Development of novel UPAR single domain antibodies

Abstract

The present invention provides anti-UPAR single domain antibody.Specifically, the present invention obtains multiple anti-UPAR single domain antibodies through phage display library screening technology.The anti-UPAR single domain antibody of the present invention can efficiently bind to UPAR, and may be applied to the treatment of related diseases.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine. In particular, the present invention relates to anti-UPAR single domain antibodies. [Background technology]

[0002] In recent years, aging has gradually attracted the attention of society, and aging is directly related to the increased incidence of diseases such as infectious diseases, cardiovascular diseases, neurodegenerative diseases, autoimmune disorders, tumors, etc. Body aging accompanied by impaired immune function is one of the important causes of the occurrence of these diseases.

[0003] In recent years, several small molecule anti-aging agents that can remove senescent cells have been developed, but their therapeutic effects are low and accompanied by severe side effects. More and more researchers are working on the development of "anti-aging agents" that can safely and effectively remove senescent cells. CAR-T therapy is currently a groundbreaking cancer treatment method that can achieve the purpose of identifying tumor cells, inducing immune responses, and efficiently killing tumor cells by introducing CAR (tumor chimeric antigen receptor) with positioning function into T cells using gene editing technology. The emergence of this treatment also provides researchers with new ideas in the research of anti-aging agents. If CAR-T cells can identify senescent cells, anti-tumor therapy can be transformed into anti-aging therapy. Traditional disease intervention methods are treatments after the occurrence of a disease, and with the development of medical technology, it will become a new trend to advance the intervention time to prevent the occurrence of aging and aging-related malignant diseases.

[0004] At present, representative drugs for aging include anti-aging agents such as rapamycin, dasatinib + quercetin, fisetin and UBX0101, but these drugs have potential side effects. First, the ability to target and eliminate senescent cells is still not precise enough, and may damage normal cells; second, if anti-aging drugs are used too early, it will cause stem cell depletion, and if used too late, it will affect the effect; third, after a large number of senescent cells die, the products left behind usually cannot be removed in a timely manner, which may be a potential risk to health. Therefore, the development of new anti-aging drugs has become a problem to be solved, and the development of CAR-T cell drugs may become a trend in treating aging. The first step in developing a method to identify senescent cells is to determine the surface characteristics of senescent cells. According to one report, by analyzing RNA sequencing data established by three independent aging models, the expression of transmembrane proteins found in aged human and mouse cells was analyzed, and it was ultimately found that urokinase-type plasminogen agonist receptor (UPAR) is the "identification card" of senescent cells. However, currently, there is a lack of research in the field on nanobodies related to UPAR that can be used to construct CAR-T cell drugs or other drugs that target UPAR.

[0005] Therefore, there is a need in the art to develop UPAR nanobodies. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a single domain antibody against UPAR and applications thereof. [Means for solving the problem]

[0007] The first aspect of the present invention provides an anti-UPAR single domain antibody, the complementarity determining region CDR of the anti-UPAR single domain antibody VHH chain being: (1) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 53, and CDR3 as set forth in SEQ ID NO: 54; (2) CDR1 as set forth in SEQ ID NO: 22, CDR2 as set forth in SEQ ID NO: 23, and CDR3 as set forth in SEQ ID NO: 24; (3) CDR1 as set forth in SEQ ID NO: 42, CDR2 as set forth in SEQ ID NO: 43, and CDR3 as set forth in SEQ ID NO: 44; (4) CDR1 as set forth in SEQ ID NO: 25, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 27; (5) CDR1 as set forth in SEQ ID NO: 28, CDR2 as set forth in SEQ ID NO: 29, and CDR3 as set forth in SEQ ID NO: 30; (6) CDR1 as set forth in SEQ ID NO: 31, CDR2 as set forth in SEQ ID NO: 32, and CDR3 as set forth in SEQ ID NO: 33; (7) CDR1 as set forth in SEQ ID NO: 34, CDR2 as set forth in SEQ ID NO: 35, and CDR3 as set forth in SEQ ID NO: 36; (8) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 39; (9) CDR1 as set forth in SEQ ID NO: 40, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 41; (10) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21; (11) CDR1 as set forth in SEQ ID NO: 45, CDR2 as set forth in SEQ ID NO: 46, and CDR3 as set forth in SEQ ID NO: 47; (12) CDR1 as set forth in SEQ ID NO: 48, CDR2 as set forth in SEQ ID NO: 49, and CDR3 as set forth in SEQ ID NO: 50; (13) CDR1 as set forth in SEQ ID NO: 51, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 52; and (14) One or more selected from the group consisting of CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18.

[0008] In another preferred embodiment, the VHH chain of said anti-UPAR single domain antibody further comprises a framework region (FR). In another preferred example, the FR has an amino acid sequence derived from any one of SEQ ID NOs: 1 to 15.

[0009] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NOs: 1-15. In another preferred embodiment, the amino acid sequence of the VHH chain of said anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NO: 3, 10 or 15.

[0010] In another preferred embodiment, said anti-UPAR single domain antibody is selected from the group consisting of a monomer, a bivalent (bivalent antibody), a multivalent antibody, or a combination thereof. In another preferred embodiment, said anti-UPAR single domain antibody is bivalent.

[0011] In another preferred embodiment, the anti-UPAR single domain antibody is selected from the group consisting of a humanized antibody, a camelid-derived antibody, and a chimeric antibody. A second aspect of the invention provides an anti-UPAR antibody, said antibody comprising one or more VHH chains of an anti-UPAR single domain antibody according to the first aspect of the invention.

[0012] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NOs: 1-15. In another preferred embodiment, the amino acid sequence of the VHH chain of said anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NO: 3, 10 or 15.

[0013] In another preferred embodiment, the anti-UPAR antibody is selected from the group consisting of a monomer, a bivalent (bivalent antibody), a multivalent antibody, or a combination thereof.

[0014] In another preferred example, the bivalent (or multivalent) refers to the amino acid sequence of the immune complex comprising two (or more) identical or different VHH chain sequences of the anti-UPAR single domain antibody described in the first aspect of the present invention.

[0015] A third aspect of the present invention provides a polynucleotide, said polynucleotide encoding a protein selected from the group consisting of an anti-UPAR single domain antibody according to the first aspect of the invention or an antibody according to the second aspect of the invention.

[0016] In another preferred embodiment, the present invention relates to a nucleic acid molecule encoding the anti-UPAR single domain antibody of the present invention. The nucleic acid of the present invention can be RNA, DNA or cDNA.

[0017] A fourth aspect of the invention provides an expression vector, said expression vector comprising a polynucleotide according to the third aspect of the invention.

[0018] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. In another preferred embodiment, the expression vector is a pcDNA3.4-hIgG1-Fc2 plasmid.

[0019] A fifth aspect of the invention provides a host cell, said host cell comprising an expression vector according to the fourth aspect of the invention or having integrated into its genome a polynucleotide according to the third aspect of the invention. In another preferred embodiment, the host cell is selected from the group consisting of a prokaryotic cell or a eukaryotic cell.

[0020] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, and mammalian cells. In another preferred embodiment, the host cell is a 293F cell.

[0021] A sixth aspect of the present invention is a method for producing a composition comprising the steps of: (a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for the production of a single domain antibody, thereby obtaining a culture comprising said anti-UPAR single domain antibody; (b) isolating and / or recovering the anti-UPAR single domain antibody from the culture; and (c) optionally purifying and / or modifying the anti-UPAR single domain antibody obtained in step (b).

[0022] A seventh aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising: (a) an anti-UPAR single domain antibody according to the first aspect of the invention, or an antibody against UPAR according to the second aspect of the invention, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0023] In another preferred embodiment, the radionuclide is (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0024] In another preferred embodiment, the coupling moiety is a detectable marker.

[0025] In another preferred embodiment, the coupling moiety is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral granule, a liposome, a nanomagnetic particle, a prodrug activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticle.

[0026] In another preferred embodiment, said immune complex comprises a multivalent (eg bivalent) VHH chain of an anti-UPAR single domain antibody according to the first aspect of the invention.

[0027] An eighth aspect of the present invention is a method for producing a composition comprising the steps of: (1) A drug for preventing and / or treating a disease associated with UPAR, (2) The present invention provides a use of an anti-UPAR single domain antibody according to the first aspect of the present invention, an antibody against UPAR according to the second aspect of the present invention, or an immune complex according to the seventh aspect of the present invention, for use in the preparation of a reagent for detecting UPAR.

