A cd25 nanobody

Abstract

The application discloses a CD25 nanobody, and the nanobody provided by the application has the advantages of high stability, easy expression and purification, small influence on an IL-2 signal path, excellent ADCC and ADCP effects, endocytosis effect and the like, and has good drug efficacy and safety.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to a CD25 nanobody and its applications. Background Technology

[0002] The IL-2 (Interleukin-2) receptor is a heterotrimer composed of three chains: α (CD25), β (CD122), and γ (CD132). CD25 is the α receptor for IL-2 (Interleukin-2 receptor subunit alpha, IL-2RA), a type I transmembrane glycoprotein. CD25, when present alone, binds to IL-2 with low affinity (K). d 10 -8 When CD25 non-covalently binds to CD122 and CD132 to form a heterotrimeric high-affinity receptor complex, it can mediate the regulation of T cell proliferation, differentiation, and survival by IL-2, and regulate the immune response by activating downstream signaling pathways such as JAK / STAT5, PI3K-AKT-mTOR, and MAPK. CD25 is mainly expressed on the surface of Treg cells (regulatory T cells), basophils, and activated T cells in tissues such as the spleen and lymph nodes. It is a specific protein molecule that is simultaneously expressed on the surface of Teff cells (effective T cells) and Treg cells.

[0003] In tumor immunology, Treg cells extensively infiltrate various solid tumor tissues, including cervical cancer, renal cell carcinoma, ovarian cancer, melanoma, pancreatic cancer, hepatocellular carcinoma, gastric cancer, and breast cancer. High levels of Treg infiltration are negatively correlated with patient survival prognosis. CD25, a characteristic molecule significantly overexpressed on the surface of Treg cells, is an ideal target for eliminating Treg cells, relieving tumor immunosuppression, and enhancing the anti-tumor activity of effector T cells, making it significant for the treatment of various solid tumors.

[0004] CD25 binding to IL-2 on the surface of effector T cells (Teff) effectively activates Teff cells and promotes their proliferation. However, on the surface of regulatory T cells (Treg), the competitive binding of CD25 to IL-2 inhibits Teff cell proliferation. IL-2 plays a crucial role in the survival and function of Teff cells. Most early CD25 antibodies targeting Treg cells blocked the IL-2 signaling pathway, thus weakening the anti-tumor activity of Teff. Conversely, CD25 monoclonal antibodies that do not interfere with IL-2 signaling (i.e., preserve normal IL-2 signal transduction in Teff cells) can induce a more robust anti-tumor immune response. Therefore, developing antibodies that do not block the IL-2 signaling pathway is particularly important. Summary of the Invention

[0005] This invention relates to a nanobody that specifically targets CD25, the nanobody comprising CDR1, CDR2, and CDR3; wherein CDR1, CDR2, and CDR3 are selected from: (1) CDR1 contains the amino acid sequence shown in SEQ ID NO: 1, CDR2 contains the amino acid sequence shown in SEQ ID NO: 2, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 3; (2) CDR1 contains the amino acid sequence shown in SEQ ID NO: 9, CDR2 contains the amino acid sequence shown in SEQ ID NO: 10, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 11; or (3) CDR1 contains the amino acid sequence shown in SEQ ID NO: 16, CDR2 contains the amino acid sequence shown in SEQ ID NO: 17, and CDR3 contains the amino acid sequence shown in SEQ ID NO: 18.

[0006] The sequence is as follows: SEQ ID NO: 1: GLTGNYA SEQ ID NO: 2: IRWSDFSR SEQ ID NO: 3: AAIIRGAYGDLWYRAQPDY SEQ ID NO: 9: GRTFSRNA SEQ ID NO: 10: ISWKGGST SEQ ID NO: 11: AQGRYDSRYSDYTFEDEYDY SEQ ID NO: 16: GFTVDDYA SEQ ID NO: 17: IMPSDGST SEQ ID NO: 18:ATECPRLGGLGYFARGF

[0007] In one aspect, the nanobody also comprises: (1) The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or The FR2 amino acid sequence as shown in SEQ ID NO: 5 or having one amino acid mutation compared to SEQ ID NO: 5, wherein the one mutation in FR2 is selected from the mutation at position 35; and / or The FR3 amino acid sequence shown in SEQ ID NO: 6 or having 1-5 amino acid mutations compared to SEQ ID NO: 6, wherein the 1-5 mutations in the FR3 are selected from mutations at positions 74, 75, 76, 82C, 83, or combinations thereof; and / or The FR4 amino acid sequence as shown in SEQ ID NO: 7 or having one amino acid mutation compared to SEQ ID NO: 7, wherein the one mutation in FR4 is selected from the mutation at position 108; or (2) The FR1 amino acid sequence as shown in SEQ ID NO: 12; and / or The FR2 amino acid sequence as shown in SEQ ID NO: 13; and / or The FR3 amino acid sequence as shown in SEQ ID NO: 14; and / or As shown in SEQ ID NO: 7, the FR4 amino acid sequence; or (3) The FR1 amino acid sequence as shown in SEQ ID NO: 19; and / or The FR2 amino acid sequence as shown in SEQ ID NO: 20; and / or The FR3 amino acid sequence as shown in SEQ ID NO: 21; and / or The FR4 amino acid sequence is shown in SEQ ID NO: 7.

[0008] Preferably, in the nanobody: (1) One mutation in FR2 is selected from the E35S mutation; and / or (2) The 1-5 mutations in FR3 are selected from A74S, R75K, A76N, M(82C)L, K83R mutations, or combinations thereof; and / or (3) One mutation in FR4 is selected from the Q108L mutation.

