Anti-CD25 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof

Abstract

An anti-CD25 antibody, an antigen-binding fragment thereof, and their medical uses are provided. Specifically, an anti-CD25 antibody and its use in preparing an anti-tumor drug are provided.

Description

[0001] This application claims the priority of a Chinese patent application (application number 202010408502.3) filed on May 14, 2020. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, particularly the field of cancer immunotherapy, including methods for treating cancer (such as solid tumors), and relates to the use of anti-CD25 antibodies or antigen-binding fragments thereof. Background Art

[0003] Cancer immunotherapy is a hot topic in the field of cancer treatment. Currently, it mainly enhances the number and activity of CD4 + and CD8 + T cells within tumors or inhibits the number and activity of inhibitory immune cells within tumors. For example, it inhibits regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), or tumor-associated macrophages. The treatment strategy of reducing or eliminating Treg cells within tumors has been clinically verified.

[0004] CD25 is a type I membrane protein composed of 272 amino acids. Its expression on T cells is regulated by TCR signals, so it is often used as a marker of T cell activation. IL-2 has an immune activation effect and is an immune regulator for cancer treatment. It acts through the interleukin-2 receptor (IL-2R) on the cell surface. IL-2R consists of three subunits, IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). The three subunits can form three receptor forms: the high-affinity receptor contains all three subunits IL-2Rα / β / γ, the medium-affinity receptor contains IL-2Rβ / γ, and the low-affinity receptor is IL-2Rα. IL-2Rα (i.e., CD25) alone has a low affinity for IL-2 (Kd is 10 -8 M) and does not transmit intracellular signals. IL-2Rβ and IL-2Rγ are necessary for IL-2 to activate downstream signaling pathways. When IL-2 binds to both IL-2Rβ and IL-2Rγ simultaneously, the two receptor subunits form a heterodimer. When IL-2Rα (i.e., CD25) forms a high-affinity receptor (Kd is 10 -11 M) with IL-2Rβ and IL-2Rγ, it can activate downstream JAK-STAT, PI3K-AKT, and MAPK signaling pathways, thereby promoting the proliferation and activity of lymphocytes such as T cells and NK cells.

[0005] Some existing anti-CD25 antibodies block or inhibit the binding of IL-2 to CD25, see, for example, WO2004 / 045512, WO2006 / 108670, WO1993 / 011238, WO1990 / 007861, and WO2017 / 174331. Basiliximab and daclizumab (DAC) are both IgG1 anti-human CD25 antibodies that inhibit the binding of IL-2 to CD25 and have been developed to reduce the activation of effector T cells (Teff) based on the immune activation function of CD25-mediated IL-2. Basiliximab is a chimeric anti-CD25 antibody currently approved for graft-versus-host disease (GVHD), while daclizumab is a humanized anti-CD25 antibody approved for the treatment of multiple sclerosis.

[0006] Since the expression abundance of CD25 on Tregs is much higher than that on other immune cells, CD25 can also be used as a Treg-specific marker for the development of antibodies to remove Treg cells inside tumors through antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), thereby relieving the inhibition of Tregs on T cells inside tumors and promoting anti-tumor immunity. However, CD25 also mediates the activation of effector T cells by IL-2. If the CD25 antibody inhibits the IL-2 signaling pathway, it will inhibit Teff and antagonize its anti-tumor activity. Some documents mention the use of anti-CD25 alone or in combination for cancer or uses related to Treg depletion (WO2004 / 074437, WO 2006 / 108670, WO 2006 / 050172, WO 2011 / 077245, WO2016 / 021720, WO 2004 / 045512). Therefore, anti-CD25 antibodies that allow IL-2 to bind to CD25 are needed. Preclinical-developed CD25 antibodies include 7D4 (rat anti-mouse CD25, see, for example, Malek et al. PNAS, 1983 Sep; 80(18):5694-8; Onizuka S et al., Cancer Res. 1999 Jul 1; 59(13):3128-33), 7G7B6 (anti-human CD25, see, for example, Zhang et al., Cancer Biother Radiopharm. 2009 Jun; 24(3):303-309), PC61 (rat anti-mouse CD25, see, for example, Yulius Y Setiady et al., Eur J Immunol. 2010 Mar; 40(3):780-6). When tested in a mouse cancer model, PC61 did not show anti-tumor activity due to its inhibitory effect on the binding of CD25 and IL2, while 7D4 showed better anti-tumor activity than PC61 because it does not affect the binding of IL2 to CD25. In addition, WO2019 / 175216 provides the anti-CD25 antibody RG6292 jointly developed by Roche and TUSK, which has entered clinical phase I to detect its safety and efficacy in solid tumors. WO2018167104 also provides anti-CD25 antibodies that do not affect the binding of IL-2 to CD25.

[0007] There is still an urgent need in the art to develop anti-CD25 antibodies with better anti-tumor effects, higher safety, that do not inhibit the binding of IL-2 to CD25, can eliminate Tregs through ADCC and ADCP effects, and at the same time avoid inhibiting the activity of Teff. Summary of the Invention

[0008] The present disclosure provides anti-CD25 antibodies, antigen-binding fragments thereof, and nucleic acids encoding the same, vectors, host cells, pharmaceutical compositions, methods for treating cancer (especially solid tumors), and pharmaceutical uses.

[0009] Anti-CD25 antibodies, antigen-binding fragments thereof

[0010] The present disclosure provides an anti-CD25 antibody or an antigen-binding fragment thereof, comprising a heavy-chain variable region (VH) and / or a light-chain variable region (VL), wherein:

[0011] 1) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:1, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:2;

[0012] 2) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:3, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:4;

[0013] 3) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:5, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:6;

[0014] 4) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:7, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:8;

[0015] 5) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:9, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:10;

[0016] 6) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:11, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:12;

[0017] 7) the VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:13, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:14;

[0018] 8) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:15, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:16;

[0019] 9) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:17, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:18;

[0020] 10) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:19, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:20;

[0021] 11) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:21, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:22;

[0022] 12) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:23, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:24;

[0023] 13) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:25, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:26; or

[0024] 14) The VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:59, and the VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:60.

[0025] The above CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; in some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0026] In some embodiments, the antibody or its antigen-binding fragment containing VH and VL as shown in 1), 2), 3), 5), or 6) blocks or partially blocks the binding of IL-2 to CD25; in other embodiments, the antibody or its antigen-binding fragment containing VH and VL as shown in 4), 7), 8), 9), 10), 11), 12), or 13) does not block, does not inhibit, or hardly blocks or inhibits the binding of IL-2 to CD25.

[0027] In some embodiments, an anti-CD25 antibody or an antigen-binding fragment thereof is provided, which comprises a VH and / or a VL, wherein:

[0028] The VH comprises an HCDR1 selected from SEQ ID NO: 27, 33, 39 or 45, and / or an HCDR2 selected from SEQ ID NO: 28, 34, 40 or 46, and / or an HCDR3 selected from SEQ ID NO: 29, 35, 41 or 47; the VL comprises an LCDR1 selected from SEQ ID NO: 30, 36, 42 or 48, and / or an LCDR2 selected from SEQ ID NO: 31, 37, 43 or 49, and / or an LCDR3 selected from SEQ ID NO: 32, 38, 44 or 50.

[0029] In some embodiments, an anti-CD25 antibody or an antigen-binding fragment thereof is provided, comprising a VH and / or a VL, wherein:

[0030] i) The VH comprises an HCDR1 as shown in SEQ ID NO: 27 or having at most 3, 2 or 1 amino acid mutations therewith, an HCDR2 as shown in SEQ ID NO: 28 or having at most 3, 2 or 1 amino acid mutations therewith, and an HCDR3 as shown in SEQ ID NO: 29 or having at most 3, 2 or 1 amino acid mutations therewith; and / or the VL comprises an LCDR1 as shown in SEQ ID NO: 30 or having at most 3, 2 or 1 amino acid mutations therewith, an LCDR2 as shown in SEQ ID NO: 31 or having at most 3, 2 or 1 amino acid mutations therewith, and an LCDR3 as shown in SEQ ID NO: 32 or having at most 3, 2 or 1 amino acid mutations therewith;

[0031] ii) The VH comprises an HCDR1 as shown in SEQ ID NO: 33 or having at most 3, 2 or 1 amino acid mutations therewith, an HCDR2 as shown in SEQ ID NO: 34 or having at most 3, 2 or 1 amino acid mutations therewith, and an HCDR3 as shown in SEQ ID NO: 35 or having at most 3, 2 or 1 amino acid mutations therewith; and / or the VL comprises an LCDR1 as shown in SEQ ID NO: 36 or having at most 3, 2 or 1 amino acid mutations therewith, an LCDR2 as shown in SEQ ID NO: 37 or having at most 3, 2 or 1 amino acid mutations therewith, and an LCDR3 as shown in SEQ ID NO: 38 or having at most 3, 2 or 1 amino acid mutations therewith;

[0032] iii) The VH comprises an HCDR1 as shown in SEQ ID NO: 39 or having at most 3, 2, or 1 amino acid mutations therefrom, an HCDR2 as shown in SEQ ID NO: 40 or having at most 3, 2, or 1 amino acid mutations therefrom, and an HCDR3 as shown in SEQ ID NO: 41 or having at most 3, 2, or 1 amino acid mutations therefrom; and / or the VL comprises an LCDR1 as shown in SEQ ID NO: 42 or having at most 3, 2, or 1 amino acid mutations therefrom, an LCDR2 as shown in SEQ ID NO: 43 or having at most 3, 2, or 1 amino acid mutations therefrom, and an LCDR3 as shown in SEQ ID NO: 44 or having at most 3, 2, or 1 amino acid mutations therefrom; or

[0033] iv) The VH comprises an HCDR1 as shown in SEQ ID NO: 45 or having at most 3, 2, or 1 amino acid mutations therefrom, an HCDR2 as shown in SEQ ID NO: 46 or having at most 3, 2, or 1 amino acid mutations therefrom, and an HCDR3 as shown in SEQ ID NO: 47 or having at most 3, 2, or 1 amino acid mutations therefrom; and / or the VL comprises an LCDR1 as shown in SEQ ID NO: 48 or having at most 3, 2, or 1 amino acid mutations therefrom, an LCDR2 as shown in SEQ ID NO: 49 or having at most 3, 2, or 1 amino acid mutations therefrom, and an LCDR3 as shown in SEQ ID NO: 50 or having at most 3, 2, or 1 amino acid mutations therefrom.

[0034] In some specific embodiments, the anti-CD25 antibody or antigen-binding fragment thereof having amino acid mutations has the same or substantially the same affinity and / or function (such as ADCC, ADCP, anti-tumor activity) for binding to human CD25 as the parental antibody or antigen-binding fragment.

[0035] In some embodiments, the foregoing anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure binds to human CD25 with a dissociation equilibrium constant equal to or less than 10-7 M. In some embodiments, it binds to human CD25 with a dissociation equilibrium constant equal to or less than 10-8 M, 10-9 M, 10-10 M, or 10-11 M.

[0036] In some embodiments, there is provided an anti-CD25 antibody or antigen-binding fragment thereof, comprising VH and / or VL, wherein:

[0037] The VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 27, 28 and 29 respectively, and the VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, 31 and 32 respectively;

[0038] The VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 33, 34 and 35 respectively, and the VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 36, 37 and 38 respectively;

[0039] The VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 39, 40 and 41 respectively, and the VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, 43 and 44 respectively; or

[0040] The VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 45, 46 and 47 respectively, and the VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 48, 49 and 50 respectively.

