Anti-CD25 nano antibody or antigen binding fragment thereof and application thereof

Abstract

The invention discloses an anti-CD25 nano antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of biology. The anti-CD25 nano antibody or the antigen binding fragment thereof can be specifically bound with CD25, and can not block the binding of IL-2 and CD25, thereby being beneficial to reducing the number of Treg cells and enhancing the anti-tumor capability, and providing new possibility for the treatment and / or prevention of tumors.

Description

[0001] Priority Claim

[0002] This application claims the priority of a Chinese patent application with the application number 202311797967.2, the filing date of December 25, 2023, and the invention title of "Anti-CD25 Nanobody or Its Antigen-Binding Fragment and Their Applications", the entire content of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to an anti-CD25 nanobody or its antigen-binding fragment and their applications. Background Art

[0004] The receptor of IL-2 (Interleukin-2) is a heterotrimer composed of three chains, namely α (CD25), β (CD122), and γ (CD132). CD25 is the α receptor of IL-2 (Interleukin-2 receptor subunit alpha), which is a 55 kDa type I transmembrane protein composed of 272 amino acids. The affinity of CD25 alone for IL-2 is relatively low (KD is 10 -8 M), and it does not transmit intracellular signals. CD122 and CD132 are necessary for IL-2 to activate downstream signaling pathways. When IL-2 binds to CD122 and CD132 simultaneously, it can promote heterodimerization, and finally form a tetramer (IL2Rα / β / γ and IL-2), and trigger the JAK / STAT5, PI3K / Akt / , and MAPK signaling pathways, which play important roles in cell growth, survival, differentiation, and immunity. CD25 is mainly expressed on the surfaces of tissues such as the spleen and lymph nodes, and cells such as Treg cells (regulatory T cells), basophils, and activated T cells. It is a class of specific protein molecules that are simultaneously expressed on the surfaces of Teff cells (effector T cells) and Treg cells.

[0005] Once CD25 binds to IL-2 on the surface of Teff, it can effectively activate T cells and promote the proliferation of T cells. When CD25 on the surface of Treg cells binds to IL-2 competitively, it can inhibit the proliferation of Teff cells. The key role of IL-2 in the survival and function of Teff cells, due to the simultaneous expression of CD25 on the surface of Teff cells, the previously developed anti-CD25 monoclonal antibodies targeting Treg cells have limited anti-cancer activity because they block the IL-2 activity in Teff cells. Anti-CD25 monoclonal antibodies that do not interfere with IL-2 signal transduction (retaining IL-2 signal transduction on Teff cells) can induce stronger anti-tumor activity. Therefore, it is very necessary to develop antibodies that do not block the IL-2 signaling pathway.

[0006] In addition, Treg cells are an important part of the body's immune regulation, helping the body achieve immune homeostasis. However, the large presence of Treg cells in tumors inhibits the activation and proliferation of effector T cells, leading to an immunosuppressive environment that promotes tumor growth. As one of the specific markers on the surface of Treg cells, the expression level of CD25 on Treg cells is much higher than that on other immune cells. Drugs targeting CD25 can be designed to kill Treg cells through ADCC and ADCP effects to play an anti-tumor role.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The object of the present invention is to provide a nanobody or its antigen-binding fragment that can specifically bind to CD25 and its application.

[0009] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0010] In the first aspect, a nanobody against CD25 or its antigen-binding fragment is provided, which comprises complementary determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region;

[0011] The complementary determining region HCDR1 comprises the amino acid sequence shown as follows: X1-Y, where X1 is N, I, M, H, L, or Y;

[0012] The complementary determining region HCDR2 comprises the amino acid sequence shown as follows: X2-S-X3-G-X4, or Y-S-D-S-S-X5, or S-W-I-G-G-S; X2 is T or R, X3 is G or D, X4 is S or T, and X5 is Y, A, or H;

[0013] The complementary determining region HCDR3 comprises the amino acid sequence shown as follows: D-F-E-D-X6-S-G-W-Y-R-R-E-P-P-L-D, or A-R-G-S-G-S-Y-Y-P-F-D-D, or H-R-G-E-Y-Y-S-D-W-R-F-D-D-M-D, where X6 is D or S.

[0014] In the second aspect, an anti-CD25 antibody or its antigen-binding fragment is further provided, and the anti-CD25 antibody or its antigen-binding fragment contains the anti-CD25 nanobody or its antigen-binding fragment of the first aspect, or competitively binds to CD25 with the anti-CD25 nanobody or its antigen-binding fragment described in the first aspect, or the epitope that binds to the CD25 antigen is the same as that of the anti-CD25 nanobody or its antigen-binding fragment described in the first aspect.

[0015] In a third aspect, the present invention provides a biomaterial, which comprises any one of the following (i) to (vi):

[0016] (i) A multispecific antibody, which contains the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect;

[0017] (ii) A chimeric antigen receptor, which comprises an extracellular region, and the extracellular region comprises an antigen-binding domain; and, the antigen-binding domain contains the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect;

[0018] (iii) A nucleic acid molecule, which encodes the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or the multispecific antibody described in (i), or the chimeric antigen receptor described in (ii);

[0019] (iv) A vector, which carries the nucleic acid molecule described in (iii);

[0020] (v) A recombinant cell, which is characterized in that it contains the nucleic acid molecule described in (iii), or the vector described in (iv), or expresses the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or expresses the multispecific antibody described in (i), or expresses the chimeric antigen receptor described in (ii);

[0021] (vi) An engineered immune effector cell, which expresses the chimeric antigen receptor described in (ii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (ii).

[0022] In a fourth aspect, a pharmaceutical composition is further provided, which contains the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or the biomaterial described in the third aspect.

[0023] In a fifth aspect, the present invention provides the use of the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or the biomaterial described in the third aspect, or the pharmaceutical composition described in the fourth aspect in any one of the following:

[0024] (Ⅰ) Detecting CD25 or cells expressing CD25;

[0025] (Ⅱ) Preparing a product for detecting CD25 or cells expressing CD25;

[0026] (Ⅲ) Depleting Treg cells;

[0027] (Ⅳ) Treating, preventing or alleviating diseases, disorders or conditions related to tumors, inflammation, autoimmune diseases, infections, immune rejection of organ transplantation.

[0028] In a sixth aspect, there is also provided a method for treating, preventing or alleviating a disease, disorder or condition, including tumors, inflammation, autoimmune diseases, infections, immune rejection caused by organ transplantation; the method includes administering to a subject a therapeutically effective amount of the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or the biomaterial of the third aspect, or the pharmaceutical composition of the fourth aspect.

[0029] In a seventh aspect, there is also provided a kit for detecting CD25, which contains the anti-CD25 nanobody or its antigen-binding fragment described in the foregoing first or second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is a schematic structural diagram of the nanobody in the embodiment of the present invention;

[0032] Figure 2 It is the binding activity of the anti-CD25 nanobodies (R2449, R2453, R2454, R2455) to C8166 cells determined by flow cytometry fluorescence-activated cell sorting technology in Example 2;

[0033] Figure 3 It is the binding activity of the anti-CD25 nanobodies (R2456, R2457, R2459, R2460) to C8166 cells determined by flow cytometry fluorescence-activated cell sorting technology in Example 2;

[0034] Figure 4 It is the binding activity of the anti-CD25 nanobodies (R2724, R2725, R2726, R2727, R2728) to C8166 cells determined by flow cytometry fluorescence-activated cell sorting technology in Example 2;

[0035] Figure 5 It is the binding activity of the anti-CD25 nanobodies (R2729, R2730, R2731, R2732, R2733) to C8166 cells determined by flow cytometry fluorescence-activated cell sorting technology in Example 2;

[0036] Figure 6The binding activity of CD25 nanobodies (R2449, R2453, R2454, R2455) to block IL-2Fc from binding to C8166 cells, as determined by flow cytometry fluorescence-activated cell sorting for Example 3;

[0037] Figure 7 The binding activity of CD25 nanobodies (R2456, R2457, R2459, R2460) to block IL-2Fc from binding to C8166 cells, as determined by flow cytometry fluorescence-activated cell sorting for Example 3;

[0038] Figure 8 The binding activity of CD25 nanobodies (R2724, R2725, R2726, R2727, R2728) to block IL-2Fc from binding to C8166 cells, as determined by flow cytometry fluorescence-activated cell sorting for Example 3;

[0039] Figure 9 The binding activity of CD25 nanobodies (R2729, R2730, R2731, R2732, R2733) to block IL-2Fc from binding to C8166 cells, as determined by flow cytometry fluorescence-activated cell sorting for Example 3;

[0040] Figure 10 The binding activity of CD25 nanobodies (R2449, R2453, R2454, R2455) to CD25-his protein, as determined by ELISA for Example 4;

[0041] Figure 11 The binding activity of CD25 nanobodies (R2456, R2457, R2459, R2460) to CD25-his protein, as determined by ELISA for Example 4;

[0042] Figure 12 The binding activity of CD25 nanobodies (R2724, R2725, R2726, R2727, R2728) to CD25-his protein, as determined by ELISA for Example 4;

[0043] Figure 13 The binding activity of CD25 nanobodies (R2729, R2730, R2731, R2732, R2733) to CD25-his protein, as determined by ELISA for Example 4;

[0044] Figure 14 The binding activity of CD25 nanobodies (R2449, R2453, R2454, R2455) to block the binding of IL-2Fc to CD25-his protein, as determined by ELISA for Example 5;

[0045] Figure 15 The binding activity of CD25 nanobodies (R2456, R2457, R2459, R2460) to block the binding of IL-2Fc to CD25-his protein as determined by ELISA technology in Example 5;

[0046] Figure 16 The binding activity of CD25 nanobodies (R2724, R2725, R2726, R2727, R2728) to block the binding of IL-2Fc to CD25-his protein as determined by ELISA technology in Example 5;

[0047] Figure 17 The binding activity of CD25 nanobodies (R2729, R2730, R2731, R2732, R2733) to block the binding of IL-2Fc to CD25-his protein as determined by ELISA technology in Example 5;

[0048] Figure 18 The binding activity of CD25 nanobodies to recombinant cells of human CD25 as determined by flow cytometry fluorescence-activated cell sorting technology in Example 6;

[0049] Figure 19 The binding activity of CD25 nanobodies to recombinant cells of murine CD25 as determined by flow cytometry fluorescence-activated cell sorting technology in Example 6;

[0050] Figure 20 The binding activity of CD25 nanobodies to recombinant cells of simian CD25 as determined by flow cytometry fluorescence-activated cell sorting technology in Example 6. Detailed implementation manners

[0051] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] The articles "a / an" and "the" in the present invention include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0053] In the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0054] In the present invention, the term "CD25" refers to the α chain of IL-2R (IL-2 receptor). Unless otherwise stated, the term includes human CD25, subtypes, and species homologs expressed by cells naturally or transfected with the CD25 gene. The term also includes artificially modified CD25, which includes but is not limited to polypeptides or proteins after mutation, truncation, or fusion with other domains, and retains the necessary antigenic epitopes for antibody binding. The anti-CD25 antibody or its antigen-binding fragment provided by the present invention can specifically bind to CD25.

