HEK293 cells) is a product of Shanghai Baiying Biotechnology Co., Ltd. CLL1 screening antigen: CLL1-6xHis (B918602) protein (derived from HEK293 cells) is a product of Shanghai Baiying Biotechnology Co., Ltd.

[0095] 1.2. Alpaca immunity Alpacas (2-3 years old, weighing approximately 50 kg) were immunized four times with the CLL1 immunizing antigen CLL1-hIgG1Fc. 0.5 mg of CLL1-hIgG1Fc was injected subcutaneously every two weeks. Five ml of blood was collected before the next immunization, and the plasma was separated to measure the antibody titer after the previous immunization. The antibody titer after the fourth immunization was measured one week after the injection.

[0096] The antibody titer in the serum was measured by indirect ELISA, and the procedure was as follows. (1) The immunizing antigen CLL1-hIgG1Fc was diluted with PBS to 2 μg / ml, coated onto an ELISA plate at 100 μL / well, and left overnight at 4° C. The control group was coated with 3% BSA. (2) The antigen solution was discarded, and the ELISA plate was washed three times with 200 μL of PBST solution (1×PBS solution containing 0.05% Tween 20) for 5 minutes each. (3) The ELISA plate was blocked with 200 μL of a blocking solution, PBSTB (PBST containing 2% BSA), at room temperature for 1.5 hours. (4) The blocking solution was discarded, and the ELISA plate was washed three times with 200 μL of PBST solution for 5 minutes each. (5) Using the blocking solution, the test serum was serially diluted to 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, and 1:10000000, and added to the ELISA plate at 100 μL / well and incubated at room temperature for 2 hours. (6) The serum was discarded, and the ELISA plate was washed four times with 200 μL of PBST solution for 5 minutes each. (7) 100 μL of goat anti-llama antibody (Goat Anti-Llama IgG H&L (HRP), Abcam, ab112786) diluted 1:10,000 in blocking solution was added to each well, and the plate was incubated at room temperature for 1 hour. (8) The detection antibody was discarded, and the ELISA plate was washed four times with 200 μL of PBST solution for 5 minutes each. (9) 100 μL of TMB substrate solution was added to each well and allowed to develop color for 2 to 3 minutes. (10) 100 μL of reaction stop solution was added to each well. (11) The absorbance at 450 nm was measured within 30 minutes using a microplate reader. Criteria for determining positive wells: OD of immune serum sample. 450 The measured OD of non-immune serum 450 Greater than the measured value and the measured value is greater than 0.5.

[0097] 1.3. Immunity Results The serum titer measurement results are shown in Table 1, with positive wells indicated in bold. After four immunizations, the serum titer of the alpaca was 10 5 reached, meaning the antibody level was 10 5 reached. Table 1. Serum titer measurement results As shown in Table 1, after the fourth immunization, the antibody levels of the alpacas were 10 5 The Ag coating was the immunogen CLL1-hIgG1Fc coating, and PBSTB was the blocking solution.

[0098] 2. Construction of phage library After successful immunization, peripheral blood mononuclear cells (PBMCs) from the alpacas were collected to construct a phage library (anti-CLL1 VHH phage antibody library).

[0099] 2.1 RNA extraction and reverse transcription Alpaca peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stored in Trizol. Total RNA was extracted from the PBMCs, and the resulting total RNA was reverse transcribed into cDNA using the Takara reverse transcription kit. The specific procedure is as follows: (1) Alpaca PBMC cells preserved in Trizol were transferred to a 1.5 mL centrifuge tube, and 1 / 5 volume of chloroform was added and mixed well, followed by standing at room temperature for 5 minutes. (2) Centrifuged at 12,000 g for 15 minutes at 4°C. (3) The supernatant was transferred to a new centrifuge tube, an equal volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. (4) Centrifuged at 12,000 g for 10 minutes at 4°C. (5) The precipitate was washed with 1 mL of 75% ethanol, centrifuged at 7500 g for 5 minutes to remove the ethanol, and dried. (6) The precipitate was dissolved in an appropriate amount of RNase-free water to obtain extracted total RNA, which was 181.2 μg (Figure 1). (7) Reverse transcription of total RNA using Takara reverse transcription kit: The total RNA sample was divided into two aliquots. One aliquot was primer-primed with the Oligo dT Primer provided with the kit, and the other aliquot was primer-primed with the Random 6-mers provided with the kit. The total RNA was reverse transcribed into cDNA according to the operating procedure described in the kit manual, yielding a total of 31.7 μg of cDNA.

[0100] 2.2. PCR of VHH target fragments of CLL1 antibody Using 8 ng cDNA as a template, the VHH target fragments were amplified by conventional PCR, yielding a total of 83 μg.

[0101] 2.3 Enzymatic Digestion and Ligation 1. pComb3XSS was used as the phage plasmid vector. The pComb3XSS vector and the VHH PCR product (VHH target fragment) were digested with the restriction enzymes SpeI and SacI, respectively, and incubated at 37°C for 4 hours. 2. The pComb3XSS vector and VHH PCR products were purified using a DNA recovery and purification kit after enzymatic digestion and stored at 4°C. 3. The pComb3XSS vector after double enzyme digestion and the VHH target fragment were ligated and recovered, yielding a total of 3.1 μg of ligation product.