[0009] In one aspect, the nanobody comprises: (1) The FR1 amino acid sequence of SEQ ID NO: 4, and / or The FR2 amino acid sequence as shown in SEQ ID NO: 5, and / or The FR3 amino acid sequence shown in SEQ ID NO: 6, and / or As shown in SEQ ID NO: 7, the FR4 amino acid sequence; or (2) The FR1 amino acid sequence of SEQ ID NO: 12, and / or The FR2 amino acid sequence shown in SEQ ID NO: 13, and / or The FR3 amino acid sequence shown in SEQ ID NO: 14, and / or As shown in SEQ ID NO: 7, the FR4 amino acid sequence; or (3) The FR1 amino acid sequence of SEQ ID NO: 19, and / or The FR2 amino acid sequence as shown in SEQ ID NO: 20, and / or The FR3 amino acid sequence shown in SEQ ID NO: 21, and / or As shown in SEQ ID NO: 7, the FR4 amino acid sequence; or (4) The FR1 amino acid sequence of SEQ ID NO: 4, and / or The FR2 amino acid sequence shown in SEQ ID NO: 23, and / or The FR3 amino acid sequence shown in SEQ ID NO: 24, and / or The FR4 amino acid sequence is shown in SEQ ID NO: 25.

[0010] The sequence is as follows: SEQ ID NO: 4: EVQLVESGGGLVQAGGSLRLSCAAS SEQ ID NO: 5: IEWFRQAPGKEREFVAA SEQ ID NO: 6: TYADSVKGRFTISRDNARATAYLQMNSMKAEDTAVYYC SEQ ID NO: 7: WGQGTQVTVSS SEQ ID NO: 12: EVQLVESGGGLVQAGDSLRLSCAHS SEQ ID NO: 13: MGWFRQAPGKEREFVAA SEQ ID NO: 14: KYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC SEQ ID NO: 19: QVQLVESGGGLVQPGGSLRLSCAAS SEQ ID NO: 20: VGWFRQAPGKEREGVSC SEQ ID NO: 21: YYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYC SEQ ID NO: 23: ISWFRQAPGKEREFVAA SEQ ID NO: 24: TYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYC SEQ ID NO: 25: WGQGTLVTVSS

[0011] In one aspect, the nanobody comprises: (1) The amino acid sequence as shown in SEQ ID NO: 8; or (2) The amino acid sequence as shown in SEQ ID NO: 15; or (3) The amino acid sequence as shown in SEQ ID NO: 22; or (4) The amino acid sequence shown in SEQ ID NO: 26.

[0012] The sequence is as follows: SEQ ID NO: 8: EVQLVESGGGLVQAGGSLRLSCAASGLTFGNYAIEWFRQAPGKEREFVAAIRWSDFSRTYADSVKGRFTISRDNARATAYLQMNSMKAEDTAVYYCAAIIRGAYGDLWYRAQPDYWGQGTQVTVSS SEQ ID NO: 15: EVQLVESGGGLVQAGDSLRLSCAHSGRTFSRNAMGWFRQAPGKEREFVAAISWKGGSTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAQGRYDSRYSDYTFEDEYDYWGQGTQVTVSS SEQ ID NO: 22: QVQLVESGGGLVQPGGSLRLSCAASGFTVDDYAVGWFRQAPGKEREGVSCIMPSDGSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCATECPRLGGLGYFARGFWGQGTQVTVSS SEQ ID NO: 26: EVQLVESGGGLVQAGGSLRLSCAASGLTFGNYAISWFRQAPGKEREFVAAIRWSDFSRTYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCAAIIRGAYGDLWYRAQPDYWGQGTLVTVSS

[0013] This invention relates to the use of the above-mentioned nanobodies or their binding fragments in the construction of monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, or recombinant proteins.

[0014] This invention relates to monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, or recombinant proteins comprising the above-mentioned nanobodies or their binding fragments.

[0015] In one aspect, the monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein includes an Fc domain. Preferably, the Fc domain is selected from IgA, IgD, IgE, IgG, and IgM. Preferably, the Fc domain is selected from IgG. Preferably, the Fc domain is selected from IgG1. The hinge region included in the Fc domain may be complete, partial, or missing.

[0016] In one aspect, the IgG1 Fc domain comprises an amino acid sequence shown in SEQ ID NO: 31, or having more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 31. SEQ ID NO: 31: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0017] This invention relates to a polynucleotide that encodes any of the nanobodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, or recombinant proteins described above.

[0018] This invention relates to a carrier comprising any of the polynucleotides described above.

[0019] This invention relates to a host cell, wherein the host cell comprises the vector described in any of the preceding claims, or wherein the genome of the host cell integrates the polynucleotides described in any of the preceding claims. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is *Escherichia coli* cells, etc. Preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell, etc.; preferably, the yeast cell is *Pichia pastoris* cells, etc. Preferably, the cell is a mammalian cell; preferably, the mammalian cell is a Chinese hamster ovary cell (CHO), human embryonic kidney cell (293), B cell, T cell, DC cell, or NK cell, etc.

[0020] This invention relates to a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments thereof described above (including but not limited to nanobodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, recombinant proteins, etc.), polynucleotides, carriers, cells or combinations thereof, and pharmaceutically acceptable carriers thereof.

[0021] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, carriers, cells or combinations thereof described above in the preparation of medicaments for treating cancer.

[0022] This invention relates to a method for treating cancer, comprising administering to a subject in need an effective amount of any of the above-described antibodies or antigen-binding fragments thereof, polynucleotides, carriers, cells or combinations thereof.

[0023] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof described above for the treatment of cancer.

[0024] The present invention relates to antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof for the treatment of cancer.

[0025] This invention relates to a kit comprising any of the antibodies or antigen-binding fragments thereof described above, polynucleotides, vectors, cells or combinations thereof.

[0026] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof as described above in the preparation of diagnostic or detection kits.

[0027] This invention relates to a method for diagnosing or detecting cancer, comprising administering an antibody or antigen-binding fragment, polynucleotide, vector, cell, or combination thereof or a kit according to any one of the present invention to a subject or sample in need.

[0028] This invention relates to the use of any of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, cells or combinations thereof, or kits described above for the detection or diagnosis of cancer.