[0055] In the present invention, the term "IL-2" refers to interleukin 2, a cytokine that regulates the activity of the immune system. Unless otherwise stated, the term includes human IL-2, subtypes, and species homologs expressed by cells naturally or transfected with the IL-2 gene. The term also includes artificially modified IL-2, which includes but is not limited to polypeptides or proteins after mutation, truncation, or fusion with other domains, and retains the necessary antigenic epitopes for antibody binding. The anti-IL-2 antibody or its antigen-binding fragment provided by the present invention can specifically bind to IL-2.

[0056] In the present invention, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, nanobodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Generally, a natural intact antibody consists of two heavy (H) chains and two light (L) chains. According to the presence of α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five main classes or isotypes: IgA, IgD, IgE, IgG, and IgM. Several main antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc. Each heavy chain is composed of a variable region (VH) and the first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively). Mammalian light chains can be divided into λ or κ, and each light chain is composed of a variable region (VL) and a constant region (CL). The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain usually contains three hypervariable regions, called "complementary determining regions (CDRs)". Among them, the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) each consist of three complementary determining regions connected by 4 framework regions (FRs). Usually, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the CDRs and FRs numbered as follows in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0057] In the present invention, the term "nanobody" is also called "single-domain antibody", which refers to a heavy-chain antibody lacking a light chain (such as: derived from camelids or sharks), and a single-domain antibody obtained by cloning its variable region. It is the smallest functional antigen-binding fragment, with a relative molecular mass (Mr) of only about 15,000. Nanobodies have the characteristics of small molecular mass, strong stability, good solubility, easy expression, and low immunogenicity.

[0058] In the present invention, the term "antigen-binding fragment" refers to a substance that contains all or part of the CDRs of an antibody, lacks at least some of the amino acids present in the full-length chains, but is still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies). The above antigen-binding fragments are capable of binding the same antigen as the parental antibody.

[0059] In the present invention, the "Fab" of an antibody refers to a part of the antibody composed of a single light chain (including the variable region and the constant region) and the variable region and the first constant region of a single heavy chain bound together by a disulfide bond. The "Fab' fragment" refers to a Fab fragment that contains a part of the hinge region. The "F(ab')2" refers to a dimer of Fab'. The "Fv fragment" is composed of the variable region of a single light chain and / or the variable region of a single heavy chain bound together. The "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by directly connecting the variable region of the light chain and the variable region of the heavy chain or by connecting them through a peptide linker sequence. The "minimal recognition unit of an antibody" refers to a structure that contains only a single CDR in the variable region. Although the minimal recognition unit has a small molecular weight and low affinity, it has the ability to bind to an antigen.

[0060] The CDR boundaries of the antibodies or antigen-binding fragments thereof in the present invention can be defined or identified according to the IMGT, Kabat, Chothia, AbM, Contact definition methods, and CDRs defined in other acceptable ways in the art also fall within the protection scope of the present invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); R.M. MacCallum et al.,. Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, A.C.R. Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig super family V-like domains, Developmental and Comparative Immunology 27 (2003) 55–77).

[0061] In the present invention, terms such as "mutant Fc region", "Fc mutant", "Fc region carrying a mutation", "mutated Fc region", "Fc region variant", "Fc variant", "variant Fc region", and "mutated Fc region" can be used interchangeably, and refer to an Fc region that contains at least one amino acid modification and is different from the native sequence Fc region / wild-type Fc region.

[0062] In the present invention, the term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs typically have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0063] In the present invention, the term "percentage identity" refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Alignment for the percentage of amino acid sequence identity can be performed in various ways in the art, such as using software well-known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment of the full length of the comparison sequences.

[0064] In the present invention, the term "specifically binds" or "specifically binding" refers to a non-random binding reaction between two molecules, for example, the reaction between an antibody and an antigen. In some specific embodiments, for example, it is determined according to fluorescence-activated cell sorting (FACS) technology.

[0065] In the present invention, the term "anti-CD25 antibody" refers to an antibody that can specifically bind to CD25. In some specific embodiments, the "anti-CD25 antibody" is an antibody that specifically binds to human or monkey CD25.

[0066] In the present invention, the term "affinity" or "avidity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen. The strength or affinity of an immunological binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The KD can be determined by using any conventional method known in the art, including but not limited to Biacore assays, Octet methods, microscale thermophoresis, HPLC-MS methods, and fluorescence-activated cell sorting techniques.

[0067] The binding of the nanobody, antibody, or any antigen-binding fragment provided by the present invention to CD25 can also be characterized by the "half maximal effective concentration (EC50)", which refers to the concentration of a drug or antibody that can achieve 50% of the maximum biological effect after a specific exposure time. Generally, the smaller the EC50, the better the affinity, indicating that binding to the target protein can occur at a lower concentration. The EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting techniques, and other binding assays).

[0068] In the present invention, the term "epitope" refers to any antigenic determinant on an antigen that is bound by the paratope of an antibody. An antigenic determinant is usually a special chemical group with a certain composition and structure. Epitopes can be linear (i.e., continuous) or conformational (i.e., including spatially separated amino acid residues, non-continuous). Epitopes define the minimum binding site of an antibody and are thus the specific targets of an antibody or its antigen-binding fragment. Epitopes can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structure determination methods (e.g., nuclear magnetic resonance spectroscopy).

[0069] In the present invention, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0070] The "chimeric antigen receptor" in the present invention, CAR, is an artificial receptor that mimics the function of TCR and is composed of an extracellular domain, a transmembrane domain, and an intracellular signaling domain connected in sequence. When the antigen (receptor) on the surface of tumor cells binds to the antibody (ligand) of the chimeric antigen receptor, signals can be transmitted to the intracellular through the hinge region and transmembrane region. The intracellular signaling domain then converts the signals into activation signals to activate effector cells. The effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also expand, further enhancing the immune killing effect. The extracellular domain generally consists of a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding antigens and a hinge region (Hinge) that plays a connecting role. The single-chain variable fragment is the antigen-binding domain of CAR, and this domain determines the specificity and function of CAR-immune cells. The hinge region is the extracellular structural region of CAR that connects the single-chain variable fragment and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells. The hinge regions of most CARs are derived from the hinge of IgG or the extracellular regions of CD8α / CD28. The type and length of the hinge region have an important impact on the functional activities of CAR. The transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8, CD28, and CD3ζ or their derivatives. The selection of the transmembrane domain affects the activation degree of the CAR structure in cell functions. The intracellular domain is composed of a co-stimulatory domain and a signal transduction domain. The co-stimulatory domain usually comes from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The signal transduction domain is usually the T cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain.

[0071] The CARs (including their functional parts and functional variants) of the present invention can be obtained by methods known in the art, for example, by any suitable method for preparing polypeptides or proteins.

[0072] In the present invention, the term "engineered immune effector cells" refers to immune effector cells expressing CAR or immune effector cells modified with CAR. Among them, "immune effector cells" generally refer to cells participating in immune responses, for example, cells promoting immune effector responses. Exemplary immune effector cells include but are not limited to T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (Natural killer T cell), dendritic cells, granulocytes, B cells, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells.

[0073] In the present invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, and nucleic acid molecules include ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical or biochemical modifications, unnatural or derivatized nucleobases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding optionally encodes the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably.

[0074] In an alternative embodiment, the nucleic acid molecule is RNA or DNA, the nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid molecule is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. It is preferably DNA when incorporated into a vector.

[0075] In the present invention, the term "vector" refers to a vehicle into which a genetic element (such as the aforementioned nucleic acid molecule) can be operatively inserted and which enables the genetic element to be expressed, for example, to produce a protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce or transfect host cells so that the genetic elements they carry are expressed in the host cells. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. Vectors can contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, vectors can also contain an origin of replication. Vectors can also include components that assist their entry into cells, including but not limited to, virus particles, liposomes or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vectors provided by the present invention (such as expression vectors) contain the nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof according to the present invention, at least one promoter operably linked to the nucleic acid sequence (for example, SV40, CMV, EF-1α), and at least one selection marker.

[0076] In the present invention, the terms "purified" or "isolated" associated with a polypeptide or nucleic acid mean that the polypeptide or nucleic acid is not in its natural medium or natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, for example, if it is naturally occurring, from its natural environment. In connection with nucleic acids, the terms isolated or purified indicate, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0077] In the present invention, the term "recombinant cell" refers to a cell that can or has been introduced with an exogenous polynucleotide and / or vector. The exogenous polynucleotide may or may not be integrated into the genome of the "recombinant cell". When a vector is contained in the recombinant cell, the vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. By culturing the recombinant cells, the corresponding antibody can be obtained. Available mammalian cells can be CHO cells, etc.

[0078] In the present invention, the term "pharmaceutical composition" is present in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered. In some specific embodiments, the antibody contained in the above pharmaceutical composition or the expressed antibody can specifically target and bind to CD25.

[0079] In the present invention, a "pharmaceutically acceptable carrier" may include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are physiologically compatible.