[0041] In some embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:

[0042] The VH is as shown in SEQ ID NO: 1 and the VL is as shown in SEQ ID NO: 2;

[0043] The VH is as shown in SEQ ID NO: 3 and the VL is as shown in SEQ ID NO: 4;

[0044] The VH is as shown in SEQ ID NO: 5 and the VL is as shown in SEQ ID NO: 6

[0045] The VH is as shown in SEQ ID NO: 7 and the VL is as shown in SEQ ID NO: 8;

[0046] The VH is as shown in SEQ ID NO: 9 and the VL is as shown in SEQ ID NO: 10;

[0047] The VH is as shown in SEQ ID NO: 11 and the VL is as shown in SEQ ID NO: 12;

[0048] The VH is as shown in SEQ ID NO: 13 and the VL is as shown in SEQ ID NO: 14;

[0049] The VH is as shown in SEQ ID NO: 15, and the VL is as shown in SEQ ID NO: 16; or

[0050] The VH is as shown in SEQ ID NO: 17, and the VL is as shown in SEQ ID NO: 18; the antibody is a murine antibody or a fragment thereof.

[0051] In some specific embodiments, provided are anti-CD25 antibody or antigen-binding fragment variants thereof, which comprise VH and / or VL, and the VH and / or VL have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VH and / or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment, respectively.

[0052] In some embodiments, the anti-CD25 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof, for example, a humanized antibody or a fragment thereof.

[0053] In some embodiments, a chimeric antibody is prepared based on a murine anti-CD25 antibody or antigen-binding fragment thereof, humanized, and then subjected to back mutation. The humanization methods in US20030040606 and US7494647 are incorporated herein by reference in their entirety.

[0054] In some embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:

[0055] The VH comprises FR1 to FR3 selected from IGHV1-46*01, and FR4 selected from IGHJ1*01, and the VL comprises FR1 to FR3 selected from IGKV4-1*01, and FR4 selected from IGKJ4*01;

[0056] The VH comprises FR1 to FR2 selected from IGHV1-18*01, FR3 selected from IGHV1-69*02, and FR4 selected from hIGHJ6*01_14, and the VL comprises FR1 selected from IGKV3-11*01, FR2 selected from IGKV5-2*01, FR3 selected from IGKV6-21*01, and FR4 selected from hIGKJ4*01_12;

[0057] The VH comprises FR1 selected from IGHV1-18*01, FR2 selected from IGHV4-31*01, FR3 selected from IGHV1-3*01, and FR4 selected from hIGHJ6*01, and the VL comprises FR1 to FR3 selected from IGKV4-1*01, and FR4 selected from hIGKJ2*01; or

[0058] The VH comprises FR1 to FR3 selected from IGHV3-23*04, and FR4 selected from IGHJ1*01, and the VL comprises FR1 to FR3 selected from IGKV2-28*01, and FR4 selected from IGKJ4*01.

[0059] In some specific embodiments, the anti-CD25 antibody or its antigen-binding fragment comprises VH and / or VL, wherein,

[0060] the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 27, 28 and 29 respectively, and FR1 to FR3 selected from IGHV1-46*01, and FR4 selected from IGHJ1*01; the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 30, 31 and 32 respectively, and FR1 to FR3 selected from IGKV4-1*01, and FR4 selected from IGKJ4*01;

[0061] the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 33, 34 and 35 respectively, and FR1 to FR2 selected from IGHV1-18*01, FR3 selected from IGHV1-69*02, and FR4 selected from hIGHJ6*01_14; the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 36, 37 and 38 respectively, and FR1 selected from IGKV3-11*01, FR2 selected from IGKV5-2*01, FR3 selected from IGKV6-21*01, and FR4 selected from hIGKJ4*01_12;

[0062] the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 39, 40 and 41 respectively, and FR1 selected from IGHV1-18*01, FR2 selected from IGHV4-31*01, FR3 selected from IGHV1-3*01, and FR4 selected from hIGHJ6*01; the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 42, 43 and 44 respectively, and FR1 to FR3 selected from IGKV4-1*01, and FR4 selected from hIGKJ2*01; or

[0063] The VH contains HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO: 45, 46 and 47 respectively, and FR1 to FR3 selected from IGHV3-23*04, and FR4 selected from IGHJ1*01; the VL contains LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO: 48, 49 and 50 respectively, and FR1 to FR3 selected from IGKV2-28*01, and FR4 selected from IGKJ4*01. Reverse mutations can be performed on the above FRs to maintain antibody activity.

[0064] In some embodiments, the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein:

[0065] The VH is as shown in SEQ ID NO: 19, and the VL is as shown in SEQ ID NO: 20;

[0066] The VH is as shown in SEQ ID NO: 21, and the VL is as shown in SEQ ID NO: 22;

[0067] The VH is as shown in SEQ ID NO: 23, and the VL is as shown in SEQ ID NO: 24;

[0068] The VH is as shown in SEQ ID NO: 25, and the VL is as shown in SEQ ID NO: 26; or

[0069] The VH is as shown in SEQ ID NO: 59, and the VL is as shown in SEQ ID NO: 60; the antibody is a humanized antibody or a fragment thereof.

[0070] In some specific embodiments, provided is a variant of an anti-CD25 antibody or antigen-binding fragment thereof, which comprises VH and / or VL, and the VH and / or VL have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VH and / or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment respectively.

[0071] In some embodiments, the anti-CD25 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region of the antibody, such as the heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region of the antibody is selected from the human antibody κ and λ chain constant regions and their conventional variants; or for example, murine IgG, such as murine IgG2a.

[0072] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V regions (diabody), disulfide-stabilized V regions (dsFv), and other antigen-binding fragments of peptides containing CDRs.

[0073] In some embodiments, there is also provided an isolated monoclonal antibody or an antigen-binding fragment thereof that competes with the anti-CD25 antibody or an antigen-binding fragment thereof described in any of the foregoing for binding to human CD25, or competes for binding to the same epitope.

[0074] In some embodiments, there is provided an anti-CD25 antibody or an antigen-binding fragment thereof that comprises VH and / or VL, wherein:

[0075] the VH is as shown in SEQ ID NO: 1 and the VL is as shown in SEQ ID NO: 2;

[0076] the VH is as shown in SEQ ID NO: 3 and the VL is as shown in SEQ ID NO: 4;

[0077] the VH is as shown in SEQ ID NO: 5 and the VL is as shown in SEQ ID NO: 6;

[0078] the VH is as shown in SEQ ID NO: 9 and the VL is as shown in SEQ ID NO: 10; or

[0079] the VH is as shown in SEQ ID NO: 11 and the VL is as shown in SEQ ID NO: 12; the antibody or an antigen-binding fragment thereof blocks or partially blocks the binding of IL-2 to CD25.

[0080] In other embodiments, there is provided an anti-CD25 antibody or an antigen-binding fragment thereof that comprises VH and / or VL, wherein:

[0081] the VH is as shown in SEQ ID NO: 7 and the VL is as shown in SEQ ID NO: 8;

[0082] the VH is as shown in SEQ ID NO: 13 and the VL is as shown in SEQ ID NO: 14;

[0083] the VH is as shown in SEQ ID NO: 15 and the VL is as shown in SEQ ID NO: 16;

[0084] the VH is as shown in SEQ ID NO: 17 and the VL is as shown in SEQ ID NO: 18;

[0085] The VH is as shown in SEQ ID NO: 19, and the VL is as shown in SEQ ID NO: 20;

[0086] The VH is as shown in SEQ ID NO: 21, and the VL is as shown in SEQ ID NO: 22;

[0087] The VH is as shown in SEQ ID NO: 23, and the VL is as shown in SEQ ID NO: 24;

[0088] The VH is as shown in SEQ ID NO: 25, and the VL is as shown in SEQ ID NO: 26; or

[0089] The VH is as shown in SEQ ID NO: 59, and the VL is as shown in SEQ ID NO: 60; the antibody or its antigen-binding fragment does not block, does not inhibit, hardly blocks, hardly inhibits, or blocks and inhibits IL-2 binding to CD25 to a low degree. For example, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody or antigen-binding fragment blocks less than about 50%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10% of IL-2 signaling, such as less than about 25% of IL-2 signaling.

[0090] In some embodiments, there is provided a variant of an anti-CD25 antibody or its antigen-binding fragment, which comprises a VH and / or a VL, and the VH and / or VL has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VH and / or VL of the aforementioned anti-CD25 antibody or its antigen-binding fragment.

[0091] In some embodiments, there is provided a variant of an anti-CD25 antibody or its antigen-binding fragment, which comprises a VH and / or a VL, and the VH and / or VL comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to the VH and / or VL of the heavy chain variable region of the aforementioned anti-CD25 antibody or its antigen-binding fragment, respectively. The amino acid changes can be conservative substitutions of amino acid residues in the variable region. In some embodiments, the antibody or antigen-binding fragment comprising the amino acid changes has the same or substantially the same affinity for human CD25 binding and / or function (such as ADCC, ADCP, anti-tumor activity) as the parental antibody or antigen-binding fragment.

[0092] In some embodiments, the anti-CD25 antibody or its antigen-binding fragment comprises a heavy chain and / or a light chain, wherein,

[0093] The heavy chain is as shown in SEQ ID NO: 51, and the light chain is as shown in SEQ ID NO: 52;

[0094] The heavy chain is as shown in SEQ ID NO: 53, and the light chain is as shown in SEQ ID NO: 54;

[0095] The heavy chain is as shown in SEQ ID NO: 55, and the light chain is as shown in SEQ ID NO: 56;

[0096] The heavy chain is as shown in SEQ ID NO: 57, and the light chain is as shown in SEQ ID NO: 58; or

[0097] The heavy chain is as shown in SEQ ID NO: 61, and the light chain is as shown in SEQ ID NO: 62.

[0098] In some embodiments, there is provided an anti-CD25 antibody or an antigen-binding fragment thereof, which comprises a heavy chain and / or a light chain, and the heavy chain and / or the light chain have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain and / or the light chain of the aforementioned anti-CD25 antibody or its antigen-binding fragment, respectively.

[0099] In some embodiments, the anti-CD25 antibody or its antigen-binding fragment of the present disclosure is of the IgG1 type, the Fc region has a defucosylation site, and has enhanced FcγRIIIa binding ability. The above anti-CD25 antibody or its antigen-binding fragment can increase the ADCC effect on Treg cells and enhance its anti-tumor activity.

[0100] In some embodiments, the anti-CD25 antibody or its antigen-binding fragment of the present disclosure is of the IgG1 type, the Fc region has a defucosylation site (such as A330I mutation), and has reduced FcγRIIb binding ability. The above anti-CD25 antibody or its antigen-binding fragment can reduce the inhibitory signal of FcγRIIb receptor-mediated ADCC / ADCP to enhance the ADCC effect on Treg cells and enhance its anti-tumor activity.

[0101] In some embodiments, the anti-CD25 antibody or antigen-binding fragment of the present disclosure has at least one of the following characteristics:

[0102] (a) The KD value of binding to human CD25 is less than 1×10-7 M;

[0103] (b) Does not inhibit (or substantially does not inhibit) the binding of IL-2 to CD25;

[0104] (c) Depletes tumor-infiltrating Treg and does not affect (or substantially does not affect) the function of Teff;

[0105] (d) binds to Fcγ receptors with an activation inhibition rate (A / I) higher than 1;

[0106] (e) binds to FcγRIIIa with a higher affinity than to FcγRI, FcγRIIc, and / or FcγRIIb, and binds to FcγRIIIa with a higher affinity than to FcγRIIb;

[0107] (f) inhibits the growth of multiple tumors.

[0108] The KD value of the CD25-binding protein or anti-CD25 antibody of the present disclosure binding to CD25 can be less than 1×10-7M, less than 1×10-8M, less than 1×10-9M, or less than 1×10-10M.

[0109] The anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure can inhibit tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%.

