Single-domain antibodies against cd25 and uses thereof

Abstract

The application discloses anti-CD25 single-domain antibodies and application thereof. Specifically, the application discloses a kind of specific single-domain antibodies for anti-human CD25. The coding sequence of the single-domain antibody or VHH chain thereof, the corresponding expression vector and host cell, and the method for producing the single-domain antibody of the application are also disclosed. The single-domain antibody of the application has high affinity and high specificity, and can be used for detection and targeted therapy for CD25.

Description

Technical Field

[0001] This invention belongs to the field of biomedical or biopharmaceutical technology, specifically relating to anti-CD25 single-domain antibodies and their applications. Background Technology

[0002] IL2RA (Interleukin-2 Receptor Alpha chain), also known as CD25, plays a crucial role in immune regulation and tumor immunotherapy. During T cell activation, CD25 expression is upregulated and plays a key role in IL-2 responsiveness, promoting T lymphocyte activation and IL-2 production. CD25 is highly expressed in regulatory T cells (Tregs) and is a biomarker for Tregs. Treg cells typically infiltrate solid tumors; targeting CD25 can regulate the balance between Teff (effector T cells) and Treg cells, thereby influencing tumor development and progression. Furthermore, CD25 is expressed at high levels in various types of hematologic malignancies, making it a potential target for cancer therapy.

[0003] Single-domain antibodies are a novel type of antibody molecule discovered in camels by the research group of Belgian immunologists Hamers-Casterman. Compared to traditional antibodies, they have a smaller molecular weight, about one-tenth the size of traditional IgG antibodies. Therefore, they have a simpler structure, are easier to modify, exhibit strong tissue infiltration, and have low immunogenicity. Furthermore, single-domain antibodies do not contain an Fc domain, so they do not mediate ADCC effects to cause cytotoxicity when used as targeting molecules. In addition, they possess advantages such as good stability, resistance to high temperatures and extreme pH environments, and low production costs. Therefore, single-domain antibodies are excellent targeting molecules with significant value for targeted drug development.

[0004] Therefore, developing a single-domain antibody targeting CD25 is of great significance for the development of anti-tumor drugs. Summary of the Invention

[0005] This invention provides a single-domain antibody targeting CD25 and its application.

[0006] In a first aspect of the invention, a single-domain antibody against CD25 is provided, said single-domain antibody having three complementarity-determining regions (CDRs) derived from the VHH chain shown in the following amino acid sequences: SEQ ID NO: 1-17;

[0007] The CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule.

[0008] In another preferred embodiment, CDR1, CDR2 and CDR3 are selected from the group consisting of:

[0009] (a1) CDR1 with amino acid sequence as shown in SEQ ID NO:18, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:20;

[0010] (a2) CDR1 with amino acid sequence as shown in SEQ ID NO:18, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:21;

[0011] (a3) CDR1 with amino acid sequence as shown in SEQ ID NO:22, CDR2 with amino acid sequence as shown in SEQ ID NO:23, and CDR3 with amino acid sequence as shown in SEQ ID NO:24;

[0012] (a4) CDR1 with amino acid sequence as shown in SEQ ID NO:25, CDR2 with amino acid sequence as shown in SEQ ID NO:23, and CDR3 with amino acid sequence as shown in SEQ ID NO:26;

[0013] (a5) CDR1 with amino acid sequence as shown in SEQ ID NO:27, CDR2 with amino acid sequence as shown in SEQ ID NO:28, and CDR3 with amino acid sequence as shown in SEQ ID NO:29;

[0014] (a6) CDR1 with amino acid sequence as shown in SEQ ID NO:30, CDR2 with amino acid sequence as shown in SEQ ID NO:31, and CDR3 with amino acid sequence as shown in SEQ ID NO:32;

[0015] (a7) CDR1 with amino acid sequence as shown in SEQ ID NO:30, CDR2 with amino acid sequence as shown in SEQ ID NO:31, and CDR3 with amino acid sequence as shown in SEQ ID NO:33;

[0016] (a8) CDR1 with amino acid sequence as shown in SEQ ID NO:34, CDR2 with amino acid sequence as shown in SEQ ID NO:35, and CDR3 with amino acid sequence as shown in SEQ ID NO:36;

[0017] (a9) CDR1 with amino acid sequence as shown in SEQ ID NO:37, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:39;

[0018] (a10) CDR1 with amino acid sequence as shown in SEQ ID NO:40, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:41;

[0019] (a11) CDR1 with amino acid sequence as shown in SEQ ID NO:37, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:42;

[0020] (a12) CDR1 with amino acid sequence as shown in SEQ ID NO:43, CDR2 with amino acid sequence as shown in SEQ ID NO:44, and CDR3 with amino acid sequence as shown in SEQ ID NO:45;

[0021] (a13) CDR1 with amino acid sequence as shown in SEQ ID NO:46, CDR2 with amino acid sequence as shown in SEQ ID NO:44, and CDR3 with amino acid sequence as shown in SEQ ID NO:47;

[0022] (a14) CDR1 with amino acid sequence as shown in SEQ ID NO:43, CDR2 with amino acid sequence as shown in SEQ ID NO:48, and CDR3 with amino acid sequence as shown in SEQ ID NO:49;

[0023] (a15) CDR1 with amino acid sequence as shown in SEQ ID NO:50, CDR2 with amino acid sequence as shown in SEQ ID NO:51, and CDR3 with amino acid sequence as shown in SEQ ID NO:52;

[0024] (a16) CDR1 with amino acid sequence as shown in SEQ ID NO:53, CDR2 with amino acid sequence as shown in SEQ ID NO:54, and CDR3 with amino acid sequence as shown in SEQ ID NO:55;

[0025] (a17) CDR1 with amino acid sequence as shown in SEQ ID NO:56, CDR2 with amino acid sequence as shown in SEQ ID NO:57, and CDR3 with amino acid sequence as shown in SEQ ID NO:58.