[0102] 2.4. Construction of bacterial library (1) 100 ng of the ligation product was electroporated into 50 μL of E. coli TG1 competent cells. (2) After recovery at 37°C for 1 hour, 100 μL of the transformed product was serially diluted 10 times and applied to a plate, followed by overnight incubation at 37°C. (3) Based on the dilution factor and the number of single colonies, the total number of transformed colonies obtained by electroporation was calculated. The volume of the bacterial library was 2.6 × 10 8 cfu. (4) Several single clones were randomly selected from the serial dilution plate and subjected to colony PCR. The results of agarose gel electrophoresis (Figure 2) showed that all the selected single clones were positive clones, with a positive rate of 100%. (5) All remaining bacterial suspensions (each serial dilution of the transformation product) were collected and evenly spread onto 20 15 cm culture plates (containing 2xYT medium, 100 μg / mL Amp, and 2% agarose), and cultured overnight at 37°C with end-over-end mixing. (6) The colonies cultured overnight were scraped off the plate using 2xYT liquid medium, transferred to a 50 mL centrifuge tube, and the OD 600 The DNA was measured at 120°C, and glycerol was added to a final concentration of 20%. The DNA was then stored at -80°C to obtain a bacterial library. (7) The sequences of the single clone-positive colonies were translated into protein sequences, and comparison of the sequence diversity showed that all sequences were independent and had good diversity. The diversity of the bacterial library met the requirements.

[0103] 2.5. Preparation of phage libraries (1) The bacterial library was inoculated into 100 mL of 2xYT liquid medium (containing 100 μg / mL of Amp) and incubated at 37°C and 250 rpm until the OD 600 The culture was continued until the pH reached 0.5 to 0.55. (2) Helper phage M13K07 was added at a ratio of bacteria number to phage number of 1:20, and the mixture was incubated at 37°C and 250 rpm for 30 minutes. (3) Kan was added to a final concentration of 50 μg / mL, and the mixture was cultured overnight at 30°C and 250 rpm. (4) The overnight cultured bacterial solution was centrifuged at 4°C and 4000 rpm for 20 minutes, and the supernatant was transferred to a new 50 mL centrifuge tube. (5) One-quarter volume of 20% PEG / 2.5M NaCl solution pre-cooled to 4°C was added, mixed well, and incubated on ice for at least 30 minutes. (6) The mixture was centrifuged at 4°C and 4000 rpm for 20 minutes, the supernatant was discarded, and the mixture was mixed by inversion for 2 minutes. (7) 1 mL of PBS was added to resuspend the sample, and the mixture was centrifuged at 12,000 rpm at 4°C for 10 minutes. (8) The supernatant was transferred to a new centrifuge tube, and 1 / 4 volume of 20% PEG / 2.5M NaCl solution pre-cooled to 4°C was added, mixed well to obtain a white suspension, and incubated on ice for at least 10 minutes. (9) The cells were centrifuged at 12,000 rpm at 4°C for 10 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of PBS and centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a new centrifuge tube and stored at -80°C. 13 A purified phage library with a titer of pfu / mL was obtained.

[0104] 3. Affinity screening Using the screening antigen (CLL1-6xHis), the phage library was subjected to three rounds of affinity screening, including titer measurement, phage eluate amplification, and phage purification. After three rounds of affinity screening, once the enrichment level of anti-CLL1 VHH-positive antibodies in the screening library reached a predetermined level, the next step, screening for positive monoclonal antibodies, could be performed. The specific procedure is as follows:

[0105] 3.1. First round screening 3.1.1. First round screening method (1) The screening antigen (CLL1-6xHis) was coated onto immunotubes (50 μg / tube, 2 mL / tube of PBS as coating solution) and slowly rotated overnight at 4°C. As a control, BSA (50 μg / tube, 2 mL / tube of PBS as coating solution) was also coated onto immunotubes. (2) The liquid in the overnight-coated immunotube was discarded, and 2 mL of PBS buffer was added and washed three times at room temperature, with rotation for 5 minutes each time. (3) 2 mL of 3% non-fat dry milk was added, and the mixture was blocked by rotating at room temperature for 2 hours. (4) Discard the liquid in the blocked immunotube, add 2 mL of PBST buffer and wash three times at room temperature, rotating each time for 5 minutes. (5) The washing solution in the immunotube was discarded, and the phage library was added as an input phage library for the first round of screening according to the following formula. PBS buffer was added to make the total volume 2 mL, and the mixture was incubated with rotation at room temperature for 1 hour. TIFF2026508333000002.tif12170 where V is the volume of phage added (unit: μL), T library is the phage titer. (6) Discard the liquid in the immunotube, add 2 mL of PBST (1x PBS plus 0.1% Tween 20; the same applies below) buffer, and wash the immunotube 20 times at room temperature, rotating each time for 5 minutes. (7) Discard the liquid in the immunotube, add 1 mL of 0.25 mg / mL trypsin solution, and rotate at room temperature for 30 minutes to elute. (8) 10 μL of 10% AEBSF (serine protease inhibitor) was added to stop the elution, and the solution in the immunotube was transferred to a new 1.5 mL centrifuge tube. The phage eluate from the first round of screening (abbreviated as "first round phage eluate") was obtained.