[0028] In another preferred embodiment, the UPAR-associated disease is selected from the group consisting of aging-associated diseases, cancer, or tumors. In another preferred embodiment, the cancer or tumor is selected from the group consisting of a hematological tumor, a lymphoma, a solid tumor, or a combination thereof.

[0029] In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0030] In another preferred embodiment, the lymphoma is selected from the group consisting of Hodgkin's lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic white cell (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL) and other complex B-cell non-Hodgkin's lymphomas.

[0031] In another preferred embodiment, the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract cancer, thyroid cancer, or a combination thereof.

[0032] In another preferred embodiment, the drug is used to treat an aging-related disease. In another preferred embodiment, the aging-related disease is selected from the group consisting of an infectious disease, a cardiovascular disease, a neurodegenerative disease, an autoimmune disorder, a tumor, or a combination thereof.

[0033] In another preferred embodiment, the drug is administered to a human or non-human mammal. In another preferred embodiment, the indicated reagent is a diagnostic reagent, preferably said diagnostic reagent is a detection sheet or a detection plate.

[0034] In another preferred embodiment, the diagnostic reagent is used to detect UPAR or a fragment thereof in a sample.

[0035] A ninth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising: (i) an anti-UPAR single domain antibody according to the first aspect of the invention, an antibody against UPAR according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention; and (ii) A pharma- ceutically acceptable carrier.

[0036] A tenth aspect of the present invention provides a recombinant protein, the recombinant protein comprising: (i) an anti-UPAR single domain antibody according to the first aspect of the invention, or an antibody against UPAR according to the second aspect of the invention, and (ii) With an optional tag sequence to aid in expression and / or purification.

[0037] In another preferred embodiment, the tag sequence is selected from the group consisting of an Fc tag, an HA tag, and a His tag. In another preferred embodiment, the recombinant protein specifically binds to UPAR.

[0038] An eleventh aspect of the present invention provides a kit, said kit comprising an anti-UPAR single domain antibody according to the first aspect of the invention, an antibody against UPAR according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention.

[0039] A twelfth aspect of the present invention provides a method for preventing and / or treating a UPAR-associated disease, said method comprising administering to a subject in need thereof an anti-UPAR single domain antibody as described in the first aspect of the invention, an antibody against UPAR as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention.

[0040] In another preferred embodiment, the subject is selected from a mammal, such as a human. In another preferred embodiment, the UPAR-associated disease is selected from cancer or an autoimmune disease.

[0041] A thirteenth aspect of the present invention provides a method for detecting UPAR or a fragment thereof in a sample in vitro, said method comprising: (1) contacting in vitro said sample with an anti-UPAR single domain antibody according to the first aspect of the invention, an antibody against UPAR according to the second aspect of the invention, or an immune complex according to the seventh aspect of the invention; (2) detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of UPAR or a fragment thereof in the sample.

[0042] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0043] A fourteenth aspect of the present invention is a method for producing a composition comprising the steps of: (i) obtaining a sample from a subject to be diagnosed and contacting said sample with an anti-UPAR single domain antibody according to the first aspect of the invention, an antibody against UPAR according to the second aspect of the invention, or an immune complex according to the seventh aspect of the invention; and (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is suffering from a UPAR-related disease.

[0044] A fifteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, said recombinant polypeptide being an anti-UPAR single domain antibody according to the first aspect of the invention, an antibody against UPAR according to the second aspect of the invention, said method comprising the steps of: (a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression; and (b) isolating the recombinant polypeptide from the culture. Effect of the Invention

[0045] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief description of the drawings]

[0046] The following drawings are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention defined by the claims. [Figure 1] 1 shows the SDS-PAGE detection results of UPAR-His recombinant protein expressed, lane 1 represents the protein band of UPAR-His, lane M represents the protein marker. [Diagram 2] 1 shows the results of activity detection of UPAR-His recombinant protein. [Diagram 3] The results of ELISA detection of immune serum are shown.

[0047] [Figure 4] 1 shows the results of PCR amplification of VHH fragments. The left panel shows the results of the first PCR, and the right panel shows the results of the second PCR. [Diagram 5] A comparison of the sequencing results of various single clones is shown. [Figure 6]The results of phage Elisa detection of solid-phase panning screening antibodies are shown.

[0048] [Figure 7A] 1 shows the flow cytometry detection results of various cloned antibodies obtained from phage display library panning. [Figure 7B] 1 shows the flow cytometry detection results of various cloned antibodies obtained from phage display library panning. [Figure 7C] 1 shows the flow cytometry detection results of various cloned antibodies obtained from phage display library panning. [Figure 7D] 1 shows the flow cytometry detection results of various cloned antibodies obtained from phage display library panning.

[0049] [Figure 8A] Flow cytometry detection results after expression and purification of UPAR antibodies of different clones are shown. [Figure 8B] Flow cytometry detection results after expression and purification of UPAR antibodies of different clones are shown. [Figure 8C] Flow cytometry detection results after expression and purification of UPAR antibodies of different clones are shown. [Figure 9] The affinity detection results of three representative UPAR antibodies (1-F09, 3-E04, 3-H02) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] After extensive and thorough research and extensive screening, the present inventors have succeeded in obtaining multiple anti-UPAR single domain antibodies. Specifically, the present invention uses UPAR-Fc antigen protein to immunize camels, and uses phage display technology to screen and pan and identify an immune single domain antibody gene library (phage display library), thereby obtaining a single domain antibody gene against UPAR. Experimental results show that the anti-UPAR single domain antibody obtained in the present invention can effectively bind to UPAR. It is expected to be applied to UPAR-related diseases, especially tumor and aging-related diseases. Based on this, the present invention is completed.

[0051] term As used herein, the terms "antibody of the invention," "antibody of the invention," "anti-UPAR single domain antibody of the invention," and "anti-UPAR single domain antibody" have the same meaning and are used interchangeably, and all refer to an antibody that specifically recognizes and binds to UPAR.

[0052] antibody As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H) with the same structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, where the constant region of the light chain faces the first constant region of the heavy chain, and the variable region of the light chain faces the variable region of the heavy chain. Certain amino acid residues form an interface between the variable regions of the light and heavy chains.

[0053] As used herein, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody" (sdAb, or nanobody) have the same meaning and are used interchangeably, and refer to cloning the variable region of an antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that naturally lacks the constant region 1 (CH1) of the light chain and heavy chain, the variable region of the antibody heavy chain is then cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0054] As used herein, the term "variable" refers to the difference in sequence of certain parts of the variable region in an antibody, which forms the binding and specificity of various specific antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called the complementarity determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved parts of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folded configuration connected by the three CDRs that form a linking ring, and can sometimes form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are involved directly in binding the antibody to an antigen, but they also exhibit various effector functions, such as those involved in antibody-dependent cellular toxicity of the antibody.

[0055] As known to those skilled in the art, immunoconjugates and fusion expression products include complexes formed by binding drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention further includes cell surface markers or antigens that bind to said Nanobodies against UPAR or fragments thereof.

[0056] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0057] In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2 and CDR3. In a preferred embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region as described above.

[0058] In the present invention, the terms "antibody of the invention," "protein of the invention," or "polypeptide of the invention" are used interchangeably and all refer to a polypeptide that specifically binds to UPAR, such as a protein or polypeptide having a heavy chain variable region, which may or may not include a starting methionine.

[0059] The present invention further provides other proteins or fusion expression products comprising the antibodies of the present invention. In particular, the present invention includes any protein or protein complex and fusion expression product (i.e., immunoconjugates and fusion expression products) of a heavy chain comprising a variable region, so long as said variable region is identical or at least 90% identical, preferably at least 95% identical, to the variable region of the heavy chain of an antibody of the present invention.

[0060] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an antigen at a concentration of about 10 -7 Less than M, e.g., about 10 -8 Less than M, 10 -9 Less than M or l0 -10 It binds with an affinity (KD) of less than or equal to M.

[0061] In general, the antigen-binding properties of an antibody can be explained by three specific regions located in the heavy chain variable region, called the variable region (CDR), and the section is divided into four framework regions (FR), and the amino acid sequences of the four FRs are relatively conserved and are not directly involved in the binding reaction. These CDRs form a cyclic structure, and the β-fold formed by the FRs between them is spatially close, and the CDRs of the heavy chain and the CDRs of the corresponding light chain constitute the antigen-binding site of the antibody. The amino acids that constitute the FR or CDR region can be determined by comparing the amino acid sequences of the same kind of antibody.