[0029] The nanobody provided by this invention has a variety of excellent effects, such as high stability, easy expression and purification, minimal impact on the IL-2 signaling pathway, excellent ADCC and ADCP effects, and endocytosis effect, and has good efficacy and safety. Attached Figure Description

[0030] Figure 1 Study on the binding of CD25 antibody with human CD-his ELISA.

[0031] Figure 2 CD25 antibody binding with cyno CD-his ELISA.

[0032] Figure 3 ELISA assay of competition between CD25 antibody and IL2.

[0033] Figure 4 pH-dependent assay of CD25 antibody.

[0034] Figure 5 Study on the binding of CD25 antibody to antigen at the cellular level.

[0035] Figure 6 Experiments on the competition between CD25 antibody and IL2 at the cellular level.

[0036] Figure 7 Effects of CD25 antibody on the intracellular STAT5 phosphorylation signaling pathway in CD4+ T cells.

[0037] Figure 8 Effects of CD25 antibody on the intracellular STAT5 phosphorylation signaling pathway in CD8+ T cells.

[0038] Figure 9 Effects of CD25 antibody on the intracellular STAT5 phosphorylation signaling pathway in Treg T cells.

[0039] Figure 10 CD25 antibody activation of T cells experiment.

[0040] Figure 11 Detection of ADCC in SU-DHL-1 cells using CD25 antibody.

[0041] Figure 12 Detection of ADCC in KARPA299 cells using CD25 antibody.

[0042] Figure 13 Detection of ADCP in SU-DHL-1 cells using CD25 antibody.

[0043] Figure 14 : Endocytosis assay of CD25 antibody.

[0044] Figure 15 CD25#16 VHH amino acid sequence position number (Kabat number).

[0045] Figure 16 :CD 25#16 First round of humanized Tm detection.

[0046] definition

[0047] Unless otherwise specifically stated otherwise, the practice of this invention will take place using conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the scope of the art, or in the same sense as commonly understood by one of ordinary skill in the art to which this invention pertains. Many of these are described below for illustrative purposes, and such techniques are well described in the literature.

[0048] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (such as any one of the three CDRs of an immunoglobulin light chain or any one of the three CDRs of an immunoglobulin heavy chain) and capable of specifically binding to an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, VHH, single-chain antibodies, chimeric antibodies, humanized antibodies, and humanized antibodies. The term "antibody" as used herein includes both naturally occurring and non-naturally occurring antibodies. Generally, naturally occurring antibodies (also called immunoglobulins) consist of two classes of polypeptide chains: a light chain and a heavy chain. The non-limiting antibody disclosed in this invention can be a complete tetraimmunoglobulin chain antibody composed of two heavy chains and two light chains. The heavy chain of an antibody can be any isoform, including IgM, IgG, IgE, IgA, or IgD, or subisoforms, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. Each heavy chain contains a variable domain (or variable region, VH) and a constant domain (or constant region, CH), which are linked together by disulfide bonds within the constant domain. Each light chain contains a variable domain (or variable region, VL) and a constant domain (or constant region, CL), which are each linked to one heavy chain by a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain it binds to. Both the light and heavy chain variable regions contain three hypervariable regions sandwiched between more conserved framework regions (FRs). These hypervariable regions, also known as complementarity-determining regions (CDRs), constitute the primary antigen-binding surface of the antibody. Determining the antibody's critical DNA sequence (CDR) by analyzing its amino acid sequence is a well-known method, and many commonly used CDR definitions exist, including but not limited to IMGT, KABAT, CHOTHIA, ABM, and CONTACT. In some embodiments, the antibody described in this invention may be a VHH or a protein containing a VHH that has antigen-binding capabilities. In some embodiments, the antibody may contain a constant region or Fc region of a human antibody. The heavy chain constant region or Fc region may be derived from IgM, IgG, IgE, IgA, or IgD or their subisotypes, such as, but not limited to, IgG1, IgG2, IgG3, and IgG4. The light chain constant region may be derived from the kappa light chain or the lambda light chain. The term antibody also includes derivatives, such as adjustments, conversions, additions, or reductions in structural / functional fragments, such as antibody-drug conjugates.

[0049] The term "nanobody" as used in this article can also be referred to as "heavy-chain single-domain antibody," "single-domain antibody (sdAb)," "VHH domain," "VHH structural domain," "VHH antibody," "VHH antibody fragment," "VHH," "Nanobody," and "Nanobody structural domain," and these names are used interchangeably. It originally derives from the antigen-binding immunoglobulin variable domain of "heavy-chain antibody" (hcAb, i.e., "antibody lacking light chains") (C. Hamers-Casterman, T. Atarhouch, et al. Naturally occurring antibodies devoid of light chains. Nature, June 3, 1993, Vol. 363, pp. 446-448). The term "VHH structural domain" distinguishes this type of variable domain from the heavy-chain variable domain (VH or VH domain) present in conventional antibodies composed of two light chains and two heavy chains (hereinafter referred to as conventional antibodies) and the light-chain variable domain (VL or VL domain) present in conventional antibodies. Nanobodies specifically bind to epitopes without requiring additional antigen-binding domains (unlike conventional antibodies, where the VH or VL domains are combined with the VH domain to recognize epitopes). Nanobodies are antigen-recognizing units formed from a single immunoglobulin domain.

[0050] The term "CDR" (complementarity-determining region) used herein refers to a hypervariable region, and the CDRs involved in this invention are defined using the IMGT system. Unless otherwise specified, the immunoglobulin residues involved in this invention are numbered using the Kabat index. The methods for defining CDRs using the IMGT system and for numbering amino acid residues using the Kabat index are well known to those skilled in the art, and can be used, for example, with tools available at "http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi" for annotation and analysis.

[0051] As used herein, the term "specificity" refers to an antigen-binding protein or antibody that selectively recognizes a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, the terms "bispecific" or "multispecific" indicate that an antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen.