[0106] 3.1.2. Determination of initial phage titer (1) A single colony of the TG1 strain stored at -80°C was streaked onto 2xYT solid medium and cultured overnight at 37°C. (2) One single colony was picked from the single colony plate, transferred to 5 mL of 2xYT medium, and cultured overnight at 37°C. (3) Transfer 50 μL of the bacterial suspension to 5 mL of 2xYT liquid medium and measure the OD at 37°C and 250 rpm. 600 The culture was continued until the pH reached 0.5 to 0.55, and a TG1 bacterial solution was obtained. (4) 10 μL of the first-round phage eluate was serially diluted 10-fold in a 1.5 mL centrifuge tube. That is, 10 μL of the first-round phage eluate was added to 90 μL of PBS, and 10 μL of the resulting solution was added to 90 μL of PBS. This process was repeated to obtain 10 -12 The solution was diluted 12 times to obtain (5) 90 μL of TG1 bacterial solution was added to each dilution centrifuge tube, shaken to mix, and incubated at 37°C for 30 minutes. (6) 5 μL of the bacterial solution was taken from each dilution centrifuge tube and added dropwise to 2×YT solid medium (Amp). After leaving it to stand for several minutes, it was mixed by inversion at 37°C overnight. (7) The number of clearly distinguishable single colonies on the plate at different dilutions was counted, and the number of phage particles per ml of phage solution, i.e., the titer of the phage library, was calculated according to the following formula: TIFF2026508333000003.tif7170 where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies on the plate at the corresponding dilution factor.

[0107] 3.1.3. Amplification of the first-round phage eluate (1) A single colony of the TG1 strain stored at -80°C was streaked onto 2xYT solid medium and cultured overnight at 37°C. (2) One single colony was picked from the single colony plate, transferred to 5 mL of 2xYT medium, and cultured overnight at 37°C. (3) Transfer 50 μL of the bacterial suspension to 5 mL of 2xYT liquid medium and measure the OD at 37°C and 250 rpm. 600 The culture was continued until the β-glucan concentration reached 0.5 to 0.55. (4) 500 μL of the first-round phage eluate was added, and the mixture was cultured at 37° C. and 250 rpm for 30 minutes. (5) All the bacterial solutions were evenly spread onto three 15 cm circular culture plates containing 100 μg / mL Amp and cultured overnight at 37°C. (6) 2xYT liquid medium was added to the surface of the culture plate, the colonies were gently scraped off with a cone rod, and the bacterial solution was collected in a 15 mL centrifuge tube to obtain the amplified bacterial sub-library. Glycerol was added to a final concentration of 20%, and the OD of the bacterial solution was measured using a spectrophotometer. 600 Measure the OD value of the eluate bacterial library 600 got the value. (7) The eluate bacterial library was transferred to 100 mL of 2xYT liquid medium (containing 100 μg / mL Amp) according to the following formula, and the initial OD 600 =0.1. TIFF2026508333000004.tif13170where V is the volume of the transferred bacterial solution (unit: μL), OD 600 is the OD of the constructed eluate bacterial library 600 is. (8) OD of bacterial solution 600 The mixture was cultured at 37°C and 250 rpm until the β-kappa bilayer thickness reached 0.5 to 0.55. (9) Helper phage M13K07 was added at a bacterial number:phage number = 1:20 according to the following formula. TIFF2026508333000005.tif13170 where V is the volume of helper phage added (unit: mL), T helper-phage is the titer of the helper phage, OD 600 is the OD of the bacterial solution 600 value. (10) The mixture was cultured at 37°C and 250 rpm for 30 minutes. (11) Kan was added to a final concentration of 50 μg / mL, and the mixture was cultured overnight at 30°C and 200 rpm.

[0108] 3.1.4. Purification of first-round phages (1) The overnight cultured bacterial solution was transferred to a new 50 mL centrifuge tube and centrifuged at 4°C and 4000 rpm for 20 minutes. (2) The supernatant was transferred to a new 50 mL centrifuge tube, and 1 / 4 volume of 20% PEG / 2.5 M NaCl solution pre-cooled to 4°C was added, mixed well, and left on ice for at least 30 minutes. (3) The mixture was centrifuged at 4°C and 4000 rpm for 20 minutes, and the supernatant was discarded. (4) 1 mL of PBS was added to resuspend and precipitate the precipitate, which was then transferred to a new 1.5 mL centrifuge tube and centrifuged at 4°C and 13,000 rpm for 15 minutes. (5) The supernatant was transferred to a new 1.5 mL centrifuge tube, and 1 / 4 volume of 20% PEG / 2.5 M NaCl solution pre-cooled to 4°C was added, mixed well, and left on ice for at least 10 minutes. (6) Centrifuge at 13,000 rpm at 4°C for 10 minutes, discard the supernatant, and add 1 mL of PBS to resuspend and precipitate the mixture. (7) Centrifuge at 13,000 rpm for 2 minutes at 4°C, and transfer the supernatant to a new 1.5 mL centrifuge tube to obtain the phage sublibrary for the first round of screening. Store at -80°C for long-term storage or at -20°C for short-term storage (1-2 weeks).