[0110] In some embodiments, the anti-CD25 antibody or antigen-binding fragment of the present disclosure binds to FcγR with high affinity, for example, binds to the activating receptor with high affinity. In some specific embodiments, the antibody of the present disclosure binds to FcγRI and / or FcγRIIA and / or FcγRIIIA with high affinity. In a specific embodiment, the antibody binds to at least one activating Fcγ receptor with a dissociation constant of less than about 10-6M, 10-7M, 10-8M, 10-9M or 10-10M.

[0111] In some embodiments, the anti-CD25 antibody or antigen-binding fragment of the present disclosure is an IgG1 antibody capable of binding to at least one Fc activating receptor. For example, the antibody can bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some specific embodiments, the antibody of the present disclosure is capable of binding to FcγRIIIA. In some embodiments, the antibody of the present disclosure is capable of binding to FcγRIIIA and FcγRIIA and optionally FcγRI. In one aspect, the antibody can bind to these receptors with high affinity, for example, with a dissociation constant of less than about 10-7M, 10-8M, 10-9M or 10-10M.

[0112] In some embodiments, the anti-CD25 antibody or antigen-binding fragment of the present disclosure binds to the inhibitory receptor FcγRIIb with low affinity. In one aspect, the antibody binds to FcγRIIb with a dissociation constant higher than about 10-7M, higher than about 10-6M, or higher than about 10-5M.

[0113] In some embodiments, the anti-CD25 antibodies or antigen-binding fragments of the present disclosure are from the IgG1 subclass and preferably have ADCC and / or ADCP activity. In other embodiments, the anti-CD25 antibodies of the present disclosure are from the IgG2 subclass.

[0114] In some embodiments, the anti-CD25 antibodies or antigen-binding fragments of the present disclosure inhibit or block less than 50% of IL-2 signaling through CD25. For example, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody or antigen-binding fragment inhibits or blocks less than about 40%, 35%, 30%, preferably less than about 25% of IL-2 signaling.

[0115] Polynucleotides and Vectors

[0116] The present disclosure provides an isolated polynucleotide encoding the anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure. The polynucleotide can be DNA or RNA.

[0117] The present disclosure provides an expression vector containing the polynucleotide as described above. The expression vector can be a eukaryotic expression vector, a prokaryotic expression vector, a viral vector, such as a plasmid, a cosmid, a phage.

[0118] Host Cells

[0119] The present disclosure provides a host cell transformed with the expression vector as described above, which can be a eukaryotic cell or a prokaryotic cell.

[0120] In some embodiments, the host cell is a bacterium, a yeast, or a mammalian cell. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells, or human embryonic kidney (HEK) 293 cells.

[0121] Preparation Methods

[0122] The present disclosure provides a method for preparing an anti-CD25 antibody or antigen-binding fragment thereof, comprising: expressing the antibody or antigen-binding fragment in the host cell as described above, and isolating the antibody or antigen-binding fragment from the host cell.

[0123] Optionally, a purification step may also be included. For example, purification is performed using an A or G Sepharose FF column with an adjusted buffer, non-specifically bound components are washed away, and then the bound antibody is eluted using a pH gradient method, detected by SDS-PAGE, and collected. Optionally, filtration and concentration are performed using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be immediately frozen, such as at -70°C, or lyophilized.

[0124] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and can be found in the prior art, such as the Antibody Experimentation Techniques Guide of Cold Spring Harbor (Chapters 5-8 and 15). For example, mice can be immunized with human FcRn or its fragments, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared by conventional methods.

[0125] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems result in glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture broth secreting the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.

[0126] Compositions

[0127] The present disclosure provides a composition, such as a pharmaceutical composition, comprising a therapeutically effective amount of the anti-CD25 antibody or its antigen-binding fragment as described above and a pharmaceutically acceptable excipient, diluent, or carrier. In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-CD25 antibody or its antigen-binding fragment per unit dosage, or the amount of the anti-CD25 antibody or its antigen-binding fragment contained in the unit dosage of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, it is 1-1000 mg.

[0128] Therapeutic Methods and Pharmaceutical Uses

[0129] The present disclosure provides any one or any combination of the anti-CD25 antibody or its antigen-binding fragment, the pharmaceutical composition containing the anti-CD25 antibody or its antigen-binding fragment, and the encoding polynucleotide, for use in methods of diagnosing, treating, preventing diseases and in the preparation of drugs and pharmaceutical compositions (for example, for treating or preventing proliferative disorders (such as cancer or tumors) or delaying the progression of related disorders).

[0130] In some embodiments, methods for preventing, treating, or alleviating a disorder in a subject are provided, which include administering to the subject an anti-CD25 antibody or an antigen-binding fragment thereof of the present disclosure, a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and / or an encoding polynucleotide. In some specific embodiments, the disorder of the subject is a proliferative disease, such as a tumor or cancer. In some embodiments, the above subject has a formed tumor, such as a solid tumor.

[0131] In some embodiments, methods for reducing the number of cells of tumor-internal or tumor-infiltrating Tregs in a subject are provided; in some embodiments, methods for eliminating or inhibiting the cell activity of tumor-internal or tumor-infiltrating Tregs in a subject are provided, both of which include administering to the subject an anti-CD25 antibody or an antigen-binding fragment thereof of the present disclosure, a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and / or an encoding polynucleotide.

[0132] In some embodiments, methods for increasing the ratio of Teff / Treg within a subject's tumor are provided, including administering to the subject an anti-CD25 antibody or an antigen-binding fragment thereof of the present disclosure, a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and / or an encoding polynucleotide. In some specific embodiments, the ratio of effector T cells to regulatory T cells (Teff / Treg) in the tumor is increased to more than 5, 10, 15, 20, 40, or 80.

[0133] In some embodiments, methods for enhancing CDC, ADCC, and / or ADCP against tumor cells in a subject are provided, including administering to the subject an anti-CD25 antibody or an antigen-binding fragment thereof of the present disclosure, a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and / or an encoding polynucleotide. In some specific embodiments, the ADCC and / or ADCP effects against tumor cells in the subject are enhanced. In some more specific embodiments, the ADCC effect against tumor cells in the subject is enhanced.

[0134] In some embodiments, the anti-CD25 antibody or an antigen-binding fragment thereof of the present disclosure, a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and / or an encoding polynucleotide are provided for pharmaceutical use in preparing a medicament for preventing, treating, or alleviating a disorder in a subject, for pharmaceutical use in preparing a medicament for reducing the number of cells of tumor-internal or tumor-infiltrating Tregs in a subject, for pharmaceutical use in preparing a medicament for eliminating or inhibiting the cell activity of tumor-internal or tumor-infiltrating Tregs in a subject, for pharmaceutical use in preparing a medicament for increasing the ratio of Teff / Treg within a subject's tumor, and for pharmaceutical use in preparing a medicament for enhancing CDC, ADCC, and / or ADCP against tumor cells in a subject.

[0135] In some specific embodiments, the disease condition of the above-mentioned subject is a proliferative disease condition (such as cancer or tumor) or the subject suffers from a proliferative disease condition (such as cancer or tumor). The tumors include, but are not limited to, carcinoma, lymphoma, leukemia, embryonal tumor, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0136] In some specific embodiments, the cancer or tumor may be a solid tumor, including but not limited to sarcoma (including cancers produced by transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinoma (including tumors produced by epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc. Cancers involving solid tumors include but are not limited to brain cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, renal cancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vaginal cancer, cervical cancer, lymphoma, etc.

[0137] In some specific embodiments, the cancer involves tumors expressing CD25, including but not limited to lymphoma, such as Hodgkin lymphoma, and lymphocytic leukemia, such as chronic lymphocytic leukemia (CLL).

[0138] Detection Uses

[0139] The present disclosure provides a detection use of an anti-CD25 antibody or an antigen-binding fragment thereof.

[0140] The present disclosure provides a reagent for detecting CD25, the reagent comprising an anti-CD25 antibody or an antigen-binding fragment thereof. The present disclosure also provides a method, system, or device for detecting CD25 in vivo or in vitro, which includes using an anti-CD25 antibody or an antigen-binding fragment thereof.

[0141] In some embodiments, an in vitro detection method, system or device may, for example, include: (1) contacting a sample with an anti-CD25 antibody or an antigen-binding fragment thereof; (2) detecting a complex formed between the anti-CD25 antibody or an antigen-binding fragment thereof and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with the antibody; and (4) determining the degree of complex formation between the antibody and the sample by comparing with the reference sample. A change in complex formation (e.g., a statistically significant change) in the sample or subject as compared to the control sample or in the subject indicates the presence of CD25 in the sample.

[0142] In some embodiments, for detection purposes, the CD25-binding protein or anti-CD25 antibody of the present disclosure can be labeled with a fluorophore and a chromophore.

[0143] In some embodiments, kits are also provided, which contain a CD25-binding protein or an anti-CD25 antibody, and may also contain instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a label or an additional diagnostic agent. For in vivo use, the antibody can be formulated as a pharmaceutical composition.

[0144] The anti-CD25 antibody or an antigen-binding fragment thereof provided in the embodiments of the present disclosure has the characteristics of high specificity, high affinity and low immunogenicity. At the same time, the antibody of the present disclosure has a good effect of inhibiting Treg, not affecting Teff, enhancing ADCC, ADCP and / or CDC in the subject, and inhibiting the occurrence and development of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1A and Figure 1B : ELISA was used to identify the activity of the recombinant protein. Among them, Figure 1A shows the detection results of the activity of recombinant human CD25 protein, Figure 1B shows the detection results of the activities of recombinant monkey and recombinant mouse CD25 proteins.

[0146] Figure 2 : FACS was used to measure the cell line 2F8 stably expressing human CD25 protein. Among them, the white peak is the CD25 positive peak, the gray peak is the control peak, and the primary antibody is a human IgG1 negative control antibody.

[0147] Figures 3A to 3C : FACS was used to detect the antibody function. Figure 3A shows the binding results of the antibodies DAC, 7G7B6, and Tab06 to CHO-K1 cells by FACS measurement, Figure 3B shows the binding results of the antibodies DAC, 7G7B6, and Tab06 to CHO-K1-CD25 cells stably expressing human CD25 protein by FACS measurement, Figure 3CResults of the binding of antibodies DAC, 7G7B6, Tab06, and Su-DHL-1 were determined by FACS, where mouse IgG or human IgG isotype was used as a negative control.

[0148] Figures 4A to 4C : Antibody binding to CD25 was detected by ELISA. Figure 4A Results of the binding of 7G7B6 and Tab06 to recombinant human CD25 protein Figure 4B Results of the binding of 7G7B6 and Tab06 to recombinant cynomolgus CD25 protein Figure 4C Results of the binding of 7D4 to recombinant mouse CD25 protein, with mouse IgG and human IgG used as negative controls.

[0149] Figures 5A to 5C : Binding of chimeric anti-CD25 antibodies to SU-DHL-1 cells was detected by FACS. Figure 5A Results of the detection of cAb001, cAb002, cAb004, and cAb006 Figure 5B Results of the detection of cAb028, cAb029, and cAb037 Figure 5C Results of the detection of cAb042 and cAb046, with Tab06 and DAC used as positive controls and human IgG (i.e., hIgG) used as a negative control.

[0150] Figures 6A to 6G : Binding of chimeric anti-CD25 antibodies to CD25 antigen on Treg cells, activated CD4 + and CD8 + effector T cells was detected by FACS. Figure 6A Results of cAb006 Figure 6B Results of cAb037 Figure 6C Results of cAb042 Figure 6D Results of cAb046 Figure 6E Results of the positive control 7G7B6 Figure 6F Results of the positive control DAC Figure 6G Results of the positive control Tab06.

[0151] Figures 7A to 7B : The effect of chimeric anti-CD25 antibodies on the binding ability to IL-2 and receptor CD25 was detected by FACS. Figure 7A Results of the detection of cAb001, cAb002, cAb028, and cAb029 Figure 7BDetection results graphs of cAb006, cAb037, cAb042, and cAb046. The anti-CD25 antibody control that blocks the binding of IL-2 and CD25 is DAC, and the anti-CD25 antibody control that does not block the binding of IL-2 and CD25 is Tab06. The negative control is human IgG (i.e., hIgG-1).