[0052] As used herein, the term "vector" refers to any construct capable of delivering one or more related polynucleotides to a host cell when introduced into the host cell. Vectors can be introduced into host cells using methods known in the art, such as electroporation, transfection, transformation, infection, and injection. Non-limiting examples of vectors include viral vectors, naked DNA or RNA, plasmids, etc. Based on vector properties, vectors can be classified as viral vectors (e.g., lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, bacteriophage vectors, etc.) and non-viral vectors (e.g., plasmids, etc.). Based on vector function, vectors can be classified as cloning vectors, expression vectors, etc. Based on the type of recipient cell into which the vector enters, vectors can be classified as eukaryotic vectors, prokaryotic vectors, shuttle vectors, etc.

[0053] As used in this article, “treatment” refers to a clinical intervention designed to alter the natural course of a treatment within an individual or cell during a clinicopathological process. Desired therapeutic effects include slowing disease progression, improving or alleviating disease states, and mitigating or improving prognosis. For example, reducing or eliminating one or more symptoms associated with the treated disease or condition (such as cancer, inflammation, or an autoimmune disease).

[0054] As used herein, the term "effective amount" refers to an amount or dose sufficient to produce a beneficial or intended effect, including preventing, slowing, delaying, or inhibiting the progression of a disease (such as cancer). In the case of cancer, the effective amounts of VHH, bispecific antibodies, multispecific antigen-binding constructs, pharmaceutical compositions, and immunoconjugates provided in this application may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably prevent) the metastasis of tumor cancer cells; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent. As understood in the clinical setting, the effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without combination with another drug, compound, or pharmaceutical composition.

[0055] The terms “subject” and “patient” as used herein are used interchangeably throughout the specification and are used to describe animals (human or non-human) to which they are treated by the method according to the invention. “Subject” is preferably a mammal, including but not limited to humans, cattle, horses, felines, canines, rodents, or primates. In some embodiments, the subject is a human. Detailed Implementation

[0056] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0057] Example

[0058] Example 1: Screening and Development of CD25-Targeted Nanobodies

[0059] Example 1.1 Molecular Characterization

[0060] Example 1.1.1 Screening and Control Construction

[0061] After screening using alpaca immunoglobulin and phage libraries, a total of 13 sequences were identified that can bind to hCD25. These 13 sequences were then artificially synthesized and constructed into the pcDNA3.4 vector, where they were fused with the hIgG1 Fc fragment for expression. The corresponding protein samples were named CD25#10~CD25#22.

[0062] One of the control antibodies used was a vopikitug analog, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 27 and the light chain amino acid sequence of which is shown in SEQ ID NO: 28.

[0063] SEQ ID NO: 27 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSLAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGSVSGTLVDFDIWG QGTMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI KPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0064] SEQ ID NO: 28 DIQMTQSPSTLSASSVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNIYPITFGGGTKVEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0065] The second control antibody uses a daclizumab analogue (BIIB-019), the heavy chain amino acid sequence of which is shown in SEQ ID NO: 29 and the light chain amino acid sequence of which is shown in SEQ ID NO: 30.

[0066] SEQ ID NO: 29 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTGYTEYNQKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0067] SEQ ID NO: 30 DIQMTQSPSTLSASSVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0068] The IgG1 Fc sequence is shown in SEQ ID NO: 31.

[0069] SEQ ID NO: 31 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0070] Example 1.1.2 Analysis of the thermostability of CD25 antibody

[0071] The Tm value of antibody samples was determined by qPCR. The system composition was: 1 µL SYPRO orange (final concentration 5x), 10 µg protein sample, and finally 1x PBS to a final volume of 20 µL. Each sample constituted one reaction. After sample preparation, the sample was scanned at 25℃ for 2 min, then the temperature was increased at 0.05℃ / s, and finally scanned at 99℃ for 2 min. The data were analyzed using a derivative method.

[0072] The results are shown in Table 1. Except for CD25#10, CD25#13, and CD25#15, whose Tm values ​​are below 55℃, the Tm values ​​of the other antibodies are all above 60℃.

[0073] Table 1. Antibody protein Tm values

[0074] Example 1.1.3 Binding of CD25 antibody to antigens of different species

[0075] The purified antibody protein was subjected to ELISA binding experiments with antigens from different species to verify the binding activity of the antibody with antigens from different species. First, antigens from different species (human CD25-his, mouse CD25-his, and cyno CD25-his) were coated overnight on 96-well ELISA plates at a concentration of 0.5 µg / mL, 100 µL / well. The plates were washed three times with 0.05% PBST, and then blocked with 200 µL of 5% milk-PBS for 1 hour at room temperature. After washing three times with 0.05% PBST, the antibody samples were diluted, starting at 50 nM and then serially diluted 5-fold eight times, with 100 µL of diluted sample added to each well. The plates were incubated at room temperature for 2 hours. After washing three times with 0.05% PBST, 100 µL of Goat pAb to Hu IgG (HRP) was added to each well at a ratio of 1:10000, and the plates were incubated at room temperature for 1 hour. After washing six times with 0.05% PBST, 100 µL of TMB was added to each well, and after 5 minutes of development, 100 µL of TMB was added to each well. The optical density of the sample was measured at an OD wavelength of 450 nm using the ELISA stop solution.

[0076] The results are as follows Figure 1-2 As shown, all 13 antibody samples exhibited good human-cyno cross-activity.