[0062] The heavy chain variable regions of the antibodies of the invention are of particular interest since at least a portion of them is involved in binding to the antigen, and therefore the invention includes molecules having an antibody heavy chain variable region with a CDR, so long as the CDR has 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein.

[0063] The present invention includes not only complete antibodies, but also immunologically active fragments of antibodies or fusion proteins formed by antibodies and other sequences. Thus, the present invention further includes fragments, derivatives and analogs of said antibodies.

[0064] As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains essentially the same biological function or activity as the antibody of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conserved amino acid residues (preferably conservative amino acid residues) have been substituted (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) a polypeptide having a substitution at one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein formed with a leader sequence or a secretory sequence or a sequence for purifying the polypeptide or a protein sequence or a 6His tag). Based on the teachings of the present specification, these fragments, derivatives, and analogs are within the purifying and known scope of those skilled in the art.

[0065] The antibody of the present invention refers to a polypeptide having UPAR binding activity and comprising the above-mentioned CDR region. The term further includes mutant forms of the polypeptide having the same function as the antibody of the present invention and comprising the above-mentioned CDR region. These mutant forms include (but are not limited to) deletion, insertion and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution by amino acids with similar or similar properties usually does not change the function of the protein. As another example, addition of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0066] Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, proteins encoded by DNA capable of hybridizing to DNA encoding an antibody of the present invention under conditions of high or low stringency, as well as polypeptides or proteins obtained using antisera against an antibody of the present invention.

[0067] The present invention further provides other polypeptides, such as fusion proteins, comprising a human antibody or a fragment thereof. In addition to most full-length polypeptides, the present invention further includes fragments of the antibodies of the present invention. Typically, the fragments will have at least about 50 contiguous amino acids of an antibody of the present invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0068] In the present invention, "conservative variants of the antibodies of the invention" refer to polypeptides formed by substitution of up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having the same or similar properties compared to the amino acid sequence of the antibodies of the invention. These conservative variant polypeptides are best generated by making amino acid substitutions according to Table A. [Table A-1] [Table A-2]

[0069] The present invention further provides a polynucleotide molecule encoding said antibody or fragment thereof or fusion protein thereof. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be coding strand or non-coding strand.

[0070] A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a coding sequence for the mature polypeptide and various additional coding sequences, a coding sequence for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

[0071] The term "polynucleotide encoding a polypeptide" includes a polynucleotide that encodes the polypeptide, or further includes a polynucleotide that contains additional coding and / or non-coding sequences.

[0072] The present invention further relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the above polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60°C, or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, or (3) hybridization that occurs only when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0073] The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombinant methods or artificial synthesis.Especially when the length of the fragment is short, a feasible method is to use artificial synthesis to synthesize the relevant sequence.Generally, multiple small fragments are first synthesized, and then connected to obtain a very long fragment of sequence.In addition, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused to form a fusion protein.

[0074] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Typically, this is achieved by cloning it into a vector, then transforming it into a cell, and then isolating the relevant sequence from the host cell that has been propagated by conventional methods. The biological molecules involved in the present invention (nucleic acids, proteins, etc.) include biological molecules that exist in isolated form.

[0075] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Additionally, mutations can be introduced into the protein sequences of the present invention by chemical synthesis.

[0076] The present invention further relates to vectors containing said appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform an appropriate host cell so as to express the protein.

[0077] The host cell may be a prokaryotic cell, such as, for example, a bacterial cell, or a lower eukaryotic cell, such as, for example, a yeast cell, or a higher eukaryotic cell, such as, for example, a mammalian cell. Representative examples include bacterial cells of E. coli, Streptomyces, Chinese hamster bacteria, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells such as CHO, COS7 and 293 cells, and the like.

[0078] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be obtained by treating with CaCl2 method after exponential growth phase, the steps used being well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation method, if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection and electroporation, liposome packaging, etc. can be selected.

[0079] The obtained transformant can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for the culture can be selected from a variety of conventional media. The culture is performed under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cells are further cultured for a certain period of time.

[0080] The recombinant polypeptide in the method can be expressed intracellularly or in the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods using physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid phase chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0081] The antibodies of the present invention can be used alone, can be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modulating moiety, or any combination of these substances.

[0082] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0083] Therapeutic agents that can be conjugated or coupled to the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biological toxins, 3. cytokines such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. nanomagnetic particles, 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

[0084] UPAR As used herein, the term "UPAR" refers to urokinase-type plasminogen activator receptor. UPAR is a multidomain glycoprotein cell surface receptor docked to the cell membrane via GPI, which binds with high affinity to urokinase-type plasminogen activator (uPA) to promote plasminogen cell activation, facilitate the degradation of extracellular matrix during fibrinolysis, wound healing, or tumorigenesis, and promote tumor cell migration, invasion, and survival. A portion of UPAR is proteolytically cleaved after ligand binding to generate soluble UPAR (sUPAR).

[0085] Examination of previously published data on protein and RNA expression in human tissues indicates that UPAR is either undetectable or present at low levels in most organs in the body, including the central nervous system, heart, and liver. However, studies have found that UPAR is highly expressed in senescent cells both in vivo and in vitro. Various models in vivo and in vitro confirm that mid-senescent cells can induce UPAR expression. In each model system, an increase in the number of UPAR-positive cells and an increase in sUPAR levels in serum are observed. These results indicate that UPAR is a very good target point for CAR-T cells targeting senescent cells. In addition, UPAR is also expressed in many solid tumor cell lines, indicating that UPAR is also an ideal target point for targeting solid tumors.

[0086] Pharmaceutical Compositions The present invention further provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or its active fragment or its fusion protein, and a pharma- ceutically acceptable carrier. Usually, these substances can be formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous vector medium, where the pH is usually about 5-8, preferably about 6-8, and the pH value varies depending on the nature of the substance to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by a conventional route, where it includes but is not limited to intraperitoneal, intravenous, or topical administration.

[0087] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody of the present invention (or a complex thereof) and a pharma- ceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight daily. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0088] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 μg / kg body weight and in most cases will not exceed about 50 mg / kg body weight, and preferably the dosage is from about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the health condition of the patient, all of which are within the skill of a skilled physician.

[0089] Anti-UPAR single domain antibody The present invention provides anti-UPAR single domain antibodies capable of specifically binding to UPAR.

[0090] One aspect of the present invention provides an anti-UPAR single domain antibody, wherein the complementarity determining region CDR of the anti-UPAR single domain antibody VHH chain is (1) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 53, and CDR3 as set forth in SEQ ID NO: 54; (2) CDR1 as set forth in SEQ ID NO: 22, CDR2 as set forth in SEQ ID NO: 23, and CDR3 as set forth in SEQ ID NO: 24; (3) CDR1 as set forth in SEQ ID NO: 42, CDR2 as set forth in SEQ ID NO: 43, and CDR3 as set forth in SEQ ID NO: 44; (4) CDR1 as set forth in SEQ ID NO: 25, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 27; (5) CDR1 as set forth in SEQ ID NO: 28, CDR2 as set forth in SEQ ID NO: 29, and CDR3 as set forth in SEQ ID NO: 30; (6) CDR1 as set forth in SEQ ID NO: 31, CDR2 as set forth in SEQ ID NO: 32, and CDR3 as set forth in SEQ ID NO: 33; (7) CDR1 as set forth in SEQ ID NO: 34, CDR2 as set forth in SEQ ID NO: 35, and CDR3 as set forth in SEQ ID NO: 36; (8) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 39; (9) CDR1 as set forth in SEQ ID NO: 40, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 41; (10) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21; (11) CDR1 as set forth in SEQ ID NO: 45, CDR2 as set forth in SEQ ID NO: 46, and CDR3 as set forth in SEQ ID NO: 47; (12) CDR1 as set forth in SEQ ID NO: 48, CDR2 as set forth in SEQ ID NO: 49, and CDR3 as set forth in SEQ ID NO: 50; (13) CDR1 as set forth in SEQ ID NO: 51, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 52; and (14) It is characterized in that it is one or more types selected from the group consisting of CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18.

[0091] In a preferred embodiment, the amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NOs: 1 to 15. More preferably, the VHH chain of the anti-UPAR single domain antibody has an amino acid sequence selected from SEQ ID NOs: 3, 10 or 15.

[0092] The nanobodies of the invention have high affinity for UPAR. -6 Less than M, preferably 10 -7 Less than M, more preferably 10 -8 It refers to binding to UPAR with an affinity (KD) of less than or equal to M.