[0152] Figure 8 : Detection of the binding of humanized anti-CD25 antibody to SU-DHL-1 cells by FACS.

[0153] Figure 9 : Detection of the effect of humanized anti-CD25 antibody on the pStat5 signaling pathway in human peripheral blood T lymphocytes by FACS.

[0154] Figure 10 : Detection of the ADCC effect of humanized anti-CD25 antibody on SU-DHL-1 cells by detecting the fluorescence activity of the ADCC reporter gene in cells.

[0155] Figure 11A and Figure 11B : Antitumor activity results of humanized anti-CD25 antibody-mediated MC38 xenografts in hCD25 mice. Among them, Figure 11A Tumor inhibition effect graph, Figure 11B is the corresponding mouse body weight graph.

[0156] Figure 12A and Figure 12B : Results of intratumoral lymphocyte analysis of humanized anti-CD25 antibody-mediated MC38 xenografts in hCD25 mice. Among them, Figure 12A is the killing result of the antibody on Treg, Figure 12B is the upregulation of the antibody on CD3. Detailed implementation mode Detailed description of the invention

[0158] To make the present disclosure easier to understand, certain technologies and sciences are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technologies and sciences used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains.

[0159] The three-letter and single-letter codes of amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0160] "CD25", "CD25 protein", or "CD25 polypeptide" may optionally include any such protein or its variants, conjugates or fragments, including (but not limited to) the known or wild-type CD25 as described herein, and any naturally occurring splice variants, amino acid variants or isotypes. The complete human CD25 sequence can be found at Uniprot accession number P01589, and amino acids 22 to 240 thereof correspond to the extracellular domain of mature human CD25.

[0161] "Binding to CD25" means being able to interact with CD25 or its epitope, and the CD25 or its epitope may be of human origin. "Antigen-binding site" refers to a three-dimensional site on an antigen that is discontinuous and recognized by the antibodies or antigen-binding fragments of the present disclosure.

[0162] "Antibody" refers to an immunoglobulin, which is a four-peptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain according to the difference in the constant region. Each of the five classes of Ig can have a κ chain or a λ chain. The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly and are variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C regions). The variable region includes 3 hypervariable regions (HVRs) and 4 relatively conserved framework regions (FRs). The 3 hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the order arranged from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In some embodiments, the antibodies of the present disclosure specifically or substantially specifically bind to CD25.

[0163] "Antibody that binds CD25" refers to an antibody that can bind to the CD25 subunit of the IL-2 receptor. This subunit is also called the α subunit of the IL-2 receptor. Such an antibody is also referred to as an "anti-CD25 antibody" herein.

[0164] The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment of the present disclosure are in number and position in accordance with the known Kabat numbering system. The EU numbering in Kabat is generally also used for constant domains and / or Fc domains.

[0165] For the determination or definition of "CDR", the definitive delineation of CDR and the identification of residues comprising the binding site of an antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of an antibody-ligand complex. This can be achieved by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is the standard for numbering residues in an antibody and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by Oxford Molecular Group for modeling antibody structures (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of an antibody from the primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on the analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the positions of CDRs can be identified as residues that make an enthalpic contribution to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, and although they may be shorter or longer according to predictions or experimental results that specific residues or groups of residues do not significantly affect antigen binding. As used herein, CDR may refer to CDRs defined by any method known in the art (including combinations of methods). The methods used herein may utilize CDRs defined according to any of these methods. The Kabat numbering rule is used to define CDRs in the embodiments of the present disclosure, but those skilled in the art will understand that CDRs can also be redefined according to any of Chothia, extended, AbM, IMGT, contact, and / or conformation definitions.

[0166] The Fc region of IgG antibodies interacts with several cellular Fcγ receptors (FcγRs) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), and one inhibitory receptor, FcγRIIb (CD32b). The communication of IgG antibodies with the immune system is controlled and mediated by FcγRs, which transmit the information sensed and collected by the antibodies to the immune system, thus providing a link between the innate and adaptive immune systems, especially in the context of biotherapy (Hayes J et al., 2016. J Inflamm Res 9:209–219). IgG subclasses differ in their ability to bind FcγRs, and this differential binding determines their ability to trigger a range of functional responses. For example, in humans, FcγRIIIa is the main receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC), and IgG1 followed by IgG3 shows the highest affinity for this receptor, reflecting their ability to effectively induce ADCC. IgG2 has weak binding to this receptor, but anti-CD25 antibodies with the human IgG2 isotype have been found to be able to effectively deplete Tregs.

[0167] "Regulatory T cells", "Tregs", or "Treg cells" refer to CD4 + T lymphocyte lineages. Generally, they regulate the activity of T cell populations, but they can also affect certain innate immune system cell types. Tregs can be identified by the expression of the biomarkers CD4, CD25, and Foxp3. Naturally occurring Treg cells usually account for about 5 to 10% of peripheral CD4 + T lymphocytes. However, within the tumor microenvironment (i.e., tumor-infiltrating Treg cells), they can account for up to the total CD4 +20 to 30% of the T lymphocyte population. Activated human Treg cells can directly kill target cells, such as Teff and APC (antigen-presenting cells), through the perforin or granzyme B-dependent pathway; cytotoxic T lymphocyte-associated antigen 4 (CTLA4 + ) Treg cells induce the expression of indoleamine 2,3-dioxygenase (IDO) through APCs, which in turn inhibit T cell activation by reducing tryptophan; Treg cells can release interleukin-10 (IL-10) and transforming growth factor (TGFβ) in vivo, thereby directly inhibiting T cell activation and inhibiting APC function by suppressing the expression of MHC molecules, CD80, CD86, and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4, which can bind to CD80 and CD86 on antigen-presenting cells and prevent the proper activation of effector T cells.

[0168] As used herein, "immune effector cells" refers to immune cells that participate in the effector phase of an immune response. Exemplary immune cells include myeloid or lymphoid cells, such as lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils.

[0169] As used herein, "not inhibit", "not block", "non-blocking", "non-IL-2 blocking", "no blocking", and the like (with respect to not blocking or inhibiting the binding of IL-2 to CD25 in the presence of an anti-CD25 antibody, e.g., "not inhibit the binding of IL-2 to CD25") include those in which the anti-CD25 antibody does not block or inhibit IL-2 signaling through CD25, particularly IL-2 signaling in cells expressing CD25. That is, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibodies of the present disclosure inhibit less than 50% of the IL-2 signaling through CD25. Preferably, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody inhibits less than about 40%, 35%, 30%, preferably less than about 25% of the IL-2 signaling.

[0170] "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (such as natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, leading to lysis of the target cells. "Antibody-dependent cell-mediated phagocytosis" (ADCP) refers to a cell-mediated reaction in which phagocytic cells expressing Fc receptors (FcR) (such as macrophages) recognize antibodies bound to target cells, leading to phagocytosis of the target cells. "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by antibodies in the presence of complement. CDC, ADCC, and ADCP can be measured using assays known and available in the art (Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6). The constant region of the antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity and phagocytosis. Thus, the isotype of the antibody can be selected based on whether the antibody is required to mediate ADCC or ADCP.

[0171] ADCC can be increased by methods that eliminate fucose moieties from the antibody glycan, such as by producing the antibody in the YB2 / 0 cell line or by introducing specific mutations (such as S298A / E333A / K334A, S239D / I332E / A330L, G236A / S239D / A330L / I332E) on the Fc portion of human IgG1 (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al. (2012) Proc Natl Acad Sci USA 109, 6181-6). ADCP can also be increased by introducing specific mutations on the Fc portion of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27).

[0172] "Murine antibody" in the present disclosure is a monoclonal antibody against human CD25 or its epitope prepared according to knowledge and skills in the art. The test subject is injected with the CD25 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a specific embodiment of the present disclosure, the murine anti-human CD25 antibody or its antigen-binding fragment may further comprise a light chain constant region of murine κ, λ chain or its variant, or may further comprise a heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or its variant.

[0173] "Chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma secreting murine specific monoclonal antibody should be selected first, and then the variable region gene is cloned from murine hybridoma cells. Next, the constant region gene of human antibody is cloned according to needs. After connecting the murine variable region gene with the human constant region gene to form a chimeric gene, it is inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of human antibody can be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or their variants, preferably including the heavy chain constant regions of human IgG2 or IgG4, or IgG1 without ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0174] "Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequences of a mouse into the framework of the variable region of a human antibody. It can overcome the strong immune response induced by chimeric antibodies due to the presence of a large amount of murine protein components. To avoid a decrease in activity while reducing immunogenicity, a minimum number of back mutations can be made to the variable region of the human antibody to maintain activity. The antibodies of the present disclosure can be affinity matured humanized antibodies, and the CDRs of the affinity matured antibody parental sequences (including all sequences) are at least 80% identical, such as 90% identical. An affinity matured antibody is an antibody having one or more altered amino acids in one or more CDRs, which results in an improvement in the affinity of the antibody for CD25 compared to the parental antibody without the altered amino acids.

[0175] "Human antibody" includes antibodies having variable and constant regions with human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto a human framework sequence (i.e., "humanized antibody").

[0176] "Antigen-binding fragment" includes single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the foregoing (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide bond-stabilized form Fab-dsFv was first disclosed in WO2010 / 035012. The antigen-binding fragments of the present disclosure also include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies can be multispecific, e.g., bispecific, or can be monospecific (see, e.g., WO92 / 22583 and WO05 / 113605), an example of the latter being the Tri-Fab (or TFM) described in WO 92 / 22583. In some embodiments, the anti-CD25 antibodies of the present disclosure encompass bispecific and multispecific antibodies.

[0177] "Epitope" refers to the site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids or by non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by adjacent amino acids generally remain after exposure to a denaturing solvent, while epitopes formed by tertiary folding generally are lost after treatment with a denaturing solvent. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0178] "Specific binding" and "selective binding" mean that an antibody binds to an epitope on a predetermined antigen. Generally, when using recombinant human CD25 or its epitope as an analyte and an antibody as a ligand, and measuring by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to the predetermined antigen or its epitope with an equilibrium dissociation constant (KD) of approximately less than 10-7M or even smaller, and its affinity for binding to the predetermined antigen or its epitope is at least twice that of its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. "Antibody that recognizes an antigen" can be used interchangeably with "antibody that specifically binds" herein.

[0179] "Binding affinity" refers to the apparent association constant or Ka. Ka is the reciprocal of the dissociation constant (Kd). For example, a binding protein may have a binding affinity for a specific target molecule of at least 10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 and 10 -11 M. A higher affinity binding of a binding ligand to a first target relative to a second target can be shown by a higher Ka (or a smaller value of Kd) for binding to the first target than for binding to the second target. In these cases, the binding protein is specific for the first target (e.g., a protein of the first conformation or its analog) relative to the second target (e.g., the same protein of the second conformation or its analog; or a second protein). The difference in binding affinity (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, 1000, or 105 times.

[0180] "Conservative substitution" means substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Thus, it will be apparent to those skilled in the art that even when substituting amino acid residues within the groups showing similar properties as described above, it will not show a specific change in properties.

[0181] "Sequence homology" or "sequence identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two sequences being compared are occupied by the same base or amino acid monomer subunit, for example if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, comparison is made when the maximum percentage of homology is obtained by aligning the two sequences.

[0182] "Cross - reactivity" refers to the ability of the antibodies of the present disclosure to bind to CD25 from different species. For example, an antibody of the present disclosure that binds human CD25 can also bind CD25 of another species. Cross - reactivity is measured by detecting specific reactivity with a purified antigen, or binding or functional interaction with cells physiologically expressing CD25 in a binding assay (such as SPR and ELISA). Methods for determining cross - reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0183] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of CD25 preferably reduces or alters the normal level or type of activity that occurs upon CD25 binding in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in CD25 binding affinity compared to CD25 not contacted with an anti - CD25 antibody when contacted with an anti - CD25 antibody.