[0077] Example 1.1.4 Competition experiment between CD25 antibody and IL2

[0078] To further verify whether the antibody samples could block IL-2, a competitive assay was performed on the antibody proteins. Human CD25-his (Cat NO: 10165-H08H, Sino) was coated overnight on a 96-well microplate at 0.5 µg / mL, 100 µL / well. The plates were washed three times with 0.05% PBST, and then each well was blocked with 200 µL of 5% milk-PBS at room temperature for 1 hour. After washing three times with 0.05% PBST, the antibody sample was initially diluted 500 nM in eight 5-fold serial dilutions. It was premixed with 2.5 µg / mL Bioteinylated IL2 (Cat NO: IL2-H82E4, ACRO) for 10 minutes and then added to the microplate. The plates were incubated at room temperature for 2 hours. After washing three times with 0.05% PBST, 100 µL of Anti-SA-HRP (Cat NO: ab7403, ...) was added to each well at a 1:5000 ratio. (abcam); wash six times with 0.05% PBST, add 100 µL TMB to each well, develop color for 5 minutes, then add 100 µL ELISA stop solution to each well, and measure the sample optical density at OD450nm.

[0079] The results are as follows Figure 3 As shown, CD25#16, CD25#17, and CD25#18 do not block the binding of IL2 to CD25.

[0080] Example 1.1.5 pH-dependent assay of CD25 antibody

[0081] To verify whether the CD25 antibody exhibits pH-dependent binding, the binding of the antibody and antigen was detected under pH 6.0 and pH 7.4 conditions, respectively. Human CD25-his (Cat NO: 10165-H08H, Sino) was coated overnight on a 96-well microplate at 0.5 µg / mL, 100 µL / well. The plates were washed three times with 0.05% PBST, then blocked with 200 µL of 5% milk-PBS for 1 hour at room temperature. After washing three times with 0.05% PBST, the antibody was diluted with 1xPBS at pH 6.0 and pH 7.4, respectively, with an initial concentration of 50 nM. Eight 5-fold serial dilutions were performed, and the plates were incubated at room temperature for 2 hours. After washing three times with 0.05% PBST, 100 µL of Goat pAb to Hu IgG (HRP) (Cat NO: ab97225, abcam) was added to each well at a ratio of 1:10000. After washing six times with 0.05% PBST, 100 µL of TMB was added to each well. After 5 minutes of development, 100 µL of TMB was added to each well. The optical density of the sample was measured at an OD wavelength of 450 nm using the ELISA stop solution.

[0082] The results are as follows Figure 4 As shown, CD25#16, CD25#17, and CD25#18 do not show obvious pH dependence.

[0083] In summary, CD25#16, CD25#17, and CD25#18 exhibit high purity, Tm values ​​above 66℃, human-cyno cross-activity, the ability to not block receptor ligand binding, and antigen binding without significant pH dependence. Therefore, these three antibodies were selected for subsequent experiments. Their sequence information is as follows:

[0084] CD25#16 (SEQ ID NO: 32; the bolded and underlined areas are CDR1-3 (IMGT system)): EVQLVESGGGLVQAGGSLRLSCAAS GLTFGNYA IEWFRQAPGKEREFVAA IRWSDFSR TYADSVKGRFTISRDNARATAYLQMNSMKAEDTAVYYC AAIIRGAYGDLWYRAQPDYWGQGTQVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0085] CDR1 of CD25#16 (SEQ ID NO: 1): GLTFGNYA

[0086] CDR2 of CD25#16 (SEQ ID NO: 2): IRWSDFSR

[0087] CDR3 of CD25#16 (SEQ ID NO: 3): AAIIRGAYGDLWYRAQPDY

[0088] FR1 of CD25#16 (SEQ ID NO: 4): EVQLVESGGGLVQAGGSLRLSCAAS

[0089] FR2 of CD25#16 (SEQ ID NO: 5): IEWFRQAPGKEREFVAA

[0090] FR3 of CD25#16 (SEQ ID NO: 6): TYADSVKGRFTISRDNARATAYLQMNSMKAEDTAVYYC

[0091] FR4 of CD25#16 (SEQ ID NO: 7): WGQGTQVTVSS

[0092] VHH of CD25#16 (SEQ ID NO: 8): EVQLVESGGGLVQAGGSLRLSCAASGLTFGNYAIEWFRQAPGKEREFVAAIRWSDFSRTYADSVKGRFTISRDNARATAYLQMNSMKAEDTAVYYCAAIIRGAYGDLWYRAQPDYWGQGTQVTVSS

[0093] CD25#17 (SEQ ID NO: 33; the bold and underlined regions are CDR1-3 (IMGT system), respectively): EVQLVESGGGLVQAGDSLRLSCAHS GRTFSRNA MGWFRQAPGKEREFVAA ISWKGGST KYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AQGRYDSRYSDYTFEDEYDY WGQGTQVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0094] CDR1 of CD25#17 (SEQ ID NO: 9): GRTFSRNA

[0095] CDR2 of CD25#17 (SEQ ID NO: 10): ISWKGGST

[0096] CDR3 of CD25#17 (SEQ ID NO: 11): AQGRYDSRYSDYTFEDEYDY

[0097] FR1 of CD25#17 (SEQ ID NO: 12): EVQLVESGGGLVQAGDSLRLSCAHS

[0098] FR2 of CD25#17 (SEQ ID NO: 13): MGWFRQAPGKEREFVAA

[0099] FR3 of CD25#17 (SEQ ID NO: 14): KYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

[0100] FR4 of CD25#17 (SEQ ID NO: 7): WGQGTQVTVSS

[0101] VHH of CD25#17 (SEQ ID NO: 15): EVQLVESGGGLVQAGDSLRLSCAHSGRTFSRNAMGWFRQAPGKEREFVAAISWKGGSTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAQGRYDSRYSDYTFEDEYDYWGQGTQVTVSS

[0102] CD25#18 (SEQ ID NO: 34; the bold and underlined regions are CDR1-3 (IMGT system), respectively): QVQLVESGGGLVQPGGSLRLSCAAS GFTVDDYA VGWFRQAPGKEREGVSC IMPSDGST YYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYC ATECPRLGGLGYFARGF WGQGTQVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0103] CDR1 of CD25#18 (SEQ ID NO: 16): GFTVDDYA