[0080] In the present invention, the term "subject" or "patient" refers to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, alpacas, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0081] In the present invention, the term "diagnosis" refers to the identification of a pathological condition, disease or disorder, such as the identification of a CD25-related disease, or the identification of a subject with a CD25-related disease who may benefit from a specific treatment regimen. In some embodiments, the diagnosis includes the identification of an abnormal level or activity of CD25. In some embodiments, the diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0082] In the present invention, the term "effective amount" refers to a therapeutically effective amount, an amount sufficient to reduce or ameliorate the severity and / or duration of a disorder and / or one or more of its symptoms; prevent the progression of a disease; cause regression of a disease; prevent the recurrence, development or progression of one or more symptoms associated with a disease; detect a disease; or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the antibody or antigen-binding fragment thereof described in the present invention depends on a variety of factors well known in the art, such as body weight, age, medical history, current treatment, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor development. Those skilled in the art (e.g., physicians or veterinarians) can proportionally reduce or increase the dose according to these or other conditions or requirements.

[0083] In a first aspect, there is provided an anti-CD25 nanobody or an antigen-binding fragment thereof, the anti-CD25 nanobody or antigen-binding fragment thereof comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region.

[0084] Complementarity determining region HCDR1 comprises the amino acid sequence shown below: X1-Y, where X1 is N, I, M, H, L or Y;

[0085] Complementarity determining region HCDR2 comprises the amino acid sequence shown below: X2-S-X3-G-X4, where X2 is T or R, X3 is G or D, X4 is S or T; or Y-S-D-S-S-X5 (SEQ ID NO.134), where X5 is Y, A or H; or S-W-I-G-G-S (SEQ ID NO.71);

[0086] Complementarity determining region HCDR3 comprises the amino acid sequence shown below: D-F-E-D-X6-S-G-W-Y-R-R-E-P-P-L-D (SEQ ID NO.135), where X6 is D or S; or A-R-G-S-G-S-Y-Y-P-F-D-D (SEQ ID NO.85), or H-R-G-E-Y-Y-S-D-W-R-F-D-D-M-D (SEQ ID NO.94).

[0087] In an alternative embodiment, the amino acid sequence of HCDR1 comprises the amino acid residues NY, exemplary HCDR1s include but are not limited to the sequences shown in any one of SEQ ID NOs. 14, 15, 16, 39 or 43.

[0088] In an alternative embodiment, the amino acid sequence of HCDR1 comprises the amino acid residues IY, exemplary HCDR1s include but are not limited to the sequences shown in any one of SEQ ID NOs. 23, 24, 25, 29, 30, 31, 32, 33, 35 or 36.

[0089] In alternative embodiments, the amino acid sequence of HCDR1 includes the amino acid residues MY, and exemplary HCDR1s include, but are not limited to, the sequences shown in any of SEQ ID NO.26, 27, 28, 38, or 42.

[0090] In alternative embodiments, the amino acid sequence of HCDR1 includes the amino acid residues HY, and exemplary HCDR1s include, but are not limited to, the sequences shown in any of SEQ ID NO.11, 12, 13, 34, or 40.

[0091] In alternative embodiments, the amino acid sequence of HCDR1 includes the amino acid residues LY, and exemplary HCDR1s include, but are not limited to, the sequences shown in any of SEQ ID NO.20, 21, 22, 37, or 41.

[0092] In alternative embodiments, the amino acid sequence of HCDR1 includes the amino acid residues YY, and exemplary HCDR1s include, but are not limited to, the sequences shown in any of SEQ ID NO.17, 18, 19, 44, or 45.

[0093] In alternative embodiments, according to the IMGT definition, the complementarity determining region HCDR1 includes the sequences shown in SEQ ID NO.12, 15, 18, 21, 24, 27, or 32.

[0094] In alternative embodiments, HCDR2 includes the amino acid sequence shown in SEQ ID NO.84 (YSDSSY), and exemplary HCDR2s include, but are not limited to, the sequences shown in any of SEQ ID NO.55, 56, 63, 81, or 84.

[0095] In alternative embodiments, HCDR2 includes the amino acid sequence shown in SEQ ID NO.77 (TSGGS), and exemplary HCDR2s include, but are not limited to, the sequences shown in any of SEQ ID NO.46, 47, 59, 60, 64, 67, 74, 75, or 77.

[0096] In alternative embodiments, HCDR2 includes the amino acid sequence shown in SEQ ID NO.76 (TSDGS), and exemplary HCDR2s include, but are not limited to, the sequences shown in any of SEQ ID NO.58, 66, 72, 73, or 76.

[0097] In alternative embodiments, HCDR2 includes the amino acid sequence shown in SEQ ID NO.71 (SWIGGS), and exemplary HCDR2s include, but are not limited to, the sequences shown in any of SEQ ID NO.48, 49, 50, 71, or 78.

[0098] In alternative embodiments, HCDR2 comprises the amino acid sequence as shown in SEQ ID NO.82 (YSDSSA), and exemplary HCDR2 includes but is not limited to the sequences shown in any one of SEQ ID NO.51, 52, 61, 79, 82.

[0099] In alternative embodiments, HCDR2 comprises the amino acid sequence as shown in SEQ ID NO.70 (RSGGT), and exemplary HCDR2 includes but is not limited to the sequences shown in any one of SEQ ID NO.57, 65, 68, 69, 70.

[0100] In alternative embodiments, HCDR2 comprises the amino acid sequence as shown in SEQ ID NO.83 (YSDSSH), and exemplary HCDR2 includes but is not limited to the sequences shown in any one of SEQ ID NO.53, 54, 62, 80 or 83.

[0101] In alternative embodiments, according to the IMGT definition, the complementarity determining region HCDR2 comprises the sequences shown in SEQ ID NO.57, 58, 59, 60, 61, 62, 63 or 78.

[0102] In alternative embodiments, HCDR3 comprises the amino acid sequence as shown in SEQ ID NO.85 (ARGSGSYYPFDD), and exemplary HCDR3 includes but is not limited to the sequences shown in any one of SEQ ID NO.86, 96, 97, 103, 104.

[0103] In alternative embodiments, HCDR3 comprises the amino acid sequence as shown in SEQ ID NO.89 (DFEDDSGWYRREPPLD), and exemplary HCDR3 includes but is not limited to the sequences shown in any one of SEQ ID NO.90, 91, 98, 99, 100.

[0104] In alternative embodiments, HCDR3 comprises the amino acid sequence as shown in SEQ ID NO.94 (HRGEYYSDWRFDDMD), and exemplary HCDR3 includes but is not limited to the sequences shown in any one of SEQ ID NO.87, 88 or 98.

[0105] In alternative embodiments, HCDR3 comprises the amino acid sequence as shown in SEQ ID NO.92 (DFEDSSGWYRREPPLD), and exemplary HCDR3 includes but is not limited to the sequences shown in any one of SEQ ID NO.93, 101 or 102.

[0106] In an alternative embodiment, according to the IMGT definition, the complementarity-determining region HCDR3 of the anti-CD25 nanobody or its antigen-binding fragment heavy chain variable region comprises the sequence shown in SEQ ID NO.88, 97, 99, 100, 102 or 104.

[0107] In an alternative embodiment, the complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the anti-CD25 nanobody or its antigen-binding fragment heavy chain variable region are selected from any one of the following combinations:

[0108] (A) HCDR1 comprises the amino acid sequence shown as follows: X1-Y, where X1 is I or L; HCDR2 comprises the amino acid sequence shown as follows: X2-S-X3-G-X4, where X2 is T or R, X3 is G or D, and X4 is S or T; HCDR3 comprises the amino acid sequence shown as follows: D-F-E-D-X6-S-G-W-Y-R-R-E-P-P-L-D (SEQ ID NO.135), where X6 is D or S.

[0109] (B) HCDR1 comprises the amino acid sequence shown as follows: X1-Y, where X1 is N, H or Y; HCDR2 comprises the amino acid sequence shown as follows: Y-S-D-S-S-X5 (SEQ ID NO.134), where X5 is Y, A or H; HCDR3 comprises the amino acid sequence shown as follows: A-R-G-S-G-S-Y-Y-P-F-D-D (SEQ ID NO.85).

[0110] (C) HCDR1 comprises the amino acid sequence shown as follows: X1-Y, where X1 is M, HCDR2 comprises the amino acid sequence shown as follows: S-W-I-G-G-S (SEQ ID NO.71); HCDR3 comprises the amino acid sequence shown as follows: H-R-G-E-Y-Y-S-D-W-R-F-D-D-M-D (SEQ ID NO.94).

[0111] In an alternative embodiment, the complementarity-determining regions of the anti-CD25 nanobody or its antigen-binding fragment heavy chain variable region are selected from the following combinations:

[0112] (a) The amino acid sequence of HCDR1 comprises the amino acid residues NY, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO.84, and the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO.85;

[0113] (b) The amino acid sequence of HCDR1 comprises the amino acid residues IY, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO.77, and the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO.89;

[0114] (c) The amino acid sequence of HCDR1 includes the amino acid residues IY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.76, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89;

[0115] (d) The amino acid sequence of HCDR1 includes the amino acid residues MY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.71, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.94;

[0116] (e) The amino acid sequence of HCDR1 includes the amino acid residues HY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.84, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85;

[0117] (f) The amino acid sequence of HCDR1 includes the amino acid residues NY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.82, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85;

[0118] (g) The amino acid sequence of HCDR1 includes the amino acid residues LY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.70, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89;

[0119] (h) The amino acid sequence of HCDR1 includes the amino acid residues IY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.77, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.92;

[0120] (i) The amino acid sequence of HCDR1 includes the amino acid residues LY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.70, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89;

[0121] (j) The amino acid sequence of HCDR1 includes the amino acid residues YY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.83, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85.

[0122] In an alternative embodiment, according to the IMGT definition: the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the anti-CD25 nanobody or its antigen-binding fragment are selected from the following combinations:

[0123] (a’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.15, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.63, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.97;

[0124] (b’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.32, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.59, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.100;

[0125] (c’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.32, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.58, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.100;

[0126] (d’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.27, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.78, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.88;

[0127] (e’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.12, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.63, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.97;

[0128] (f’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.15, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.61, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.104;

[0129] (g’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.21, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.57, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.99;

[0130] (h’) The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.24, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.60, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.102;

[0131] (i’) The amino acid sequence of HCDR1 comprises the sequence shown in SEQ ID NO.21, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO.57, and the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO.100;

[0132] (j’) The amino acid sequence of HCDR1 comprises the sequence shown in SEQ ID NO.18, the amino acid sequence of HCDR2 comprises the sequence shown in SEQ ID NO.62, and the amino acid sequence of HCDR3 comprises the sequence shown in SEQ ID NO.97.