[0026] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO:1 to 17.

[0027] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

[0028] In another preferred embodiment, the amino acid sequence of the single-domain antibody includes any of the sequences shown in SEQ ID NO:1 to 3.

[0029] In another preferred embodiment, the nanobody is a humanized antibody comprising the sequence shown in any one of SEQ ID NO:59 to 77.

[0030] In another preferred embodiment, the nanobody is a humanized antibody comprising the sequence shown in any one of SEQ ID NO: 59-68 and 70-77.

[0031] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0032] In another preferred embodiment, any of the amino acid sequences described above further includes a derivative sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining CD25 binding affinity.

[0033] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0034] In another preferred embodiment, the VHH chain of the nanobody further includes a framework region (FR).

[0035] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0036] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0037] In another preferred embodiment, the nanobody is bound to human, mouse, or monkey CD25.

[0038] In another preferred embodiment, the antibody is a heavy chain antibody, which includes heavy chain constant regions CH2 and CH3 (Fc segment).

[0039] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0040] In another preferred embodiment, the VHH chain of the CD25-targeting nanobody has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the amino acid sequences shown in SEQ ID NO:1-17.

[0041] In another preferred embodiment, the VHH chain of the anti-CD25 nanobody has one or more amino acid sequences as shown in SEQ ID NO:1-17.

[0042] In another preferred embodiment, the anti-CD25 nanobody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0043] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of any one of the sequences shown in SEQ ID NO:1 to 17.

[0044] In a second aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0045] (i) the single-domain antibody described in the first aspect of the present invention; and

[0046] (ii) Optional tag sequences to assist in expression and / or purification.

[0047] In another preferred embodiment, the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.

[0048] In another preferred embodiment, the recombinant protein specifically binds to CD25.

[0049] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0050] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0051] In another preferred embodiment, the tag sequence is an Fc tag.

[0052] In another preferred embodiment, the recombinant protein comprises a fusion protein.

[0053] In another preferred embodiment, the recombinant protein is a fusion protein, and the fusion protein has a structure from the N-terminus to the C-terminus as shown in Formula I:

[0054] Z1-L-Z2 (Formula I)

[0055] In the formula,

[0056] Z1 is the VHH chain of the anti-CD25 single-domain antibody as described in the first aspect of the present invention;

[0057] L represents the connector sequence;

[0058] Z2 is the Fc segment of an immunoglobulin.

[0059] In a third aspect of the invention, a polynucleotide is provided that encodes the single-domain antibody described in the first aspect of the invention.

[0060] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0061] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the third aspect of the invention.

[0062] 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. Preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0063] In another preferred embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0064] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or the genome containing the polynucleotide described in the third aspect of the invention.

[0065] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0066] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0067] In a sixth aspect of the invention, a method for generating an anti-CD25 single-domain antibody is provided, comprising the steps of:

[0068] (a) Culturing the host cells described in the fifth aspect of the invention under conditions suitable for generating single-domain antibodies, thereby obtaining a culture containing the anti-CD25 single-domain antibody; and

[0069] (b) Isolate or recover the anti-CD25 nanobody from the culture.

[0070] In another preferred embodiment, the anti-CD25 single-domain antibody has an amino acid sequence as shown in any of SEQ ID NO:1 to 17.

[0071] In another preferred embodiment, the anti-CD25 single-domain antibody has an amino acid sequence as shown in any of SEQ ID NO:1 to 3.

[0072] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0073] (a) A single-domain antibody against CD25 as described in the first aspect of the invention; and

[0074] (b) A conjugation portion conjugated to the single-domain antibody, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0075] In another preferred embodiment, the single-domain antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0076] In another preferred embodiment, the coupling portion is a drug or toxin.

[0077] In another preferred embodiment, the drug is a cytotoxic drug.

[0078] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0079] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0080] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0081] In another preferred embodiment, the coupling portion is a detectable marker.

[0082] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antisense oligonucleotides, small interfering RNA, microRNAs, nucleic acid aptamers, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanoran, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0083] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) anti-CD25 single-domain antibody as described in the first aspect of the invention. The multivalent meaning is that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-CD25 single-domain antibodies as described in the first aspect of the invention.

[0084] In another preferred embodiment, the term "multivalent" means that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-CD25 single-domain antibodies as described in the first aspect of the invention.

[0085] In an eighth aspect of the invention, the use of a single-domain antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention, for the preparation of pharmaceuticals, reagents, detection plates, or kits is provided.

[0086] The reagents, detection plates, or kits are used to detect CD25 in samples;

[0087] The agent is used to prevent and / or treat CD25-overexpressing tumors, organ transplant rejection, and autoimmune diseases.

[0088] In another preferred embodiment, the tumor includes both solid tumors and non-solid tumors.

[0089] In another preferred embodiment, the tumor is selected from the group consisting of: cutaneous T-cell lymphoma, hairy cell leukemia, lymphoma, refractory Hodgkin lymphoma, head and neck tumors, melanoma, or combinations thereof.

[0090] In a ninth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0091] (i) a single-domain antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention; and

[0092] (ii) Pharmaceutically acceptable carriers.

[0093] In another preferred embodiment, the carrier is an excipient or a diluent.

[0094] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0095] In a tenth aspect of the present invention, a CD25 protein detection reagent is provided, the detection reagent comprising:

[0096] (i) the anti-CD25 single-domain antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the immunoconjugate described in the seventh aspect of the present invention; and

[0097] (ii) A detectable carrier.

[0098] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0099] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0100] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0101] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0102] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0103] In an eleventh aspect of the present invention, a reagent kit is provided, the reagent kit comprising the detection reagents described in the tenth aspect of the present invention.