[0104] CDR2 of CD25#18 (SEQ ID NO: 17): IMPSDGST

[0105] CDR3 of CD25#18 (SEQ ID NO: 18): ATECPRLGGLGYFARGF

[0106] FR1 of CD25#18 (SEQ ID NO: 19): QVQLVESGGGLVQPGGSLRLSCAAS

[0107] FR2 of CD25#18 (SEQ ID NO: 20): VGWFRQAPGKEREGVSC

[0108] FR3 of CD25#18 (SEQ ID NO: 21): YYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYC

[0109] CD25#18 FR4 (SEQ ID NO: 7): WGQGTQVTVSS

[0110] VHH of CD25#18 (SEQ ID NO: 22): QVQLVESGGGLVQPGGSLRLSCAASGFTVDDYAVGWFRQAPGKEREGVSCIMPSDGSTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCATECPRLGGLGYFARGFWGQGTQVTVSS

[0111] Example 1.2 Functional Verification

[0112] Example 1.2.1 Binding of CD25 antibody to antigen at the cellular level

[0113] To verify the binding ability of antibodies to CD25 on cells, the binding of antibody samples was tested using two cell lines: SU-DHL-1 and KARPA299. First, SU-DHL-1 and KARPA299 cells were collected, and cell numbers were counted. 100 µL of each cell line was seeded into 96-well plates, with a cell count of 2E5. The cells were washed three times with 1xPBS, centrifuged, and the supernatant was discarded. Antibody samples were added, with an initial concentration of 50 nM. Eight 5-fold serial dilutions were performed, with 100 µL of the diluted sample added to each well and mixed thoroughly. The plates were incubated at 4°C for 1 hour. After washing three times with 1xPBS, centrifuged, and the supernatant was discarded. 100 µL of Alexa Fluor® 647-AffiniPure Goat Anti-Human IgG (Cat NO. 109-605-098, Jackson) diluted 1:500 was added to each well, and the plates were incubated at 4°C for 1 hour. The plates were then washed six times with 1xPBS, and antibody binding was detected using FACS.

[0114] The results are as follows Figure 5 As shown, CD25#16, CD25#17, and CD25#18 showed good binding in both SU-DHL-1 and KARPA299 cell lines.

[0115] Example 1.2.2 Competition experiment between CD25 antibody and IL2 at the cellular level

[0116] To verify whether the antibody does not block the binding of IL2 to CD25 when it binds to CD25 on cells, a competition experiment at the cellular level can be performed. First, collect SU-DHL-1 cells, count the cell number, and seed 100 µL of cells into each well of a 96-well plate (2E5 cells per well). Wash three times with 1xPBS, centrifuge and discard the supernatant. Dilute the antibody sample to a final concentration of 1000 nM in the first well, perform eight 5-fold serial dilutions, and premix with 5 µg / mL Biotinylated IL-2 for 10 minutes. Add 100 µL of the premixed sample to each well of the 96-well plate containing the cells and incubate at 4°C for 1 hour. Wash three times with 1xPBS, centrifuge and discard the supernatant. Add 100 µL of PE anti-biotin (Cat NO: 409004) to each well and incubate at 4°C for 1 hour. Centrifuge and discard the supernatant. Wash the cells with 1% FBS + PBS, then resuspend the cells in 150 µL of 1% FBS + PBS before FACSLyric assay.

[0117] The results are as follows Figure 6 As shown, CD25#16, CD25#17, and CD25#18 do not block the binding of IL2 to CD25.

[0118] Example 1.2.3 Experiment on the effect of CD25 antibody on STAT5 phosphorylation signaling pathway

[0119] The STAT5 signaling pathway in CD4+, CD8+, and Treg cells was measured separately to identify the nanobody among CD25#16, CD25#17, and CD25#18 that has the least impact on the IL-2 activation of the STAT5 phosphorylation signaling pathway at the signaling pathway level. First, 1000 nM antibody sample was co-cultured with CD4+, CD8+, and Treg cells at 37°C for 30 min. Then, 10 IU / mL IL-2 was added, and the cells were co-cultured at 37°C for 15 min. Next, 1X Foxp3 fixation / permeablization (Cat NO: 00-5523-00, Invitrogen) was added, and the cells were incubated at room temperature in the dark for 40 min. Each well was washed once with 200 µL of 1×rupture solution, then centrifuged at 400g for 5 min and the supernatant was discarded. Phosfow™ Perm Bufer III (Cat NO: 558050, BD Biosciences) was added on ice, and the cells were incubated at 4°C for 30 min. The cells were washed once with 1xPBS, centrifuged, and the supernatant was discarded. Cells were then stained with Alexa488 pSTAT5 (Cat NO: 562075, BDBiosciences) at room temperature for 40 min. Finally, 1%... After washing once with FBS and PBS, centrifuge and discard the supernatant. Resuspend the cells in 1% FBS and PBS and use FACS Lyric for detection.

[0120] The results are as follows Figures 7-9 As shown, CD25#16 has the least impact on the IL-2 activation of the STAT5 phosphorylation signaling pathway.

[0121] Example 1.2.4 CD25 antibody activation of T cells experiment

[0122] To further verify the effects of CD25#16, CD25#17, and CD25#18 on the IL2-activated T cell signaling pathway, Human CD8+ T cells were first activated using CD3 / CD28 beads in the presence of different antibody concentrations. Then, 1xFoxp3 fixation / permeablization (Cat NO: 00-5523-00, Invitrogen) was added and the cells were incubated at room temperature in the dark for 40 minutes. 200µL of 1×rupture solution was added to each well, and the cells were centrifuged at 400g for 5 minutes, washed once, and the supernatant was discarded. The cells were then washed once with 1xPBS, and the supernatant was discarded. The cells were then stained with APC Ki67 (Cat NO: 350514, Biolegend) and FITC Granzyme B (Cat NO: 515403, Biolegend) at room temperature for 40 minutes. The cells were washed once with 1% FBS+PBS, the supernatant was discarded, and the cells were resuspended in 1% FBS+PBS and analyzed using FACS Lyric.