[0093] The nanobody of the present invention has the advantages of nanobody, such as small molecular weight, fast tissue penetration, high solubility and stability, high antigen binding specificity, weak immunogenicity, etc. In addition, because nanobody drugs have small molecular weight, nanobody can also identify some hidden antigen epitopes that monoclonal antibody drugs cannot identify, and furthermore, in vivo, nanobody may be less immunogenic than mouse antibody. Therefore, using nanobody sequence to construct antibody drug or CAR-T drug has a great advantage over using mouse single chain antibody sequence to construct antibody drug or CAR-T drug.

[0094] The UPAR nanobodies of the present invention can be used to construct UPAR nanobody drugs or CAR-T drugs based on UPAR nanobody sequences that target the treatment of aging-related diseases or tumors.

[0095] Another aspect of the present invention further provides an anti-UPAR antibody, which may be monovalent or polyvalent and may comprise one or more identical or different VHH chains of the anti-UPAR single domain antibody of the present invention, preferably the VHH chains having the amino acid sequence shown in SEQ ID NO:3, 10 or 15.

[0096] Detection Method The present invention further relates to a method for detecting UPAR, the steps of which are broadly as follows: obtaining a cell and / or tissue sample, lysing the sample in a medium, and detecting the level of UPAR in the solubilized sample.

[0097] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample preserved in a cell preservation solution.

[0098] kit The present invention further provides a kit comprising the antibody (or fragment thereof) or detection plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, an instruction manual, a buffer, and the like.

[0099] The present invention further provides a detection kit for detecting UPAR levels, which includes an antibody that identifies UPAR protein, a dissolution medium for dissolving the sample, and generally reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

[0100] application As described above, the antibody of the present invention has broad biological and clinical application value, and its application involves multiple fields such as diagnosis and treatment of UPAR-related diseases, basic medical research, biological research, etc. The preferred application is clinical diagnosis, prevention and treatment of UPAR-related diseases.

[0101] The main advantages of the present invention are: 1) The antibody of the present invention can specifically bind to UPAR. 2) The antibody of the present invention has high affinity for UPAR. 3) The UPAR nanobody of the present invention has the advantages of small molecular weight, fast tissue penetration, high solubility and stability, high antigen-binding specificity, weak immunogenicity, etc., and can be used in the development of anti-aging or cancer therapeutic drugs.

[0102] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, the experimental methods that do not show specific conditions are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight percentages and parts by weight.

[0103] Laboratory reagents, consumables and equipment: Main Reagents: Agar (Sigma, CAT#A1296), Peptone (Sigma, CAT#93926), Yeast Extract (OXOID, CAT#:LP0021), Sodium Chloride (Aladdin, CAT#:C111533), Potassium Chloride (Aladdin, CAT#:P112133), Magnesium Sulfate (Sinopharm, CAT#:10013018), Magnesium Chloride (Sinopharm, CAT#:10012818), Glucose (sangon, CAT#:GT1991), SfiI (NEB, CAT#:R0123L), T4 DNA Ligase (TaKaRa, CAT#:2011A), PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B), NuHi Power Mix (power mix) (nuhighbio, CAT#:NH9303), 3M sodium acetate (pH 5.2-6) (Sigma, CAT#:126-96-5), DNA fragment recovery kit (TakaRa, CAT#:9761), gel recovery kit (Qiagen, CAT#:28706), tiangen plasmid extraction kit (tiangen, CAT#:DP117), HRP-M13 (Sino Biolo, CAT#:11973-MM05), PE anti-human IgG (eBioscience, CAT#:12-4998-82), Rabbit anti-llama IgG (H+L) secondary antibody [HRP] (Novus, CAT#NBP1-75095), SS320 competent (iCarTab), pComF phage display vector (iCarTab), NHS-biotin (APExBIO, CAT#:A8002), HRP-Streptavidin (Boster, CAT#:BA1088), Streptavidin magnetic beads (NEB, CAT#:S14205), Antibody affinity detection buffer:HBS-EP+10X (GE, CAT#BR100669), Amino coupling kit (GE, CAT#BR100050), 10mM Glycine 2.5 (GE, CAT#BR100356), S-series CM5 chip (GE, Cat#29149603), PBS (Gbico, CAT#14190-250), DMEM (Gbico, CAT#41965-062), RPMI1640 (Gbico, CAT#61870044), FBS (Gbico, CAT#10099-141), Genomic DNA Purification Kit (Lifetech, CAT#K0512), Polybrene (Sigma, CAT#107689-10G), LVtransm transfection reagent (iCarTab, CAT#LVTran100), Lymphocyte isolation solution (Stem Cell, CAT#18051), X-Vivo15 serum-free medium (Lonza, CAT#04-418Q), DynabeadsR Human T-Expander CD3 / CD28 (Thermo, CAT#11141D), IL-2 (Beijing Yuance Pharmacy, National Drug Approval Number S10980067), IL-7 (PrimeGene, CAT#101-07), IL-15 (PrimeGene, CAT#101-15), Bright-GloTM Luciferase Assay System (Promega, CAT#E2610), IL-2 ELISA Kit (R&D, CAT#D2050), IFN-γ ELISA Kit (R&D, CAT#DIF50).

[0104] Key consumables: 50mL Falcon centrifuge tubes (Corning, CAT#352070), electric shock cups (Bio-Rad 0.2cm), RNase-free 1.5ml EP tubes (QSP, CAT#:509-GRD-Q), 200μL RNase-free PCR tubes (Axygen, PCR-02D-C), T125 shake flasks (Corning, CAT#431143), 15mL Falcon centrifuge tubes (Corning, CAT#430052), 6-well plates (Corning, CAT#3516), 96-well plates (Corning, CAT#3365).

[0105] Main equipment: electroporation device (Eppendorf Multiporator), centrifuge (cence H1650R), constant temperature incubator (Shanghai Jinghong DNP-9052), constant temperature shaking incubator (Longvolt DZ-85A), ultra-clean workbench (Suzhou Aritech SW-CJ-1FD), PCR device (Applied Biosystems ABI2720), biological safety cabinet (Haier, HR40-IIA2), flow cytometer (Thermo Attune Nxt flow cytometer), Thermo 3111 CO2 incubator, BiaCore T200.

[0106] Experimental Method 1. Antigen Preparation 1) The UPAR extracellular segment (23AA-303AA) sequence is gene-synthesized, a His tag is added to the C-terminus, and the sequence is subcloned into a eukaryotic expression vector to construct an antigen expression vector. 2) The constructed UPAR-His protein expression vector is extracted from the plasmid and transiently transfected into 293 cells, which are then continuously cultured for 8 days. The culture supernatant is collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate is transferred to a sterile centrifuge tube and purified using a Ni-NTA column. 3) uPA is used to detect biological activity against the purified antigen.

[0107] 2. Immunization of llamas with antigens The antigen prepared above was used to immunize one alpaca, subcutaneously at multiple sites for three times, and the process was as shown in the table below. [Table 1]

[0108] 3. Detection of Immune Titers 1) 5 ml of peripheral blood was collected and the centrifuge tube in which the blood sample was collected was placed in a 37°C incubator for 1 hour, and then the blood sample was transferred to 4°C and placed overnight. 2) Transfer the serum to a new sterile centrifuge tube, centrifuge at 5000 rpm for 20 minutes, and detect the immune titer using ELISA.

[0109] 4. PBMC Isolation 100 ml of peripheral blood is collected and PBMCs are selected using lymphocyte separation fluid. RNA is extracted and reverse transcribed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit to prepare cDNA. The method is as follows.

[0110] Prepare reaction mixture MIX1 according to Table 2 for 200 µL PCR. [Table 2]

[0111] After incubating at 65°C for 5 minutes, the mixture was quickly cooled on ice. Prepare the reaction mixture in Table 3 in the PCR tube described above. [Table 3]

[0112] After pipetting and mixing evenly, dispense 80 μL / tube and place in the PCR machine at 42 °C for 1 h, heat inactivate at 70 °C for 15 min, and finally place the cDNA samples on ice or store at -20 °C for long term.

[0113] 5. Amplification of VHH fragments Arrange the first PCR reaction system (50 μL / tube) as shown in Table 4. [Table 4]

[0114] After setting up the PCR reaction system, set up the PCR machine according to the program in Table 5 below. [Table 5]

[0115] The PCR product is electrophoretically analyzed using 1% agarose to separate a fragment with a molecular weight of approximately 750 bp. The PCR product is recovered using a gel recovery kit, and the concentration is measured using a NanoDrop. Arrange two-round PCR reactions (50 µL / tube) according to Table 6.