[0104] In a twelfth aspect of the present invention, a method for detecting CD25 protein in a sample is provided, the method comprising the steps of:

[0105] (1) Contact the sample with the single-domain antibody described in the first aspect of the present invention;

[0106] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD25 protein in the sample.

[0107] In another preferred embodiment, the method is an in vitro method.

[0108] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0109] In a thirteenth aspect of the invention, a method for treating tumors is provided, the method comprising administering to a desired subject a single-domain antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention.

[0110] In another preferred embodiment, the object includes mammals, such as humans.

[0111] In another preferred embodiment, the tumor includes both solid tumors and non-solid tumors.

[0112] In another preferred embodiment, the tumor is selected from the group consisting of: cutaneous T-cell lymphoma, hairy cell leukemia, lymphoma, refractory Hodgkin lymphoma, head and neck tumors, melanoma, or combinations thereof.

[0113] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0114] Figure 1 The results show the cell binding titers of serum for the three- and four-immune vaccines as detected by FACS.

[0115] Figure 2 The positive rate of the yeast display enrichment library of anti-CD25 single-domain antibodies was shown by flow cytometry analysis.

[0116] Figure 3 The binding ability of the anti-CD25 single-domain antibody to CHO-K1 cells with high hCD25 expression was demonstrated by FACS detection.

[0117] Figure 4 This demonstrates the ability of ELISA to detect the binding of anti-CD25 single-domain antibody to human CD25 antigen.

[0118] Figure 5 This demonstrates the ability of ELISA to detect the binding of anti-CD25 single-domain antibody to monkey CD25 antigen.

[0119] Figure 6 This demonstrates the ability of ELISA to detect the binding of anti-CD25 single-domain antibody to mouse CD25 antigen.

[0120] Figure 7 The results showed that ELISA validation of the ligand IL-2 did not affect the binding of the anti-CD25 single-domain antibody to the CD25 antigen.

[0121] Figure 8 This study demonstrates the IL-2 blocking activity assay of anti-CD25 single-domain antibodies using ELISA.

[0122] Figure 9 The study showed that Nb097-MA11-MMAE inhibited the growth of karpas 299 tumor cells.

[0123] Figure 10 The binding activity of the humanized antibody to Karpas 299 cells with high hCD25 expression was demonstrated.

[0124] Figure 11 The binding activity of the humanized antibody to MJ cells with high hCD25 expression was shown. Detailed Implementation

[0125] Through extensive and in-depth research and screening, the inventors have, for the first time, developed a single-domain antibody specifically targeting CD25 and its humanized antibody. Experimental results show that the single-domain antibody and its humanized antibody obtained in this invention have specific high affinity for CD25. The single-domain antibody of this invention exhibits good endocytic activity and both possess IL-2 ligand blocking activity. The single-domain antibody of this invention can specifically bind to human CD25 protein (including CD25 on the cell surface) and also exhibits cross-binding activity between humans, mice, and monkeys. Furthermore, the single-domain antibody of this invention can achieve effective killing of tumor cells through conjugation with a toxin. Based on these findings, this invention has been completed.

[0126] the term

[0127] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0128] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0129] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0130] The term "EC50" refers to the concentration for 50% of the maximal effect, which is the concentration at which the maximal effect is achieved.

[0131] The term "IC50" refers to the half-inhibitory concentration of the antagonist being measured.

[0132] CD25

[0133] IL2RA (Interleukin-2 Receptor Alpha chain), also known as CD25, plays an important role in immune regulation and tumor immunotherapy. During T cell activation, CD25 expression is upregulated and plays a key role in IL-2 responsiveness, promoting T lymphocyte activation and IL-2 production.

[0134] The single-domain antibody of the present invention

[0135] As used herein, the terms "single-domain antibody of the present invention," "CD25 single-domain antibody of the present invention," "nanobody of the present invention," "anti-human CD25 nanobody of the present invention," and "single-domain antibody targeting CD25 of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to CD25 (including human CD25). Particularly preferred are nanobodies with the amino acid sequences of the VHH chain as shown in SEQ ID NO:1-17.

[0136] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. 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; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0137] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0138] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together 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)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0139] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates 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 also includes cell surface markers or antigens that bind to the described anti-CD25 antibody or fragments thereof.

[0140] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0141] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0142] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0143] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0144] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the CD25 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0145] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0146] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0147] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0148] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0149] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0150] The antibody of this invention refers to a polypeptide containing the aforementioned CDR region and having CD25 protein-binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0151] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0152] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0153] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0154] Table A

[0155]

[0156]

[0157] Polynucleotides, vectors and host cells

[0158] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0159] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0160] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0161] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0162] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0163] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0164] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0165] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0166] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0167] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0168] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0169] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0170] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0171] 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 detectable products.

[0172] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0173] Immunoconjugates

[0174] The present invention also provides immunoconjugates (ADCs) based on the antibodies of the present invention, preferably nanobody-drug conjugates (NDCs).

[0175] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of the following: a toxic protein, a chemotherapeutic agent, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), or a radionuclide.

[0176] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0177] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0178] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, an NDC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0179] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0180] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0181] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0182] The immunoconjugated drug of the present invention can also be a radionuclide conjugated drug (RDC), which is composed of the antibody of the present invention conjugated with a radionuclide.

[0183] In this invention, the drug-linker can be used to form NDC in a simple step. In other embodiments, bifunctional linker compounds can be used to form NDC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form NDC.

[0184] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0185] The present invention also provides a method for preparing NDC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (NDC).

[0186] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0187] In some embodiments, the structure of the immunoconjugate, preferably a single-domain antibody-drug conjugate NDC, is shown in the following molecular formula:

[0188]

[0189] in:

[0190] nAb refers to the single-domain antibody targeting CD25, the heavy-chain antibody targeting CD25, or the multispecific antibody; LU is the adapter / linker.