[0109] 3.1.5. Titering the Phage Sublibrary from the First Round of Screening The method was the same as for measuring the initial titer of phage in step 3.1.2.

[0110] 3.2. Second round screening 3.2.1. Second-round screening method The screening method was the same as that of the first round of screening in step 3.1.1. The phage sublibrary obtained by amplification and purification in the first round of screening (the phage sublibrary of the first round of screening) was used as the input phage library for the second round of screening, and the phage eluate for the second round of screening was obtained.

[0111] 3.2.2 Titering the second-round phage eluate The measurement method was the same as that for measuring the initial phage titer in step 3.1.2.

[0112] 3.2.3. Amplification and purification of the second-round phage eluate The method was the same as the amplification and purification of the first-round phage eluate in steps 3.1.3 and 3.1.4, to obtain the phage sublibrary for the second-round screening.

[0113] 3.2.4. Titering the Phage Sublibrary from the Second Round of Screening The method was the same as for titering the phage sublibrary from the first round of screening in step 3.1.5.

[0114] 3.3 Third round screening 3.3.1. Third-round screening method The screening method was the same as that of the first round of screening in step 3.1.1. The phage sublibrary obtained by amplification and purification in the second round of screening (the phage sublibrary of the second round of screening) was used as the input phage library for the third round of screening, and the phage eluate for the third round of screening was obtained.

[0115] 3.3.2. Titering of the phage eluate from the third round The measurement method was the same as that for measuring the initial phage titer in step 3.1.2.

[0116] 3.3.3. Amplification of the third round of eluate The method was the same as that for amplifying the first-round phage eluate in step 3.1.3. The third-round amplified phage eluate was obtained.

[0117] 3.4. Results of the third round of screening The results of three rounds of affinity screening are shown in Table 2. Table 2. Results of three rounds of affinity screening TIFF2026508333000006.tif32170Here, *1Enrichment level is the screening titer divided by the input titer. The larger the value, the higher the enrichment level of antibodies in the input library. *2Fold difference is the screening titer divided by the control titer. The larger the value, the higher the content of positive antibodies in the screening library.

[0118] The results showed that after three rounds of screening, the enrichment level was 1.3 × 10 -2 The monoclonality of the clones was confirmed by ELISA.

[0119] 4. Selection of CLL1 VHH-positive single clones 4.1. ELISA detection of CLL1 VHH-positive single clones (1) A suitable dilution of bacterial suspension was taken from the phage eluate amplified in the third round, and the suspension was uniformly applied to a solid medium plate containing 100 μg / mL of Amp and cultured overnight at 37°C. (2) 192 single clone colonies were randomly picked and transferred to a 96-well cell culture plate (P1-P2), and 200 μL of 2xYT medium (containing 100 μg / mL Amp) was added to each well and cultured overnight at 37°C. (3) 5 μL of the overnight cultured bacterial solution was transferred to a new 96-well cell culture plate with 200 μL of 2xYT liquid medium (containing 100 μg / mL Amp) added to each well, and cultured at 37°C for 5 hours. (4) Helper phage M13K07 was added at a bacterial number:phage number = 1:20 according to the following formula. TIFF2026508333000007.tif13170 where V is the volume of helper phage added (unit: mL), T helper-phage is the titer of the helper phage. (5) After incubation at 37°C for 30 minutes, Kan was added to a final concentration of 50 μg / mL, and the mixture was cultured overnight at 30°C. (6) The overnight cultured bacterial solution was centrifuged at 4°C and 4000 rpm for 10 minutes to obtain the supernatant, which was then stored at 4°C. (7) The screening antigen was diluted to 1 μg / ml with PBS, coated onto an ELISA plate at 100 μL / well, and left overnight at 4° C. The control group was coated with 3% BSA. (8) The antigen solution was discarded, 200 μL of PBS buffer was added to each well, and the ELISA plate was washed three times at room temperature for 10 minutes each. (9) 200 μL of blocking solution PBSTB (PBST containing 2% BSA) was added to each well, and the ELISA plate was blocked at room temperature for 1 hour. (10) The blocking solution was discarded, and 200 μL of PBST (1×PBS containing 0.1% Tween 20) buffer was added to each well, and the ELISA plate was washed three times at room temperature for 10 minutes each. (11) 100 μL of blocking solution was added to each well, and then 100 μL of the supernatant obtained by centrifugation in the above step (6) was added, followed by incubation at room temperature for 2 hours. (12) The liquid in the ELISA plate was discarded, and 200 μL of PBST buffer was added to each well, followed by washing three times for 10 minutes each. (13) M13 Bacteriophage HRP Antibody, Mouse Mab (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number 11973-MM05T-H) diluted 1:30,000 in blocking solution was added to each well at 100 μL / well and incubated at room temperature for 1 hour. (14) The liquid in the ELISA plate was discarded, and 200 μL of PBST buffer was added to each well, followed by washing six times for 5 minutes each. (15) Add 100 μL of TMB single component coloring solution to each well, allow to develop in the dark for 1 to 3 minutes, add 100 μL of 1M HCl to each well to stop the coloring, and measure the OD using a microplate reader. 450 The value was read. OD 450 A value of >0.5 is considered positive, and the resulting positive clone is a phage containing a single domain antibody capable of binding to the CLL1 protein.