[0184] "Inhibit growth" (e.g., as related to cells) is intended to include any measurable decrease in cell growth.

[0185] Methods for producing and purifying antibodies and antigen - binding fragments are well - known and can be found in the prior art, such as the Antibody Engineering Laboratory Manual from Cold Spring Harbor (Chapters 5 - 8 and 15). For example, mice can be immunized with human CD25 or a fragment thereof, and the resulting antibodies can be renatured, purified, and their amino acid sequences can be determined by conventional methods. Antigen - binding fragments can also be prepared by conventional methods. The antibodies or antigen - binding fragments of the invention are engineered to add one or more human FR regions to the non - human CDR regions. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) at http: / / imgt.cines.fr, or from the Journal of Immunology, 2001 ISBN 012441351.

[0186] The antibodies of the present disclosure can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable for multiple embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" generally refers to products such as cells or nucleic acids, proteins, or vectors, indicating that the cell, nucleic acid, protein, or vector has been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cell is derived from such modified cells. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, under-expressed, or not expressed at all.

[0187] "Monoclonal antibody" or "mAb" refers to an antibody obtained from a single clone of cells, and the cell line is not limited to eukaryotic, prokaryotic, or phage clone cell lines. Monoclonal antibodies or antigen-binding fragments can be recombinantly obtained using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic techniques (such as CDR-grafting), or other existing techniques.

[0188] The antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. The positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture broth secreting the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be immediately frozen, such as at -70 °C, or lyophilized.

[0189] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding to an antigen. High-throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Publication WO03 / 48731. Thus, conventional techniques known to those skilled in the art can be used to obtain antibodies and their antigen-binding fragments that compete with the antibody molecules of the present disclosure for binding to the same epitope on CD25. The method for detecting antibody competitive binding in WO2018167104 is hereby incorporated by reference in its entirety.

[0190] "Administering", "applying", and "treating", when applied to an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid, mean the contacting of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "applying", and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treating a cell includes contacting the cell with a reagent, and contacting the reagent with a fluid that contacts the cell. "Administering", "applying", and "treating" also mean treating a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "Treating", when applied to a human, veterinary, or research subject, means a therapeutic treatment, a prophylactic or preventive measure, research, and diagnostic applications.

[0191] "Treatment" means administering to a subject a therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments or conjugates thereof of the present disclosure, either internally or externally, to a subject who has, is suspected of having, or is predisposed to having one or more diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect on such symptoms. Generally, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, either by inducing regression of such symptoms or by inhibiting the development of such symptoms to any clinically measurable degree. The amount of the therapeutic agent effective to alleviate any particular disease symptom (also referred to as "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a physician or other professional healthcare provider to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (such as a treatment method or article) may be ineffective in alleviating the symptoms of the target disease in a particular subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0192] The term "treating cancer" as used herein includes (a) inhibiting cancer, i.e., preventing its development, including but not limited to blocking or delaying the progression of cancer, blocking or delaying the metastasis of cancer; and / or (b) alleviating cancer, i.e., causing the regression of cancer, including but not limited to alleviating or relieving one or more symptoms associated with the cancer, alleviating or relieving metastatic cancer, and / or reducing or eliminating the tumor.

[0193] "Prevention" as used herein refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (whereby the disease does not occur) or only effective in certain individuals or for a limited time.

[0194] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical disorder. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the disorder to be treated, the overall health of the subject, the method of administration, the route and dosage, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0195] "Cell", "cell line", and "cell culture" are used interchangeably and all such designations include their progeny. It should also be understood that due to either deliberate or inadvertent mutation, all progeny may not be precisely identical in their DNA content. Included are mutant progeny having the same function or biological activity as screened for in the original transformed cell.

[0196] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and this description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 - 3 variable regions of an antibody heavy chain" means that the variable regions of the antibody heavy chain of a particular sequence may but need not be present.

[0197] "Pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen - binding fragments described herein or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0198] "Tumor" applies to subjects diagnosed or suspected of having a tumor. Cancer refers to any malignant or potentially malignant neoplasm or tissue mass, regardless of size, and includes primary tumors and secondary neoplasms. "Cancer", "malignant tumor", "neoplasm", "tumor", and "cancer" are also used interchangeably herein to refer to tumors and tumor cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, and thus exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Generally, the target cells for detection or treatment include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. "Solid tumor" is an abnormal growth or mass of tissue that generally does not contain cysts or fluid regions, particularly tumors and / or metastases (wherever located) other than leukemia or non-solid lymphomas. Solid tumors can be either benign or malignant. Different types of solid tumors are named after the cell types that form them and / or the tissue or organ in which they are located. Examples of solid tumors include, but are not limited to, sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), carcinomas (including tumors derived from epithelial cells), melanomas, lymphomas, mesotheliomas, neuroblastomas, and retinoblastomas.

[0199] Unless otherwise specified, the articles "a" and "an" as used herein refer to one or more than one (i.e., at least one / kind) of the grammatical objects of the said articles. For example, "an element" refers to one or more than one element. Detailed Description of the Invention

[0201] The following examples are used for further description, but these examples do not limit the scope.

[0202] For experimental methods without specific conditions indicated in the examples or test examples, they are generally carried out under conventional conditions, or according to the conditions recommended by the raw material or commodity manufacturers. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents without specific sources indicated are conventional reagents purchased from the market.

[0203] Example 1. Activity Detection of Recombinant CD25 Protein

[0204] Recombinant human CD25-Fc fusion protein (purchased from ACRO Biosystems) is a fusion of human IgG1 Fc-tagged protein at the C-terminus of human CD25 protein (GenBank accession number: NP_000408.1). Recombinant human CD25-His protein (purchased from Sino Biological Inc.) is a fusion of multiple histidine tags at the C-terminus of human CD25 protein (GenBank accession number: NP_000408.1) at Met1-Cys213. Recombinant cynomolgus CD25-His protein (purchased from Sino Biological Inc.) is a fusion of His-tag at the C-terminus of cynomolgus CD25 protein (GenBank accession number: NP_001270633.1) at Met1-Arg213. Recombinant mouse CD25-His protein (purchased from Sino Biological Inc.) is a fusion of His-tag at the C-terminus of mouse CD25 protein (GenBank accession number: NP_032393.3) at Met1-Lys236. A series of quality control tests, such as activity verification, were performed on the purchased recombinant proteins using anti-CD25 antibody DAC (Efalizumab).

[0205] The activity of the recombinant proteins was verified by ELISA (enzyme-linked immunosorbent assay). Specifically: The recombinant human CD25-Fc fusion protein, human CD25-His protein, cynomolgus CD25-His protein, and mouse CD25-His protein were each diluted to 1 μg / mL with PBS and added to the ELISA microplate, 100 μL per well, and incubated overnight at 4°C; ELISA blocking solution (PBS phosphate buffer containing 1% BSA (w / v), pH 7.4) was added and blocked at 37°C for 2 hours. Then, serially diluted detection antibodies DAC and 7D4 (anti-mouse CD25 antibody) were added in sequence and incubated at 37°C for 1 hour. The plate was washed 2 - 3 times with the wash buffer; HRP-labeled secondary antibody was added and incubated at 37°C for 1 hour. The plate was washed 2 - 3 times with the wash buffer. 100 μL of TMB substrate was added to each well and incubated at room temperature for 15 minutes. Then, 50 μL of stop solution (2 M HCl) was added to each well. The OD450nm value was read using an ELISA plate reader (SpectraMax M5e), and the results are as Figure 1A and 1B shown.

[0206] The results showed that both recombinant human CD25 proteins and recombinant cynomolgus CD25-His protein could bind to DAC, and the detection signal changed with the concentration of the detection antibody; recombinant mouse CD25-His protein could not bind to DAC but could bind to 7D4, demonstrating that the above recombinant CD25 proteins were all active.

[0207] Example 2. Establishment of a stable cell line expressing recombinant human CD25 protein

[0208] After transfecting CHO-K1 cells with a plasmid encoding recombinant human CD25 protein, a cell line stably expressing recombinant human CD25 protein was obtained through screening. The nucleotide sequence encoding human CD25 was cloned and ligated into the pCDNA3.4 vector (purchased from Invitrogen) to prepare the plasmid. The CHO-K1 cell line (all purchased from Invitrogen) was transfected with Lipofectamine 2000, selectively cultured in CD-CHO medium containing G418 for 2 weeks, subcloned by the limiting dilution method in a 96-well culture plate, and placed in an incubator at 37 °C containing 5% CO 2 (v / v) for culturing. After 2 weeks, some monoclonal wells were selected and amplified into a 24-well plate, and then amplified into a 6-well plate for culturing. The amplified clones were detected and screened by flow cytometry analysis. Figure 2 The results showed that the screened cell line 2F8 could stably express human CD25 protein.

[0209] Example 3. Functional verification of detection antibodies

[0210] DAC, 7G7B6, Tab06, and 7D4 are all anti-CD25 antibodies. The sequence of DAC is listed in US5530101, which is incorporated herein by reference. 7G7B6 has been proposed as a targeting moiety for targeting radionuclides to lymphomas expressing CD25 (Zhang et al, 2009, Cancer Biother Radiopharm 24(3), 303-309). Tab06 is an anti-CD25 antibody that does not inhibit the binding of IL-2 to CD25, and its sequence is listed in WO2018167104 (sequences 27 and 29 of this patent), which is incorporated herein by reference. 7D4 is a rat IgM anti-mouse CD25 antibody, and 7D4 has been widely used to detect CD25-positive cells in the presence of PC61 or after treatment with PC61 or in the presence of antibodies with similar binding properties (Malek, 1983, Immunology, Vol. 80, pp. 5694-5698; Onizuka S et al., 1999. Canc Res. 59, 3128-3133).

[0211] Functional verification of the detection antibodies DAC, 7G7B6, Tab06, and 7D4 was performed by FACS. Specifically: SU-DHL-1 cells, CHO-K1 cells, and CHO-K1 cells stably expressing human CD25 (CHO-K1-CD25 cells) in the logarithmic growth phase were collected by centrifugation, washed with PBS, and centrifuged at 200 g for 5 minutes. Use 100 μL containing 2x 10 5Plate the cells in the culture medium. After centrifuging at 400 g for 5 minutes, add the detection antibody (100 nM, serially diluted), and the control antibody is human IgG isotype. Incubate on ice for 1 h, wash away the excess antibody with PBS, and centrifuge at 400 g for 5 minutes. Add the secondary antibody Alexa Fluor 488 goat anti-human (Fc) antibody (purchased from Life Technology, catalog number A11013), and incubate for 1 hour in an ice bath. Wash away the excess secondary antibody with PBS, and centrifuge at 400 g for 5 minutes. Add 200 μL of flow cytometry detection solution to each well, and then perform the detection on the machine.

[0212] As Figures 3A - 3C shown, in the binding experiment with Su-DHL-1 cells and CHO-K1 cells stably expressing human CD25 (CHO-K1-CD25 cells) (i.e., monoclonal 2F8), the signal of DAC was the strongest, followed by Tab06, and the signal of 7G7B6 was the weakest; all three antibodies could bind to Su-DHL-1 cells and CHO-K1 cells stably expressing human CD25, and none of them could bind to CHO-K1 cells.