[0191] D is a drug;

[0192] And the subscript p is a value selected from 1 to 10.

[0193] Pharmaceutical Composition

[0194] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0195] The pharmaceutical compositions of the present invention can be directly used to bind to CD25 protein molecules, and therefore can be used to treat allergies. In addition, other therapeutic agents can be used simultaneously.

[0196] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0197] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0198] Labeled nanobodies

[0199] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0200] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, anti-CD25 nanobodies are labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0201] The anti-human CD25 nanobody of the present invention has excellent specificity and high potency.

[0202] Detection methods

[0203] This invention also relates to a method for detecting CD25 protein. The method generally involves the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of CD25 protein in the dissolved samples.

[0204] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0205] Reagent test kit

[0206] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0207] This invention also provides a detection kit for detecting CD25 levels, comprising an antibody that recognizes the CD25 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0208] application

[0209] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of CD25-related diseases, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy against CD25.

[0210] In another preferred embodiment, the CD25-related diseases include: CD25-overexpressing tumors, organ transplant rejection, and autoimmune diseases.

[0211] In another preferred embodiment, the tumor includes both solid tumors and non-solid tumors.

[0212] In another preferred embodiment, the tumor is selected from the group consisting of: cutaneous T-cell lymphoma, hairy cell leukemia, lymphoma, refractory Hodgkin lymphoma, head and neck tumors, melanoma, or combinations thereof.

[0213] The main advantages of this invention include:

[0214] (a) The present invention provides humanized single-domain antibodies that reduce the immunogenicity of the antibodies and improve their in vivo safety.

[0215] (b) The single-domain antibody of the present invention has good endocytosis activity and can mediate the endocytosis of the target, which is beneficial to the construction and use of ADC.

[0216] (c) This invention provides a single-domain antibody with cross-binding activity with human, mouse and monkey species.

[0217] (d) The production of the single-domain antibody of the present invention is simple.

[0218] (e) The single-domain antibody of the present invention has ligand blocking activity.

[0219] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0220] Example 1: Production of anti-CD25 single-domain antibodies induced by immunization of alpacas and detection of serum titer

[0221] For the initial immunization, 0.5 mg of human CD25-Fc antigen (ACRO, Cat#ILA-H5251) was mixed with an equal volume of complete Freund's adjuvant (CFA) and subcutaneously injected into two healthy alpacas to stimulate the alpaca's immune system to produce corresponding anti-CD25 antibodies. Subsequent immunizations were administered every 21 days for a total of four immunizations. The second and third immunizations used 2 x 10-1 doses. 7 MJ cells were mixed with an equal volume of manganese adjuvant and subcutaneously injected into the alpaca. For the fourth immunization, 0.25 mg of human CD25-Fc antigen was mixed with an equal volume of incomplete Freund's adjuvant (IFA) and subcutaneously injected. Serum titers of the target antibody were measured one week after the second, third, and fourth immunizations, before immunization.

[0222] ELISA potency testing methods:

[0223] 1) Dilute CD25-his (ACRO, Cat#ILA-H52H9) with 0.05M carbonate buffer (pH 9.6) to 2 μg / mL, and coat overnight at 4°C at a rate of 100 μL / well.

[0224] 2) Discard the coating solution, wash 3 times with PBST, add 300 μL of 5% skim milk to each well, and block at 37°C for 1 h;

[0225] 3) Wash 3 times with PBST, add 100 μL / well of serum dilution buffer (start with serial dilution from 1:2000), and incubate at 37°C for 45 min;

[0226] 4) Wash 5 times with PBST, and incubate 100 μL / well with goat anti-alpaca IgG (H+L) antibody diluted 10000 times (AlpVHHs, Cat#: 053-404-005) at 37℃ for 45 min.

[0227] 5) Wash the plate 5 times with PBST. Add TMB colorimetric solution (100 μL / well) for color development, incubate at 37°C for 5 min, then add stop solution (50 μL / well) to terminate the reaction. Measure the optical density at 450 nm.

[0228] The results of serum ELISA titers for the three and four immunizations are shown in Table 1.

[0229] Table 1 - Serum ELISA titer detection for triple and quadruple immune vaccinations

[0230]

[0231] FACS valence testing method:

[0232] 1) Collect Jurkat cells and CHO-K1 cells in the logarithmic growth phase, centrifuge, wash, and resuspend in PBS;

[0233] 2) Trypan blue staining to count live cells;

[0234] 3) Adjust the cell density to 4x10 using 1% horse serum / PBS (washing buffer). 6 / ml;

[0235] 4) Take 50 μL of cell suspension (containing 200,000 cells) into a 1.5 mL EP tube; add a certain volume of serum (two gradients: 1:100 and 1:1000) to the cells and incubate at 4°C for 1 h;

[0236] 5) Add washing solution, centrifuge, and discard the supernatant;

[0237] 6) Resuspend the cells in 50 μL of iFluor647-labeled goat anti-alpaca IgG (H+L) antibody (AlpVHHs, Cat#053-404-009) and incubate at 4°C for 1 h.

[0238] 7) Wash the cells twice, resuspend them, and place them at 4°C for analysis.

[0239] The results of serum FACS titer detection for the three- and four-immune vaccinations are shown in Table 2. Figure 1 .

[0240] Table 2 - Serum FACS titer detection for triple and quadruple immunizations

[0241]

[0242] The results above show that the serum collected from alpacas A and B met the library construction standards in terms of both protein and cell binding levels, and can be used for the subsequent construction of yeast display libraries.