[0120] 4.2. ELISA Secondary Validation of CLL1 VHH-Positive Single Clones To eliminate false-positive results, clones that initially tested positive were subjected to ELISA secondary validation using the same method as in step 4.1.

[0121] 4.3. Sequencing of CLL1 VHH-positive single clones Five microliters of the 181 positive clones from the secondary ELISA plate were inoculated into 1 mL of 2xYT medium (containing 100 μg / mL of Amp), and the OD was measured at 37°C and 250 rpm. 600 The culture was continued until the antibody titer reached 0.8 to 1.0, and 0.5 mL of the bacterial solution was sampled and sequenced to obtain different antibody sequences.

[0122] 5. Preparation of anti-CLL1 single domain antibodies 5.1. Structure and sequence of anti-CLL1 single domain antibodies The antibody gene sequences obtained by sequencing the positive single clones in step 4.3 were selected and used to produce anti-CLL1 single-domain antibodies (CLL1-VHH) by genetic engineering. One of the antibodies was designated CLL1-VHH-1.

[0123] A single domain antibody has only the variable region of a heavy chain antibody (VHH), and is composed of a framework region FR1, a complementarity-determining region CDR1, a framework region FR2, a complementarity-determining region CDR2, a framework region FR3, a complementarity-determining region CDR3, and a framework region FR4, in this order. The specific sequence information is as follows: The amino acid sequence of the single domain antibody CLL1-VHH-1 is SEQ ID NO. 1. In SEQ ID NO. 1, positions 1 to 25 represent the framework region FR1, positions 26 to 35 represent the complementarity-determining region CDR1, positions 36 to 49 represent the framework region FR2, positions 50 to 59 represent the complementarity-determining region CDR2, positions 60 to 98 represent the framework region FR3, positions 99 to 118 represent the complementarity-determining region CDR3, and positions 119 to 129 represent the framework region FR4. The nucleotide sequence of a nucleic acid molecule encoding the single domain antibody CLL1-VHH-1 (CLL1-VHH-1 gene) is shown in SEQ ID NO. 2.

[0124] In this regard, positions 1 to 75 of SEQ ID NO. 2 are the coding sequence of framework region FR1, positions 76 to 105 are the coding sequence of complementarity-determining region CDR1, positions 106 to 147 are the coding sequence of framework region FR2, positions 148 to 177 are the coding sequence of complementarity-determining region CDR2, positions 178 to 294 are the coding sequence of framework region FR3, positions 295 to 354 are the coding sequence of complementarity-determining region CDR3, and positions 355 to 387 are the coding sequence of framework region FR4.

[0125] Amino acid sequence of single domain antibody CLL1-VHH-1: 5'-DVQLQESGGGLVQPGGSLRLSCAAS GFTFGDYDMT WVRQAPGKGPEWVS AINSGGGSTY YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAT IDRLSTVVAGTYQPSDEYDY WGQGTQVTVSS-3' (SEQ ID No. 1). The three underlined regions are CDR1, CDR2, and CDR3, respectively.

[0126] Nucleotide sequence of the CLL1-VHH-1 gene: 5'-GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTCGGAGACTATGACATGACC TGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGTGGGTCTCA GCTATTAATAGTGGCGGTGGTAGCACATAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAACT ATCGACCGCCTTAGTACGGTAGTAGCTGGTACGTACCAGCCGTCCGATGAGTATGACTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC-3' (SEQ ID No. 2). The three underlined parts are the coding sequences for CDR1, CDR2, and CDR3, respectively.

[0127] The sequences of the complementarity determining regions are defined according to the Kabat numbering system.

[0128] The single domain antibody is a single domain antibody that specifically binds to the CLL1 protein.

[0129] 5.2. Expression and Purification of Anti-CLL1 Single Domain Antibodies The eukaryotic expression vector pcDNA3.1 was used to express the anti-CLL1 single domain antibody CLL1-VHH-1.

[0130] 5.2.1. Construction of recombinant vectors To facilitate purification, a human IgG1 Fc gene (hFc gene, nucleotide sequence of which is SEQ ID No. 3 and encoded amino acid sequence of which is SEQ ID No. 4) was added to the 3' end of the gene encoding the single-domain antibody CLL1-VHH-1 (SEQ ID No. 2), and the resulting fusion gene was designated CLL1-VHH-1-hFc. Next, a BamHI site was added before the initiation codon at the 5' end of the fusion gene, and an EcoRI site was added after the termination codon at the 3' end. This fusion gene was then cloned between the BamHI and EcoRI sites of the eukaryotic expression vector pcDNA3.1 to obtain the recombinant eukaryotic expression vector pcDNA3.1-CLL1-VHH-1-hFc, which expresses the single-domain antibody CLL1-VHH-1 fused to hFc (SEQ ID No. 4) at its C-terminus.