[0213] Function verification of 7G7B6 and Tab06 was performed by ELISA. Specifically: Dilute recombinant human CD25-His protein, recombinant monkey CD25-His protein, and recombinant mouse CD25-his protein to 1 μg / mL with PBS, add them to the ELISA microplate, 100 μL per well, and incubate overnight at 4 °C; add the ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4), block at 37 °C for 2 hours, then add the serially diluted detection antibodies in sequence, and incubate at 37 °C for 1 hour; wash the plate 2 - 3 times with the washing solution; add the secondary antibody labeled with horseradish peroxidase (HRP), incubate at 37 °C for 1 hour, and wash the plate 2 - 3 times with the washing solution. Add 100 μL of TMB substrate to each well, incubate at room temperature for 15 minutes, then add 50 μL of the stop solution 2 M HCl to each well, and read the OD450nm value.

[0214] As Figures 4A - 4C shown, 7G7B6 and Tab06 could bind to human CD25 protein and monkey CD25 protein respectively, indicating that 7G7B6 and Tab06 had human-monkey cross-reactivity; 7D4 could bind to mouse CD25 protein, indicating that 7D4 had the binding activity to mouse CD25 protein.

[0215] Example 4. Obtaining murine anti-CD25 monoclonal antibody by immunizing mice with recombinant CD25 protein

[0216] Balb / c and SJL / J mice at 6 - 8 weeks of age were immunized with recombinant human CD25 - Fc as the immunogen, and SJL / J mice were immunized with recombinant human CD25 - His as the immunogen. The primary immunization dose was 50 μg per mouse. Two weeks after the primary immunization, a booster immunization was given, and the immunization dose was 25 μg per mouse. Thereafter, each booster immunization was spaced 3 weeks apart. Serum samples were collected one week after each booster immunization, and the antibody activity in the mouse serum was detected by ELISA. The plate was coated with 1 μg / mL recombinant human CD25 - His overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed 3 times. The mouse serum was diluted 10 - fold gradient starting from 1:100 in the blocking buffer, incubated at 37°C for 1 hour, washed 3 times, and incubated with the secondary antibody anti - mouse IgG - Fc - HRP for 1 hour. After washing 3 times with PBST, 100 μL of TMB substrate was added to each well, and the reaction was terminated with 2M HCl after 15 minutes. The absorbance at 450 nm was read using a microplate reader. The sera of mice immunized with the immunogen recombinant human CD25 - Fc showed different degrees of binding to the immunogen, and the highest dilution factor of the serum was 1:10 5 when the antigen - antibody reaction could still be presented.

[0217] For the last immunization, 100 μg of recombinant human CD25 - Fc and recombinant human CD25 - His were injected intraperitoneally. Five days later, the mice were sacrificed, and the spleens were taken and ground to collect splenocytes. NH 4 OH with a final concentration of 1% (w / w) was added to lyse the red blood cells mixed in the splenocytes to obtain a splenocyte suspension, and the cells were washed 3 times by centrifugation at 1000 rpm. Mouse spleen cells and mouse myeloma cells SP2 / 0 were mixed at a ratio of 5:1 by the number of live cells, and cell fusion was carried out using the high - efficiency electrofusion method. The fused cells were diluted into a 96 - well cell culture plate with DMEM medium containing 20% fetal bovine serum and 1×HAT (w / w), 200 μL per well, a total of 1×10 5 cells, and placed in a 37°C incubator with 5% (v / v) CO 2 . Fourteen days later, the fused cells were screened by ELISA. The positive clones with OD450nm > 1.0 were amplified to a 24 - well cell plate and further expanded in DMEM medium containing 10% (w / w) HT fetal bovine serum under the conditions of 37°C and 5% (v / v) CO 2 . Three days later, the supernatant was collected by centrifuging the culture solution in the 24 - well cell plate, and the binding activities to recombinant human CD25 protein and CD25 - positive cells were determined by ELISA and FACS. After two rounds of monoclonalization, excellent monoclonal strains were screened using the above - mentioned ELISA detection method and flow cytometry detection method. The amino acid sequence of the antibody after sequencing is shown in Table 1, and the underlined part is the complementarity - determining region CDR sequence (using the Kabat numbering system).

[0218] Table 1. Mouse-derived anti-CD25 antibody sequences

[0219]

[0220]

[0221] Example 5. Preparation of recombinant chimeric anti-CD25 antibody and humanization process

[0222] For chimeric antibodies, recombinant chimeric antibodies were obtained by replacing the constant regions of mouse monoclonal antibodies. Subsequently, the nucleotide sequences encoding the variable regions of mouse monoclonal antibodies were cloned into the pTT5 vector containing the protein sequences encoding human heavy and light chain constant regions (Human IgG1, kappa), and then transfected into HEK293 cells. That is, the heavy and light chain variable regions of the obtained chimeric antibodies are the same as those of the mouse antibodies. For humanized antibodies: After predicting the structure of mouse monoclonal antibodies by homology modeling, the mouse anti-CDRs were chimerized onto a suitable human GermLine framework (Bioinformation. 2014; 10(4): 180–186; Methods Mol Biol. 2019; 1904: 213-230). Subsequently, back mutations were introduced at sites that might affect antibody-antigen binding. Finally, the nucleotide sequences encoding the variable regions of humanized monoclonal antibodies were cloned into the pTT5 vector containing the protein sequences encoding human heavy and light chain constant regions (Human IgG1, kappa), and then transfected into HEK293 cells. After 5 days, the cells were removed by centrifugation, and the cell culture medium was collected and filtered. After adjusting the pH to 7.0, the supernatant of the harvested cell culture medium was loaded onto a Protein A column (MabSelect SuRe, GE), and the bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris.

[0223] The selection of antibody variable region frameworks is shown in Table 2, the antibody variable region sequences are shown in Table 3, the heavy chain CDRs (HCDR1\HCDR2\HCDR3) and light chain CDRs (LCDR1\LCDR2\LCDR3) are shown in Table 4, and the full-length antibody amino acid sequences are shown in Table 5. Among them, PR006 is the humanized antibody of cAb006, PR071 is the humanized antibody of cAb037, PR031 is the humanized antibody of cAb042, and PR058 and PR157 are the humanized antibodies of cAb046.

[0224] Table 2. Selection of antibody variable region frameworks

[0225]

[0226]

[0227] Table 3. Variable region sequences of humanized anti-CD25 antibodies that partially do not block IL-2 function

[0228]

[0229] Table 4. Variable region sequences of humanized anti-CD25 antibodies that partially do not block IL-2 function (Kabat numbering system)

[0230]

[0231]

[0232] Table 5. Full-length amino acid sequences of heavy and light chains of humanized antibodies

[0233]

[0234]

[0235]

[0236] Example 6. Affinity detection of chimeric anti-CD25 antibodies for human and cynomolgus monkey CD25 proteins

[0237] The binding activity of chimeric anti-CD25 antibodies to CD25 recombinant proteins was detected by ELISA. A 96-well plate was coated with 50 μL per well at a concentration of 1 μg / mL human CD25-His / cynomolgus monkey CD25-His. The unbound antigen was washed off with the washing solution (washing solution: 1×PBST). After washing, it was blocked with a 1% BSA blocking solution prepared with 1×PBST at 37°C for 1 hour. After washing the plate 3 times with the washing solution, different diluted concentrations of the chimeric antibody to be tested were added and incubated in an incubator at 37°C for 1 hour. After washing the plate 3 times with the washing solution, 100 μL of a 1:5000 diluted goat anti-human IgG secondary antibody was added and incubated in an incubator at 37°C for 0.5 hour. After washing the plate, a TMB chromogenic solution A and B were mixed at a 1:1 ratio for color development. The color development reaction was terminated with 1 M hydrochloric acid after 15 minutes, and the fluorescence value at 450 nm was detected. The results showed that all the chimeric anti-CD25 antibodies tested could bind to human and cynomolgus monkey CD25 proteins, and the half-maximal effective concentration (EC 50 ) is shown in Tables 6 and 7.

[0238] Table 6. EC 50 (ELISA) of chimeric anti-CD25 antibodies binding to cynomolgus monkey CD25

[0239] Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> cAb001 0.3991 cAb029 0.1145 cAb028 0.2295 cAb002 0.5244 cAb037 0.1467 DAC 0.3973 cAb004 0.3106 cAb042 0.3486 Tab06 0.6144 cAb006 0.2338 cAb046 5.569

[0240] Table 7. EC 50 (ELISA) of chimeric anti-CD25 antibodies binding to human CD25

[0241] Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> cAb001 0.2284 cAb028 0.3444 cAb046 0.3547 cAb002 0.2738 cAb029 0.1661 DAC 0.2319 cAb004 0.2552 cAb037 0.1653 Tab06 0.4032 cAb006 0.2004 cAb042 0.224

[0242] Example 7. Binding of Chimeric Anti-CD25 Antibody to SU-DHL-1 Cells

[0243] Since the lymphoma cell line SU-DHL-1 highly expresses CD25, we used flow cytometry to detect the binding of the chimeric anti-CD25 antibody to SU-DHL-1 cells. SU-DHL-1 cells in the logarithmic growth phase were selected, collected, washed with PBS and centrifuged. 100 μl of 2×10 5 cells were plated per well and centrifuged at 400 g for 5 minutes. Different concentrations of the antibody to be tested were added, incubated on ice for 1 h, washed with PBS, and centrifuged at 400 g for 5 min. Alexa Fluor 488, a goat anti-human secondary antibody with a fluorescent group, was added and incubated on ice for 1 h, washed with PBS and then detected on the machine. As Figures 5A - 5C shown, the detected chimeric anti-CD25 antibodies of the present disclosure can all bind to SU-DHL-1 cells. Table 8 shows the EC 50 values of the binding.

[0244] Table 8. EC of the Binding of Chimeric Anti-CD25 Antibody to SU-DHL-1 Cells 50

[0245] Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> Antibody Number <![CDATA[EC 50 (nM)]]> cAb001 0.1799 cAb028 0.1319 cAb046 0.3455 cAb002 0.1001 cAb029 0.1193 DAC 0.3407 cAb004 0.1681 cAb037 0.1502 Tab06 0.8323 cAb006 0.3286 cAb042 0.501

[0246] Example 8. Binding of Chimeric Anti-CD25 Antibody to Treg Cells / Activated CD4 + T Cells / CD8 + T Cells

[0247] Since CD25 is expressed in both activated Tregs and activated effector T cells, in order to detect whether there is a difference in the binding of the anti-CD25 antibody of the present disclosure to CD25 expressed on Tregs and CD4 + and CD8 + effector T cells, the following method of FACS was used to compare the binding ability of the antibody to Tregs and activated effector T cells.

[0248] Human peripheral blood mononuclear cells (PBMCs) were taken and natural CD4 + cells were isolated and extracted by magnetic bead method, and the medium for inducing Tregs ((AIM V + 10% FBS + human IL-2 (400 U / mL) + Rapamycin (0.1 μg / mL) or TGFβ (20 ng / mL) + atRA (200 nM)) was added, and magnetic beads coated with anti-CD3 / CD28 antibody were added at the same time. During the culture, the cell density was maintained at 2×10 6 / mL, 37°C, 5% CO 2 Cultured under these conditions. After 7 days of culture, Dynabeads TM ReguLatory CD4 + / CD25 + T Cell Kit (Thermo Fisher, 11363D) was used to isolate cells to obtain activated CD4 + CD25 + Treg cells, and Treg cells with a FOXP3 positive rate greater than 90% were obtained.

[0249] Fresh PBMC was enriched respectively using CD4 + Cell Negative Selection Enrichment Kit cells and CD8 + T Cell Screening Kit for enrichment, and highly pure CD4 + and CD8 + effector T cells were isolated, with a cell purity greater than 90%. And magnetic beads coated with anti-CD3 / CD28 antibodies were added to activate and expand the cells. After 3 days, effector CD4 + T cells with high CD25 expression and effector CD8 + T cells were obtained.