[0243] Example 2: Construction and screening of anti-CD25 single-domain antibody yeast display library

[0244] Peripheral blood mononuclear cells (PBMCs) were isolated from alpaca peripheral blood collected one week after the completion of the third and fourth vaccinations, and total RNA was extracted using RNAiso Plus reagent. PrimeScript was used for further analysis. TM II. The 1st StrandcDNA Synthesis Kit (Takara, Cat#6210A) was used to reverse transcribe 5 μg of total RNA into cDNA, following the kit instructions. Using the cDNA as a template, 2.5 μl of a 5-fold diluted cDNA was used for the first round of nested PCR. A 750 bp fragment of the PCR product was gel-recycled as the template for the second round of nested PCR. After two rounds of nested PCR amplification, a single-domain antibody fragment was obtained. The PCR product was purified using the Cycle-Puer Kit and used to construct a yeast display library. Based on the number of transformants, insertion rate, and diversity sequencing analysis, the size of the anti-CD25 single-domain antibody yeast display library constructed from alpaca peripheral blood A was determined to be 3.05 x 10⁻⁶. 8 The yeast display library of anti-CD25 single-domain antibodies constructed from alpaca peripheral blood has a size of 2 x 10⁻⁶. 8 .

[0245] Libraries A and B were merged and then screened. The first round of enrichment used 1000 nM human CD25-His antigen, performed using magnetic bead sorting. The second round of enrichment was performed on the same magnetic bead-enriched library, using 500 nM human CD25-Fc antigen, and flow cytometry was used to identify and sort the bipositive cell populations. The third round of enrichment was performed on the second round of sorting libraries, using 200 nM monkey CD25-His antigen (ACRO, Cat#ILA-C52H8) and 200 nM mouse CD25-His antigen (ACRO, Cat#ILA-M52H9), respectively, and flow cytometry was used to identify and sort the bipositive cell populations. The fourth round of enrichment was performed on the mouse antigen sorting library from the third round. Using 100 nM mouse CD25-hFc antigen (SinoBiological, Cat#50292-M02H), double-positive cell populations were identified and sorted by flow cytometry. The positivity rate of each enriched library was validated by flow cytometry analysis. Figure 2 ).

[0246] Subsequently, 88 monoclonal antibodies were selected from the third round of monkey antigen enrichment library and the fourth round of mouse antigen enrichment library and sent for sequencing. Sequence alignment analysis revealed 17 positive sequences for human and monkey CD25 antigen binding, of which 3 sequences had mouse CD25 antigen binding ability (Table 3), involving 13 CDR1 sequences, 12 CDR2 sequences and 17 CDR3 sequences (Table 4).

[0247] Table 3 - Sequences of human CD25 antigen-binding positive single-domain antibodies

[0248]

[0249]

[0250]

[0251] Table 4 - Human CD25 antigen-binding positive CDR sequences

[0252]

[0253]

[0254] Example 3: Binding activity analysis of anti-CD25 single-domain antibody with human CD25-overexpressing cells

[0255] The C-terminus of the VHH sequence was fused with a human 6*his tag. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for expression for 7 days, and VHH was purified by affinity chromatography.

[0256] hCD25 CHO-K1 cells were obtained by stable transfection of CHO-K1 cells with the pIRES-Neo3 vector expressing the human CD25 gene (NM_000417.3). Cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 1 × 10⁻⁶ cells / year. 6 Cells / mL, add 180 μL / well of cell suspension to a 96-well U-shaped plate. Dilute the test sample stock solution with flow cytometry buffer to prepare serially diluted 10* concentration antibody solutions. Add 20 μL of the above solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, add 200 μL / well of 1:1000 diluted iFluor647-labeled rabbit anti-camel VHH antibody (Genscript, Cat#A02019) solution, vortex to mix, and incubate the 96-well plate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μL / well of flow cytometry buffer. Measure the average fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated. Figure 3 Experimental results showed that all candidate antibodies could bind to hCD25 expressed on the cell surface.

[0257] Example 4: Binding activity analysis of anti-CD25 single-domain antibody with recombinant CD25 protein in humans, monkeys, and mice.

[0258] Ten candidate sequences with significant CDR3 sequence differences were selected, and the binding activity of human, monkey, and mouse CD25 recombinant proteins was verified using ELISA.

[0259] Prepare human, monkey, and mouse CD25 antigen solutions separately using PBS to a final concentration of 1 μg / mL. Add 25 μL / well to each well of a 384-well ELISA plate and incubate overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 50 μL / well of blocking buffer (PBST + 3% BSA), and block at room temperature for 1 hour. Wash three times with PBST, add 25 μL / well of serially diluted single-domain antibody solution, and incubate at room temperature for 1 hour. Wash three times with PBST, add 25 μL / well of 1:5000 diluted horseradish peroxidase-labeled rabbit anti-camel VHH antibody (Genscript, Cat#A02016), and incubate at room temperature for 1 hour. Wash three times with PBST, blot dry, add 25 μL / well of TMB substrate solution (Beyotime, Cat#P0209), and incubate at room temperature for 5 to 30 minutes. Then add 25 μL / well of stop solution (Beyotime, Cat#P0215). Measure the absorbance at 450 nm in each well using a Molecular Devices (SpectraMax i3x) microplate reader. Calculate the binding EC50 values ​​using a 4-parameter S-curve equation fitted with GraphPad Prism 10 software (Table 5).

[0260] Experimental results showed that all candidate antibodies possessed cross-binding activity with human and monkey CD25. Figure 4-5 Three candidate antibodies, Nb097-MA7, Nb097-MA11, and Nb097-MC10, exhibited strong binding activity to mouse CD25 antigen. Figure 6 ).

[0261] Table 5 - EC50 of single-domain antibody binding to human, monkey, and mouse CD25 protein

[0262]

[0263] Surface plasmon resonance (SPR) was used to further verify the binding activity of candidate antibodies Nb097-MA11 and Nb097-MC10 human, monkey, and mouse CD25 recombinant proteins.