[0131] 5.2.2. Expression of Anti-CLL1 Single Domain Antibodies The eukaryotic expression vector pcDNA3.1-CLL1-VHH-1-hFc obtained in step 5.2.1 was introduced into HEK293 cells by conventional electroporation for expression.

[0132] 5.2.3 Purification of anti-CLL1 single domain antibodies Because Protein A has the activity of binding to the Fc fragment of IgG, anti-CLL1 single domain antibodies expressed in HEK293 cells were purified using a Protein A column. Finally, a purified anti-CLL1 single domain antibody fused with hFc (CLL1-VHH-1-hFc) was obtained.

[0133] Example 2: Performance characterization of anti-CLL1 single domain antibodies 1. Measurement of the affinity constant between antigen and antibody Test antibody: anti-CLL1 single domain antibody fused with hFc (CLL1-VHH-1-hFc) prepared in step 5.2.3.

[0134] Procedure for determining affinity constant: (1) Pierce the test antibody CLL1-VHH-1-hFc. TM The antibody was labeled with the NHS-Fluorescein Antibody Labeling Kit to obtain the FITC-labeled antibody FITC-CLL1-VHH-1-hFc for flow cytometry. (2) CLL1-positive cells K562-CLL1 and U937 cells 2 × 10 6 The cells were resuspended at a density of 100 μL / mL, and 100 μL of the cell suspension was transferred to each 96-well V-bottom plate, centrifuged, the supernatant discarded, and the cells were collected (K562 cells alone were used as a control for CLL1-negative cells). (3) 40 μL of single domain antibody FITC-CLL1-VHH-1-hFc diluted in different gradients (see Table 3 for specific groupings) was mixed with the above cells and incubated at room temperature in the dark for 10 minutes. Table 3. Grouping of affinity tests for CLL1-VHH-1-hFc antibodies TIFF2026508333000008.tif19170(4) The cells were washed twice with 200 μL of FACS buffer by centrifugation, resuspended in 200 μL of FACS buffer, and immediately measured for mean fluorescence intensity (MFI) using flow cytometry. (5) The equilibrium dissociation constant (KD) of the binding between the CLL1 VHH antibody and the target cells was calculated according to the formula to determine affinity. The smaller the KD value, the stronger the affinity. 1 / (MFI-Con)=1 / Fmax+(KD / Fmax)(1 / [CLL1-scfv IgG Fc]). Here, MFI-Con represents the relative mean fluorescence intensity, which is the value obtained by subtracting the mean fluorescence intensity of the background from the mean fluorescence intensity of the experimental group, and CLL1-scfv IgG Fc represents the amount of antibody used (unit: ng). A standard curve was plotted with the reciprocal of the antibody amount used on the horizontal axis and the reciprocal of the relative mean fluorescence intensity on the vertical axis, and a linear regression equation was obtained. The intersection of the standard curve and the vertical axis was calculated using the linear regression equation, where 1 / Fmax is the value, and KD is the value obtained by multiplying the slope of the line by Fmax.

[0135] The calculated equilibrium dissociation constants are shown in Table 4. Table 4. Equilibrium dissociation constant (KD) of CLL1-VHH-1-hFc and K562-CLL1 cells TIFF2026508333000009.tif13170

[0136] The results showed that the single domain antibody (CLL1-VHH-1) obtained by the alpaca immunization and screening of the present invention had an affinity constant of 48.3 nM for K562-CLL1 cells and high affinity for the CLL1 protein. In the present invention, we screened for a high-affinity anti-CLL1 single domain antibody CLL1-VHH-1.

[0137] 2. Detection of the specificity of the single domain antibody CLL1-VHH-1 FITC-labeled CLL1-VHH-1 antibody (FITC-CLL1-VHH-1-hFc) was used to detect CLL1 expression on various tumor cells. The procedure is as follows. (1) CLL1-negative cell lines (control group): K562 cells, U266 cells, and Jurkat cells. CLL1-positive cell lines (experimental group): U937 cells, THP-1 cells, and the CLL1-overexpressing cell line K562-CLL1. They were recovered at 37°C, added to cell culture flasks, cultured at 37°C and 5% CO2, and passaged twice before being used for detection of the anti-CLL1 single-domain antibody (FITC-CLL1-VHH-1-hFc). (2) The concentration of each cell was increased to 2 × 10 6The solution was adjusted to cells / mL, added to a 96-well plate at 200 μL / well, and centrifuged at 1500 rpm for 5 minutes. (3) Wash with FACS buffer and then centrifuged. (4) 20 μL of diluted FITC-CLL1-VHH-1-hFc antibody (1:400) was added and mixed well, followed by incubation at room temperature for 10 minutes. (5) FACS buffer was added, and the mixture was centrifuged to remove the antibody. The mixture was then resuspended in buffer and measured using a flow cytometer.

[0138] Flow cytometry results showed that the FITC-CLL1-VHH-1-hFc antibody could specifically bind to the CLL1 antigen molecule expressed on tumor cells (Figure 3).