[0133] In a second aspect, there is provided an anti-CD25 nanobody or an antigen-binding fragment thereof, wherein the anti-CD25 nanobody or an antigen-binding fragment thereof contains complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy-chain variable region, and the complementarity-determining regions HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3 of the heavy-chain variable region shown in any one of SEQ ID NOs. 1 to 10;

[0134] The above HCDR1, HCDR2 and HCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition or the IMGT definition.

[0135] In an alternative embodiment, HCDR1, HCDR2 and HCDR3 of the anti-CD25 nanobody or an antigen-binding fragment thereof can be independently selected from Tables 1 to 10. Tables 1 to 10 show the CDR amino acid sequences defined according to different definition methods of exemplary nanobodies R2453, R2459, R2460, R2455, R2724, R2725, R2726, R2728, R2730 and R2732 (the amino acid sequences of the heavy-chain variable regions are shown in SEQ ID NOs. 1 to 10 respectively).

[0136] Table 1 CDR amino acid sequences of R2453 defined according to different definition methods

[0137]

[0138] Table 2 CDR amino acid sequences of R2459 defined according to different definition methods

[0139]

[0140] Table 3 CDR amino acid sequences of R2460 defined according to different definition methods

[0141]

[0142] Table 4 CDR Amino Acid Sequences Defined by Different Definition Methods for R2455

[0143]

[0144]

[0145] Table 5 CDR Amino Acid Sequences Defined by Different Definition Methods for R2724

[0146]

[0147] Table 6 CDR Amino Acid Sequences Defined by Different Definition Methods for R2725

[0148]

[0149] Table 7 CDR Amino Acid Sequences Defined by Different Definition Methods for R2726

[0150]

[0151] Table 8 CDR Amino Acid Sequences Defined by Different Definition Methods for R2728

[0152]

[0153]

[0154] Table 9 CDR Amino Acid Sequences Defined by Different Definition Methods for R2730

[0155]

[0156] Table 10 CDR Amino Acid Sequences Defined by Different Definition Methods for R2732

[0157]

[0158] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment contains a heavy chain framework region, and the heavy chain framework region is derived from at least one of a camelid-derived antibody, a murine-derived antibody, a human-derived antibody, a primate-derived antibody, or a mutant thereof.

[0159] In an alternative embodiment, the three CDRs derived from the heavy chain variable region of the anti-CD25 nanobody or its antigen-binding fragment are separated by flanking portions called framework regions (including HFR1, HFR2, HFR3, and HFR4), and the framework regions are more highly conserved than the CDRs and form a scaffold to support the highly variable loops.

[0160] In an alternative embodiment, the HFR1 comprises the amino acid sequence shown below: D-V-Q-L-Q-E-S-G-G-G-L-X7-Q-X8-G-G-S-L-R-L-S-C-A-A-S (SEQ ID NO.136), where X7 is V or A, and X8 is P or A.

[0161] In an alternative embodiment, the amino acid sequence of the HFR1 comprises the sequence shown in SEQ ID NO.105, 106 or 107.

[0162] In an alternative embodiment, the HFR2 comprises the amino acid sequence shown below:

[0163] W-X9-R-X 10 -A-P-X 11 -K-X 12 -X 13 -E (SEQ ID NO.137), where X9 is A, Y, F or L, X 10 is Q or R, X 11 is G or E,, X 12 is G, Q or E, X 13 is L or R.

[0164] In an alternative embodiment, the amino acid sequence of the HFR2 comprises the sequence shown in SEQ ID NO.115, 116, 117, 118 or 119.

[0165] In an alternative embodiment, the HFR3 comprises the amino acid sequence shown below:

[0166] R-F-T-I-S-R-D-N-A-K-N-T-X 14 -Y-L-X 15 -M-N-X 16 -L-K-P-E-D-T-A-X 17 -Y-Y-C (SEQID NO.138), X 14 is L or V, X 15 is Q or L, X 16 is S or T, X 17 is L or V.

[0167] In an alternative embodiment, the amino acid sequence of the HFR3 comprises the sequence shown in SEQ ID NO.125, 126, 127, 128 or 129.

[0168] In an alternative embodiment, the HFR4 comprises the amino acid sequence shown below: X 18 -G-X 19 -G-T-Q-V-T-V-S-S, X18 is R or W, X 19 is Q or K (SEQ ID NO.139).

[0169] In an alternative embodiment, the amino acid sequence of HFR4 includes, for example, SEQ ID NO.132, 133 or 122.

[0170] In an alternative embodiment, HFR1, HFR2, HFR3 and HFR4 comprise HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region shown in any one of SEQ ID NOs.1-10. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition or IMGT definition in Tables 1-10, and the FR regions of the corresponding definition methods are obtained according to the structure of the heavy chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3 and HFR4 are selected from any one of the following combinations:

[0171] (a”) HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy chain variable region shown in SEQ ID NO.1, and comprise the amino acid sequences shown in SEQ ID NOs.107, 113, 130 and 132 respectively.

[0172] (b”) HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy chain variable region shown in SEQ ID NO.2, and comprise the amino acid sequences shown in SEQ ID NOs.107, 112, 123 and 122 respectively.

[0173] (c”) HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy chain variable region shown in SEQ ID NO.3, and comprise the amino acid sequences shown in SEQ ID NOs.107, 109, 121 and 122 respectively.

[0174] (d”) HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy chain variable region shown in SEQ ID NO.4, and comprise the amino acid sequences shown in SEQ ID NOs.106, 108, 131 and 133 respectively.

[0175] (e”) HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy chain variable region shown in SEQ ID NO.5, and comprise the amino acid sequences shown in SEQ ID NOs.105, 113, 130 and 132 respectively.

[0176] (f”) HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable regions shown in SEQ ID NO.6 and respectively contain the amino acid sequences shown in SEQ ID NO.105, 114, 130, and 122.

[0177] (g”) HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable regions shown in SEQ ID NO.7 and respectively contain the amino acid sequences shown in SEQ ID NO.107, 111, 124, and 122.

[0178] (h”) HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable regions shown in SEQ ID NO.8 and respectively contain the amino acid sequences shown in SEQ ID NO.107, 110, 120, and 122.

[0179] (i”) HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable regions shown in SEQ ID NO.9 and respectively contain the amino acid sequences shown in SEQ ID NO.107, 111, 124, and 122.

[0180] (j”) HFR1, HFR2, HFR3, and HFR4 are selected from the framework regions of the heavy chain variable regions shown in SEQ ID NO.10 and respectively contain the amino acid sequences shown in SEQ ID NO.105, 113, 130, and 132.

[0181] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment contains an appropriate framework region (FR) sequence, provided that the antibody or its antigen-binding fragment can specifically bind to CD25. For example, the CDR sequences shown in Tables 1-10 above are obtained from alpaca antibodies, but can be grafted onto any suitable FR sequence of any suitable species (such as mouse, human, rat, rabbit, and others) using suitable methods well known in the art (such as recombinant techniques).

[0182] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment has a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs. 1-10 or having at least 83% (such as 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 1-10.

[0183] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment further comprises a partial or full sequence of a constant region derived from at least one of camelid-derived antibodies, murine antibodies, human antibodies, primate-derived antibodies or their mutants.

[0184] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment is derived from the constant region of a heavy chain.

[0185] In an alternative embodiment, the heavy chain constant region contains the sequence of a partial or full constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, preferably the sequence of a partial or full constant region of IgG1.

[0186] In an alternative embodiment, the sequence of a partial or full constant region of any one of the above IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD contains a mutated Fc region.

[0187] In an alternative embodiment, the heavy chain constant region contains a mutated Fc region of an IgG1-type antibody.

[0188] In an alternative embodiment, the constant region of the anti-CD25 nanobody or its antigen-binding fragment contains a mutated Fc region of an IgG1-type antibody, and the mutation type is DLE mutation, i.e., including S239D, I332E and A330L mutations. Tregs exert immunosuppressive effects by producing inhibitory cytokines such as IL-10, TGF-β, etc., which is the main mechanism for iTregs to induce immunosuppression. Tregs can also secrete granzyme A / B and perforin into the cell gap in contact with effector T cells, and mediate the lysis of target cells with the help of CD27. CD4 + CD25 + Foxp3 + The highly expressed CD25 on Tregs can bind to IL-2 in the microenvironment, block the metabolism of target cells by consuming the IL-2 required by effector T cells, thereby triggering apoptosis. Antibodies with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) / ADCP (antibody-dependent cell phagocytosis) effects after DLE mutation can better clear CD4 in the immune environment + CD25 + Foxp3 + Tregs, increase the proportion of effector T cells, and enhance the anti-tumor effect.

[0189] In an alternative embodiment, the amino acid sequence of the constant region of the anti-CD25 nanobody or its antigen-binding fragment is as shown in SEQ ID NO.140, which is the constant region of a DLE-mutated IgG1-type antibody.

[0190] In an alternative embodiment, the anti-CD25 nanobody or its antigen-binding fragment has one or more of the following properties (i)-(v):

[0191] (i) binds to isolated CD25 molecules and / or cells expressing CD25, and does not block the binding of IL-2 to isolated CD25 molecules;

[0192] (ii) binds to isolated CD25 molecules and / or cells expressing CD25, and does not block the binding of IL-2 to cells expressing CD25;

[0193] (iii) binds to isolated CD25 molecules and cells expressing CD25, and does not block the binding of IL-2 to isolated CD25 molecules, nor does it block the binding of IL-2 to cells expressing CD25;

[0194] (iv) binds to isolated human CD25 molecules and / or cells expressing CD25;

[0195] (vi) does not bind to isolated murine CD25 molecules and / or cells expressing CD25.