[0264] Human CD25 (ACRO, Cat#ILA-H52H9), monkey CD25 (ACRO, Cat#ILA-C52H8), and mouse CD25 (ACRO, Cat#ILA-M52H9) were immobilized on CM5 chips. Serially diluted single-domain antibody solutions were flowed through the chip surface at a flow rate of 30 μl / min, with a binding time of 90 s. Then, running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA) with 0.005% Tween-20 (pH 7.4) was injected to dissociate the complexes, with a dissociation time of 210 s. The instruments used in the above experiments were Biacore 8K (Cytiva). The affinity data obtained through fitting calculations are summarized in the following table (Table 6).

[0265] Table 6 - Species cross-binding activity of SPR single-domain antibodies

[0266]

[0267] The experimental results showed that Nb097-MA11 and Nb097-MC10 possess cross-species binding activity, exhibiting good binding activity with human and monkey antigens, but relatively weaker binding activity with mouse antigens. These results are consistent with the ELISA validation results.

[0268] Example 5: Ligand IL-2 does not affect the binding of anti-Nb097-MC10 to CD25.

[0269] Prepare Nb097-MC10 solution with PBS to a final concentration of 1 μg / mL, add 100 μL / well to a 96-well microplate, and incubate overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μL / well of blocking buffer (PBST + 3% BSA), and block at room temperature for 1 hour. Simultaneously, mix serially diluted Human CD25 Protein, Fc Tag (ACRO, Cat#ILA-H5251) with 200 nM Human IL-2 Protein, Tag Free (ACRO, Cat#IL2-H5215), or 200 nM Nb097-MA11, or 200 nM Nb097-MC10, and incubate at room temperature for half an hour. Wash the microplate once with PBST, then add 100 μL / well of serially diluted Human CD25 Protein, Fc Tag (ACRO, Cat#ILA-H5251) or the incubated mixture. Incubate at room temperature for 0.5 hours, then wash three times with PBST. Next, add 100 μL / well of Mouse Anti-Human IgG Fc Antibody (50B4A9) [HRP] mAb solution (1:5000 dilution) and incubate at room temperature for 0.5 hours. Wash three times with PBST, blot dry, add 100 μL / well of TMB substrate solution (Beyotime, Cat#P0209), and incubate at room temperature for 5 to 30 minutes. Then add 100 μL / well of stop solution (Beyotime, Cat#P0215). Measure the absorbance at 450 nm in each well using a Molecular Devices (SpectraMax i3x) microplate reader. The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated. Figure 7 ).

[0270] Experimental results show that ligand IL-2 does not affect the binding of Nb097-MC10 to CD25, while Nb097-MA11 does affect the binding of Nb097-MC10 to CD25, indicating that Nb097-MA11 and Nb097-MC10 are in a competitive relationship.

[0271] Example 6: Anti-CD25 single-domain antibody blocks the binding of ligand IL-2 to CD25.

[0272] Prepare a human Goat Anti-Human IgG-Fc Secondary Antibody (Sino, Cat#SSA015) solution with PBS to a final concentration of 2 μg / mL. Add 100 μL / well to each well of a 96-well microplate and incubate overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 200 μL / well of blocking buffer (PBST + 3% BSA), and block at room temperature for 1 hour. Wash once with PBST, add 0.2 μg / 100 μL / well of Human CD25 Protein, Fc Tag (ACRO, Cat#ILA-H5251) solution, incubate at room temperature for 0.5 hours, and wash three times with PBST. Next, add 100 μL of a serially diluted solution of 200 nM anti-CD25 single-domain antibody mixed with biotinylated human IL-2 protein, His, and Avitag (ACRO, Cat#IL2-H82E4) and incubate at room temperature for 0.5 hours. Wash three times with PBST. Then add 100 μL / well of Streptavidin-HRP (Mabtech, Cat#3310-9) solution (1:1000 dilution) and incubate at room temperature for 0.5 hours. Wash three times with PBST, blot dry, add 100 μL / well of TMB substrate solution (Beyotime, Cat#P0209), and incubate at room temperature for 5 to 30 minutes. Then add 100 μL / well of stop solution (Beyotime, Cat#P0215). Measure the absorbance at 450 nm in each well using a Molecular Devices (SpectraMax i3x) microplate reader. The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated. Figure 8 ).

[0273] Experimental results show that, except for candidate antibody Nb097-CB4, other candidate antibodies Nb097-MA7, Nb097-MA11, Nb097-MC10, Nb097-MA5, Nb097-CG10, Nb097-CH10, Nb097-CG11, Nb097-CH3, and Nb097-CF6 all have ligand IL-2 blocking activity.

[0274] Example 7: Preparation of anti-CD25 single-domain antibody-toxin conjugate and in vitro tumor cell killing experiment

[0275] The purified Nb097-MA11 from Example 3 was nonspecifically labeled with the toxin MMAE (MedChemExpress, Cat#HY-100566) using the active group NHS ester, and then Nb097-MA11-MMAE was purified using PD10. An irrelevant antibody, Nb032-645-MMAE (which does not bind to the karpas299 surface receptor), was used as a negative control.

[0276] Karpas 299 cells with high CD25 expression were seeded into 96-well plates at a density of 5000 cells / 90 μl / well, with triplet wells, and incubated at 37°C. Test sample stock solutions were diluted with complete culture medium to prepare serially diluted 10-fold concentrations of test sample solutions. 10 μl / well of the above solution was added to each well of the cell plate, and the plates were incubated at 37°C. After 72 hours, cells were treated with the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit (Vazyme, Cat#D1101), and the chemiluminescence intensity of each well was measured using a microplate reader. The cell-killing effect of the drug was calculated.

[0277] Experimental results showed that Nb097-MA11-MMAE could be internalized into Karpas 299 cells, mediating effective tumor cell killing, while the negative control could not be internalized by Karpas 299 cells and could not mediate tumor cell killing. Figure 9 ).