[0139] Example 3: Detection of anti-tumor activity via anti-CLL1 single domain antibodies 1. Measurement of NK cell effector function (IFNγ) Effector cells were primary PBMC cells from healthy donors and were calculated as a percentage of NK cells. Target cells were CLL1 + Cell lines U937 and CLL1 - The cell line was MM1.S. The effector function of NK cells mediated by anti-CLL1 single domain antibodies was evaluated by measuring IFNγ secretion, and the measurement procedure was as follows. (1) Effector cells (NK cells: 1 × 10 5 After mixing at a ratio of 1:1, 0, 2, 10, or 50 μg / mL of CLL1-VHH-1 (VHH-hFC) antibody was added, and the cells were co-incubated in a 96-well flat-bottom plate for 4 hours. (2) After co-incubation, the 96-well plate was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and 20 μl of diluted anti-human CD3-PE antibody (1:100, Biolegend) and 20 μl of diluted anti-human CD56 PE-Cy7 antibody (1:200, Biolegend) were added and mixed well, followed by incubation at room temperature for 10 minutes. (3) 200 μl of FACS buffer was added to wash the cells, followed by centrifugation. 50 μl / well of a permeabilization solution, Cytofix (BD Biosciences, catalog number 554722), was added, and the cells were permeabilized at room temperature for 15 minutes. (4) 200 μl of Perm buffer was added, the mixture was washed twice, and centrifuged. 20 μl of diluted anti-human IFNg-APC antibody (1:50, Biolegend) was added, and the mixture was incubated at room temperature for 20 minutes. (5) After washing with Perm buffer, the expression of IFNγ in NK cells was detected by flow cytometry.

[0140] As shown in Figure 4, the antibody CLL1-VHH-1 (VHH-hFc) of the present invention effectively promoted IFNγ secretion from NK cells and activated the effector function of NK cells. The higher the concentration of CLL1-VHH-1 antibody, the higher the concentration of IFNγ secreted from NK cells. This indicates that the antibody CLL1-VHH-1 of the present invention effectively promoted IFNγ secretion from NK cells and activated the effector function of NK cells. + It has been shown to significantly enhance the potency of NK cells against target cells and improve antitumor activity.

[0141] 2. Measurement of ADCC activity by CLL1-VHH-1 antibody Effector cells were primary PBMCs from healthy donors, calculated as a percentage of NK cells. Target cells were CLL1 cells stably expressing luciferase. + The cell line U937 (U937-Luc cell line) and CLL1 stably expressing luciferase - The cell line was MM1.S (MM1.S-Luc cell line). The measurement procedure was as follows. (1) Effector cells (NK cells): Target cells (2 × 10 4 The cells were mixed at a ratio of 10:1 (10:1 / 100uL), and 6.25, 12.5, 25, or 50µg / mL of CLL1-VHH-1 (VHH-hFC) antibody was added, followed by co-incubation for 4 hours in a 96-well flat-bottom plate. (2) Immediately after adding fluorescein sodium salt (10 μg / mL), the fluorescence value was measured using a TECAN spark microplate reader, and the specific killing activity (cytotoxicity) of NK cells was calculated using the average value of three replicate wells.

[0142] The analytical formula is as follows: Specific lysis%=100-100×(E exp -E min ) / (T max -T min ) T min : RLU value (Relative luciferase activity, relative light units) under conditions of maximum killing rate E exp : RLU value when effector cells and target cells are co-cultured T max : RLU value of spontaneous death of target cells in the absence of effector cells E min : RLU value of spontaneous death of effector cells in the absence of cells

[0143] As shown in Figure 5, the antibody CLL1-VHH-1 of the present invention was able to mediate strong ADCC activity, and the higher the concentration of the CLL1-VHH-1 antibody, the stronger the specific killing activity of NK cells, and at a concentration of 50 μg / mL, the specific killing activity of NK cells reached 60%. This indicates that the antibody CLL1-VHH-1 of the present invention has a strong ability to significantly induce NK cell cytotoxicity (ADCC) and specifically kill CLL1-positive cells.

[0144] The present invention has been described in detail above. Those skilled in the art can practice the present invention to a broader extent under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation. Although the present invention has been described with specific examples, it is understood that the present invention can be further improved. In short, based on the principles of the present invention, the present application is intended to cover any changes, uses, or improvements to the present invention that depart from the scope disclosed herein, including improvements made using conventional techniques known in the art. Some basic features may be applied within the scope of the following appended claims. [Industrial Applicability]

[0145] The anti-CLL1 single domain antibody (CLL1-VHH-1) of the present invention has high affinity and excellent specificity, and can specifically target CLL1-positive cells (cells expressing the CLL1 protein). The antibody CLL1-VHH-1 of the present invention more effectively promotes IFNγ secretion from NK cells, activates the effector function of NK cells, and inhibits CLL1. + The single domain antibody of the present invention enhances the efficacy of NK cells against target cells, improves antitumor activity, significantly induces NK cell cytotoxicity (ADCC), and has a strong ability to specifically kill CLL1-positive cells. The single domain antibody of the present invention can be used to produce specific antibody drugs clinically used for the prevention and treatment of CLL1-targeted diseases (acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), as well as to produce CLL1-targeting CAR cells and CLL1 protein detection kits. The single domain antibody drug of the present invention has a stable structure, small molecule, is easy to recombinantly express, and has low production costs. It can be used alone or as a drug delivery system for delivering related drugs, and has very broad prospects and important significance in fields such as pharmaceutical applications and clinical diagnosis.