[0250] The in vitro-induced Treg cells and in vitro-activated CD4 + and CD8 + effector T cells were respectively added to different dilution concentrations of the test antibody and incubated on ice for 1 h, then washed with PBS. Then, goat anti-human antibody Alexa Fluor488 was added for ice bath staining for 1 h, washed with PBS, and 200 μL of flow cytometry detection solution was added to each well for simultaneous detection on the machine under the same conditions.

[0251] The results were as Figures 6A - 6G shown. All the detected chimeric antibodies could bind to the CD25 antigen on Treg cells, activated CD4 + and CD8 + effector T cells. However, in terms of the binding degree, the binding ability to Treg was much greater than that to CD4 + T cells, and the binding to CD4 + T cells was slightly greater than that to CD8 + T cells.

[0252] Example 9. Effect of Chimeric Anti-CD25 Antibody on the Binding Ability of IL-2 and Its Receptor

[0253] Collect SU-DHL-1 cells in the logarithmic growth phase and incubate them with 200 nM IL-2-biotin and different concentrations of the chimeric anti-CD25 antibody to be tested at 4°C for 1 h. Then wash the cells once with PBS. Add Alexa Fluor 488 Streptavidin (1:1000) and incubate at 4°C for 1 h. Remove the excess secondary antibody with PBS and detect the fluorescent cells by FACS. If the antibody affects the binding of IL-2 and CD25, the fluorescence intensity weakens.

[0254] Use the following formula to calculate the inhibition rate:

[0255] Inhibition rate % = 100 × (maximum binding fluorescence intensity without antibody - fluorescence intensity after antibody binding) / binding intensity without antibody.

[0256] The results are as Figures 7A - 7B shown. DAC, cAb001, cAb002, cAb028, and cAb029 inhibit the binding of IL-2 to SU-DHL-1 to varying degrees, indicating that the above chimeric antibodies inhibit the IL-2 signaling pathway. However, cAb006, cAb037, cAb042, and cAb046 do not inhibit the binding of IL-2 to SU-DHL-1, similar to Tab006, indicating that the above chimeric antibodies are non-antagonistic antibodies.

[0257] Example 10. Cell binding of humanized anti-CD25 antibody to SUDH-L1

[0258] Use the flow cytometry method in Example 7 to detect the binding of the chimeric anti-CD25 antibody to SU-DHL-1 cells. Figure 8 It shows that the humanized anti-CD25 antibodies RP006, PR031, PR058, and PR071 of the present disclosure all have good SU-DHL-1 binding activity.

[0259] Example 11. Effect of humanized anti-CD25 antibody on the pStat5 signaling pathway of human peripheral blood T lymphocytes

[0260] As the α chain of the IL-2 cytokine receptor, CD25 forms a high-affinity receptor with the β and γ chains. After binding to IL-2, it activates the downstream JAK-STAT, PI3K-AKT, and MAPK signaling pathways. Therefore, further detect the effect of the chimeric antibody on the upregulation of phosphorylated STAT-5 by IL-2 in PBMC.

[0261] Resuscitate cryopreserved PBMC and co-incubate the humanized anti-CD25 antibody with PBMC (37°C, 5% CO 2)。After 30 minutes, 100 U / mL of IL-2 was added respectively and induced for 10 minutes. After activation, the PBMCs were fixed, 200 μL of staining solution (PBS containing 2% FBS) was added to each well, 5 μL of V450 mouse anti-human CD3 was added to label T cells. After washing away the unbound antibody, 300 μl of pre-cooled permeabilization agent Perm Buffer III was added, vortexed and mixed well, and incubated on ice for 30 minutes. Then stained with Alexa 647 mouse anti-Stat5 for 60 minutes. Remove the excess antibody and detect by flow cytometry.

[0262] As Figure 9 shown, DAC can significantly inhibit the phosphorylation of Stat5 in CD3 cells induced by IL-2, while Tab06 cannot inhibit the phosphorylation of Stat5 induced by IL-2. PR006 can weakly inhibit the phosphorylation of Stat5 at 300 nM and 100 nM, and PR031, PR058, and PR071 do not inhibit the phosphorylation of Stat5 mediated by IL-2.

[0263] Example 12. ADCC effect of humanized anti-CD25 antibody mediated on SU-DHL-1 cells

[0264] Antibody-mediated cytotoxic killing (ADCC) is an important function of antibody therapy. It is our expectation to activate tumor immunity by killing Treg cells with high expression of CD25 through the activity of ADCC. The ADCC activity of the humanized anti-CD25 antibody of the present disclosure was detected as follows.

[0265] ADCC reporter gene cells (Promega G7010 ADCC Bioassay E ector Cells) were mixed with SU-DHL-1 cells at a ratio of 1:1, and after incubation with different concentrations of antibody for 6 hours, the fluorescence activity of the cells was detected. From Figure 10 and Table 9, it can be seen that PR006, PR031, PR058, and PR071 all have good ADCC activity. Among them, the activities of PR058 and PR071 are significantly stronger than those of antibody 686.

[0266] The sequence of antibody 686 can be found in Sequence 5 and Sequence 9 of US20190284287A.

[0267] Table 9. ADCC activity of humanized antibodies

[0268]

[0269] Example 13. Antitumor activity of humanized anti-CD25 antibody mediated on the MC38 xenograft tumor model of hCD25 mice

[0270] The anti-tumor activity of the humanized anti-CD25 antibody was evaluated in a MC38 colon cancer mouse model of B-hIL2RA humanized mice. The variable regions of the aforementioned PR058 and 686 were ligated to murine IgG2a to construct PR058 mIgG2a and 686 mIgG2a antibodies for animal experiments.

[0271] MC38 colon cancer cells resuspended in PBS were inoculated subcutaneously into the right side of B-hIL2RA humanized mice at a concentration of 5×10 5 cells / 0.1 mL and a volume of 0.1 mL / mouse. When the average tumor volume reached approximately 113 mm 3 , 48 mice with appropriate individual tumor volumes were randomly divided into 6 groups of 8 mice each. The groups were: G1 isotype mIgG2a (3 mg / kg) control group, G2 PR058 mIgG2a (3 mg / kg) group, and G3 686 mIgG2a (3 mg / kg) group. The administration route for all groups was intraperitoneal injection, once a week for 3 consecutive times, and the experiment ended 7 days after the last administration. The tumor volume and body weight were measured 2 times a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the animals were euthanized, the tumors were dissected, weighed, photographed, and the relative tumor inhibition rate (TGI%) was calculated.

[0272] The research results are as Figure 11A shown. At the 3 mg / kg dose level, PR058 had the optimal inhibitory effect on the growth of MC38 subcutaneous xenografts, which was superior to 686. Figure 11B It was shown that the experimental animals were in good condition in terms of activity and food intake during the drug administration period, and the body weights of the experimental animals all increased to a certain extent, indicating that the test drug did not have obvious toxic effects on the experimental animals and had good safety. As Figure 12A , Figure 12B the results of intratumoral lymphocyte analysis showed that PR058 could more strongly mediate Treg killing and upregulation of the number of CD3. Among them, * indicates p < 0.05, with statistical difference compared with the mIgG2a group; ** indicates p < 0.01, with significant statistical difference compared with the mIgG2a group; *** indicates p < 0.001, with extremely significant statistical difference compared with the mIgG2a group; ns indicates no statistical difference compared with the mIgG2a group.