[0196] The anti-CD25 nanobody or its antigen-binding fragment provided in the above aspect has the following effects:

[0197] The anti-CD25 nanobody or its antigen-binding fragment provided in the above aspect can specifically bind to the CD25 protein, including specifically binding to isolated CD25 molecules and human T lymphocyte leukemia cells expressing CD25, while not blocking the binding of IL-2 to isolated CD25 molecules, which is beneficial to reducing the number of Treg cells without affecting the IL-2 activation signaling pathway, ensuring the effective activation and proliferation of T cells, enhancing the anti-tumor ability, and providing new possibilities for the treatment and / or prevention of tumors.

[0198] In a third aspect, an anti-CD25 antibody or its antigen-binding fragment is also provided, which contains the anti-CD25 nanobody or its antigen-binding fragment of the first or second aspect, or competitively binds to CD25 with the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect, or the epitope of its binding to the CD25 antigen is the same as that of the anti-CD25 nanobody or its antigen-binding fragment described in the first or second aspect.

[0199] In an alternative embodiment, the anti-CD25 antibody or its antigen-binding fragment further contains complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region.

[0200] In an alternative embodiment, the complementarity determining regions of the light chain variable region of the anti-CD25 antibody or its antigen-binding fragment target CD25.

[0201] In an alternative embodiment, the complementarity determining regions of the light chain variable region of the anti-CD25 antibody or its antigen-binding fragment target other antigens, such as immune checkpoints, exemplary immune checkpoints including but not limited to PD-1, CTLA-4, BTLA, KIR, LAG3, VISTA, TIGIT, TIM3, PD-L1, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS.

[0202] In an alternative embodiment, the anti-CD25 antibody or its antigen-binding fragment comprises one or more of a full-length antibody, F(ab’)2, Fab’, Fab, Fv, scFv, dsFv, and antibody minimal recognition unit.

[0203] In an alternative embodiment, the anti-CD25 antibody or its antigen-binding fragment contains the sequence of part or all of the constant region, and the constant region is derived from at least one of camelid-derived antibodies, murine antibodies, human antibodies, primate-derived antibodies, or mutants thereof.

[0204] In an alternative embodiment, the heavy chain constant region contains the sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0205] In an alternative embodiment, the light chain constant region is a κ or λ chain.

[0206] In an alternative embodiment, the species from which the constant region is derived includes one or more combinations of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0207] In a fourth aspect, the present invention provides a multispecific antibody comprising the anti-CD25 nanobody or its antigen-binding fragment as shown in any one of the foregoing.

[0208] In an alternative embodiment, the multispecific antibody is a bispecific antibody, and the bispecific antibody comprises a first antigen-binding part and a second antigen-binding part. The first antigen-binding part is the nanobody or its antigen-binding fragment of the first or second aspect.

[0209] In an alternative embodiment, the second antigen-binding part is an antibody or its antigen-binding fragment that specifically binds to a target molecule.

[0210] In an alternative embodiment, the second antigen-binding portion specifically binds to a tumor surface antigen or an immune checkpoint.

[0211] In an alternative embodiment, the tumor surface antigens include, but are not limited to, MUC16, VEGFR-2 (KDR / FIK-1), K-RAS, CD2, CD3, CD19, CD19, CD20, CD21, CD22, CD30, CD32B, CD33, CD38, CD39, CD40, CD45, CD52, CD70, CD80, CD60, CD62, CD72, CD79a, CD79B, CD123, EGFR, HER2, HER3, HER4, GPC3, CLL1, TGF-β, TGF-beta RII, VEGF, GD3, CCR4, CCR5, BCMA, CTLA4, mesothelin, OX40.

[0212] In an alternative embodiment, the immune checkpoints include, but are not limited to, PD-1, PD-L1, PD-L2, TIGIT, CD47, CD27, CD28, CD40, CD122, CD137, CD94 / NKG2A, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, LAG3, TIM-3, VISTA, GARP, PS, CSF1R, TDO, CTLA-4, IDO, KIR, GITR, TNFR, FasR / DcR, and CEACAM1.

[0213] In a fifth aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular region, the extracellular region comprising an antigen-binding domain; and the antigen-binding domain contains the anti-CD25 nanobody or an antigen-binding fragment thereof as shown in any one of the foregoing.

[0214] In an alternative embodiment, the antigen-binding domain contains a single-chain antibody scFv, wherein the heavy-chain variable region has the complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the anti-CD25 nanobody or an antigen-binding fragment thereof as shown in any one of the foregoing.

[0215] In an alternative embodiment, the antigen-binding domain contains a single-chain antibody scFv, wherein the heavy-chain variable region has the heavy-chain variable region of the anti-CD25 nanobody or an antigen-binding fragment thereof as shown in any one of the foregoing.

[0216] In a sixth aspect, the present invention provides a nucleic acid molecule encoding the anti-CD25 nanobody or antigen-binding fragment thereof described in the first or second aspect, or the anti-CD25 antibody or antigen-binding fragment thereof described in the third aspect, or the multispecific antibody of the fourth aspect, or the chimeric antigen receptor of the fifth aspect.

[0217] In an alternative embodiment, the nucleic acid molecule is an isolated nucleic acid molecule.

[0218] In a seventh aspect, the present invention provides a vector carrying the nucleic acid molecule of the seventh aspect.

[0219] In an eighth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule, or the aforementioned vector, or expressing the anti-CD25 nanobody or antigen-binding fragment thereof described in the first or second aspect, or expressing the anti-CD25 antibody or antigen-binding fragment thereof described in the third aspect, or expressing the multispecific antibody of the fourth aspect, or expressing the chimeric antigen receptor of the fifth aspect.

[0220] In a ninth aspect, the present invention provides an engineered immune effector cell expressing the chimeric antigen receptor of the fifth aspect or containing a nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0221] In a tenth aspect, the present invention provides the anti-CD25 nanobody or antigen-binding fragment thereof described in the first or second aspect, or the anti-CD25 antibody or antigen-binding fragment thereof described in the third aspect, or the multispecific antibody of the fourth aspect, or the chimeric antigen receptor of the fifth aspect, or the nucleic acid molecule of the sixth aspect, or the vector of the seventh aspect, or the recombinant cell of the eighth aspect, or the engineered immune effector cell of the ninth aspect for use in any one of the following:

[0222] (I) Detecting CD25 or cells expressing CD25;

[0223] (II) Preparing a product for detecting CD25 or cells expressing CD25;

[0224] (III) Depleting Treg cells;

[0225] (IV) Preparing a pharmaceutical composition for treating, preventing or alleviating a disease, disorder or condition associated with tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation;

[0226] (V) Treating, preventing or alleviating a disease, disorder or condition associated with tumor, inflammation, autoimmune disease, infection, immune rejection of organ transplantation.

[0227] In alternative embodiments, the anti-CD25 nanobody or antigen-binding fragment thereof, or any type of antibody or antigen-binding fragment containing the same, that can be utilized by the above applications can be used to detect CD25 or cells expressing CD25 based on immunoassay techniques. For example, when the antibody or antigen-binding fragment is an immunoconjugate, such as conjugated with a fluorophore, a fluorescence detection device can be used to localize or detect CD25 in real time. For example, it can be used in immunoblotting, immunoprecipitation, flow cytometry, etc., which involve using the specific binding property between the CD25 antigen and the antibody to detect CD25 or cells expressing CD25. Correspondingly, in the above-mentioned aspect (II), those skilled in the art can select the reagent composition in the kit according to the actual detection means, including but not limited to antagonists, anti-CD25 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. The kit can also be used to detect diseases, disorders, or conditions related to CD25.

[0228] In alternative embodiments, the detection of CD25 or cells expressing CD25 is for non-diagnostic and non-therapeutic purposes.

[0229] In alternative embodiments, the purpose of depleting Treg cells is for non-diagnostic and non-therapeutic purposes.

[0230] In alternative embodiments, the purpose of depleting Treg cells is for preventing, treating, or alleviating diseases, disorders, or conditions, including but not limited to solid tumors, hematological tumors, inflammation, or immune diseases.

[0231] In alternative embodiments, the application is for depleting tumor-infiltrating Treg cells, and the application includes treating solid tumors.

[0232] In the eleventh aspect, the present invention provides a pharmaceutical composition containing the anti-CD25 nanobody or antigen-binding fragment thereof described in the first or second aspect, or the anti-CD25 antibody or antigen-binding fragment thereof in the third aspect, or the multispecific antibody in the fourth aspect, or the chimeric antigen receptor in the fifth aspect, or the nucleic acid molecule in the sixth aspect, or the vector in the seventh aspect, or the recombinant cell in the eighth aspect, or the engineered immune effector cell in the ninth aspect.

[0233] In alternative embodiments, the above pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipients can be any carrier and / or excipient known and conventional in the art. Examples of carriers include but are not limited to any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.; examples of excipients include but are not limited to fillers, disintegrants, preservatives, solubilizers, and emulsifiers, etc.

[0234] In an alternative embodiment, the pharmaceutical composition further comprises one or more pharmaceutically active ingredients having other therapeutic effects, including but not limited to one or a combination of several of chemotherapeutic agents, anti-cancer drugs, radiotherapy agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormonal therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0235] In an alternative embodiment, the pharmaceutical composition is used for treating, preventing or alleviating diseases, disorders or conditions related to tumors, inflammation, autoimmune diseases, infections, immune rejection associated with organ transplantation.

[0236] In a twelfth aspect, there is also provided a method for treating, preventing or alleviating a disease, disorder or condition, said disease, disorder or condition including tumors, inflammation, autoimmune diseases, infections, immune rejection caused by organ transplantation; the method comprises administering to a subject a therapeutically effective amount of the anti-CD25 nanobody or an antigen-binding fragment thereof according to the first or second aspect, or the anti-CD25 antibody or an antigen-binding fragment thereof according to the third aspect, or the multispecific antibody according to the fourth aspect, or the chimeric antigen receptor according to the fifth aspect, or the nucleic acid molecule according to the sixth aspect, or the vector according to the seventh aspect, or the recombinant cell according to the eighth aspect, or the engineered immune effector cell according to the ninth aspect, or the pharmaceutical composition according to the eleventh aspect.