Claims

1. A single domain antibody, comprising a heavy chain variable region, the heavy chain variable region comprising a complementarity determining region (CDR1) having the amino acid sequence of positions 26 to 35 of SEQ ID No. 1, a complementarity determining region (CDR2) having the amino acid sequence of positions 50 to 59 of SEQ ID No. 1, and a complementarity determining region (CDR3) having the amino acid sequence of positions 99 to 118 of SEQ ID No.

1.

2. 2. The single domain antibody of claim 1, wherein the heavy chain variable region has an amino acid sequence of SEQ ID No. 1 or an amino acid sequence having at least 80% identity and the same function as SEQ ID No.

1.

3. A biomaterial related to the single domain antibody of claim 1 or 2, characterized in that it is any one of the following C1) to C4): C1) A nucleic acid molecule encoding the heavy chain variable region of the single domain antibody of claim 1 or 2; C2) an expression cassette comprising the nucleic acid molecule according to C1); C3) A recombinant vector comprising the nucleic acid molecule according to C1), or a recombinant vector comprising the expression cassette according to C2); C4) A recombinant host cell comprising a nucleic acid molecule according to C1), or a recombinant host cell comprising an expression cassette according to C2), or a recombinant host cell comprising a recombinant vector according to C3).

4. 4. The biomaterial of claim 3, wherein the nucleic acid molecule is one of the following: D1) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2; D2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in D1) and having the same function.

5. 10. Any one of the following uses of a single domain antibody according to claim 1 or 2 or a biomaterial according to claim 3 or 4: E1) Use in the manufacture of a medicament for the prevention or treatment of tumors, or use in the prevention or treatment of tumors; E2) Use in the manufacture of a medicament for the prevention or treatment of a CLL1 target-related disease, or use in the prevention or treatment of a CLL1 target-related disease; E3) Use in the manufacture of a product for screening, diagnosing or aiding in diagnosis of a CLL1 target-related disease, or use in screening, diagnosing or aiding in diagnosis of a CLL1 target-related disease; E4) Use in the detection of CLL1 protein or in the manufacture of a product for the detection of CLL1 protein; E5) Use in the manufacture of a product for binding to CLL1 protein; E6) Use in the drug-mediated specific recognition of tumors expressing the CLL1 antigen or in the manufacture of a product for the drug-mediated specific recognition of tumors expressing the CLL1 antigen; E7) Use in in vivo imaging of CLL1 protein or in the manufacture of a product for in vivo imaging of CLL1 protein; E8) Use in the manufacture of products targeting CLL1.

6. The use according to claim 5, characterized in that the CLL1 target-related disease is a CLL1-positive cancer.

7. The use according to claim 6, characterized in that the CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

8. 3. A pharmaceutical composition comprising a single domain antibody according to claim 1 or 2, or a cell modified with a chimeric antigen receptor containing a single domain antibody according to claim 1 or 2.

9. A reagent or kit comprising a single domain antibody according to claim 1 or 2 and having one of the following uses: G1) Use in detecting CLL1 protein; G2) Use in screening, diagnosing or aiding in diagnosis of CLL1 target-related diseases; G3) Use in in vivo imaging of CLL1 protein.

10. 10. A method for producing a single domain antibody according to claim 1 or 2, comprising: constructing a recombinant expression vector containing the nucleic acid molecule according to claim 3 or 4; introducing the recombinant expression vector into a host cell to obtain a recombinant cell that expresses the single domain antibody; culturing the recombinant cell; and obtaining the single domain antibody by separation and purification.

11. A method for diagnosing or assisting in the diagnosis of a CLL1 target-related disease, the method comprising: separating a sample from a subject, detecting the content of CLL1 protein in the sample using a single domain antibody described in claim 1 or 2, and diagnosing or assisting in the diagnosis of a CLL1 target-related disease based on the content of the CLL1 protein.

12. A method for screening for a CLL1 target-related disease, characterized in that the method comprises separating a sample from a subject, detecting the content of CLL1 protein in the sample using a single domain antibody described in claim 1 or 2, and screening for a CLL1 target-related disease based on the content of the CLL1 protein.

13. A method for preventing or treating a CLL1 target-related disease, characterized in that the method comprises administering a single domain antibody described in claim 1 or 2 or a pharmaceutical composition described in claim 8 to a subject suffering from a CLL1 target-related disease.

14. 14. The method according to any one of claims 11 to 13, characterized in that the CLL1 target-associated disease is a CLL1 target-associated tumor.

15. 15. The method of claim 14, wherein the CLL1 target-associated tumor is a CLL1-positive cancer.

16. 16. The method of claim 15, wherein the CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

17. A method for detecting CLL1 protein in vitro, characterized in that the method comprises detecting CLL1 protein using a single domain antibody described in claim 1 or 2 or a reagent or kit described in claim 9.

18. An antibody-drug conjugate comprising an antibody portion and a binding portion, wherein the antibody portion comprises a single domain antibody according to claim 1 or 2.

19. 19. The antibody-drug conjugate of claim 18, wherein the binding moiety comprises a detectable label, a chemical drug, or a cytotoxin.

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