[0116] [Table 6]

[0117] After setting up the PCR reaction system, set up the PCR machine according to the program in Table 7. [Table 7]

[0118] The PCR products are electrophoretically analyzed using 1% agarose to separate the VHH fragments with a molecular weight size of approximately 400 bp. The VHH PCR products are recovered using a gel recovery kit and the concentration is measured by NanoDrop.

[0119] 6. Phage Display Library Construction Construction of phage display vector: 1) Digest the pCom F vector and the VHH PCR gel recovered products obtained above using SfiI, respectively, and digest overnight at 50°C. 2) Separate the pCom F vector fragment using a 1% agarose gel, excise the 5000bp vector fragment and recover the gel, and at the same time purify the PCR digestion product using a DNA fragment recovery kit and measure the concentration by NanoDrop. 3) Ligate the digested pCom F vector and the VHH fragment with T4 ligase and incubate at 16° C. overnight.

[0120] Electrotransformation of E. coli with phage ligation products: 1) Prepare the electroporation cup, ligation product, and electroporation competent tubes and place them on ice to pre-cool. 2) Take the pre-chilled library construction ligation products and add them to the electroporation competent, place on ice for 1 minute, add 70 μL of DNA / competent mix to each electroporation cup, place the electroporation cup on ice, 3) Perform electroporation at 2500V for 5ms. 4) After the electric shock, immediately add SOC medium equilibrated to room temperature to resuspend the bacteria, and culture in a shaker at 37°C for 1 hour. 5) Perform phage rescue directly with 15 mL of the bacterial solution, and add an equal volume of 50% glycerol to the remaining 5 mL of electroporation product, mix uniformly, and then store at -80 °C. 6) Take another 20 μL of the bacterial solution and dilute it by adding 980 μL of 2YT medium, then take 100 μL of the diluted product and add 900 μL of 2YT medium to perform a second dilution, take 50 μL and evenly spread it on an ampicillin-containing LB plate and culture it at 37°C overnight. 7) The next day, remove the plates and calculate the number of clones produced in each ligation and calculate the library volume. 8) At the same time, pick out 20 single clones from the plate and place them in ampicillin-containing 2YT medium, shake and culture at 37 °C for about 6 to 8 hours, sequence the bacterial solution (sequencing universal primer M13R), and calculate the diversity of the library.

[0121] 7. Panning of Phage Display Libraries The procedures for revival and rescue of the phage display library and phage precipitation are as follows.

[0122] 1) Dilute the transformed product after electroporation with 2YT to an OD600 of approximately 0.2, add ampicillin to a final concentration of 100 ug / mL, place in a thermostatic shaker, and culture at 37°C and 225 rpm. Stop when the OD600 reaches 0.5. 2) Add M13KO7, shake evenly, and then leave to stand at 37°C for 30 minutes, and then culture at 37°C and 225 rpm for 1 hour. 3) M13KO7 volume = 10 x volume x OD600 x 5 x 108 / M13KO7 titer 4) The bacterial solution was centrifuged at 6000 rpm for 10 minutes, then resuspended in 2YT-AK medium and cultured overnight at 25°C and 200 rpm. 5) Centrifuge the bacterial solution at 10,000 rpm for 15 minutes; 6) Discard the precipitate and transfer the supernatant to a new centrifuge tube. Add PEG / NaCl in an amount equal to 1 / 5 the volume of the bacterial solution to the tube. Mix uniformly and then leave to stand at 4°C for 2 hours. 7) The precipitated phage supernatant was centrifuged at 10,000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the precipitate (phage) in each 50 ml centrifuge tube was resuspended in 1 ml of sterile PBS. 8) Transfer the resuspended phages to a 1.5 mL EP tube, place in a centrifuge, and centrifuge at 4°C and 12,000 g for 5 minutes. 9) The supernatant was transferred to a new 1.5 ml EP tube, 250 μl of PEG / NaCl was added to each tube, and the tubes were mixed uniformly and then allowed to stand at 4°C for 10 minutes. 10) Centrifuge at 12,000 g for 10 minutes, discard the supernatant, add 1 ml of PBS and resuspend. 11) Centrifuge at 12,000 g for 5 minutes, discard the precipitate, and transfer the supernatant to a new 1.5 ml EP tube. 12) Centrifuge at 12,000 g for 5 minutes and transfer the supernatant to a new 1.5 ml EP tube to obtain the original phage library. 13) Take 10 μl of the precipitate and add it to 90 μl of 2YT medium. -1 Record it as 10 in order. -9 Dilute 10 times to 10 -7 , 10 -8 , 10 -9Take 20μl of diluted samples from the three gradients, add 200μl of pre-prepared ER2738 with an OD600 of 0.5, mix evenly, place in a 37℃ water bath, and leave for 10 minutes. Spread one LB-AMP solid plate per 108μl, keep at 37℃ overnight, and count the spots the next day to determine the titer. 14) Titer calculation: Select a plate with a spot count between 30 and 300, take the average of the two plates, multiply the spot count by the dilution factor, and then multiply it by 100 to get the titer.

[0123] 8. Panning of phage display libraries The procedure for solid-phase panning of a phage display library is as follows.

[0124] ELISA plates are used to coat the target protein, and after several elution steps, the recombinant phages bound to the immobilized antigen are eluted using TEA and amplified. After 3-4 rounds of panning, single clones are selected and sequenced.

[0125] 1) Dilute the antigen in PBS to 50 μg / ml, 150 μl / well, coat a total of 3 wells, and coat overnight at 4°C. 2) Aspirate the target protein and seal with 3% MPBS for 1 hour at room temperature. 3) Dilute the lib phage or the amplification precipitate from the previous round in 450 μl of 3% MPBS and dilute to 6 × 10 11 Pfu phages (2 x 10 per well 11 Pfu) was added to wells with control proteins, 150 μl of diluted phage was added to each well and incubated at room temperature for 1 hour. 4) Aspirate off the MPBS containing the target protein, transfer the phages in the control wells to the wells coated with the target protein, and incubate at room temperature for 1 to 1.5 hours. 5) Aspirate off the phage, wash with 0.05% PBST 8-10 times for 2-3 minutes each time, wash with PBS 4-5 times for 2-3 minutes each time, and at the same time wash the pre-sealed 1.5 ml EP tube with PBS. 6) Elute with 1x TEA for 6-8 minutes at 200 μl per well, collect the eluted products in pre-sealed EP tubes, and neutralize by adding 100 μl of Tris-HCl to each well. 7) Take 10 μl of the output product and add it to 90 μl of 2YT medium, record it as 100, and repeat in order. -2 Dilute 10 times to 10 1 , 10 0 , 10 -1 , 10 -2 Take 20 μl of diluted samples from each of the four gradients and add 200 μl of pre-prepared ER2738 with an OD600 of 0.5 (10 1 add 20 μl of undiluted product directly to ER2738), mix evenly, place in a 37°C water bath, let stand for 10 min, spread one LB-AMP solid plate per 108 μl, keep at 37°C overnight, and count the spots the next day to determine the titer. 8) Titer calculation: Select a plate with a spot count between 30 and 300, take the average of the two plates, multiply the spot count by the dilution factor, and then multiply it by the elution volume.

[0126] The procedure for cell-based panning of the phage display library is as follows.

[0127] Using a recombinant cell line that overexpresses the target protein, the phage library is incubated sequentially with empty cells and cells that overexpress the target protein, and then washed multiple times to remove non-specifically bound phages. The recombinant phages bound to the cell surface are eluted and amplified using glycine or TEA, and after 3 to 4 rounds of panning, single clones are selected and detected by ELISA.

[0128] 1) One day before, fill a 1.5 ml EP tube with 1% PBSA, seal it, and keep it at 4°C overnight. 2) Target cells and control cells were each cultured at 1 × 10 7 Harvested, washed 3 times with PBS, resuspended in 10 mL of 1% PBSA, sealed and destained on a shaker at low speed for 1 hour at room temperature. 3) 1 × 10 control cells 11 of phage was added and incubated for 1 hour, allowing the target cells to continue to seal. 4) Place the cells after incubation in a centrifuge and centrifuge at 1000g for 5 minutes, discard the target cell supernatant (1% PBSA), carefully aspirate the control cell supernatant, add it to the target cell tube, resuspend it, then place it on a shaker and incubate at low speed for 1 hour; 5) Place the target cells in a centrifuge and centrifuge at 1000g for 5 minutes (at the same time, wash the 1.5ml EP tube sealed yesterday with PBS three times), discard the supernatant of the target cells, add 4ml of PBS to resuspend, dispense into a sealed 1.5ml EP tube, centrifuge with PBS to wash five times, transfer the cells to another four EP tubes, wash five times in succession, then transfer the cells to the same EP tube; 6) Resuspend the cells in 200 μl of PBS, then add 200 μl of 2×TEA and quickly pipette until the solution becomes viscous, and neutralize by adding 200 μl of Tris-HCl to obtain the product. 7) As with the solid-phase panning scheme, the output library is titered.