[0237] In an alternative embodiment, the treatment, prevention or alleviation of a disease, disorder or condition related to a tumor in any of the above embodiments is achieved by depleting tumor-infiltrating Treg cells.

[0238] In an alternative embodiment, the tumors in any of the above embodiments include solid tumors, exemplary solid tumors including but not limited to one or more of gastric cancer, esophageal cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanoma, breast cancer, myeloma, squamous cell carcinoma, urogenital cancer, rectal cancer, sarcoma, testicular cancer, cervical cancer, mast cell tumor, hemangioma, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, rectal cancer, uterine cancer, brain cancer, and glioma.

[0239] In alternative embodiments, the tumor in any of the above embodiments includes hematological tumors. Exemplary hematological tumors include, but are not limited to, hematological tumors such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML), B-cell malignancies, lymphomas (Hodgkins Lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myeloma), and one or more of myeloproliferative disorders.

[0240] In alternative embodiments, the therapeutically effective dose in any of the above embodiments is between about 0.001 mg / kg and about 1000 mg / kg. In some embodiments, the dosage administered can vary during the course of treatment. For example, in some embodiments, the initial dosage administered can be higher than subsequent dosages. In some embodiments, the dosage administered is adjusted during the course of treatment based on the response of the subject being administered.

[0241] In alternative embodiments, the pharmaceutical composition as described in the present invention can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a hormone therapeutic agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).

[0242] In alternative embodiments, when the pharmaceutical composition as described in the present invention is used in combination with one or more additional therapeutic agents, it can be administered simultaneously with the one or more additional therapeutic agents. In certain such embodiments, the pharmaceutical composition and the additional therapeutic agent can be administered simultaneously as part of the same composition. However, a pharmaceutical composition "used in combination" with another therapeutic agent does not require simultaneous administration or administration in the same composition as that therapeutic agent. The meaning of "used in combination" in the present invention also includes that a pharmaceutical composition administered before or after another therapeutic agent is also considered to be "used in combination" with that therapeutic agent, i.e., the pharmaceutical composition and the second substance are administered by different routes of administration.

[0243] In a thirteenth aspect, there is also provided a kit for detecting CD25, which comprises the anti-CD25 nanobody or antigen-binding fragment thereof described in the first or second aspect as described above, or the anti-CD25 antibody or antigen-binding fragment thereof in the third aspect.

[0244] In alternative embodiments, for the product in any of the above aspects, for example, the anti-CD25 antibody or antigen-binding fragment thereof in a reagent, a kit, a blocker, and a pharmaceutical composition can be conjugated with at least one diagnostic agent and / or therapeutic agent to form an immunoconjugate.

[0245] The diagnostic agent is selected from one or more of radiocontrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent markers, ultrasound contrast agents, and photosensitizers;

[0246] The therapeutic agent is selected from one or more of cytotoxin agents, drugs, radionuclides, boron atoms, immunomodulators, anti-apoptosis reagents, photosensitive therapeutic agents, immunoconjugates, and oligonucleotides.

[0247] Radionuclides include but are not limited to 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb and 83 one or more of Sr.

[0248] Paramagnetic ions include but are not limited to one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0249] Fluorescent labels include, but are not limited to, one or more of Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green488, Oregon Green 500, Oregon Green514, PacificBlue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl violet acetate, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptate, tris-bipyridyl diamine europium, europium cryptate or chelate, diamine, dicarbocyanine, La Jolla blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red.

[0250] Oligonucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0251] Drugs include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxine alkaloid, and L-asparaginase.

[0252] Immunomodulators include, but are not limited to, one or more of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factors (CSF), interferons, erythropoietin, thrombopoietin, tumor necrosis factor (TNF), interleukins (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and stem cell growth factor.

[0253] Radionuclides include but are not limited to 111 In, 111 At, 177 Lu, 211 Bi, 212 Bi, 213 Bi, 211 At, 62 Cu, 67 Cu, 90 Y, 125 I, 131 I, 133 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 153 Sm, 161 Tb, 152 Dy, 166 Dy, 161 Ho, 166 Ho, 186 Re, 188 Re, 189 Re, 211 Pb, 212 Pb, 223 Ra, 225 Ac, 77 As, 89 Sr, 99 Mo, 105 Rh, 149 Pm, 169 Er, 194 Ir, 58 Co, 80 mBr, 99 mTc, 103 mRh, 109 Pt, 119 Sb, 189 mOs, 192 Ir, 219 Rn, 215 Po, 221 Fr, 255 Fm, 11 C, 13 N, 15 O, 75 Br, 198 Au, 199 Au, 224 Ac, 77 Br, 113 mIn, 95 Ru, 97 Ru,103 Ru, 105 Ru, 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 142 Pr, 143 Pr, 161 Tb, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 76 Br and 169 one or more of Yb.

[0254] The present invention will be further illustrated by specific embodiments below. However, it should be understood that these embodiments are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0255] The nanobody structure used in the following examples is as Figure 1 shown, which comprises a heavy chain variable region and a constant region of the hIgG1 subtype containing the DLE mutation (S239D / I332E / A330L). The DLE mutation can enhance the ADCC effect, and the amino acid sequence of the constant region is as shown in SEQ ID NO.140:

[0256] EPKSSDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.140)

[0257] In the following examples, the positive controls are all full-length antibodies. The numbers and protein names of the positive controls are shown in Table 11, and the amino acid sequences of the positive controls are shown in Table 12. Among them, R1980 and R2133 have the same heavy chain variable region and light chain variable region, R1722 and R2134 have the same heavy chain variable region and light chain variable region, and the constant regions of the positive controls are all constant regions of the hIgG1 subtype. Among them, the constant regions of R2133 and R2134 have a DLE mutation (S239D / I332E / A330L mutation).

[0258] Table 11

[0259]

[0260]

[0261] Table 12

[0262]

[0263] Example 1. Generation of Nanobodies Against CD25

[0264] (I) Bacterial Library Construction:

[0265] PBMC was isolated from the blood of alpacas immunized with CD25 antigen, RNA was extracted from it and reverse transcribed into cDNA. After amplifying the cDNA fragment with specific primers, a band of about 450 bp was recovered by cutting the gel. Then, the VHH target fragment was amplified with NcoI-VHH upstream primer and SalI-JH primer. After column recovery of the PCR product, it was double digested with restriction enzymes NOCI and SalI and then electrophoresed to cut the gel and recover. Then, it was ligated with the PCGMT phage vector that had been double digested with NOCI and SalI in advance. After column recovery and purification of the ligation product, it was electrotransformed into Escherichia coli TG1 competent cells. After electrotransformation, the TG1 cells were cultured in a shaker for 1 hour, and the bacterial solution was evenly spread on an ampicillin-resistant plate and cultured overnight at 37°C. Then, the colonies on the plate were carefully scraped off, and the bacterial library was labeled and frozen and aliquoted and stored in a -80°C refrigerator.

[0266] (II) Bacterial Library Amplification:

[0267] Take 1 mL of the bacterial library and add it to a shake flask containing 500 mL of 2YT + A + G medium. The OD600 of the library medium is 0.05 - 0.1. Incubate with shaking at 37°C for 1 hour until the OD600 of the bacterial solution reaches 0.6 - 0.8. Add helper phage M13KO7 (multiplicity of infection is 20), and let it stand and infect at 37°C in an incubator for 45 minutes. Then continue to incubate with shaking for 1 hour. Collect the bacterial solution, centrifuge at 4500 rpm for 10 minutes, discard the supernatant, resuspend with the medium (2×YT + 100 μg / mL carbenicillin + 50 μg / mL kanamycin). After making up the corresponding volume, incubate with shaking in a shaker overnight. Centrifuge the overnight bacterial solution at 10000 g at 4°C for 20 minutes, collect the supernatant containing phage particles, and add 1 / 4 volume of PEG6000 - NaCl (20% PEG in 2.5 M NaCl solution, which has been autoclaved). Place it on ice for 3 hours, then centrifuge at 10000 g at 4°C for 20 minutes. Collect the phage precipitate, resuspend the precipitate with sterile PBS buffer, and centrifuge at 10000 g at 4°C for 5 minutes. Collect the supernatant, continue to add 1 / 4 volume of PEG - NaCl solution, place it on ice for 30 minutes, centrifuge at 10000 g at 4°C for 5 minutes, collect the phage precipitate. Finally, resuspend the precipitate with 20 mL of PBS buffer containing 30% glycerol, measure the phage titer to be 1.68E14 pfu / mL, name it FPX025N - 2 - R0 - P, and aliquot and store it in a -80°C refrigerator for later use.

[0268] (III) Phage panning:

[0269] 1. Adopt the solid - phase panning method. Coat the antigens R1712 and R1916 with sterile CBS solution overnight. Use the R1916 protein as an irrelevant protein with the same his - tag for reverse screening. The coating concentrations are: S1: 25 μg / mL, S2: 12.5 μg / mL, S3: 6 μg / mL, S4: 2 μg / mL; coat 10 wells per protocol, 100 μL per well. After coating, wash twice with PBST containing 0.05% Tween, 250 μL per well.

[0270] 2. Blocking: Block the well plates and the phage library with 5% casein (5% casein = casein + PBS). Block the well plates in an incubator at 37°C for 1 hour, and at the same time block the phage library with a rotator for 30 minutes.

[0271] 3. Reverse screening: After blocking, discard the blocking solution. First add the pre - blocked phage to column B (reverse - screening column) for reverse screening for 30 minutes, 100 μL per well; then mix the liquid in column B well and add it to column A (positive - screening column) for positive screening for 1 hour.

[0272] 4. Washing: Discard the well - plate liquid, first wash with 1×PBST containing 0.05% Tween, and then wash with PBS (sterile).

[0273] 5. Elution: Elute with TEA for 8 minutes.

[0274] 6. Termination: After thoroughly mixing the eluted phage on the ELISA plate, aspirate it into a 1.5 mL EP tube, add 550 μL of 1 M Tris-HCl pH 7.4 (half the volume of TEA) to terminate, obtaining the panned phage, and measure the titer value.