[0278] Example 8: Humanization of Anti-CD25 Single-Domain Antibody

[0279] Humanization of anti-CD25 single-domain antibodies involves comparing the parent sequence with the human Germline database, defining the parent antibody's CDR and framework regions, and then designing sequences with different degrees of humanization based on the differences in the framework regions (Tables 7-8).

[0280] Table 7 - Summary of Nb097-MA11 Humanization Sequences

[0281]

[0282] Table 8 - Summary Table of Humanization Sequences for Nb097-MC10

[0283]

[0284] Example 9: Stability Analysis of Humanized Anti-CD25 Single-Domain Antibody

[0285] The C-terminus of the humanized VHH sequence was fused with a 6*his tag or a flag tag. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for expression for 7 days, and VHH was purified by affinity chromatography.

[0286] The aggregate formation tendency of humanized VHH was analyzed by HPLC-SEC. The chromatographic column used in the experiment was an XBridge BEH. The SEC filter was 3.5 μm and 7.8 × 300 mm (Waters, 186007640), and the flow rate was set to 0.8 mL / min. The detection wavelength was 280 nm. The detection results are shown in Table 9.

[0287] Table 9 - Summary of Results from SEC-HPLC for Humanized Antibodies

[0288]

[0289] Note: " / " represents none.

[0290] The thermal stability of humanized VHH was further analyzed using differential scanning fluorescence (DSF). An ABI 7500 Fast Real-Time PCR instrument was used, with the experiment selected as melting curve analysis in continuous mode, and the scanning temperature ranging from 30℃ to 95℃. The temperature corresponding to the first peak and trough of the melting curve derivative was determined as the denaturation temperature Tm1 of the protein, the temperature corresponding to the second peak and trough as Tm2, ​​and the temperature corresponding to the third peak and trough as Tm3. The detection results are shown in Table 10.

[0291] The results showed that, except for Nb097-hMA11-VHH2, Nb097-hMA11-VHH4, and Nb097-hMA11-VHH5, T m1 Aside from slightly higher than the parent, other humanized VHHs have T m1 All showed varying degrees of reduction.

[0292] Table 10 - Summary of DSF Results for Humanized Antibodies

[0293] Sample Name Tm 1 (℃) Tm 2 (℃) Tm 3 (℃) Nb097-MA11 66.47 / / Nb097-hMA11-VHH1 65.73 / / Nb097-hMA11-VHH2 67.4 / / Nb097-hMA11-VHH3 53.36 69.01 / Nb097-hMA11-VHH4 66.85 / / Nb097-hMA11-VHH5 66.87 / / Nb097-hMA11-VHH6 64.96 / / Nb097-hMA11-VHH7 61.13 / / Nb097-hMA11-VHH8 62.77 / / Nb097-hMA11-VHH9 56.78 / / Nb097-hMA11-VHH10 58.66 / / Nb097-MC10 62.36 / / Nb097-hMC10-VHH1 53.6 / / Nb097-hMC10-VHH2 54.62 / / Nb097-hMC10-VHH3 59.45 / / Nb097-hMC10-VHH4 57.75 / / Nb097-hMC10-VHH5 59.51 / / Nb097-hMC10-VHH6 59.95 / / Nb097-hMC10-VHH7 56.14 / / Nb097-hMC10-VHH8 49.23 / /

[0294] Example 10: Analysis of the human CD25 protein binding activity of the humanized anti-CD25 single-domain antibody

[0295] The binding activity of humanized VHH to human CD25 protein (ACRO, Cat#ILA-H5251) was analyzed by surface plasmon resonance (SPR). The instrument used was a biacore T200 (Cytiva). The results are shown in Table 11. Affinity tests showed that all humanized VHH bound to human CD25 protein. Except for Nb097-hMA11-VHH5, whose binding activity was slightly lower than the parent antibody, the binding activities of the other antibodies were comparable to or higher than the parent antibody.

[0296] Table 11 - Summary of SPR Results

[0297]

[0298]

[0299] Example 11: Cell binding activity analysis of humanized anti-CD25 single-domain antibody

[0300] Cell binding assays used Karpas 299 or MJ cells with high hCD25 expression as target cells, and humanized VHH cells were tagged with a flag at the C-terminus.

[0301] The experimental method for VHH-flag-form humanized antibody cell binding assay is as follows:

[0302] 1) Cell Plating: Transfer Karpas299 or MJ cells (both tumor cell lines highly express CD25) from culture flasks to centrifuge tubes. Centrifuge to remove supernatant, resuspend in culture medium, count, and adjust cell density to 1×10⁶ cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a pipette. Centrifuge at 300 g / min for 5 minutes. Discard the supernatant.

[0303] 2) Addition of candidate antibody dilution buffer: Dilute the antibody with FACS buffer containing 2% FBS to prepare eight concentration gradients of 20 μg / ml. Use a 12-channel 100 μL pipette to add 100 μL of antibody dilution buffer to each well of a 96-well cell plate. Mix well and incubate at 4°C for 1 hour.

[0304] 3) Addition of secondary antibody: Dilute the PE-labeled anti-DYKDDDK tag antibody (Biolegend, Cat#637310) 1:100 with FACS buffer containing 2% FBS. Centrifuge the cell culture plate to remove the supernatant. Using a 100 μL 12-channel pipette, add 100 μL of the secondary antibody dilution buffer to the cell culture plate. Incubate the cell culture plate at 4°C for 30 minutes. Centrifuge to remove the supernatant, wash the plate twice with FACS buffer, and resuspend the cells in 120 μL of FACS buffer in each well.

[0305] 4) Data acquisition: Turn on the flow cytometer and after the instrument has finished cleaning, measure the average fluorescence intensity of each sample.