[0123] The results are as follows Figure 10 As shown, the three antibodies CD25#16, CD25#17, and CD25#18 can all activate T cells, and their effects on the IL2 signaling pathway are all smaller than those of the Daclizumab analogue. Among them, CD25#16 and CD25#18 have a smaller effect on the IL2 signaling pathway than CD25#17.

[0124] Example 1.2.5 ADCC and ADCP experiments of CD25 antibody

[0125] To verify the antibody-dependent cell-mediated cytotoxicity of the antibody samples, ADCC assays were performed. First, SU-DHL-1 and KARPA299 cells were seeded into 96-well plates, resulting in 2E4 cells per well. The antibody samples were diluted, with an initial concentration of 300 nM, followed by eight 5-fold serial dilutions, with 50 µL of diluted antibody sample added to each well. Then, 25 µL of analytical medium (RPMI 1640 + 2% FBS) containing 1E5 Jurkat-FcγIIIa-NFAT cells was added to each well, resulting in an effector cell to target cell ratio of 5:1. The assay plate was incubated at 37°C and 5% CO2 for 6 hours. Finally, 100 µL of luminescent reagent (Cat NO: DD1204-02, Vazyme) was added, and relative luciferase units (RLUs) were read using a microplate reader.

[0126] The results are as follows Figures 11-12As shown, CD25#16, CD25#17, and CD25#18 all exhibited stronger ADCC effects than Daclizumab and RG6292 analogs in SU-DHL-1 and KARPA299 cells, with CD25#18 showing the strongest ADCC effect.

[0127] To verify the antibody-dependent cell-mediated phagocytosis of the antibody samples, ADCP detection was performed. First, SU-DHL-1 cells were seeded into 96-well plates, resulting in 2E4 cells per well. The antibody samples were diluted, with an initial concentration of 1000 nM, followed by six 3-fold serial dilutions. 50 µL of the diluted antibody sample was added to each well to resuspend the cells and premix for 15 minutes. Then, 25 µL of analytical medium (RPMI 1640 + 2% FBS) containing 1.6E5 Jurkat-FcγIIIa-NFAT cells was added to each well. The plate was incubated at 37°C for 6 hours. Finally, 100 µL of luminescent reagent (Cat NO: DD1204-02, Vazyme) was added, and relative luciferase units (RLUs) were read using a microplate reader.

[0128] The results are as follows Figure 13 As shown, CD25#16, CD25#17, and CD25#18 all have stronger ADCP effects than Daclizumab analogs and RG6292 analogs, with CD25#16 having the best ADCP effect.

[0129] The corresponding antibodies exhibit excellent ADCC and ADCP effects, suggesting that the antibody molecules can be used for the development of bispecific / polyclonal antibodies.

[0130] Example 1.2.6 Internalization assay of CD25 antibody

[0131] To verify whether the antibody sample exhibited endocytosis, the endocytosis effect was assessed. First, SU-DHL-1 cells were seeded into 96-well plates with 1E5 cells per well. After centrifugation and discarding the supernatant, 100 µL of diluted antibody sample was added to each well. The initial concentration of the sample in each well was 500 nM, with eight 5-fold serial dilutions. After resuspending the cells, they were incubated at 4°C for 30 minutes. After centrifugation and discarding the supernatant, the cells were washed three times with 1xPBS, and then 100 µL of pHrodo Red labeled anti-human IgG (Cat NO: P35356, Invitrogen) was added. The cells were then incubated at 37°C for 16 hours. After washing the cells with FACS buffer and resuspending them, FACSLyric assays were performed.

[0132] The results showed that CD25#16, CD25#17, and CD25#18 all had good endocytosis effects. Figure 14 This suggests that antibodies can be used in the development of ADCs.

[0133] In summary, CD25#16 was selected for humanization modification.

[0134] Example 2: Humanization and Optimization of CD25#16

[0135] The humanized templates are IGHV3-23*04 and IGHJ4*01. The CDRs from the camel source are transplanted into their humanized templates to obtain the corresponding humanized templates.

[0136] Example 2.1 First round of humanization

[0137] As needed, key amino acids in the FR region of the humanized antibody were reverse-mutated to the corresponding amino acids of the camel-derived antibody to maintain the original affinity. The specific reverse-mutation design is shown in Table 2. The reverse-mutated amino acids are numbered using the Kabat code, and the amino acid sequence positions are as follows: Figure 15 As shown.

[0138] Table 2. Mutation design of CD25#16 humanized antibody Note: CD25#23 is the sequence of the camel-derived antibody CDR transplanted into the FR region of the human template (where human amino acids 37, 44, 45, and 47 have been replaced with the camel-derived amino acids, namely V37F, G44E, L45R, and W47F); for molecules CD25#24 to CD25#35, the mutations listed in the mutation column in Table 2 are the mutations made to the corresponding molecules relative to CD25#23. For example, P14A means that the P at position 14 of CD25#23 is mutated to A, and so on.

[0139] Eukaryotic expression plasmids were constructed, transfected with 293SQ, purified, and then their functions were validated. Protein stability was analyzed. Figure 16 The Tm values ​​of CD25#25, CD25#27, and CD25#32 were relatively lower, indicating that I34, A49, and A78 affect protein stability. The binding ability of CD25#32 to the antigen human CD25 is much weaker than that of CD25#16 to the antigen human CD25, indicating that A78 plays an important role in maintaining antigen binding activity after humanization (Table 3).

[0140] Table 3. Functional validation of CD25#16 in the first round of humanized antibody testing

[0141] Example 2.2 Second round of humanization

[0142] Based on the preliminary experimental results and protein simulation analysis of CD25#16, humanized design was performed. Eukaryotic expression plasmids were constructed, transfected with 293SQ, purified, and then their functions were validated. Specific reversion mutations and detection results are shown in Table 4.