[0129] 9. Phage ELISA 1) Dispense 500μl of 2YT-Amp medium into a 96-well deep well plate, select a single clone on the output plate, and culture at 37℃ and 225rpm until OD600 reaches 0.5. Do not select a clone in the last two wells H11 and H12, but put only medium as a blank control. 2) At the same time, coat the antigen onto an ELISA plate using CBS at a concentration of 1 μg / ml, 100 μl / well, and coat for 2 hours at 37°C; 3) Take another 96-well deep well plate, dispense 500 μl of 2YT-A medium per well, and use a volley gun to sequentially aspirate 10 μl of bacterial solution with an OD600 of 0.5 into the newly dispensed 96-well plate, place at 37 ° C, and culture overnight at 225 rpm, which is the sample feeding sequencing bacterial solution. 4) Add M13KO7 to a bacterial solution with an OD600 of 0.5, mix evenly, place at 37°C, and leave to stand for 15 minutes. 5) M13KO7 volume = 10 x volume x OD600 x 5 x 10 8 / M13KO7 titer 6) After infection, place the bacterial solution on a shaker and incubate at 37°C and 225 rpm for 45 minutes. 7) Place the bacterial solution in a centrifuge and centrifuge at 4000 rpm for 10 minutes, discard the supernatant, resuspend in 2YT-AK medium at 800 μl / well, place in a shaker and culture overnight at 30°C and 210 rpm. 8) At the same time, discard the antigen from the ELISA plate, wash it three times with PBST washing solution, seal it with 250 μl / well of 3% MPBS, keep it at 4 ° C overnight, and seal the blank plate as a blank additionally. 9) The next day, place the 96-well deep well plate in the centrifuge and centrifuge at 4000 rpm for 10 minutes, discard the milk in the ELISA plate, wash with 200 μL PBST 4 times, add 50 μL PBST to each well first, then add 50 μL of phage supernatant after centrifugation one by one correspondingly, incubate at 4 ° C for 1 hour, discard the supernatant, wash with PBST 5 times, dilute HRP-anti-M13 secondary antibody with PBST at 100 μl / well, incubate at 4 ° C for 45 minutes, wash off the secondary antibody, wash with PBST 5 times, TMB develop at room temperature for 10 minutes, stop with hydrochloric acid, read, select the clone with the large S / N ratio, and measure the preserved bacterial solution.

[0130] 10. Amplification, Transient Transfection, and Detection of Overlap PCR Products 10.1.1st PCR: Amplification of CMV, VHH and FC 1) Set up a PCR reaction system [50 μL system / reaction] (CMV and FC fragments can be prepared in large quantities) (CMV fragment amplification primers: CMV-F and PCom-R1, VHH fragment amplification primers: PCom-F1 and PCom-R2, FC fragment amplification primers: PCom-F2 and PGK-R) Components and doses: 2 µL of upstream primer (5 µM), 2 µL of downstream primer (5 µM), 25 µL of NuHi power mix (2x), 1 µL of template (plasmid or bacterial solution), supplement with sterile water to 50 µL. The PCR reaction program was as follows: 95°C for 10 minutes, (95°C for 15 seconds, 56°C for 30 seconds, 68°C for 60 seconds) x 25 cycles, 68°C for 10 minutes. 2) 50 μL of the PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and electrophoretic analysis was performed using 1% agarose. The band sizes of CMV and FC were approximately 750 bp, and the band size of VHH was approximately 560 bp. 3) Cut out the target band from the gel, purify the PCR product, and measure the concentration with a NanoDrop (if the concentration is too high, it can be diluted and used to perform subsequent reactions).

[0131] 10.2. Second PCR: Overlap Extension PCR Ligation of CMV, VHH and FC 1) PCR reaction system layout: Ingredients and doses: 50 ng CMV primary product, 50 ng VHH primary product, 50 ng FC primary product, 25 μL NuHi Power Mix (2×), supplemented to 46 μL with sterile water. The PCR reaction program was as follows: 95°C for 10 minutes, (95°C for 15 seconds, 60°C for 30 seconds, 68°C for 120 seconds) x 15 cycles. Add 2 ul each of primers CMV-F and PGK-R. The PCR reaction program was as follows: 95°C for 10 minutes, (95°C for 15 seconds, 60°C for 30 seconds, 68°C for 120 seconds) x 20 cycles, 68°C for 10 minutes. 4) Use TakaRa's DNA fragment recovery kit to purify the overlap PCR product, measure the concentration with NanoDrop, and at least 10 μg of PCR product is required for subsequent cell transfection verification. 5) The transfection procedure is the same as that of the eukaryotic expression vector.

[0132] 11. Transient Transfection Expression of Single Domain Antibodies 1. Take out the LVTransm transfection reagent and antibody expression vector pcDNA3.4-hIgG1-Fc2 or overlap PCR product from the refrigerator, thaw at room temperature, then mix thoroughly by pipetting up and down with a pipette. Take out the PBS buffer and warm it to room temperature. Take 500μL of PBS into one well of a 24-well plate, add 4μg of pcDNA3.4-hIgG1-Fc2, mix thoroughly by pipetting up and down with a pipette, then add 12μL of LVTransm, immediately pipette up and down with a pipette to mix evenly, and leave at room temperature for 10 minutes. The mixture here is referred to as DNA / LVTransm complex.

[0133] 2. Add 532μL of the DNA / LVTransm complex to 1.5mL of 293F cells and mix thoroughly by shaking gently. Place the cells in a 37℃, 5% CO2 incubator and incubate at 130RPM for 6-8 hours, then add 1.5mL of fresh 293 medium and return the cells to the incubator to continue culturing.

[0134] 3. After 3 days of continuous cultivation, collect the medium supernatant by centrifugation, filter through a 0.45 μm filter membrane, transfer the filtrate into a sterile centrifuge tube, and perform subsequent flow cytometry and ELISA detection.

[0135] 12. Construction of VHH eukaryotic expression vector Based on the results of phage monoclonal Elisa detection, positive clones are selected and sequenced to obtain the VHH antibody sequences. The VHH antibody sequences obtained through the analysis are gene synthesized respectively and subcloned into the expression vector pcDNA3.4-hIgG1-Fc2 in parallel with human IgG1Fc. After the vectors are confirmed to be correct by sequencing, endotoxin-free plasmids are prepared and stockpiled using the Qiagen Plasmid Extraction Kit.

[0136] 13. Flow Cytometric Detection of Binding of Recombinant Antibodies to Target Proteins 1. Recover CHO-S and CHO-S / UPAR cell lines from liquid nitrogen and adjust the cell state to logarithmic growth phase. 2.Two types of cells are divided into several parts, and the number of cells in each part is 5×10^5 cells. 3. Incubate each of the expressed antibodies with the target cells, mix thoroughly, and then incubate at room temperature for 1 hour. 4. Centrifuge at 800xg for 5 minutes at room temperature, remove the antibody-containing supernatant and wash the cells three times with PBS. Add 5.1 μL of PE-labeled anti-human IgG, mix thoroughly, and then incubate at room temperature in the dark for 30 minutes. 6. Centrifuge at 800xg for 5 minutes at room temperature, remove the secondary antibody-containing supernatant and wash the cells three times with PBS. 7. Resuspend the cells in 500uL of PBS and perform flow cytometry analysis.

[0137] 14. Expression and Purification of Single Domain Antibodies 1. Take out the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and then mix thoroughly by pipetting up and down with a pipette. Take out the PBS or HBSS buffer and warm it to room temperature. Take 2mL of PBS into one well of a 6-well plate, add 130μg of pcDNA3.4-hIgG1-Fc2 each, mix thoroughly by pipetting up and down with a pipette, then add 400μL of LVTransm, immediately pipette up and down with a pipette to mix evenly, and leave at room temperature for 10 minutes.

[0138] 2. Add the above DNA / LVTransm complex to 50mL of 293F cells and shake gently to mix thoroughly. Place the cells in a 37℃, 5% CO2 incubator and culture at 130 RPM for 6-8 hours, then add 50mL of fresh 293 medium and return the cells to the incubator to continue culturing.