[0306] The S-curve was fitted with a 4-parameter equation using GraphPad Prism 10 software, and the combined EC50 value was calculated. The results are as follows: Figure 10-11 As shown, the experimental results indicate that all humanized antibodies can bind to human CD25 expressed on the cell surface. Among them, the cell-binding activities of Nb097-hMA11-VHH1, Nb097-hMA11-VHH7, Nb097-hMA11-VHH8, Nb097-hMA11-VHH9, and Nb097-hMA11-VHH10 are comparable to those of the parent antibody Nb097-MA11; while the cell-binding activities of Nb097-hMC10-VHH1, Nb097-hMC10-VHH2, Nb097-hMC10-VHH3, Nb097-hMC10-VHH4, and Nb097-hMC10-VHH6 are better than those of the parent antibody Nb097-MC10.

[0307] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A single-domain antibody against CD25, characterized in that, The single-domain antibody has three complementarity-determining regions (CDRs) derived from the VHH chain shown in the following amino acid sequences: SEQ ID NO: 1-17; The CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule.

2. The single-domain antibody as described in claim 1, characterized in that, CDR1, CDR2 and CDR3 are selected from the following group: (a1) CDR1 with amino acid sequence as shown in SEQ ID NO:18, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:20; (a2) CDR1 with amino acid sequence as shown in SEQ ID NO:18, CDR2 with amino acid sequence as shown in SEQ ID NO:19, and CDR3 with amino acid sequence as shown in SEQ ID NO:21; (a3) CDR1 with amino acid sequence as shown in SEQ ID NO:22, CDR2 with amino acid sequence as shown in SEQ ID NO:23, and CDR3 with amino acid sequence as shown in SEQ ID NO:24; (a4) CDR1 with amino acid sequence as shown in SEQ ID NO:25, CDR2 with amino acid sequence as shown in SEQ ID NO:23, and CDR3 with amino acid sequence as shown in SEQ ID NO:26; (a5) CDR1 with amino acid sequence as shown in SEQ ID NO:27, CDR2 with amino acid sequence as shown in SEQ ID NO:28, and CDR3 with amino acid sequence as shown in SEQ ID NO:29; (a6) CDR1 with amino acid sequence as shown in SEQ ID NO:30, CDR2 with amino acid sequence as shown in SEQ ID NO:31, and CDR3 with amino acid sequence as shown in SEQ ID NO:32; (a7) CDR1 with amino acid sequence as shown in SEQ ID NO:30, CDR2 with amino acid sequence as shown in SEQ ID NO:31, and CDR3 with amino acid sequence as shown in SEQ ID NO:33; (a8) CDR1 with amino acid sequence as shown in SEQ ID NO:34, CDR2 with amino acid sequence as shown in SEQ ID NO:35, and CDR3 with amino acid sequence as shown in SEQ ID NO:36; (a9) CDR1 with amino acid sequence as shown in SEQ ID NO:37, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:39; (a10) CDR1 with amino acid sequence as shown in SEQ ID NO:40, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:41; (a11) CDR1 with amino acid sequence as shown in SEQ ID NO:37, CDR2 with amino acid sequence as shown in SEQ ID NO:38, and CDR3 with amino acid sequence as shown in SEQ ID NO:42; (a12) CDR1 with amino acid sequence as shown in SEQ ID NO:43, CDR2 with amino acid sequence as shown in SEQ ID NO:44, and CDR3 with amino acid sequence as shown in SEQ ID NO:45; (a13) CDR1 with amino acid sequence as shown in SEQ ID NO:46, CDR2 with amino acid sequence as shown in SEQ ID NO:44, and CDR3 with amino acid sequence as shown in SEQ ID NO:47; (a14) CDR1 with amino acid sequence as shown in SEQ ID NO:43, CDR2 with amino acid sequence as shown in SEQ ID NO:48, and CDR3 with amino acid sequence as shown in SEQ ID NO:49; (a15) CDR1 with amino acid sequence as shown in SEQ ID NO:50, CDR2 with amino acid sequence as shown in SEQ ID NO:51, and CDR3 with amino acid sequence as shown in SEQ ID NO:52; (a16) CDR1 with amino acid sequence as shown in SEQ ID NO:53, CDR2 with amino acid sequence as shown in SEQ ID NO:54, and CDR3 with amino acid sequence as shown in SEQ ID NO:55; (a17) CDR1 with amino acid sequence as shown in SEQ ID NO:56, CDR2 with amino acid sequence as shown in SEQ ID NO:57, and CDR3 with amino acid sequence as shown in SEQ ID NO:

58.

3. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the single-domain antibody of claim 1; and (ii) Optional tag sequences to assist in expression and / or purification.

4. A polynucleotide, characterized in that, The polynucleotide encodes the single-domain antibody of claim 1.

5. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or the genome is integrated with the polynucleotide of claim 4.

7. A method for generating anti-CD25 single-domain antibodies, characterized in that, Including the following steps: (a) Culturing the host cells of claim 6 under conditions suitable for generating single-domain antibodies to obtain a culture containing the anti-CD25 single-domain antibody; and (b) Isolate or recover the anti-CD25 single-domain antibody from the culture.

8. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The anti-CD25 single-domain antibody as described in claim 1; and (b) A conjugation portion conjugated to the single-domain antibody, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

9. The use of the single-domain antibody as claimed in claim 1, the recombinant protein as claimed in claim 3, or the immunoconjugate as claimed in claim 8, characterized in that, Used in the preparation of pharmaceuticals, reagents, test plates, or kits; The reagents, detection plates, or kits are used to detect CD25 in samples; The agent is used to prevent and / or treat CD25-overexpressing tumors, organ transplant rejection, and autoimmune diseases.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the single-domain antibody as claimed in claim 1, the recombinant protein as claimed in claim 3, or the immunoconjugate as claimed in claim 8; and (ii) Pharmaceutically acceptable carriers.

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