[0143] Table 4. Second Round Humanized Antibody Mutation Design and Results for CD25#16

[0144] Based on a comprehensive evaluation of yield, Tm value, and EC50 value of affinity for the CD25 antigen, CD25#41 was found to be the best-performing molecule. Therefore, CD25#41 was selected as the lead molecule after humanization of CD25#16.

[0145] CD25#41 contains the same CDRs as CD25#16, with the following sequence:

[0146] CD25#41 (SEQ ID NO: 35; the bolded and underlined areas are CDR1-3 (IMGT system)): EVQLVESGGGLVQAGGSLRLSCAAS GLTFGNYA ISWFRQAPGKEREFVAA IRWSDFSR TYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYC AAIIRGAYGDLWYRAQPDY WGQGTLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0147] FR1 of CD25#41 (SEQ ID NO: 4): EVQLVESGGGLVQAGGSLRLSCAAS

[0148] FR2 of CD25#41 (SEQ ID NO: 23): ISWFRQAPGKEREFVAA

[0149] FR3 of CD25#41 (SEQ ID NO:24): TYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYC

[0150] CD25#41 FR4 (SEQ ID NO: 25): WGQGTLVTVSS

[0151] VHH of CD25#41 (SEQ ID NO: 26): EVQLVESGGGLVQAGGSLRLSCAASGLTFGNYAISWFRQAPGKEREFVAAIRWSDFSRTYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCAAIIRGAYGDLWYRAQPDYWGQGTLVTVSS

[0152] Example 2.3 Verification of Cyno Binding in Humanized Molecules

[0153] The binding of CD25#16 to cyno CD25 before and after humanization was verified by ELISA binding. The results are shown in Table 5. Both CD25#16 and CD25#41 bind to cyno CD25.

[0154] Table 5. Detection of CD25#16 humanized molecule cyno CD25 binding.

[0155] The above description discloses only some embodiments of the present invention and is not intended to limit the present invention in any way. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described above. For those skilled in the art, various improvements and modifications can be made to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A nanobody specifically targeting CD25, comprising CDR1, CDR2 and CDR3; wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

2. The nanobody according to claim 1, wherein, The nanobody also comprises: The FR1 amino acid sequence as shown in SEQ ID NO: 4; and / or The FR2 amino acid sequence as shown in SEQ ID NO: 5 or having one amino acid mutation compared to SEQ ID NO: 5, wherein the one mutation in FR2 is selected from the mutation at position 35; and / or The FR3 amino acid sequence shown in SEQ ID NO: 6 or having 1-5 amino acid mutations compared to SEQ ID NO: 6, wherein the 1-5 mutations in the FR3 are selected from mutations at positions 74, 75, 76, 82C, 83, or combinations thereof; and / or The FR4 amino acid sequence is shown in SEQ ID NO: 7 or has a mutation of 1 amino acid compared to SEQ ID NO: 7, wherein the mutation in FR4 is selected from the mutation at position 108.

3. The nanobody according to claim 2, wherein: (1) One mutation in FR2 is selected from the E35S mutation; and / or (2) The 1-5 mutations in FR3 are selected from A74S, R75K, A76N, M(82C)L, K83R mutations, or combinations thereof; and / or (3) One mutation in FR4 is selected from the Q108L mutation.

4. The nanobody according to claim 3, wherein, The nanobody comprises: (1) The FR1 amino acid sequence of SEQ ID NO: 4, and / or The FR2 amino acid sequence as shown in SEQ ID NO: 5, and / or The FR3 amino acid sequence shown in SEQ ID NO: 6, and / or As shown in SEQ ID NO: 7, the FR4 amino acid sequence; or (2) The FR1 amino acid sequence of SEQ ID NO: 4, and / or The FR2 amino acid sequence shown in SEQ ID NO: 23, and / or The FR3 amino acid sequence shown in SEQ ID NO: 24, and / or The FR4 amino acid sequence is shown in SEQ ID NO:

25.

5. The nanobody according to claim 4, wherein, The nanobody comprises: (1) The amino acid sequence as shown in SEQ ID NO: 8; or (2) The amino acid sequence shown in SEQ ID NO:

26.

6. The use of any of the nanobodies described in claims 1-5 in the construction of monospecific antibodies, bispecific antibodies, multispecific antibodies, antibody-drug conjugates, or recombinant proteins.

7. A monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein comprising any of the nanobodies described in claims 1-5.

8. The monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein according to claim 7, comprising an Fc domain, wherein the Fc domain is selected from IgA, IgD, IgE, IgG, or IgM.

9. The monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein according to claim 8, wherein, The Fc domain is selected from IgG1, wherein the hinge region contained in the Fc can be complete, partial or missing.

10. The monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate, or recombinant protein according to claim 9, wherein, The Fc domain of the IgG1 contains an amino acid sequence that is identical to or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the same as SEQ ID NO:

31.

11. A polynucleotide, said polynucleotide encoding the nanobody of claims 1-5 or the monospecific antibody, bispecific antibody, multispecific antibody or recombinant protein of claims 7-10.

12. A vector comprising the polynucleotide of claim 11.

13. A host cell comprising the vector of claim 12, or having the polynucleotide of claim 11 integrated into its genome, wherein the cell is a bacterial cell, a fungal cell, or a mammalian cell.

14. The cell according to claim 13, wherein the cell is an Escherichia coli cell, a Pichia pastoris cell, a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, or an NK cell.

15. A pharmaceutical composition comprising the nanobody of claims 1-5 or the monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate or recombinant protein of claims 7-10, the polynucleotide of claim 11, the carrier of claim 12, the cell of claims 13-14 or a combination thereof, and a pharmaceutically acceptable carrier thereof.

16. Use of the nanobody of claims 1-5, or the monospecific antibody, bispecific antibody, multispecific antibody, antibody-drug conjugate or recombinant protein of claims 7-10, the polynucleotide of claim 11, the carrier of claim 12, the cell of claims 13-14, or a combination thereof, in the preparation of a medicament for treating cancer.

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