[0275] 7. Amplification: Take 900 μL of phage to infect 5 mL of TG1 bacterial solution (OD600 = 0.8), let it stand in a 37 °C incubator for 30 min, then add 12 mL of 2×YT and 2 mL of 20% glucose, shake and culture at 37 °C for 30 min, then add carbenicillin (final concentration is 100 μg / mL) and M13KO7 helper phage (multiplicity of infection is 20). After standing in a 37 °C incubator for 30 minutes, shake and culture on a 37 °C shaker for 1.5 hours. Collect the bacterial solution and centrifuge at 4500 rpm for 10 min, discard the supernatant, add 40 mL of 2YT / carbenicillin / kanamycin medium to it, shake and culture overnight at 30 °C. Collect the overnight bacterial solution, and the purification method is the same as the above-mentioned bacterial library amplification and purification steps. Measure the titer value. The purified and collected amplification product is used as the input for the next round of panning. After 4 rounds of panning, infect TG1 with the output products of each round of each scheme, coat them on the plate, randomly select monoclonal plaques on the plate, add induction medium, and induce expression overnight in the shaker.

[0276] (IV) ELISA Screening and Detection

[0277] R1712 and R1916 control proteins were coated on the ELISA plate overnight, blocked with 5% casein at 37°C for 1h, washed with PBST, and 50μL PBST was added to each well to dilute the monoclonal bacterial supernatant. The overnight expression bacterial solution was centrifuged at 4500rpm for 10 minutes, 50μL supernatant was aspirated and added to the ELISA plate, incubated at 37°C for 1 hour, washed 6 times with PBST, and then 100μL / well of Anti-M13 Antibody, Mouse Monoclonal (1:5000 dilution) was added, incubated at 37°C for 1 hour, washed 6 times with PBST, and then 100μL / well of Peroxidase-Goat Anti-Mouse IgG, Fcy Fragment Specific (1:20000 dilution) was added, incubated at 37°C for 30 minutes, washed 18 times with PBST, and finally the color development solution was added to detect the binding of phages, and the specific binding samples were selected for sequencing after repeated ELISA test. By analyzing the aligned sequences, 18 unique sequences were screened out and numbered as R2449, R2453, R2454, R2455, R2456, R2457, R2459, R2460, R2724, R2725, R2726, R2727, R2728, R2729, R2730, R2731, R2732, and R2733.

[0278] Example 2. Binding activity of CD25 nanobody to C8166 cells

[0279] C8166 cells (human T lymphocyte leukemia cells) that naturally express CD25 in good condition were collected, centrifuged at 350G for 5 minutes, resuspended and counted, and the cell density was adjusted to 2E6 / mL with 3% BSA. The cells were plated on a V-shaped 96-well plate at 100μL / well, and gradiently diluted CD25 antibodies and control antibodies were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200μL of 3% BSA was added to each well to wash the cells, and the supernatant was discarded after centrifugation. Then PE Goat anti human IgG Fc (1:200 dilution) was added at 100μL / well, the cells were resuspended and mixed, and incubated at 4°C for 30 minutes. After centrifugation, 200μL of 3% BSA was added to each well to wash the cells, and then 150μL of PBS was added to each well to resuspend the cells and use a Cytoflex flow cytometer (Beckman Countler) to evaluate the binding differences of CD25 antibodies to C8166 cells. The binding curve is shown in the figure. Figures 2 to 5As shown, except for R2456 and R2457, they all have varying degrees of binding activity with C8166 cells and are all superior to the positive control R2016 (RG6292). The binding EC50 of R2449, R2454, R2455, R2727, R2724, R2729, R2731, R2733 with C8166 cells is lower, being superior to all positive controls R2133DLE (BT942), R2134 DLE (Daclizumab), R2016 (RG6296), R1722 (Daclizumab).

[0280] Example 3. Binding activity of CD25 nanobody blocking IL-2Fc with C8166 cells

[0281] Collect C8166 cells in good condition, centrifuge at 350G for 5 minutes, resuspend and count. Adjust the cell density to 2E6 / mL with 3% BSA, plate at 100 μL / well into a V-shaped 96-well plate, and add gradient-diluted CD25 antibody and control antibody at 50 μL / well respectively, and incubate at 4°C for 30 minutes. Add the prepared IL-2Fc to the cells at 50 μL / well and incubate at 4°C for 30 minutes. After centrifuging at 350G for 5 minutes, add 200 μL of 3% BSA to wash the cells once per well, centrifuge and discard the supernatant. Then add SA-PE (diluted 1:50) at 100 μL / well, resuspend and mix the cells evenly and incubate at 4°C for 30 minutes. After centrifuging, add 200 μL of 3% BSA to wash the cells once per well, and then add 150 μL of PBS to resuspend the cells and evaluate the difference in the binding of CD25 antibody blocking IL-2Fc with C8166 cells using a Cytoflex flow cytometer (Beckman Countler). The blocking curve is as Figures 6 to 9 shown, R2453, R2455, R2459, R2460 have binding activity and do not disrupt the binding of IL-2 to CD25.

[0282] Example 4. Binding activity of CD25 nanobody with CD25-his protein

[0283] Coat the ELISA plate with coating buffer (Na2CO3 / NaHCO3) at pH 9.6, dilute the CD25-His protein at a dilution concentration of 1.25 μg / mL, add 100 μL / well to the ELISA plate, and place it at 4°C overnight. Prepare PBST (5‰ Tween 20), wash the ELISA plate twice with PBST (300 μL / well), add 1% BSA solution for blocking, 200 μL / well, and incubate at 37°C for 1 hour. Wash the ELISA plate twice with PBST (300 μL / well); dilute the antibody sample with 1% BSA in gradient, add the diluted sample to the ELISA plate at 100 μL / well, and incubate at 37°C for 1 hour. Wash the ELISA plate four times with PBST (300 μL / well); dilute Goat anti hIgG-HRP with 1% BSA at a dilution factor of 1:15K, 100 μL / well, and incubate at 37°C for 0.5 hour; wash the ELISA plate five times with PBST (300 μL / well). Add TMB, 100 μL / well, and place it at room temperature in the dark for 10 min; add the stop solution, 50 μL / well. Read the absorbance at 450 nM using an iX3 ELISA reader (Molecular Device), and analyze and plot the graph using GraphPad 7.0. The binding curves are as Figures 10 to 13 shown, and all the tested antibodies have binding activity.

[0284] Example 5. CD25 Nanobody Blocks the Binding Activity of IL-2Fc to CD25-his Protein

[0285] Coat the ELISA plate with coating buffer (Na2CO3 / NaHCO3) at pH 9.6, dilute the CD25-His protein at a dilution concentration of 1.25 μg / mL, add 100 μL / well to the ELISA plate, and place it at 4 °C overnight. Prepare PBST (5‰ Tween 20), wash the ELISA plate twice with PBST (300 μL / well), add 1% BSA solution for blocking, 200 μL / well, and place it at 37 °C for 1 hour. Wash the ELISA plate twice with PBST (300 μL / well); dilute the antibody and ligand IL-2Fc with 1% BSA in gradients, add the diluted antibody and ligand to the ELISA plate at 100 μL / well respectively, and incubate at 37 °C for 2 hours. Wash the ELISA plate four times with PBST (300 μL / well); dilute SA-HRP with 1% BSA at a dilution factor of 1:10K, 100 μL / well, and incubate at 37 °C for 0.5 hour; wash the ELISA plate five times with PBST (300 μL / well). Add TMB, 100 μL / well, and place it at room temperature in the dark for 10 min; add the stop solution, 50 μL / well. Read the absorbance at 450 nM with an iX3 microplate reader (Molecular Device), and analyze and plot the graph with GraphPad 7.0. The blocking curve is as Figures 14 to 17 shown, and R2453, R2455, R2456, R2457, R2459, R2460, R2724, R2725, R2726, R2728, R2730, R2732 have no blocking activity.

[0286] Example 6. Cross-detection of CD25 nanobodies in humans, mice and monkeys

[0287] Collect recombinant cells expressing human, mouse and monkey CD25 respectively, centrifuge at 350G for 5 minutes, resuspend and count, adjust the cell density to 2E6 / mL with 3% BSA, plate 100 μL / well into a V-bottom 96-well plate, add the diluted CD25 antibody respectively, and incubate at 4 °C for 30 minutes. After centrifugation, add 200 μL of 3% BSA to each well to wash the cells once, centrifuge and discard the supernatant. Then add 100 μL / well of PE Goat anti human IgG Fc (diluted 1:200), resuspend and mix the cells, and incubate at 4 °C for 30 minutes. After centrifugation, add 200 μL of 3% BSA to each well to wash the cells once, then add 150 μL of PBS to each well to resuspend the cells, and evaluate the binding of the CD25 antibody to the cells with a Cytoflex flow cytometer (Beckman Coulter). The binding curves are respectively as Figure 18 、 Figure 19 and Figure 20 shown, among which R2455 cross-reacts with mice, and the remaining antibodies do not cross-react with mice but cross-react with monkeys.

[0288] Example 7. Sequencing of CD25 Nanobody

[0289] After screening, R2453, R2459, R2460, and R2455 have the ability to bind to both CD25-expressing cells and the CD25 molecule, and do not block the binding of IL-2 to CD25-expressing cells or the binding of IL-2 to the CD25 molecule; R2455, R2724, R2725, R2726, R2728, R2730, and R2732 have the ability to bind to both CD25-expressing cells and the CD25 molecule, and do not block the binding of IL-2 to the CD25 molecule. The heavy-chain variable regions of the screened nanobodies were sequenced, and the results are shown in Table 13. The amino acid sequences of the CDR regions are shown in Tables 1 to 10.

[0290] Table 13 Sequencing Results of the Heavy-Chain Variable Region (VH) of CD25 Nanobody

[0291]

[0292]

[0293] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-CD25 nanobody or an antigen-binding fragment thereof, characterized in that: Complementarity determining regions comprising heavy chain variable region HCDR1, HCDR2 and HCDR3; The complementary determining region HCDR1 comprises an amino acid sequence as shown below: X1-Y, X1 is N, I, M, H, L or Y; The complementary determining region HCDR2 comprises an amino acid sequence as follows: X2-S-X3-G-X4, or YSDSS-X5 (SEQ ID NO.134), or SWIGGS (SEQ ID NO.71); X2 is T or R, X3 is G or D, X4 is S or T, and X5 is Y, A or H; The complementary determining region HCDR3 comprises an amino acid sequence as shown below: DFED-X6-SGWYRREPPLD (SEQ ID NO.135), or ARGSGSYYPFDD (SEQ ID NO.85), or HRGEYYSDWRFDDMD (SEQ ID NO.94), X6 is D or S.