[0139] 3. After 7 days of continuous culture, collect the culture supernatant by centrifugation, filter through a 0.45 μm filter membrane, transfer the filtrate into a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0140] 15. Affinity Detection of Recombinant Antibodies The UPAR recombinant protein is immobilized on a CM5 chip using 10 mM acetate buffer, and each of the prepared single domain antibodies is used as the mobile phase to detect the binding ability of the candidate single domain antibodies to the target protein UPAR.

[0141] Example 1. Expression and purification of UPAR-His recombinant protein 1.1. Detection of UPAR-His recombinant protein activity The above-mentioned experimental method 1 is used to prepare UPAR-His antigen, and the expression of UPAR-His protein is detected by SDS-PAGE. The results are shown in Figure 1. Lane 1 is the band of UPAR-His protein, the theoretical molecular weight is 32.3 kDa, and the SDS-PAGE detection is about 45 kDa.

[0142] 2 μg / ml uPA (novoprotein, C393) was coated on ELISA plates, and a 5-fold gradient dilution of biotin-UPAR-His was used as the primary antibody, and the secondary antibody was streptavidin-HRP.

[0143] Results: The results of ELISA detection showed that UPAR-His can bind to its natural ligand uPA (see Figure 2), has biological activity, and can be used for alpaca immunity and antibody screening.

[0144] 1.2.Immunotiter ELISA detection The above-mentioned experimental method 2 is used to immunize llamas, and the immune titer is detected using experimental method 3. Specifically, the serum of alpaca after three immunizations is collected, limiting dilution is performed according to the dilution gradient shown in Figure 3, and ELISA detection is performed using a 96-well plate precoated with UPAR-His antigen.

[0145] Results: According to the ELISA detection effect (see Figure 3), the immune serum binds to UPAR-His, and shows a gradient change with the serum dilution gradient OD450 value, which indicates that the immunization was successful. Detection using IgG, subclass 2+3 secondary antibody also gives a higher immune titer. Collect 100ml of peripheral blood and arrange for library construction.

[0146] 1.3. Amplification of VHH fragments The above-mentioned experimental methods 4 and 5 are used to separate PBMCs and amplify VHH fragments. Specifically, total RNA is extracted from the peripheral blood of the alpaca immunized in Example 1.2, reverse transcribed into cDNA, and then two rounds of nested PCR are performed using single domain antibody amplification primers.

[0147] Results: Figure 4 shows the results of agarose gel electrophoresis of the PCR products. In the first PCR, two PCR bands of approximately 1000 bp and 750 bp are obtained, and the 750 bp fragment is recovered from the gel as a template for the second round of PCR. In the second PCR, a band of approximately 450 bp, which is the VHH fragment, is obtained.

[0148] 1.4. Diversity analysis of phage display libraries The above-mentioned experimental method 6 is used to carry out the construction and diversity analysis of the phage display library. Specifically, the VHH fragment amplified in Example 1.3 is digested with SfiI and then subcloned into the phage display vector pComF, and the ligation product is electrotransformed into SS320 E. coli competent cells to construct a single domain antibody phage display library. By calculation, the capacity of the phage display library is 2.53E8. From the phage display library, 20 single clones are randomly selected and sequenced to analyze the diversity of the constructed phage display library.

[0149] Results: As shown in Figure 5. Comparison of the sequencing results shows that the empty rate and antibody overlap rate of the phage display library are less than 10%.

[0150] 1.5. Solid-phase panning of phage display libraries The above-mentioned experimental method 7 is used to carry out solid-phase panning of the phage display library. Specifically, UPAR-His and Fc recombinant proteins are coated respectively, and the constructed phage display library is screened and enriched four times to enrich the positive clones.

[0151] 1.6. Phage ELISA of solid-phase panning products of phage display libraries From the phage positive clones enriched in Example 1.5, select a single clone of phage and perform phage ELISA identification using Experimental Method 8 above.

[0152] Specifically, the first, second and third rounds of solid-phase panning outputs were selected to perform phage ELISA experiments, respectively coated with UPAR-His antigen protein, and phage Elisa detection was performed, and the control groups were directly sealed well plates and wells coated with Fc.

[0153] Results: The results of phage ELISA detection are shown in Figure 6. The clones with S / N ratios of 5 or more and not binding to Fc are selected and sequenced to analyze the sequence antibodies. A total of 18 different antibodies are obtained by antigen solid-phase panning.

[0154] 1.7. Flow cytometry analysis of transient transfection products and detection of overlap products In this example, vector construction and expression detection are carried out for the 18 VHH antibody sequences screened and sequenced in Example 1.6. CMV promoter, signal peptide and human IgG1 Fc tag are added to the N-terminus and C-terminus of the candidate antibody sequences by overlap PCR, respectively, and the purified PCR products are transiently transfected into 293 cells to express the antibodies and perform flow cytometry detection.

[0155] Results: The FACS detection results are shown in Figures 7A-7D. From the 18 clones obtained by panning the phage display library, a total of 15 suitable clones are selected for expression vector synthesis. Two clones (1-A2 and 1-D2) that bind very weakly to the UPAR overexpressing cell line and a clone (3-F4) that has high similarity to the known sequence are not synthesized as expression vectors.

[0156] 1.8. Expression and purification of candidate antibodies, flow cytometry detection Candidate antibody expression vectors are transiently transfected into HEK293, and antibodies expressed in the supernatant are purified with Protein A, protein quantified, and detected by SDS-PAGE. 15 candidate single domain antibodies are detected by flow cytometry with the overexpressing cell line CHO-S / UPAR and its parental cell line.

[0157] Results: Flow cytometry detection results show that all 15 candidate single domain antibodies can specifically bind to CHO-S / UPAR (see Figures 8A-8C).