[0273] Although the specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are only examples. Without departing from the principles and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope of the present disclosure is defined by the appended claims. Sequence Listing <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Shengdi Pharmaceutical Co., Ltd. <120> Anti-CD25 Antibody, Its Antigen-Binding Fragment and Pharmaceutical Use <130> 721051CPCT <150> CN202010408502.3 <151> 2020-05-14 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CAB001-VH <400> 1 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Phe 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Val Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Thr Thr Glu Val Gly Asn Tyr Asp Val Trp Gly Met 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 108 <212> PRT <213> Artificial sequence <220> <223> CAB001-VL <400> 2 Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Gly Ile Thr Ser Asn 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ile Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CAB002-VH <400> 3 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe 20 25 30 Gly Leu Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala Gln Ile Trp Trp Asp Asp Asp Glu Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Asn Val Asp Thr Gly Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Met Gly Asp Thr Tyr Trp Tyr Phe Asp Val Trp Gly Thr 100 105 110 Gly Thr Thr Val Ala Val Ser Ser 115 120 <210> 4 <211> 108 <212> PRT <213> Artificial sequence <220> <223> CAB002-VL <400> 4 Glu Ile Val Val Thr Gln Ser Pro Ala Leu Met Ala Val Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Leu Ser Ser Gly Ile Thr Ser Ser 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser 50 55 60 Gly Ser Gly Ser Lys Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> CAB004-VH <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Asp 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Thr Phe Tyr Ala Ser Asn Tyr Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CAB004-VL <400> 6 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Leu Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 119 <212> PRT <213> Synthetic Sequence <220> <223> CAB006-VH <400> 7 Gln Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Gly His 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asn Lys Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Glu Thr Ser Ser Asn Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Gly Pro His Trp Pro Phe Ala Phe Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ala 115 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> CAB006-VL <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asn Val His 20 25 30 Gly Ala His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Ile Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Phe Cys Gln Gln Thr Ile 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CAB028-VH <400> 9 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asn Asp 20 25 30 Tyr Val His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Asp Asn Gly Asp Thr Glu Tyr Ala Ser Lys Phe 50 55 60 Leu Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Gly Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Thr Asp Asp Thr Ser Tyr Gly Met Phe Ala Cys Trp Gly Leu Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CAB028-VL <400> 10 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CAB029-VH <400> 11 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Glu Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Thr Arg Ile Met Leu Thr Thr Gly Tyr Phe Asp Val Trp Gly Thr 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 108 <212> PRT <213> Artificial sequence <220> <223> CAB029-VL <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Thr Val Ser Ser Gly Ile Thr Ser Ser 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Phe Thr Phe Gly Ser Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 13 <211> 122 <212> PRT <213> Artificial sequence <220> <223> CAB037-VH <400> 13 Gln Val Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Phe Pro Gly Asn Gly Asn Thr Tyr Phe Asn Glu Asn Phe 50 55 60 Lys Asp Lys Ala Thr Leu Ser Val Asp Lys Phe Ser Ser Thr Ala Phe 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ala Thr Tyr Asp His Gly Gly Tyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 14 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> CAB037-VL <400> 14 Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser His Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Val Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Asn Ser Phe Thr Phe Gly 85 90 95 Gly Gly Thr Glu Leu Glu Leu Lys 100 <210> 15 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CAB042-VH <400> 15 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Asp Ile Asn Trp Met Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Glu Asp Thr Ser Ser Asn Thr Ala Phe 65 70 75 80 Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Arg Ile Tyr Asp Gly Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 16 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> CAB042-VL <400> 16 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Ser Met Arg Cys Lys Ser Ser Gln Arg Leu Phe Asn Ser 20 25 30 Arg Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ile Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 17 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CAB046-VH <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser His Leu Lys Ser Glu Asp Thr Ala Met Tyr Leu Cys 85 90 95 Val Arg Asp Asn Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Thr 115 <210> 18 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CAB046-VL <400> 18 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Arg Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 19 <211> 119 <212> PRT <213> Artificial sequence <220> <223> PR0006-VH <400> 19 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly His 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asn Lys Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Gly Pro His Trp Pro Phe Ala Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 20 <211> 111 <212> PRT <213> Artificial sequence <220> <223> PR0006-VL <400> 20 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asn Val His 20 25 30 Gly Ala His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Ile Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Ile 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 21 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> PR0071-VH <400> 21 Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Ala Trp Ile Phe Pro Gly Asn Gly Asn Thr Tyr Phe Asn Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Phe Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Thr Tyr Asp His Gly Gly Tyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 22 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> PR0071-VL <400> 22 Glu Ile Val Leu Ser Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser His Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Val Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Asn Ser Phe Thr Phe Gly 85 90 95 Gly Gly Thr Lys Val Glu Ile Lys 100 <210> 23 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> PR0031-VH <400> 23 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Asp Ile Asn Trp Met Lys Gln His Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Glu Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ile Tyr Asp Gly Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 24 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> PR0031-VL <400> 24 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Arg Leu Phe Asn Ser 20 25 30 Arg Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ile Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 25 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> PR0058-VH <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Val Arg Asp Asn Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> PR0058-VL <400> 26 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 27 <211> 5 <212> PRT <213> Artificial sequence <220> <223> PR0006-HCDR1 <400> 27 Gly His Trp Ile Glu 1 5 <210> 28 <211> 17 <212> PRT <213> Artificial sequence <220> <223> PR0006-HCDR2 <400> 28 Glu Ile Leu Pro Gly Ser Asn Lys Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> PR0006-HCDR3 <400> 29 Gly Gly Gly Pro His Trp Pro Phe Ala Phe 1 5 10 <210> 30 <211> 15 <212> PRT <213> Artificial sequence <220> <223> PR0006-LCDR1 <400> 30 Arg Ala Ser Glu Ser Val Asn Val His Gly Ala His Leu Met His 1 5 10 15 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> PR0006-LCDR2 <400> 31 Ala Ala Ser Ile Leu Glu Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> PR0006-LCDR3 <400> 32 Gln Gln Thr Ile Glu Asp Pro Arg Thr 1 5 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <220> <223> PR0071-HCDR1 <400> 33 Asp Tyr Tyr Ile Asn 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <220> <223> PR0071-HCDR2 <400> 34 Trp Ile Phe Pro Gly Asn Gly Asn Thr Tyr Phe Asn Glu Asn Phe Lys 1 5 10 15 Asp <210> 35 <211> 13 <212> PRT <213> Artificial sequence <220> <223> PR0071-HCDR3 <400> 35 Ala Thr Tyr Asp His Gly Gly Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> PR0071-LCDR1 <400> 36 Arg Ala Ser Ser Ser Val Ser His Ile His 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> PR0071-LCDR2 <400> 37 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> PR0071-LCDR3 <400> 38 Gln His Trp Asn Ser Phe Thr 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial sequence <220> <223> PR0031-HCDR1 <400> 39 Thr Tyr Asp Ile Asn 1 5 <210> 40 <211> 17 <212> PRT <213> Artificial sequence <220> <223> PR0031-HCDR2 <400> 40 Trp Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Ala Lys Phe Lys 1 5 10 15 Gly <210> 41 <211> 10 <212> PRT <213> Artificial sequence <220> <223> PR0031-HCDR3 <400> 41 Glu Arg Ile Tyr Asp Gly Ser Phe Asp Tyr 1 5 10 <210> 42 <211> 17 <212> PRT <213> Artificial sequence <220> <223> PR0031-LCDR1 <400> 42 Lys Ser Ser Gln Arg Leu Phe Asn Ser Arg Asp Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 43 <211> 7 <212> PRT <213> Artificial sequence <220> <223> PR0031-HCDR2 <400> 43 Gly Ala Ser Thr Arg Glu Ser 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> PR0031-LCDR3 <400> 44 Gln Asn Asp His Ile Tyr Pro Leu Thr 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> PR0058-HCDR1 <400> 45 Ser Tyr Ala Met Ser 1 5 <210> 46 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PR0058-HCDR2 <400> 46 Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> PR0058-HCDR3 <400> 47 Asp Asn Pro Trp Phe Ala Tyr 1 5 <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> PR0058-LCDR1 <400> 48 Arg Ser Ser Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> PR0058-LCDR2 <400> 49 Lys Val Ser Lys Arg Phe Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> PR0058-LCDR3 <400> 50 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 <210> 51 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> PR0006-H <400> 51 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly His 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asn Lys Ile Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Gly Pro His Trp Pro Phe Ala Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 52 <211> 218 <212> PRT <213> Artificial sequence <220> <223> PR0006-L <400> 52 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asn Val His 20 25 30 Gly Ala His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Ile Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Ile 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 53 <211> 452 <212> PRT <213> Artificial sequence <220> <223> PR0071-H <400> 53 Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Ala Trp Ile Phe Pro Gly Asn Gly Asn Thr Tyr Phe Asn Glu Asn Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Phe Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Thr Tyr Asp His Gly Gly Tyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 54 <211> 211 <212> PRT <213> Artificial sequence <220> <223> PR0071-L <400> 54 Glu Ile Val Leu Ser Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser His Ile 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Val Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Asn Ser Phe Thr Phe Gly 85 90 95 Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 100 105 110 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 115 120 125 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 130 135 140 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 145 150 155 160 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 165 170 175 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 180 185 190 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 195 200 205 Gly Glu Cys 210 <210> 55 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> PR0031-H <400> 55 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Asp Ile Asn Trp Met Lys Gln His Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Arg Asp Gly Ser Thr Lys Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Glu Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ile Tyr Asp Gly Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 56 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> PR0031-L <400> 56 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Arg Leu Phe Asn Ser 20 25 30 Arg Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ile Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 57 <211> 446 <212> PRT <213> Artificial sequence <220> <223> PR0058-H <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Val Arg Asp Asn Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 58 <211> 219 <212> PRT <213> Artificial sequence <220> <223> PR0058-L <400> 58 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 59 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> PR0157-VH <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Val Arg Asp Asn Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 60 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> PR0157-VL <400> 60 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 61 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> PR0157-H <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Glu Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys 85 90 95 Val Arg Asp Asn Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 62 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> PR0157-L <400> 62 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. An anti-CD25 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: 1) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 17, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 18; 2) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2; 3) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 3, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 4; 4) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 5, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 6; 5) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 7, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 8; 6) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 9, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 10; 7) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 11, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 12; 8) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 13, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 14; or 9) the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 15, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 16; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

2. An anti-CD25 antibody or antigen-binding fragment thereof, comprising VH and VL, wherein: 1) the VH comprises HCDR1, HCDR2, HCDR3 shown as SEQ ID NO: 45, 46, 47 respectively, and the VL comprises LCDR1, LCDR2, LCDR3 shown as SEQ ID NO: 48, 49, 50 respectively; 2) The VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 27, 28, and 29 respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31, and 32 respectively; 3) The VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 33, 34, and 35 respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 36, 37, and 38 respectively; or 4) The VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 39, 40, and 41 respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 42, 43, and 44 respectively.

3. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the anti-CD25 antibody is a murine antibody, a chimeric antibody, a human antibody, or a humanized antibody.

4. The anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein: 1) The VH comprises FR1 to FR3 selected from IGHV1-46*01 and FR4 selected from IGHJ1*01, and the VL comprises FR1 to FR3 selected from IGKV4-1*01 and FR4 selected from IGKJ4*01; 2) The VH comprises FR1 to FR2 selected from IGHV1-18*01, FR3 selected from IGHV1-69*02, and FR4 selected from hIGHJ6*01_14, and the VL comprises FR1 selected from IGKV3-11*01, FR2 selected from IGKV5-2*01, FR3 selected from IGKV6-21*01, and FR4 selected from hIGKJ4*01_12; 3) The VH comprises FR1 selected from IGHV1-18*01, FR2 selected from IGHV4-31*01, FR3 selected from IGHV1-3*01, and FR4 selected from hIGHJ6*01, and the VL comprises FR1 to FR3 selected from IGKV4-1*01 and FR4 selected from hIGKJ2*01; or 4) The VH comprises FR1 to FR3 selected from IGHV3-23*04 and FR4 selected from IGHJ1*01, and the VL comprises FR1 to FR3 selected from IGKV2-28*01 and FR4 selected from IGKJ4*01.

5. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: the VH is as shown in SEQ ID NO: 25 or has at least 90% sequence identity therewith, and the VL is as shown in SEQ ID NO: 26 or has at least 90% sequence identity therewith; The VH is as shown in SEQ ID NO: 59 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 60 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 1 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 2 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 3 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 4 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 5 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 6 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 7 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 8 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 9 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 10 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 11 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 12 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 13 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 14 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 15 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 16 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 17 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 18 or has at least 90% sequence identity thereto; The VH is as shown in SEQ ID NO: 19 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 20; The VH is as shown in SEQ ID NO: 21 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 22 or has at least 90% sequence identity thereto; or The VH is as shown in SEQ ID NO: 23 or has at least 90% sequence identity thereto, and the VL is as shown in SEQ ID NO: 24 or has at least 90% sequence identity thereto.

6. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is a scFv, Fv, Fab or Fab' fragment.

7. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-CD25 antibody is an IgG antibody.

8. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 7, wherein the anti-CD25 antibody has a defucosylation site in the Fc region to enhance the binding ability to FcγRIIIa and / or reduce the binding ability to FcγRIIb.

9. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 8, wherein the defucosylation site is at position 297.

10. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain and a light chain, wherein: 1) The heavy chain is as shown in SEQ ID NO: 51 or has at least 80% sequence identity therewith, and the light chain is as shown in SEQ ID NO: 52 or has at least 80% sequence identity therewith; 2) The heavy chain is as shown in SEQ ID NO: 53 or has at least 80% sequence identity therewith, and the light chain is as shown in SEQ ID NO: 54 or has at least 80% identity therewith; 3) The heavy chain is as shown in SEQ ID NO: 55 or has at least 80% sequence identity therewith, and the light chain is as shown in SEQ ID NO: 56 or has at least 80% sequence identity therewith; 4) The heavy chain is as shown in SEQ ID NO: 57 or has at least 80% sequence identity therewith, and the light chain is as shown in SEQ ID NO: 58 or has at least 80% sequence identity therewith; or 5) The heavy chain is as shown in SEQ ID NO: 61 or has at least 80% sequence identity therewith, and the light chain is as shown in SEQ ID NO: 62 or has at least 80% sequence identity therewith.

11. The anti-CD25 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, the anti-CD25 antibody or antigen-binding fragment thereof has at least one of the following characteristics: (a) The KD value of the combination with human CD25 is less than 1×10 -7 M; (b) Does not inhibit or substantially does not inhibit the binding of IL-2 to CD25; (c) Depletes tumor-infiltrating Tregs without affecting or substantially not affecting the function of Teffs; (d) Binds to Fcγ receptors with an activation inhibition rate (A / I) higher than 1; (e) Binds to FcγRIIIa with a higher affinity than binding to FcγRI, FcγRIIc, and / or FcγRIIb; (f) Inhibits tumor growth; and (g) Induces an enhanced CDC, ADCC, and / or ADCP response.

12. A polynucleotide encoding the anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1-11.

13. A vector containing the polynucleotide according to claim 12.

14. A host cell comprising the vector according to claim 13.

15. The host cell according to claim 14, wherein, the host cell is a bacterium, yeast, or mammalian cell.

16. The host cell according to claim 14, wherein, The host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

17. A method for preparing an anti-CD25 antibody or an antigen-binding fragment thereof, comprising: expressing the anti-CD25 antibody or an antigen-binding fragment thereof according to any one of claims 1-11 in the host cell according to any one of claims 14-16, and isolating the anti-CD25 antibody or an antigen-binding fragment thereof from the host cell.

18. A pharmaceutical composition, comprising: the anti-CD25 antibody or an antigen-binding fragment thereof according to any one of claims 1-11; and one or more pharmaceutically acceptable excipients, diluents or carriers.

19. Use of any one selected from the following or any combination thereof in the preparation of a drug or a pharmaceutical composition for treating cancer: the anti-CD25 antibody or an antigen-binding fragment thereof according to any one of claims 1-11 and the pharmaceutical composition according to claim 18.

20. The use according to claim 19, wherein the cancer or tumor is selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), gastrointestinal cancer, renal cancer, ovarian cancer, lymphocytic leukemia, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, cervical cancer, brain cancer, bladder cancer, liver cancer, breast cancer and head and neck cancer.

21. A kit for detecting CD25, comprising the anti-CD25 antibody or antigen-binding fragment thereof according to any one of claims 1-11.

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