2. The anti-CD25 nanobody or antigen-binding fragment thereof according to claim 1, characterized in that: It contains complementary determining regions HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region, wherein the complementary determining regions HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NOs. 1 to 10; Preferably, HCDR1, HCDR2 and HCDR3 are defined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition or the IMGT definition; Preferably, the complementarity determining regions HCDR1, HCDR2 and HCDR3 are selected from any one of the following combinations (A) to (C): (A) the HCDR1 comprises the following amino acid sequence: X1-Y, X1 is I or L; HCDR2 comprises the following amino acid sequence: X2-S-X3-G-X4, X2 is T or R, X3 is G or D, and X4 is S or T; HCDR3 comprises the following amino acid sequence: DFED-X6-SGWYRREPPLD (SEQ ID NO.135), X6 is D or S; (B) the HCDR1 comprises the following amino acid sequence: X1-Y, X1 is N, H or Y; HCDR2 comprises the following amino acid sequence: YSDSS-X5 (SEQ ID NO.134), X5 is Y, A or H; HCDR3 comprises the following amino acid sequence: ARGSGSYYPFDD (SEQ ID NO.85); (C) the HCDR1 comprises the following amino acid sequence: X1-Y, X1 is M, HCDR2 comprises the following amino acid sequence: SWIGGS (SEQ ID NO.71); HCDR3 comprises the following amino acid sequence: HRGEYYSDWRFDDMD (SEQ ID NO.94); Preferably, according to the IMGT definition, the complementary determining region HCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 12, 15, 18, 21, 24, 27 or 32; And / or, according to the IMGT definition, the complementary determining region HCDR2 comprises an amino acid sequence as shown in SEQ ID NO. 57, 58, 59, 60, 61, 62, 63 or 78; And / or, according to the IMGT definition, the complementary determining region HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 88, 97, 99, 100, 102 or 104; Preferably, the complementarity determining regions HCDR1, HCDR2 and HCDR3 are selected from any one of the following combinations (a) to (j): (a) the amino acid sequence of HCDR1 includes the amino acid residue NY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.84, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85; (b) the amino acid sequence of HCDR1 includes amino acid residue IY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.77, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89; (c) the amino acid sequence of HCDR1 includes amino acid residue IY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.76, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89; (d) the amino acid sequence of HCDR1 includes the amino acid residue MY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.71, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.94; (e) the amino acid sequence of HCDR1 includes the amino acid residue HY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.84, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85; (f) the amino acid sequence of HCDR1 includes the amino acid residue NY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.82, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.85; (g) the amino acid sequence of HCDR1 includes the amino acid residue LY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.70, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89; (h) the amino acid sequence of HCDR1 includes amino acid residue IY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.77, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.92; (i) the amino acid sequence of HCDR1 includes the amino acid residue LY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.70, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.89; (j) the amino acid sequence of HCDR1 includes amino acid residue YY, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.83, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.

85.

3. The anti-CD25 nanobody or antigen-binding fragment thereof according to claim 2, characterized in that: According to the IMGT definition: the complementary determining regions HCDR1, HCDR2 and HCDR3 of the anti-CD25 nanobody or its antigen-binding fragment are selected from any one of (a')-(j'): (a') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.15, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.63, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.97; (b') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.32, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.59, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.100; (c') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.32, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.58, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.100; (d') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.27, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.78, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.88; (e') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.12, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.63, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.97; (f') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.15, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.61, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.104; (g') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.21, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.57, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.99; (h') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.24, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.60, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.102; (i') the amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.21, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.57, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.100; (j') The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO.18, the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO.62, and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO.

97.

4. The anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The heavy chain variable region contains a heavy chain framework region, and the heavy chain framework region is derived from at least one of a camelid antibody, a mouse antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, the framework region of the heavy chain variable region includes HFR1, HFR2, HFR3 and HFR4; The HFR1 comprises the amino acid sequence shown below: DVQLQESGGGL-X7-Q-X8-GGSLRLSCAAS (SEQ ID NO. 136), X7 is V or A, X8 is P or A; And / or, the HFR2 comprises the amino acid sequence shown below: W-X9-RX 10 -APX 11 -KX 12 -X 13 -E (SEQ ID NO.137), X9 is A, Y, F or L, X 10 It is Q or R, X 11 It is G or E, X 12 is G, Q or E, X 13 is L or R; And / or, the HFR3 comprises the amino acid sequence shown below: RFTISRDNAKNTX 14 -YLX 15 -MNX 16 -LKPEDTAX 17 -YYC (SEQ ID NO. 138), X 14 It is L or V, X 15 It is Q or L, X 16 Is S or T, X 17 is L or V; Preferably, taking the IMGT definition as an example, HFR1, HFR2, HFR3 and HFR4 are selected from any one of the following combinations (a") to (j"): (a") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.1, and comprise the amino acid sequences shown in SEQ ID NO.107, 113, 130 and 132, respectively; (b") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO. 2, and comprise the amino acid sequences shown in SEQ ID NO. 107, 112, 123 and 122, respectively; (c") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.3, and comprise the amino acid sequences shown in SEQ ID NO.107, 109, 121 and 122, respectively; (d") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO. 4, and comprise the amino acid sequences shown in SEQ ID NO. 106, 108, 131 and 133, respectively; (e") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.5, and comprise the amino acid sequences shown in SEQ ID NO.105, 113, 130 and 132, respectively; (f") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.6, and comprise the amino acid sequences shown in SEQ ID NO.105, 114, 130 and 122, respectively; (g") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.7, and comprise the amino acid sequences shown in SEQ ID NO.107, 111, 124 and 122, respectively; (h") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO. 8, and comprise the amino acid sequences shown in SEQ ID NO. 107, 110, 120 and 122, respectively; (i") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.9, and comprise the amino acid sequences shown in SEQ ID NO.107, 111, 124 and 122, respectively; (j") HFR1, HFR2, HFR3 and HFR4 are selected from the framework region of the heavy chain variable region shown in SEQ ID NO.10, and comprise the amino acid sequences shown in SEQ ID NO.105, 113, 130 and 132, respectively.

5. The anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: A heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 10 or having at least 83% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs. 1 to 10.

6. The anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: It also comprises a partial or complete sequence of a constant region, wherein the constant region is derived from at least one of a camelid antibody, a mouse antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, the anti-CD25 nanobody or antigen-binding fragment thereof is derived from the constant region of the heavy chain; Preferably, the heavy chain constant region contains a sequence of a partial or entire constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Preferably, the heavy chain constant region is a sequence of part or all of the constant region of IgG1; Preferably, the sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD contains a mutated Fc region; Preferably, the mutated Fc region comprises a mutated Fc region of an IgG1 antibody, and the mutation sites comprise S239D, I332E and A330L mutations; Preferably, the amino acid sequence of the constant region of the anti-CD25 nanobody or its antigen-binding fragment is as shown in SEQ ID NO.

140.

7. The anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The anti-CD25 nanobody or antigen-binding fragment thereof has one or more of the following properties (i)-(v): (i) binds to isolated CD25 molecules and / or cells expressing CD25, and does not block the binding of IL-2 to isolated CD25 molecules; (ii) binds to isolated CD25 molecules and / or cells expressing CD25, and does not block the binding of IL-2 to cells expressing CD25; (iii) binds to isolated CD25 molecules and cells expressing CD25, and does not block the binding of IL-2 to isolated CD25 molecules or to cells expressing CD25; (iv) binding to isolated human CD25 molecules and / or cells expressing CD25; (v) does not bind to isolated murine CD25 molecules and / or cells expressing CD25.

8. An anti-CD25 antibody or an antigen-binding fragment thereof, characterized in that: Contains the anti-CD25 nanobody or its antigen-binding fragment according to any one of claims 1 to 7; or competes with the anti-CD25 nanobody or its antigen-binding fragment according to any one of claims 1 to 7 for binding to CD25, or its epitope binding to the CD25 antigen is the same as the epitope of the anti-CD25 nanobody or its antigen-binding fragment according to any one of claims 1 to 7.

9. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 8, characterized in that: The antibodies include full-length antibodies and nanobodies; the antigen-binding fragments include one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv and the minimum recognition unit of an antibody.

10. A biomaterial, characterized in that: The biological material comprises any one of the following (i) to (vi): (i) a multispecific antibody, the multispecific antibody comprising the anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 9; (ii) a chimeric antigen receptor, comprising an extracellular region, the extracellular region comprising an antigen binding domain; and the antigen binding domain comprises the anti-CD25 nanobody or antigen binding fragment thereof according to any one of claims 1 to 9; (iii) a nucleic acid molecule encoding the anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to (i), or the chimeric antigen receptor according to (ii); (iv) a vector carrying the nucleic acid molecule described in (iii); (v) a recombinant cell, characterized in that it comprises the nucleic acid molecule described in (iii), or the vector described in (iv), or expresses the anti-CD25 nanobody or antigen-binding fragment thereof described in any one of claims 1 to 9, or expresses the multispecific antibody described in (i), or expresses the chimeric antigen receptor described in (ii); (vi) An engineered immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor described in (ii) or contains a nucleic acid molecule encoding the chimeric antigen receptor of (ii).

11. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

12. Use of the anti-CD25 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the biomaterial according to claim 10, or the pharmaceutical composition according to claim 11 in any of the following: (Ⅰ) Detection of CD25 or cells expressing CD25; (II) preparing products for detecting CD25 or cells expressing CD25; (III) depleting Treg cells, preferably, depleting tumor-infiltrating Treg cells; (IV) Treating, preventing or alleviating diseases, disorders or conditions associated with tumors, inflammation, autoimmune diseases, infections, and immune rejection of organ transplantation.

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