[0158] The sequences obtained by sequencing the 15 antibodies are as follows: 1)pcDNA3.4-SDAB20-7-26-2-1-B4-hFC QVQLVESGGGLVQPGGSLRLSCAAS GFTLDYYA IGWFRQAPGKEREGVSC ISSTGGST NYADSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAADTHYLTVCYDRLGFDY WGQGTQVTVSS (SEQ ID NO: 1), 2)pcDNA3.4-SDAB20-7-26-2-1-F1-hFC EVQLAESGGGLVQPGGSLRLSCAAS GFTFSSYT MSWVRQAPGkgLEWVSA IDSDGGVT SYADSVkgRFTISRDNAKNTLYLQMNSLKPEDTAV YYCANGGYCSGYGCYPRL LSQGTLVTVSS(SEQ ID NO:2), 3)pcDNA3.4-SDAB20-7-26-2-1-F9-hFC QSLLVESGGGLVQPGGSLRLSCAAS GFSLDYYA IGWFRQAPGKEREEVSC ISSGGGST NYADSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAAKRNFMLYLVQCPYEYDY WGQGTQVTVSS (SEQ ID NO: 3), 4)pcDNA3.4-SDAB20-7-26-2-1-F10-hFC QVQLVESGGGLVQPGGSLRLSCAAS GSILSIRA VGWYRQAPGKQRELVA LITSDGTT HYALSVkgRFTISRDNAKTTVYLQMNSLKPEDTAV YYCNREDYARRY WGQGTQVTVSS(SEQ ID NO:4)、 5)pcDNA3.4-SDAB20-7-26-2-1-G1-hFC QVQLVESGGGLVQPGGSLRLSCAAS GNSLRRYA IGWFRQAPGKEREGVVC ISTVGGSS YYVDSVkgRFTISEDNVKNTAYLQMNSLEPEDTAV YYCAAANRLYCPAYGSAGYD YWGQGTQVTVSS(SEQ ID NO:5)、 6)pcDNA3.4-SDAB20-7-26-2-1-G2-hFC QSQLAESGGGLVQPGGSLRLSCVAS RFSLDAYA IAWFRQAPGKEREGVSC ISSIGGRT NYADPVkgRFTISRDNAKNTAYLEMNSLQPEDTAV YYCAAVQRLFGPCLLSGGMDY WGkgTPVTVSS(SEQ ID NO:6)、 7)pcDNA3.4-SDAB20-7-26-2-1-H2-hFC QVQLVESGGGLVQPGGSLRLSCAAS GSTLDYA IAWFRQAPGkgREGVSC ISSSGGRT NYADSVkgRFTVSRDNVKNTVYLQMNSLKPEDTAV YNCAAGGDLSCYGTPTSIWQYDL WGQGTQVTVAS(SEQ ID NO:7)、 8)pcDNA3.4-SDAB20-7-26-2-1-H8-hFC QRQLVESGGGLVQPGGSLRLSCAVS EFTLDYYA IGWFRQAPGKEREGVSC ISSSGGST VYADSVkgRFTISADNAKNTVYLQMNSLKPEDTAV YYCAAGSLLLFRLCVSRLYEYDY WGQGTQVTVSS(SEQ ID NO:8)、 9)pcDNA3.4-SDAB20-7-26-2-3-E03-hFC AVQLVDSGGGLVQAGGSLRLSCAAS GIRLSSNA MAWYRQAPGKQRGLVA LITSDGTT NYVDSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCKREPYLSTRGY WGQGTQVTVSS(SEQ ID NO:9)、 10)pcDNA3.4-SDAB20-7-26-2-3-E04-hFC QLRLVESGGGLVQPGGSLRLSCAAS GFTLDYYD IGWFRQAPGKEREGVSC IGRISGDT DYAESVkgRFTASRDNAKNTVYLQMNSLKPEDTAV YYCAAERRFFGGGCRRSVDNMDS WGkgTLVTVSS(SEQ ID NO:10)、 11)pcDNA3.4-SDAB20-7-26-2-3-E06-hFC EVQLVESGGGLVQAGGSLTLSCV ASASGSILRL NAMGWYRQAPAKQRELVA LITRDGST NYPDSVkgRFTISRDNAKNTVYLQMSSLSPEDTAV YYCYVQNHYSNY WGQGTQVTVSS(SEQ ID NO:11)、 12)pcDNA3.4-SDAB20-7-26-2-3-E09-hFC AVQLVDSGGGLVQPGGSLRLSCAAS GFTFSSYA MGWARQVPGkgLEWVS IIYRDGNT YYTDSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAPVETVAARRRDY WGQGTQVNVSS(SEQ ID NO:12)、 13)pcDNA3.4-SDAB20-7-26-2-3-E10-hFC AVQLVDSGGGLVQPGGSLRLSCAAS GFTFSSYA MGWARQVPGkgLEWVS IIYRDGNT YYTDSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAPVETVAARRRDY WGQGTQVNVSS(SEQ ID NO:13)、 14)pcDNA3.4-SDAB20-7-26-2-3-G07-hFC RLQLVESGGGLVQPGGSLRLSCAVS GKALDYYS IGWFRQAPRKQREGVSC ISSSGGST NYADSVkgRFTISRDNAKDTVYLQMNSLKPEDTAD YYCAVVSTVLCGLGIYEYDY WGQGTLVTVSS (SEQ ID NO: 14), 15)pcDNA3.4-SDAB20-7-26-2-3-H02-hFC QLTLVESGGGLVQPGGSLRLSCAAS EFTLDYYA IGWFRQAPGKEREGVSC ISSSDGST YYADSVkgRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAIGGLLLLRLWERGNYEFDY WGQGTQVTVSS (SEQ ID NO: 15). Here, the CDR regions of each VHH chain are underlined below.

[0159] The amino acid sequences of the CDR regions of each antibody are shown in Table 9. [Table 9-1] [Table 9-2]

[0160] Example 2. Detection of antibody affinity Three representative antibodies (1-F09, 3-E04, 3-H02) with the highest binding rates were selected and antibody affinity was detected using the experimental method described above15.

[0161] Results: As shown in FIG. 9 and Table 10. [Table 10]

[0162] This example demonstrates that three representative antibodies of the present invention, 1-F09, 3-E04, and 3-H02, all have high affinity for UPAR.

[0163] All documents mentioned in the present invention are incorporated by reference in this application as if each document was incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents are also included in the scope defined by the appended claims of this application.

Claims

1. 1. An anti-UPAR single domain antibody, The complementarity determining regions (CDRs) of the anti-UPAR single domain antibody VHH chain are (1) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 53, and CDR3 as set forth in SEQ ID NO: 54; (2) CDR1 as set forth in SEQ ID NO: 22, CDR2 as set forth in SEQ ID NO: 23, and CDR3 as set forth in SEQ ID NO: 24; (3) CDR1 as set forth in SEQ ID NO: 42, CDR2 as set forth in SEQ ID NO: 43, and CDR3 as set forth in SEQ ID NO: 44; (4) CDR1 as set forth in SEQ ID NO: 25, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 27; (5) CDR1 as set forth in SEQ ID NO: 28, CDR2 as set forth in SEQ ID NO: 29, and CDR3 as set forth in SEQ ID NO: 30; (6) CDR1 as set forth in SEQ ID NO: 31, CDR2 as set forth in SEQ ID NO: 32, and CDR3 as set forth in SEQ ID NO: 33; (7) CDR1 as set forth in SEQ ID NO: 34, CDR2 as set forth in SEQ ID NO: 35, and CDR3 as set forth in SEQ ID NO: 36; (8) CDR1 as set forth in SEQ ID NO: 37, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 39; (9) CDR1 as set forth in SEQ ID NO: 40, CDR2 as set forth in SEQ ID NO: 26, and CDR3 as set forth in SEQ ID NO: 41; (10) CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and CDR3 as set forth in SEQ ID NO: 21; (11) CDR1 as shown in SEQ ID NO: 45, CDR2 as shown in SEQ ID NO: 46, and CDR3 as shown in SEQ ID NO: 47; (12) CDR1 as set forth in SEQ ID NO: 48, CDR2 as set forth in SEQ ID NO: 49, and CDR3 as set forth in SEQ ID NO: 50; (13) CDR1 as set forth in SEQ ID NO: 51, CDR2 as set forth in SEQ ID NO: 38, and CDR3 as set forth in SEQ ID NO: 52; and (14) The anti-UPAR single domain antibody, characterized in that it has one or more CDRs selected from the group consisting of CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO:

18.

2. The amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NO: 1 to 15. The anti-UPAR single domain antibody of claim 1.

3. The amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NO. 3, 10 or 15. The anti-UPAR single domain antibody of claim 1.

4. The anti-UPAR single domain antibody is characterized in that it is selected from the group consisting of a humanized antibody, a camelid-derived antibody, and a chimeric antibody. The anti-UPAR single domain antibody of claim 1.

5. An anti-UPAR antibody, The anti-UPAR antibody, characterized in that the antibody comprises one or more VHH chains of the anti-UPAR single domain antibody described in claim 1.

6. The amino acid sequence of the VHH chain of the anti-UPAR single domain antibody is selected from the sequences shown in SEQ ID NO: 1 to 15. The anti-UPAR antibody according to claim 5 .

7. A polynucleotide comprising: The polynucleotide, characterized in that it encodes a protein selected from the group consisting of the anti-UPAR single domain antibody of claim 1 or the antibody of claim 5.

8. An expression vector comprising: The expression vector, characterized in that it comprises the polynucleotide according to claim 7.

9. A host cell comprising: The host cell is characterized in that it contains the expression vector described in claim 8 or has the polynucleotide described in claim 7 integrated into its genome.

10. 1. A method for producing an anti-UPAR single domain antibody, comprising: The method comprises: (a) culturing the host cell of claim 9 under conditions suitable for the production of a single domain antibody, thereby obtaining a culture comprising said anti-UPAR single domain antibody; (b) isolating and / or recovering said anti-UPAR single domain antibody from said culture; and (c) optionally purifying and / or modifying the anti-UPAR single domain antibody obtained in step (b).

11. 1. An immune complex comprising: The immune complex is (a) an anti-UPAR single domain antibody according to claim 1 or an anti-UPAR antibody according to claim 5, and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

12. Use of an anti-UPAR single domain antibody according to any one of claims 1 to 4, an anti-UPAR antibody according to claim 5, or an immunoconjugate according to claim 11, (1) A drug for preventing and / or treating a disease associated with UPAR, (2) Use of the anti-UPAR single domain antibody according to any one of claims 1 to 4, the anti-UPAR antibody according to claim 5, or the immune complex according to claim 11, characterized in that it is used for the preparation of a reagent for detecting UPAR.

13. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (i) an anti-UPAR single domain antibody according to claim 1, an anti-UPAR antibody according to claim 5, or an immune complex according to claim 11, and (ii) a pharma- ceutically acceptable carrier.

14. A method for preventing and / or treating a UPAR-associated disease, comprising: The method is characterized in that it comprises administering to a subject in need thereof an anti-UPAR single domain antibody described in claim 1, an anti-UPAR antibody described in claim 5, an immunoconjugate described in claim 11, or a pharmaceutical composition described in claim 13.

15. The UPAR-related disease is selected from the group consisting of aging-related diseases, cancer, and tumors. The method of claim 14.

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